Dynamics of memory cell subsets in human tonsils with age: impact on the functional reconfiguration of the organ

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Paula de la Guardia, Ignacio Rojas Campión, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8643189/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background. The palatine tonsils are lymphoepithelial structures in which immune responses to inhaled and ingested pathogens are mounted. While we have previously shown that tonsillar immune effector function declines with age, the impact of such erosion on the local viral reservoirs or in specific memory responses remains to be defined. Methods. We performed phenotypic and functional characterization of tonsillar mononuclear cells (TMC) and tissue biopsies from a cohort of around 80 patients (ages 1–45). We quantified the proportion of particular lymphoid subsets associated with ageing through flow cytometry. We also assessed the expression of LMP-1 and EBNA-2, latency proteins of Epstein-Barr virus (EBV), via immunohistochemistry. Finally, we functionally tested the specific response of tonsillar memory B cells distantly induced by tetanus toxoid (TT) injection, through ELISA on the supernatant of TT- stimulated TMC cultures. Results. We demonstrated a significant age-dependent decline in the B cell proportion balanced by a concomitant rise in the T cell fraction. This shift was complemented by the accumulation of particular phenotypes associated with aged B cells and senescent T cells. Despite these alterations, tonsillar samples from older patients presented a more effective repression of EBV viral antigens (Ag) than those of younger children. We also established that TMC from all patients tested harboured responsive TT specific memory B cells and moreover, anti-TT Ig production correlated with the time since the last vaccination. Conclusion . Our findings suggest that tonsillar involution constitutes a functional transition from a germinal centre driven secondary lymphoid organ in childhood to a memory dominated, immunoregulatory reservoir in adulthood. This reconfiguration preserves local viral control and serves to integrate both local and systemic immunological memory. tonsils mucosa immunity aging memory latency Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background The palatine tonsils are positioned at the confluence of the digestive and the respiratory tracts. They function as secondary lymphoid organs that coordinate the first response to inhaled and ingested pathogens. The percentage of germinal centre (GC) lymphoid populations is reduced in tonsils of adolescents and adults when compared to those from younger children. Additionally, younger tonsils render a higher proportion of proliferative immune cells than those from older ones ( 1 ). These observations are associated with poor protective immunity in infections which in turn reflects in a temporal pattern in the cause of surgery. Tonsillar hypertrophy (TH) is the major motive for tonsillectomy in children below 10 years old while recurrent tonsillitis (RT) stands as the primary reason for the indication of surgery from puberty onwards ( 2 ). The clinical significance of TH stems from obstruction. These children present a failure of immune homeostatic mechanisms caused by the local loss of the capacity to discriminate between commensals and pathogens that leads to persistent inflammation and follicular hyperplasia ( 3 ) ( 4 ). Conversely, RT is defined by a specific number of annual infections presenting with systemic and local symptoms including fever, sore throat and tonsillar exudate. The teenagers and young adults undergoing surgery due to RT usually have a history of TH but not at a score to cause obstruction. Organisms have evolved mechanisms to regulate local immunity upon years of constant inflammation in a particular tissue, to keep the physiological function of the latter and alleviate immunopathology ( 5 ). We have previously characterized two of these tonsillar age-associated regulatory mechanisms. Aging correlated with an increment in the percentage of tonsillar B cells secreting IL10, the classical regulatory B cell population (Breg) ( 3 ) and with a non-classical Breg subset, phenotyped as CD20 + CD39 high CD73 + , as well ( 1 ). Hence, we hypothesized that there is a gradual re-configuration of the physiological function of the tonsils as the patients age, from representing a major site of B cell effector mechanisms targeted to reduce pathogen burden in childhood to a memory reservoir where regulatory mechanisms become prevalent beyond puberty. The coordinated cellular shifts underlying such transition were partly determined, and mainly focused on germinal centre (GC) dynamics ( 1 ). It is unclear how the relative proportions of the major tonsillar lymphoid subsets along with their various memory and activated phenotypes, change from childhood until adulthood. Moreover, the impact of such an evolving microenvironment on the functional capacity to maintain control over widespread local latent infections, such as those caused by the Epstein-Barr Virus (EBV), has not yet been studied. In the context of the tonsils’ role as a memory B cell pool, it remains to be determined whether they preserve immunological memory initially generated through parenteral vaccination, and the duration of this maintenance. In this study, we addressed these issues through phenotypic and functional characterization of tonsillar mononuclear cells (TMC) and tissue biopsies across a broad age cohort (1 to 45 years old). We found a significant age-dependent decline in the overall B cell proportion balanced by the concomitant rise in the T cell compartment. This shift was accompanied by the accumulation of specific phenotypes associated with immune aging and chronic activation, including the so-called aged or atypical memory B cells (ABC) and HLA-DR + CD3 + cells. Despite the rise of these senescent subsets, our data did not provide evidence of a failure of local viral control. Finally, we demonstrated that the tonsils function as an accessory immune reservoir harbouring memory B cells’ specific for a parenterally administered antigen (tetanus toxoid, TT), underscoring their role in integrating both local and systemic immunity even after the onset of the decline in effector immune mechanisms (around 10 years old). Taken together, these findings corroborate that the well documented tonsillar involution with age represents in fact a functional transition towards an immunoregulatory microenvironment, memory-dominated vigilant state. Material and methods Patient recruitment and collection of samples Tonsillar mononuclear cells (TMC) were purified from patients aged from 1 to 45 years old undergoing tonsillectomy. Patients under 18 years old were recruited from Clinical Hospital “José de San Martín”, whereas adults were enrolled from the Institute of Otolaryngology Arauz. The number of patients enrolled in each experiment are detailed from Table 1 to Table 9. Patients who presented any kind of immunodeficiency, or had undergone medical treatment (antibiotics, corticoids) one month prior to the surgery were excluded. The institutional ethics committee (Clinical Hospital, School of Medicine, Buenos Aires and Institute of Otolaryngology Arauz, Buenos Aires) approved the collection and use of clinical material, conformed to the provisions of the Declaration of Helsinki (as revised in Edinburgh 2000). Informed consent was obtained from all participants and/or their legal guardian/s. Isolation of tonsillar mononuclear cells The organs were collected in saline solution (0.9% NaCl). Prior to cell isolation, a tissue section was cut off and formaldehyde-fixated in order to perform immunostainings. TMC were recovered from the organ by injecting phosphate saline buffer supplemented with 10% inactivated fetal calf serum (supplemented PBS) through the tonsils. TMC were then isolated using density gradient centrifugation employing Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden). The collected TMC from the halo were washed and cells counted in a Neubauer camera. Viable cells proportion, determined by Trypan Blue staining, was always greater than 95%. Flow cytometry of fresh isolated cells Cells were assessed for their expression of different surface markers using flow cytometry. 1x10 6 cells were stained with fluorochrome conjugated monoclonal antibodies: CD19 (PerCP; BioLegend, clone SJ25C1), CD20 (FITC; BioLegend, clone L27), CD27 (APC; BD Pharmigen, clone M-271), CD11c (PE; BioLegend, clone 3.9), CD3 (APC-Cy7; BD Pharmigen, clone SK7, or PE-CF594; BD Horizon, clone UCHT1), HLA-DR (FITC; BioLegend, clone LN3), CD4 (PerCP; BioLegend, clone SK3), CD8a (Pacific Blue; BioLegend, clone HIT8a), CD45RA (APC; BD Pharmigen clone HI100), CD45RO (PE; BD Pharmigen, clone UCHL1), CXCR5 (Alexa Fluor 488; BD Pharmigen, clone RF8B2) and PD-1 (Brilliant Violet 711; BioLegend, clone EH12.2H7). Parallely, corresponding compensation and isotype controls were performed with monoclonal antibodies purchased from BD Biosciences (CA, USA) and Biolegend (CA, USA). After a 30 minutes incubation, cells were washed and resuspended in flow cytometry buffer (10% fetal calf serum in PBS 1x). Intracellular cytometry was performed to detect T-bet transcription factor, cells were incubated with Fixation/Permeabilization (eBioscience FOXP3/Transcription, Invitrogen) for 45 min and washed with Permeabilization Buffer (eBioscience FOXP3/Transcription, Invitrogen). Then, the cells were stained with an anti-T-bet antibody (Brilliant Violet 650; BioLegend, clone 4B10). Cells were acquired using FACSAria II (BD Biosciences, CA, USA) and analyzed with FlowJo™ software version 10.0 (Treestar, OR, USA). Single stained controls were used to set compensation parameters. Fluorescence minus one and isotype-matched Ab controls were used to set analysis gates. Gating strategy for each cell population is depicted either in supplementary or respective figures. Immunohistochemistry for lymphoid populations Formalin fixated and paraffin embedded (FFPE) tissue sections were cut in 5 µm slices and mounted in silane coated glasses. Slides were stained for immunohistochemistry. Sections were deparaffinized and re-hydrated via a xylene/ethanol gradient, consisting of a pair of 10 minute incubations in xylene, followed by sequential 3 minute incubations in ethanol 100%, 96%, 70% and distilled water. Slides were then washed twice for 5 minutes in saline phosphate buffer (PBS); all the subsequent washing steps were performed in the same way. Antigen retrieval was then performed: samples were soaked in Sodium Citrate Buffer (0,01 M, pH 6) and heated up to 95º C in a microwave (500–600 w), for 30 minutes. Once cold, tissue sections were washed and permeabilized for 20 minutes employing 0,5% Triton X-100 (Sintorgan, Buenos Aires, Argentina) in PBS 1X. Slides were then washed, and endogenous peroxidase was blocked by incubating the slides in 3% H 2 O 2 in methanol, for 30 min in darkness. After incubation, sections were washed and nonspecific binding sites were blocked with Bovine Serum Albumin (BSA. Sigma, MO, USA) 1% in PBS. Samples were incubated overnight with antibodies for human CD3 and CD20 (clones MRQ-39 and SP32, respectively, Cell Marque, Darmstadt, Germany) lymphocyte surface markers and for Epstein-Barr proteins LMP-1 (clones CS.1–4, Dako, CA, USA) and EBNA-2 (clone PE2, Abcam, Cambridge, UK). After incubation, slides were washed and the corresponding HRP-conjugated secondary antibodies were added. Finally, after 45 minutes, tissue sections were incubated with 3, 3’-diaminobenzidine tetrahydrochloride (DAB. Sigma, MO, USA) until brown precipitate was detected. Incubation was stopped with tap water, and slides were counterstained with hematoxylin. Samples were dehydrated with an ethanol:xylene gradient, and mounted with synthetic Canada balsam (Stanton, Buenos Aires, Argentina). In parallel, controls for unspecific binding were performed, replacing primary antibodies isotype controls diluted in blocking buffer. Slides were examined using a Leica DM500 (Leica, Germany), for CD20 and CD3 stainings, or with a Zeiss Axio Scope A1 optical microscope (Carl Zeiss, Germany) for LMP-1 and EBNA-2. Pictures were acquired with a Leica ICC50 W camera at 40X, 100X and 400X magnifications, or a 503 color Axiocam camera, at 200X, 400X and 1000X magnifications, respectively. For follicular area measurement, images were analyzed employing Image J software. Areas are expressed in square millimeters. Enumeration of LMP-1 and EBNA-2 expressing cells FFPE tissue sections were processed like above. Images were analyzed at 200X, 400X and 1000X magnifications. Positive cells for each viral protein were counted employing the corresponding microscopes softwares, LAS EZ 3.6 and ZEN 4.3. For LMP-1, a cell was considered positive only if the mark was observed within the cytoplasm, whereas for