Cytometric analysis of CD3+CD4+T populations and activation and regulation status of naïve and memory CD4+CD45RA T cells in immunocompetent patients with neurocryptococcosis

Cytometric analysis of CD3+CD4+T populations and activation and regulation status of naïve and memory CD4+CD45RA T cells in immunocompetent patients with neurocryptococcosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cytometric analysis of CD3+CD4+T populations and activation and regulation status of naïve and memory CD4+CD45RA T cells in immunocompetent patients with neurocryptococcosis Isabel Alves Feitosa Maciel, Juliana Ruiz Ruiz, Renata Buccheri de Oliveira de Oliveira, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6844830/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Dec, 2025 Read the published version in Mycopathologia → Version 1 posted 6 You are reading this latest preprint version Abstract Neurocryptococcosis is a serious disease that mainly affects individuals with compromised immune systems. However, “immunocompetent” individuals are also affected by this condition even without any known underlying disease or compromised immune system. In this study, we evaluated the CD4 + T lymphocyte population and subpopulations in the peripheral blood of eight hospitalized patients with neurocryptococcosis and eight healthy control individuals. Thus, our objective was to contribute to this understanding by characterizing the T lymphocyte population (CD3 + CD4+) and subpopulations, with analyses of the activation and regulation status of responsive T cells in naïve (N), central memory (TMC), effector memory (TME) and terminally differentiated effector (TEMRA) in apparently immunocompetent patients and healthy control individuals. Our results showed a significant increase in CD4 + γδ T subpopulations, CD4 + CD25 + CD127low, CD4 + CD25 + CD127 + high regulatory T cells, CD4 + CD45RA + CCR7- terminally differentiated effector memory (TEMRA) T cells and CD4 + CD45RA-CCR7- effector memory (TME) T cells. We also observed a significant decrease in total lymphocytes, CD4 + CD45RA + CCR7+ (naïve) T cells and CD4 + CD45RA-CCR7 + central memory (TMC) T cells. CD4 + T and CD4 + αβ T cells did not show statistically significant differences between the study groups. These results suggest that the immune response of these patients is undergoing alterations in the maturation and differentiation of T lymphocytes and may be related to the virulence factors of the fungus that interfere in several mechanisms of the cells of both the innate and adaptive immune response, as well as with possible regulation disorders of T helper subsets immune responses during Cryptococcus infection. Cryptococcal meningitis Virulence factors Epidemiology of cryptococcosis Cryptococcus neoformans Cryptococcus gattii Immunocompetent patients Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Cryptococcal meningitis is an opportunistic infection caused by the pathogen Cryptococcus spp. that mainly affects immunosuppressed individuals and is frequently associated with HIV-AIDS infection [ 1 , 2 ]. Neurocryptococcosis has a higher incidence in this group, in addition to being associated with 181,000 deaths each year [ 1 , 2 ]. However, cases of neurocryptococcosis have been reported in “apparently immunocompetent” individuals in the last decade [ 3 ]. Immunocompromised patients or those undergoing immunosuppressive therapies are more susceptible to infection by the C. neoformans strain, while C. gattii generally affects apparently healthy individuals, with no previous history of disease or any other form of apparent immune system impairment [ 2 , 3 ]. Therefore, in order to better understand the severity of the disease, more specific studies need to be conducted and focused mainly on the real participation of T lymphocytes in the course of the infection. Materials and Methods 2.1. Separation of the peripheral blood mononuclear cells (PBMCs) Peripheral blood mononuclear cells (PBMCs) were obtained by acollecting 40 mL of heparinized blood and diluting them in sterile 0.9% saline solution at a ratio of 1:1. The blood diluted in saline was transferred to tubes containing 50 mL of Ficoll-Hypaque solution (GE Healthcare Bio-Sciences AB) at a ratio of 1:3 and then centrifuged at 900 G for 20 minutes at 18°C. The cells obtained were centrifuged once with RPMI-1640 culture medium supplemented with 10% fetal bovine serum (FBS) for 10 minutes at 250 G (GIBCO). The PBMCs were washed again in 1 mL of RPMI-1640 with (FBS) and gently resuspended dropwise in 1 mL of the freezing solution (FBS with 10% DMSO). They were then stored in the freezer at − 80°C until use. To assess viability, the cells were tested with trypan blue and counted in a Neubauer chamber. 2.2. Thawing of peripheral blood mononuclear cells and Immunophenotyping of Total lymphocytes and CD4 + subpopulations PBMCs were thawed in a water bath at 37°C and centrifuged once with 10 mL of RPMI-1640 with 10% (FBS) at 250 G for 10 min. They were then resuspended in 1 mL of RPMI-1640 for counting and transferred to the incubator at 37°C and 5% CO₂ in culture medium with RPMI-1640 with 10% (FBS) at 1 mL of medium for each 1x10 6 cells. After the 24-hour period, the cells were centrifuged once in 1 mL of RPMI-1640 with 10% (FBS) for 10 min at 250 G. They were then resuspended in 1 mL of PBS and incubated with monoclonal antibodies (BD Biosciences) for 25 min at room temperature protected from light (Table 1). After that, the cells were washed once with 1 mL of Isoton (PBS-azide) for five minutes at 250 G and resuspended with 300 µL of Isoton. 1x106 cells were acquired on a Fortessa flow cytometer (BD LSR-FortessaTM). 2.3. Adjustment of parameters on the cytometer The acquisition of patient and control samples was performed by multiparametric cytometry on a flow cytometer (BD LSR-FortessaTM). The acquisition was adjusted to 100,000 events in the lymphocyte window. Immunophenotyping was performed with monoclonal antibodies for surface antigens on 1x10 6 PBMCs. To properly assess compensation and determine the positive population by flow cytometry, the Fluorescence Minus One (FMO) control was performed. The phenotypic characterization of the total lymphocyte population and CD4 + T cell subpopulations was performed by means of their characteristics regarding size (FSC) and granularity (SSC) (Fig. 1). The results were analyzed using the FlowJo™ Software (version 10). From the selection of T lymphocytes, CD3 + T cells and CD3 + CD4 + T cells were selected. Then, within the CD3 + CD4 + T compartment, the subpopulations were defined as: — naïve CD4 + CD45RA + CCR7+ (N); – effector memory CD4 + CD45RA-CCR7- (TEM); – central memory CD4 + CD45RA-CCR7+ (TCM); and – terminally differentiated effector memory CD4 + CD45RA + CCR7- (TEMRA). The other cell subpopulations were defined according to their activation state: effector CD25 + CD127 + high and regulatory CD25 + CD127low. 2.4. Casuistry The study was conducted with a sample of eight “immunocompetent” patients with neurocryptococcosis, hospitalized at the Hospital das Clínicas and Instituto de Infectologia Emílio Ribas. The control group was composed of eight healthy individuals. Both groups, patients and controls, had no history of other associated infections or underlying diseases, as shown in (Table 2). No patient was undergoing therapy with corticosteroids or other immunosuppressive medication. Blood samples from patients were collected between the 15th and 30th day of hospitalization, in parallel with the samples from the control group. 3. Statistical Analysis The statistical analyses of the results obtained were performed using GraphPad Prism® Software (version 5.0). All data related to the immunophenotyping of CD4 + T lymphocytes were tested to assess the normality profile using the D'Agostino & Pearson test. For those that presented normal distribution between the groups, the T test was applied. For those that did not present normal distribution, the nonparametric Mann-Whitney test was performed. All values that presented P < 0.05 were considered statistically significant. Results 4.1. Demographic analysis Demographics showed a prevalence of cryptococcosis in males (75%), with a slightly higher mean and median for the patient group, around 39.5 (Table 2). 