+1 Frameshifting as a novel mechanism to generate a cryptic cytotoxic T lymphocyte epitope derived from human interleukin 10.

Recent data indicate that some cytotoxic T cells (CTLs) recognize so-called cryptic epitopes, encoded by nonprimary open reading frame (ORF) sequences or other nonclassical expression pathways. We describe here a novel mechanism leading to generation of a cryptic CTL epitope. We isolated from the synovial fluid of a patient suffering from a Reiter's syndrome an autoreactive T cell clone that recognized cellular IL-10 in the HLA-B*2705 context. The minimal IL-10 sequence corresponding to nucleotides 379-408 was shown to activate this clone, upon cotransfection into COS cells with the DNA encoding HLA-B*2705, but the synthetic peptide deduced from this sequence did not stimulate the clone. Using a site-directed mutagenesis approach, we found that this clone recognized a transframe epitope generated by an internal +1 frameshifting in the IL-10 sequence and so derived partly from ORF1, partly from ORF2. We defined that +1 frameshifting was induced by a specific heptamer sequence. These observations illustrate the variety of mechanisms leading to generation of cryptic epitopes and suggest that frameshifting in normal cellular genes may be more common than expected.

Selection of high-avidity CD8 T cells correlates with control of hepatitis C virus infection.

UNLABELLED: Both strong antigenic avidity and acquisition of proper effector functions contribute to the efficacy of antiviral T cell responses. To correlate these parameters with the outcome of hepatitis C virus (HCV) infection, we characterized HCV-specific CD8 T cell lines isolated after immunomagnetic sorting of peripheral blood mononuclear cells from human leukocyte antigen A*02 (HLA-A*02) individuals with various HCV serological statuses, using recombinant HLA-A*0201 multimers loaded with three immunodominant HCV genotype 1-derived epitopes. CD8 T cells specific for these three epitopes were derived from most HLA-A*0201 individuals, regardless of their HCV serology or clinical outcome. Donors recovered from genotype 1 HCV infection were enriched for high-avidity T cells with enhanced interferon gamma (IFN-gamma), tumor necrosis factor alpha, and cytotoxic T lymphocyte responses, when compared with seronegative donors and seropositive patients infected with irrelevant HCV genotypes. Patients chronically infected with genotype 1 strain yielded almost exclusively low-avidity T cells, whose hyporesponsiveness was primarily attributable to low T cell receptor (TCR) avidity rather than intrinsic functional defects. CONCLUSION: This study suggests that strong IFN-gamma responses associated with efficient viral clearance primarily result from Ag-driven selection/survival of HCV-specific T cells expressing high-avidity TCR. It also suggests a link between the quality of the initial HCV-specific T cell repertoire and susceptibility to chronic infection.

The human T cell immune response to Epstein-Barr virus.

The Epstein-Barr virus (EBV) is a gamma-herpes virus which establishes latent, life-long infection in more than 95% of the human adult population. Despite its growth transforming capacity, most carriers control EBV associated malignacies efficiently and remain free of EBV+ tumors. Though EBV is controlled by a potent immune response, this virus uses latency to persist in vivo. This review summarizes work which has been done to characterize T cell responses to EBV. The CD8 T cell responses are rather well characterized and have been shown by several groups to be highly focused towards early lytic antigens. Much less is known about CD4 T cell epitopes, due to the small size of the CD4 compartment. However, recent data indicate a control of lytic and latent cycles of EBV by specific CD4+ T cells. A clear understanding of the T cell response to EBV is important with a view to developing immunotherapies for the virus and its related malignancies.

HLA-E-restricted cross-recognition of allogeneic endothelial cells by CMV-associated CD8 T cells: a potential risk factor following transplantation.

Although association between CMV infection and allograft rejection is well admitted, the precise mechanisms involved remain uncertain. Here, we report the characterization of an alloreactive HLA-E-restricted CD8 T cell population that was detected in the PBL of a kidney transplant patient after its CMV conversion. This monoclonal CD8 T cell population represents a sizable fraction in the blood (3% of PBL) and is characterized by an effector-memory phenotype and the expression of multiple NK receptors. Interestingly, these unconventional T cells display HLA-E-dependent reactivity against peptides derived from the leader sequences of both various HCMV-UL40 and allogeneic classical HLA-I molecules. Consequently, while HLA-E-restricted CD8 T cells have potential to contribute to the control of CMV infection in vivo, they may also directly mediate graft rejection through recognition of peptides derived from allogeneic HLA-I molecules on graft cells. Therefore, as HLA-E expression in nonlymphoid organs is mainly restricted to endothelial cells, we investigated the reactivity of this HLA-E-restricted T cell population towards allogeneic endothelial cells. We clearly demonstrated that CMV-associated HLA-E-restricted T cells efficiently recognized and killed allogeneic endothelial cells in vitro. Moreover, our data indicate that this alloreactivity is tightly regulated by NK receptors, especially by inhibitory KIR2DL2 that strongly prevents TCR-induced activation through recognition of HLA-C molecules. Hence, a better evaluation of the role of CMV-associated HLA-E-restricted T cells in transplantation and of the impact of HLA-genotype, especially HLA-C, on their alloreactivity may determine whether they indeed represent a risk factor following organ transplantation.

