Large TCR diversity of virus-specific CD8 T cells provides the mechanistic basis for massive TCR renewal after antigen exposure.

Ex vivo analysis of virus-specific CD8 T cell populations by anchored PCR has shown that the CD8 TCR repertoire was less oligoclonal (seven to nine clonotypes per individual epitope) than previously thought. In the current study, TCR diversity was investigated by assessing both the overall TCR β-chain variable regions usage as well as the CDR3 regions in ex vivo-isolated CMV- and EBV-specific CD8 T cells from 27 healthy donors. The average number of clonotypes specific to most single viral epitopes comprised between 14 and 77. Changes in the CD8 TCR repertoire were also longitudinally assessed under conditions of HIV-1 chronic infection (i.e., in patients with suppressed virus replication and after treatment interruption and Ag re-exposure). The results showed that a large renewal (≤80%) of the TRB repertoire occurred after Ag re-exposure and was eventually associated with an increased T cell recognition functional avidity. These results demonstrate that the global CD8 TCR repertoire is much more diverse (≤9-fold) than previously estimated and provide the mechanistic basis for supporting massive repertoire renewal during chronic virus infection and Ag re-exposure.

Specific Silencing of the REST Target Genes in Insulin-Secreting Cells Uncovers Their Participation in Beta Cell Survival.

The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST) in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice), and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP-REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knock-down in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival.

Notch1 expression on T cells is not required for CD4+ T helper differentiation.

Notch1 proteins are involved in binary cell fate decisions. To determine the role of Notch1 in the differentiation of CD4(+) Th1 versus Th2 cells, we have compared T helper polarization in vitro in naive CD4(+) T cells isolated from mice in which the N1 gene is specifically inactivated in all mature T cells. Following activation, Notch1-deficient CD4(+) T cells transcribed and secreted IFN-gamma under Th1 conditions and IL-4 under Th2 conditions at levels similar to that of control CD4(+) T cells. These results show that Notch1 is dispensable for the development of Th1 and Th2 phenotypes in vitro. The requirement for Notch1 in Th1 differentiation in vivo was analyzed following inoculation of Leishmania major in mice with a T cell-specific inactivation of the Notch1 gene. Following infection, these mice controlled parasite growth at the site of infection and healed their lesions. The mice developed a protective Th1 immune response characterized by high levels of IFN-gamma mRNA and protein and low levels of IL-4 mRNA with no IL-4 protein in their lymph node cells. Taken together, these results indicate that Notch1 is not critically involved in CD4(+) T helper 1 differentiation and in resolution of lesions following infection with L. major.

Transgenic expression of Ly49A on T cells impairs a specific antitumor response.

Inhibitory MHC receptors determine the reactivity and specificity of NK cells. These receptors can also regulate T cells by modulating TCR-induced effector functions such as cytotoxicity, cytokine production, and proliferation. Here we have assessed the capacity of mouse T cells expressing the inhibitory MHC class I receptor Ly49A to respond to a well-defined tumor Ag in vivo using Ly49A transgenic mice. We find that the presence of Ly49A on the vast majority of lymphocytes prevents the development of a significant Ag-specific CD8+ T cell response and, consequently, the rejection of the tumor. Despite minor alterations in the TCR repertoire of CD8+ T cells in the transgenic lines, precursors of functional tumor-specific CD8+ T cells exist but could not be activated most likely due to a lack of appropriate CD4+ T cell help. Surprisingly, all of these effects are observed in the absence of a known ligand for the Ly49A receptor as defined by its ability to regulate NK cell function. Indeed, we found that the above effects on T cells may be based on a weak interaction of Ly49A with Kb or Db class I molecules. Thus, our data demonstrate that enforced expression of a Ly49A receptor on conventional T cells prevents a specific immune response in vivo and suggest that the functions of T and NK cells are differentially sensitive to the presence of inhibitory MHC class I receptors.

New generation vaccine induces effective melanoma-specific CD8+ T cells in the circulation but not in the tumor site.

Although increasing evidence suggests that CTL are important to fight the development of some cancers, the frequency of detectable tumor-specific T cells is low in cancer patients, and these cells have generally poor functional capacities, compared with virus-specific CD8(+) T cells. The generation with a vaccine of potent CTL responses against tumor Ags therefore remains a major challenge. In the present study, ex vivo analyses of Melan-A-specific CD8(+) T cells following vaccination with Melan-A peptide and CpG oligodeoxynucleotides revealed the successful induction in the circulation of effective melanoma-specific T cells, i.e., with phenotypic and functional characteristics similar to those of CTL specific for immunodominant viral Ags. Nonetheless, the eventual impact on tumor development in vaccinated melanoma donors remained limited. The comprehensive study of vaccinated patient metastasis shows that vaccine-driven tumor-infiltrating lymphocytes, although activated, still differed in functional capacities compared with blood counterparts. This coincided with a significant increase of FoxP3(+) regulatory T cell activity within the tumor. The consistent induction of effective tumor-specific CD8(+) T cells in the circulation with a vaccine represents a major achievement; however, clinical benefit may not be achieved unless the tumor environment can be altered to enable CD8(+) T cell efficacy.

