Discordant restoration of TCR expression and function by CD247 somatic reversions

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Briones, Ana V Marin, Rebeca Chaparro-García, Marta López-Nevado, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5741291/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Jul, 2025 Read the published version in Journal of Clinical Immunology → Version 1 posted 8 You are reading this latest preprint version Abstract Background: The CD247 chain of the T-cell receptor (TCR) is essential for normal T cell development and function. Reported CD247-deficient patients showed severe immunodeficiency despite the presence of two populations of peripheral T cells, most with low TCR levels carrying the germline variant and a few with higher TCR levels due to somatic reversion. However, the revertant T cells remained a minority and did not improve the patients’ clinical status. Purpose: To compare the capability of somatic reversions of CD247 germline changes (p.M1T and p.Q70X) to restore TCR expression and function. Methods: CD247 wild-type (WT) and p.Q70L/W/Y somatic revertants were individually introduced in CD247-deficient mouse (MA5.8), human mutant (PM1T), and CRISPR/Cas9-generated Jurkat (ZKO) T cell lines by nucleofection or transduction. Results: MA5.8 mouse T cells do not accurately model human CD247 deficiencies, as Q70X restores TCR expression in MA5.8 but not in human cells. In human cell models, all somatic revertant variants restored TCR expression with varying degrees (WT=Q70L>Q70W>Q70Y). However, rescue of TCR-induced activation events, including ZAP-70 phosphorylation and CD69/CD25 upregulation, did not match such hierarchy (WT=Q70W>Q70L=Q70Y). Conclusion: Somatic reversions, such as those detected in patients with pathogenic CD247 germinal changes, display a discordant capability to rescue TCR expression versus function. These findings shed light on the role of CD247 in TCR expression and function during human T cell development, with implications for immunodeficiencies, as well as for the biological consequences of CD247 somatic mosaicism. CD247 CD3Z TCR immunodeficiency somatic reversions Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The CD247 chain, also known as CD3ζ, is crucial for T cell development and function [ 1 ]. It plays a key role in T-cell receptor (TCR) assembly and surface expression by cooperating with other invariant CD3 molecules and in TCR development and function by transducing signals from the TCR to initiate the activation and response of T cells for thymus selection or antigen recognition [ 2 , 3 ]. CD247 deficiency is a rare early-onset primary immunodeficiency disorder characterized by variants in the CD247 gene ( Table S1 ). These variants can result in a lack of functional CD247 chain expression in T cells causing severe defects of TCR expression and function, which, in turn, may affect T cell development and function and, thus, immune responses. All reported patients showed T cell lymphopenia, suffered infections, and required stem cell transplantation to survive. Older patients also showed autoimmune features, suggesting poor T-cell selection. Revertant somatic mosaicism is a phenomenon where spontaneous genetic corrections or compensations occur in a subset of cells, partially restoring the function of an altered germ-line gene [ 4 ]. CD247 deficiency frequently associates with revertant somatic mosaicism (4 of 5 patients, Table S1 ), likely due to the propensity of CD247 to vary [ 5 ]. This can lead to improvements in TCR expression (partial phenotypic reversion) in a minute subset of T cells, but it is unclear if phenotypic reversion leads to functional reversion, as CD247 protein domains involved in the former may not be sufficient for the latter. The clinical impact of CD247 somatic mosaicism also remains unclear, as it did not improve the clinical status of any reported patient. T-cell models are invaluable tools for studying human T-cell immunodeficiencies [ 6 ]. However, a human T cell model to study CD247 somatic mosaicism has not been reported. MA5.8, a murine CD247-deficient T cell line [ 7 ], has been widely used to establish the role of CD247 in TCR assembly [ 8 , 9 ] and expression [ 5 ], as well as to study CD247 splicing variants [ 10 ]. As human and murine invariant TCR-associated molecules show differential roles in each species [ 11 , 12 ], murine T cell models may show limitations to predict human CD247 role in TCR expression and function. Here, we compared the capacity of CD247 somatic variants in two immunodeficiency cases with severe CD247 germline changes (p.M1T and p.Q70X, Fig. S1 ) to restore TCR expression and function in different T cell models, both human and murine. The somatic variants in each case were a wild-type (WT) reversion of p.M1T and three missense compensating variants (p.Q70L, p.Q70W, and p.Q70Y) of the germinal p.Q70X. Materials and methods Cell lines and culture HTLV-1 transformed p.M1T T-cell line was derived from peripheral blood mononuclear cells (PBMC) of a CD247-deficient patient [ 13 ] as described in the Online Repository. The Jurkat wild-type T-cell line (J77cl20 clone) was provided by Dr B. Rubin (Centre National de la Recherche Scientifique, Centre Hospitalier Universitaire, Purpan, Toulouse, France). The Jurkat CD247-deficient cell line, ZKO, was generated by CRISPR/Cas9 gene editing [ 14 ]. All human T-cell lines were grown in RPMI-1640 (Lonza, Basel, Switzerland), supplemented with 10% FBS, 1x L-Glutamine 200mM, and 1x Antibiotic Antimycotic from Gibco (Bethesda, MD, USA). In addition, 100 U/mL recombinant human IL-2 (provided by Craig W. Reynolds, Frederick Cancer Research and Development Center, National Cancer Institute, National Institutes of Health, Frederick, MD, USA) was added to p.M1T cells. The HEK293T and Phoenix-Ampho (ATCC® CRL-3213™) packaging cell lines were grown under standard conditions in Iscove’s Modified Dulbecco’s Medium (Lonza). The murine T-cell lines, including parental 2B4 and its CD247-deficient derivative MA5.8 [ 7 ], were gifted by Balbino Alarcón (Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain). These cell lines were cultured in the same conditions as above but with 5% FBS. All cell lines were maintained at 37°C and 5% CO 2 in a humidified incubator. Plasmids, nucleofection, retroviral and lentiviral transduction The pEGFP-N1 vector (Clontech, Mountain View, CA, USA) containing the human CD247 transcript variant or isoform 2 sequence (NM_000734; hCD247-WT from now on) or germline p.Q70X change (X for short) was a kind gift from Hugh T. Reyburn (National Centre for Biotechnology, Madrid, Spain). The somatic variants p.Q70L, p.Q70W and p.Q70Y were introduced by site-directed mutagenesis. For nucleofection assays, 1.5 x 10 6 immortalized HTLV-1 or murine T cells were nucleofected with 2 µg of pEGFP-N1 plasmid, carrying each CD247 variant, using the Cell Line Nucleofector Kits V or R, respectively, and the Amaxa Nucleofector 2b device (Lonza, Walkersville, MD, USA) according to the manufacturer’s instructions. Twenty hours post-nucleofection, cells were collected, and the transfection efficiency was analyzed through flow cytometry by counting the fraction of green fluorescent protein (GFP) - expressing cells. For retroviral transduction hCD247-WT, along with germline and somatic CD247 variants were introduced into the pLZRS-IRES-ΔNGFR retroviral plasmid and transfected into Phoenix-AMPHO cells as described [ 15 ]. Both the pLZRS-IRES-ΔNGFR retroviral plasmid and the Phoenix-AMPHO cells were provided by Rubén Martínez-Barricarte (Vanderbilt Institute for Infection, Vanderbilt University Medical Center, Nashville, TN). For lentiviral transduction, the studied CD247 variants were cloned into the pHRSIN-C56W-UbEM lentiviral plasmid (gifted by Hugh T Reyburn). Viral particle generation and cell transduction were performed following the previously reported protocol [ 14 ]. Transduced cells were selected by flow cytometry. Those transduced with the pLZRS-IRES-ΔNGFR plasmid were positive for the CD271 marker, whereas those transduced with the pHRSIN-C56W-UbEM plasmid were positive for GFP. Flow cytometry analysis Standard extracellular flow cytometry was performed with monoclonal antibodies (mAbs) against human CD3ε (clone UCHT1) from Beckman Coulter (Brea, CA) or mouse CD3ε (clone 145-2C11) from eBioscience (San Diego, CA). In addition, a mAb against CD271 (clone C40-1457) from BD Biosciences (San Jose, CA) was also employed. Intracellular stainings were done with the FOXP3/Transcription factor staining buffer set from Invitrogen. For intracellular quantification, human CD247 mAb (Clone 6B10.2) from BioLegend (San Diego, CA) was used. To analyze surface TCR complex expression, mAb against TCR𝛼ꞵ (clone IP26) from Thermo Fisher Scientific, and TCR Vꞵ8 (clone 56C5.2) from Beckman Coulter, were used. Data were acquired with a FACSCalibur flow cytometer (BD Biosciences) and analyzed with FlowJo software from TreeStar (Ashland, OR). Cell sorting was carried out to purify specific cell populations using a FACSAria™ III sorter (BD Life Sciences, San Jose, CA). In all cases, mean fluorescence intensity (MFI) stands for geoMFI. Functional studies CD247 plays a critical role in T cell activation at the initiation of TCR signaling [ 16 ]. Proliferation, in contrast, is a late consequence of T cell activation and occurs distally from CD247. Also, the proliferation of transformed T cells (as the ones used in our study) following TCR stimulation does not differ significantly from that of unstimulated cells because of the intrinsic proliferative phenotype, which is usually associated with cell transformation. For these reasons, we chose to assess immediate (ZAP-70 tyrosine phosphorylation) and early (CD69/CD25 surface upregulation) events of TCR-mediated T cell activation to dissect the differential impact of CD247 variants. To measure CD69 upregulation after TCR engagement, CD247-deficient human T cell lines (HTLV-1 or Jurkat-derived) were stimulated for 24 hours with 1 µg/ml of plastic-coated anti-CD3ε (clone OKT3) from eBioscience or 10 ng/ml Phorbol 12-myristate 13-acetate (PMA) plus 1 µM Ionomycin from Sigma-Aldrich. To analyze T-cell function in a more physiological way (namely, superantigen recognition, WT or ZKO-transduced Jurkat T cells expressing different CD247 variants were co-cultured with Raji cells (Ratio 1:1) preloaded with Staphylococcal Enterotoxin E (SEE) (0,5 µg/mL) (Sigma-Aldrich) for 18 hours in round-bottom 96-well plates. Cells were then collected and stained with anti-CD19 (clone HIB19; BD Biosciences) to discriminate Jurkat from Raji cells. CD69 and CD25 induction in response to TCR activation were evaluated essentially as published [ 14 ]. ZAP-70 phosphorylation was determined by intracellular flow cytometry. 