Allergen‐specific naïve and memory CD4+ T cells exhibit functional and phenotypic differences between individuals with or without allergy

Although allergen‐specific CD4⁺ T cells are detectable in the peripheral blood of both individuals with or without allergy, their frequencies and phenotypes within the memory as well as naïve repertoires are incompletely known. Here, we analyzed the DRB1*0401‐restricted responses of peripheral blood‐derived memory (CD4⁺CD45RO⁺) and naïve (CD4⁺CD45RA⁺) T cells from subjects with or without allergy against the immunodominant epitope of the major cow dander allergen Bos d 2 by HLA class II tetramers in vitro. The frequency of Bos d 2_127–142‐specific memory T cells in the peripheral blood‐derived cultures appeared to be higher in subjects with allergy than those without, whereas naïve Bos d 2_127–142‐specific T cells were detectable in the cultures of both groups at nearly the same frequency. Surprisingly, the TCR avidity of Bos d 2_127–142‐specific T cells of naïve origin, as assessed by the intensity of HLA class II tetramer staining, was found to be higher in individuals with allergy. Upon restimulation, long‐term Bos d 2_127–142‐specific T‐cell lines generated from both memory and naïve T‐cell pools from individuals with allergy proliferated more strongly, produced more IL‐4 and IL‐10, and expressed higher levels of CD25 but lower levels of CXCR3 than the T‐cell lines from individuals without allergy, demonstrating differences also at the functional level. Collectively, our current results suggest that not only the memory but also the naïve allergen‐specific T‐cell repertoires differ between individuals with or without allergy.     

Persistent viral infection in humans can drive high frequency low-affinity T-cell expansions

CD8 T cells that recognize cytomegalovirus (CMV) ‐encoded peptides can be readily detected by staining with human leucocyte antigen (HLA) –peptide tetramers. These cells are invariably highly differentiated effector memory cells with high avidity T‐cell receptors (TCR). In this report we demonstrate an HLA‐A*0201 restricted CMV‐specific CD8 T‐cell response (designated YVL) that represents several percent of the CD8 T‐cell subset, yet fails to bind tetrameric major histocompatibility complex (MHC) ligands. However, these tetramer‐negative cells are both phenotypically and functionally similar to other CMV‐specific CD8 T cells. YVL peptide‐specific CD8 T‐cell clones were generated and found to be of high avidity in both cytotoxicity and interferon‐γ (IFN‐γ) assays, and comparable with other CMV peptide‐specific CD8 T‐cell clones. However, under conditions of CD8 blockade, the response was almost nullified even at very high ligand concentrations. This was also the case in IFN‐γ experiments using peripheral blood mononuclear cells stimulated with peptide ex vivo. In contrast, all other CMV specificities (tetramer‐positive) displayed minimal or only partial CD8 dependence. This suggests that YVL‐specific responses depict a low‐affinity TCR–MHC–peptide interaction, that is compensated by substantial CD8 involvement for functional purposes, yet cannot engage multivalent soluble ligands for ex vivo analysis. It is interesting that such a phenomenon is apparent in the face of a persistent virus infection such as CMV, where the responding cells represent an immunodominant response in that individual and may present a highly differentiated effector phenotype.     

Strong and sustained effector function of memory‐ versus naïve‐derived T cells upon T‐cell receptor RNA transfer: Implications for cellular therapy

Current protocols used to select CMV‐specific T cells for adoptive immunotherapy focus on virus‐specific memory T cells from seropositive donors. However, this strategy is not feasible in patients undergoing allogeneic haematopoietic stem‐cell transplantation (HSCT) from CMV‐seronegative donors. Here, we redirected T cells of CMV‐seronegative donors with a human genetically engineered TCR recognizing an HLA‐A*0201‐binding peptide epitope of CMVpp65. To facilitate clinical translation of this approach, we used a non‐viral expression system based on in vitro transcribed RNA and electroporation. Although memory and naïve‐derived T‐cell subsets were both efficiently transfected by TCR‐RNA, memory‐derived T cells showed much stronger levels of HLA‐A*0201‐restricted cytolytic activity to CMV‐infected fibroblasts and maintained acquired function for 5–10 days. In addition to redirection of CD8⁺ cytotoxic T cells, TCR‐RNA transfection was capable of redirecting CD4⁺ T cells into potent Ag‐specific Th cells that efficiently triggered maturation of DCs. Our data suggest that memory rather than naïve‐derived T cells are the preferred subset for transient TCR expression by RNA electroporation, providing more efficient and sustained virus‐specific CD4⁺ and CD8⁺ T‐cell function. CMV TCR‐RNA may represent a suitable therapeutic ‘off‐the‐shelf’ reagent to be used in severe CMV infections of HSCT patients when endogenous CMV‐specific T‐cell immunity is insufficient.     

