Different development of myelin basic protein agonist- and antagonist-specific human TCR transgenic T cells in the thymus and periphery.

Myelin basic protein (MBP)-specific T cells are thought to play a role in the development of multiple sclerosis. MBP residues 111-129 compose an immunodominant epitope cluster restricted by HLA-DRB1*0401. The sequence of residues 111-129 of MBP (MBP(111-129)) differs in humans (MBP122:Arg) and mice (MBP122:Lys) at aa 122. We previously found that approximately 50% of human MBP(111-129) (MBP122:Arg)-specific T cell clones, including MS2-3C8 can proliferate in response to mouse MBP(111-129) (MBP122:Lys). However, the other half of T cell clones, including HD4-1C2, cannot proliferate in response to MBP(111-129) (MBP122:Lys). We found that MBP(111-129) (MBP122:Lys) is an antagonist for HD4-1C2 TCR, therefore, MS2-3C8 and HD4-1C2 TCRs are agonist- and antagonist-specific TCRs in mice, respectively. Therefore, we examined the development of HD4-1C2 TCR and MS2-3C8 TCR transgenic (Tg) T cells in the thymus and periphery. We found that dual TCR expression exclusively facilitates the development of MBP(111-129) TCR Tg T cells in the periphery of HD4-1C2 TCR/HLA-DRB1*0401 Tg mice although it is not required for their development in the thymus. We also found that MS2-3C8 TCR Tg CD8(+) T cells develop along with MS2-3C8 TCR Tg CD4(+) T cells, and that dual TCR expression was crucial for the development of MS2-3C8 TCR Tg CD4(+) and CD8(+) T cells in the thymus and periphery, respectively. These results suggest that thymic and peripheral development of MBP-specific T cells are different; however, dual TCR expression can facilitate their development.

Use of a lentiviral vector encoding a HCMV-chimeric IE1-pp65 protein for epitope identification in HLA-Transgenic mice and for ex vivo stimulation and expansion of CD8(+) cytotoxic T cells from human peripheral blood cells.

H2-deleted, HLA-A2, or HLA-B7 transgenic mice were used to identify new human cytomegalovirus (HCMV)-derived major histocompatibility complex class I-restricted epitopes. Three different approaches for mice immunization were compared for their ability to induce a cytotoxic CD8(+) T cell (CTL) response: (1). inoculation of infectious HCMV, (2). injection of immunogenic synthetic peptides, and (3). infection with a newly designed HIV-derived central DNA flap positive lentiviral vector encoding the chimeric IE1-pp65 protein (Trip-IE1-pp65). Targets pulsed with either known immunogenic peptides or computer predicted ones were used to characterize CTL. Most of the mice immunized with the pp65 (495-NLVPMVATV-503) immunodominant peptide responded after one injection whereas only two of six mice responded to two successive inoculations with HCMV. Infection of mice with Trip-IE1-pp65 induced activation and expansion of CTL directed against peptides from both pp65 and IE1 and allowed identification of new epitopes. We further demonstrated the high capacity of monocyte-macrophage cells transduced with Trip-IE1-pp65 to activate and expand CTL directed against pp65 from peripheral blood mononuclear cells of HCMV-seropositive donors. Altogether these results suggest that Trip-IE1-pp65 is a powerful construct both to characterize new epitopes in combination with human leukocyte antigen-transgenic mice immunization and to provide an alternative to the use of known infectious and noninfectious approaches to expand effector T cells for adoptive immunotherapy.

Genomic absence of the gene encoding T cell receptor Vbeta7.2 is linked to the presence of autoantibodies in Sjögren's syndrome.