EBNA-2, the mark had to be located on the nucleus. In neither case clustered cells were counted. For each protein, a patient with a minimal count of two positive cells per slide was considered positive. Positive controls were performed on biopsies from Hondgkin’s lymphoma for LMP-1, and from post-transplant lymphoproliferative disorders for EBNA-2. Cell cultures Cells were cultured at 1x10 6 cells/mL in IMDM medium supplemented with 10% heat-inactivated fetal calf serum, 2mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES), 1 mM sodium pyruvate and 50 µM 2-mercaptoethanol (all from Invitrogen, CA, USA). B lymphocytes’ survival signals, human IL4 (20 ng/ml; R&D Systems, MN, USA) and human recombinant CD40L (250 ng/ml; R&D Systems, MN, USA) were added, and stimulation with recombinant Tetanus toxoid (5 µg/ml; Argentinian Biological Institute S.A.I.C., Buenos Aires, Argentina) was performed, when indicated. Cells were seeded in 48 well flat bottom plates (0,5 mL/well), and cultured at 37ºC, 5% CO 2 , humid atmosphere for three days. After incubation, culture supernatants were recovered and stored at -20ºC, for the following determinations. ELISA assay for specific antibodies against Tetanus toxoid . In order to measure the specific antibodies against Tetanus toxoid, a home-made ELISA was set up. 96 well high binding plates were coated with Tetatus toxoid (5 µg/ml; Argentinian Biological Institute S.A.I.C., Buenos Aires, Argentina) and incubated over-night at 4ºC. Positive controls assessing total IgGs were performed by coating with anti-IgG (5 µg/ml; BD Biosciences). Afterwards, plates were repeatedly washed with a 0,05% Tween-20 in PBS 1x buffer, and blocked with 10% ultra-low IgG fetal calf serum in PBS 1x, pH 7 (Sigma, MO, USA). All the subsequent incubations were followed by the same washing step described. After 1 hour at RT, supernatants were incubated for 2 hours at 37ºC. In parallel, negative controls were performed adding blocking buffer. For the detection step, biotinylated anti-kappa light chain antibody was added (5 µg/ml; BD Biosciences, CA, USA) and incubated for 1 hour at RT. Then, plates were incubated in darkness for 30 minutes with streptavidin conjugated horseradish peroxidase (HRP. BD OptEIA™, CA, USA). Lastly, the assay was revealed with 3,3',5,5'-Tetramethylbenzidine (TMB. BD OptEIA™, CA, USA): once light blue color had developed, the enzymatic reaction was stopped with sulfuric acid, and absorbance at 450 nm was measured. In every case, blank absorbance at 450 nm was subtracted from the raw measurement. For every patient, two measurements were performed. Statistical analysis The data were analyzed using Graphpad Prism 8.0.2 software. Prior to any analysis, Shapiro-Wilk’s normality test was performed. Pearson parametric or Spearman non-parametric correlation tests were employed according to normality test results. For demographic description of each study population, mean and standard deviation were calculated. Comparisons between two groups from different patients were performed with a parametric unpaired t test, whereas a parametric paired t test was performed when two groups with matching samples were analyzed. All tests were two-tailed, and p < 0.05 was considered statistically significant. Results Dynamics of tonsillar lymphocytes subsets with age We have recently shown that the tonsillar GC B cell population steadily decreases with increasing age in the surgical samples we work with ( 1 ). In order to extend our findings, we qualitatively assessed immunohistochemistry staining for CD20 and CD3 expression in biopsies of different ages (Fig. 1 A, B). We observed a greater size of B cell follicles in children´s biopsies as compared to the adult´s ones (Fig. 1 C). Conversely, older biopsies displayed a relative volume up of the inter or extra-follicular areas (EF) stained with anti-CD3 relative to the younger ones (Fig. 1 A, B). To quantify such changes in cellularity, the TMC extracted from the samples of patients aged between 1 and 45 years old (N = 88, Table 1) were analyzed by flow cytometry (gating strategy in Suppl. Figure 1) and CD20 + cells were scored and plotted against age (Fig. 2 ). As expected, the proportion of CD20 + tonsillar B cells declined with age (Fig. 2 A, r=-0.4, p < 0.001) whereas the frequency of CD3 + tonsillar T cells increased accordingly (Fig. 2 B, r = 0.5, p < 0.0001). The following measurements were not performed on all 88 patients due to different circumstances with some samples; the exact N is designated in each figure legend and a number of tables. Being Tfh cells the primary drivers of T cell–dependent GC responses, we also determined the age-related evolution of the different Tfh subsets in terms of their histological localization (germinal center, Tfh GC ; mantle Tfh M and extrafollicular Tfh EF ; gating strategy as in ( 1 )) in the cohort of patients indicated in Table 2. We found a significant negative correlation with age in the proportion of Tfh GC defined as CD3 + CD4 + CXCR5 high PD-1 high cells (Fig. 2 C, r=-0.5, p < 0.0001). On the other hand, Tfh M (CD3 + CD4 + CXCR5 int PD-1 int ) cells (Fig. 2 D, r = 0.6, p < 0.0001) increased with increasing age. Tfh EF (CD3 + CD4 + CXCR5 − PD-1 − ) displayed a tendency towards an increment (Fig. 2 E, r = 0.2, p = 0.1). Moreover, CD3 + CD4 + as a whole population displayed a positive correlation with age (Fig. 3 , r = 0.5, p < 0.0001; Table 3). On the other hand, the proportion of CD3 + CD8 + in TMC decreased with increasing age (Fig. 3 , r=-0.3, p < 0.05; gating strategy in Suppl. Figure 1). Therefore, we confirmed the age-dependent reduction of the B cell mass in this location with a concomitant reduction of the histological compartments occupied by this cell population and the accessory T cell ones. Diverse memory cell subsets accumulate in samples from older patients Tonsillar B mem (defined as CD3 − CD20 + CD10 low CD44 high CD27 + CD38 + as in ( 6 )) increase with age ( 1 ). It has been reported that ABC accumulation contributes to impaired B lymphopoiesis in old mice. We then sought to determine whether there was an age-related increase of the tonsillar ABC population. Thus, TMC from patients at different ages were tested by flow cytometry for ABC identification (see Table 4). ABC were defined as CD3 − CD19 high CD27 + CD11c + Tbet + cells. Single lymphocytes were gated based on forward/side scatter properties. Tonsillar B cells were gated as CD19 + cells and within that region, we scored the CD27 + CD11c + Tbet + cells (Fig. 4 A). We found that the proportion of ABC population increased with increasing age. We established a positive correlation between ABC percentage and the age of the patients (r = 0.5, p < 0.0001, Fig. 4 B). These findings further validate the role of CD19 high CD27 + CD11c + Tbet + cells as a B cell population associated with aging. We have investigated T cell memory as well. We used CD45RA and CD45R0 expression to differentiate naïve and memory T cells (Table 5, Fig. 4 C). While we detected a moderate positive correlation of memory CD8 + T cells with age (r = 0.4, p < 0.005, Fig. 4 D), we did not detect significant changes of memory CD4 + T cells associated to the tonsillar aging process. Tonsillar HLADR + T cells steadily increase with increasing age CD3 + HLADR + are considered as activated T cells with regulatory properties given the fact that they are upregulated in blood in autoimmune disorders ( 7 ) and chronic viral infections ( 8 ). We have previously shown that the declining of the tonsillar GC reaction with ageing and chronic inflammation, correlated with an increment in the proportion of different B cell phenotypes associated with immunosuppressive activity ( 3 ) ( 1 ). In this context, we assessed the tonsillar CD3 + HLADR + cell population in our cohort of patients (Table 6). Single lymphocytes were gated based on forward/side scatter properties, and tonsillar activated T cells were identified as CD3 + HLA DR + cells within the live singlets and lymphocyte gate (Fig. 5 A). We determined that CD3 + HLA DR + increased with age (r = 0.3, p < 0.05; Fig. 5 B). This observation complements our previous findings in relation to the decline of tonsillar effector immune responses over time from puberty onwards ( 1 ) as expression of HLA DR on CD3 + cells has long been related to immune senescence ( 9 ). Impact of the age-related tonsillar lymphocyte shift on a local latent infection Tonsils are replication sites for the EBV virus and viral DNA can be detected by PCR in around 70% of the samples from adults ( 10 ). In children, it has been shown an association between the host age and EBV DNA detection rate ( 11 ) ( 12 ) as most infections occur at this stage of life. In order to test whether the age-related changes in the proportion of tonsillar B and T cell subpopulations have an impact on EBV latency, we determined the expression of the viral proteins LMP-1 and EBNA-2 by immunohistochemistry on biopsies of a subset of samples of different ages (Table 7 and Fig. 6 A). LMP-1 and EBNA-2 constitute the minimal set of EBV proteins sufficient for B cell transformation ( 13 ). We found expression of these viral proteins in 21 samples of the 25 analyzed (84.0%, Table 8). We did not serologically test the samples; hence we were unable to determine the infectious status of those 4 tonsillar samples in which we did not detect neither LMP-1 nor EBNA-2. Within the 12 samples positive for viral Ag from children, 11 of them (91.7%) co-expressed LMP-1 and EBNA-2 (Table 8). The remainder positive sample expressed only EBNA-2. Ultimately, we found that samples with co-expression of viral proteins clustered in the first and second decade of life (94% of all samples expressing LMP1 and EBNA2, Fig. 6 B). On the other hand, those expressing a single viral protein grouped in the third and fourth decades (60% of the samples expressing a single viral Ag belonged to the older group, Fig. 6 B). As we were not able to ascertain whether the younger patients were primary infected or EBV carriers, we can only conclude that older patients displayed a higher repression on viral Ag than the younger ones. These results underscore the efficacy of the local specific memory cell pool, once established and consolidated, to keep EBV at bay at later stages of life despite the raise in the fraction of immune cell phenotypes associated with immunosuppressive activity in ageing tonsils. All the cells positive for LMP1 were scored in the interfollicular T-cell zone of the tonsillar biopsies. Nearly all the cells positive for EBNA2 (85%) were located in the same region (Fig. 6 A). Two of the remainder samples with EBNA-2 expression displayed positive cells in follicles as well as in the T-cell zone. The third sample, from a 6-year-old patient, exhibited numerous cells EBNA-2 + scattered in a single germinal centre and nowhere else, a situation on which we will not speculate further due to the lack of serologic data (Fig. 6 C). These results indicate that EBV latency is not affected by the changes in the proportion of lymphoid populations that occurs with age progression. Tonsillar B mem accumulation does not depend exclusively on age We have already mentioned that tonsillar B mem increase with age ( 1 ). Theoretically, B mem circulate from their site of induction through blood to distant lymphoid organs. Consequently, B mem generated elsewhere via parenteral vaccination, for instance, could contribute to the tonsillar accrual of this population, a situation more dependent on the date of last inoculation or number of them, rather than on age. Conversely, whether tonsils harbour B mem induced at distal sites in humans, has been a matter of debate. While some researchers demonstrated that tonsils function independently of the systemic immune apparatus ( 14 ), others could find tonsillar B mem with specificity for extra-tonsillar Ag ( 15 ). Thus, we decided to examine whether we could detect TT + B mem within TMC. To do so, we optimized our own assay. Briefly, we cultured TMC from vaccinated individuals (N = 23, children and adolescents, Table 9) for 3 days in media supplemented with CD40L, IL4 and TT and tested for anti-TT Ig in the culture supernatant. Hence, we used anti-TT Ig as a read-out of the differentiation of TT + B mem in ASC in culture by specific stimulation. We found that TMC harbor TT + B mem . Moreover, we established a strong correlation between anti-TT Ig OD from TMC cultures and the time from the last dose of parenteral vaccination (r=-0.7; p < 0.0001; Fig. 7 A and B). Control non-stimulated cultures were also set up with a subset of samples to confirm the increase in specific anti-TT Ig in response to stimulation (Supl Fig. 2 ). There was no correlation with the number of doses or age. We concluded that a fraction of the B mem recovered from TMC were generated by Ag exposure at distal locations. As there are no reliable phenotypic markers able to discriminate between circulating