4.2. Phenotypic characterization of total lymphocytes and CD3 + CD4 + subpopulations The study of the total population of T lymphocytes in blood samples in the study groups showed statistical differences (P = 0.007) with a mean of 15.88 ± 5.748 and 24.26 ± 5.046 in the patient and control groups, respectively (Fig. 2a). Our results demonstrate that subpopulations within the CD3 + CD4 + T compartment, the subpopulation of CD4 + TCR-γδ cells is increased in the patient group, with significant statistical differences (P < 0.001) (Fig. 2b), however the CD4 + TCR-αꞵ population did not present statistical differences between the groups analyzed. The mean CD3 + CD4 + T population in the patient group was 45.99 ± 23.11 and in the control group 54.43 ± 12.50 (Fig. 2c). The CD4 + TCR-αꞵ cell population showed a mean in the patient group of 93.50 ± 2.419 and in the control group of 93.20 ± 2.169 as can be seen in (Fig. 2d). Both CD4 + CD25 + CD127 + low regulatory T cells, as well as CD25 + CD127 + high effector T cells showed statistically significant differences, that is, P = 0.013 and P = 0.001, respectively as shown in (Fig. 3a and 3b). The results regarding the memory state of CD4 + T lymphocytes show significant statistical differences between the subpopulations for the naïve (P = 0.001) and TCM (P < 0.001) phenotypes in the patient group (Fig. 4a and b), respectively. The TEM phenotype differs statistically (P < 0.001) with a mean in the patient group of 57.44 ± 19.05 and controls 27.30 ± 6.825 as can be seen in (Fig. 4c), respectively. The analyses were performed based on the expression of the CD45RA and CCR7[ 4 – 7 ] markers. The TEMRA phenotype showed a statistical difference (P = 0.005) with a mean of 22.25 ± 11.43 for patients and controls, a mean of 7.979 ± 4.607 (Fig.d). Discussion In this study, we analyzed apparently “immunocompetent” individuals with neurocryptococcosis, together with a control group (healthy individuals), to characterize the population of T lymphocytes (CD3 + CD4+) and subpopulations by analyzing the activation and regulation status of responsive T cells in naïve (N), central memory (TCM), effector memory (TEM) and terminally differentiated effector (TEMRA). Thus, our objective was to contribute to this understanding by characterizing the population of T lymphocytes (CD3 + CD4+) and subpopulations after collecting peripheral blood between the 15th and 30th day of hospitalization of the patients, for both groups. Although the incidence of cryptococcal meningitis is related to immunosuppression, studies/reports have demonstrated an increase in these occurrences in individuals considered immunocompetent with infection by both the C. neoformans and C. gattii strains [ 3 , 8 , 9 ]. A recent study demonstrates that individuals are continually exposed to fungi, and constant exposure to fungal load may be a risk factor even among healthy individuals [ 6 , 10 ]. Cryptococcal infection may be asymptomatic and remain latent for years, or even manifest years later. Furthermore, prolonged exposure to the fungus may impair the memory immune response, should the individual contract the infection [ 8 , 12 ]. Our results showed significant differences in the total lymphocyte population and γδ T cells in the patient group. However, there was no statistically significant difference for the CD4 + T population when compared with healthy individuals. Similar results are observed during infection and treatment period of disseminated cryptococcosis by C. neoformans and/or C. gattii as described by Ruan et al. (2017) in an adult patient with disseminated disease by C. neoformans and normal CD4 + T cell count. Similar results were described by Suchitha et al. (2012) in an adult patient affected by C. gattii [ 11 , 12 ]. A Chinese retrospective study involving 154 HIV-negative patients with disseminated cryptococcosis demonstrated that 103 patients without comorbidity associated with the disease had normal CD4 + T lymphocyte counts [ 9 ]. Lymphopenia observed during disseminated infection in cryptococcosis may be related to high concentrations of circulating Glucuronoxylomannan (GXM). Fungal polysaccharides interfere with the release of pro-inflammatory cytokines such as TNF-α, IL-1-β and IFN-γ, inhibiting the release of IL-12 and stimulating a very significant increase in IL-10 [ 13 , 14 ]. In addition, several factors may contribute to the suppression state observed during neurocryptococcosis, such as fungal virulence factors, prevalence of anti-inflammatory cytokines and intensity of stimuli conferred by APCs to T lymphocytes. γδ T cells are a distinct subset of T cells, which express Vγ Vδ T cell receptors (TCRs). In human blood, these cells constitute 2 to 10% of the total T cell pool. (Vδ1 + T) cells are predominant in the mucosa and skin, while cells expressing the Vγ2Vδ2 TCR called (Vδ2 + T) cells are predominant in the peripheral blood. When activated, Vδ2 + T cells exhibit phenotypic characteristics of professional APCs, as well as several effector functions. They interact with innate immune cells, T cells and B cells, secrete chemokines, cytokines and promote the lysis of target cells. The presence of γδ T cells is associated with protective immune responses, since in the absence of neutrophils they can generate rapid immune responses to contain the infection [ 15 , 16 ]. Research indicates that γδ T cells play an important role in adaptive immunity, through B cell and dendritic cell maturation. They also have a memory function. Furthermore, when activated, these cells can stimulate CD8 + αβ T cells to multiply and differentiate into cytotoxic T lymphocytes. γδ T cells recognize small, non-peptide antigens that do not require processing by antigen-presenting cells (APCs) and can recognize multiple MHC-related and -unrelated T cell receptor (TCR) ligands in diverse pathophysiological processes [ 17 , 18 ]. However, unlike dendritic cells (DCs), γδ T cells require activation before they can begin to take up antigen [ 19 ]. They are tissue lymphocytes that provide a first line of defense against extracellular and intracellular pathogens, and can translate stimuli that lead to the appropriate functional polarization of αβ T and B cell responses in diverse pathophysiological processes. [ 17 , 19 – 22 ]. There is an additional hypothesis, namely, that γδ T cells may exert negative regulation for Th1 responses during C. neoformans infection by developing an immunoregulatory role in exacerbated Th1 pathway responses, thus possibly acting in the balance between Th1 and Th2 responses during the course of infections [ 15 – 17 , 23 ]. Although we cannot yet clarify the significant increase in γδ T cells and the decrease in total lymphocytes observed in our results, these characteristics may be related to both the effector and regulatory role of these cells during infection. To date, there are few studies and much controversy regarding the effector and regulatory role of γδ T cells during Cryptococcus spp. infection [ 15 , 16 ]. Recently, studies have shown that γδ T cells can increase their expression in response to various pathogens early in the infection. However, as the disease progresses, there is a decrease in both the number of these cells and their function [ 17 , 18 ]. Another important function to be considered is the role of Treg cells in the context of infection. Tregs express the transcription factor Forkhead/winged helix P3 (FOXP3) and perform a homeostatic function to ensure the modulation of immune responses to various autoimmune diseases and fungal infections. Naturally occurring cells have their maintenance promoted by IL-2 [ 15 , 24 – 26 ]. Studies on the role of these cells show that they develop a mediating function and that the regulatory effects are possibly due to the intense Th2 immune responses during Cryptococcus infection [ 15 , 25 , 26 ]. The regulatory T profile demonstrated in our results, with a significant increase in the population (CD4 + CD25 + CD127low) in the group of patients, may be evidence of this regulation, as well as the significant increase demonstrated by the activation phenotype (CD4 + CD25 + CD127 + high) in the control group. Although we did not perform Foxp3 labeling, we can state that CD25 + CD127 low cells present a phenotype with a regulatory profile based on the expression and labeling of CD127 [ 27 ]. The nuclear factor FoxP3 is an important protein involved in the negative regulation of the expression of the CD127 molecule [ 27 ]. The results of the studies by Liu et al. (2006) showed that FoxP3 is present in 86.6% of classical cells (CD25 + CD127low/-), in 22.9% of cells (CD25 + CD127+) and only 1.8% in the phenotype (CD127 + CD25–) [ 27 ]. It is important to emphasize that, according to studies presented by Venet et al. (2009), patients with sepsis develop a regulatory profile with an abundant increase in the population (CD25 + CD127low) and a decrease in the subpopulation (CD25-CD127 + high) in CD4 + T lymphocytes [ 28 , 29 ]. Another regulatory mechanism involves the surface molecule, such as cytotoxic antigen 4 (CTLA-4), characterized as a negative regulator of cytotoxic T cells. However, Chan et al. (2013) showed for the first time that CTLA-4 expression is significantly higher in CD4 + T cells than in CD8 + T cells, although this regulatory mediation is not yet fully understood [ 30 ]. CTLA-4 is constitutively expressed in naturally occurring regulatory cells, and its expression is controlled by the transcription factor Foxp3. CTLA-4 has the characteristic of binding with high affinity to the CD80 and CD86 molecules present in antigen-presenting cells [ 31 ]. When CTLA-4 interacts with CD80 and CD86 molecules present on dendritic cells, it induces the expression of the molecule indoleamine 2,3-dioxygenase (IDO) to release degradation products resulting in the inhibition of T lymphocytes [ 31 ]. Recent studies support the idea of ​​a mutual interaction between plasmacytoid dendritic cells (pDC) and regulatory T cells (Treg) in the initiation and maintenance of immunological tolerance. The implications of indoleamine 2,3-dioxygenase (IDO) in immunoregulation in fungal infections are numerous [ 32 , 33 ]. IDO promotes the generation of regulatory T cells (Tregs) with anti-inflammatory and tolerogenic activities. IDO may be involved in the catabolism of tryptophan and the high levels of IL-10 production may be a consequence of this activation caused by the fungus, which impairs Th1 responses. Tryptophan deprivation can suppress T cell responses, favoring persistent infection. [ 33 , 34 ] Furthermore, pathogens can express IDO and even exploit an environment regulated by this molecule to evade host defense mechanisms. As observed in experimental models in which IDO blockade significantly increased fungal infection, eliminating resistance to reinfection, it was possible to verify that IDO blockade increased fungal infection in experimental models. According to the authors, this suggests that the TGF-β1 signaling pathway may be involved in the induction of both γδ-type Tregs and αβ-type Tregs [ 33 , 34 ]. Inflammation regulated by different pathways is important for defense against a variety of mucosal pathogens, but recent studies indicate a detrimental side of the IL-17/IL-23 inflammatory pathway. IL-23 and the Th17 pathway, which negatively regulate tryptophan catabolism, may instead favor pathology, contributing to the seemingly contradictory association between chronic inflammation and fungal persistence [ 33 , 34 ]. Cells that specifically produce IL-17A play an important role in regulating inflammation in the lungs, acting in several ways. However, excessive production of IL-17A and IL-17F can increase airway inflammation and cause lung hyperreactivity, which may impair the body’s ability to fight infection.