Expansion of activated human naïve T-cells precedes effector function

Naïve T‐cells divide and mature, both functionally and phenotypically, upon stimulation through the T‐cell receptor. Although much is known about the overall changes that occur in naïve cells upon TCR stimulation, and the different memory/effector populations that arise following stimulation, the relationship between cell division and functional and phenotypical changes that occur after activation is poorly understood. Here, we examine the early stages of human naïve and antigen‐experienced T‐cell activation, and the relationship between cell division and acquisition of effector function during the transition from resting antigen‐experienced or naïve T‐cells into effector cells. Stimulated naïve T‐cells proliferate prior to acquisition of effector function, as measured by cytokine production and expression of effector‐associated cell surface molecules. Additionally, we show that interlukin‐7 (IL‐7) can drive proliferation of naïve T‐cells without TCR:MHC peptide interactions. IL‐7 alone does not, however, drive the proliferation of antigen‐experienced T‐cells. Memory T‐cells will divide in response to exogenous IL‐7 but only in the presence of naïve T‐cells and IL‐2. This study contributes to the current understanding of the mechanistic differences between naïve and memory T‐cell responses by defining the functional and phenotypic changes that occur to T‐cells after stimulation.     

Peripheral CD4+ T‐cell tolerance is induced in vivo by rare antigen‐bearing B cells in follicular,marginal zone,and B‐1 subsets

B cells are efficient APCs when they internalize antigen via BCR‐mediated uptake. Adoptively transferred antigen‐presenting B cells can induce T‐cell tolerance to foreign and self antigens; however, it is unknown whether endogenous B cells presenting self‐peptides interact with naïve T cells and contribute to peripheral T‐cell self‐tolerance. Moreover, the relative abilities of mature B‐cell subsets to induce T‐cell tolerance have not been examined. To address these questions, we created a new mouse model wherein a very small fraction of B cells expresses an antigen transgene that cannot be transferred to other APCs. We limited antigen expression to follicular, marginal zone, or B‐1 B‐cell subsets and found that small numbers of each subset interacted with naïve antigen‐specific T cells. Although antigen expressed by B‐1 B cells induced the most T‐cell division, divided T cells subsequently disappeared from secondary lymphoid tissues. Independent of which B‐cell subset presented antigen, the remaining T cells were rendered hypo‐responsive, and this effect was not associated with Foxp3 expression. Our data show that physiologically relevant proportions of B cells can mediate peripheral T‐cell tolerance, and suggest that the mechanisms of tolerance induction might differ among follicular, marginal zone, and B‐1 B‐cell subsets.     

Frequent occurrence of high affinity T cells against MELOE‐1 makes this antigen an attractive target for melanoma immunotherapy

We recently showed that the infusion of tumor infiltrating lymphocytes specific for the MELOE‐1 antigen was associated with a prolonged relapse‐free survival for HLA‐A2⁺ melanoma patients who received tumor infiltrating lymphocytes therapy. Here, we characterized the MELOE‐1/A2‐specific T‐cell repertoire in healthy donors and melanoma patients to further support an immunotherapy targeting this epitope. Using tetramer enrichment followed by multicolor staining, we found that MELOE‐1‐specific T cells were present in the blood of healthy donors and patients at similar frequencies (around 1 in 1×10⁵ CD8⁺ cells). These cells mainly displayed a naïve phenotype in 4/6 healthy donors and 3/6 patients, whereas high proportions of memory cells were observed in the remaining individuals of both groups. There was a recurrent usage of the Vα12.1 chain for 17/18 MELOE‐1‐specific T‐cell clones derived from healthy donors or patients, associated with diverse Vβ chains and V(D)J junctional sequences. All clones derived from melanoma patients (9/9) were reactive against the MELOE‐1_36–44 peptide and against HLA‐A2⁺ melanoma cell lines. This study documents the existence of a large TCR repertoire specific for the MELOE‐1/A2 epitope and its capacity to give rise to antitumor CTL that supports the development of immunotherapies targeting this epitope.     