Effect of vitamin D on EBV-specific CD8+T cells in patients with early multiple sclerosis

Introduction: Infection with Epstein-Barr Virus (EBV) and a lack invitamin D are emerging as the twomost significant environmental triggersof multiple sclerosis (MS). Sincewe and others have shown that CD8+T cells are important immune mediatorsof the inflammatory response inMS, we examined whether vitamin Ddirectly affects the CD8+ T cell response.We also explored if vitaminDmodulates the EBV-specific CD8+ Tcell response. Methods: PBMC of 10patients with early MS and 10 healthycontrols (HC) were stimulated eitherwith a pool of EBVimmunodominantpeptides or anti-CD3/anti-CD28 beads.Cytokine secretion was assessed witha Cytometric Beads Array (CBA),ELISA and intracellular cytokinestaining. To examine whether vitaminD could directly modulate CD8+ Tcell immune responses, we depletedCD4+ T cells using a negative selection.Results: We found that vitaminD-treated PBMC stimulated eitherwith the EBV peptide pool or anti-CD3/anti-CD28 beads adopted ananti-inflammatory profile: significantdecrease in IFN-and TNF secretion,contrasting with a significant increasein IL-5 and TGF-secretion. At baseline,but also after vitamin D stimulation,IL-5 was significantly less producedby stimulated CD8+ T cells ofearly MS than HC. Finally, using depletionof CD4+ T cells, we couldshow that vitaminDcan directlymodulateCD8+ T cells. Discussion: Ourdata suggest that vitaminDconfers ananti-inflammatory profile to CD8+ Tcells, without the help of CD4+ Tcells. Even if vitamin D has a significanteffect on CD8+ T cells of earlyMS patients, this "rescuing" effect isof smaller magnitude than in HC subjects.Finally, vitamin D does influencethe CD8+ T cell response toEBV in early MS patients, suggestingthat there is an interplay betweenthese two major environmental factorsof MS.

Establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on MPSS and array expression data.

INTRODUCTION: Diverse microarray and sequencing technologies have been widely used to characterise the molecular changes in malignant epithelial cells in breast cancers. Such gene expression studies to identify markers and targets in tumour cells are, however, compromised by the cellular heterogeneity of solid breast tumours and by the lack of appropriate counterparts representing normal breast epithelial cells. METHODS: Malignant neoplastic epithelial cells from primary breast cancers and luminal and myoepithelial cells isolated from normal human breast tissue were isolated by immunomagnetic separation methods. Pools of RNA from highly enriched preparations of these cell types were subjected to expression profiling using massively parallel signature sequencing (MPSS) and four different genome wide microarray platforms. Functional related transcripts of the differential tumour epithelial transcriptome were used for gene set enrichment analysis to identify enrichment of luminal and myoepithelial type genes. Clinical pathological validation of a small number of genes was performed on tissue microarrays. RESULTS: MPSS identified 6,553 differentially expressed genes between the pool of normal luminal cells and that of primary tumours substantially enriched for epithelial cells, of which 98% were represented and 60% were confirmed by microarray profiling. Significant expression level changes between these two samples detected only by microarray technology were shown by 4,149 transcripts, resulting in a combined differential tumour epithelial transcriptome of 8,051 genes. Microarray gene signatures identified a comprehensive list of 907 and 955 transcripts whose expression differed between luminal epithelial cells and myoepithelial cells, respectively. Functional annotation and gene set enrichment analysis highlighted a group of genes related to skeletal development that were associated with the myoepithelial/basal cells and upregulated in the tumour sample. One of the most highly overexpressed genes in this category, that encoding periostin, was analysed immunohistochemically on breast cancer tissue microarrays and its expression in neoplastic cells correlated with poor outcome in a cohort of poor prognosis estrogen receptor-positive tumours. CONCLUSION: Using highly enriched cell populations in combination with multiplatform gene expression profiling studies, a comprehensive analysis of molecular changes between the normal and malignant breast tissue was established. This study provides a basis for the identification of novel and potentially important targets for diagnosis, prognosis and therapy in breast cancer.

