Opposing peripheral fates of tissue-restricted self antigen-specific conventional and regulatory CD4+ T cells

The development of self antigen-specific T cells is influenced by how the self antigen is expressed. Here, we created a mouse in which a model self antigen is conditionally expressed in different tissue environments. Using peptide:MHCII tetramer-based cell enrichment methods, we examined the development of corresponding endogenous self antigen-specific CD4⁺ T cell populations. While ubiquitous self antigen expression resulted in efficient deletion of self antigen-specific T cells in the thymus, some tissue-restricted expression patterns resulted in partial deletion of the population in peripheral lymphoid organs. Deletion specifically affected Foxp3⁻ conventional T cells (Tconv) with a bias towards high avidity TCR expressing cells in the case of thymic, but not peripheral deletion. In contrast, Foxp3⁺ Treg exhibited elevated frequencies with increased TCR avidity. T cells surviving deletion were functionally impaired, with Tconv cells exhibiting more impairment than Tregs. Collectively, our results illustrate how postthymic recognition of tissue-restricted self antigens results in opposing developmental fates for Tconv and Treg cell subsets.     

Weakly self‐reactive T‐cell clones can homeostatically expand when present at low numbers

T‐cell division is central to maintaining a stable T‐cell pool in adults. It also enables T‐cell expansion in neonates, and after depletion by chemotherapy, bone marrow transplantation, or infection. The same signals required for T‐cell survival in lymphoreplete settings, IL‐7 and T‐cell receptor (TCR) interactions with self‐peptide MHC (pMHC), induce division when T‐cell numbers are low. The strength of reactivity for self‐pMHC has been shown to correlate with the capacity of T cells to undergo lymphopenia‐induced proliferation (LIP), in that weakly self‐reactive T cells are unable to divide, implying that T‐cell reconstitution would significantly skew the TCR repertoire toward TCRs with greater self‐reactivity and thus compromise T‐cell diversity. Here, we show that while CD4⁺ T cells with low self‐pMHC reactivity experience more intense competition, they are able to divide when present at low enough cell numbers. Thus, at physiological precursor frequencies CD4⁺ T cells with low self‐pMHC reactivity are able to contribute to the reconstitution of the T‐cell pool.     

On integrity in immunity during ontogeny or how thymic regulatory T cells work

The Standard model of T cell recognition asserts that T cell receptor (TCR) specificities are positively and negatively selected during ontogeny in the thymus and that peripheral T cell repertoire has mild self‐major histocompatibility complex (MHC) reactivity, known as MHC restriction of foreign antigen. Thus, the TCR must bind both a restrictive molecule (MHC allele) and a peptide reclining in its groove (pMHC ligand) in order to transmit signal into a T cell. The Standard and Cohn's Tritope models suggest contradictory roles for complementarity‐determining regions (CDRs) of the TCRs. Here, I discuss both concepts and propose a different solution to ontogenetic mechanism for TCR‐MHC–conserved interaction. I suggest that double (CD4⁺CD8⁺)‐positive (DP) developing thymocytes compete with their αβTCRs for binding to self‐pMHC on cortical thymic epithelial cells (cTECs) that present a selected set of tissue‐restricted antigens. The competition between DPs involves TCR editing and secondary rearrangements, similar to germinal‐centre B cell somatic hypermutation. These processes would generate cells with higher TCR affinity for self‐pMHC, facilitating sufficiently long binding to cTECs to become thymic T regulatory cells (tTregs). Furthermore, CD4⁺ Foxp3⁺ tTregs can be generated by mTECs via Aire‐dependent and Aire‐independent pathways, and additionally on thymic bone marrow–derived APCs including thymic Aire‐expressing B cells. Thymic Tregs differ from the induced peripheral Tregs, which comprise the negative feedback loop to restrain immune responses. The implication of thymocytes’ competition for the highest binding to self‐pMHC is the co‐evolution of species‐specific αβTCR V regions with MHC alleles.     