0.3 x 10 6 WT or ZKO-transduced Jurkat T cells were stimulated with 20 µg/mL of anti-CD3ε mAb (clone OKT3) for 30 minutes at 4ºC. Then, anti-CD3ε was crosslinked with 10 µg/mL goat F(ab’) 2 anti-mouse Ig (H + L) for 5 minutes at 37 ºC. The reaction was stopped by adding cold PBS and centrifuging at 10.000 rpm for 5 seconds. Cells were then fixed/permeabilized with eBioscience™ Foxp3 / Transcription Factor Fixation/Permeabilization Concentrate and Diluent (Invitrogen), according to the manufacturer’s instructions, and finally stained with anti-ZAP70/Syk (Tyr319, Tyr352) mAb (clone n3kobu5) APC-labelled from eBioscience. Statistical analysis To determine the statistical significance of the obtained results, either one-sample t-test or one-way ANOVA test was performed. The test used in each case is indicated in the figure legend. The error bars represent the standard error of the mean (SEM). Results Partial surface TCR reconstitution after transfection of the PM1T cell line with hCD247-WT After confirming that the PM1T cell line lacks CD247 protein, as evidenced by its impairment in TCR expression and function ( Fig. S2 ), it was selected as the cellular model to study the impact of different CD247 variants on TCR assembly and signaling. First, human CD247-WT (hCD247-WT) was cloned into the pEGFP-N1 vector and then nucleofected into the CD247-deficient human (PM1T) or mouse (MA5.8) cell lines. Our results showed that hCD247-WT poorly restored surface TCR expression in PM1T, only increasing it from 4–9% (relative to HTLV-1 control cell line levels) (Fig. 1 A). However, introducing hCD247-WT in MA5.8 significantly improved surface TCR expression, which increased from 12–75% (compared to the 2B4 parental cell line) (Fig. 1 B). Human, but not mouse, CD247-deficient cell lines recapitulate CD24 7 variants’ effects in vivo. For the study of the impact of CD247 variants on surface TCR expression and function, we selected the variants reported in a 10-month-old child with severe combined Immunodeficiency (SCID) caused by CD247 deficiency [ 17 ]. These include a CD247 homozygous nonsense germline change, Q70X (which generates a protein lacking all three ITAMs), and was identified in 90% of patients’ T cells that exhibited low TCR expression. Additionally, the study identified three somatic missense reversions, namely, Q70W, Q70L and Q70Y ( Fig. 2 A ) , in the remaining 10% of the child’s T cells. These mixed reversions, purified by their higher surface CD3 expression, allowed normal TCR expression but were jointly considered poorly functional by the authors, based on the lack of ZAP-70 phosphorylation upon CD3 engagement [ 17 ]. Interestingly, the in silico predictors PolyPhen, SIFT or Saphetor classified the Q70X variant as strongly pathogenic, and Q70W and Q70Y variants as probably damaging, whereas Q70L was considered benign. Nucleofection results showed that the Q70X change did not restore TCR expression in human PM1T, mimicking the patient’s conditions where it was described. Surprisingly, this change fully restored TCR levels in MA5.8 (Fig. 2 B, C). Therefore, this result suggests that PM1T is a more suitable model for studying human CD247 variants than the mouse MA5.8 cell line. Nucleofection assays with plasmids encoding for somatic reversions, Q70W and Q70L, rescued almost up to 100% surface TCR levels in p.M1T (relative to those obtained with the WT gene). However, the Q70Y variant was not able to reach the same level of restoration (61%, compared to those obtained with the WT gene) (Fig. 2 B). Similar results were obtained in MA5.8 with Q70W and Q70L variants (Fig. 2 C). However, in MA5.8, Q70Y showed a more significant defect in recovering surface TCR than in PM1T (35% vs. 61%, respectively). Finally, we also found that the nucleofection of Q70X in control Jurkat T cells results in a decrease in surface TCR expression. However, this effect was not observed in control mouse T cells (2B4) ( Fig. S3 ). Transduction of CD247-defective cell lines significantly improves TCR surface reconstitution values To improve the TCR reconstitution levels obtained by nucleofection, we cloned the analyzed CD247 variants into the retroviral plasmid pLZRS-IRES-ΔNGFR. Our results showed that transduction with hCD247-WT resulted in a significant increase in TCR expression in PM1T, from 4–52%. Meanwhile, the TCR recovery in MA5.8 was almost complete, reaching up to 96%, which is similar to the parental 2B4 cell line levels (Fig. 3 A). When the ability of transduced CD247 somatic reversions to recover TCR expression in PM1T was tested, Q70W rendered similar reconstitution levels to hCD247-WT (100%), whilst Q70L and Q70Y recovered 85% and 57%, respectively. In contrast, Q70X only re-established 9,8% of surface TCR expression (Fig. 3 B). The analysis of CD3 MFI values showed that reconstitution levels observed among hCD247-WT and Q70Y/Q70X were statistically significant (Fig. 3 C). According to our data, we have confirmed that transduction is a more efficient method than nucleofection for restoring surface TCR levels in CD247-deficient cell lines. Unfortunately, the retroviral transduction efficiency was very low (8%), which greatly hindered our ability to conduct functional assays with the variants of interest. As one of the objectives of this work was to test the functional performance of the different CD247 variants, we generated a CD247-deficient Jurkat cell line (ZKO) by employing CRISPR/Cas9 technology. As reported, the ZKO cell line lacked intracellular CD247 and showed a significant reduction in extracellular CD3 expression [ 14 ]. For additional details on the characteristics of the ZKO cell line, see Figs. S4 and S5 . CD247 variants display contrasting phenotypic and functional TCR reconstitution levels in ZKO cells. At first, we examined TCR expression following ZKO transfection with EGFP-tagged constructions. We discovered that transfection of hCD247-WT resulted in TCR levels that were comparable to control values. Additionally, the increase in surface CD3 expression was directly proportional to the amount of EGFP expressed by the cell (Figure S6 ). Unfortunately, we were unable to assess the functional recovery of these cells due to damage caused by transfection. To overcome this issue, we cloned all CD247 variants into the pHRSIN-C56W-UbEM plasmid and utilized a lentiviral transduction protocol to carry out these assays. When hCD247-WT was introduced into the ZKO cell line through lentiviral transduction, surface TCR expression, measured with UCHT1 mAb, was completely restored (100%, compared with the Jurkat WT cell line). However, the other CD247 variants showed varying abilities to recover TCR expression, as seen in previous experiments (Fig. 4 A). The quantification of these results showed that the somatic variants Q70L and Q70W had the highest reconstitution levels, with Q70L recovering 100% and Q70W recovering 70% of TCR expression when compared to hCD247-WT. On the other hand, the somatic reversion Q70Y and the germline Q70X change only recovered 37% and 7% of surface TCR levels, respectively (Fig. 4 B). These results are comparable to those obtained from transfection experiments and confirmed that CD247 somatic reversions have different abilities to allow a correct TCR assembly and expression at the plasma membrane. In addition to UCHT1, the TCR complex surface expression in the transduced ZKO cell lines was also measured with IP26 and VB8 monoclonal antibodies, which recognize the alpha-beta chains or the β-chain variable region (Vβ8) of the TCR, respectively. In general, the results obtained with IP26 and VB8 were similar to those obtained with UCHT1, as indicated by the following ranking: WT > Q70L > Q70W > Q70Y > Q70X. However, in all the variants, we observed slightly lower IP26 values than those obtained with VB8 (Fig. 4 B). Besides evaluating phenotypic TCR reconstitution, we assessed the functional reconstitution of transduced ZKO cells by observing the upregulation of T-cell activation markers upon stimulation with SEE-loaded Raji cell conjugates. Concerning late T-cell activation markers, specifically CD69 upregulation, our results showed that transduction of the ZKO cell line with hCD247-WT restored TCR-dependent induction of this marker after TCR engagement. Surprisingly, Q70W variant performed better than hCD247-WT in inducing CD69; whereas Q70L, which fully recovered TCR expression, was not as efficient in upregulating CD69. Q70Y, only partially induced CD69 expression, while Q70X completely blocked TCR-dependent CD69 induction (Fig. 4 C). The performance of the different variants to upregulate CD69 is abbreviated as follows: Q70W > WT > Q70L > Q70Y > Q70X. Similarly, for CD25 induction, transduction with hCD247-WT recovered the ability of the ZKO cell line to express this activation marker. Once again, the Q70W reversion resulted in better CD25 upregulation compared to Q70L. Interestingly, Q70X induced CD25 at similar levels to Q70Y (Fig. 4 C). In summary, the order of CD25 induction efficiency is Q70W = WT > Q70L > Q70Y = Q70X. Regarding the early activation marker, p-ZAP70, we observed a significant increase only in hCD247-WT transduced ZKO cells. However, we also noticed a slight upregulation in Q70L, Q70Y and Q70W, while Q70X was unable to induce it ( Fig. S7 ). Based on our findings, we determined that the TCR phenotypical reconstitution ability of the studied CD247 variants differs from their functional reconstitution ability. Our data also showed that the CD247 somatic reversions had a poorer functional performance at short times (p-ZAP-70) but improved over time (CD69 and CD25). To explain our results, we used AlphaFold to generate in silico 3D models of CD247. While the transmembrane region showed reliable predictions, the cytoplasmic region, including Q70, was intrinsically disordered, preventing accurate structural modeling (data not shown). To assess whether the Q70Y variant affects CD247-ZAP70 and CD247-LCK interactions, we performed additional in silico analyses. Our results showed that both Q70 and Y70 are positioned outside the ZAP70 interaction interface, suggesting no impact on CD247-ZAP70 binding (data not shown). For the CD247-LCK interactions, accurate modeling of ITAM1 and ITAM2 was not possible, but we successfully modeled ITAM3, where G139 is the position equivalent to Q70 in ITAM3, relative to the first tyrosine of ITAM3 (Y141). Introducing the G139Q maintained Y141 within the LCK active site, whereas the G139Y variant repositioned Y141 outside ( Fig. S8 ). Although these results support the notion that the Y70 variant reduces CD247 phosphorylation by LCK, a similar result was observed with the CD247 L70 variant (data not shown). These findings suggest that Q70 plays a critical role in stabilizing or enhancing CD247-LCK interactions. Discussion Somatic reversion, a phenomenon observed in various primary immunodeficiencies, significantly modifies the clinical outcomes of these pathologies [ 4 ]. It can be positive, neutral or negative, depending on the gene. Interestingly, despite the detection of immune cell subsets expressing functional proteins and exhibiting restored functionality, somatic reversion improved clinical outcomes only in certain conditions [ 4 , 18 ]. For instance, it has a positive clinical impact on ADA deficiency, Wiskott-Aldrich syndrome (WAS), Fanconi anemia, and in variants affecting DOCK8, ITGB2, or CXCR4 [ 18 ]. However, it negatively impacts patients with somatic variants in CARD11, RAG1, as well as in a reported case of IL2RG reversion in tissue infiltrating T-cells, all associated with Omenn syndrome [ 4 , 18 ]. Additionally, one patient with a reversion in NEMO, developed refractory inflammatory colitis, as his revertant T cells activated NF-kB in response to growth signals and had a growth advantage over cells carrying the germline change [ 19 ]. In other cases, somatic reversion can be neutral, as seen in CD247 or Ligase IV [ 20 ] deficiencies, where WT reversions were unable to modify the clinical or immunological phenotype. It has been hypothesized that somatic revertant variants are common in proliferative tissues, like the hematopoietic system, but are limited by the need for functional protein restoration [ 21 , 22 ]. In the case of CD247 deficiency, only five patients with homozygous germline variants have been reported. Among these, four exhibited a small population of revertant T cells ( Table S1 ), but this did not correlate with improved clinical outcomes as all experienced life-threatening infections, with only one surviving post-hematopoietic stem-cell transplantation (HSCT). Since no functional studies have explored the potential effect of CD247 somatic reversions, one main objective of our study was to understand, at the molecular level, the impact of different variants on TCR surface expression and CD247-dependent T-cell functions. To this end, we chose the main CD247 somatic reversions (WT, p.Q70W, p.Q70L, and p.Q70Y) in comparison to the germline change (p.M1T, and p.Q70X) reported by Marin [ 13 ] and Rieux-Laucat [ 17 ], respectively. We found varying degrees of surface TCR expression restoration among the revertants (WT > Q70L > Q70W > Q70Y > > Q70X), which was consistent across different staining antibodies. As expected, the