[ 34 ] Although some inflammation is necessary for IL-17A to exert its protective effects, persistent or excessive inflammation caused by dysregulation of several cytokines, such as IL-1, which activate Th17 cells, can give rise to a vicious cycle that sustains a chronic and/or difficult-to-treat infection. The complexity of the effects of cytokines, which converge on IL-17A, with a dual function at the host/fungal interface, is still unclear. [ 34 ] The exact role of γδ T cells in these processes is still unclear, but studies have shown that the production of IL-17A by these cells is essential for the control of pulmonary infection caused by Mycobacterium tuberculosis in mice. [ 33 , 34 ] IL-17A, produced by γδ T cells, may play an important role in initiating a rapid and early neutrophil response against mucosal infections. Furthermore, IL-17A may be one of the mechanisms by which γδ T cells help in the defense of the organism until the activation of adaptive immune cells. During fungal pneumonia, γδ T cells increase rapidly but are transient, negatively regulating the inflammation caused by the Th1 response to infection. [ 33 , 34 ] Research indicates that the interaction of γδ cells, which are present in all vertebrates, with tryptophan catabolism may be an important point in the evolution of the innate and acquired immune system and represent a milestone in the adequate control of infection [ 33 , 34 ]. CD4 + T cells have been subdivided into subsets based on expression of CD45RA and CCR7. Naïve cells express CCR7 (CD45RA + and CCR7+), which is predominant in lymphoid tissues. Central memory cells (CD45RA- CCR7+) travel to lymphoid tissues, while effector memory cells (CD45RA- and CCR7-) and TEMRA (CD45RA + and CCR7-) can migrate to various peripheral tissue sites. In humans, memory T cells are distinguished by the CD45RO isoform and the absence of CD45RA isoform expression [ 35 ]. When we analyzed the subpopulations in the CD4 + T compartment for memory status, we observed a significant increase in the expression of TEM cells, TEMRA, and a subsequent decrease in TCM and naïve cells in the patient group. There are several hypotheses about the origin of memory T cells. Some studies argue that they are distinct subgroups and that differentiation occurs linearly, where the stages of differentiation are defined based on the strength of the signal via TCR and antigen concentration, starting from the TCM population to TEM and then TEMRA [ 5 ]. TEMRA are cells with inefficient and highly differentiated effector function. They secrete TNF and IL-6, have low replication, and in the final stages of differentiation, reexpress CD45RA+ [ 36 ]. They are possibly derived from TEM and are more present in older individuals as a result of the numerous infections experienced throughout life [ 5 , 36 ]. The significant increase in TEMRA observed in our results, in the group of patients, may be related to the prolonged exposure of T lymphocytes to the fungus, which could lead to exhaustion and low effector function of these cells. Alternatively, this increase may be evidence of the migration state of this subpopulation to peripheral tissues during infection in these patients [ 6 , 10 ]. Recent studies have shown that TEMRA cells can also follow a death pathway and, the result may be related to increased proliferation and exposure to these antigens, which we did not investigate in the proposal and characterization objectives of this study [ 35 ]. TEM cells emerge at the beginning of the infection, have an immediate effector memory function, secrete IFN-γ and IL-2, and are present in inflamed peripheral tissues [ 5 , 7 ]. TCM are cells with a longer lifespan and ability to renew themselves, have a high proliferative potential, and secrete mainly IL-2. They do not have an effective function, however, when they express the CCR7 + receptor, TCM migrate to the lymph nodes rapidly, where they proliferate and differentiate into effector cells in response to the known antigenic stimulus. They are cells that are more sensitive to antigen stimuli and less dependent on co-stimulation, as well as having a greater capacity for interaction with (DCs) and B lymphocytes [ 4 , 5 ]. Some studies demonstrate that weak stimuli possibly deprive T lymphocytes from acquiring the TEM phenotype and favor the origin of TCM lymphocytes [ 4 , 5 , 7 ]. According to these investigations, the differentiation of memory T cells throughout the immune response is attributed to the complex heterogeneity of APCs, since in the initial stages of infection they assume characteristics that are distinct from local APCs. Thus, they begin to make less antigen available and reduce the production of cytokines, as well as the expression of co-stimulatory molecules [ 5 , 7 ]. In persistent infections, weak signaling directed at lymphocytes may be essential to avoid exhaustion of T lymphocytes and important in the generation of both TEM and TCM cells. Therefore, a balance between stimuli appears to be crucial in both the acute and chronic phases of infection for the generation of both TEM and TCM cell populations [ 5 , 7 ]. Based on these investigations into the mechanisms and development of memory T cells, the increased expression of TEM observed in the group of patients appears to be the result of constant stimuli caused by the fungal load. Furthermore, the decrease in central memory T cells (TCM) may be related to the need for these cells to fulfill their protective functions and maintain memory in situations where there is greater elimination of fungi and control of infection. Inflammation is essential for the body's defense against pathogens, but it is important to balance pro-inflammatory and anti-inflammatory signals and stimuli. Thus, the interactions between the innate and adaptive immune systems can effectively combat fungi. Conclusion Neurocryptococcosis is a serious infection. Both C. neoformans and C. gattii confer persistence in the host through virulence factors and diverse interactions with interference in both innate and adaptive immune response pathways, which can alter both homeostatic and effector function, affecting the immune response of T lymphocytes during infection. The mechanisms and determination of memory T cells have been a difficult challenge and still remain undefined in determining the signals and activation pathways of these cells in neurocryptococcosis. Therefore, monitoring cytokines, Treg populations and other T lymphocyte subpopulations during neurocryptococcosis may be important to guide the choice of the best therapy to adopt for these apparently immunocompetent patients during neurocryptococcosis. Declarations This study received a grant from the Coordination for the Improvement of Higher Education Personnel (CAPES) during the development period of Isabel Alves Feitosa Maciel doctoral thesis under the Beneficiary Program, [n o . 33002010060P2], in the area of Medicine, the Department of Dermatology. Process [n o . 88882.377985/2019-01]. “The authors declare that they did not receive any funding or any other type of subsidy during the preparation of this manuscript”. Conflict of interest All the authors declare no conflict of interest. Author declaration All the authors contributed to the conception and design of this study. The material was prepared by [Isabel Alves Feitosa Maciel], [Roseli Santos de Freitas-Xavier], and [Juliana Ruiz]. Patient data was collected by [Isabel Alves Feitosa Maciel], [Renata Buccheri de Oliveira], [Roseli Santos de Freitas-Xavier], and [Victor Angelo Folgosi]. The data was organized by [Isabel Alves Feitosa Maciel], [Roseli Santos de Freitas-Xavier], and [Victor Angelo Folgosi]. All the data was analyzed by [Isabel Alves Feitosa Maciel], [Juliana Ruiz], [Dewton Morais Vasconcelos], [Paula Ordonhez Rigato], and [Roseli Santos de Freitas-Xavier]. All preliminary versions of the manuscript were written by [Isabel Alves Feitosa Maciel] and [Roseli Santos de Freitas-Xavier]. The final revision of the manuscript was carried out by [Isabel Alves Feitosa Maciel], [Dewton Morais Vasconcelos], [Roseli Santos de Freitas-Xavier], and [Paula Ordonhez Rigato]. All the authors have read and approved the final text. Ethical approval The protocol was evaluated and approved by the Ethics Committee for the Evaluation of Research Projects [CAPPesq] of the the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. [n o . 1.483.420/2016] and the Institute of Infectious Diseases of the Emílio Ribas Hospital [n o .1.569.194/2016]. Consent to Participate All the patients or their legal guardians signed an informed consent form. References Ashton PM, Thanh LT, Trieu PH, Van Anh D, Trinh NM, Beardsley J, et al. Three phylogenetic groups have driven the recent population expansion of Cryptococcus neoformans. Nat Commun. 