CD8+ CD122+ regulatory T cells contain clonally expanded cells with identical CDR3 sequences of the T‐cell receptor β‐chain

We identified CD8⁺ CD122⁺ regulatory T cells (CD8⁺ CD122⁺ Treg cells) and reported their importance in maintaining immune homeostasis. The absence of CD8⁺ CD122⁺ Treg cells has been shown to lead to severe systemic autoimmunity in several mouse models, including inflammatory bowel diseases and experimental autoimmune encephalomyelitis. The T‐cell receptors (TCRs) expressed on CD8⁺ CD122⁺ Treg cells recognize the target cells to be regulated. To aid in the identification of the target antigen(s) recognized by TCRs of CD8⁺ CD122⁺ Treg cells, we compared the TCR diversity of CD8⁺ CD122⁺ T cells with that of conventional, naive T cells in mice. We analysed the use of TCR‐Vβ in the interleukin 10‐producing population of CD8⁺ CD122⁺ T cells marked by high levels of CD49d expression, and found the significantly increased use of Vβ13 in these cells. Immunoscope analysis of the complementarity‐determining region 3 (CDR3) of the TCR β‐chain revealed remarkable skewing in a pair of Vβ regions, suggesting the existence of clonally expanded cells in CD8⁺ CD122⁺ T cells. Clonal expansion in Vβ13⁺ cells was confirmed by determining the DNA sequences of the CDR3s. The characteristic TCR found in this study is an important building block for further studies to identify the target antigen recognized by CD8⁺ CD122⁺ Treg cells.     

Clonotype‐specific avidity influences the dynamics and hierarchy of virus‐specific regulatory and effector CD4+ T‐cell responses

A key component of immunity against viruses, CD4⁺ T cells expand and differentiate into functional subsets upon primary infection, where effector (Teff) cells facilitate infection control and regulatory (Treg) cells mitigate immunopathology. After secondary infection, Teff cells mount a robust response from the memory pool. Here, we show that Treg‐cell responses are diminished upon secondary infection, and Treg‐cell response dynamics are associated more with T‐cell receptors (TCRs) repertoire and avidity than with epitope specificity. In the murine model, the IA^bM₂₀₉ epitope of respiratory syncytial virus is recognized by both CD4⁺ Treg and Teff cells, while the IA^bM2₂₆ epitope is recognized almost exclusively by CD4⁺ Teff cells expressing high avidity TCR Vβ8.1/8.2 and dominating the CD4⁺ T‐cell response during primary and secondary infections. IA^bM₂₀₉‐Teff cells express relatively low avidity TCRs during early primary infection, but high avidity TCR Vβ7‐expressing IA^bM₂₀₉‐Teff cells emerge during the late phase, and become dominant after secondary infection. The emerging high avidity IA^bM₂₀₉‐Teff cells outcompete IA^bM₂₀₉‐Treg cells that share the same epitope, but have low avidity and are restricted to TCR Vβ2 and Vβ6 subpopulations. These data indicate that MHC‐peptide‐TCR interactions can produce different kinetic and functional profiles in CD4⁺ T‐cell populations even when responding to the same epitope.     

Activation of CD4+ CD25+ regulatory T cell suppressor function by analogs of the selecting peptide

CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells can undergo both thymic selection and peripheral expansion in response to self peptides that are agonists for their T cell receptors (TCR). However, the specificity by which these TCR must recognize peptide:MHC complexes to activate Treg cell function is not known. We show that CD4⁺CD25⁺Foxp3⁺ Treg cells can mediate suppression in response to peptides that are only weakly cross‐reactive with the self peptide that induced their formation in vivo. Moreover, suppression could be efficiently activated by peptide analogs that were inefficient at inducing CD69 up‐regulation, and that also induced little or no proliferation of naïve CD4⁺CD25^–Foxp3^– T cells expressing the same TCR. These findings provide evidence that self peptide‐specific CD4⁺CD25⁺Foxp3⁺ Treg cells can exert regulatory function in response to self‐ and/or pathogen‐derived peptides with which they are only weakly cross‐reactive.     

Diverse CD1d-restricted reactivity patterns of human T cells bearing “invariant” AV24BV11 TCR

Human and murine natural T (NT) cells, also referred to as NK1.1⁺ or NK T cells, express TCR with homologous V regions (hAV24/BV11 and mAV14/BV8, respectively) and conserved “invariant” TCR AVAJ junctional sequences, suggesting recognition of closely related antigens. Murine NT cells recognize CD1-expressing cells and are activated in a CD1-restricted fashion by several synthetic α-glycosylceramides, such as α-GalCer. Here we studied the reactivity of human T cells against CD1d⁺ cells pulsed or not with α-GalCer and other related ceramides. CD1d-restricted recognition of α-GalCer was a general and specific feature of T cell clones expressing both BV11 and canonical AV24AJ18 TCR chains. Besides, human and murine NT cells showed the same reactivity patterns against a set of related glycosylceramides, suggesting a highly conserved mode of recognition of these antigens in humans and rodents. We also identified several AV24BV11 T cell clones self reactive against CD1⁺ cells of both hemopoietic and nonhemopoietic origin, suggesting the existence of distinct NT cell subsets differing by their ability to recognize self CD1d molecules.     