Antibody-dependent cell-mediated cytolysis of human colon carcinoma cells induced by specific antisera against carcinoembryonic antigen.

Antibody-dependent lymphocyte cytotoxicity against human colon carcinoma cells grown in vitro was demonstrated with rabbit anti-carcinoembryonic antigen (CEA) antisera and normal human lymphocytes. The same antisera produced no tumor cell lysis in a complement-dependent cytotoxicity test. The specificity of the reaction was demonstrated by the inhibition of antibody-dependent lymphocyte cytotoxicity after the addition of increasing amounts of purified CEA to the antiserum and by the fact that only tumor cell lines expressing CEA on their surface were lysed. Antibody-dependent lymphocyte cytotoxicity was also observed against two colon carcinoma cell lines that expressed Blood Group A antigen, using a human serum containing anti-Blood Group A antibodies of the immunoglobulin G class. This reaction was specifically inhibited by absorption with Blood Group A red cells, whereas the anti-CEA-dependent cytotoxicity was not inhibited by absorption with red cells of different blood groups.

Lymphocyte specificity to protein antigens. V. Conformational dependence of activation of cytochrome c-specific T cells.

Variations in the immunogenic and antigenic properties of native and denatured forms of cytochrome c were observed depending on the strain of mouse tested. In C57BL/6 and (C57BL/6 X DBA/2)F1 (BDF1) mice, priming with either native or denatured cytochrome c (apocytochrome c) gave rise to T cell blasts responding in a similar fashion to the two forms of the antigen and to different peptides derived from CNBr cleavage of the protein when tested for proliferation in the presence of C57BL/6 or BDF1 accessory cells. A different pattern of proliferation was observed when apocytochrome c-specific DBA/2 or BDF1 T cell blasts were tested with DBA/2 accessory cells. In this case, no response was obtained to heme peptide 1-65. This was not due to an inability of DBA/2 macrophages to process and present heme peptide 1-65, as they were able to present this antigen to native cytochrome c-specific BDF1 T cell blasts. Thus, it seems that different sets of clones are generated upon priming BDF1 mice with denatured cytochrome c which are able to recognize different sets of peptides depending on the nature of the accessory cells. The results obtained are consistent with the hypothesis that degradation and presentation of native and denatured cytochrome c by macrophages is dependent on the three-dimensional conformation of the protein.

T helper 1 (Th1) and Th2 characteristics start to develop during T cell priming and are associated with an immediate ability to induce immunoglobulin class switching.

The respective production of specific immunoglobulin (Ig)G2a or IgG1 within 5 d of primary immunization with Swiss type mouse mammary tumor virus [MMTV(SW)] or haptenated protein provides a model for the development of T helper 1 (Th1) and Th2 responses. The antibody-producing cells arise from cognate T cell B cell interaction, revealed by the respective induction of Cgamma2a and Cgamma1 switch transcript production, on the third day after immunization. T cell proliferation and upregulation of mRNA for interferon gamma in response to MMTV(SW) and interleukin 4 in response to haptenated protein also starts during this day. It follows that there is minimal delay in these responses between T cell priming and the onset of cognate interaction between T and B cells leading to class switching and exponential growth. The Th1 or Th2 profile is at least partially established at the time of the first cognate T cell interaction with B cells in the T zone. The addition of killed Bordetella pertussis to the hapten-protein induces nonhapten-specific IgG2a and IgG1 plasma cells, whereas the anti-hapten response continues to be IgG1 dominated. This indicates that a Th2 response to hapten-protein can proceed in a node where there is substantial Th1 activity.

Ex vivo characterization of allo-MHC-restricted T cells specific for a single MHC-peptide complex.

Alloreactive T cells are thought to be a potentially rich source of high-avidity T cells with therapeutic potential since tolerance to self-Ags is restricted to self-MHC recognition. Given the particularly high frequency of alloreactive T cells in the peripheral immune system, we used numerous MHC class I multimers to directly visualize and isolate viral and tumor Ag-specific alloreactive CD8 T cells. In fact, all but one specificities screened were undetectable in ex vivo labeling. In this study, we report the occurrence of CD8 T cells specifically labeled with allo-HLA-A*0201/Melan-A/MART-1(26-35) multimers at frequencies that are in the range of 10(-4) CD8 T cells and are thus detectable ex vivo by flow cytometry. We report the thymic generation and shaping of tumor Ag-specific, alloreactive T cells as well as their fate once seeded in the periphery. We show that these cells resemble their counterparts in HLA-A*0201-positive individuals, based on their structural and functional attributes.

Long-term persistence of HIV-1 vaccine-induced CD4+CD45RA-CD62L-CCR7- memory T-helper cells.