OBJECTIVE: It is not yet known whether the absence of certain T cell receptor V(beta) (TCRBV) genes (e.g., due to genomic deletion) has functional significance. We examined this question in relation to a known 21.6-kb insertion/deletion-related polymorphism (IDRP) in the human BV locus. METHODS: New polymerase chain reaction (PCR) genotyping methods were used. Monoclonal antibodies to TCRBV gene products were used to confirm the absence of the relevant proteins. Patients with Sjögren's syndrome (SS) or systemic lupus erythematosus (SLE) were compared with normal controls with regard to TCR genotypes and serologic profiles. RESULTS: There are 3 known haplotypes (I, D1, D2) and 6 possible genotypes related to the 21.6-kb IDRP. Novel PCR-based methods were used to define these genotypes. In subjects with deleted/deleted (D/D) genotypes, T cells could not express V(beta)7.2 TCRs, as assayed with a new antibody specific for V(beta)7.2. This was the sole significant difference between subjects without the insertion and those with either 1 or 2 copies. Surprisingly, we found that the D/D genotype was associated with primary SS, but only when pathogenic autoantibodies were present. CONCLUSION: These results suggest that T cells expressing TCRs with V(beta)7.2 are protective against a pathogenic immune response in SS. Thus, genomic polymorphism of TCR genes (along with the correct HLA alleles) determines whether T cells can direct a pathogenic autoimmune response.

Longitudinal analysis of Mycobacterium tuberculosis 19-kDa antigen-specific T cells in patients with pulmonary tuberculosis: association with disease activity and cross-reactivity to a peptide from HIVenv gp120.

CD8(+) T cells play a central role in immune protection against infection with Mycobacterium tuberculosis. One of the target epitopes for anti-M. tuberculosis directed CD8(+) T cells is the HLA-A2-restricted 19-kDa lipoprotein peptide VLTDGNPPEV. T cell clones directed against this epitope recognized not only the nominal peptide ligand, but also a closely related peptide (VPTDPNPPEV) from the HIV envelope gp120 (HIV(env) gp120) protein characterized by IFN-gamma release. This cross-reactivity was confirmed in ex vivo in M. tuberculosis 19-kDa tetramer-sorted T cells from patients with tuberculosis and in HIVgp120 tetramer-reactive T cells sorted from HIV(+) patients. M. tuberculosis 19-kDa antigen-reactive T cells were present in HLA-A2(+) patients (10/10) with HIV infection with no evidence of M. tuberculosis infection, but they are absent in peripheral blood lymphocytes from healthy HLA-A2(+) individuals (10/10). M. tuberculosis 19-kDa antigen-reactive T cells were elevated in acute pulmonary tuberculosis, declined with response to therapy (7/10 patients) and resided in the terminally differentiated CD8(+) T cell subset. CD8(+) cross-reactive T cells recognizing HIV(env) or M. tuberculosis 19-kDa antigens may contribute to pathogenesis in individuals co-infected with both pathogens and may also present a marker for active tuberculosis.

Characterization of antigen-specific repertoire diversity following in vitro restimulation by a recombinant adenovirus expressing human cytomegalovirus pp65.

Human cytomegalovirus (HCMV) and adenovirus cause significant morbidity and mortality in immunocompromised hosts undergoing allogeneic stem cell transplantation. We have previously established a procedure for the generation of polyclonal CTL with specificity against adenovirus and HCMV using a recombinant adenovirus encoding the HCMV pp65 protein (RAdpp65). However, specific CTL expanded after in vitro culture steps were subjected to several in vitro restimulations and, depending on the protocol adopted, this could lead to a selection bias, compromising the clinical benefit. To determine which part of the memory repertoire is selected after in vitro restimulation, we have followed the specificity and clonal composition of pp65-peptide-specific CD8(+) T cells in HLA-A*201 individuals before and after repeated in vitro restimulation of cells with RAdpp65, combining HLA tetrameric complexes and immunoscope analysis. Tetramer staining showed that, after in vitro restimulation, up to 60% of CD8(+) T cells were virus-specific. Immunoscope analysis showed that the predominant TCRBV diversity of pp65-specific clones was conserved, demonstrating that the memory repertoire was preserved all along the procedure. Altogether, these results suggest that the use of RAdpp65 to induce CMV- and adenovirus-specific CTL maybe appropriate for immunotherapy.

Impact of antigen presentation on TCR modulation and cytokine release: implications for detection and sorting of antigen-specific CD8+ T cells using HLA-A2 wild-type or HLA-A2 mutant tetrameric complexes.