Effect of Antihuman T Lymphocyte Globulin on Immune Recovery after Myeloablative Allogeneic Stem Cell Transplantation with Matched Unrelated Donors: Analysis of Immune Reconstitution in a Double-Blind Randomized Controlled Trial

We recently conducted a randomized double-blind study in which we demonstrated that moderate/severe chronic graft-versus-host disease (cGVHD) but not cGVHD-free survival was reduced in patients receiving anti-T lymphocyte globulin (ATLG) versus placebo. In a companion study we performed immunophenotypic analysis to determine the impact of ATLG on immune reconstitution (IR) and to correlate IR with clinical outcomes. The randomized study (n?=?254) included patients (aged 18 to 65 years) who underwent myeloablative transplants for acute myeloid leukemia, myelodysplastic syndrome, or acute lymphoblastic leukemia from HLA-matched unrelated donors. Ninety-one patients consented for the companion IR study (ATLG?=?44, placebo?=?47). Blood samples were collected on days 30, 100, 180, and 360 after hematopoietic cell transplantation (HCT), and multiparameter flow cytometry was performed in a blinded fashion. Reconstitution of CD3⁺ and CD4⁺ T cells was delayed up to 6 months post-HCT in the ATLG arm, whereas absolute regulatory T cell (Treg) (CD4⁺25⁺127-) numbers were lower only in the first 100 days. Analysis of the CD4⁺ Treg and conventional T cells (Tconv) (CD4⁺25^–127⁺) compartments showed a profound absence of naive Tregs and Tconv in the first 100 days post-HCT, with very slow recovery for 1 year. B cell and natural killer cell recovery were similar in each arm. Higher absolute counts of CD3⁺, CD4⁺, CD8⁺ T, Tregs, and Tconv were associated with improved overall survival, progression-free survival, and nonrelapse mortality but not moderate/severe cGVHD. Although ATLG delays CD3⁺ and CD4⁺ T cell recovery post-transplant, it has a relative Treg sparing effect after the early post-HCT period, with possible implications for protection from cGVHD. ATLG severely compromises the generation of naive CD4⁺ cells (Treg and Tconv), potentially affecting the diversity of the TCR repertoire and T cell responses against malignancy and infection.     

T Cell Receptor Repertoire in Rheumatoid Arthritis

CD4⁺ T cells are a major component of the inflammatory infiltrate in rheumatoid synovitis. Within synovial lesions, clonal CD4⁺ T cell populations are detectable, supporting the notion of an antigen specific recognition event in the joint. In general, the clonal size of individual T cell clones is small and does not lead to a marked distortion of the synovial T cell receptor (TCR) repertoire. Comparison of TCR sequences derived from different patients has not provided evidence for common sequences. Either multiple antigens are recognized or the TCR repertoire is sufficiently plastic with a multitude of different TCR structures responding to the same antigen(s). However, within one individual, the repertoire of clonal T cell populations is restricted. Identical T cell clones can be identified in different joints and at different timepoints of the disease, emphasizing that the spectrum of antigens recognized is conserved over time and that the T cell response pattern is not subject to evolution. Characterization of antigens involved in the latter stages of the disease may thus provide critical information on disease-initiating events.

Recent data have led to the new concept that the role of T cells in rheumatoid arthritis (RA) is not limited to synovial inflammation. Evidence has been provided that the premorbid TCR repertoires of RA patients and normal controls can be distinguished. The T cell repertoire in RA patients is prone to recognize certain microbial products and autoantigens. The selection of this response pattern can only partially be attributed to the disease associated HLA-DRB1 alleles. Additional factors common in RA patients but not in HLA-DR matched control individuals seem to be important in shaping the TCR repertoire. Furthermore, the repertoire of mature T cells in RA patients is characterized by oligoclonality which involves T cells in the peripheral blood compartment. Possibly, these clonal T cell populations react to widespread autoantigens, raising the possibility that RA patients have a defect in controlling peripheral tolerance and an anomaly of lymphoproliferation. In contrast to joint residing CD4⁺T cells, expanded clonotypes isolated from the blood of different patients have been described to share TCRβ chain structures. How these characteristic features of the global TCR repertoire in RA patients translate into mechanisms of disease remains to be elucidated.     