2019; 10(1): 2035; https://doi.org/10.1038/s41467-019-10092-5. Pizani AT, Almeida MTG. 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Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 1995; 155(3): 1151–64. Schulze B, Piehler D, Eschke M, von Buttlar H, Köhler G, Sparwasser T, et al. CD4(+) FoxP3(+) regulatory T cells suppress fatal T helper 2 cell immunity during pulmonary fungal infection. Eur J Immunol. 2014; 44(12): 3596–604. https://doi.org/10.1002/eji.201444963 Wiesner DL, Smith KD, Kotov DI, Nielsen JN, Bohjanen PR, Nielsen K. Regulatory T Cell Induction and Retention in the Lungs Drives Suppression of Detrimental Type 2 Th Cells During Pulmonary Cryptococcal Infection. J Immunol. 2016 Jan 1; 196(1): 365–74. J Immunol. 2016; 196 (1): 365-74. https://doi.org/10.4049/jimmunol.1501871 Liu W, Putnam AL, Xu-Yu Z, Szot GL, Lee MR, Zhu S, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med. 2006 Jul; 203(7): 1701–11. https://doi.org/10.1084/jem.20060772 Siqueira-Batista R, Gomes AP, Azevedo SFM, Vitorino RR, Mendonça EG de, Sousa FO de et al. CD4+CD25+ T lymphocytes and regulation of the immune system: perspectives for a pathophysiological understanding of sepsis. Rev Bras Ter Intensiva. 2012; 24(3): 294–301. Venet F, Chung CS, Kherouf H, Geeraert A, Malcus C, Poitevin F, et al. Increased circulating regulatory T cells (CD4+CD25 +CD127-) contribute to lymphocyte anergy in septic shock patients. Intensive Care Med. 2009; 35(4): 678–86. https://doi.org/10.1007/s00134-008-1337-8 Chan D V, Gibson HM, Aufiero BM, Wilson AJ, Hafner MS, Mi QS, et al. Differential CTLA-4 expression in human CD4+ versus CD8+ T cells is associated with increased NFAT1 and inhibition of CD4+ proliferation. Genes Immun. 2014; 15(1): 25–32. https://doi.org/10.1038/gene.2013.57 Fallarino F, Grohmann U, Hwang KW, Orabona C, Vacca C, Bianchi R, et al. Modulation of tryptophan catabolism by regulatory T cells. Nat Immunol. 2003; 4(12): 1206–12. https://doi.org/10.1038/ni1003 Fallarino F, Grohmann U, Hwang KW, Orabona C, Vacca C, Bianchi R, et al. Modulation of tryptophan catabolism by regulatory T cells. Nat Immunol. 2003; 4(12): 1206–12. https://www.nature.com/articles/ni1003 Zelante T, Fallarino F, Bistoni F, Puccetti P, Romani L. Indoleamine 2,3-dioxygenase in infection: the paradox of an evasive strategy that benefits the host. Microbes and Infection. 2009; 11(1): 133-41. https://doi.org/10.1016/j.micinf.2008.10.007 Romani L, Zelante T, De Luca A, Fallarino F, Puccetti P. IL-17 and therapeutic kynurenines in pathogenic inflammation to fungi. J Immunol. 2008; 180 (8): 5157-62. https://doi.org/10.4049/jimmunol.180.8.5157 Farber DL, Yudanin NA, Restifo NP. Human memory T cells: generation, compartmentalization and homeostasis. Nat Rev Immunol. 2014; 14(1): 24–35. https://doi.org/10.1038/nri3567 Di Mitri D, Azevedo RI, Henson SM, Libri V, Riddell NE, Macaulay R, et al. Reversible Senescence in Human CD4+CD45RA+CD27− Memory T Cells. The Journal of Immunology. 2011; 187(5): 2093–100. https://doi.org/10.4049/jimmunol.1100978 Tables Table 1 Monoclonal antibodies for surface antigens (BD Biosciences) A-Monoclonals, B-Fluorochromes and C-Clones Table 2 Demographic analysis of patients with neurocryptococcosis and controls Cite Share Download PDF Status: Published Journal Publication published 12 Dec, 2025 Read the published version in Mycopathologia → Version 1 posted Editorial decision: Minor revisions 07 Oct, 2025 Reviewers agreed at journal 30 Jul, 2025 Reviewers invited by journal 30 Jul, 2025 Editor invited by journal 25 Jun, 2025 Editor assigned by journal 21 Jun, 2025 First submitted to journal 20 Jun, 2025 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. 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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-6844830","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":493192506,"identity":"d445aff5-6760-4498-9631-0269cce87037","order_by":0,"name":"Isabel Alves Feitosa Maciel","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-3515-7214","institution":"University of Sao Paulo: Universidade de Sao Paulo","correspondingAuthor":true,"prefix":"","firstName":"Isabel","middleName":"Alves Feitosa","lastName":"Maciel","suffix":""},{"id":493192507,"identity":"6ddcb4d9-4dc7-4d8a-b725-4927a5d7a730","order_by":1,"name":"Juliana Ruiz Ruiz","email":"","orcid":"","institution":"University of Sao Paulo: Universidade de Sao Paulo","correspondingAuthor":false,"prefix":"","firstName":"Juliana","middleName":"Ruiz","lastName":"Ruiz","suffix":""},{"id":493192508,"identity":"077e9748-bf3e-4acd-9083-67792bb5f366","order_by":2,"name":"Renata Buccheri de Oliveira de Oliveira","email":"","orcid":"","institution":"University of California San Francisco","correspondingAuthor":false,"prefix":"","firstName":"Renata","middleName":"Buccheri de Oliveira","lastName":"de Oliveira","suffix":""},{"id":493192509,"identity":"afacb63c-50a4-4f68-95b7-0d7ade30e872","order_by":3,"name":"Paula Ordonhez Rigato Rigato","email":"","orcid":"","institution":"Adolfo Lutz Institute: Instituto Adolfo Lutz","correspondingAuthor":false,"prefix":"","firstName":"Paula","middleName":"Ordonhez Rigato","lastName":"Rigato","suffix":""},{"id":493192510,"identity":"eb9a9a00-d9a9-492b-b8c3-bb0395a6a513","order_by":4,"name":"Victor Victor Angelo Folgosi","email":"","orcid":"","institution":"University of Sao Paulo: Universidade de Sao Paulo","correspondingAuthor":false,"prefix":"","firstName":"Victor","middleName":"Victor Angelo","lastName":"Folgosi","suffix":""},{"id":493192511,"identity":"b2e20839-2988-45cc-b032-d5f9ae67f83d","order_by":5,"name":"Roseli Santos de Freitas-Xavier Freitas-Xavier","email":"","orcid":"","institution":"University of Sao Paulo: Universidade de Sao Paulo","correspondingAuthor":false,"prefix":"","firstName":"Roseli","middleName":"Santos de Freitas-Xavier","lastName":"Freitas-Xavier","suffix":""},{"id":493192512,"identity":"1459c51c-7e6b-4692-afec-3cb00867a34c","order_by":6,"name":"Dewton de Moraes Vasconcelos Vasconcelos","email":"","orcid":"","institution":"University of Sao Paulo: Universidade de Sao Paulo","correspondingAuthor":false,"prefix":"","firstName":"Dewton","middleName":"de Moraes Vasconcelos","lastName":"Vasconcelos","suffix":""}],"badges":[],"createdAt":"2025-06-07 23:16:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6844830/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6844830/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11046-025-01030-9","type":"published","date":"2025-12-12T15:58:43+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88307606,"identity":"17722fcf-0ce9-49ab-9bb4-65cb21313295","added_by":"auto","created_at":"2025-08-05 06:11:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":161204,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis strategy. (a) Singles (FSC-A) versus (FSC-H); (b) Total population of T lymphocytes based on size (FSC) versus granularity (SSC) parameters; (c) Population of T lymphocytes (positive for CD3); (d) Population of helper T lymphocytes, expressing CD3+ and CD4+; (e) Histogram for the CD3+ marker; (f) Histogram for the CD4+ marker\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6844830/v1/8b640c7db192f55cb117296e.png"},{"id":88307611,"identity":"c1461033-4d05-48ea-a701-4fa10a26b4cb","added_by":"auto","created_at":"2025-08-05 06:11:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5383511,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Total lymphocytes (**P=0.007); (b) CD4+TCR-γδ(***P\u0026lt;0.001); (c) CD3+CD4+(N. S) (d)CD4+TCR-αꞵ (N.S). Not Significant (N.S)\u003c/p\u003e","description":"","filename":"02LayoutTREGs.png","url":"https://assets-eu.researchsquare.com/files/rs-6844830/v1/5cf4d88c19335f296c967347.png"},{"id":88307620,"identity":"24f2bbc2-1c7d-4462-a70d-9708fe303071","added_by":"auto","created_at":"2025-08-05 06:11:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8702222,"visible":true,"origin":"","legend":"\u003cp\u003e(a) CD4+CD25+CD127 low (*P=0.013); (b) CD4+CD25+CD127+high (**P=0.001)\u003c/p\u003e","description":"","filename":"03LayoutLTCD4AlfaeBeta.png","url":"https://assets-eu.researchsquare.com/files/rs-6844830/v1/4edae7f684102bff1460e64e.png"},{"id":88307619,"identity":"0a6ef1c1-b6c9-4223-9009-dde82f9cf3c9","added_by":"auto","created_at":"2025-08-05 06:11:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9368267,"visible":true,"origin":"","legend":"\u003cp\u003e(a) naïve (**P=0.001); (b) CD4+ TCM (***P\u0026lt;0.001); (c) CD4+TEM (***P\u0026lt;0.001) ;(d) CD4+TEMRA (**P=0.005)\u003c/p\u003e","description":"","filename":"04LayoutMEMORIACD45.png","url":"https://assets-eu.researchsquare.com/files/rs-6844830/v1/091735626f8c06cb1c7892a1.png"},{"id":98244812,"identity":"05be4e84-4766-4ba0-a1df-a66a6376eed3","added_by":"auto","created_at":"2025-12-15 16:15:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":24541229,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6844830/v1/5bd09e13-c9a7-4e31-812b-2ff3348659f2.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eCytometric analysis of CD3+CD4+T populations and activation and regulation status of naïve and memory CD4+CD45RA T cells in immunocompetent patients with neurocryptococcosis\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCryptococcal meningitis is an opportunistic infection caused by the pathogen \u003cem\u003eCryptococcus spp.