and resident tonsillar B mem yet, whether the accumulation is driven by tonsillar resident cells B mem remains unresolved in light of these findings. Discussion The tonsils serve as the initial barrier against inhaled or ingested pathogens. Despite this recognized protective role, many questions persist about their exact nature. One of them is why do they peak in size near the age of puberty, before they begin to gradually diminish. These results provide new evidence regarding the age-related transition of the human tonsillar immune microenvironment, documenting a shift from a highly proliferative, germinal center (GC)-driven situation in children toward a steady, memory-dominated, and exhausted state in older patients. This transition appears to be a coordinated process involving a marked decreased in the proportion of the B cell population as a whole, compensated by the envisaged increment of the T cell subset alongside the accumulation of diverse memory and activated cell subsets of both major lymphoid populations. Interestingly, the decline of the overall GC-forming mass and the rise of senescent phenotypes does not translate into failed local viral control, as suggested by the efficient repression of EBV viral antigens (LMP-1 and EBNA-2). Finally, we demonstrated that TMC of all ages tested, harboured B mem cells specific to a parenterally administered Ag, underscoring the tonsils significance as an accessory immune reservoir. These results build upon our earlier research which established a link between the shifts in the subsets of tonsillar lymphocytes with age and the temporal pattern in the cause of surgery ( 1 ). We have now established a strong positive correlation between the ABC percentage and the age of the patients. Such correlation is consistent with previous reports in the spleen, a systemic B-cell organ. ABC comprise a GC-independent memory subset capable of rapid and robust recall response ( 16 ), an observation that is compatible with the notion that tonsils contribute to the memory reserve pool of an individual, despite the declination of their GC formation capacity. Moreover, the linear increase in total CD3 + HLA-DR + T cells points toward a state of local immune senescence or activation, typical of chronic infection control. The expression of HLA-DR on CD3 + T cells has long been associated with immune aging and/or chronic viral persistence, such as in CMV or EBV infection ( 17 ) ( 13 ). Indeed, we showed EBV persistence in most of the samples that were analysed. Despite the accumulation of B and T cell phenotypes associated with immune senescence and chronic activation, we observed that older patients display a higher repression of EBV viral antigens (LMP-1 and EBNA-2). Therefore, the rise of senescent phenotypes did not translate into failed local viral control in the samples we worked with. We speculate that the specific, locally consolidated memory pool remains highly effective and capable of keeping EBV latent in tonsils, as it is well known that some EBV latent proteins are good immunogens ( 18 ). As for the age-related increase of tonsillar B mem populations, we found that is not solely due to local Ag exposure but it is also influenced by systemic memory trafficking and retention. This dynamic exchange further confirms the tonsil's role as an immune hub that integrates both local and systemic immunity. Conclusions The work presented here further demonstrate that tonsils undergo reformations during ageing that do not impact on the latency of local EBV. These insights may help to predict variations in the immune responses to locally administered Ag based on the age of the patient. Finally, they confirm that parenterally induced B mem home to oropharyngeal tissue, highlighting the extent of their mucosal infiltration. Declarations Acknowledgments We are grateful to Ariel Billordo and Plácida Baz for their technical assistance with the flow cytometry assays. Finally, we are grateful to the anonymous patients and/or their parents that consent for the donation of samples for this research project. Ethical Approval and Consent to participate. The institutional ethics committee (Clinical Hospital, School of Medicine, Buenos Aires and Institute of Otolaryngology Arauz, Buenos Aires) approved the collection and use of clinical material, conformed to the provisions of the Declaration of Helsinki (as revised in Edinburgh 2000). Informed consent was obtained from all participants and/or their legal guardian/s. Consent for publication. All authors of this manuscript concur with its submission. Availability of supporting data. The authors confirm that the data supporting the findings of this study are available within the article. In case of need of further details, Eloísa Arana would provide them upon reasonable request. Funding L.D, R.P, S.L.C and M.E.A were the recipients of a CONICET postgraduate scholarship each one. P. G and I.R.C were the recipients of an UBA undergraduate scholarship each one. This research was partially funded by the following Argentinean governmental agencies: ANPCyT (BID PICT 2019-0068, BID PICT 2021 00718), UBA (UBACYT 20720220100005BA), CONICET (PIP 0474) granted to E. Arana. However, the government halted funding of all the BID PICT projects in 2024. These studies were then completed through the personal financial efforts of main authors and solidarity in sharing reagents among the senior authors. Conflict of interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Author Contributions L.D, P.G, I.R.C, R.P, S.L.C and M.E.A optimized, performed and initially analyzed data. While L.D, P.G, R.P, and S.L.C were involved with FACS and IHQ of lymphoid subsets in particular, I.R.C and M.E.A took on board all EBV IHQ. L.D also set up, optimized and performed the cultures and ELISA TT assays starting from TMC of vaccinated individuals whose legal guardians she needed to personally contact in order to get documented proof of the last vaccinations dates. B.P, E.C and A.B provided samples and aided L.D with the patient’s contacts and medical history. D.H provided reagents and assisted in curating as well as analyzing data of vaccination. G.B, N.S and A.P.L performed cryosections and H&E stainings. S.C assisted with FACS. EA and PCh were responsible for the conceptualization, study design and secure funding. E.A and L.D wrote the original manuscript. Edition, revision and approval of the manuscript, all authors. References Pastor R, Puyssegur J, de la Guardia MP, Varón LS, Beccaglia G, Spada N, et al. Role of germinal center and CD39highCD73 + B cells in the age-related tonsillar involution. Immun Ageing. 2024;21(1):24. Mattila PS, Tahkokallio O, Tarkkanen J, Pitkäniemi J, Karvonen M, Tuomilehto J. Causes of tonsillar disease and frequency of tonsillectomy operations. Arch Otolaryngol Head Neck Surg. 2001;127(1):37–44. Sarmiento Varon L, De Rosa J, Machicote A, Billordo LA, Baz P, Fernández PM, et al. Characterization of tonsillar IL10 secreting B cells and their role in the pathophysiology of tonsillar hypertrophy. Sci Rep. 2017;7(1):11077. Sarmiento Varón L, De Rosa J, Rodriguez R, Fernández PM, Billordo LA, Baz P, et al. Role of Tonsillar Chronic Inflammation and Commensal Bacteria in the Pathogenesis of Pediatric OSA. Front Immunol. 2021;12:648064. Medzhitov R, Schneider DS, Soares MP. Disease tolerance as a defense strategy. Science. 2012;335(6071):936–41. Pascual M, Alignani D, Vilas-Zornoza A, Delgado JA, Vázquez I, Malumbres R, et al. Use of human pharyngeal and palatine tonsils as a reservoir for the analysis of B-cell ontogeny in 10 paired samples. Clinical otolaryngology: official journal of ENT-UK ; official journal of Netherlands Society for Oto-Rhino-Laryngology. Cervico-Facial Surg. 2016;41(5):606–11. Zhou H, Li B, Li J, Wu T, Jin X, Yuan R, et al. Dysregulated T Cell Activation and Aberrant Cytokine Expression Profile in Systemic Lupus Erythematosus. Mediators Inflamm. 2019;2019:8450947. Bogner JR, Matuschke A, Heinrich B, Schreiber MA, Nerl C, Goebel FD. Expansion of activated T lymphocytes (CD3 + HLA/DR +) detectable in early stages of HIV-1 infection. Klinische Wochenschrift. 1990;68(8):393–6. Rea IM, McNerlan SE, Alexander HD. CD69, CD25, and HLA-DR activation antigen expression on CD3 + lymphocytes and relationship to serum TNF-alpha, IFN-gamma, and sIL-2R levels in aging. Exp Gerontol. 1999;34(1):79–93. Seishima N, Kondo S, Wakisaka N, Kobayashi E, Imoto T, Moriyama-Kita M, et al. EBV infection is prevalent in the adenoid and palatine tonsils in adults. J Med Virol. 2017;89(6):1088–95. Gonzalez-Lucano LR, Vasquez-Armenta GV, Pereira-Suarez AL, Ramirez-de Arellano A, Ramirez-de Los Santos S, Lopez-Pulido EI. Prevalence of Epstein-Barr virus DNA in tonsillar tissue from patients with chronic tonsillitis in Mexican population. J Infect developing Ctries. 2019;13(8):764–7. Kalantari S, Zadheidar S, Heydarifard Z, Nejati A, Sadeghi K, Shatizadeh Malekshahi S et al. Epstein-Barr virus in tonsillar tissue of Iranian children with tonsillar hypertrophy: Quantitative measurement by real-time PCR. World Journal of Otorhinolaryngology - Head and Neck Surgery.n/a(n/a). Hofstee MI, Cevirgel A, de Zeeuw-Brouwer M-L, de Rond L, van der Klis F, Buisman A-M. Cytomegalovirus and Epstein–Barr virus co-infected young and middle-aged adults can have an aging-related T-cell phenotype. Sci Rep. 2023;13(1):10912. Quiding-Järbrink M, Granström G, Nordström I, Holmgren J, Czerkinsky C. Induction of compartmentalized B-cell responses in human tonsils. Infect Immun. 1995;63(3):853–7. Cao Y, Gordic M, Kobold S, Lajmi N, Meyer S, Bartels K, et al. An optimized assay for the enumeration of antigen-specific memory B cells in different compartments of the human body. J Immunol Methods. 2010;358(1–2):56–65. Song W, Antao OQ, Condiff E, Sanchez GM, Chernova I, Zembrzuski K, et al. Development of Tbet- and CD11c-expressing B cells in a viral infection requires T follicular helper cells outside of germinal centers. Immunity. 2022;55(2):290–e3075. Amarillo ME, Lindl K, Lapido V, Rojas Campión IE, Collado MS, Speratti J, et al. Co-expression of PD1 + and HLA-DR + in CD8 + T cells is increased in tonsils of children with EBV primary and persistent infection. Front Immunol. 2025;16:1653165. Niller HH, Wolf H, Minarovits J. Regulation and dysregulation of Epstein-Barr virus latency: implications for the development of autoimmune diseases. Autoimmunity. 2008;41(4):298–328. Tables Tables 1 to 9 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables1.pdf Tables2.pdf Tables3.pdf Tables4.pdf Tables5.pdf Tables6.pdf Tables7.pdf Tables8.pdf Tables9.pdf FinalSupplFig1.png Supplementary Figure 1: Flow chart depicting the major lymphoid subsets gating strategy. Fresh TMC were stained for surface CD20, CD3, CD4 and CD8. Singlets were gated by plotting FSC-H vs FSC-A for each sample (top left panel). Within the singlet population, lymphoid gate was determined through SSC-A vs FSC-A according to their size and granularity (top right panel). Within the lymphocyte gate, CD20 + CD3 - and CD20 - CD3 + gates were drawn (bottom left panel). Within the CD20 - CD3 + gate, the T cell subsets were determined (bottom right panel). SuplFig2.png Supplementary Figure 2: TT specific B mem cells differentiate into anti-TT antibody secreting cells under specific TT stimulation. TMC were cultured in presence (TT supplemented culture) or absence (control culture) of TT, and specific anti-TT Igs were measured in the supernatants of both situations. Dot-plot representing absorbance at 450 nm for both conditions. (Parametric paired t test. p<0.05 (*), N = 7). Each point represents the mean between two measurements, and each pair of matching points belongs to the same patient. In every case, blank absorbance at 450 nm was subtracted. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8643189","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":580908849,"identity":"e3b17033-ad05-4d64-8bb6-17838495cdc5","order_by":0,"name":"Lucía Donnoli","email":"","orcid":"","institution":"University of Buenos Aires (UBA), National Council for Scientific and Technological Research (CONICET)","correspondingAuthor":false,"prefix":"","firstName":"Lucía","middleName":"","lastName":"Donnoli","suffix":""},{"id":580908850,"identity":"416df00a-22ff-4b5e-8069-f6677085eb51","order_by":1,"name":"M. Paula de la Guardia","email":"","orcid":"","institution":"University of Buenos Aires (UBA), National Council for Scientific and Technological Research (CONICET)","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"Paula de la","lastName":"Guardia","suffix":""},{"id":580908851,"identity":"18e7c834-6059-4dc9-954d-4a53b991e9c0","order_by":2,"name":"Ignacio Rojas Campión","email":"","orcid":"","institution":"University of Buenos Aires (UBA), National Council for Scientific and Technological Research (CONICET)","correspondingAuthor":false,"prefix":"","firstName":"Ignacio","middleName":"Rojas","lastName":"Campión","suffix":""},{"id":580908852,"identity":"35ad38a5-1259-4381-8d2f-4aa03881f1f0","order_by":3,"name":"Rocío Pastor","email":"","orcid":"","institution":"University of Buenos Aires (UBA), National Council for Scientific and Technological Research (CONICET)","correspondingAuthor":false,"prefix":"","firstName":"Rocío","middleName":"","lastName":"Pastor","suffix":""},{"id":580908854,"identity":"56b5ae58-381b-4b93-aeca-c29c928d2e43","order_by":4,"name":"Sofía López Cardoso","email":"","orcid":"","institution":"University of Buenos Aires (UBA), National Council for Scientific and Technological Research (CONICET)","correspondingAuthor":false,"prefix":"","firstName":"Sofía","middleName":"López","lastName":"Cardoso","suffix":""},{"id":580908856,"identity":"039f9c10-da71-48dc-8760-f094f9b5ba17","order_by":5,"name":"Gladys Beccaglia","email":"","orcid":"","institution":"Clinical Hospital ‘Jose de San Martín’","correspondingAuthor":false,"prefix":"","firstName":"Gladys","middleName":"","lastName":"Beccaglia","suffix":""},{"id":580908858,"identity":"bc8ebe7e-9850-4389-985e-802e52e4f99b","order_by":6,"name":"Nicolás Spada","email":"","orcid":"","institution":"Clinical Hospital ‘Jose de San Martín’","correspondingAuthor":false,"prefix":"","firstName":"Nicolás","middleName":"","lastName":"Spada","suffix":""},{"id":580908861,"identity":"695ed374-046a-4ecf-b938-e586b1d3a97f","order_by":7,"name":"Andrea Paes Lima","email":"","orcid":"","institution":"Clinical Hospital ‘Jose de San Martín’","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"Paes","lastName":"Lima","suffix":""},{"id":580908863,"identity":"8a9718d2-e7e5-462b-a5c4-f606ecc4099f","order_by":8,"name":"M. Soledad Collado","email":"","orcid":"","institution":"University of Buenos Aires (UBA), National Council for Scientific and Technological Research (CONICET)","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"Soledad","lastName":"Collado","suffix":""},{"id":580908864,"identity":"788ef8a1-f337-4ccf-8bf6-09a7b101a411","order_by":9,"name":"Andrés Blanco","email":"","orcid":"","institution":"University of Buenos Aires (UBA)","correspondingAuthor":false,"prefix":"","firstName":"Andrés","middleName":"","lastName":"Blanco","suffix":""},{"id":580908865,"identity":"2336b98e-07c6-4cc8-ba6d-dfd0a8405a90","order_by":10,"name":"Estefany Cáceres","email":"","orcid":"","institution":"Clinical Hospital ‘Jose de San Martín’","correspondingAuthor":false,"prefix":"","firstName":"Estefany","middleName":"","lastName":"Cáceres","suffix":""},{"id":580908866,"identity":"486e3408-5e9e-4872-a1d5-7bb0d433b61a","order_by":11,"name":"Bibiana Paoli","email":"","orcid":"","institution":"Clinical Hospital ‘Jose de San Martín’","correspondingAuthor":false,"prefix":"","firstName":"Bibiana","middleName":"","lastName":"Paoli","suffix":""},{"id":580908869,"identity":"de3cfb2a-ed34-4b43-a5f4-8bc45bb58f81","order_by":12,"name":"Daniela Hozbor","email":"","orcid":"","institution":"University of La Plata (UNLP), National Council for Scientific and Technological Research (CONICET)","correspondingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"","lastName":"Hozbor","suffix":""},{"id":580908872,"identity":"0cb2278d-8772-4950-9dd6-a58f26fe72aa","order_by":13,"name":"María Eugenia Amarillo","email":"","orcid":"","institution":"National Council for Scientific and Technological Research (CONICET)-GCBA, Ricardo Gutiérrez Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Eugenia","lastName":"Amarillo","suffix":""},{"id":580908873,"identity":"51d883c4-ad5d-4866-b285-9014af15ac7a","order_by":14,"name":"Paola Chabay","email":"","orcid":"","institution":"National Council for Scientific and Technological Research (CONICET)-GCBA, Ricardo Gutiérrez Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Paola","middleName":"","lastName":"Chabay","suffix":""},{"id":580908874,"identity":"c901c9ae-bd65-46a6-9304-ab10a7b44bd4","order_by":15,"name":"Eloísa I. Arana","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYDACdijNz8DARqQWZigt2UCyFoMDxGrhZ2Y++Lmi5o688Y3kZw8+VDDI84sdwK9FspktWfLMsWeG226kmRvOOMNgOHN2An4tBod5DCQb2A4zbruRYCbN28aQYHCbgBb7wzzGPxv+HbbfPCP9G3FaDJh5zCQb2w4nbpDIIdIWicNsaZaNfYeTZ5x5UyY544wEYb/wtzcfvtnw7bBtf3v6NokPFTby/NIEtCCAAFilBLHKwfYdIEX1KBgFo2AUjCQAABYvQPFFfa4aAAAAAElFTkSuQmCC","orcid":"","institution":"University of Buenos Aires (UBA), National Council for Scientific and Technological Research (CONICET)","correspondingAuthor":true,"prefix":"","firstName":"Eloísa","middleName":"I.","lastName":"Arana","suffix":""}],"badges":[],"createdAt":"2026-01-19 22:08:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8643189/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8643189/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101753295,"identity":"69e8a9cd-ecde-4bd3-84bd-6facce41f889","added_by":"auto","created_at":"2026-02-03 10:39:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9788283,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in tonsillar histology across different age groups. A)-B) Immunohistochemistry (IHQ) of tonsillar fixed sections. A) CD20 staining. B) CD3 staining. Left panels, tonsillar sections of a 6 years old. Right panels, tonsillar sections of a 39 years old. Ages of the donors are indicated on the top and yo stands for years old. LF stands for Lymphoid Follicles, EF for Extra-Follicular and MZ stands for Mantle Zone. Samples were examined with 40X magnification, and analyzed with a Leica Microscope DM500. Scale bar, 500 μm. (C) The negative correlation between the size of lymphoid follicles and age. Each point represents the mean of three measurements from one patient (Pearson correlation test. r = -0.6, p\u0026lt;0.005 (**), N = 22). p \u0026lt; 0.05 was considered significant.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/9518f42f7dec15fe16e06755.png"},{"id":101753251,"identity":"249ea3de-3219-4993-a085-48d99fbb37ce","added_by":"auto","created_at":"2026-02-03 10:39:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":679306,"visible":true,"origin":"","legend":"\u003cp\u003eAge-dependent distribution of tonsillar cell populations. A)-B) Frequencies of B (CD20\u003csup\u003e+\u003c/sup\u003e) and T (CD3\u003csup\u003e+\u003c/sup\u003e) cells were assessed. A) Negative correlation between frequency of B cells and age. (Pearson correlation test. r=-0.4, p\u0026lt;0.001 (***), N = 88) (B) Positive correlation between frequency of T cells and age (Spearman correlation test, r = 0.5, p\u0026lt;0.0001 (****), N = 88).\u0026nbsp; (C-E) T cell subsets were identified by flow cytometry, based on its CXCR5 and PD1 expression. Percentages of germinal center (Tfh\u003csub\u003eGC\u003c/sub\u003e, CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCXCR5\u003csup\u003ehigh\u003c/sup\u003ePD1\u003csup\u003ehigh\u003c/sup\u003e); mantle zone (Tfh\u003csub\u003eM\u003c/sub\u003e, CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCXCR5\u003csup\u003eint\u003c/sup\u003ePD1\u003csup\u003eint\u003c/sup\u003e) and extra-follicular (Tfh\u003csub\u003eEF\u003c/sub\u003e, CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCXCR5\u003csup\u003elow\u003c/sup\u003ePD1\u003csup\u003elow\u003c/sup\u003e) T cells were obtained. (C) Negative correlation between frequency of Thf\u003csub\u003eGC\u003c/sub\u003e and age (Pearson correlation test. r = -0.5, p\u0026lt;0.0001 (****), N = 83) (D) Positive correlation between frequency of Tfh\u003csub\u003eM\u003c/sub\u003e and age (Pearson correlation test, r = 0.6, p\u0026lt;0.0001 (****), N = 83). (E) Correlation between frequency of Tfh\u003csub\u003eEF\u003c/sub\u003e and age (Pearson correlation test r = 0.2, p = 0.1, N = 83). (A-E) Each point represents one patient. p\u0026lt;0.05 was considered significant.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/d6752384bcb600a47496c5cb.png"},{"id":101640660,"identity":"dc92ece8-b4dd-423a-a47a-2763accc384c","added_by":"auto","created_at":"2026-02-02 07:29:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":336922,"visible":true,"origin":"","legend":"\u003cp\u003eDynamics of the major tonsillar T cell subsets with age. A) Positive correlation between the percentage of helper T cells (CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e) and age. (Spearman correlation test r = 0.5, p\u0026lt;0.0001 (****), N = 76). B) Negative correlation between the percentage of cytotoxic T cells (CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e) and age (Spearman correlation test. r = -0.3, p\u0026lt;0.05 (*), N = 76). A)-B) Each point represents one patient. p\u0026lt;0.05 was considered significant.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/b66f79816217ccad1e15dd62.png"},{"id":101640665,"identity":"258e7972-9c84-4789-a5a7-4a6506c73f65","added_by":"auto","created_at":"2026-02-02 07:29:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":727115,"visible":true,"origin":"","legend":"\u003cp\u003eMemory cell populations increased with ageing. A) Flow chart partially depicting ABC (CD3\u003csup\u003e-\u003c/sup\u003eCD19\u003csup\u003ehigh\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eTbet\u003csup\u003e+\u003c/sup\u003e) gating strategy. Fresh TMC were stained for surface CD19, CD3, CD27, CD11c and T-bet. Singlets were gated by plotting FSC-H vs FSC-A for each sample (not shown). Within the singlet population, lymphoid gate was determined through SSC-A vs FSC-A according to their size and granularity (not shown). Within the lymphocyte gate, the successive gates used were indicated on the top of each panel. B) Positive correlation between the percentage of ABC and age (Spearman correlation test. r = 0.5, p\u0026lt;0,0001. N = 79). C) Flow chart partially depicting cytotoxic memory cells (CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e-\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eCD45R0\u003csup\u003e+\u003c/sup\u003eCD45RA\u003csup\u003e-\u003c/sup\u003e) gating strategy. Within the singlet population, lymphoid gate was determined through SSC-A vs FSC-A according to their size and granularity (not shown). Within the lymphocyte gate, the successive gates used were indicated on the top of each panel. D) Positive correlation between the percentage of CD8 memory cells and age (Pearson correlation test. r = 0.4, p\u0026lt;0.005 (**), N = 70). (B-D) Each point represents one patient. p\u0026lt;0.05 was considered significant.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/6b97a5f5995e0aacf2214c4d.png"},{"id":101640661,"identity":"c5fd3d40-0623-4114-a1e1-950ba42f6804","added_by":"auto","created_at":"2026-02-02 07:29:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":329254,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of a T cell phenotype associated with senescence. A) Senescent T cells were identified within the lymphocyte gate as CD3\u003csup\u003e+\u003c/sup\u003eHLA-DR\u003csup\u003e+\u003c/sup\u003e (indicated by an hexagon shape). B) Positive correlation between the percentage of HLA-DR\u003csup\u003e+\u003c/sup\u003e T cells and age (Spearman correlation test. r = 0.3, p\u0026lt;0.05 (*), N = 82). Each point represents one patient. p\u0026lt;0.05 was considered significant.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/e12717b04076ac77462c180f.png"},{"id":101753022,"identity":"0f66b263-9a6a-4549-8fed-65465f4e4a09","added_by":"auto","created_at":"2026-02-03 10:38:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8984934,"visible":true,"origin":"","legend":"\u003cp\u003eTonsillar expression of EBV latency viral proteins. A) Representative images of positive IHC staining in tonsils from patients infected with EBV of LMP-1 (top panel) and EBNA-2 (bottom panel). Three magnifications are shown for each protein. LF stands for Lymphoid Follicles, EF for Extra-Follicular and MZ stands for Mantle Zone. Arrows indicate infected cells. Scale bar is indicated in each picture. B) Graph bar representing samples LMP-1\u003csup\u003e+\u003c/sup\u003eEBNA-2\u003csup\u003e+ \u003c/sup\u003e(left bar, co-expression) and samples LMP-1\u003csup\u003e+\u003c/sup\u003eEBNA-2\u003csup\u003e-\u003c/sup\u003e or LMP-1\u003csup\u003e-\u003c/sup\u003eEBNA-2\u003csup\u003e+ \u003c/sup\u003e(right bar, single expression). The colours of the bars represent the different age groups (black for children and adolescents and grey for adults). Fisher’s test. p\u0026lt;0.05 (*), N = 21. C) Patient with a single follicle with several EBNA-2\u003csup\u003e+\u003c/sup\u003e cells. Two magnifications are shown. LF stands for Lymphoid Follicles, EF for Extra-Follicular and MZ stands for Mantle Zone. Arrows indicate infected cells. Scale bar is indicated in each picture.