TCR sequences and tissue distribution discriminate the subsets of naïve and activated/memory Treg cells in mice

Analyses of the regulatory T (Treg) cell TCR repertoire should help elucidate the nature and diversity of their cognate antigens and thus how Treg cells protect us from autoimmune diseases. We earlier identified CD44^hiCD62L^low activated/memory (am) Treg cells as a Treg‐cell subset with a high turnover and possible self‐specificity. We now report that amTreg cells are predominantly distributed in lymph nodes (LNs) draining deep tissues. Multivariate analyses of CDR3 spectratyping first revealed that amTreg TCR repertoire is different from that of naïve Treg cells (nTreg cells) and effector T (Teff) cells. Furthermore, in deep‐ versus superficial LNs, TCR‐β deep sequencing further revealed diversified nTreg‐cell and amTreg‐cell repertoires, although twofold less diverse than that of Teff cells, and with repertoire richness significantly lower in deep‐LN versus superficial‐LN Treg cells. Importantly, expanded clonotypes were mostly detected in deep‐LN amTreg cells, some accounting for 20% of the repertoire. Strikingly, these clonotypes were absent from nTreg cells, but found at low frequency in Teff cells. Our results, obtained in nonmanipulated mice, indicate different antigenic targets for naïve and amTreg cells and that amTreg cells are self‐specific. The data we present are consistent with an instructive component in Treg‐cell differentiation.     

Diversity and clonotypic composition of influenza‐specific CD8+ TCR repertoires remain unaltered in the absence of Aire

TCR repertoire diversity is important for the protective efficacy of CD8⁺ T cells, limiting viral escape and cross‐reactivity between unrelated epitopes. The exact mechanism for selection of restricted versus diverse TCR repertoires is far from clear, although one thought is that the epitopes resembling self‐peptides might select a limited array of TCR due to the deletion of autoreactive TCR. The molecule Aire promotes the expression of tissue‐specific Ag on thymic medullary epithelial cells and the deletion of autoreactive cells, and in the absence of Aire autoreactive cells persist. However, the contribution of Aire‐dependent peptides to the selection of the Ag‐specific TCR repertoire remains unknown. In this study, we dissect restricted (D^bNP₃₆₆%⁺CD8⁺) and diverse (D^bPA₂₂₄%⁺CD8⁺, K^dNP₁₄₇%⁺CD8⁺) TCR repertoires responding to three influenza‐derived peptides in Aire‐deficient mice on both B6 and BALB/c backgrounds. Our study shows that the number, qualitative characteristics and TCR repertoires of all influenza‐specific, D^bNP₃₆₆%⁺CD8⁺, D^bPA₂₂₄%⁺CD8⁺ and K^dNP₁₄₇%⁺CD8⁺ T cells are not significantly altered in the absence of Aire. This provides the first demonstration that the selection of an Ag‐specific T‐cell repertoire is not significantly perturbed in the absence of Aire.     

Central tolerance spares the private high‐avidity CD4+ T‐cell repertoire specific for an islet antigen in NOD mice

Although central tolerance induces the deletion of most autoreactive T cells, some autoreactive T cells escape thymic censorship. Whether potentially harmful autoreactive T cells present distinct TCRαβ features remains unclear. Here, we analyzed the TCRαβ repertoire of CD4⁺ T cells specific for the S100β protein, an islet antigen associated with type 1 diabetes. We found that diabetes‐resistant NOD mice deficient for thymus specific serine protease (TSSP), a protease that impairs class II antigen presentation by thymic stromal cells, were hyporesponsive to the immunodominant S100β_1‐15 epitope, as compared to wild‐type NOD mice, due to intrathymic negative selection. In both TSSP‐deficient and wild‐type NOD mice, the TCRαβ repertoire of S100β‐specific CD4⁺ T cells though diverse showed a specific bias for dominant TCRα rearrangements with limited CDR3α diversity. These dominant TCRα chains were public since they were found in all mice. They were of intermediate‐ to low‐avidity. In contrast, high‐avidity T cells expressed unique TCRs specific to each individual (private TCRs) and were only found in wild‐type NOD mice. Hence, in NOD mice, the autoreactive CD4⁺ T‐cell compartment has two major components, a dominant and public low‐avidity TCRα repertoire and a private high‐avidity CD4⁺ T‐cell repertoire; the latter is deleted by re‐enforced negative selection.     