Cross-reactivity of herpesvirus-specific CD8 T cell lines toward allogeneic class I MHC molecules.

Although association between persistent viral infection and allograft rejection is well characterized, few examples of T-cell cross-reactivity between self-MHC/viral and allogeneic HLA molecules have been documented so far. We appraised in this study the alloreactivity of CD8 T cell lines specific for immunodominant epitopes from human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). CD8 T cell lines were generated after sorting with immunomagnetic beads coated with either pp65(495-503)/A*0201, BMLF1(259-267)/A*0201, or BZLF1(54-64)/B*3501 multimeric complexes. Alloreactivity of the CD8 T cell lines against allogeneic class I MHC alleles was assessed by screening of (i) TNF-alpha production against COS-7 cells transfected with as many as 39 individual HLA class I-encoding cDNA, and (ii) cytotoxicity activity toward a large panel of HLA-typed EBV-transformed B lymphoblastoid cell lines. We identified several cross-reactive pp65/A*0201-specific T cell lines toward allogeneic HLA-A*3001, A*3101, or A*3201. Moreover, we described here cross-recognition of HLA-Cw*0602 by BZLF1/B*3501-specific T cells. It is noteworthy that these alloreactive CD8 T cell lines showed efficient recognition of endothelial cells expressing the relevant HLA class I allele, with high level TNF-alpha production and cytotoxicity activity. Taken together, our data support the notion that herpes virus-specific T cells recognizing allo-HLA alleles may promote solid organ rejection.

Pattern of DAP12 expression in leukocytes from both healthy and systemic lupus erythematosus patients.

DAP12 is an ITAM-bearing transmembrane adaptor originally identified on the surface of Natural Killer cells. A broad expression among other immune cells was later found in myeloid and lymphoid cells. However, data on DAP12 expression pattern rely only on immunoblot and microarray analysis. Here, we describe the generation and the characterization of an anti-DAP12 monoclonal antibody. Using this novel reagent, we show that DAP12 expression is restricted to innate immune cells in basal condition. Since a decreased expression of DAP12 has been suggested in NK cells of systemic lupus erythematosus patients, we have further investigated the NK cell receptor repertoire and leukocyte expression of DAP12 in these patients and no major changes were detectable when compared to controls.

Impact of CD8-MHC class I interaction in detection and sorting efficiencies of antigen-specific T cells using MHC class I/peptide multimers: contribution of pMHC valency.

Recombinant soluble multimeric forms of MHC class I molecules loaded with antigenic peptides (pMHC) have turned out to be particularly useful to detect and isolate specific T cells. These applications both rely on the oligomerization of pMHC monomers in order to compensate for their inherent low affinity for the TCR. In this study, we have evaluated the precise contribution of CD8-pMHC interaction on the specificity and sorting efficiency of pMHC multimers according to their degree of oligomerization. To this end several wild-type versus mutated pMHC complexes (A*0201, B*0701, B*0801, B*3501) carrying point mutations known to reduce (245V mutants) or to abrogate (227,8KA mutants) CD8-pMHC interaction and showing various degrees of valency have been used. We show that irrespective of the HLA allele tested, 245V mutation strongly improves the binding specificity and immunomagnetic sorting efficiency of pMHC multimers. Our results also indicate that the contribution of CD8 to the binding of pMHC multimers to specific CD8+ T cells is inversely correlated to the degree of pMHC oligomerization. Consequently, efficient staining or specific sorting of high-affinity T cells (i.e. CD8 independent) can only be achieved using 227,8KA pMHC complexes with low-order oligomerization.

EBV-specific CD4+ T cell clones exhibit vigorous allogeneic responses.

Alloreactive T cells play a key role in mediating graft-vs-host disease and allograft rejection, and recent data suggest that most T cell alloreactivity resides within the CD4 T cell subset. Particularly, T cell responses to herpesvirus can shape the alloreactive repertoire and influence transplantation outcomes. In this study, we describe six distinct EBV-specific CD4(+) T cell clones that cross-reacted with EBV-transformed lymphoblastoid cell lines (LCLs), dendritic cells, and endothelial cells expressing MHC class II alleles commonly found in the population. Allorecognition showed exquisite MHC specificity. These CD4(+) T cell clones efficiently killed dendritic cells or LCLs expressing the cross-reactive allogeneic MHC class II molecules, whereas they did not kill autologous LCLs. Endothelial cells expressing the proper allogeneic MHC molecules were poorly killed, but they induced high-level TNF-alpha production by the EBV-specific CD4(+) T cell clones. As already proposed, the strong alloreactivity toward LCLs suggest that these cells could be used for selective depletion of alloreactive T cells.

