A novel IFN-gamma regulated human melanoma associated antigen gp33-38 defined by monoclonal antibody Me14/D12. I. Identification and immunochemical characterization.

A novel melanoma-associated differentiation Ag whose surface expression can be enhanced or induced by IFN-gamma was identified by mAb Me14/D12. Testing of numerous tumor cell lines and tumor tissue sections showed that Me14/D12-defined Ag was present not only on melanoma but also on other tumor lines of neuroectodermal origin such as gliomas and neuroblastomas and on some lymphoblastic B cell lines, on monocytes and macrophages. Immunoprecipitation by mAb Me14/D12 of lysates from [35S]methionine-labeled melanoma cells analyzed by SDS-PAGE revealed two polypeptide chains of 33 and 38 KDa, both under reducing and nonreducing conditions. Cross-linking experiments indicated that the two chains were present at the cell surface as a dimeric structure. Two-dimensional gel electrophoresis showed that the two chains of 33 and 38 KDa had isoelectric points of 6.2 and 5.7, respectively. Treatment of the melanoma cells with tunicamycin, an inhibitor of N-linked glycosylation, resulted in a reduction of the Mr from 33 to 24 KDa and from 38 to 26 KDa. Peptide maps obtained after Staphylococcus aureus V8 protease digestion showed no shared peptides between the two chains. Although biochemical data indicate that Me14/D12 molecules do not correspond to any known MHC class II Ag, their dimeric structure, tissue distribution, and regulation of IFN-gamma suggest that they could represent a new member of the MHC class II family.

Human melanoma-specific CD8(+) T-cells from metastases are capable of antigen-specific degranulation and cytolysis directly ex vivo.

The relatively low frequencies of tumor Ag-specific T-cells in PBMC and metastases from cancer patients have long precluded the analysis of their direct ex vivo cytolytic capacity. Using a new composite technique that works well with low cell numbers, we aimed at determining the functional competence of melanoma-specific CD8(+) T-cells. A multiparameter flow cytometry based technique was applied to assess the cytolytic function, degranulation and IFNγ production by tumor Ag-specific CD8(+) T-cells from PBMC and tumor-infiltrated lymph nodes (TILN) of melanoma patients. We found strong cytotoxicity by T-cells not only when they were isolated from PBMC but also from TILN. Cytotoxicity was observed against peptide-pulsed target cells and melanoma cells presenting the naturally processed endogenous antigen. However, unlike their PBMC-derived counterparts, T-cells from TILN produced only minimal amounts of IFNγ, while exhibiting similar levels of degranulation, revealing a critical functional dichotomy in metastatic lesions. Our finding of partial functional impairment fits well with the current knowledge that T-cells from cancer metastases are so-called exhausted, a state of T-cell hyporesponsiveness also found in chronic viral infections. The identification of responsible mechanisms in the tumor microenvironment is important for improving cancer therapies.

Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

Immunogenicity of a long 20-mer NY-ESO-1f peptide vaccine was evaluated in a lung cancer patient TK-f01, immunized with the peptide with Picibanil OK-432 and Montanide ISA-51. We showed that internalization of the peptide was necessary to present CD8 T-cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T-cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01-restricted CD8 T-cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02-restricted CD8 T-cell clones were defined and peptide/HLA tetramers were produced. NY-ESO-1 91-101 on A*24:02, NY-ESO-1 92-102 on B*35:01, NY-ESO-1 96-104 on B*52:01 and NY-ESO-1 96-104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T-cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20-mer NY-ESO-1f peptide harboring multiple CD8 T-cell epitopes as an NY-ESO-1 vaccine. Characterization of CD8 T-cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T-cell responses comprised the dominant response.

Different antigen-presenting cells differ in their capacity to induce lymphokine production and proliferation of an apo-cytochrome c-specific T cell clone.

The activation of an apo-cytochrome c-specific T cell clone was found to differ, depending on the antigen-presenting cell population. Whereas total syngeneic spleen cells and bone marrow macrophages could be shown to trigger proliferation, IL 2, and MAF production by the T cell clone, a B cell lymphoma only induced MAF secretion. Further studies demonstrated that this effect was not due to a different antigen processing by the B lymphoma or to limiting amounts of Ia and antigen molecules on the B lymphoma cell surface. The dissociation of induction of MAF production from IL-2 production/proliferation found with the different antigen-presenting cells indicates strongly that molecules other than Ia and antigen may be required for the complete functional activation of antigen-specific T cell clones.