T cell receptor binding affinity governs the functional profile of cancer-specific CD8+ T cells

Antigen-specific T cell receptor (TCR) gene transfer via patient-derived T cells is an attractive approach to cancer therapy, with the potential to circumvent immune regulatory networks. However, high-affinity tumour-specific TCR clonotypes are typically deleted from the available repertoire during thymic selection because the vast majority of targeted epitopes are derived from autologous proteins. This process places intrinsic constraints on the efficacy of T cell-based cancer vaccines and therapeutic strategies that employ naturally generated tumour-specific TCRs. In this study, we used altered peptide ligands and lentivirus-mediated transduction of affinity-enhanced TCRs selected by phage display to study the functional properties of CD8⁺ T cells specific for three different tumour-associated peptide antigens across a range of binding parameters. The key findings were: (i) TCR affinity controls T cell antigen sensitivity and polyfunctionality; (ii) supraphysiological affinity thresholds exist, above which T cell function cannot be improved; and (iii) T cells transduced with very high-affinity TCRs exhibit cross-reactivity with self-derived peptides presented by the restricting human leucocyte antigen. Optimal system-defined affinity windows above the range established for natural tumour-specific TCRs therefore allow the enhancement of T cell effector function without off-target effects. These findings have major implications for the rational design of novel TCR-based biologics underpinned by rigorous preclinical evaluation.     

TCR function analysis using a novel system reveals the multiple unconventional tumor-reactive T cells in human breast cancer-infiltrating lymphocytes

Tumor-infiltrating lymphocytes (TILs) are a potent source for obtaining tumor-reactive T cell receptors (TCRs). Although comprehensive methods to analyze the TCR repertoire in TILs have been reported, the evaluation system for TCR-reactivity to endogenously expressed antigen in tumor cells remains laborious and time consuming. Consequently, very limited numbers of TCRs in TILs have been analyzed for their reactivity to tumor cells. In this study, we developed an efficient evaluation system for TCR function designated c-FIT (c omprehensive f unctional i nvestigation of T CRs) to analyze TCR reactivity. The c-FIT system enabled us to analyze up to 90 TCRs for their reactivity to tumor cells by a single assay within a month. Using c-FIT, we analyzed 70 TCRs of CD8⁺ TILs derived from two breast cancer patients and obtained 23 TCRs that reacted to tumor cells. Surprisingly, although two TCRs were HLA class I-restricted, the remaining 21 TCRs were non-HLA-restricted. Thus, c-FIT can be applied for monitoring multiple conventional and unconventional antigen-specific killer T cells in TILs, leading to the development of new designs for more effective T-cell-based immunotherapies.     

Diverse CD1d-restricted T cells: diverse phenotypes, and diverse functions

Invariant CD1d-restricted T cells express NK cell markers and use a limited TCR repertoire. Here, we describe a second CD1d-restricted T cell subset that uses a diverse TCR repertoire. These T cells can also express NK cell markers and function similarly to invariant T cells. The antigens recognized by the diverse subset are likely to be different from those recognized by invariant TCRs. The variable NK1.1 antigen expression on these T cell populations limits its usefulness in identifying CD1d-restricted T cells. Lastly, the discovery of antigens recognized by diverse CD1d-restricted T cells will provide insight into their role in normal and pathological immune responses.     

Positive selection of self‐antigen‐specific CD8+ T cells by hematopoietic cells

In contrast to thymic epithelial cells, which induce the positive selection of conventional CD8⁺ T cells, hematopoietic cells (HCs) select innate CD8⁺ T cells whose Ag specificity is not fully understood. Here we show that CD8⁺ T cells expressing an H‐Y Ag‐specific Tg TCR were able to develop in mice in which only HCs expressed MHC class I, when HCs also expressed the H‐Y Ag. These HC‐selected self‐specific CD8⁺ T cells resemble innate CD8⁺ T cells in WT mice in terms of the expression of memory markers and effector functions, but are phenotypically distinct from the thymus‐independent CD8⁺ T‐cell population. The peripheral maintenance of H‐Y‐specific CD8⁺ T cells required presentation of the self‐Ag and IL‐15 on HCs. HC‐selected CD8⁺ T cells in mice lacking the Tg TCR also showed these features. Furthermore, by using MHC class I tetramers with a male Ag peptide, we found that self‐Ag‐specific CD8⁺ T cells in TCR non‐Tg mice could develop via HC‐induced positive selection, supporting results obtained from H‐Y TCR Tg mice. These findings indicate the presence of self‐specific CD8⁺ T cells that are positively selected by HCs in the peripheral T‐cell repertoire.     