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/32bed77182585f48e873aeec.png"},{"id":101753794,"identity":"a4a798ce-03e2-4dd3-b009-46906f902a92","added_by":"auto","created_at":"2026-02-03 10:40:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":217751,"visible":true,"origin":"","legend":"\u003cp\u003eTonsils harbour TT specific B\u003csub\u003emem\u003c/sub\u003e capable of differentiating into anti-TT antibody secreting cells. A) Scheme illustrating TT\u003csup\u003e+\u003c/sup\u003e B\u003csub\u003emem\u003c/sub\u003e cells differentiation. TMC were cultured for three days, specifically stimulated with TT, in addition to general survival and activation signals, IL-4 and CD40L. B) Anti-TT Igs in the culture supernatant were measured by indirect ELISA. Negative correlation between absorbance at 450 nm and time from last dose. (Pearson correlation test. r = -0.7, p\u0026lt;0.0001 (****), N = 23). Each point represents the mean between two measurements. In every case, blank absorbance at 450 nm was subtracted.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/b63340a25fca25494be97501.png"},{"id":102014747,"identity":"b7d83bff-1af2-4dff-93be-4366556c4533","added_by":"auto","created_at":"2026-02-06 07:12:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23494650,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/8ac211c8-1bc3-4477-a2bd-41e300b67b5d.pdf"},{"id":101640658,"identity":"50addaaf-c832-45c0-92f9-02212dc2ebb2","added_by":"auto","created_at":"2026-02-02 07:29:13","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":35495,"visible":true,"origin":"","legend":"","description":"","filename":"Tables1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/c50d9bd67d78e16b4b16ae7e.pdf"},{"id":101640667,"identity":"e60199da-be10-4151-ae47-3f46708328bc","added_by":"auto","created_at":"2026-02-02 07:29:14","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":35508,"visible":true,"origin":"","legend":"","description":"","filename":"Tables2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/c0b7d7730654dc166b8a36d5.pdf"},{"id":101752985,"identity":"ec756638-5189-4ccf-bac8-e289993adf66","added_by":"auto","created_at":"2026-02-03 10:38:38","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":35506,"visible":true,"origin":"","legend":"","description":"","filename":"Tables3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/f0ec3f5ef45a498a71310277.pdf"},{"id":101640664,"identity":"c76961a4-f472-4833-a46c-3fae0f0951e7","added_by":"auto","created_at":"2026-02-02 07:29:13","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":35510,"visible":true,"origin":"","legend":"","description":"","filename":"Tables4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/e803c2f17ed8ca04ea660386.pdf"},{"id":101753274,"identity":"8428126c-b075-4f2d-876d-098164ef3cc1","added_by":"auto","created_at":"2026-02-03 10:39:35","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":35504,"visible":true,"origin":"","legend":"","description":"","filename":"Tables5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/817d323adc012bd927ccfdfd.pdf"},{"id":101640674,"identity":"c11e0d95-aafe-4556-ae2c-50e330bb8e03","added_by":"auto","created_at":"2026-02-02 07:29:14","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":35483,"visible":true,"origin":"","legend":"","description":"","filename":"Tables6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/31890adb1408fdcd94013497.pdf"},{"id":101640670,"identity":"f0ecb324-f028-47fe-aeda-ce56aab17297","added_by":"auto","created_at":"2026-02-02 07:29:14","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":36431,"visible":true,"origin":"","legend":"","description":"","filename":"Tables7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/f615010638a452155ff1d6b9.pdf"},{"id":101640668,"identity":"40dfa265-24e3-43fb-bc91-78f9e4137c1a","added_by":"auto","created_at":"2026-02-02 07:29:14","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":36271,"visible":true,"origin":"","legend":"","description":"","filename":"Tables8.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/45b7f675727657132d373bbc.pdf"},{"id":101753309,"identity":"1246ab82-185d-44d2-9ea1-32399301646a","added_by":"auto","created_at":"2026-02-03 10:39:40","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":35479,"visible":true,"origin":"","legend":"","description":"","filename":"Tables9.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/6c3511d5ae63d300bf437d80.pdf"},{"id":101640675,"identity":"8b902524-875f-40b1-b7d7-eeb00d5dbdab","added_by":"auto","created_at":"2026-02-02 07:29:14","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1938578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1\u003c/strong\u003e: Flow chart depicting the major lymphoid subsets gating strategy. Fresh TMC were stained for surface CD20, CD3, CD4 and CD8. Singlets were gated by plotting FSC-H vs FSC-A for each sample (top left panel). Within the singlet population, lymphoid gate was determined through SSC-A vs FSC-A according to their size and granularity (top right panel). Within the lymphocyte gate, CD20\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e- \u003c/sup\u003eand CD20\u003csup\u003e-\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e gates were drawn (bottom left panel). Within the CD20\u003csup\u003e-\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e gate, the T cell subsets were determined (bottom right panel).\u003c/p\u003e","description":"","filename":"FinalSupplFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/c4ca3914127ed5822fa5d21f.png"},{"id":101640673,"identity":"8743c6fd-63a8-40a2-a245-2de59bf70c04","added_by":"auto","created_at":"2026-02-02 07:29:14","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":257272,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2:\u003c/strong\u003e TT specific B\u003csub\u003emem\u003c/sub\u003e cells differentiate into anti-TT antibody secreting cells under specific TT stimulation. TMC were cultured in presence (TT supplemented culture) or absence (control culture) of TT, and specific anti-TT Igs were measured in the supernatants of both situations. Dot-plot representing absorbance at 450 nm for both conditions. (Parametric paired t test. p\u0026lt;0.05 (*), N = 7). Each point represents the mean between two measurements, and each pair of matching points belongs to the same patient. In every case, blank absorbance at 450 nm was subtracted.\u003c/p\u003e","description":"","filename":"SuplFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8643189/v1/6a37995cf8eb3cc7934d2339.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dynamics of memory cell subsets in human tonsils with age: impact on the functional reconfiguration of the organ","fulltext":[{"header":"Background","content":"\u003cp\u003eThe palatine tonsils are positioned at the confluence of the digestive and the respiratory tracts. They function as secondary lymphoid organs that coordinate the first response to inhaled and ingested pathogens. The percentage of germinal centre (GC) lymphoid populations is reduced in tonsils of adolescents and adults when compared to those from younger children. Additionally, younger tonsils render a higher proportion of proliferative immune cells than those from older ones (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). These observations are associated with poor protective immunity in infections which in turn reflects in a temporal pattern in the cause of surgery. Tonsillar hypertrophy (TH) is the major motive for tonsillectomy in children below 10 years old while recurrent tonsillitis (RT) stands as the primary reason for the indication of surgery from puberty onwards (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe clinical significance of TH stems from obstruction. These children present a failure of immune homeostatic mechanisms caused by the local loss of the capacity to discriminate between commensals and pathogens that leads to persistent inflammation and follicular hyperplasia (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Conversely, RT is defined by a specific number of annual infections presenting with systemic and local symptoms including fever, sore throat and tonsillar exudate. The teenagers and young adults undergoing surgery due to RT usually have a history of TH but not at a score to cause obstruction.\u003c/p\u003e \u003cp\u003eOrganisms have evolved mechanisms to regulate local immunity upon years of constant inflammation in a particular tissue, to keep the physiological function of the latter and alleviate immunopathology (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). We have previously characterized two of these tonsillar age-associated regulatory mechanisms. Aging correlated with an increment in the percentage of tonsillar B cells secreting IL10, the classical regulatory B cell population (Breg) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) and with a non-classical Breg subset, phenotyped as CD20\u003csup\u003e+\u003c/sup\u003eCD39\u003csup\u003ehigh\u003c/sup\u003eCD73\u003csup\u003e+\u003c/sup\u003e, as well (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Hence, we hypothesized that there is a gradual re-configuration of the physiological function of the tonsils as the patients age, from representing a major site of B cell effector mechanisms targeted to reduce pathogen burden in childhood to a memory reservoir where regulatory mechanisms become prevalent beyond puberty.\u003c/p\u003e \u003cp\u003eThe coordinated cellular shifts underlying such transition were partly determined, and mainly focused on germinal centre (GC) dynamics (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It is unclear how the relative proportions of the major tonsillar lymphoid subsets along with their various memory and activated phenotypes, change from childhood until adulthood. Moreover, the impact of such an evolving microenvironment on the functional capacity to maintain control over widespread local latent infections, such as those caused by the Epstein-Barr Virus (EBV), has not yet been studied. In the context of the tonsils\u0026rsquo; role as a memory B cell pool, it remains to be determined whether they preserve immunological memory initially generated through parenteral vaccination, and the duration of this maintenance.\u003c/p\u003e \u003cp\u003eIn this study, we addressed these issues through phenotypic and functional characterization of tonsillar mononuclear cells (TMC) and tissue biopsies across a broad age cohort (1 to 45 years old). We found a significant age-dependent decline in the overall B cell proportion balanced by the concomitant rise in the T cell compartment. This shift was accompanied by the accumulation of specific phenotypes associated with immune aging and chronic activation, including the so-called aged or atypical memory B cells (ABC) and HLA-DR\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003ecells. Despite the rise of these senescent subsets, our data did not provide evidence of a failure of local viral control. Finally, we demonstrated that the tonsils function as an accessory immune reservoir harbouring memory B cells\u0026rsquo; specific for a parenterally administered antigen (tetanus toxoid, TT), underscoring their role in integrating both local and systemic immunity even after the onset of the decline in effector immune mechanisms (around 10 years old).\u003c/p\u003e \u003cp\u003eTaken together, these findings corroborate that the well documented tonsillar involution with age represents in fact a functional transition towards an immunoregulatory microenvironment, memory-dominated vigilant state.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient recruitment and collection of samples\u003c/h2\u003e \u003cp\u003eTonsillar mononuclear cells (TMC) were purified from patients aged from 1 to 45 years old undergoing tonsillectomy. Patients under 18 years old were recruited from Clinical Hospital \u0026ldquo;Jos\u0026eacute; de San Mart\u0026iacute;n\u0026rdquo;, whereas adults were enrolled from the Institute of Otolaryngology Arauz. The number of patients enrolled in each experiment are detailed from Table\u0026nbsp;1 to Table\u0026nbsp;9. Patients who presented any kind of immunodeficiency, or had undergone medical treatment (antibiotics, corticoids) one month prior to the surgery were excluded. The institutional ethics committee (Clinical Hospital, School of Medicine, Buenos Aires and Institute of Otolaryngology Arauz, Buenos Aires) approved the collection and use of clinical material, conformed to the provisions of the Declaration of Helsinki (as revised in Edinburgh 2000). Informed consent was obtained from all participants and/or their legal guardian/s.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIsolation of tonsillar mononuclear cells\u003c/h3\u003e\n\u003cp\u003eThe organs were collected in saline solution (0.9% NaCl). Prior to cell isolation, a tissue section was cut off and formaldehyde-fixated in order to perform immunostainings. TMC were recovered from the organ by injecting phosphate saline buffer supplemented with 10% inactivated fetal calf serum (supplemented PBS) through the tonsils. TMC were then isolated using density gradient centrifugation employing Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden). The collected TMC from the halo were washed and cells counted in a Neubauer camera. Viable cells proportion, determined by Trypan Blue staining, was always greater than 95%.