TLR9 stimulation drives naïve B cells to proliferate and to attain enhanced antigen presenting function

Mechanisms that regulate naïve B cell proliferation and function are incompletely defined. In this study, we test the hypothesis that naïve B cell expansion, survival and ability to present antigen to T lymphocytes can be directly modulated by Toll‐like receptor (TLR) agonists. In the absence of B cell receptor stimulation, CpG oligonucleotide, a TLR9 agonist, was particularly efficient in inducing naïve B cell proliferation and survival. Although the expanded naïve B cells did not mature into CD27⁺ or IgG⁺ memory B cells, these cells did differentiate into IgM‐secreting cells with increased surface expression of HLA‐DR, CD40 and CD80. This was associated with an increased potential for these B cells to activate allogeneic T cells. We propose that the activation and expansion of naïve B cells induced by TLR9 agonists could enhance the potential of these cells to interact with cognate antigens and facilitate cell‐mediated immune responses.     

Monocyte‐derived dendritic cells can induce autoreactive CD4+ T cells showing myeloid lineage directed reactivity in healthy individuals

T cells against self‐antigens can be detected in peripheral blood of healthy individuals, although intrathymic negative selection removes most high‐avidity T cells specific for self‐antigens from the peripheral repertoire. Moreover, spontaneous T‐cell proliferation following stimulation with autologous monocyte‐derived dendritic cells (autoDCs) has been observed in vitro. In this study, we characterized the nature and immunological basis of the autoDC reactivity in the T‐cell repertoire of healthy donors. We show that a minority of naive and memory CD4⁺ T cells within the healthy human T‐cell repertoire mediates HLA‐restricted reactivity against autoDCs, which behave like a normal antigen‐specific immune response. This reactivity appeared to be primarily directed against myeloid lineage cells. Although cytokine production by the reactive T cells was observed, this did not coincide with overt cytotoxic activity against autoDCs. AutoDC reactivity was also observed in the CD8⁺ T‐cell compartment, but this appeared to be mainly cytokine‐induced rather than antigen‐driven. In conclusion, we show that the presence of autoreactive T cells harboring the potential to react against autologous and HLA‐matched allogeneic myeloid cells is a common phenomenon in healthy individuals. These autoDC‐reactive T cells may help the induction of primary T‐cell responses at the DC priming site.     

Cytomegalovirus‐seropositivity has a profound influence on the magnitude of major lymphoid subsets within healthy individuals

Cytomegalovirus (CMV) infects most individuals and elicits a strong CMV‐specific immune response. We have studied the influence of CMV‐seropositivity on the size of lymphoid subsets in healthy donors and demonstrate that the virus substantially modulates the peripheral lymphoid pool. CD8⁺ T cell numbers are increased in all CMV‐seropositive individuals because of a striking 60% increment in the CD8⁺ T cell memory pool. The CD45RA⁺ resting memory pool is doubled after CMV infection and increases further with age. The magnitude of the naïve CD8⁺ T cell pool is dramatically reduced in CMV‐seropositive individuals at all ages, and this accelerates the physiological decline by approximately 40 years. The number of CD4⁺ effector memory T cells is increased in CMV‐seropositive individuals and is differentially accommodated by a reduction in the number of naïve and central memory CD4⁺ T cells in young and elderly donors respectively. CMV‐seropositivity also increases the total number of B cells in older donors and suppresses the number of CD5⁺ B cells. These data reveal that CMV has a profound influence on the immune system of all healthy individuals and add to growing concern regarding the clinical and immunomodulatory significance of CMV infection in healthy donors.     

Regulation of CD4 T‐cell receptor diversity by vaccine adjuvants

New vaccines based on soluble recombinant antigens (Ags) require adjuvants to elicit long‐lasting protective humoral and cellular immunity. Despite the importance of CD4 T helper cells for the generation of long‐lived memory B and CD8 T cells, the impact of adjuvants on CD4 T‐cell responses is still poorly understood. Adjuvants are known to promote dendritic cell (DC) maturation and migration to secondary lymphoid organs where they present foreign peptides bound to class II major histocompatibility complex molecules (pMHCII) to naïve CD4 T cells. Random and imprecise rearrangements of genetic elements during thymic development ensure that a vast amount of T‐cell receptors (TCRs) are present in the naïve CD4 T‐cell repertoire. Ag‐specific CD4 T cells are selected from this vast pre‐immune repertoire based on the affinity of their TCR for pMHCII. Here, we review the evidence demonstrating a link between the adjuvant and the specificity and clonotypic diversity of the CD4 T‐cell response, and consider the potential mechanisms at play.     