\u003c/em\u003e that mainly affects immunosuppressed individuals and is frequently associated with HIV-AIDS infection [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Neurocryptococcosis has a higher incidence in this group, in addition to being associated with 181,000 deaths each year [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, cases of neurocryptococcosis have been reported in \u0026ldquo;apparently immunocompetent\u0026rdquo; individuals in the last decade [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eImmunocompromised patients or those undergoing immunosuppressive therapies are more susceptible to infection by the \u003cem\u003eC. neoformans\u003c/em\u003e strain, while \u003cem\u003eC. gattii\u003c/em\u003e generally affects apparently healthy individuals, with no previous history of disease or any other form of apparent immune system impairment [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTherefore, in order to better understand the severity of the disease, more specific studies need to be conducted and focused mainly on the real participation of T lymphocytes in the course of the infection.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Separation of the peripheral blood mononuclear cells (PBMCs)\u003c/h2\u003e\n \u003cp\u003ePeripheral blood mononuclear cells (PBMCs) were obtained by acollecting 40 mL of heparinized blood and diluting them in sterile 0.9% saline solution at a ratio of 1:1. The blood diluted in saline was transferred to tubes containing 50 mL of Ficoll-Hypaque solution (GE Healthcare Bio-Sciences AB) at a ratio of 1:3 and then centrifuged at 900 G for 20 minutes at 18\u0026deg;C. The cells obtained were centrifuged once with RPMI-1640 culture medium supplemented with 10% fetal bovine serum (FBS) for 10 minutes at 250 G (GIBCO). The PBMCs were washed again in 1 mL of RPMI-1640 with (FBS) and gently resuspended dropwise in 1 mL of the freezing solution (FBS with 10% DMSO). They were then stored in the freezer at \u0026minus;\u0026thinsp;80\u0026deg;C until use. To assess viability, the cells were tested with trypan blue and counted in a Neubauer chamber.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Thawing of peripheral blood mononuclear cells and Immunophenotyping of Total lymphocytes and CD4\u0026thinsp;+\u0026thinsp;subpopulations\u003c/h2\u003e\n \u003cp\u003ePBMCs were thawed in a water bath at 37\u0026deg;C and centrifuged once with 10 mL of RPMI-1640 with 10% (FBS) at 250 G for 10 min. They were then resuspended in 1 mL of RPMI-1640 for counting and transferred to the incubator at 37\u0026deg;C and 5% CO₂ in culture medium with RPMI-1640 with 10% (FBS) at 1 mL of medium for each 1x10\u003csup\u003e6\u003c/sup\u003e cells. After the 24-hour period, the cells were centrifuged once in 1 mL of RPMI-1640 with 10% (FBS) for 10 min at 250 G. They were then resuspended in 1 mL of PBS and incubated with monoclonal antibodies (BD Biosciences) for 25 min at room temperature protected from light (Table\u0026nbsp;1). After that, the cells were washed once with 1 mL of Isoton (PBS-azide) for five minutes at 250 G and resuspended with 300 \u0026micro;L of Isoton. 1x106 cells were acquired on a Fortessa flow cytometer (BD LSR-FortessaTM).\u003c/p\u003e\n \n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Adjustment of parameters on the cytometer\u003c/h2\u003e\n \u003cp\u003eThe acquisition of patient and control samples was performed by multiparametric cytometry on a flow cytometer (BD LSR-FortessaTM). The acquisition was adjusted to 100,000 events in the lymphocyte window. Immunophenotyping was performed with monoclonal antibodies for surface antigens on 1x10\u003csup\u003e6\u003c/sup\u003e PBMCs. To properly assess compensation and determine the positive population by flow cytometry, the Fluorescence Minus One (FMO) control was performed. The phenotypic characterization of the total lymphocyte population and CD4\u0026thinsp;+\u0026thinsp;T cell subpopulations was performed by means of their characteristics regarding size (FSC) and granularity (SSC) (Fig.\u0026nbsp;1). The results were analyzed using the FlowJo\u0026trade; Software (version 10).\u003c/p\u003e\n \u003cp\u003eFrom the selection of T lymphocytes, CD3\u0026thinsp;+\u0026thinsp;T cells and CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;T cells were selected. Then, within the CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;T compartment, the subpopulations were defined as: \u0026mdash; na\u0026iuml;ve CD4\u0026thinsp;+\u0026thinsp;CD45RA\u0026thinsp;+\u0026thinsp;CCR7+ (N); \u0026ndash; effector memory CD4\u0026thinsp;+\u0026thinsp;CD45RA-CCR7- (TEM); \u0026ndash; central memory CD4\u0026thinsp;+\u0026thinsp;CD45RA-CCR7+ (TCM); and \u0026ndash; terminally differentiated effector memory CD4\u0026thinsp;+\u0026thinsp;CD45RA\u0026thinsp;+\u0026thinsp;CCR7- (TEMRA). The other cell subpopulations were defined according to their activation state: effector CD25\u0026thinsp;+\u0026thinsp;CD127\u0026thinsp;+\u0026thinsp;high and regulatory CD25\u0026thinsp;+\u0026thinsp;CD127low.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Casuistry\u003c/h2\u003e\n \u003cp\u003eThe study was conducted with a sample of eight \u0026ldquo;immunocompetent\u0026rdquo; patients with neurocryptococcosis, hospitalized at the Hospital das Cl\u0026iacute;nicas and Instituto de Infectologia Em\u0026iacute;lio Ribas. The control group was composed of eight healthy individuals. Both groups, patients and controls, had no history of other associated infections or underlying diseases, as shown in (Table\u0026nbsp;2). No patient was undergoing therapy with corticosteroids or other immunosuppressive medication. Blood samples from patients were collected between the 15th and 30th day of hospitalization, in parallel with the samples from the control group.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e3. Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eThe statistical analyses of the results obtained were performed using GraphPad Prism\u0026reg; Software (version 5.0). All data related to the immunophenotyping of CD4\u0026thinsp;+\u0026thinsp;T lymphocytes were tested to assess the normality profile using the D\u0026apos;Agostino \u0026amp; Pearson test. For those that presented normal distribution between the groups, the T test was applied. For those that did not present normal distribution, the nonparametric Mann-Whitney test was performed. All values that presented P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e4.1. Demographic analysis\u003c/h2\u003e\n \u003cp\u003eDemographics showed a prevalence of cryptococcosis in males (75%), with a slightly higher mean and median for the patient group, around 39.5 (Table 2).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e4.2. Phenotypic characterization of total lymphocytes and CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;subpopulations\u003c/h2\u003e\n \u003cp\u003eThe study of the total population of T lymphocytes in blood samples in the study groups showed statistical differences (P\u0026thinsp;=\u0026thinsp;0.007) with a mean of 15.88\u0026thinsp;\u0026plusmn;\u0026thinsp;5.748 and 24.26\u0026thinsp;\u0026plusmn;\u0026thinsp;5.046 in the patient and control groups, respectively (Fig.\u0026nbsp;2a).\u003c/p\u003e\n \u003cp\u003eOur results demonstrate that subpopulations within the CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;T compartment, the subpopulation of CD4\u0026thinsp;+\u0026thinsp;TCR-\u0026gamma;\u0026delta; cells is increased in the patient group, with significant statistical differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;2b), however the CD4\u0026thinsp;+\u0026thinsp;TCR-\u0026alpha;ꞵ population did not present statistical differences between the groups analyzed. The mean CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;T population in the patient group was 45.99\u0026thinsp;\u0026plusmn;\u0026thinsp;23.11 and in the control group 54.43\u0026thinsp;\u0026plusmn;\u0026thinsp;12.50 (Fig.\u0026nbsp;2c). The CD4\u0026thinsp;+\u0026thinsp;TCR-\u0026alpha;ꞵ cell population showed a mean in the patient group of 93.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.419 and in the control group of 93.20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.169 as can be seen in (Fig.\u0026nbsp;2d).\u003c/p\u003e\n \u003cp\u003eBoth CD4\u0026thinsp;+\u0026thinsp;CD25\u0026thinsp;+\u0026thinsp;CD127\u0026thinsp;+\u0026thinsp;low regulatory T cells, as well as CD25\u0026thinsp;+\u0026thinsp;CD127\u0026thinsp;+\u0026thinsp;high effector T cells showed statistically significant differences, that is, P\u0026thinsp;=\u0026thinsp;0.013 and P\u0026thinsp;=\u0026thinsp;0.001, respectively as shown in (Fig.\u0026nbsp;3a and 3b).\u003c/p\u003e\n \u003cp\u003eThe results regarding the memory state of CD4\u0026thinsp;+\u0026thinsp;T lymphocytes show significant statistical differences between the subpopulations for the na\u0026iuml;ve (P\u0026thinsp;=\u0026thinsp;0.001) and TCM (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) phenotypes in the patient group (Fig.\u0026nbsp;4a and b), respectively. The TEM phenotype differs statistically (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) with a mean in the patient group of 57.44\u0026thinsp;\u0026plusmn;\u0026thinsp;19.05 and controls 27.30\u0026thinsp;\u0026plusmn;\u0026thinsp;6.825 as can be seen in (Fig.\u0026nbsp;4c), respectively. The analyses were performed based on the expression of the CD45RA and CCR7[\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e] markers. The TEMRA phenotype showed a statistical difference (P\u0026thinsp;=\u0026thinsp;0.005) with a mean of 22.25\u0026thinsp;\u0026plusmn;\u0026thinsp;11.43 for patients and controls, a mean of 7.979\u0026thinsp;\u0026plusmn;\u0026thinsp;4.607 (Fig.d).