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry of fresh isolated cells\u003c/h3\u003e\n\u003cp\u003eCells were assessed for their expression of different surface markers using flow cytometry. 1x10\u003csup\u003e6\u003c/sup\u003e cells were stained with fluorochrome conjugated monoclonal antibodies: CD19 (PerCP; BioLegend, clone SJ25C1), CD20 (FITC; BioLegend, clone L27), CD27 (APC; BD Pharmigen, clone M-271), CD11c (PE; BioLegend, clone 3.9), CD3 (APC-Cy7; BD Pharmigen, clone SK7, or PE-CF594; BD Horizon, clone UCHT1), HLA-DR (FITC; BioLegend, clone LN3), CD4 (PerCP; BioLegend, clone SK3), CD8a (Pacific Blue; BioLegend, clone HIT8a), CD45RA (APC; BD Pharmigen clone HI100), CD45RO (PE; BD Pharmigen, clone UCHL1), CXCR5 (Alexa Fluor 488; BD Pharmigen, clone RF8B2) and PD-1 (Brilliant Violet 711; BioLegend, clone EH12.2H7). Parallely, corresponding compensation and isotype controls were performed with monoclonal antibodies purchased from BD Biosciences (CA, USA) and Biolegend (CA, USA). After a 30 minutes incubation, cells were washed and resuspended in flow cytometry buffer (10% fetal calf serum in PBS 1x).\u003c/p\u003e \u003cp\u003eIntracellular cytometry was performed to detect T-bet transcription factor, cells were incubated with Fixation/Permeabilization (eBioscience FOXP3/Transcription, Invitrogen) for 45 min and washed with Permeabilization Buffer (eBioscience FOXP3/Transcription, Invitrogen). Then, the cells were stained with an anti-T-bet antibody (Brilliant Violet 650; BioLegend, clone 4B10).\u003c/p\u003e \u003cp\u003eCells were acquired using FACSAria II (BD Biosciences, CA, USA) and analyzed with FlowJo\u0026trade; software version 10.0 (Treestar, OR, USA). Single stained controls were used to set compensation parameters. Fluorescence minus one and isotype-matched Ab controls were used to set analysis gates.\u003c/p\u003e \u003cp\u003eGating strategy for each cell population is depicted either in supplementary or respective figures.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry for lymphoid populations\u003c/h3\u003e\n\u003cp\u003eFormalin fixated and paraffin embedded (FFPE) tissue sections were cut in 5 \u0026micro;m slices and mounted in silane coated glasses. Slides were stained for immunohistochemistry. Sections were deparaffinized and re-hydrated via a xylene/ethanol gradient, consisting of a pair of 10 minute incubations in xylene, followed by sequential 3 minute incubations in ethanol 100%, 96%, 70% and distilled water. Slides were then washed twice for 5 minutes in saline phosphate buffer (PBS); all the subsequent washing steps were performed in the same way. Antigen retrieval was then performed: samples were soaked in Sodium Citrate Buffer (0,01 M, pH 6) and heated up to 95\u0026ordm; C in a microwave (500\u0026ndash;600 w), for 30 minutes. Once cold, tissue sections were washed and permeabilized for 20 minutes employing 0,5% Triton X-100 (Sintorgan, Buenos Aires, Argentina) in PBS 1X. Slides were then washed, and endogenous peroxidase was blocked by incubating the slides in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in methanol, for 30 min in darkness. After incubation, sections were washed and nonspecific binding sites were blocked with Bovine Serum Albumin (BSA. Sigma, MO, USA) 1% in PBS. Samples were incubated overnight with antibodies for human CD3 and CD20 (clones MRQ-39 and SP32, respectively, Cell Marque, Darmstadt, Germany) lymphocyte surface markers and for Epstein-Barr proteins LMP-1 (clones CS.1\u0026ndash;4, Dako, CA, USA) and EBNA-2 (clone PE2, Abcam, Cambridge, UK). After incubation, slides were washed and the corresponding HRP-conjugated secondary antibodies were added. Finally, after 45 minutes, tissue sections were incubated with 3, 3\u0026rsquo;-diaminobenzidine tetrahydrochloride (DAB. Sigma, MO, USA) until brown precipitate was detected. Incubation was stopped with tap water, and slides were counterstained with hematoxylin. Samples were dehydrated with an ethanol:xylene gradient, and mounted with synthetic Canada balsam (Stanton, Buenos Aires, Argentina). In parallel, controls for unspecific binding were performed, replacing primary antibodies isotype controls diluted in blocking buffer.\u003c/p\u003e \u003cp\u003eSlides were examined using a Leica DM500 (Leica, Germany), for CD20 and CD3 stainings, or with a Zeiss Axio Scope A1 optical microscope (Carl Zeiss, Germany) for LMP-1 and EBNA-2. Pictures were acquired with a Leica ICC50 W camera at 40X, 100X and 400X magnifications, or a 503 color Axiocam camera, at 200X, 400X and 1000X magnifications, respectively. For follicular area measurement, images were analyzed employing Image J software. Areas are expressed in square millimeters.\u003c/p\u003e\n\u003ch3\u003eEnumeration of LMP-1 and EBNA-2 expressing cells\u003c/h3\u003e\n\u003cp\u003eFFPE tissue sections were processed like above. Images were analyzed at 200X, 400X and 1000X magnifications. Positive cells for each viral protein were counted employing the corresponding microscopes softwares, LAS EZ 3.6 and ZEN 4.3. For LMP-1, a cell was considered positive only if the mark was observed within the cytoplasm, whereas for EBNA-2, the mark had to be located on the nucleus. In neither case clustered cells were counted. For each protein, a patient with a minimal count of two positive cells per slide was considered positive. Positive controls were performed on biopsies from Hondgkin\u0026rsquo;s lymphoma for LMP-1, and from post-transplant lymphoproliferative disorders for EBNA-2.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell cultures\u003c/h2\u003e \u003cp\u003eCells were cultured at 1x10\u003csup\u003e6\u003c/sup\u003e cells/mL in IMDM medium supplemented with 10% heat-inactivated fetal calf serum, 2mM L-glutamine, 100 U/ml penicillin, 100 \u0026micro;g/ml streptomycin, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES), 1 mM sodium pyruvate and 50 \u0026micro;M 2-mercaptoethanol (all from Invitrogen, CA, USA). B lymphocytes\u0026rsquo; survival signals, human IL4 (20 ng/ml; R\u0026amp;D Systems, MN, USA) and human recombinant CD40L (250 ng/ml; R\u0026amp;D Systems, MN, USA) were added, and stimulation with recombinant \u003cem\u003eTetanus toxoid\u003c/em\u003e (5 \u0026micro;g/ml; Argentinian Biological Institute S.A.I.C., Buenos Aires, Argentina) was performed, when indicated. Cells were seeded in 48 well flat bottom plates (0,5 mL/well), and cultured at 37\u0026ordm;C, 5% CO\u003csub\u003e2\u003c/sub\u003e, humid atmosphere for three days. After incubation, culture supernatants were recovered and stored at -20\u0026ordm;C, for the following determinations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eELISA assay for specific antibodies against\u003c/b\u003e \u003cb\u003eTetanus toxoid\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn order to measure the specific antibodies against Tetanus toxoid, a home-made ELISA was set up. 96 well high binding plates were coated with \u003cem\u003eTetatus toxoid\u003c/em\u003e (5 \u0026micro;g/ml; Argentinian Biological Institute S.A.I.C., Buenos Aires, Argentina) and incubated over-night at 4\u0026ordm;C. Positive controls assessing total IgGs were performed by coating with anti-IgG (5 \u0026micro;g/ml; BD Biosciences). Afterwards, plates were repeatedly washed with a 0,05% Tween-20 in PBS 1x buffer, and blocked with 10% ultra-low IgG fetal calf serum in PBS 1x, pH 7 (Sigma, MO, USA). All the subsequent incubations were followed by the same washing step described. After 1 hour at RT, supernatants were incubated for 2 hours at 37\u0026ordm;C. In parallel, negative controls were performed adding blocking buffer. For the detection step, biotinylated anti-kappa light chain antibody was added (5 \u0026micro;g/ml; BD Biosciences, CA, USA) and incubated for 1 hour at RT. Then, plates were incubated in darkness for 30 minutes with streptavidin conjugated horseradish peroxidase (HRP. BD OptEIA\u0026trade;, CA, USA). Lastly, the assay was revealed with 3,3',5,5'-Tetramethylbenzidine (TMB. BD OptEIA\u0026trade;, CA, USA): once light blue color had developed, the enzymatic reaction was stopped with sulfuric acid, and absorbance at 450 nm was measured. In every case, blank absorbance at 450 nm was subtracted from the raw measurement. For every patient, two measurements were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data were analyzed using Graphpad Prism 8.0.2 software. Prior to any analysis, Shapiro-Wilk\u0026rsquo;s normality test was performed. Pearson parametric or Spearman non-parametric correlation tests were employed according to normality test results. For demographic description of each study population, mean and standard deviation were calculated. Comparisons between two groups from different patients were performed with a parametric unpaired t test, whereas a parametric paired t test was performed when two groups with matching samples were analyzed. All tests were two-tailed, and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDynamics of tonsillar lymphocytes subsets with age\u003c/h2\u003e \u003cp\u003eWe have recently shown that the tonsillar GC B cell population steadily decreases with increasing age in the surgical samples we work with (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In order to extend our findings, we qualitatively assessed immunohistochemistry staining for CD20 and CD3 expression in biopsies of different ages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). We observed a greater size of B cell follicles in children\u0026acute;s biopsies as compared to the adult\u0026acute;s ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Conversely, older biopsies displayed a relative volume up of the inter or extra-follicular areas (EF) stained with anti-CD3 relative to the younger ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). To quantify such changes in cellularity, the TMC extracted from the samples of patients aged between 1 and 45 years old (N\u0026thinsp;=\u0026thinsp;88, Table\u0026nbsp;1) were analyzed by flow cytometry (gating strategy in Suppl. Figure\u0026nbsp;1) and CD20\u003csup\u003e+\u003c/sup\u003e cells were scored and plotted against age (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As expected, the proportion of CD20\u003csup\u003e+\u003c/sup\u003e tonsillar B cells declined with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, r=-0.4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) whereas the frequency of CD3\u003csup\u003e+\u003c/sup\u003e tonsillar T cells increased accordingly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, r\u0026thinsp;=\u0026thinsp;0.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The following measurements were not performed on all 88 patients due to different circumstances with some samples; the exact N is designated in each figure legend and a number of tables. Being Tfh cells the primary drivers of T cell\u0026ndash;dependent GC responses, we also determined the age-related evolution of the different Tfh subsets in terms of their histological localization (germinal center, Tfh\u003csub\u003eGC\u003c/sub\u003e ; mantle Tfh\u003csub\u003eM\u003c/sub\u003e and extrafollicular Tfh\u003csub\u003eEF\u003c/sub\u003e; gating strategy as in (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)) in the cohort of patients indicated in Table\u0026nbsp;2. We found a significant negative correlation with age in the proportion of Tfh\u003csub\u003eGC\u003c/sub\u003e defined as CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCXCR5\u003csup\u003ehigh\u003c/sup\u003ePD-1\u003csup\u003ehigh\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, r=-0.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). On the other hand, Tfh\u003csub\u003eM\u003c/sub\u003e (CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCXCR5\u003csup\u003eint\u003c/sup\u003ePD-1\u003csup\u003eint\u003c/sup\u003e) cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, r\u0026thinsp;=\u0026thinsp;0.6, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) increased with increasing age. Tfh\u003csub\u003eEF\u003c/sub\u003e (CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCXCR5\u003csup\u003e\u0026minus;\u003c/sup\u003ePD-1\u003csup\u003e\u0026minus;\u003c/sup\u003e) displayed a tendency towards an increment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, r\u0026thinsp;=\u0026thinsp;0.2, p\u0026thinsp;=\u0026thinsp;0.1). Moreover, CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e as a whole population displayed a positive correlation with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, r\u0026thinsp;=\u0026thinsp;0.