p.Q70X germline variant showed no restoration (Fig. 4 A, 4 B and Table S2 ). This suggests that the revertants can partially or fully restore TCR expression, whereas the germline variant is incapable of doing so. These differences might indicate that the specific amino acid changes in each revertant differentially affect the efficiency of TCR complex assembly and surface expression. Notably, by testing murine and human cell lines side-by-side, we learned that CD247-deficient mouse T cells MA5.8 cannot be used to model human CD247 deficiencies, since Q70X, which is expectedly unable to restore TCR expression in human CD247-deficient T cells (PM1T or ZKO), does so in MA5.8 (Fig. 2 and Table S2 ). Therefore, previous reports using such murine cell lines should be reinterpreted in light of our findings [ 5 , 23 ]. A potential limitation of this conclusion is that they are drawn from a truncated protein (Q70X) that is translated upon transfection and transduction, respectively, from a cDNA, and thus may not exist in primary patient’s T cells due to nonsense-mediated RNA decay which is prevented when using cDNA. However, Rieux-Laucat et al. reported in the discussion that Q70X CD247 was detected in small amounts in the cytoplasm but not on the membrane in primary patient’s T cells [ 17 ], although no data were included to support that contention. We have detected a different truncated CD247 in primary patient’s T cells which did not undergo nonsense-mediated RNA decay either (Y154X, unpublished), suggesting that this may be the case also with Q70X. Genome-edited cell lines with Q70X are in progress to address this question in more detail in the future. Interestingly, analysis of the impact of revertant variants on TCR function, by measuring surface expression of CD69 and CD25 after stimulation with SEE-loaded Raji cells, revealed patterns of CD69 upregulation (Q70W > WT > Q70L > Q70Y > > Q70X) (Fig. 4 C, left ) and CD25 + cells (Q70W > WT > Q70Y = Q70X > Q70L) (Fig. 4 C, right ) which did not correlate with surface TCR expression. The p.Q70W variant showed the highest functional restoration, while the p.Q70L variant, despite good TCR expression recovery, was less effective in inducing CD25 expression upon TCR engagement. These results indicate that the p.Q70W variant, although not the best at restoring TCR expression, is the most effective in triggering T cell activation, as shown by higher TCR-mediated CD69 upregulation. We hypothesize that Q70W outperforms wild-type (WT) and Q70L in CD69 and CD25 upregulation due to its aromatic ring, which may facilitate stronger interactions with signaling partners, enhancing ITAM phosphorylation and activation. In contrast, Q70L hydrophobic nature likely hinders essential hydrogen bonding, impairing ITAM accessibility and phosphorylation. This lack of interactions may impair early activation events, including CD69 upregulation and sustained CD25 induction. This further suggests that certain CD247 reversions may enhance signaling pathways downstream of the TCR, leading to more robust T-cell activation. In silico predictors PolyPhen, SIFT or Saphetor classified Q70X as strongly pathogenic, Q70W and Q70Y as probably damaging, while Q70L is considered benign. Therefore, the expected clinical hierarchy based on these predictors would be (WT = Q70L > Q70W = Q70Y > > Q70X). Our phenotypic results generally confirm but also refine such predictions (WT > Q70L > Q70W > Q70Y > > Q70X). The functional results add further layers of complexity, as expression is required for function: Q70W > WT > Q70L > Q70Y > > Q70X (by CD69 upregulation), Q70W > WT > Q70Y = Q70X > Q70L (by CD25 + cells) and WT > > Q70L = Q70W = Q70Y > > Q70X (by Zap70 phosphorylation, as reported by Rieux-Laucat [ 17 ]). We thus believe that our cellular model to interrogate variants is more informative than purely in silico predictors In addition, the discrepancy between the TCR expression levels and the degree of TCR activation, suggests that Q70 substitutions impact CD247 function beyond surface expression. Q70, a polar residue, likely stabilizes CD247 via hydrogen bonding. Replacing it with hydrophobic (leucine and tryptophan) or amphipathic (tyrosine) residues may alter conformation and signaling. Q70L supports TCR expression but weakens activation, likely due to the loss of hydrogen bonding crucial for ITAM phosphorylation. Q70W disrupts surface expression but enhances signaling, possibly by facilitating stronger ITAM interactions through its aromatic ring. Q70Y impairs both expression and early activation, likely because its bulky aromatic ring and polar hydroxyl group disrupt the structural integrity or proper folding of CD247. However, its hydroxyl group may allow limited interactions that support moderate CD25 expression, indicating partial preservation of sustained signaling pathways. Given the crucial role of CD247 in TCR selection and tolerance, the clinical implications of the results obtained with the Q70W and Q70L variants are significant. The Q70W heightened induction of CD69 and CD25 could potentially lead to excessive T-cell activation, disrupting the immune balance and increasing the risk of autoimmune diseases and chronic inflammation. Conversely, the Q70L variant, with its impaired T-cell activation, may result in immunodeficiency, increasing susceptibility to infections and related complications. In addition, none of the CD247 revertants or the germline change induced ZAP-70 phosphorylation after TCR stimulation with an anti-CD3 mAb ( Fig. S7 and Table S2 ). This can be attributed to the fact that ZAP-70 phosphorylation is an immediate (very early) event during T cell activation, whilst CD69 and CD25 upregulation are early events, relatively distal from TCR signaling initiation (which peak at 24 and 48 hours post-stimulation, respectively); thus, allowing the cell to respond to cumulative TCR signaling from CD247 somatic variants, or, alternatively, from CD3 receptor subunits [ 24 ]. Overall, these findings highlight that restoring TCR expression does not always lead to functional recovery, and each revertant shows a unique restoration profile, underlining the importance of evaluating multiple functional markers to fully understand the impact of somatic reversions. However, we are fully aware that for missense variants, overexpression from strong promoters can compensate for the functional defects of the protein, so it would be advisable for this kind of experiments, to generate cell lines that express the variants of interest from the endogenous gene and not generating knock-out cell lines that are transduced with a cDNA that is overexpressed. Concerning the discordant effects on expression and function of the studied variants, Kaiser et al. [ 21 ] reported a CD247-deficient patient (Table S1 ) with a frameshift mutation in the CD247 leader peptide, exhibiting more than 30 non-WT somatic mutations that could, to varying degrees, restore surface TCR expression. Some of them persisted for months and some others did not, irrespectively of surface TCR expression levels, suggesting the existence of discordant effects on expression and function (measured as T cell survival in vivo) , although such effects were not operating in TCR assembly interactions, but rather in leader peptide functions. In other genes, some examples have been reported, too. Reconstitution assays in vitro with PreTCRα variants found in patients showed that some restored surface preTCRα expression partially but were fully impaired functionally [ 25 ]. Similarly, expression of a mutant NEMO protein was not markedly reduced by flow cytometer, but the activity of mutant NEMO was defective, as confirmed by a mutant NEMO-NF-κB luciferase reporter assay [ 19 ]. In conclusion, our results indicate that somatic mosaicism in CD247 is a common, random event in T cells. However, its capacity to restore TCR expression does not match TCR function. Additionally, none of the tested revertants, including WT, improved the patients’ survival, likely because these events took place too late in T cell development to have a clinical impact, as suggested by Kaiser [ 21 ] and Attardi [ 22 ]. These findings may be relevant to understand the role of CD247 in TCR structure and function during human T cell development in vivo and its impact on human immunodeficiencies. Declarations Acknowledgements The authors are grateful to Balbino Alarcon for supplying the 448 polyclonal antibody; Hugh T. Reyburn for providing the CD247- pEGFP-N1 construction and the pHRSIN vector, and Rubén Martínez Barricarte for the pLZRS-IRES-ΔNGFR vector and the Phoenix-AMPHO cell line. Authors contribution Alejandro C. Briones, Paula P. Cardenas and José R. Regueiro conceived and designed the study. Alejandro C. Briones, Rebeca Chaparro-García, Marta López-Nevado, Iván Estevez-Benito and Daniel Chacón-Arguedas conducted the experiments. Alejandro C. Briones, Iván Estevez-Benito, Daniel Chacón-Arguedas and Paula P. Cárdenas analyzed the experimental data. Ana V. Marin generated the PM1T cell line and assisted with manuscript editing. David Abia made the 3D models prediction. Paula P. Cardenas and José R. Regueiro wrote the manuscript. Edgar Fernández-Malavé provided input on the study design and critically revised the manuscript. All authors reviewed and approved the final manuscript. Funding This study was supported by grants from the Ministerio de Economía y Competitividad (MINECO RED2022-134750-T, PID2021-125501OB-I00, and RTI2018-095673-B-I00), the Comunidad Autónoma de Madrid (P2022/BMD-7278, PR38/21-13 ANTICIPA-CM and CAM B2017/BMD3673), and the Asociación Española Contra el Cáncer (AECC PROYE20084REGU). A.C.B. was supported by Complutense University scholarship Q16 (CT27/16 and CT31/21). P.P.C. was supported by the MINECO Juan de la Cierva - Incorporación fellowship (IJCI-2014-19262). Data availability The data used or analyzed during the current study is provided within the manuscript and supplementary information files, or are available upon reasonable request from the corresponding authors. Ethical Approval: This study was reviewed and approved by CEIm Hospital Clínico San Carlos. Consent to Participate: The patient’s legal guardian provided written informed consent to participate in this study. Consent to Publication: Not applicable. Competing interests: The authors declare no competing interests References Call ME, Wucherpfennig KW. The T cell receptor: critical role of the membrane environment in receptor assembly and function. Annu Rev Immunol. 2005;23:101–25. https://doi.org/10.1146/annurev.immunol.23.021704.115625 . Love PE, Shores EW, Johnson MD, Tremblay ML, Lee EJ, Grinberg A, et al. T cell development in mice that lack the zeta chain of the T cell antigen receptor complex. Science. 1993;261:918–21. 10.1126/science.7688481 . https://www.science.org/doi/ . Love PE, Hayes SM. ITAM-mediated signaling by the T-cell antigen receptor. Cold Spring Harb Perspect Biol. 2010;2:a002485. https://cshperspectives.cshlp.org/content/2/6/a002485 . Miyazawa H, Wada T. Reversion Mosaicism in Primary Immunodeficiency Diseases. Front Immunol. 2021;12:783022. https://doi.org/10.3389/fimmu.2021.783022 . Blázquez-Moreno A, Pérez-Portilla A, Agúndez-Llaca M, Dukovska D, Valés-Gómez M, Aydogmus C, et al. Analysis of the recovery of CD247 expression in a PID patient: insights into the spontaneous repair of defective genes. Blood. 2017;130:1205–8. https://doi.org/10.1182/blood-2017-01-762864 . Marin AVM, Garcillán B, Jiménez-Reinoso A, Muñoz-Ruiz M, Briones AC, Fernández-Malavé E, et al. Human congenital T-cell receptor disorders. LymphoSign J. 2015;2:3–19. https://doi.org/10.14785/lpsn-2014-0012 . Sussman JJ, Bonifacino JS, Lippincott-Schwartz J, Weissman AM, Saito T, Klausner RD, et al. Failure to synthesize the T cell CD3-zeta chain: structure and function of a partial T cell receptor complex. Cell. 1988;52:85–95. https://doi.org/10.1016/0092-8674(88)90533-8 . Dietrich J, Kastrup J, Lauritsen JP, Menné C, von Bülow F, Geisler C. TCRzeta is transported to and retained in the Golgi apparatus independently of other TCR chains: implications for TCR assembly. Eur J Immunol. 