On stand by: host genetics of HIV control.

The impact of host genetic variation on determining the differential outcomes after HIV infection has been studied by two approaches: targeting of candidate genes and genome-wide association studies (GWASs). The overlap in genetic variants that has been identified by these two means has essentially been restricted to variants near to the human leukocyte antigen (HLA) class I genes, although variation in the CCR5 locus, which was first shown to have an effect on HIV outcomes using the candidate gene approach, does reach significance genome-wide when very large samples sizes (i.e. thousands) are used in GWAS. Overall, many of the variants identified by the candidate gene approach are likely to be spurious, as no additional variants apart from a novel variant near the HLA-C gene have been consistently identified by GWAS. Variants with low frequency and/or low impact on HIV outcomes are likely to exist in the genome and there could be many of them, but these are not identifiable, given current GWAS sample sizes. Several loci centrally involved in the immune response, including the immunoglobulin genes, T-cell receptor loci, or leukocyte receptor complex, are either poorly covered on the GWAS chips or difficult to interpret due to their repetitive nature and/or the presence of insertion/deletion polymorphisms in the region. These loci warrant further interrogation, but genetic characterization of these regions across a range of individuals will first be required. Finally, synergistic interactions between loci may affect outcome after infection, as suggested by associations of specific, functionally relevant HLA and killer cell immunoglobulin-like receptor variants with HIV disease outcomes, and these require further consideration as well.

T-cell hybridoma specific for a cytochrome c peptide: specific antigen binding and interleukin 2 production.

T-cell hybridomas were obtained after fusion of BW 5147 thymoma and long-term cultured T cells specific for cytochrome c peptide 66-80 derivatized with a 2,4-dinitroaminophenyl (DNAP) group. The resulting hybridomas were selected for their capacity to specifically bind to soluble radiolabeled peptide antigen. One T-cell hybrid was positive for antigen binding. This hybrid T cell exhibits surface phenotypic markers of the parent antigen-specific T cells. The binding could be inhibited either by an excess of unlabeled homologous antigen or by cytochrome c peptide 11-25 derivatized with a 2-nitrophenylsulfenyl group. Several other peptide antigens tested failed to inhibit binding of the radioactive peptide. This suggests that a specific amino acid sequence, modified by a DNAP group, is the antigenic structure recognized by the putative T-cell receptor. In addition, direct interaction of DNAP-66-80 peptide with the hybridoma cell line induced production of the T-cell growth factor interleukin 2. Furthermore, supernatants derived from syngeneic macrophages pulsed with the relevant peptide also induced the antigen-specific hybridoma to produce interleukin 2.

Requirement for CARMA1 in antigen receptor-induced NF-kappa B activation and lymphocyte proliferation.

Ligation of antigen receptors (TCR, BCR) on T and B lymphocytes leads to the activation of new transcriptional programs and cell cycle progression. Antigen receptor-mediated activation of NF-kappa B, required for proliferation of B and T cells, is disrupted in T cells lacking PKC theta and in B and T cells lacking Bcl10, a caspase recruitment domain (CARD)-containing adaptor protein. CARMA1 (also called CARD11 and Bimp3), the only lymphocyte-specific member in a family of membrane-associated guanylate kinase (MAGUK) scaffolding proteins that interact with Bcl10 by way of CARD-CARD interactions, is required for TCR-induced NF-kappa B activation in Jurkat T lymphoma cells. Here we show that T cells from mice lacking CARMA1 expression were defective in recruitment of Bcl10 to clustered TCR complexes and lipid rafts, in activation of NF-kappa B, and in induction of IL-2 production. Development of CD5(+) peritoneal B cells was disrupted in these mice, as was B cell proliferation in response to both BCR and CD40 ligation. Serum immunoglobulin levels were also markedly reduced in the mutant mice. Together, these results show that CARMA1 has a central role in antigen receptor signaling that results in activation and proliferation of both B and T lymphocytes.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications.

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Immune response to HIV.

