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.     

The proteasome immunosubunits,PA28 and ER‐aminopeptidase 1 protect melanoma cells from efficient MART‐126‐35‐specific T‐cell recognition

The immunodominant MART‐1_26(27)‐35 epitope, liberated from the differentiation antigen melanoma antigen recognized by T cells/melanoma antigen A (MART‐1/Melan‐A), has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits β5i/LMP7 (LMP, low molecular weight protein) or β1i/LMP2 and β5i/LMP7 interfere with MART‐1_26‐35 epitope generation in tumor cells. Here, we demonstrate that in addition the IFN‐γ‐inducible proteasome subunit β2i/MECL‐1 (multicatalytic endopeptidase complex‐like 1), proteasome activator 28 (PA28), and ER‐resident aminopeptidase 1 (ERAP1) impair MART‐1_26‐35 epitope generation. β2i/MECL‐1 and PA28 negatively affect C‐ and N‐terminal cleavage and therefore epitope liberation from the proteasome, whereas ERAP1 destroys the MART‐1_26‐35 epitope by overtrimming activity. Constitutive expression of PA28 and ERAP1 in melanoma cells indicate that both interfere with MART‐1_26‐35 epitope generation even in the absence of IFN‐γ. In summary, our results provide first evidence that activities of different antigen‐processing components contribute to an inefficient MART‐1_26‐35 epitope presentation, suggesting the tumor cell's proteolytic machinery might have an important impact on the outcome of epitope‐specific immunotherapies.     

Distinct activation phenotype of a highly conserved novel HLA‐B57‐restricted epitope during dengue virus infection

Variation in the sequence of T‐cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T‐cell responses during second heterologous infections. We identified a highly conserved, novel, HLA‐B57‐restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57‐NS1_26–34‐specific CD8⁺ T cells in peripheral blood mononuclear cells from individuals undergoing secondary rather than primary DENV infection. However, high tetramer‐positive T‐cell frequencies during acute infection were seen in only one of nine subjects with secondary infection. B57‐NS1_26–34‐specific and other DENV epitope‐specific CD8⁺ T cells, as well as total CD8⁺ T cells, expressed an activated phenotype (CD69⁺ and/or CD38⁺) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope‐specific CD8⁺ T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen‐specific T‐cell activation.     

T‐cell receptors: Tugging on the anchor for a tighter hold on the tumor‐associated peptide

Although it has been shown that human tumor‐associated, HLA anchor residue modified “heteroclitic” peptides may induce stronger immune responses than wild‐type peptides in cancer vaccine trials, it has also been shown that some T cells primed with these heteroclitic peptides subsequently fail to recognize the natural, tumor‐expressed peptide efficiently. This may provide a molecular reason for why clinical trials of these peptides have been thus far unsuccessful. In this issue of the European Journal of Immunology, Madura et al. [Eur. J. Immunol. 2015. 45: 584–591] highlight a novel twist on T‐cell receptor (TCR) recognition of HLA–peptide complexes. Tumor‐associated peptides often lack canonical anchor residues, which can be substituted for the optimal residue to improve their antigenicity. T‐cell cross‐reactivity between the natural and modified (heteroclitic) peptides is essential for this approach to work and depends on whether the anchor residue substitution influences peptide conformation. The Melan‐A/MART‐1_26‐35 peptide epitope is an example where T cells can make this distinction, with the natural peptide stimulating higher affinity CD8⁺ T cells than the heteroclitic peptide, despite the heteroclitic peptide's more stable association with HLA‐A2. The molecular basis for peptide discrimination is identified through the structure of the TCR bound to the natural peptide; TCR engagement of the natural peptide “lifts” its amino‐terminus partly away from the HLA peptide binding groove, forming a higher affinity interface with the TCR than is formed with the anchor residue “optimized” heteroclitic peptide, which cannot be “pulled” from the HLA groove.     