OBJECTIVE: To determine in chimpanzees if candidate HIV-1 subunit protein vaccines were capable of eliciting long-lasting T-cell memory responses in the absence of viral infection, and to determine the specific characteristics of these responses. DESIGN: A longitudinal study of cell-mediated immune responses induced in three chimpanzees following immunization with subunit envelope glycoproteins of either HIV-1 or herpes simplex virus (HSV)-2. Following these pre-clinical observations, four human volunteers who had been immunized 7 years previously with the same HIV-1 vaccine candidate donated blood for assessment of immune responses. METHODS: Responses were monitored by protein and peptide based ELISpot assays, lymphocyte proliferation, and intracellular cytokine staining. Humoral responses were assessed by enzyme-linked immunosorbent assay and virus neutralization assays. RESULTS: Although antigen (Ag)-specific CD4 T-cell responses persisted for at least 5 years in chimpanzees, CD8 T-cell responses were discordant and declined within 2 years. Detailed cellular analyses revealed that strong Th1 in addition to Th2 type responses were induced by AS2/gp120 and persisted, whereas CD8 T-cell memory declined in peripheral blood. The specificity of both Th and cytotoxic T-lymphocyte responses revealed that the majority of responses were directed to conserved epitopes. The remarkable persistence of Ag-specific CD4 T-cell memory was characterized as a population of the CD45RA-CD62L-CCR7- "effector phenotype" producing the cytokines IFNgamma, IL-2 and IL-4 upon epitope-specific recognition. Importantly, results in chimpanzees were confirmed in peripheral blood of one of four human volunteers studied more than 7 years after immunization. CONCLUSION: These studies demonstrate that epitope-specific Th1 and Th2 cytokine-dependent Th responses can be induced and maintained for longer than 5 years by immunization with subunit proteins of HIV-1.

Exhaustion of tumor-specific CD8⁺ T cells in metastases from melanoma patients.

In chronic viral infections, CD8⁺ T cells become functionally deficient and display multiple molecular alterations. In contrast, only little is known of self- and tumor-specific CD8⁺ T cells from mice and humans. Here we determined molecular profiles of tumor-specific CD8⁺ T cells from melanoma patients. In peripheral blood from patients vaccinated with CpG and the melanoma antigen Melan-A/MART-1 peptide, we found functional effector T cell populations, with only small but nevertheless significant differences in T cells specific for persistent herpesviruses (EBV and CMV). In contrast, Melan-A/MART-1-specific T cells isolated from metastases from patients with melanoma expressed a large variety of genes associated with T cell exhaustion. The identified exhaustion profile revealed extended molecular alterations. Our data demonstrate a remarkable coexistence of effector cells in circulation and exhausted cells in the tumor environment. Functional T cell impairment is mediated by inhibitory receptors and further molecular pathways, which represent potential targets for cancer therapy.

HLA-B7-Restricted EBV-Specific CD8+ T Cells Are Dysregulated in Multiple Sclerosis.

It was hypothesized that the EBV-specific CD8(+) T cell response may be dysregulated in multiple sclerosis (MS) patients, possibly leading to a suboptimal control of this virus. To examine the CD8(+) T cell response in greater detail, we analyzed the HLA-A2-, HLA-B7-, and HLA-B8-restricted EBV- and CMV-specific CD8(+) T cell responses in a high number of MS patients and control subjects using tetramers. Content in cytolytic granules, as well as cytotoxic activity, of EBV- and CMV-specific CD8(+) T cells was assessed. We found that MS patients had a lower or a higher prevalence of HLA-A2 and HLA-B7, respectively. Using HLA class I tetramers in HLA-B7(+) MS patients, there was a higher prevalence of MS patients with HLA-B*0702/EBV(RPP)-specific CD8(+) T cells ex vivo. However, the magnitude of the HLA-B*0702/EBV(RPP)-specific and HLA-B*0702/CMV(TPR)-specific CD8(+) T cell response (i.e., the percentage of tetramer(+) CD8(+) T cells in a study subject harboring CD8(+) T cells specific for the given epitope) was lower in MS patients. No differences were found using other tetramers. After stimulation with the HLA-B*0702/EBV(RPP) peptide, the production of IL-2, perforin, and granzyme B and the cytotoxicity of HLA-B*0702/EBV(RPP)-specific CD8(+) T cells were decreased. Altogether, our findings suggest that the HLA-B*0702-restricted viral (in particular the EBV one)-specific CD8(+) T cell response is dysregulated in MS patients. This observation is particularly interesting knowing that the HLA-B7 allele is more frequently expressed in MS patients and considering that EBV is associated with MS.