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we analyzed apparently \u0026ldquo;immunocompetent\u0026rdquo; individuals with neurocryptococcosis, together with a control group (healthy individuals), to characterize the population of T lymphocytes (CD3\u0026thinsp;+\u0026thinsp;CD4+) and subpopulations by analyzing the activation and regulation status of responsive T cells in na\u0026iuml;ve (N), central memory (TCM), effector memory (TEM) and terminally differentiated effector (TEMRA). Thus, our objective was to contribute to this understanding by characterizing the population of T lymphocytes (CD3\u0026thinsp;+\u0026thinsp;CD4+) and subpopulations after collecting peripheral blood between the 15th and 30th day of hospitalization of the patients, for both groups. Although the incidence of cryptococcal meningitis is related to immunosuppression, studies/reports have demonstrated an increase in these occurrences in individuals considered immunocompetent with infection by both the \u003cem\u003eC. neoformans\u003c/em\u003e and \u003cem\u003eC. gattii\u003c/em\u003e strains [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A recent study demonstrates that individuals are continually exposed to fungi, and constant exposure to fungal load may be a risk factor even among healthy individuals [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Cryptococcal infection may be asymptomatic and remain latent for years, or even manifest years later. Furthermore, prolonged exposure to the fungus may impair the memory immune response, should the individual contract the infection [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOur results showed significant differences in the total lymphocyte population and γδ T cells in the patient group. However, there was no statistically significant difference for the CD4\u0026thinsp;+\u0026thinsp;T population when compared with healthy individuals.\u003c/p\u003e\u003cp\u003eSimilar results are observed during infection and treatment period of disseminated cryptococcosis by \u003cem\u003eC. neoformans\u003c/em\u003e and/or \u003cem\u003eC. gattii\u003c/em\u003e as described by Ruan et al. (2017) in an adult patient with disseminated disease by \u003cem\u003eC. neoformans\u003c/em\u003e and normal CD4\u0026thinsp;+\u0026thinsp;T cell count. Similar results were described by Suchitha et al. (2012) in an adult patient affected by \u003cem\u003eC. gattii\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA Chinese retrospective study involving 154 HIV-negative patients with disseminated cryptococcosis demonstrated that 103 patients without comorbidity associated with the disease had normal CD4\u0026thinsp;+\u0026thinsp;T lymphocyte counts [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLymphopenia observed during disseminated infection in cryptococcosis may be related to high concentrations of circulating Glucuronoxylomannan (GXM). Fungal polysaccharides interfere with the release of pro-inflammatory cytokines such as TNF-α, IL-1-β and IFN-γ, inhibiting the release of IL-12 and stimulating a very significant increase in IL-10 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In addition, several factors may contribute to the suppression state observed during neurocryptococcosis, such as fungal virulence factors, prevalence of anti-inflammatory cytokines and intensity of stimuli conferred by APCs to T lymphocytes.\u003c/p\u003e\u003cp\u003eγδ T cells are a distinct subset of T cells, which express Vγ Vδ T cell receptors (TCRs). In human blood, these cells constitute 2 to 10% of the total T cell pool. (Vδ1\u0026thinsp;+\u0026thinsp;T) cells are predominant in the mucosa and skin, while cells expressing the Vγ2Vδ2 TCR called (Vδ2\u0026thinsp;+\u0026thinsp;T) cells are predominant in the peripheral blood. When activated, Vδ2\u0026thinsp;+\u0026thinsp;T cells exhibit phenotypic characteristics of professional APCs, as well as several effector functions. They interact with innate immune cells, T cells and B cells, secrete chemokines, cytokines and promote the lysis of target cells. The presence of γδ T cells is associated with protective immune responses, since in the absence of neutrophils they can generate rapid immune responses to contain the infection [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Research indicates that γδ T cells play an important role in adaptive immunity, through B cell and dendritic cell maturation. They also have a memory function. Furthermore, when activated, these cells can stimulate CD8\u0026thinsp;+\u0026thinsp;αβ T cells to multiply and differentiate into cytotoxic T lymphocytes. γδ T cells recognize small, non-peptide antigens that do not require processing by antigen-presenting cells (APCs) and can recognize multiple MHC-related and -unrelated T cell receptor (TCR) ligands in diverse pathophysiological processes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, unlike dendritic cells (DCs), γδ T cells require activation before they can begin to take up antigen [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. They are tissue lymphocytes that provide a first line of defense against extracellular and intracellular pathogens, and can translate stimuli that lead to the appropriate functional polarization of αβ T and B cell responses in diverse pathophysiological processes. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. There is an additional hypothesis, namely, that γδ T cells may exert negative regulation for Th1 responses during \u003cem\u003eC. neoformans\u003c/em\u003e infection by developing an immunoregulatory role in exacerbated Th1 pathway responses, thus possibly acting in the balance between Th1 and Th2 responses during the course of infections [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough we cannot yet clarify the significant increase in γδ T cells and the decrease in total lymphocytes observed in our results, these characteristics may be related to both the effector and regulatory role of these cells during infection. To date, there are few studies and much controversy regarding the effector and regulatory role of γδ T cells during \u003cem\u003eCryptococcus spp.\u003c/em\u003e infection [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Recently, studies have shown that γδ T cells can increase their expression in response to various pathogens early in the infection. However, as the disease progresses, there is a decrease in both the number of these cells and their function [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAnother important function to be considered is the role of Treg cells in the context of infection. Tregs express the transcription factor Forkhead/winged helix P3 (FOXP3) and perform a homeostatic function to ensure the modulation of immune responses to various autoimmune diseases and fungal infections. Naturally occurring cells have their maintenance promoted by IL-2 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Studies on the role of these cells show that they develop a mediating function and that the regulatory effects are possibly due to the intense Th2 immune responses during Cryptococcus infection [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe regulatory T profile demonstrated in our results, with a significant increase in the population (CD4\u0026thinsp;+\u0026thinsp;CD25\u0026thinsp;+\u0026thinsp;CD127low) in the group of patients, may be evidence of this regulation, as well as the significant increase demonstrated by the activation phenotype (CD4\u0026thinsp;+\u0026thinsp;CD25\u0026thinsp;+\u0026thinsp;CD127\u0026thinsp;+\u0026thinsp;high) in the control group. Although we did not perform Foxp3 labeling, we can state that CD25\u0026thinsp;+\u0026thinsp;CD127 low cells present a phenotype with a regulatory profile based on the expression and labeling of CD127 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe nuclear factor FoxP3 is an important protein involved in the negative regulation of the expression of the CD127 molecule [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The results of the studies by Liu et al. (2006) showed that FoxP3 is present in 86.6% of classical cells (CD25\u0026thinsp;+\u0026thinsp;CD127low/-), in 22.9% of cells (CD25\u0026thinsp;+\u0026thinsp;CD127+) and only 1.8% in the phenotype (CD127\u0026thinsp;+\u0026thinsp;CD25\u0026ndash;) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt is important to emphasize that, according to studies presented by Venet et al. (2009), patients with sepsis develop a regulatory profile with an abundant increase in the population (CD25\u0026thinsp;+\u0026thinsp;CD127low) and a decrease in the subpopulation (CD25-CD127\u0026thinsp;+\u0026thinsp;high) in CD4\u0026thinsp;+\u0026thinsp;T lymphocytes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAnother regulatory mechanism involves the surface molecule, such as cytotoxic antigen 4 (CTLA-4), characterized as a negative regulator of cytotoxic T cells. However, Chan et al. (2013) showed for the first time that CTLA-4 expression is significantly higher in CD4\u0026thinsp;+\u0026thinsp;T cells than in CD8\u0026thinsp;+\u0026thinsp;T cells, although this regulatory mediation is not yet fully understood [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. CTLA-4 is constitutively expressed in naturally occurring regulatory cells, and its expression is controlled by the transcription factor Foxp3. CTLA-4 has the characteristic of binding with high affinity to the CD80 and CD86 molecules present in antigen-presenting cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. When CTLA-4 interacts with CD80 and CD86 molecules present on dendritic cells, it induces the expression of the molecule indoleamine 2,3-dioxygenase (IDO) to release degradation products resulting in the inhibition of T lymphocytes [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Recent studies support the idea of ​​a mutual interaction between plasmacytoid dendritic cells (pDC) and regulatory T cells (Treg) in the initiation and maintenance of immunological tolerance. The implications of indoleamine 2,3-dioxygenase (IDO) in immunoregulation in fungal infections are numerous [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. IDO promotes the generation of regulatory T cells (Tregs) with anti-inflammatory and tolerogenic activities. IDO may be involved in the catabolism of tryptophan and the high levels of IL-10 production may be a consequence of this activation caused by the fungus, which impairs Th1 responses. Tryptophan deprivation can suppress T cell responses, favoring persistent infection. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] Furthermore, pathogens can express IDO and even exploit an environment regulated by this molecule to evade host defense mechanisms. As observed in experimental models in which IDO blockade significantly increased fungal infection, eliminating resistance to reinfection, it was possible to verify that IDO blockade increased fungal infection in experimental models. According to the authors, this suggests that the TGF-β1 signaling pathway may be involved in the induction of both γδ-type Tregs and αβ-type Tregs [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Inflammation regulated by different pathways is important for defense against a variety of mucosal pathogens, but recent studies indicate a detrimental side of the IL-17/IL-23 inflammatory pathway. IL-23 and the Th17 pathway, which negatively regulate tryptophan catabolism, may instead favor pathology, contributing to the seemingly contradictory association between chronic inflammation and fungal persistence [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCells that specifically produce IL-17A play an important role in regulating inflammation in the lungs, acting in several ways. However, excessive production of IL-17A and IL-17F can increase airway inflammation and cause lung hyperreactivity, which may impair the body\u0026rsquo;s ability to fight infection.[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] Although some inflammation is necessary for IL-17A to exert its protective effects, persistent or excessive inflammation caused by dysregulation of several cytokines, such as IL-1, which activate Th17 cells, can give rise to a vicious cycle that sustains a chronic and/or difficult-to-treat infection. The complexity of the effects of cytokines, which converge on IL-17A, with a dual function at the host/fungal interface, is still unclear. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eThe exact role of γδ T cells in these processes is still unclear, but studies have shown that the production of IL-17A by these cells is essential for the control of pulmonary infection caused by \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e in mice. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] IL-17A, produced by γδ T cells, may play an important role in initiating a rapid and early neutrophil response against mucosal infections. Furthermore, IL-17A may be one of the mechanisms by which γδ T cells help in the defense of the organism until the activation of adaptive immune cells. During fungal pneumonia, γδ T cells increase rapidly but are transient, negatively regulating the inflammation caused by the Th1 response to infection. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eResearch indicates that the interaction of γδ cells, which are present in all vertebrates, with tryptophan catabolism may be an important point in the evolution of the innate and acquired immune system and represent a milestone in the adequate control of infection [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCD4\u0026thinsp;+\u0026thinsp;T cells have been subdivided into subsets based on expression of CD45RA and CCR7. Na\u0026iuml;ve cells express CCR7 (CD45RA\u0026thinsp;+\u0026thinsp;and CCR7+), which is predominant in lymphoid tissues. Central memory cells (CD45RA- CCR7+) travel to lymphoid tissues, while effector memory cells (CD45RA- and CCR7-) and TEMRA (CD45RA\u0026thinsp;+\u0026thinsp;and CCR7-) can migrate to various peripheral tissue sites.\u003c/p\u003e\u003cp\u003eIn humans, memory T cells are distinguished by the CD45RO isoform and the absence of CD45RA isoform expression [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhen we analyzed the subpopulations in the CD4\u0026thinsp;+\u0026thinsp;T compartment for memory status, we observed a significant increase in the expression of TEM cells, TEMRA, and a subsequent decrease in TCM and na\u0026iuml;ve cells in the patient group. There are several hypotheses about the origin of memory T cells. Some studies argue that they are distinct subgroups and that differentiation occurs linearly, where the stages of differentiation are defined based on the strength of the signal via TCR and antigen concentration, starting from the TCM population to TEM and then TEMRA [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTEMRA are cells with inefficient and highly differentiated effector function. They secrete TNF and IL-6, have low replication, and in the final stages of differentiation, reexpress CD45RA+ [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThey are possibly derived from TEM and are more present in older individuals as a result of the numerous infections experienced throughout life [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe significant increase in TEMRA observed in our results, in the group of patients, may be related to the prolonged exposure of T lymphocytes to the fungus, which could lead to exhaustion and low effector function of these cells. Alternatively, this increase may be evidence of the migration state of this subpopulation to peripheral tissues during infection in these patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecent studies have shown that TEMRA cells can also follow a death pathway and, the result may be related to increased proliferation and exposure to these antigens, which we did not investigate in the proposal and characterization objectives of this study [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTEM cells emerge at the beginning of the infection, have an immediate effector memory function, secrete IFN-γ and IL-2, and are present in inflamed peripheral tissues [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTCM are cells with a longer lifespan and ability to renew themselves, have a high proliferative potential, and secrete mainly IL-2. They do not have an effective function, however, when they express the CCR7\u0026thinsp;+\u0026thinsp;receptor, TCM migrate to the lymph nodes rapidly, where they proliferate and differentiate into effector cells in response to the known antigenic stimulus. They are cells that are more sensitive to antigen stimuli and less dependent on co-stimulation, as well as having a greater capacity for interaction with (DCs) and B lymphocytes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSome studies demonstrate that weak stimuli possibly deprive T lymphocytes from acquiring the TEM phenotype and favor the origin of TCM lymphocytes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. According to these investigations, the differentiation of memory T cells throughout the immune response is attributed to the complex heterogeneity of APCs, since in the initial stages of infection they assume characteristics that are distinct from local APCs. Thus, they begin to make less antigen available and reduce the production of cytokines, as well as the expression of co-stimulatory molecules [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In persistent infections, weak signaling directed at lymphocytes may be essential to avoid exhaustion of T lymphocytes and important in the generation of both TEM and TCM cells. Therefore, a balance between stimuli appears to be crucial in both the acute and chronic phases of infection for the generation of both TEM and TCM cell populations [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Based on these investigations into the mechanisms and development of memory T cells, the increased expression of TEM observed in the group of patients appears to be the result of constant stimuli caused by the fungal load. Furthermore, the decrease in central memory T cells (TCM) may be related to the need for these cells to fulfill their protective functions and maintain memory in situations where there is greater elimination of fungi and control of infection. Inflammation is essential for the body's defense against pathogens, but it is important to balance pro-inflammatory and anti-inflammatory signals and stimuli. Thus, the interactions between the innate and adaptive immune systems can effectively combat fungi.