High expression of CD26 accurately identifies human bacteria‐reactive MR1‐restricted MAIT cells

Mucosa‐associated invariant T (MAIT) cells express the semi‐invariant T‐cell receptor TRAV1–2 and detect a range of bacteria and fungi through the MHC‐like molecule MR1. However, knowledge of the function and phenotype of bacteria‐reactive MR1‐restricted TRAV1–2⁺ MAIT cells from human blood is limited. We broadly characterized the function of MR1‐restricted MAIT cells in response to bacteria‐infected targets and defined a phenotypic panel to identify these cells in the circulation. We demonstrated that bacteria‐reactive MR1‐restricted T cells shared effector functions of cytolytic effector CD8⁺ T cells. By analysing an extensive panel of phenotypic markers, we determined that CD26 and CD161 were most strongly associated with these T cells. Using FACS to sort phenotypically defined CD8⁺ subsets we demonstrated that high expression of CD26 on CD8⁺ TRAV1–2⁺ cells identified with high specificity and sensitivity, bacteria‐reactive MR1‐restricted T cells from human blood. CD161^hi was also specific for but lacked sensitivity in identifying all bacteria‐reactive MR1‐restricted T cells, some of which were CD161^dim. Using cell surface expression of CD8, TRAV1–2, and CD26^hi in the absence of stimulation we confirm that bacteria‐reactive T cells are lacking in the blood of individuals with active tuberculosis and are restored in the blood of individuals undergoing treatment for tuberculosis.     

Differential CD4+ T‐cell responses of allergic and non‐allergic subjects to the immunodominant epitope region of the horse major allergen Equ c 1

The responses of allergen‐specific CD4⁺ T cells of allergic and healthy individuals are still incompletely understood. Our objective was to investigate the functional and phenotypic properties of CD4⁺ T cells of horse‐allergic and healthy subjects specific to the immunodominant epitope region of the major horse allergen Equ c 1. Specific T‐cell lines (TCLs) and clones were generated from peripheral blood mononuclear cells with Equ c 1_143–160, the peptide containing the immunodominant epitope region of Equ c 1. The frequency, proliferative response, cytokine production and HLA restriction of the cells were examined. The frequency of Equ c 1‐specific CD4⁺ T cells was low (approximately 1 per 10⁶ CD4⁺ T cells) in both allergic and non‐allergic subjects. The cells of allergic subjects had a stronger proliferative capacity than those of non‐allergic subjects, and they predominantly emerged from the memory T‐cell pool and expressed the T helper type 2 cytokine profile, whereas the cells of non‐allergic subjects emerged from the naive T‐cell pool and produced low levels of interferon‐γ and interleukin‐10. T‐cell response to Equ c 1_143–160 was restricted by several common HLA class II molecules from both DQ and DR loci. As the phenotypic and functional properties of Equ c 1‐specific CD4⁺ T cells differ between allergic and non‐allergic subjects, allergen‐specific T cells appear to be tightly implicated in the development of diseased or healthy outcome. Restriction of the specific CD4⁺ T‐cell response by multiple HLA alleles suggests that Equ c 1_143–160 is a promising candidate for peptide‐based immunotherapy.     

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.     

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.     

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.     

The CD4+ T‐cell epitope‐binding register is a critical parameter when generating functional HLA‐DR tetramers with promiscuous peptides