1999;29:1719–28. https://doi.org/10.1002/(SICI)1521-4141(199905)29:053.0.CO;2-M . Delgado P, Alarcón B. An orderly inactivation of intracellular retention signals controls surface expression of the T cell antigen receptor. J Exp Med. 2005;201:555–66. https://doi.org/10.1084/jem.20041133 . Atkinson TP, Hall CG, Goldsmith J, Kirkham PM. Splice variant in TCRζ links T cell receptor signaling to a G-protein-related signaling pathway. Biochem Biophys Res Commun. 2003;310:761–6. https://doi.org/10.1016/j.bbrc.2003.09.073 . Siegers GM, Swamy M, Fernández-Malavé E, Minguet S, Rathmann S, Guardo AC, et al. Different composition of the human and the mouse γδ T cell receptor explains different phenotypes of CD3γ and CD3δ immunodeficiencies. J Exp Med. 2007;204:2537–44. https://doi.org/10.1084/jem.20070782 . Fernández-Malavé E, Wang N, Pulgar M, Schamel WWA, Alarcón B, Terhorst C. Overlapping functions of human CD3δ and mouse CD3γ in αβ T-cell development revealed in a humanized CD3γ-deficient mouse. Blood. 2006;108:3420–7. https://doi.org/10.1182/blood-2006-03-010850 . Marin AV, Jiménez-Reinoso A, Briones AC, Muñoz-Ruiz M, Aydogmus C, Pasick LJ, et al. Primary T-cell immunodeficiency with functional revertant somatic mosaicism in CD247. J Allergy Clin Immunol. 2017;139:347–e3498. https://doi.org/10.1016/j.jaci.2016.06.020 . Briones AC, Megino RF, Marin AV, Chacón-Arguedas D, García-Martinez E, Balastegui-Martín H, et al. Nonsense CD247 mutations show dominant-negative features in T-cell receptor expression and function. J Allergy Clin Immunol. 2024;154:1022–32. https://doi.org/10.1016/j.jaci.2024.06.019 . Martínez-Barricarte R, De Jong SJ, Markle J, De Paus R, Boisson‐Dupuis S, Bustamante J et al. Transduction of Herpesvirus saimiri ‐Transformed T Cells with Exogenous Genes of Interest. CP in Immunology. 2016. Available from: https://currentprotocols.onlinelibrary.wiley.com/doi/ 10.1002/cpim.15 Dexiu C, Xianying L, Yingchun H, Jiafu L. Advances in CD247. Scand J Immunol. 2022l;96(1):e13170. https://doi.org/10.1111/sji.13170 . Rieux-Laucat F, Hivroz C, Lim A, Mateo V, Pellier I, Selz F, et al. Inherited and somatic CD3zeta mutations in a patient with T-cell deficiency. N Engl J Med. 2006;354:1913–21. 10.1056/NEJMoa053750 . https://www.nejm.org/doi/full/ . Revy P, Kannengiesser C, Fischer A. Somatic genetic rescue in Mendelian haematopoietic diseases. Nat Rev Genet. 2019;20:582–98. https://doi.org/10.1038/s41576-019-0139-x . Mizukami T, Obara M, Nishikomori R, Kawai T, Tahara Y, Sameshima N, et al. Successful treatment with infliximab for inflammatory colitis in a patient with X-linked anhidrotic ectodermal dysplasia with immunodeficiency. J Clin Immunol. 2012;32:39–49. https://doi.org/10.1007/s10875-011-9600-0 . Jiang J, Tang W, An Y, Tang M, Wu J, Qin T. Molecular and immunological characterization of DNA ligase IV deficiency. Clin Immunol. 2016;163:75–83. https://doi.org/10.1016/j.clim.2015.12.016 . Kaiser FMP, Reisli I, Pico-Knijnenburg I, Langerak AW, Kavelaars FG, Artac H, et al. Protein functionality as a potential bottleneck for somatic revertant variants. J Allergy Clin Immunol. 2021;147:391–e3938. https://doi.org/10.1016/j.jaci.2020.04.045 . Attardi E, Corey SJ, Wlodarski MW. Clonal hematopoiesis in children with predisposing conditions. Semin Hematol. 2024;61:35–42. https://doi.org/10.1053/j.seminhematol.2024.01.005 . Roberts JL, Lauritsen JPH, Cooney M, Parrott RE, Sajaroff EO, Win CM, et al. T-B + NK + severe combined immunodeficiency caused by complete deficiency of the CD3zeta subunit of the T-cell antigen receptor complex. Blood. 2007;109:3198–206. Hermans MH, Malissen B. The cytoplasmic tail of the T cell receptor zeta chain is dispensable for antigen-mediated T cell activation. Eur J Immunol. 1993;23:2257–62. https://doi.org/10.1002/eji.1830230931 . Materna M, Delmonte OM, et al. The immunopathological landscape of human pre-TCRα deficiency: From rare to common variants. Science. 2024;383(6686):eadh4059. https://doi.org/10.1126/science.adh4059 . Additional Declarations Competing interest reported. José R. Regueiro is an editorial board member of the Journal of Clinical Immunology. Supplementary Files SupplementaryinformationBriones.pdf Cite Share Download PDF Status: Published Journal Publication published 25 Jul, 2025 Read the published version in Journal of Clinical Immunology → Version 1 posted Editorial decision: Revision requested 09 May, 2025 Reviews received at journal 08 May, 2025 Reviews received at journal 29 Apr, 2025 Reviewers agreed at journal 29 Apr, 2025 Reviewers agreed at journal 21 Apr, 2025 Reviewers invited by journal 21 Apr, 2025 Submission checks completed at journal 06 Apr, 2025 First submitted to journal 04 Apr, 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. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5741291","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":445687492,"identity":"33b9d7be-91da-4058-9f3f-eb51d88eb1b0","order_by":0,"name":"Alejandro C. Briones","email":"","orcid":"","institution":"Complutense University School of Medicine and 12 de Octubre Health Research Institute (imas12)","correspondingAuthor":false,"prefix":"","firstName":"Alejandro","middleName":"C.","lastName":"Briones","suffix":""},{"id":445687494,"identity":"14b6827a-3963-495d-a71c-6ffe6a44e6ef","order_by":1,"name":"Ana V Marin","email":"","orcid":"","institution":"Complutense University School of Medicine and 12 de Octubre Health Research Institute (imas12)","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"V","lastName":"Marin","suffix":""},{"id":445687495,"identity":"a4847baf-ceda-45cd-9168-fe8d0b7e78b4","order_by":2,"name":"Rebeca Chaparro-García","email":"","orcid":"","institution":"Complutense University School of Medicine and 12 de Octubre Health Research Institute (imas12)","correspondingAuthor":false,"prefix":"","firstName":"Rebeca","middleName":"","lastName":"Chaparro-García","suffix":""},{"id":445687496,"identity":"6950386d-bf8b-4c2b-a5c8-6a875ea0fde0","order_by":3,"name":"Marta López-Nevado","email":"","orcid":"","institution":"Complutense University School of Medicine and 12 de Octubre Health Research Institute (imas12)","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"López-Nevado","suffix":""},{"id":445687497,"identity":"5d986222-20a1-4153-b24f-1c4ff5002f7b","order_by":4,"name":"David Abia","email":"","orcid":"","institution":"Centro de Biología Molecular Severo Ochoa","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Abia","suffix":""},{"id":445687498,"identity":"80626906-1817-4209-af4b-1f71a96f7528","order_by":5,"name":"Iván Estevez-Benito","email":"","orcid":"","institution":"Complutense University School of Medicine and 12 de Octubre Health Research Institute (imas12)","correspondingAuthor":false,"prefix":"","firstName":"Iván","middleName":"","lastName":"Estevez-Benito","suffix":""},{"id":445687499,"identity":"394b43f2-3c35-42d1-b379-5c8b32774922","order_by":6,"name":"Daniel Chacón-Arguedas","email":"","orcid":"","institution":"Complutense University School of Medicine and 12 de Octubre Health Research Institute (imas12)","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Chacón-Arguedas","suffix":""},{"id":445687500,"identity":"92946f69-a6c8-4e4c-8668-5cb80467ad60","order_by":7,"name":"Edgar Fernández-Malavé","email":"","orcid":"","institution":"Complutense University School of Medicine and 12 de Octubre Health Research Institute (imas12)","correspondingAuthor":false,"prefix":"","firstName":"Edgar","middleName":"","lastName":"Fernández-Malavé","suffix":""},{"id":445687501,"identity":"8b7a6206-7c03-42e6-a0a3-981070df7991","order_by":8,"name":"Paula P. Cardenas","email":"","orcid":"","institution":"Complutense University School of Medicine and 12 de Octubre Health Research Institute (imas12)","correspondingAuthor":false,"prefix":"","firstName":"Paula","middleName":"P.","lastName":"Cardenas","suffix":""},{"id":445687502,"identity":"5f6ce386-1301-4e6e-93a9-f84fe60940f6","order_by":9,"name":"José R. Regueiro","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYNACAwYGfiAlQZoWyQbStIB0HSBWi3l7+zWJDwX35IyvHT54g7HNLpqBvf0BXi0yZ86USc4wKDY2u52WbMHYlpzbwHPGAK8WCYmcNGkeg4TEbbdzzCQY2w7kNkjk4HeYhPybNOk/QC2bZ+d/g2iRf47fYRIS7MekGYBaNkjnsEFtYSDgMJ4cZssegwRjidtpxhYJ55Jz23hyCGhhP/7wxo8/CXL8s5Mf3vhQZpfbz34cv8MYGHiQzEwAYjYC6oGAnZCZo2AUjIJRMOIBAEEaQYs3HeGzAAAAAElFTkSuQmCC","orcid":"","institution":"Complutense University School of Medicine and 12 de Octubre Health Research Institute (imas12)","correspondingAuthor":true,"prefix":"","firstName":"José","middleName":"R.","lastName":"Regueiro","suffix":""}],"badges":[],"createdAt":"2024-12-31 10:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5741291/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5741291/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10875-025-01908-9","type":"published","date":"2025-07-25T15:58:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81513762,"identity":"04687e8a-d6a3-4224-9da3-ca6ada4cac4c","added_by":"auto","created_at":"2025-04-28 06:42:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1298806,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRecovery of surface TCR expression after introducing hCD247-WT into PM1T or MA5.8 cell lines.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e The human CD247-deficient PM1T cell line was transfected by nucleofection with either an empty (Mock) or hCD247-WT carrying construction. A CD247-sufficient HTLV-1 immortalized cell line was used as control of TCR expression. \u003cstrong\u003e(B)\u003c/strong\u003e Murine CD247-deficient MA5.8 cell line was transfected by nucleofection with either an empty (Mock) or hCD247-WT construction. The CD247-sufficient 2B4 parental cell line was used as a control for TCR expression. In \u003cstrong\u003e(A)\u003c/strong\u003e and \u003cstrong\u003e(B)\u003c/strong\u003e surface TCR levels were measured on pre-gated GFP+ cells by flow cytometry using mAbs against CD3ε (clones UCHT1 or 2C11 for human or mouse cell lines, respectively). The vertical line represents the isotype control. The graphics represent one experiment (from 2 independent experiments).\u003c/p\u003e","description":"","filename":"Figure1Briones.png","url":"https://assets-eu.researchsquare.com/files/rs-5741291/v1/8669116c068058ba6877d7a7.png"},{"id":81513761,"identity":"50b348e0-3a54-456e-8997-b17641f2d04a","added_by":"auto","created_at":"2025-04-28 06:42:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3148414,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTCR reconstitution ability of the studied hCD247 variants in CD247-deficient human or mouse cell lines. (A) \u003c/strong\u003eSchematic representation of the domain organization of hCD247-WT, somatic reversions Q70L, Q70W and Q70Y, as well as germline change Q70X. The leader peptide (LP), transmembrane region (TM), and immunoreceptor tyrosine-based activation motif (ITAM) are depicted. \u003cstrong\u003e(B) \u003c/strong\u003eRecovery of TCR surface expression after transfection with the studied hCD247 variants in transfected (GFP+)PM1T\u003cstrong\u003e \u003c/strong\u003eor\u003cstrong\u003e (C) \u003c/strong\u003eMA5.8 cell lines. In \u003cstrong\u003e(B)\u003c/strong\u003eand \u003cstrong\u003e(C),\u003c/strong\u003e surface CD3 quantified as MFI% relative to hCD247-WT expression was analyzed with mAbs against hCD3ε (clone UCHT1) or mCD3ε (clone 2C11). The vertical line corresponds to the isotype control. The graphics correspond to one representative experiment (from 3 independent experiments).