PURPOSE OF REVIEW: Major advances have been made in the delineation of HIV-specific immune response and in the mechanisms of virus escape. The kinetics of the immunological and virological events occurring during primary HIV infection indicate that the establishment of the latent HIV reservoir, the major obstacle to HIV eradication likely occurs during the very early stages of primary infection, that is, the 'eclipse phase', prior to the development of the HIV-specific immune response which has limited efficacy in the control of the early events of infection. Therefore, the window of opportunity to develop effective interventions either to clear HIV during primary infection or to prevent rebound of HIV in patients successfully treated who stop antiretroviral therapy is very narrow. RECENT FINDINGS: Genetic factors most strongly associated with nonprogressive infection are human leukocyte antigen (HLA) class I alleles and particularly HLA-B5701. CD4 and CD8 T-cell responses with polyfunctional profile are associated with nonprogressive infection. Broader neutralizing antibodies are detected 3-4 years after infection, generated only in 20% of individuals but show no efficacy in the control of HIV replication. SUMMARY: In the present review, we shall discuss the different components of the HIV-specific immune response elicited by the infection, the kinetics of these responses during primary infection and the changes following transition to the chronic phase of infection, and the functional profile of 'effective' versus 'noneffective' HIV-specific immune responses.

Adjuvants that improve the ratio of antigen-specific effector to regulatory T cells enhance tumor immunity.

Antitumor immunity is strongly influenced by the balance of tumor antigen-specific effector and regulatory T cells. However, the impact that vaccine adjuvants have in regulating the balance of antigen-specific T cell populations is not well understood. We found that antigen-specific T regulatory cells (Treg) were induced following subcutaneous vaccination with either OVA or melanoma-derived peptides, with a restricted expansion of effector T cells. Addition of the adjuvants CpG-ODN or Poly(I:C) preferentially amplified effector T cells over Tregs, dramatically increasing the antigen-specific T effector:Treg ratios and inducing polyfunctional effector cells. In contrast, two other adjuvants, imiquimod and Quil A saponin, favored an expansion of antigen-specific Tregs and failed to increase effector T cell:Treg ratios. Following therapeutic vaccination of tumor-bearing mice, high ratios of tumor-specific effector T cells:Tregs in draining lymph nodes were associated with enhanced CD8+ T cell infiltration at the tumor site and a durable rejection of tumors. Vaccine formulations of peptide+CpG-ODN or Poly(I:C) induced selective production of pro-inflammatory Type I cytokines early after vaccination. This environment promoted CD8+ and CD4+ effector T cell expansion over that of antigen-specific Tregs, tipping the effector T cell to Treg balance to favor effector cells. Our findings advance understanding of the influence of different adjuvants on T cell populations, facilitating the rational design of more effective cancer vaccines.

Systems analysis of MVA-C induced immune response reveals its significance as a vaccine candidate against HIV/AIDS of clade C.

Based on the partial efficacy of the HIV/AIDS Thai trial (RV144) with a canarypox vector prime and protein boost, attenuated poxvirus recombinants expressing HIV-1 antigens are increasingly sought as vaccine candidates against HIV/AIDS. Here we describe using systems analysis the biological and immunological characteristics of the attenuated vaccinia virus Ankara strain expressing the HIV-1 antigens Env/Gag-Pol-Nef of HIV-1 of clade C (referred as MVA-C). MVA-C infection of human monocyte derived dendritic cells (moDCs) induced the expression of HIV-1 antigens at high levels from 2 to 8 hpi and triggered moDCs maturation as revealed by enhanced expression of HLA-DR, CD86, CD40, HLA-A2, and CD80 molecules. Infection ex vivo of purified mDC and pDC with MVA-C induced the expression of immunoregulatory pathways associated with antiviral responses, antigen presentation, T cell and B cell responses. Similarly, human whole blood or primary macrophages infected with MVA-C express high levels of proinflammatory cytokines and chemokines involved with T cell activation. The vector MVA-C has the ability to cross-present antigens to HIV-specific CD8 T cells in vitro and to increase CD8 T cell proliferation in a dose-dependent manner. The immunogenic profiling in mice after DNA-C prime/MVA-C boost combination revealed activation of HIV-1-specific CD4 and CD8 T cell memory responses that are polyfunctional and with effector memory phenotype. Env-specific IgG binding antibodies were also produced in animals receiving DNA-C prime/MVA-C boost. Our systems analysis of profiling immune response to MVA-C infection highlights the potential benefit of MVA-C as vaccine candidate against HIV/AIDS for clade C, the prevalent subtype virus in the most affected areas of the world.