Detection of CD4⁺ T cells specific for tumor‐associated antigens is critical to investigate the spontaneous tumor immunosurveillance and to monitor immunotherapy protocols in patients. We investigated the ability of HLA‐DR^*1101 multimers to detect CD4⁺ T cells specific for three highly promiscuous MAGE‐A3 derived peptides: MAGE‐A3_191–205 (p39), MAGE‐A3_281–295 (p57) and MAGE‐A3_286–300 (p58). Tetramers stained specific CD4⁺ T cells only when loaded with p39, although all peptides activated the specific T cells when presented by plastic‐bound HLA‐DR^*1101 monomers. This suggested that tetramer staining ability was determined by the mode rather than the affinity of peptide binding to HLA‐DR^*1101. We hypothesized that peptides should bear a single P1 anchor residue to bind all arms of the multimer in a homogeneous register to generate peptide‐HLA‐DR conformers with maximal avidity. Bioinformatics analysis indicated that p39 contained one putative P1 anchor residue, whereas the other two peptides contained multiple ones. Designing p57 and p58 analogues containing a single anchor residue generated HLA‐DR^*1101 tetramers that stained specific CD4⁺ T cells. Producing HLA‐DR^*1101 monomers linked with the optimized MAGE‐A3 analogues, but not with the original epitopes, further improved tetramer efficiency. Optimization of CD4⁺ T‐cell epitope‐binding registers is thus critical to generate functional HLA‐DR tetramers.     

The impact of HLA class I and EBV latency‐II antigen‐specific CD8+ T cells on the pathogenesis of EBV+ Hodgkin lymphoma

In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein–Barr virus (EBV) latency‐II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV⁺cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA‐A*02 is protective in EBV⁺cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA‐A*02^– versus HLA‐A*02⁺ EBV⁺cHL patients, suggesting that LMP2A‐specific CD8⁺ T cell anti‐tumoral immunity may be relatively ineffective in HLA‐A*02^– EBV⁺cHL. To ascertain the impact of HLA class I on EBV latency antigen‐specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV⁺cHL, the magnitude of ex‐vivo LMP1/2A‐specific CD8⁺ T cell responses was elevated in HLA‐A*02⁺ patients. Furthermore, in a controlled in‐vitro assay, LMP2A‐specific CD8⁺ T cells from healthy HLA‐A*02 heterozygotes expanded to a greater extent with HLA‐A*02‐restricted compared to non‐HLA‐A*02‐restricted cell lines. In an extensive analysis of HLA class I‐restricted immunity, immunodominant EBNA3A/3B/3C‐specific CD8⁺ T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A‐specific responses were confined largely to HLA‐A*02. Our results demonstrate that HLA‐A*02 mediates a modest, but none the less stronger, EBV‐specific CD8⁺ T cell response than non‐HLA‐A*02 alleles, an effect confined to EBV latency‐II antigens. Thus, the protective effect of HLA‐A*02 against EBV⁺cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency‐II antigen‐specific CD8⁺ T cell hierarchies.     

Identification of a dengue virus‐specific HLA‐A*0201‐restricted CD8+ T cell epitope

In this study, a combination of epitope‐prediction programs and in vitro assays was used to identify dengue virus (DENV)‐specific CD8⁺ T cell epitopes. Peripheral blood mononuclear cells (PBMCs) isolated from patients who recovered from dengue fever were stimulated with candidate epitope peptides derived from DENV, which were predicted by using SYFPEITHI and RANKpep epitope‐prediction programs. The IFN‐γ ELISpot results and the results of intracellular staining of IFN‐γ showed that peptides NS4b_40 (TLYAVATTI), E_256 (QEGAMHTAL), NS3_205 (LPAIVREAI), NS5_210 (SRNSTHEMY), and NS3_207 (AIVREAIKR) could induce the recall response of CD8⁺ T cells. Furthermore, the results of the MHC–peptide complex stabilization assay revealed that peptide NS4b_40 (TLYAVATTI) has a high affinity for HLA‐A*0201 molecules. The IFN‐γ ELISpot results and staining of intracellular IFN‐γ confirmed that this peptide could induce high‐level CD8⁺ T cell response in HLA‐A*0201 positive PBMCs. Peptide NS4b_40 (TLYAVATTI) was identified as a novel DENV‐specific HLA‐A*0201‐restricted CD8⁺ T cell epitope. J. Med. Virol. 82:642–648, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