\u003c/p\u003e","description":"","filename":"Figure2Briones.png","url":"https://assets-eu.researchsquare.com/files/rs-5741291/v1/d153837ec7b7a747578cf5fa.png"},{"id":81514198,"identity":"2d2876e5-98bb-42b5-87bd-62a72670b7db","added_by":"auto","created_at":"2025-04-28 06:50:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3700157,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRetroviral transduction of CD247-deficient human (PM1T) or mouse (MA5.8) cell lines. (A) \u003c/strong\u003eSurface TCR complex expression on PM1T or MA5.8 transduced cells (CD271+) lines was analyzed with mAb against human (UCHT1) or mouse (2C11) CD3, respectively. The vertical line represents the isotype control. \u003cstrong\u003e(B)\u003c/strong\u003e Surface TCR expression of PM1T cell line transduced with a retroviral plasmid carrying hCD247-WT, Q70L/W/Y somatic reversions and Q70X change. In \u003cstrong\u003e(A)\u003c/strong\u003e and \u003cstrong\u003e(B),\u003c/strong\u003e the graphics are representative of a single experiment (from 5 independent experiments). \u003cstrong\u003e(C) \u003c/strong\u003eQuantification of hCD3 expression from (B). MFI values were normalized as: (test-mock)/(WT-mock) (n=3). \u003cstrong\u003e*\u003c/strong\u003ep-value \u0026lt;0.05. Statistical significance was calculated using a one-sample t-test analysis comparing the different variants with respect to hCD247-WT.\u003c/p\u003e","description":"","filename":"Figure3Briones.png","url":"https://assets-eu.researchsquare.com/files/rs-5741291/v1/fe613ce7145e1db25af60f6f.png"},{"id":81514199,"identity":"a5877cae-a733-4701-80e5-d99e6d98013b","added_by":"auto","created_at":"2025-04-28 06:50:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2793654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypical and functional TCR reconstitution analysis by the CD247 variants expressed in the ZKO cell line. (A) \u003c/strong\u003eRepresentative\u003cstrong\u003e \u003c/strong\u003eflow cytometry analysis of\u003cstrong\u003e \u003c/strong\u003esurface TCR complex reconstitution in Jurkat WT (control) or ZKO cell lines transduced with the pHRSIN-C56W-UbEM plasmid carrying either nothing (Mock) or hCD247-WT (left) or the different CD247 variants (right) using anti-CD3ε (UCHT1) mAb. Transduced cells were pre-gated as GFP+. The name of each variant is indicated above its corresponding histogram (right). The vertical line represents the isotype control. \u003cstrong\u003e(B)\u003c/strong\u003eQuantification of hCD3 expression from A (mean MFI values, n=6). \u003cstrong\u003e(C)\u003c/strong\u003e Quantification of surface CD69 induction (% MFI) and percentage of CD25+ cell induction (n=3) after stimulation with SEE-loaded Raji cell conjugates. Transduced cells were gated as CD19-, GFP+. In \u003cstrong\u003e(B)\u003c/strong\u003e and \u003cstrong\u003e(C)\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003evalues are relative to the highest for each analyzed parameter and were normalized as (test - mock)/(highest variant - mock). In all cases, WT corresponds to hCD247-WT, X to Q70X, Y to Q70Y, W to Q70W and L to Q70L. Statistical significance was calculated using a one-way ANOVA test,\u003cstrong\u003e *\u003c/strong\u003ep-value \u0026lt;0.05, **p-value \u0026lt;0.01, ***p-value \u0026lt;0.001,****p-value \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure4Briones.png","url":"https://assets-eu.researchsquare.com/files/rs-5741291/v1/87873b3d578618acaf613477.png"},{"id":88506173,"identity":"8995626c-bc91-4971-9939-899a394e3a00","added_by":"auto","created_at":"2025-08-07 07:32:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11378857,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5741291/v1/4e8ada72-c184-493a-afe9-16d45ee3cf97.pdf"},{"id":81513764,"identity":"f17056e6-5814-44f2-8d7c-0305be7e3761","added_by":"auto","created_at":"2025-04-28 06:42:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1318732,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationBriones.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5741291/v1/9e384a27d1d83349a236c5f2.pdf"}],"financialInterests":"Competing interest reported. José R. Regueiro is an editorial board member of the Journal of Clinical Immunology.","formattedTitle":"Discordant restoration of TCR expression and function by CD247 somatic reversions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe CD247 chain, also known as CD3ζ, is crucial for T cell development and function [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It plays a key role in T-cell receptor (TCR) assembly and surface expression by cooperating with other invariant CD3 molecules and in TCR development and function by transducing signals from the TCR to initiate the activation and response of T cells for thymus selection or antigen recognition [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCD247 deficiency is a rare early-onset primary immunodeficiency disorder characterized by variants in the \u003cem\u003eCD247\u003c/em\u003e gene (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). These variants can result in a lack of functional CD247 chain expression in T cells causing severe defects of TCR expression and function, which, in turn, may affect T cell development and function and, thus, immune responses. All reported patients showed T cell lymphopenia, suffered infections, and required stem cell transplantation to survive. Older patients also showed autoimmune features, suggesting poor T-cell selection.\u003c/p\u003e \u003cp\u003eRevertant somatic mosaicism is a phenomenon where spontaneous genetic corrections or compensations occur in a subset of cells, partially restoring the function of an altered germ-line gene [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. CD247 deficiency frequently associates with revertant somatic mosaicism (4 of 5 patients, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), likely due to the propensity of CD247 to vary [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This can lead to improvements in TCR expression (partial phenotypic reversion) in a minute subset of T cells, but it is unclear if phenotypic reversion leads to functional reversion, as CD247 protein domains involved in the former may not be sufficient for the latter. The clinical impact of CD247 somatic mosaicism also remains unclear, as it did not improve the clinical status of any reported patient.\u003c/p\u003e \u003cp\u003eT-cell models are invaluable tools for studying human T-cell immunodeficiencies [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, a human T cell model to study CD247 somatic mosaicism has not been reported. MA5.8, a murine CD247-deficient T cell line [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], has been widely used to establish the role of CD247 in TCR assembly [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and expression [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], as well as to study CD247 splicing variants [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. As human and murine invariant TCR-associated molecules show differential roles in each species [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], murine T cell models may show limitations to predict human CD247 role in TCR expression and function.\u003c/p\u003e \u003cp\u003eHere, we compared the capacity of CD247 somatic variants in two immunodeficiency cases with severe CD247 germline changes (p.M1T and p.Q70X, \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) to restore TCR expression and function in different T cell models, both human and murine. The somatic variants in each case were a wild-type (WT) reversion of p.M1T and three missense compensating variants (p.Q70L, p.Q70W, and p.Q70Y) of the germinal p.Q70X.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and culture\u003c/h2\u003e \u003cp\u003eHTLV-1 transformed p.M1T T-cell line was derived from peripheral blood mononuclear cells (PBMC) of a CD247-deficient patient [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] as described in the Online Repository. The Jurkat wild-type T-cell line (J77cl20 clone) was provided by Dr B. Rubin (Centre National de la Recherche Scientifique, Centre Hospitalier Universitaire, Purpan, Toulouse, France). The Jurkat CD247-deficient cell line, ZKO, was generated by CRISPR/Cas9 gene editing [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. All human T-cell lines were grown in RPMI-1640 (Lonza, Basel, Switzerland), supplemented with 10% FBS, 1x L-Glutamine 200mM, and 1x Antibiotic Antimycotic from Gibco (Bethesda, MD, USA). In addition, 100 U/mL recombinant human IL-2 (provided by Craig W. Reynolds, Frederick Cancer Research and Development Center, National Cancer Institute, National Institutes of Health, Frederick, MD, USA) was added to p.M1T cells.\u003c/p\u003e \u003cp\u003eThe HEK293T and Phoenix-Ampho (ATCC\u0026reg; CRL-3213\u0026trade;) packaging cell lines were grown under standard conditions in Iscove\u0026rsquo;s Modified Dulbecco\u0026rsquo;s Medium (Lonza).\u003c/p\u003e \u003cp\u003eThe murine T-cell lines, including parental 2B4 and its CD247-deficient derivative MA5.8 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], were gifted by Balbino Alarc\u0026oacute;n (Centro de Biolog\u0026iacute;a Molecular Severo Ochoa, Consejo Superior de Investigaciones Cient\u0026iacute;ficas, Universidad Aut\u0026oacute;noma de Madrid, Madrid, Spain). These cell lines were cultured in the same conditions as above but with 5% FBS. All cell lines were maintained at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified incubator.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmids, nucleofection, retroviral and lentiviral transduction\u003c/h3\u003e\n\u003cp\u003eThe pEGFP-N1 vector (Clontech, Mountain View, CA, USA) containing the human CD247 transcript variant or isoform 2 sequence (NM_000734; hCD247-WT from now on) or germline p.Q70X change (X for short) was a kind gift from Hugh T. Reyburn (National Centre for Biotechnology, Madrid, Spain). The somatic variants p.Q70L, p.Q70W and p.Q70Y were introduced by site-directed mutagenesis. For nucleofection assays, 1.5 x 10\u003csup\u003e6\u003c/sup\u003e immortalized HTLV-1 or murine T cells were nucleofected with 2 \u0026micro;g of pEGFP-N1 plasmid, carrying each CD247 variant, using the Cell Line Nucleofector Kits V or R, respectively, and the Amaxa Nucleofector 2b device (Lonza, Walkersville, MD, USA) according to the manufacturer\u0026rsquo;s instructions. Twenty hours post-nucleofection, cells were collected, and the transfection efficiency was analyzed through flow cytometry by counting the fraction of green fluorescent protein (GFP) - expressing cells.\u003c/p\u003e \u003cp\u003eFor retroviral transduction hCD247-WT, along with germline and somatic CD247 variants were introduced into the pLZRS-IRES-ΔNGFR retroviral plasmid and transfected into Phoenix-AMPHO cells as described [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Both the pLZRS-IRES-ΔNGFR retroviral plasmid and the Phoenix-AMPHO cells were provided by Rub\u0026eacute;n Mart\u0026iacute;nez-Barricarte (Vanderbilt Institute for Infection, Vanderbilt University Medical Center, Nashville, TN).\u003c/p\u003e \u003cp\u003eFor lentiviral transduction, the studied CD247 variants were cloned into the pHRSIN-C56W-UbEM lentiviral plasmid (gifted by Hugh T Reyburn). Viral particle generation and cell transduction were performed following the previously reported protocol [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Transduced cells were selected by flow cytometry. Those transduced with the pLZRS-IRES-ΔNGFR plasmid were positive for the CD271 marker, whereas those transduced with the pHRSIN-C56W-UbEM plasmid were positive for GFP.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry analysis\u003c/h3\u003e\n\u003cp\u003eStandard extracellular flow cytometry was performed with monoclonal antibodies (mAbs) against human CD3ε (clone UCHT1) from Beckman Coulter (Brea, CA) or mouse CD3ε (clone 145-2C11) from eBioscience (San Diego, CA). In addition, a mAb against CD271 (clone C40-1457) from BD Biosciences (San Jose, CA) was also employed. Intracellular stainings were done with the FOXP3/Transcription factor staining buffer set from Invitrogen. For intracellular quantification, human CD247 mAb (Clone 6B10.2) from BioLegend (San Diego, CA) was used.\u003c/p\u003e \u003cp\u003eTo analyze surface TCR complex expression, mAb against TCR\u0026#120572;ꞵ (clone IP26) from Thermo Fisher Scientific, and TCR Vꞵ8 (clone 56C5.2) from Beckman Coulter, were used.