Long-term MHC class II presentation of the EBV lytic protein BHRF1 by EBV latently infected b cells following capture of BHRF1 antigen.

Although T lymphocytes are considered essential for the control of EBV infection, it remains uncertain how this control occurs. We previously reported unexpected killing of EBV-transformed B-lymphoblastoid cells (LCLs) that did not express BHRF1 by CD4+ T cells specific for BHRF1, an EBV lytic cycle protein. Using LCLs transformed with an EBV mutant, in which the BHRF1 gene was deleted, we showed that killing of latently infected cells through the recognition of a protein produced during the lytic cycle is due to transfer of BHRF1 from lytically infected to latently infected cells, which occurs in culture. Accordingly, LCLs efficiently presented exogenous BHRF1 protein. Furthermore, we present evidence for persistence of captured BHRF1 Ag for several days. Due to this long-term persistence, repeated loading of suboptimal amounts of BHRF1 led to accumulation of BHRF1 Ags in LCLs and, ultimately, to their optimal recognition by BHRF1-specific CD4+ T cells. These results unveil an MHC class II-dependent pathway that could be important for the control of EBV latent infection through recognition of lytic cycle Ags.

Search for correlation of CD8 T cell response to Epstein-Barr virus with clinical status in rheumatoid arthritis: a 15 month followup pilot study.

OBJECTIVE: To assess in a longitudinal 15 month followup study the CD8 T cell response to immunodominant Epstein-Barr virus (EBV) antigens of 17 patients with rheumatoid arthritis (RA); and to seek an association between these responses and both clinical activity/severity of RA and a qualitative PCR for EBV in peripheral blood. METHODS: At each patient's visit every 3 months: (1) RA activity was assessed for Disease Activity Score (DAS-28); (2) a qualitative PCR for EBV was performed; (3) CD8 T cell response to EBV epitopes was screened in peripheral blood, using an autopresentation assay of 13 EBV peptides previously identified as immunodominant targets in RA synovia. Activation of anti-EBV CD8 T cells was evaluated by measuring the release of tumor necrosis factor-a. RESULTS: The semiquantitative CD8 T cell response to EBV roughly paralleled RA clinical activity in only 4/17 patients. No clear association could be found between positive PCR for EBV (performed at least once in 10/17 patients) and RA activity/severity or fatigue. Reactivity was not qualitatively broader in samples where PCR for EBV proved positive, and most often focused on one or 2 EBV antigens. However, these antigens differed between patients, as did the magnitude of CD8 T cell response to immunodominant antigens at different timepoints for the same patient. CONCLUSION: The CD8 T cell response to EBV paralleled clinical activity in only 4/17 patients. Our pilot study does not support the hypothesis that this CD8 response contributes to RA activity/flares, although the quantitative variations in the pattern of this reactivity over time confirmed that control of EBV manifestations was difficult in most patients with RA.

Direct killing of Epstein-Barr virus (EBV)-infected B cells by CD4 T cells directed against the EBV lytic protein BHRF1.

Due to their low frequency, CD4 T-cell responses to Epstein-Barr virus (EBV) lytic antigens are, so far, poorly characterized. Human peptide major histocompatibility complex (MHC) class II multimers provide a means to detect and characterize such rare T cells. Along a screening of T-cell responses to lytic or latent EBV antigens within peripheral blood leukocyte (PBL)- or synovial-derived CD4 T-cell lines, we identified an human leukocyte antigen-DR*0401 (HLA-DR*0401)-restricted epitope derived from BHRF1 (BamHI fragment H rightward open reading frame 1), a viral protein produced during the early stages of the lytic cycle. We show here that T-cell responses to this particular BHRF1 epitope are shared by most EBV-infected DR*0401(+) individuals, as BHRF1-specific CD4 T cells could be sorted out from all the DRB*0401 T-cell lines analyzed, using magnetic beads coated with recombinant BHRF1/DR*0401 complexes. Sorting with these peptide MHC class II multimers was very efficient, as the yield of recovery of BHRF1-specific T cells was nearly 100%. Functional analysis of a large number of clones responding to BHRF1/DR*0401 demonstrated their cytolytic action against autologous and allogeneic DR*0401(+) EBV-transformed B-lymphoblastoid cell lines (B-LCLs), with 40% to 80% killing efficiency and potent interferon gamma production, thus suggesting that this CD4 T-cell population contributes to the control of EBV replication. B-LCL lysis by these T-cell clones was DR*0401 dependent, EBV dependent, and was not merely due to bystander killing. Taken together, these data provide the first demonstration that a lytic antigen can induce a direct cytolytic response against EBV-infected cells.