The CD8+HLA‐DR+ T cells expanded in HIV‐1 infection are qualitatively identical to those from healthy controls

HIV‐induced immune activation leads to expansion of a subset of human CD8⁺ T cells expressing HLA‐DR antigens. Expansion of CD8⁺HLA‐DR⁺ T cells can be also observed in non‐HIV settings including several autoimmune diseases and aging. Although these cells are felt to represent “immune exhaustion” and/or to be anergic, their precise role in host defense has remained unclear. Here, we report that this subset of cells exhibits a restricted repertoire, shows evidence of multiple rounds of division, but lacks markers of recent TCR engagement. Detailed cell cycle analysis revealed that compared with their CD8⁺HLA‐DR^? counterpart, the CD8⁺HLA‐DR⁺ T‐cell pool contained an increased fraction of cells in S‐phase with elevated levels of the G2/M regulators: cyclin A2, CDC25C, Cdc2 (CDK1), indicating that these cells are not truly anergic but rather experiencing proliferation in vivo. Together, these data support a hypothesis that antigen stimulation leads to the initial expansion of a CD8⁺ pool of cells in vivo that undergo further expansion independent of ongoing TCR engagement. No qualitative differences were noted between CD8⁺HLA‐DR⁺ cells from HIV⁺ and HIV^? donors, indicating that the generation of CD8⁺HLA‐DR⁺ T cells is a part of normal immune regulation that is exaggerated in the setting of HIV‐1 infection.     

CTL recognition of HIV‐1‐infected cells via cross‐recognition of multiple overlapping peptides from a single 11‐mer Pol sequence

It is known that overlapping HIV‐1 peptides of different lengths can be presented by a given HLA class I molecule. However, the role of those peptides in CD8⁺ T cells recognition of HIV‐1‐infected cells remains unclear. Here we investigated the recognition of overlapping 8‐mer to 11‐mer peptides of Pol 155–165 by HLA‐B*54:01‐restricted CD8⁺ T cells. The analysis of ex vivo T cells using ELISPOT and tetramer binding assays showed that there were different patterns of CD8⁺ T‐cell responses to these peptides among chronically HIV‐1‐infected HLA‐B*54:01⁺ individuals, though the response to the 9‐mer peptide was the strongest among them. CD8⁺ T‐cell clones with TCRs specific for the 9‐mer, 10‐mer, and/or 11‐mer peptides effectively killed HIV‐1‐infected cells. Together, these results suggest that the 9‐mer and 10‐mer peptides could be predominantly presented by HLA‐B*54:01, though it remains possible that the 11‐mer peptide was also presented by this HLA allele. The present study demonstrates effective CD8⁺ T‐cell recognition of HIV‐1‐infected cells via presentation of multiple overlapping HIV‐1 peptides and cross‐recognition by the CD8⁺ T cells.     

Analysis of nucleophosmin–anaplastic lymphoma kinase (NPM‐ALK)‐reactive CD8+ T cell responses in children with NPM‐ALK+ anaplastic large cell lymphoma

Cellular immune responses against the oncoantigen anaplastic lymphoma kinase (ALK) in patients with ALK‐positive anaplastic large cell lymphoma (ALCL) have been detected using peptide‐based approaches in individuals preselected for human leucocyte antigen (HLA)‐A*02:01. In this study, we aimed to evaluate nucleophosmin (NPM)‐ALK‐specific CD8⁺ T cell responses in ALCL patients ensuring endogenous peptide processing of ALK antigens and avoiding HLA preselection. We also examined the HLA class I restriction of ALK‐specific CD8⁺ T cells. Autologous dendritic cells (DCs) transfected with in‐vitro‐transcribed RNA (IVT‐RNA) encoding NPM–ALK were used as antigen‐presenting cells for T cell stimulation. Responder T lymphocytes were tested in interferon‐gamma enzyme‐linked immunospot (ELISPOT) assays with NPM–ALK‐transfected autologous DCs as well as CV‐1 in Origin with SV40 genes (COS‐7) cells co‐transfected with genes encoding the patients’ HLA class I alleles and with NPM–ALK encoding cDNA to verify responses and define the HLA restrictions of specific T cell responses. NPM–ALK‐specific CD8⁺ T cell responses were detected in three of five ALK‐positive ALCL patients tested between 1 and 13 years after diagnosis. The three patients had also maintained anti‐ALK antibody responses. No reactivity was detected in samples from five healthy donors. The NPM–ALK‐specific CD8⁺ T cell responses were restricted by HLA‐C‐alleles (C*06:02 and C*12:02) in all three cases. This approach allowed for the detection of NPM–ALK‐reactive T cells, irrespective of the individual HLA status, up to 9 years after ALCL diagnosis.     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.     