\u003c/p\u003e \u003cp\u003eData were acquired with a FACSCalibur flow cytometer (BD Biosciences) and analyzed with FlowJo software from TreeStar (Ashland, OR). Cell sorting was carried out to purify specific cell populations using a FACSAria\u0026trade; III sorter (BD Life Sciences, San Jose, CA). In all cases, mean fluorescence intensity (MFI) stands for geoMFI.\u003c/p\u003e\n\u003ch3\u003eFunctional studies\u003c/h3\u003e\n\u003cp\u003eCD247 plays a critical role in T cell activation at the initiation of TCR signaling [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Proliferation, in contrast, is a late consequence of T cell activation and occurs distally from CD247. Also, the proliferation of transformed T cells (as the ones used in our study) following TCR stimulation does not differ significantly from that of unstimulated cells because of the intrinsic proliferative phenotype, which is usually associated with cell transformation. For these reasons, we chose to assess immediate (ZAP-70 tyrosine phosphorylation) and early (CD69/CD25 surface upregulation) events of TCR-mediated T cell activation to dissect the differential impact of CD247 variants.\u003c/p\u003e \u003cp\u003eTo measure CD69 upregulation after TCR engagement, CD247-deficient human T cell lines (HTLV-1 or Jurkat-derived) were stimulated for 24 hours with 1 \u0026micro;g/ml of plastic-coated anti-CD3ε (clone OKT3) from eBioscience or 10 ng/ml Phorbol 12-myristate 13-acetate (PMA) plus 1 \u0026micro;M Ionomycin from Sigma-Aldrich.\u003c/p\u003e \u003cp\u003eTo analyze T-cell function in a more physiological way (namely, superantigen recognition, WT or ZKO-transduced Jurkat T cells expressing different CD247 variants were co-cultured with Raji cells (Ratio 1:1) preloaded with Staphylococcal Enterotoxin E (SEE) (0,5 \u0026micro;g/mL) (Sigma-Aldrich) for 18 hours in round-bottom 96-well plates. Cells were then collected and stained with anti-CD19 (clone HIB19; BD Biosciences) to discriminate Jurkat from Raji cells. CD69 and CD25 induction in response to TCR activation were evaluated essentially as published [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eZAP-70 phosphorylation was determined by intracellular flow cytometry. 0.3 x 10\u003csup\u003e6\u003c/sup\u003e WT or ZKO-transduced Jurkat T cells were stimulated with 20 \u0026micro;g/mL of anti-CD3ε mAb (clone OKT3) for 30 minutes at 4\u0026ordm;C. Then, anti-CD3ε was crosslinked with 10 \u0026micro;g/mL goat F(ab\u0026rsquo;)\u003csub\u003e2\u003c/sub\u003e anti-mouse Ig (H\u0026thinsp;+\u0026thinsp;L) for 5 minutes at 37 \u0026ordm;C. The reaction was stopped by adding cold PBS and centrifuging at 10.000 rpm for 5 seconds. Cells were then fixed/permeabilized with eBioscience\u0026trade; Foxp3 / Transcription Factor Fixation/Permeabilization Concentrate and Diluent (Invitrogen), according to the manufacturer\u0026rsquo;s instructions, and finally stained with anti-ZAP70/Syk (Tyr319, Tyr352) mAb (clone n3kobu5) APC-labelled from eBioscience.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eTo determine the statistical significance of the obtained results, either one-sample t-test or one-way ANOVA test was performed. The test used in each case is indicated in the figure legend. The error bars represent the standard error of the mean (SEM).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePartial surface TCR reconstitution after transfection of the PM1T cell line with hCD247-WT\u003c/h2\u003e \u003cp\u003eAfter confirming that the PM1T cell line lacks CD247 protein, as evidenced by its impairment in TCR expression and function (\u003cb\u003eFig. S2\u003c/b\u003e), it was selected as the cellular model to study the impact of different CD247 variants on TCR assembly and signaling.\u003c/p\u003e \u003cp\u003eFirst, human CD247-WT (hCD247-WT) was cloned into the pEGFP-N1 vector and then nucleofected into the CD247-deficient human (PM1T) or mouse (MA5.8) cell lines.\u003c/p\u003e \u003cp\u003eOur results showed that hCD247-WT poorly restored surface TCR expression in PM1T, only increasing it from 4\u0026ndash;9% (relative to HTLV-1 control cell line levels) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). However, introducing hCD247-WT in MA5.8 significantly improved surface TCR expression, which increased from 12\u0026ndash;75% (compared to the 2B4 parental cell line) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHuman, but not mouse, CD247-deficient cell lines recapitulate CD24 7 variants\u0026rsquo; effects in vivo.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor the study of the impact of CD247 variants on surface TCR expression and function, we selected the variants reported in a 10-month-old child with severe combined Immunodeficiency (SCID) caused by CD247 deficiency [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These include a CD247 homozygous nonsense germline change, Q70X (which generates a protein lacking all three ITAMs), and was identified in 90% of patients\u0026rsquo; T cells that exhibited low TCR expression. Additionally, the study identified three somatic missense reversions, namely, Q70W, Q70L and Q70Y \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, in the remaining 10% of the child\u0026rsquo;s T cells. These mixed reversions, purified by their higher surface CD3 expression, allowed normal TCR expression but were jointly considered poorly functional by the authors, based on the lack of ZAP-70 phosphorylation upon CD3 engagement [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Interestingly, the in silico predictors PolyPhen, SIFT or Saphetor classified the Q70X variant as strongly pathogenic, and Q70W and Q70Y variants as probably damaging, whereas Q70L was considered benign.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNucleofection results showed that the Q70X change did not restore TCR expression in human PM1T, mimicking the patient\u0026rsquo;s conditions where it was described. Surprisingly, this change fully restored TCR levels in MA5.8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). Therefore, this result suggests that PM1T is a more suitable model for studying human CD247 variants than the mouse MA5.8 cell line.\u003c/p\u003e \u003cp\u003eNucleofection assays with plasmids encoding for somatic reversions, Q70W and Q70L, rescued almost up to 100% surface TCR levels in p.M1T (relative to those obtained with the WT gene). However, the Q70Y variant was not able to reach the same level of restoration (61%, compared to those obtained with the WT gene) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Similar results were obtained in MA5.8 with Q70W and Q70L variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). However, in MA5.8, Q70Y showed a more significant defect in recovering surface TCR than in PM1T (35% vs. 61%, respectively).\u003c/p\u003e \u003cp\u003eFinally, we also found that the nucleofection of Q70X in control Jurkat T cells results in a decrease in surface TCR expression. However, this effect was not observed in control mouse T cells (2B4) (\u003cb\u003eFig. S3\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTransduction of CD247-defective cell lines significantly improves TCR surface reconstitution values\u003c/h3\u003e\n\u003cp\u003eTo improve the TCR reconstitution levels obtained by nucleofection, we cloned the analyzed CD247 variants into the retroviral plasmid pLZRS-IRES-ΔNGFR. Our results showed that transduction with hCD247-WT resulted in a significant increase in TCR expression in PM1T, from 4\u0026ndash;52%. Meanwhile, the TCR recovery in MA5.8 was almost complete, reaching up to 96%, which is similar to the parental 2B4 cell line levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen the ability of transduced CD247 somatic reversions to recover TCR expression in PM1T was tested, Q70W rendered similar reconstitution levels to hCD247-WT (100%), whilst Q70L and Q70Y recovered 85% and 57%, respectively. In contrast, Q70X only re-established 9,8% of surface TCR expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The analysis of CD3 MFI values showed that reconstitution levels observed among hCD247-WT and Q70Y/Q70X were statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eAccording to our data, we have confirmed that transduction is a more efficient method than nucleofection for restoring surface TCR levels in CD247-deficient cell lines. Unfortunately, the retroviral transduction efficiency was very low (8%), which greatly hindered our ability to conduct functional assays with the variants of interest.\u003c/p\u003e \u003cp\u003eAs one of the objectives of this work was to test the functional performance of the different CD247 variants, we generated a CD247-deficient Jurkat cell line (ZKO) by employing CRISPR/Cas9 technology. As reported, the ZKO cell line lacked intracellular CD247 and showed a significant reduction in extracellular CD3 expression [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For additional details on the characteristics of the ZKO cell line, see \u003cb\u003eFigs. S4 and S5\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCD247 variants display contrasting phenotypic and functional TCR reconstitution levels in ZKO cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAt first, we examined TCR expression following ZKO transfection with EGFP-tagged constructions. We discovered that transfection of hCD247-WT resulted in TCR levels that were comparable to control values. Additionally, the increase in surface CD3 expression was directly proportional to the amount of EGFP expressed by the cell \u003cb\u003e(Figure S6\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eUnfortunately, we were unable to assess the functional recovery of these cells due to damage caused by transfection. To overcome this issue, we cloned all CD247 variants into the pHRSIN-C56W-UbEM plasmid and utilized a lentiviral transduction protocol to carry out these assays.\u003c/p\u003e \u003cp\u003eWhen hCD247-WT was introduced into the ZKO cell line through lentiviral transduction, surface TCR expression, measured with UCHT1 mAb, was completely restored (100%, compared with the Jurkat WT cell line). However, the other CD247 variants showed varying abilities to recover TCR expression, as seen in previous experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The quantification of these results showed that the somatic variants Q70L and Q70W had the highest reconstitution levels, with Q70L recovering 100% and Q70W recovering 70% of TCR expression when compared to hCD247-WT. On the other hand, the somatic reversion Q70Y and the germline Q70X change only recovered 37% and 7% of surface TCR levels, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results are comparable to those obtained from transfection experiments and confirmed that CD247 somatic reversions have different abilities to allow a correct TCR assembly and expression at the plasma membrane.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition to UCHT1, the TCR complex surface expression in the transduced ZKO cell lines was also measured with IP26 and VB8 monoclonal antibodies, which recognize the alpha-beta chains or the β-chain variable region (Vβ8) of the TCR, respectively. In general, the results obtained with IP26 and VB8 were similar to those obtained with UCHT1, as indicated by the following ranking: WT\u0026thinsp;\u0026gt;\u0026thinsp;Q70L\u0026thinsp;\u0026gt;\u0026thinsp;Q70W\u0026thinsp;\u0026gt;\u0026thinsp;Q70Y\u0026thinsp;\u0026gt;\u0026thinsp;Q70X. However, in all the variants, we observed slightly lower IP26 values than those obtained with VB8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eBesides evaluating phenotypic TCR reconstitution, we assessed the functional reconstitution of transduced ZKO cells by observing the upregulation of T-cell activation markers upon stimulation with SEE-loaded Raji cell conjugates.