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Table\u0026nbsp;3). On the other hand, the proportion of CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e in TMC decreased with increasing age (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, r=-0.3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; gating strategy in Suppl. Figure\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTherefore, we confirmed the age-dependent reduction of the B cell mass in this location with a concomitant reduction of the histological compartments occupied by this cell population and the accessory T cell ones.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDiverse memory cell subsets accumulate in samples from older patients\u003c/h2\u003e \u003cp\u003eTonsillar B\u003csub\u003emem\u003c/sub\u003e (defined as CD3\u003csup\u003e\u0026minus;\u003c/sup\u003eCD20\u003csup\u003e+\u003c/sup\u003eCD10\u003csup\u003elow\u003c/sup\u003eCD44\u003csup\u003ehigh\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e+\u003c/sup\u003e as in (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)) increase with age (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It has been reported that ABC accumulation contributes to impaired B lymphopoiesis in old mice. We then sought to determine whether there was an age-related increase of the tonsillar ABC population. Thus, TMC from patients at different ages were tested by flow cytometry for ABC identification (see Table\u0026nbsp;4). ABC were defined as CD3\u003csup\u003e\u0026minus;\u003c/sup\u003eCD19\u003csup\u003ehigh\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eTbet\u003csup\u003e+\u003c/sup\u003e cells. Single lymphocytes were gated based on forward/side scatter properties. Tonsillar B cells were gated as CD19\u003csup\u003e+\u003c/sup\u003e cells and within that region, we scored the CD27\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eTbet\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We found that the proportion of ABC population increased with increasing age. We established a positive correlation between ABC percentage and the age of the patients (r\u0026thinsp;=\u0026thinsp;0.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These findings further validate the role of CD19\u003csup\u003ehigh\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eTbet\u003csup\u003e+\u003c/sup\u003e cells as a B cell population associated with aging.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe have investigated T cell memory as well. We used CD45RA and CD45R0 expression to differentiate na\u0026iuml;ve and memory T cells (Table\u0026nbsp;5, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). While we detected a moderate positive correlation of memory CD8\u003csup\u003e+\u003c/sup\u003e T cells with age (r\u0026thinsp;=\u0026thinsp;0.4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.005, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), we did not detect significant changes of memory CD4\u003csup\u003e+\u003c/sup\u003e T cells associated to the tonsillar aging process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eTonsillar HLADR\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eT cells steadily increase with increasing age\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eCD3\u003csup\u003e+\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003e are considered as activated T cells with regulatory properties given the fact that they are upregulated in blood in autoimmune disorders (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) and chronic viral infections (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). We have previously shown that the declining of the tonsillar GC reaction with ageing and chronic inflammation, correlated with an increment in the proportion of different B cell phenotypes associated with immunosuppressive activity (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In this context, we assessed the tonsillar CD3\u003csup\u003e+\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003e cell population in our cohort of patients (Table\u0026nbsp;6). Single lymphocytes were gated based on forward/side scatter properties, and tonsillar activated T cells were identified as CD3\u003csup\u003e+\u003c/sup\u003eHLA DR\u003csup\u003e+\u003c/sup\u003e cells within the live singlets and lymphocyte gate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We determined that CD3\u003csup\u003e+\u003c/sup\u003eHLA DR\u003csup\u003e+\u003c/sup\u003e increased with age (r\u0026thinsp;=\u0026thinsp;0.3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis observation complements our previous findings in relation to the decline of tonsillar effector immune responses over time from puberty onwards (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) as expression of HLA DR on CD3\u003csup\u003e+\u003c/sup\u003e cells has long been related to immune senescence (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImpact of the age-related tonsillar lymphocyte shift on a local latent infection\u003c/h2\u003e \u003cp\u003eTonsils are replication sites for the EBV virus and viral DNA can be detected by PCR in around 70% of the samples from adults (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In children, it has been shown an association between the host age and EBV DNA detection rate (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) as most infections occur at this stage of life. In order to test whether the age-related changes in the proportion of tonsillar B and T cell subpopulations have an impact on EBV latency, we determined the expression of the viral proteins LMP-1 and EBNA-2 by immunohistochemistry on biopsies of a subset of samples of different ages (Table\u0026nbsp;7 and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). LMP-1 and EBNA-2 constitute the minimal set of EBV proteins sufficient for B cell transformation (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). We found expression of these viral proteins in 21 samples of the 25 analyzed (84.0%, Table\u0026nbsp;8). We did not serologically test the samples; hence we were unable to determine the infectious status of those 4 tonsillar samples in which we did not detect neither LMP-1 nor EBNA-2. Within the 12 samples positive for viral Ag from children, 11 of them (91.7%) co-expressed LMP-1 and EBNA-2 (Table\u0026nbsp;8). The remainder positive sample expressed only EBNA-2. Ultimately, we found that samples with co-expression of viral proteins clustered in the first and second decade of life (94% of all samples expressing LMP1 and EBNA2, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). On the other hand, those expressing a single viral protein grouped in the third and fourth decades (60% of the samples expressing a single viral Ag belonged to the older group, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). As we were not able to ascertain whether the younger patients were primary infected or EBV carriers, we can only conclude that older patients displayed a higher repression on viral Ag than the younger ones. These results underscore the efficacy of the local specific memory cell pool, once established and consolidated, to keep EBV at bay at later stages of life despite the raise in the fraction of immune cell phenotypes associated with immunosuppressive activity in ageing tonsils. All the cells positive for LMP1 were scored in the interfollicular T-cell zone of the tonsillar biopsies. Nearly all the cells positive for EBNA2 (85%) were located in the same region (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Two of the remainder samples with EBNA-2 expression displayed positive cells in follicles as well as in the T-cell zone. The third sample, from a 6-year-old patient, exhibited numerous cells EBNA-2\u003csup\u003e+\u003c/sup\u003e scattered in a single germinal centre and nowhere else, a situation on which we will not speculate further due to the lack of serologic data (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results indicate that EBV latency is not affected by the changes in the proportion of lymphoid populations that occurs with age progression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTonsillar B\u003csub\u003emem\u003c/sub\u003e accumulation does not depend exclusively on age\u003c/h2\u003e \u003cp\u003eWe have already mentioned that tonsillar B\u003csub\u003emem\u003c/sub\u003e increase with age (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Theoretically, B\u003csub\u003emem\u003c/sub\u003e circulate from their site of induction through blood to distant lymphoid organs. Consequently, B\u003csub\u003emem\u003c/sub\u003e generated elsewhere via parenteral vaccination, for instance, could contribute to the tonsillar accrual of this population, a situation more dependent on the date of last inoculation or number of them, rather than on age. Conversely, whether tonsils harbour B\u003csub\u003emem\u003c/sub\u003e induced at distal sites in humans, has been a matter of debate. While some researchers demonstrated that tonsils function independently of the systemic immune apparatus (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), others could find tonsillar B\u003csub\u003emem\u003c/sub\u003e with specificity for extra-tonsillar Ag (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Thus, we decided to examine whether we could detect TT\u003csup\u003e+\u003c/sup\u003eB\u003csub\u003emem\u003c/sub\u003e within TMC. To do so, we optimized our own assay. Briefly, we cultured TMC from vaccinated individuals (N\u0026thinsp;=\u0026thinsp;23, children and adolescents, Table\u0026nbsp;9) for 3 days in media supplemented with CD40L, IL4 and TT and tested for anti-TT Ig in the culture supernatant. Hence, we used anti-TT Ig as a read-out of the differentiation of TT\u003csup\u003e+\u003c/sup\u003eB\u003csub\u003emem\u003c/sub\u003e in ASC in culture by specific stimulation. We found that TMC harbor TT\u003csup\u003e+\u003c/sup\u003eB\u003csub\u003emem\u003c/sub\u003e. Moreover, we established a strong correlation between anti-TT Ig OD from TMC cultures and the time from the last dose of parenteral vaccination (r=-0.7; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and B). Control non-stimulated cultures were also set up with a subset of samples to confirm the increase in specific anti-TT Ig in response to stimulation (Supl Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). There was no correlation with the number of doses or age. We concluded that a fraction of the B\u003csub\u003emem\u003c/sub\u003e recovered from TMC were generated by Ag exposure at distal locations. As there are no reliable phenotypic markers able to discriminate between circulating and resident tonsillar B\u003csub\u003emem\u003c/sub\u003e yet, whether the accumulation is driven by tonsillar resident cells B\u003csub\u003emem\u003c/sub\u003e remains unresolved in light of these findings.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe tonsils serve as the initial barrier against inhaled or ingested pathogens. Despite this recognized protective role, many questions persist about their exact nature. One of them is why do they peak in size near the age of puberty, before they begin to gradually diminish. These results provide new evidence regarding the age-related transition of the human tonsillar immune microenvironment, documenting a shift from a highly proliferative, germinal center (GC)-driven situation in children toward a steady, memory-dominated, and exhausted state in older patients. This transition appears to be a coordinated process involving a marked decreased in the proportion of the B cell population as a whole, compensated by the envisaged increment of the T cell subset alongside the accumulation of diverse memory and activated cell subsets of both major lymphoid populations. Interestingly, the decline of the overall GC-forming mass and the rise of senescent phenotypes does not translate into failed local viral control, as suggested by the efficient repression of EBV viral antigens (LMP-1 and EBNA-2). Finally, we demonstrated that TMC of all ages tested, harboured B\u003csub\u003emem\u003c/sub\u003e cells specific to a parenterally administered Ag, underscoring the tonsils significance as an accessory immune reservoir.