Presence of HLA‐DR Molecules and HLA‐DRB1 mRNA in Circulating CD4+ T Cells

The human major histocompatibility complex class II isotype HLA‐DR is currently used as an activation marker for T cells. However, whether an endogenous protein expression or a molecular acquisition accounts for the presence of HLA‐DR on T cells remains undetermined and still controversial. To further characterize this phenomenon, we compared several aspects of the presence of the HLA‐DR protein to the presence of associated mRNA (HLA‐DRB1), focusing on human T cells from peripheral blood of healthy individuals. Using a flow cytometric approach, we determined that the HLA‐DR observed on CD4⁺ T cells was almost exclusively cell surface‐associated, while for autologous CD19⁺ B cells, the protein could be located in the plasma membrane as well as in the cytoplasm. Moreover, negligible expression levels of HLA‐DRB1 were found in CD4⁺ T cells, using an HLA‐DRB1 allele‐specific qPCR assay. Finally, the presence of HLA‐DR was not confined to activated CD4⁺ and CD8⁺ T cells, as evaluated by the co‐expression of CD25. The functional role of the HLA‐DR molecule on T cells remains enigmatic; however, this study presents evidence of fundamental differences for the presence of HLA‐DR on T cells from HLA‐DR in the context of antigen‐presenting cells, which is a well‐known phenomenon. Although an inducible endogenous protein expression cannot be excluded for the T cells, our findings suggest that a re‐evaluation of the HLA‐DR as a T cells activation marker is warranted.     

CD8‐β ADP‐ribosylation affects CD8+ T‐cell function

The CD8αβ coreceptor is crucial for effective peptide: MHC‐I recognition by the TCR of CD8⁺ T cells. Adenosine diphosphate ribosyl transferase 2.2 (ART2.2) utilizes extracellular NAD⁺ to transfer ADP‐ribose to arginine residues of extracellular domains of surface proteins. Here, we show that in the presence of extracellular NAD⁺, ART2.2 caused ADP‐ribosylation of CD8‐β on murine CD8⁺ T cells in vitro and in vivo. Treatment with NAD⁺ prevented binding of anti‐CD8‐β mAb YTS156.7.7 but not of mAb H35–17.2, indicating that NAD⁺ caused modification of certain epitopes and not a general loss of CD8‐β. Loss of antibody binding was strictly dependent on ART2.2, because it was not observed on ART2‐deficient T cells or in the presence of inhibitory anti‐ART2.2 single‐domain antibodies. ADP‐ribosylation of CD8‐β occurred during cell isolation, particularly when cells were isolated from CD38‐deficient mice. Incubation of ART2‐expressing, but not of ART2‐deficient, OVA‐specific CD8⁺ T cells with NAD⁺ interfered with binding of OVA_257–264:MHC‐I tetramers. In line with this result, treatment of WT mice with NAD⁺ resulted in reduced CD8⁺ T‐cell mediated cytotoxicity in vivo. We propose that ADP‐ribosylation of CD8‐β can regulate the coreceptor function of CD8 in the presence of elevated levels of extracellular NAD⁺.