\u003c/p\u003e \u003cp\u003eConcerning late T-cell activation markers, specifically CD69 upregulation, our results showed that transduction of the ZKO cell line with hCD247-WT restored TCR-dependent induction of this marker after TCR engagement. Surprisingly, Q70W variant performed better than hCD247-WT in inducing CD69; whereas Q70L, which fully recovered TCR expression, was not as efficient in upregulating CD69. Q70Y, only partially induced CD69 expression, while Q70X completely blocked TCR-dependent CD69 induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The performance of the different variants to upregulate CD69 is abbreviated as follows: Q70W\u0026thinsp;\u0026gt;\u0026thinsp;WT\u0026thinsp;\u0026gt;\u0026thinsp;Q70L\u0026thinsp;\u0026gt;\u0026thinsp;Q70Y\u0026thinsp;\u0026gt;\u0026thinsp;Q70X.\u003c/p\u003e \u003cp\u003eSimilarly, for CD25 induction, transduction with hCD247-WT recovered the ability of the ZKO cell line to express this activation marker. Once again, the Q70W reversion resulted in better CD25 upregulation compared to Q70L. Interestingly, Q70X induced CD25 at similar levels to Q70Y (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In summary, the order of CD25 induction efficiency is Q70W\u0026thinsp;=\u0026thinsp;WT\u0026thinsp;\u0026gt;\u0026thinsp;Q70L\u0026thinsp;\u0026gt;\u0026thinsp;Q70Y\u0026thinsp;=\u0026thinsp;Q70X.\u003c/p\u003e \u003cp\u003eRegarding the early activation marker, p-ZAP70, we observed a significant increase only in hCD247-WT transduced ZKO cells. However, we also noticed a slight upregulation in Q70L, Q70Y and Q70W, while Q70X was unable to induce it (\u003cb\u003eFig. S7\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eBased on our findings, we determined that the TCR phenotypical reconstitution ability of the studied CD247 variants differs from their functional reconstitution ability. Our data also showed that the CD247 somatic reversions had a poorer functional performance at short times (p-ZAP-70) but improved over time (CD69 and CD25).\u003c/p\u003e \u003cp\u003eTo explain our results, we used AlphaFold to generate \u003cem\u003ein silico\u003c/em\u003e 3D models of CD247. While the transmembrane region showed reliable predictions, the cytoplasmic region, including Q70, was intrinsically disordered, preventing accurate structural modeling (data not shown). To assess whether the Q70Y variant affects CD247-ZAP70 and CD247-LCK interactions, we performed additional \u003cem\u003ein silico\u003c/em\u003e analyses. Our results showed that both Q70 and Y70 are positioned outside the ZAP70 interaction interface, suggesting no impact on CD247-ZAP70 binding (data not shown). For the CD247-LCK interactions, accurate modeling of ITAM1 and ITAM2 was not possible, but we successfully modeled ITAM3, where G139 is the position equivalent to Q70 in ITAM3, relative to the first tyrosine of ITAM3 (Y141). Introducing the G139Q maintained Y141 within the LCK active site, whereas the G139Y variant repositioned Y141 outside (\u003cb\u003eFig. S8\u003c/b\u003e). Although these results support the notion that the Y70 variant reduces CD247 phosphorylation by LCK, a similar result was observed with the CD247 L70 variant (data not shown). These findings suggest that Q70 plays a critical role in stabilizing or enhancing CD247-LCK interactions.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSomatic reversion, a phenomenon observed in various primary immunodeficiencies, significantly modifies the clinical outcomes of these pathologies [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. It can be positive, neutral or negative, depending on the gene. Interestingly, despite the detection of immune cell subsets expressing functional proteins and exhibiting restored functionality, somatic reversion improved clinical outcomes only in certain conditions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. For instance, it has a positive clinical impact on ADA deficiency, Wiskott-Aldrich syndrome (WAS), Fanconi anemia, and in variants affecting DOCK8, ITGB2, or CXCR4 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, it negatively impacts patients with somatic variants in CARD11, RAG1, as well as in a reported case of IL2RG reversion in tissue infiltrating T-cells, all associated with Omenn syndrome [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, one patient with a reversion in NEMO, developed refractory inflammatory colitis, as his revertant T cells activated NF-kB in response to growth signals and had a growth advantage over cells carrying the germline change [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In other cases, somatic reversion can be neutral, as seen in CD247 or Ligase IV [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] deficiencies, where WT reversions were unable to modify the clinical or immunological phenotype. It has been hypothesized that somatic revertant variants are common in proliferative tissues, like the hematopoietic system, but are limited by the need for functional protein restoration [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the case of CD247 deficiency, only five patients with homozygous germline variants have been reported. Among these, four exhibited a small population of revertant T cells (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), but this did not correlate with improved clinical outcomes as all experienced life-threatening infections, with only one surviving post-hematopoietic stem-cell transplantation (HSCT).\u003c/p\u003e \u003cp\u003eSince no functional studies have explored the potential effect of CD247 somatic reversions, one main objective of our study was to understand, at the molecular level, the impact of different variants on TCR surface expression and CD247-dependent T-cell functions. To this end, we chose the main CD247 somatic reversions (WT, p.Q70W, p.Q70L, and p.Q70Y) in comparison to the germline change (p.M1T, and p.Q70X) reported by Marin [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and Rieux-Laucat [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], respectively. We found varying degrees of surface TCR expression restoration among the revertants (WT\u0026thinsp;\u0026gt;\u0026thinsp;Q70L\u0026thinsp;\u0026gt;\u0026thinsp;Q70W\u0026thinsp;\u0026gt;\u0026thinsp;Q70Y\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;Q70X), which was consistent across different staining antibodies. As expected, the p.Q70X germline variant showed no restoration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB \u003cb\u003eand Table S2\u003c/b\u003e). This suggests that the revertants can partially or fully restore TCR expression, whereas the germline variant is incapable of doing so. These differences might indicate that the specific amino acid changes in each revertant differentially affect the efficiency of TCR complex assembly and surface expression.\u003c/p\u003e \u003cp\u003eNotably, by testing murine and human cell lines side-by-side, we learned that CD247-deficient mouse T cells MA5.8 cannot be used to model human CD247 deficiencies, since Q70X, which is expectedly unable to restore TCR expression in human CD247-deficient T cells (PM1T or ZKO), does so in MA5.8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003eand Table S2\u003c/b\u003e). Therefore, previous reports using such murine cell lines should be reinterpreted in light of our findings [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. A potential limitation of this conclusion is that they are drawn from a truncated protein (Q70X) that is translated upon transfection and transduction, respectively, from a cDNA, and thus may not exist in primary patient\u0026rsquo;s T cells due to nonsense-mediated RNA decay which is prevented when using cDNA. However, Rieux-Laucat et al. reported in the discussion that Q70X CD247 was detected in small amounts in the cytoplasm but not on the membrane in primary patient\u0026rsquo;s T cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], although no data were included to support that contention. We have detected a different truncated CD247 in primary patient\u0026rsquo;s T cells which did not undergo nonsense-mediated RNA decay either (Y154X, unpublished), suggesting that this may be the case also with Q70X. Genome-edited cell lines with Q70X are in progress to address this question in more detail in the future.\u003c/p\u003e \u003cp\u003eInterestingly, analysis of the impact of revertant variants on TCR function, by measuring surface expression of CD69 and CD25 after stimulation with SEE-loaded Raji cells, revealed patterns of CD69 upregulation (Q70W\u0026thinsp;\u0026gt;\u0026thinsp;WT\u0026thinsp;\u0026gt;\u0026thinsp;Q70L\u0026thinsp;\u0026gt;\u0026thinsp;Q70Y\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;Q70X) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cb\u003eleft\u003c/b\u003e) and CD25\u0026thinsp;+\u0026thinsp;cells (Q70W\u0026thinsp;\u0026gt;\u0026thinsp;WT\u0026thinsp;\u0026gt;\u0026thinsp;Q70Y\u0026thinsp;=\u0026thinsp;Q70X\u0026thinsp;\u0026gt;\u0026thinsp;Q70L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cb\u003eright\u003c/b\u003e) which did not correlate with surface TCR expression. The p.Q70W variant showed the highest functional restoration, while the p.Q70L variant, despite good TCR expression recovery, was less effective in inducing CD25 expression upon TCR engagement. These results indicate that the p.Q70W variant, although not the best at restoring TCR expression, is the most effective in triggering T cell activation, as shown by higher TCR-mediated CD69 upregulation. We hypothesize that Q70W outperforms wild-type (WT) and Q70L in CD69 and CD25 upregulation due to its aromatic ring, which may facilitate stronger interactions with signaling partners, enhancing ITAM phosphorylation and activation. In contrast, Q70L hydrophobic nature likely hinders essential hydrogen bonding, impairing ITAM accessibility and phosphorylation. This lack of interactions may impair early activation events, including CD69 upregulation and sustained CD25 induction. This further suggests that certain CD247 reversions may enhance signaling pathways downstream of the TCR, leading to more robust T-cell activation.\u003c/p\u003e \u003cp\u003eIn silico predictors PolyPhen, SIFT or Saphetor classified Q70X as strongly pathogenic, Q70W and Q70Y as probably damaging, while Q70L is considered benign. Therefore, the expected clinical hierarchy based on these predictors would be (WT\u0026thinsp;=\u0026thinsp;Q70L\u0026thinsp;\u0026gt;\u0026thinsp;Q70W\u0026thinsp;=\u0026thinsp;Q70Y\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;Q70X). Our phenotypic results generally confirm but also refine such predictions (WT\u0026thinsp;\u0026gt;\u0026thinsp;Q70L\u0026thinsp;\u0026gt;\u0026thinsp;Q70W\u0026thinsp;\u0026gt;\u0026thinsp;Q70Y\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;Q70X). The functional results add further layers of complexity, as expression is required for function: Q70W\u0026thinsp;\u0026gt;\u0026thinsp;WT\u0026thinsp;\u0026gt;\u0026thinsp;Q70L\u0026thinsp;\u0026gt;\u0026thinsp;Q70Y\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;Q70X (by CD69 upregulation), Q70W\u0026thinsp;\u0026gt;\u0026thinsp;WT\u0026thinsp;\u0026gt;\u0026thinsp;Q70Y\u0026thinsp;=\u0026thinsp;Q70X\u0026thinsp;\u0026gt;\u0026thinsp;Q70L (by CD25\u0026thinsp;+\u0026thinsp;cells) and WT\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;Q70L\u0026thinsp;=\u0026thinsp;Q70W\u0026thinsp;=\u0026thinsp;Q70Y\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;Q70X (by Zap70 phosphorylation, as reported by Rieux-Laucat [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]). We thus believe that our cellular model to interrogate variants is more informative than purely in silico predictors\u003c/p\u003e \u003cp\u003eIn addition, the discrepancy between the TCR expression levels and the degree of TCR activation, suggests that Q70 substitutions impact CD247 function beyond surface expression. Q70, a polar residue, likely stabilizes CD247 via hydrogen bonding. Replacing it with hydrophobic (leucine and tryptophan) or amphipathic (tyrosine) residues may alter conformation and signaling. Q70L supports TCR expression but weakens activation, likely due to the loss of hydrogen bonding crucial for ITAM phosphorylation. Q70W disrupts surface expression but enhances signaling, possibly by facilitating stronger ITAM interactions through its aromatic ring. Q70Y impairs both expression and early activation, likely because its bulky aromatic ring and polar hydroxyl group disrupt the structural integrity or proper folding of CD247. However, its hydroxyl group may allow limited interactions that support moderate CD25 expression, indicating partial preservation of sustained signaling pathways.