Minor Histocompatibility Antigen UTY as Target for Graft‐versus‐Leukemia and Graft‐versus‐Haematopoiesis in the Canine Model

Male patients with female‐stem‐cell donors have better prognosis compared to female‐to‐male combinations due to Y‐encoded minor histocompatibility antigens recognized by female‐alloimmune‐effector lymphocytes in the context of a graft‐versus‐leukemia (GvL) effect. We provide data in a dog‐model that the minor histocompatibility antigen UTY might be a promising target to further improve GvL‐immune reactions after allogeneic‐stem‐cell transplantations. Female‐canine‐UTY‐specific T cells (CTLs) were stimulated in vitro using autologous‐DCs loaded with three HLA‐A2‐restricted‐UTY‐derived peptides (3‐fold‐expansion), and specific T cell responses were determined in 3/6 female dogs. CTLs specifically recognized/lysed autologous‐female‐peptide‐loaded DCs, but not naïve‐autologous‐female DCs and monocytes. They mainly recognized bone‐marrow (BM) and to a lower extent DCs, monocytes, PBMCs and B‐cells from DLA‐identical‐male littermates and peptide‐loaded T2‐cells in an MHC‐I‐restricted manner. A UTY‐/male‐specific reactivity was also obtained in vivo after stimulation of a female dog with DLA‐identical‐male PBMCs. In summary, we demonstrated natural UTY processing and presentation in dogs. We showed that female‐dog CTLs were specifically stimulated by HLA‐A2‐restricted‐UTY peptides, thereby enabling recognition of DLA‐identical‐male cells, mainly BM cells. These observations suggest UTY as a promising candidate‐antigen to improve GvL‐reactions in the course of immunotherapy.     

Homeostatic proliferation and survival of naïve and memory T cells

The immune system relies on homeostatic mechanisms in order to adapt to the changing requirements encountered during steady‐state existence and activation by antigen. For T cells, this involves maintenance of a diverse repertoire of naïve cells, rapid elimination of effector cells after pathogen clearance, and long‐term survival of memory cells. The reduction of T‐cell counts by either cytotoxic drugs, irradiation, or certain viruses is known to lead to lymphopenia‐induced proliferation and restoration of normal T‐cell levels. Such expansion is governed by the interaction of TCR with self‐peptide/MHC (p/MHC) molecules plus contact with cytokines, especially IL‐7. These same ligands, i.e. p/MHC molecules and IL‐7, maintain naïve T lymphocytes as resting cells under steady‐state T‐cell‐sufficient conditions. Unlike naïve cells, typical “central” memory T cells rely on a combination of IL‐7 and IL‐15 for their survival in interphase and for occasional cell division without requiring signals from p/MHC molecules. Other memory T‐cell subsets are less quiescent and include naturally occurring activated memory‐phenotype cells, memory cells generated during chronic viral infections, and effector memory cells. These subsets of activated memory cells differ from central memory T cells in their requirements for homeostatic proliferation and survival. Thus, the factors controlling T‐cell homeostasis can be seen to vary considerably from one subset to another as described in detail in this review.     

Memory B cells from a subset of treatment‐naïve relapsing‐remitting multiple sclerosis patients elicit CD4+ T‐cell proliferation and IFN‐γ production in response to myelin basic protein and myelin oligodendrocyte glycoprotein

Recent evidence suggests that B‐ and T‐cell interactions may be paramount in relapsing‐remitting MS (RRMS) disease pathogenesis. We hypothesized that memory B‐cell pools from RRMS patients may specifically harbor a subset of potent neuro‐APC that support neuro‐Ag reactive T‐cell proliferation and cytokine secretion. To test this hypothesis, we compared CD80 and HLA‐DR expression, IL‐10 and lymphotoxin‐α secretion, neuro‐Ag binding capacity, and neuro‐Ag presentation by memory B cells from RRMS patients to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HD). We identified memory B cells from some RRMS patients that elicited CD4⁺ T‐cell proliferation and IFN‐γ secretion in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Notwithstanding the fact that the phenotypic parameters that promote efficient Ag presentation were observed to be similar between RRMS and HD memory B cells, a corresponding capability to elicit CD4⁺ T‐cell proliferation in response to myelin basic protein and myelin oligodendrocyte glycoprotein was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B‐cell pool in RRMS harbors neuro‐Ag specific B cells that can activate T cells.     