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eNeurocryptococcosis is a serious infection. Both \u003cem\u003eC. neoformans\u003c/em\u003e and \u003cem\u003eC. gattii\u003c/em\u003e confer persistence in the host through virulence factors and diverse interactions with interference in both innate and adaptive immune response pathways, which can alter both homeostatic and effector function, affecting the immune response of T lymphocytes during infection. The mechanisms and determination of memory T cells have been a difficult challenge and still remain undefined in determining the signals and activation pathways of these cells in neurocryptococcosis. Therefore, monitoring cytokines, Treg populations and other T lymphocyte subpopulations during neurocryptococcosis may be important to guide the choice of the best therapy to adopt for these apparently immunocompetent patients during neurocryptococcosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThis study received a grant from the Coordination for the Improvement of Higher Education Personnel (CAPES) during the development period of Isabel Alves Feitosa Maciel doctoral thesis under the Beneficiary Program, [n\u003csup\u003eo\u003c/sup\u003e. 33002010060P2], in the area of Medicine, the Department of Dermatology. Process [n\u003csup\u003eo\u003c/sup\u003e. 88882.377985/2019-01]. \u0026ldquo;The authors declare that they did not receive any funding or any other type of subsidy during the preparation of this manuscript\u0026rdquo;.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All the authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All the authors contributed to the conception and design of this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The material was prepared by [Isabel Alves Feitosa Maciel], [Roseli Santos de Freitas-Xavier], and [Juliana Ruiz]. Patient data was collected by [Isabel Alves Feitosa Maciel], [Renata Buccheri de Oliveira], [Roseli Santos de Freitas-Xavier],\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand\u003csup\u003e\u0026nbsp;\u003c/sup\u003e[Victor Angelo Folgosi]. The data was organized by [Isabel Alves Feitosa Maciel], [Roseli Santos de Freitas-Xavier], and [Victor Angelo Folgosi]. All the data was analyzed by [Isabel Alves Feitosa Maciel], [Juliana Ruiz], [Dewton Morais Vasconcelos], [Paula Ordonhez Rigato], and [Roseli Santos de Freitas-Xavier]. All preliminary versions of the manuscript were written by [Isabel Alves Feitosa Maciel] and [Roseli Santos de Freitas-Xavier]. The final revision of the manuscript was carried out by [Isabel Alves Feitosa Maciel], [Dewton Morais Vasconcelos], [Roseli Santos de Freitas-Xavier], and [Paula Ordonhez Rigato]. All the authors have read and approved the final text.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe protocol was evaluated and approved by the Ethics Committee for the Evaluation of Research Projects [CAPPesq] of the the Hospital das Cl\u0026iacute;nicas da Faculdade de Medicina da Universidade de S\u0026atilde;o Paulo. [n\u003csup\u003eo\u003c/sup\u003e. 1.483.420/2016] and the Institute of Infectious Diseases of the Em\u0026iacute;lio Ribas Hospital [n\u003csup\u003eo\u003c/sup\u003e.1.569.194/2016].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll the patients or their legal guardians signed an informed consent form.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAshton PM, Thanh LT, Trieu PH, Van Anh D, Trinh NM, Beardsley J, et al. Three phylogenetic groups have driven the recent population expansion of Cryptococcus neoformans. Nat Commun. 2019; 10(1): 2035; https://doi.org/10.1038/s41467-019-10092-5.\u003c/li\u003e\n\u003cli\u003ePizani AT, Almeida MTG. Criptococose em pacientes HIV positivos: revis\u0026atilde;o sistem\u0026aacute;tica da literatura. 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Indoleamine 2,3-dioxygenase in infection: the paradox of an evasive strategy that benefits the host. Microbes and Infection. 2009; 11(1): 133-41. https://doi.org/10.1016/j.micinf.2008.10.007\u003c/li\u003e\n\u003cli\u003eRomani L, Zelante T, De Luca A, Fallarino F, Puccetti P. IL-17 and therapeutic kynurenines in pathogenic inflammation to fungi. J Immunol. 2008; 180 (8): 5157-62. https://doi.org/10.4049/jimmunol.180.8.5157\u003c/li\u003e\n\u003cli\u003eFarber DL, Yudanin NA, Restifo NP. Human memory T cells: generation, compartmentalization and homeostasis. Nat Rev Immunol. 2014; 14(1): 24\u0026ndash;35. https://doi.org/10.1038/nri3567\u003c/li\u003e\n\u003cli\u003eDi Mitri D, Azevedo RI, Henson SM, Libri V, Riddell NE, Macaulay R, et al. Reversible Senescence in Human CD4+CD45RA+CD27\u0026minus; Memory T Cells. The Journal of Immunology. 2011; 187(5): 2093\u0026ndash;100. https://doi.org/10.4049/jimmunol.1100978\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Monoclonal antibodies for surface antigens (BD Biosciences) A-Monoclonals, B-Fluorochromes and C-Clones\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Demographic analysis of patients with neurocryptococcosis and controls\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"mycopathologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"myco","sideBox":"Learn more about [Mycopathologia](https://www.springer.com/journal/11046)","snPcode":"11046","submissionUrl":"https://submission.nature.com/new-submission/11046/3","title":"Mycopathologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cryptococcal meningitis, Virulence factors, Epidemiology of cryptococcosis, Cryptococcus neoformans, Cryptococcus gattii, Immunocompetent patients","lastPublishedDoi":"10.21203/rs.3.rs-6844830/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6844830/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeurocryptococcosis is a serious disease that mainly affects individuals with compromised immune systems. However, \u0026ldquo;immunocompetent\u0026rdquo; individuals are also affected by this condition even without any known underlying disease or compromised immune system. In this study, we evaluated the CD4\u0026thinsp;+\u0026thinsp;T lymphocyte population and subpopulations in the peripheral blood of eight hospitalized patients with neurocryptococcosis and eight healthy control individuals. Thus, our objective was to contribute to this understanding by characterizing the T lymphocyte population (CD3\u0026thinsp;+\u0026thinsp;CD4+) and subpopulations, with analyses of the activation and regulation status of responsive T cells in na\u0026iuml;ve (N), central memory (TMC), effector memory (TME) and terminally differentiated effector (TEMRA) in apparently immunocompetent patients and healthy control individuals. Our results showed a significant increase in CD4\u0026thinsp;+\u0026thinsp;γδ T subpopulations, CD4\u0026thinsp;+\u0026thinsp;CD25\u0026thinsp;+\u0026thinsp;CD127low, CD4\u0026thinsp;+\u0026thinsp;CD25\u0026thinsp;+\u0026thinsp;CD127\u0026thinsp;+\u0026thinsp;high regulatory T cells, CD4\u0026thinsp;+\u0026thinsp;CD45RA\u0026thinsp;+\u0026thinsp;CCR7- terminally differentiated effector memory (TEMRA) T cells and CD4\u0026thinsp;+\u0026thinsp;CD45RA-CCR7- effector memory (TME) T cells. We also observed a significant decrease in total lymphocytes, CD4\u0026thinsp;+\u0026thinsp;CD45RA\u0026thinsp;+\u0026thinsp;CCR7+ (na\u0026iuml;ve) T cells and CD4\u0026thinsp;+\u0026thinsp;CD45RA-CCR7\u0026thinsp;+\u0026thinsp;central memory (TMC) T cells. CD4\u0026thinsp;+\u0026thinsp;T and CD4\u0026thinsp;+\u0026thinsp;αβ T cells did not show statistically significant differences between the study groups. These results suggest that the immune response of these patients is undergoing alterations in the maturation and differentiation of T lymphocytes and may be related to the virulence factors of the fungus that interfere in several mechanisms of the cells of both the innate and adaptive immune response, as well as with possible regulation disorders of T helper subsets immune responses during Cryptococcus infection.\u003c/p\u003e","manuscriptTitle":"Cytometric analysis of CD3+CD4+T populations and activation and regulation status of naïve and memory CD4+CD45RA T cells in immunocompetent patients with neurocryptococcosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-05 06:11:04","doi":"10.21203/rs.3.rs-6844830/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2025-10-07T08:21:41+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-07-30T19:11:24+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-30T12:59:35+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Mycopathologia","date":"2025-06-25T13:59:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-21T14:13:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mycopathologia","date":"2025-06-20T16:55:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mycopathologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"myco","sideBox":"Learn more about [Mycopathologia](https://www.springer.com/journal/11046)","snPcode":"11046","submissionUrl":"https://submission.nature.com/new-submission/11046/3","title":"Mycopathologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8d7d3896-db76-41bc-82fc-11b94ce3298a","owner":[],"postedDate":"August 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T16:08:19+00:00","versionOfRecord":{"articleIdentity":"rs-6844830","link":"https://doi.org/10.1007/s11046-025-01030-9","journal":{"identity":"mycopathologia","isVorOnly":false,"title":"Mycopathologia"},"publishedOn":"2025-12-12 15:58:43","publishedOnDateReadable":"December 12th, 2025"},"versionCreatedAt":"2025-08-05 06:11:04","video":"","vorDoi":"10.1007/s11046-025-01030-9","vorDoiUrl":"https://doi.org/10.1007/s11046-025-01030-9","workflowStages":[]},"version":"v1","identity":"rs-6844830","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6844830","identity":"rs-6844830","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}