Soluble MHC class I molecules loaded with antigenic peptides are available either to detect and to enumerate or, alternatively, to sort and expand MHC class I-restricted and peptide-reactive T cells. A defined number of MHC class I/peptide complexes can now be implemented to measure T cell responses induced upon Ag-specific stimulation, including CD3/CD8/zeta-chain down-regulation, pattern, and quantity of cytokine secretion. As a paradigm, we analyzed the reactivity of a Melan-A/MART-1-specific and HLA-A2-restricted CD8(+) T cell clone to either soluble or solid-phase presented peptides, including the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or the peptide analog ELAGIGILTV presented either by the HLA-A2 wild-type (wt) or mutant (alanineright arrowvaline aa 245) MHC class I molecule, which reduces engagement of the CD8 molecule with the HLA-A2 heavy chain. Soluble MHC class I complexes were used as either monomeric or tetrameric complexes. Soluble monomeric MHC class I complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8 and TCR zeta-chain down-regulation, but did not induce measurable cytokine release. In general, differences pertaining to CD3/CD8/zeta-chain regulation and cytokine release, including IL-2, IFN-gamma, and GM-CSF, were associated with 1) the format of Ag presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide). These differences are to be considered if T cells are exposed to recombinant MHC class I Ags loaded with peptides implemented for detection, activation, or sorting of Ag-specific T cells.

Improved detection of melanoma antigen-specific T cells expressing low or high levels of CD8 by HLA-A2 tetramers presenting a Melan-A/Mart-1 peptide analogue.

MHC class I tetramers containing peptide epitopes are sensitive tools for detecting antigen-specific CD8(+) T-cell responses. We demonstrate here that binding of HLA-A2 tetramers to CD8(+) T cells specific for the melanoma-associated antigen Melan-A/MART-1 can be fine-tuned by altering either the bound peptide epitope or residues in the alpha 3 domain of HLA-A2, which is important for CD8 binding. Antigen-specific T cells expressing high levels of CD8 could be detected using HLA-A2 tetramers containing the peptide AAGIGILTV, an epitope which is naturally processed and presented from Melan-A/MART-1. In contrast, low CD8-expressing, antigen-specific T cells could be detected efficiently only by using a mutated HLA-A2 tetramer with an altered CD8 binding site or, less efficiently, using the wild-type HLA-A2 tetramer loaded with the peptide analogue ELAGIGILTV, which is superior in stimulating antigen-specific T-cell responses. Our results suggest ways to optimize the identification and expansion of antigen-specific T cells with different requirements for the costimulatory CD8 molecule in facilitating T-cell receptor binding to peptide variants.

Human CD8 co-receptor is strictly involved in MHC-peptide tetramer-TCR binding and T cell activation.

Although there has been extensive analysis on the capacity of MHC-peptide tetramers to bind antigen-specific TCR, there have been comparatively few studies regarding the role of the CD4 and CD8 co-receptors in binding and activation by these multimeric molecules. Here, we start from the observation that different antibodies against human CD8 exert opposite effects on MHC-peptide tetramer binding to the TCR: tetramer staining was enhanced by OKT8 antibody, while it was blocked with SK1 antibody. We used these different anti-CD8 antibodies to modulate CD8 function during tetramer staining of Melan-A/MART1-specific CTL clones. We show that CD8 action could be variably modulated during all the phases of interaction, indicating that CD8 participates in both the initial association of the TCR with MHC-peptide tetramers and the stability of this interaction. While the blocking effect of anti-CD8 antibodies was mostly exerted during the initial binding of the TCR with MHC-peptide tetramers, the enhancing effect was exerted by augmenting the duration of this interaction. Blocking anti-CD8 antibodies were also capable of preventing tetramer-mediated T cell activation. The possibility of variably affecting MHC-peptide tetramer binding and T cell activation using anti-CD8 antibodies confirms the critical role exerted by the CD8 co-receptor in this interaction and supports the notion that TCR engagement by MHC-peptide ligands typically involves CD8.