Primed B cells present type-II collagen to T cells

Development of type‐II collagen (CII)‐induced arthritis (CIA) is dependent on a T‐cell mediated activation of autoreactive B cells. However, it is still unclear if B cells can present CII to T cells. To investigate the role of B cells as antigen‐presenting cells (APCs) for CII, we purified B cells from lymph nodes of immunized and nonimmunized mice. These B cells were used as APC for antigen‐specific T‐cell hybridomas. B cells from naïve mice did present native, triple‐helical, CII (nCII) but also ovalbumin (OVA) and denatured CII (dCII) to antigen‐specific T‐cell hybridomas. In addition, B cells primed with nCII or OVA, but not dCII, activated the antigen‐specific T‐cell hybridomas two to three times better than naïve B cells. We conclude that antigen‐primed B cells have the capacity to process and present CII to primed T cells, and antigen‐primed antigen‐specific B cells are more efficient as APC than naïve B cells. We further conclude that B cells have the potential to play an important role as APC in the development of CIA.     

Cardiolipin activates antigen‐presenting cells via TLR2‐PI3K‐PKN1‐AKT/p38‐NF‐kB signaling to prime antigen‐specific naïve T cells in mice

Mitochondrial defects and antimitochondrial cardiolipin (CL) antibodies are frequently detected in autoimmune disease patients. CL from dysregulated mitochondria activates various pattern recognition receptors, such as NLRP3. However, the mechanism by which mitochondrial CL activates APCs as a damage‐associated molecular pattern to prime antigen‐specific naïve T cells, which is crucial for T‐cell‐dependent anticardiolipin IgG antibody production in autoimmune diseases is unelucidated. Here, we show that CL increases the expression of costimulatory molecules in CD11c⁺ APCs both in vitro and in vivo. CL activates CD11c⁺ APCs via TLR2‐PI3K‐PKN1‐AKT/p38MAPK‐NF‐κB signaling. CD11c⁺ APCs that have been activated by CL are sufficient to prime H‐Y peptide‐specific naïve CD4⁺ T cells and OVA‐specific naïve CD8⁺ T cells. TLR2 is necessary for anti‐CL IgG antibody responses in vivo. Intraperitoneal injection of CL does not activate CD11c⁺ APCs in CD14 KO mice to the same extent as in wild‐type mice. CL binds to CD14 (Kd = 7 × 10^?7 M). CD14, but not MD2, plays a role in NF‐kB activation by CL, suggesting that CD14⁺ macrophages contribute to recognizing CL. In summary, CL activates signaling pathways in CD11c⁺ APCs through a mechanism similar to gram (+) bacteria and plays a crucial role in priming antigen‐specific naïve T cells.     

Selection of CMV-specific CD8+ and CD4+ T cells by mini-EBV-transformed B cell lines

Efficient protocols to generate cytomegalovirus (CMV)-specific T cells are required for adoptive immunotherapy. Recombinant Epstein-Barr virus (EBV) vectors called mini-EBV can be used to establish permanent B cell lines in a single step, which present the CMV antigen pp65 in a constitutive manner. These B cell lines, coined pp65 mini-LCL, were successfully used to reactivate and expand CMV-specific cytotoxic T cells. Here we evaluate this pp65 mini-EBV system in closer detail, focusing on (1) the quantification of T cells with specific effector function and (2) the identification of CMV-specific CD4(+) helper T cells. The co-expansion of various functional CMV epitope specificities was demonstrated by IFN-gamma enzyme-linked immunospot assay (ELISPOT) assays and HLA-peptide tetramer staining. Single-cell cloning resulted in both CD4(+) and CD8(+) T cell clones, the majority of which was CMV specific. Thus, mini-LCL present the pp65 antigen on HLA class I and II, mobilizing both arms of the T cell response. Using a peptide library covering the pp65 sequence for further analysis of T cell clones, we identified new pp65 CD8(+) and CD4(+) T cell epitopes.     