Molecular basis of lipid antigen presentation by CD1d and recognition by natural killer T cells

Together with peptides, T lymphocytes respond to hydrophobic molecules, mostly lipids, presented by the non-classical CD1 family (CD1a–e). These molecules have evolved complex and diverse binding grooves in order to survey different cellular compartments for self and exogenous antigens, which are then presented for recognition to T-cell receptors (TCRs) on the surface of T cells. In particular, most CD1d-presented antigens are recognized by a population of lymphocytes denominated natural killer T (NKT) cells, characterized by a strong immunomodulatory potential. Among NKT cells, two major subsets (type I and type II NKT cells) have been described, based on their TCR repertoire and antigen specificity. Here we review recent structural and biochemical studies that have shed light on the molecular details of CD1d-mediated antigen recognition by type I and II NKT cells, which are in many aspects distinct from what has been observed for peptide major histocompatibility complex-reactive TCRs.     

Peripheral tolerance of CD4 T cells following local activation in adolescent mice

In addition to thymic T cell selection, post-thymic mechanisms of tolerance induction are required to eliminate autoreactive T cells with specificities for peripheral self antigens. While CD8⁺ T cells can recognize their target antigen on a wide variety of cell types, CD4⁺ T cells generally depend on the presence of specialized antigen-presenting cells. Because of this fundamental difference in antigen recognition peripheral tolerance of CD4⁺ T cells appears more difficult to achieve than of CD8⁺ T cells. Utilizing T cell receptor (TCR)-transgenic mice in which CD4⁺ T cells specific for a pancreatic β cell neoantigen (the simian virus 40 T antigen) are constantly generated at low frequency, we have now established a mouse model of peripheral, tissue-specific CD4⁺ T cell tolerance. In these animals, tolerance is preceded by a phase of activation of the autoreactive T cells as characterized by up-regulation of CD69 and CD44, and down-regulation of the L-selectin lymph node homing receptor. T antigen-specific T cells bearing this phenotype can be detected in the local lymphoid environment of the pancreas but not in more remote locations like axillary or inguinal lymph nodes. The proportion of activated, autoreactive T cells is maximal at 2–3 weeks of age, after which these cells are gradually deleted from the peripheral lymphocyte pool. We further demonstrate that deletion of the autoreactive T cells does not occur in TCR-tansgenic mice bred to the RAG-1-deficient background in which the transgenic T cells represent the only functional lymphocyte population.     

Reconstitution and Phenotype of Tregs in CMV Reactivating Patients Following Allogeneic Hematopoietic Stem Cell Transplantation

In experimental and clinical settings Tregs prevent graft-versus-host disease (GvHD) by inhibiting the proliferation and function of conventional T cells (Tconv). The suppressive potency of Tregs might also lead to the inhibition of protective antiviral T cell responses. As the control of CMV reactivation is important to improve the clinical outcome in allogeneic HSCT, we analyzed the Treg reconstitution in CMV reactivating patients with and without GvHD (n=47) in the first 6 months following transplantation. Most importantly, CMV reactivation does not correlate with the numerical reconstitution of CD4⁺CD25^highCD127^? Tregs. During CMV reactivation the proportion of Tregs within the CD4⁺ T cell population decreased significantly independent of GvHD manifestation. A comprehensive FACS analysis was performed in order to characterize the phenotype of Tregs and Tconv cells in greater detail for activation, co-stimulation, proliferation, suppressive function and migratory capability. Interestingly, Tregs of patients with CMV reactivation showed a significantly higher CXCR3 expression. CD4⁺ Tconv cells expressed significantly higher protein levels of the proliferation marker Ki67 correlating with a numerical increase of CD4⁺ T cells. Our results indicate that Tregs are not inhibiting pathogen clearance by Tconv following HSCT, which is of high relevance for future Treg cell-based clinical trials in allogeneic HSCT.