\u003c/p\u003e \u003cp\u003eThese results build upon our earlier research which established a link between the shifts in the subsets of tonsillar lymphocytes with age and the temporal pattern in the cause of surgery (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). We have now established a strong positive correlation between the ABC percentage and the age of the patients. Such correlation is consistent with previous reports in the spleen, a systemic B-cell organ. ABC comprise a GC-independent memory subset capable of rapid and robust recall response (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), an observation that is compatible with the notion that tonsils contribute to the memory reserve pool of an individual, despite the declination of their GC formation capacity. Moreover, the linear increase in total CD3\u0026thinsp;+\u0026thinsp;HLA-DR\u0026thinsp;+\u0026thinsp;T cells points toward a state of local immune senescence or activation, typical of chronic infection control. The expression of HLA-DR on CD3\u003csup\u003e+\u003c/sup\u003e T cells has long been associated with immune aging and/or chronic viral persistence, such as in CMV or EBV infection (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Indeed, we showed EBV persistence in most of the samples that were analysed. Despite the accumulation of B and T cell phenotypes associated with immune senescence and chronic activation, we observed that older patients display a higher repression of EBV viral antigens (LMP-1 and EBNA-2). Therefore, the rise of senescent phenotypes did not translate into failed local viral control in the samples we worked with. We speculate that the specific, locally consolidated memory pool remains highly effective and capable of keeping EBV latent in tonsils, as it is well known that some EBV latent proteins are good immunogens (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs for the age-related increase of tonsillar B\u003csub\u003emem\u003c/sub\u003e populations, we found that is not solely due to local Ag exposure but it is also influenced by systemic memory trafficking and retention. This dynamic exchange further confirms the tonsil's role as an immune hub that integrates both local and systemic immunity.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe work presented here further demonstrate that tonsils undergo reformations during ageing that do not impact on the latency of local EBV. These insights may help to predict variations in the immune responses to locally administered Ag based on the age of the patient. Finally, they confirm that parenterally induced B\u003csub\u003emem\u003c/sub\u003e home to oropharyngeal tissue, highlighting the extent of their mucosal infiltration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Ariel Billordo and Pl\u0026aacute;cida Baz for their technical assistance with the flow cytometry assays. Finally, we are grateful to the anonymous patients and/or their parents that consent for the donation of samples for this research project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to participate.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe institutional ethics committee (Clinical Hospital, School of Medicine, Buenos Aires and Institute of Otolaryngology Arauz, Buenos Aires) approved the collection and use of clinical material, conformed to the provisions of the Declaration of Helsinki (as revised in Edinburgh 2000). Informed consent was obtained from all participants and/or their legal guardian/s.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors of this manuscript concur with its submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of supporting data.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article. In case of need of further details, Elo\u0026iacute;sa Arana would provide them upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.D, R.P, S.L.C and M.E.A were the recipients of a CONICET postgraduate scholarship each one. P. G and I.R.C were the recipients of an UBA undergraduate scholarship each one. This research was partially funded by the following Argentinean governmental agencies: ANPCyT (BID PICT 2019-0068, BID PICT 2021 00718), UBA (UBACYT 20720220100005BA), CONICET (PIP 0474) granted to E. Arana. However, the government halted funding of all the BID PICT projects in 2024. These studies were then completed through the personal financial efforts of main authors and solidarity in sharing reagents among the senior authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.D, P.G, I.R.C, R.P, S.L.C and M.E.A optimized, performed and initially analyzed data. While L.D, P.G, R.P, and S.L.C were involved with FACS and IHQ of lymphoid subsets in particular, I.R.C and M.E.A took on board all EBV IHQ. L.D also set up, optimized and performed the cultures and ELISA TT assays starting from TMC of vaccinated individuals whose legal guardians she needed to personally contact in order to get documented proof of the last vaccinations dates. B.P, E.C and A.B provided samples and aided L.D with the patient\u0026rsquo;s contacts and medical history. D.H provided reagents and assisted in curating as well as analyzing data of vaccination. G.B, N.S and A.P.L performed cryosections and H\u0026amp;E stainings. S.C assisted with FACS. EA and PCh were responsible for the conceptualization, study design and secure funding. E.A and L.D wrote the original manuscript. Edition, revision and approval of the manuscript, all authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePastor R, Puyssegur J, de la Guardia MP, Var\u0026oacute;n LS, Beccaglia G, Spada N, et al. Role of germinal center and CD39highCD73\u0026thinsp;+\u0026thinsp;B cells in the age-related tonsillar involution. Immun Ageing. 2024;21(1):24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMattila PS, Tahkokallio O, Tarkkanen J, Pitk\u0026auml;niemi J, Karvonen M, Tuomilehto J. Causes of tonsillar disease and frequency of tonsillectomy operations. Arch Otolaryngol Head Neck Surg. 2001;127(1):37\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarmiento Varon L, De Rosa J, Machicote A, Billordo LA, Baz P, Fern\u0026aacute;ndez PM, et al. Characterization of tonsillar IL10 secreting B cells and their role in the pathophysiology of tonsillar hypertrophy. Sci Rep. 2017;7(1):11077.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarmiento Var\u0026oacute;n L, De Rosa J, Rodriguez R, Fern\u0026aacute;ndez PM, Billordo LA, Baz P, et al. Role of Tonsillar Chronic Inflammation and Commensal Bacteria in the Pathogenesis of Pediatric OSA. Front Immunol. 2021;12:648064.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMedzhitov R, Schneider DS, Soares MP. Disease tolerance as a defense strategy. Science. 2012;335(6071):936\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePascual M, Alignani D, Vilas-Zornoza A, Delgado JA, V\u0026aacute;zquez I, Malumbres R, et al. Use of human pharyngeal and palatine tonsils as a reservoir for the analysis of B-cell ontogeny in 10 paired samples. Clinical otolaryngology: official journal of ENT-UK ; official journal of Netherlands Society for Oto-Rhino-Laryngology. Cervico-Facial Surg. 2016;41(5):606\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou H, Li B, Li J, Wu T, Jin X, Yuan R, et al. Dysregulated T Cell Activation and Aberrant Cytokine Expression Profile in Systemic Lupus Erythematosus. Mediators Inflamm. 2019;2019:8450947.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBogner JR, Matuschke A, Heinrich B, Schreiber MA, Nerl C, Goebel FD. Expansion of activated T lymphocytes (CD3\u0026thinsp;+\u0026thinsp;HLA/DR +) detectable in early stages of HIV-1 infection. Klinische Wochenschrift. 1990;68(8):393\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRea IM, McNerlan SE, Alexander HD. CD69, CD25, and HLA-DR activation antigen expression on CD3\u0026thinsp;+\u0026thinsp;lymphocytes and relationship to serum TNF-alpha, IFN-gamma, and sIL-2R levels in aging. Exp Gerontol. 1999;34(1):79\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeishima N, Kondo S, Wakisaka N, Kobayashi E, Imoto T, Moriyama-Kita M, et al. EBV infection is prevalent in the adenoid and palatine tonsils in adults. J Med Virol. 2017;89(6):1088\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonzalez-Lucano LR, Vasquez-Armenta GV, Pereira-Suarez AL, Ramirez-de Arellano A, Ramirez-de Los Santos S, Lopez-Pulido EI. Prevalence of Epstein-Barr virus DNA in tonsillar tissue from patients with chronic tonsillitis in Mexican population. J Infect developing Ctries. 2019;13(8):764\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalantari S, Zadheidar S, Heydarifard Z, Nejati A, Sadeghi K, Shatizadeh Malekshahi S et al. Epstein-Barr virus in tonsillar tissue of Iranian children with tonsillar hypertrophy: Quantitative measurement by real-time PCR. World Journal of Otorhinolaryngology - Head and Neck Surgery.n/a(n/a).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHofstee MI, Cevirgel A, de Zeeuw-Brouwer M-L, de Rond L, van der Klis F, Buisman A-M. Cytomegalovirus and Epstein\u0026ndash;Barr virus co-infected young and middle-aged adults can have an aging-related T-cell phenotype. Sci Rep. 2023;13(1):10912.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuiding-J\u0026auml;rbrink M, Granstr\u0026ouml;m G, Nordstr\u0026ouml;m I, Holmgren J, Czerkinsky C. Induction of compartmentalized B-cell responses in human tonsils. Infect Immun. 1995;63(3):853\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao Y, Gordic M, Kobold S, Lajmi N, Meyer S, Bartels K, et al. An optimized assay for the enumeration of antigen-specific memory B cells in different compartments of the human body. J Immunol Methods. 2010;358(1\u0026ndash;2):56\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong W, Antao OQ, Condiff E, Sanchez GM, Chernova I, Zembrzuski K, et al. Development of Tbet- and CD11c-expressing B cells in a viral infection requires T follicular helper cells outside of germinal centers. Immunity. 2022;55(2):290\u0026ndash;e3075.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmarillo ME, Lindl K, Lapido V, Rojas Campi\u0026oacute;n IE, Collado MS, Speratti J, et al. Co-expression of PD1\u0026thinsp;+\u0026thinsp;and HLA-DR\u0026thinsp;+\u0026thinsp;in CD8\u0026thinsp;+\u0026thinsp;T cells is increased in tonsils of children with EBV primary and persistent infection. Front Immunol. 2025;16:1653165.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiller HH, Wolf H, Minarovits J. Regulation and dysregulation of Epstein-Barr virus latency: implications for the development of autoimmune diseases. Autoimmunity. 2008;41(4):298\u0026ndash;328.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 9 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":true,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"tonsils, mucosa, immunity, aging, memory, latency","lastPublishedDoi":"10.21203/rs.3.rs-8643189/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8643189/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground.\u003c/h2\u003e \u003cp\u003eThe palatine tonsils are lymphoepithelial structures in which immune responses to inhaled and ingested pathogens are mounted. While we have previously shown that tonsillar immune effector function declines with age, the impact of such erosion on the local viral reservoirs or in specific memory responses remains to be defined.\u003c/p\u003e\u003ch2\u003eMethods.\u003c/h2\u003e \u003cp\u003eWe performed phenotypic and functional characterization of tonsillar mononuclear cells (TMC) and tissue biopsies from a cohort of around 80 patients (ages 1\u0026ndash;45). We quantified the proportion of particular lymphoid subsets associated with ageing through flow cytometry. We also assessed the expression of LMP-1 and EBNA-2, latency proteins of Epstein-Barr virus (EBV), via immunohistochemistry. Finally, we functionally tested the specific response of tonsillar memory B cells distantly induced by tetanus toxoid (TT) injection, through ELISA on the supernatant of TT- stimulated TMC cultures.\u003c/p\u003e\u003ch2\u003eResults.\u003c/h2\u003e \u003cp\u003eWe demonstrated a significant age-dependent decline in the B cell proportion balanced by a concomitant rise in the T cell fraction. This shift was complemented by the accumulation of particular phenotypes associated with aged B cells and senescent T cells. Despite these alterations, tonsillar samples from older patients presented a more effective repression of EBV viral antigens (Ag) than those of younger children. We also established that TMC from all patients tested harboured responsive TT specific memory B cells and moreover, anti-TT Ig production correlated with the time since the last vaccination.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003e. Our findings suggest that tonsillar involution constitutes a functional transition from a germinal centre driven secondary lymphoid organ in childhood to a memory dominated, immunoregulatory reservoir in adulthood. This reconfiguration preserves local viral control and serves to integrate both local and systemic immunological memory.\u003c/p\u003e","manuscriptTitle":"Dynamics of memory cell subsets in human tonsils with age: impact on the functional reconfiguration of the organ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-02 07:29:08","doi":"10.21203/rs.3.rs-8643189/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"65109590-6348-45b7-b00e-e91d7b03262d","owner":[],"postedDate":"February 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-06T07:11:25+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-02 07:29:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8643189","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8643189","identity":"rs-8643189","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}