\u003c/p\u003e \u003cp\u003eGiven the crucial role of CD247 in TCR selection and tolerance, the clinical implications of the results obtained with the Q70W and Q70L variants are significant. The Q70W heightened induction of CD69 and CD25 could potentially lead to excessive T-cell activation, disrupting the immune balance and increasing the risk of autoimmune diseases and chronic inflammation. Conversely, the Q70L variant, with its impaired T-cell activation, may result in immunodeficiency, increasing susceptibility to infections and related complications.\u003c/p\u003e \u003cp\u003eIn addition, none of the CD247 revertants or the germline change induced ZAP-70 phosphorylation after TCR stimulation with an anti-CD3 mAb (\u003cb\u003eFig. S7 and Table S2\u003c/b\u003e). This can be attributed to the fact that ZAP-70 phosphorylation is an immediate (very early) event during T cell activation, whilst CD69 and CD25 upregulation are early events, relatively distal from TCR signaling initiation (which peak at 24 and 48 hours post-stimulation, respectively); thus, allowing the cell to respond to cumulative TCR signaling from CD247 somatic variants, or, alternatively, from CD3 receptor subunits [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOverall, these findings highlight that restoring TCR expression does not always lead to functional recovery, and each revertant shows a unique restoration profile, underlining the importance of evaluating multiple functional markers to fully understand the impact of somatic reversions. However, we are fully aware that for missense variants, overexpression from strong promoters can compensate for the functional defects of the protein, so it would be advisable for this kind of experiments, to generate cell lines that express the variants of interest from the endogenous gene and not generating knock-out cell lines that are transduced with a cDNA that is overexpressed.\u003c/p\u003e \u003cp\u003eConcerning the discordant effects on expression and function of the studied variants, Kaiser et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] reported a CD247-deficient patient (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) with a frameshift mutation in the CD247 leader peptide, exhibiting more than 30 non-WT somatic mutations that could, to varying degrees, restore surface TCR expression. Some of them persisted for months and some others did not, irrespectively of surface TCR expression levels, suggesting the existence of discordant effects on expression and function (measured as T cell survival \u003cem\u003ein vivo)\u003c/em\u003e, although such effects were not operating in TCR assembly interactions, but rather in leader peptide functions. In other genes, some examples have been reported, too. Reconstitution assays in vitro with PreTCRα variants found in patients showed that some restored surface preTCRα expression partially but were fully impaired functionally [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Similarly, expression of a mutant NEMO protein was not markedly reduced by flow cytometer, but the activity of mutant NEMO was defective, as confirmed by a mutant NEMO-NF-κB luciferase reporter assay [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, our results indicate that somatic mosaicism in CD247 is a common, random event in T cells. However, its capacity to restore TCR expression does not match TCR function. Additionally, none of the tested revertants, including WT, improved the patients\u0026rsquo; survival, likely because these events took place too late in T cell development to have a clinical impact, as suggested by Kaiser [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and Attardi [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These findings may be relevant to understand the role of CD247 in TCR structure and function during human T cell development \u003cem\u003ein vivo\u003c/em\u003e and its impact on human immunodeficiencies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAcknowledgements\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to Balbino Alarcon for supplying the 448 polyclonal antibody; Hugh T. Reyburn for providing the CD247- pEGFP-N1 construction and the pHRSIN vector, and Rub\u0026eacute;n Mart\u0026iacute;nez Barricarte for the pLZRS-IRES-\u0026Delta;NGFR vector and the Phoenix-AMPHO cell line.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAuthors contribution\u003c/u\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlejandro C. Briones, Paula P. Cardenas and Jos\u0026eacute; R. Regueiro conceived and designed the study. Alejandro C. Briones, Rebeca Chaparro-Garc\u0026iacute;a, Marta L\u0026oacute;pez-Nevado, Iv\u0026aacute;n Estevez-Benito and Daniel Chac\u0026oacute;n-Arguedas conducted the experiments. \u0026nbsp; Alejandro C. Briones, Iv\u0026aacute;n Estevez-Benito, Daniel Chac\u0026oacute;n-Arguedas and Paula P. C\u0026aacute;rdenas analyzed the experimental data. Ana V. Marin generated the PM1T cell line and assisted with manuscript editing. David Abia made the 3D models prediction. Paula P. Cardenas and Jos\u0026eacute; R. Regueiro wrote the manuscript. Edgar Fern\u0026aacute;ndez-Malav\u0026eacute; provided input on the study design and critically revised the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eFunding\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the Ministerio de Econom\u0026iacute;a y Competitividad (MINECO RED2022-134750-T, PID2021-125501OB-I00, and RTI2018-095673-B-I00), the Comunidad Aut\u0026oacute;noma de Madrid (P2022/BMD-7278, PR38/21-13 ANTICIPA-CM and CAM B2017/BMD3673), and the Asociaci\u0026oacute;n Espa\u0026ntilde;ola Contra el C\u0026aacute;ncer (AECC PROYE20084REGU). A.C.B. was supported by Complutense University scholarship Q16 (CT27/16 and CT31/21). P.P.C. was supported by the MINECO Juan de la Cierva - Incorporaci\u0026oacute;n fellowship (IJCI-2014-19262).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eData availability\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used or analyzed during the current study is provided within the manuscript and supplementary information files, \u0026nbsp;or are available upon reasonable request from the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval:\u0026nbsp;\u003c/strong\u003eThis study was reviewed and approved by CEIm Hospital Cl\u0026iacute;nico San Carlos.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate:\u003c/strong\u003e The patient\u0026rsquo;s legal guardian provided written informed consent to participate in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publication:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCall ME, Wucherpfennig KW. 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Science. 2024;383(6686):eadh4059. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.adh4059\u003c/span\u003e\u003cspan address=\"10.1126/science.adh4059\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-clinical-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joci","sideBox":"Learn more about [Journal of Clinical Immunology](https://www.springer.com/journal/10875)","snPcode":"10875","submissionUrl":"https://submission.nature.com/new-submission/10875/3","title":"Journal of Clinical Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"CD247, CD3Z, TCR, immunodeficiency, somatic reversions","lastPublishedDoi":"10.21203/rs.3.rs-5741291/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5741291/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The CD247 chain of the T-cell receptor (TCR) is essential for normal T cell development and function. Reported CD247-deficient patients showed severe immunodeficiency despite the presence of two populations of peripheral T cells, most with low TCR levels carrying the germline variant and a few with higher TCR levels due to somatic reversion. However, the revertant T cells remained a minority and did not improve the patients’ clinical status.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePurpose: \u003c/strong\u003eTo compare the capability of somatic reversions of CD247 germline changes (p.M1T and p.Q70X) to restore TCR expression and function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eCD247 wild-type (WT) and p.Q70L/W/Y somatic revertants were individually introduced in CD247-deficient mouse (MA5.8), human mutant (PM1T), and CRISPR/Cas9-generated Jurkat (ZKO) T cell lines by nucleofection or transduction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e MA5.8 mouse T cells do not accurately model human CD247 deficiencies, as Q70X restores TCR expression in MA5.8 but not in human cells. In human cell models, all somatic revertant variants restored TCR expression with varying degrees (WT=Q70L\u0026gt;Q70W\u0026gt;Q70Y). However, rescue of TCR-induced activation events, including ZAP-70 phosphorylation and CD69/CD25 upregulation, did not match such hierarchy (WT=Q70W\u0026gt;Q70L=Q70Y).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eSomatic reversions, such as those detected in patients with pathogenic CD247 germinal changes, display a discordant capability to rescue TCR expression versus function. These findings shed light on the role of CD247 in TCR expression and function during human T cell development, with implications for immunodeficiencies, as well as for the biological consequences of CD247 somatic mosaicism.\u003c/p\u003e","manuscriptTitle":"Discordant restoration of TCR expression and function by CD247 somatic reversions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-28 06:42:31","doi":"10.21203/rs.3.rs-5741291/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-10T00:50:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-08T14:28:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-29T21:18:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"203940622554378643409042196050277407025","date":"2025-04-29T10:15:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31403453104917344080351570855480767607","date":"2025-04-21T09:57:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-21T09:53:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-07T02:33:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Clinical Immunology","date":"2025-04-04T15:02:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-clinical-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joci","sideBox":"Learn more about [Journal of Clinical Immunology](https://www.springer.com/journal/10875)","snPcode":"10875","submissionUrl":"https://submission.nature.com/new-submission/10875/3","title":"Journal of Clinical Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ea14db5f-5171-48ba-8285-45315623d137","owner":[],"postedDate":"April 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-07T07:19:16+00:00","versionOfRecord":{"articleIdentity":"rs-5741291","link":"https://doi.org/10.1007/s10875-025-01908-9","journal":{"identity":"journal-of-clinical-immunology","isVorOnly":false,"title":"Journal of Clinical Immunology"},"publishedOn":"2025-07-25 15:58:09","publishedOnDateReadable":"July 25th, 2025"},"versionCreatedAt":"2025-04-28 06:42:31","video":"","vorDoi":"10.1007/s10875-025-01908-9","vorDoiUrl":"https://doi.org/10.1007/s10875-025-01908-9","workflowStages":[]},"version":"v1","identity":"rs-5741291","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5741291","identity":"rs-5741291","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}