Role of the MHC restriction during maturation of antigen‐specific human T cells in the thymus

In the thymus, a T‐cell repertoire able to confer protection against infectious and noninfectious agents in a peptide‐dependent, self‐MHC‐restricted manner is selected. Direct detection of Ag‐specific thymocytes, and analysis of the impact of the expression of the MHC‐restricting allele on their frequency or function has never been studied in humans because of the extremely low precursor frequency. Here, we used a tetramer‐based enrichment protocol to analyze the ex vivo frequency and activation‐phenotype of human thymocytes specific for self, viral and tumor‐antigens presented by HLA‐A*0201 (A2) in individuals expressing or not this allele. Ag‐specific thymocytes were quantified within both CD4CD8 double or single‐positive compartments in every donor. Our data indicate that the maturation efficiency of Ag‐specific thymocytes is poorly affected by HLA‐A2 expression, in terms of frequencies. Nevertheless, A2‐restricted T‐cell lines from A2⁺ donors reacted to A2⁺ cell lines in a highly peptide‐specific fashion, whereas their alloreactive counterparts showed off‐target activity. This first ex vivo analysis of human antigen‐specific thymocytes at different stages of human T‐cell development should open new perspectives in the understanding of the human thymic selection process.     

Generation of β cell‐specific human cytotoxic T cells by lentiviral transduction and their survival in immunodeficient human leucocyte antigen‐transgenic mice

Several β cell antigens recognized by T cells in the non‐obese diabetic (NOD) mouse model of type 1 diabetes (T1D) are also T cell targets in the human disease. While numerous antigen‐specific therapies prevent diabetes in NOD mice, successful translation of rodent findings to patients has been difficult. A human leucocyte antigen (HLA)‐transgenic mouse model incorporating human β cell‐specific T cells might provide a better platform for evaluating antigen‐specific therapies. The ability to study such T cells is limited by their low frequency in peripheral blood and the difficulty in obtaining islet‐infiltrating T cells from patients. We have worked to overcome this limitation by using lentiviral transduction to ‘reprogram’ primary human CD8 T cells to express three T cell receptors (TCRs) specific for a peptide derived from the β cell antigen islet‐specific glucose‐6‐phosphatase catalytic subunit‐related protein (IGRP_265–273) and recognized in the context of the human class I major histocompatibility complex (MHC) molecule HLA‐A2. The TCRs bound peptide/MHC multimers with a range of avidities, but all bound with at least 10‐fold lower avidity than the anti‐viral TCR used for comparison. One exhibited antigenic recognition promiscuity. The β cell‐specific human CD8 T cells generated by lentiviral transduction with one of the TCRs released interferon (IFN)‐γ in response to antigen and exhibited cytotoxic activity against peptide‐pulsed target cells. The cells engrafted in HLA‐A2‐transgenic NOD‐scid IL2rγ^null mice and could be detected in the blood, spleen and pancreas up to 5 weeks post‐transfer, suggesting the utility of this approach for the evaluation of T cell‐modulatory therapies for T1D and other T cell‐mediated autoimmune diseases.     

T cell receptor alpha variable 12‐2 bias in the immunodominant response to Yellow fever virus

The repertoire of human αβ T‐cell receptors (TCRs) is generated via somatic recombination of germline gene segments. Despite this enormous variation, certain epitopes can be immunodominant, associated with high frequencies of antigen‐specific T cells and/or exhibit bias toward a TCR gene segment. Here, we studied the TCR repertoire of the HLA‐A*0201‐restricted epitope LLWNGPMAV (hereafter, A2/LLW) from Yellow Fever virus, which generates an immunodominant CD8⁺ T cell response to the highly effective YF‐17D vaccine. We discover that these A2/LLW‐specific CD8⁺ T cells are highly biased for the TCR α chain TRAV12‐2. This bias is already present in A2/LLW‐specific naïve T cells before vaccination with YF‐17D. Using CD8⁺ T cell clones, we show that TRAV12‐2 does not confer a functional advantage on a per cell basis. Molecular modeling indicated that the germline‐encoded complementarity determining region (CDR) 1α loop of TRAV12‐2 critically contributes to A2/LLW binding, in contrast to the conventional dominant dependence on somatically rearranged CDR3 loops. This germline component of antigen recognition may explain the unusually high precursor frequency, prevalence and immunodominance of T‐cell responses specific for the A2/LLW epitope.