A novel population of human melanoma-specific CD8 T cells recognizes Melan-AMART-1 immunodominant nonapeptide but not the corresponding decapeptide.

HLA-A2-restricted cytolytic T cells specific for the immunodominant human tumor Ag Melan-A(MART-1) can kill most HLA-matched melanoma cells, through recognition of two naturally occurring antigenic variants, i.e., Melan-A nonamer AAGIGILTV and decamer EAAGIGILTV peptides. Several previous studies have suggested a high degree of TCR cross-reactivity to the two peptides. In this study, we describe for the first time that some T cell clones are exclusively nonamer specific, because they are not labeled by A2/decamer-tetramers and do not recognize the decamer when presented endogenously. Functional assays with peptides gave misleading results, possibly because decamers were cleaved by exopeptidases. Interestingly, nonapeptide-specific T cell clones were rarely Valpha2.1 positive (only 1 of 19 clones), in contrast to the known strong bias for Valpha2.1-positive TCRs found in decamer-specific clones (59 of 69 clones). Molecular modeling revealed that nonapeptide-specific TCRs formed unfavorable interactions with the decapeptide, whereas decapeptide-specific TCRs productively created a hydrogen bond between CDR1alpha and glutamic acid (E) of the decapeptide. Ex vivo analysis of T cells from melanoma metastases demonstrated that both nonamer and decamer-specific T cells were enriched to substantial frequencies in vivo, and representative clones showed efficient tumor cell recognition and killing. We conclude that the two peptides should be regarded as distinct epitopes when analyzing tumor immunity and developing immunotherapy against melanoma.

Antigen binding to secretory immunoglobulin A results in decreased sensitivity to intestinal proteases and increased binding to cellular Fc receptors.

In intestinal secretions, secretory IgA (SIgA) plays an important sentinel and protective role in the recognition and clearance of enteric pathogens. In addition to serving as a first line of defense, SIgA and SIgA x antigen immune complexes are selectively transported across Peyer's patches to underlying dendritic cells in the mucosa-associated lymphoid tissue, contributing to immune surveillance and immunomodulation. To explain the unexpected transport of immune complexes in face of the large excess of free SIgA in secretions, we postulated that SIgA experiences structural modifications upon antigen binding. To address this issue, we associated specific polymeric IgA and SIgA with antigens of various sizes and complexity (protein toxin, virus, bacterium). Compared with free antibody, we found modified sensitivity of the three antigens assayed after exposure to proteases from intestinal washes. Antigen binding further impacted on the immunoreactivity toward polyclonal antisera specific for the heavy and light chains of the antibody, as a function of the antigen size. These conformational changes promoted binding of the SIgA-based immune complex compared with the free antibody to cellular receptors (Fc alphaRI and polymeric immunoglobulin receptor) expressed on the surface of premyelocytic and epithelial cell lines. These data reveal that antigen recognition by SIgA triggers structural changes that confer to the antibody enhanced receptor binding properties. This identifies immune complexes as particular structural entities integrating the presence of bound antigens and adds to the known function of immune exclusion and mucus anchoring by SIgA.

Protection against cutaneous leishmaniasis in outbred vervet monkeys using a recombinant histone H1 antigen.

Infection with Leishmania major parasites results in the development of cutaneous ulcerative lesions on the skin. We investigated the protective potential of a single, recombinant histone H1 antigen against cutaneous leishmaniasis in an outbred population of vervet monkeys, using Montanide adjuvant. Protection was assessed by challenging the animals with a mixture of vector sand fly salivary-gland lysate and a low dose of in vitro-derived parasites, thus more closely mimicking natural infection induced by L. major. The course of infection in immunized monkeys was compared with that of animals that had healed from a primary infection and were immune. The monkeys immunized with recombinant histone H1 showed a reduced development of lesion size, compared with controls. Our study therefore illustrates the potential use of histone H1 as a vaccine candidate against cutaneous leishmaniasis in humans.