Brigitte Birebent

Suppressive properties of human CD4+CD25+ regulatory T cells are dependent on CTLA-4 expression.

It has been demonstrated that T cells with regulatory properties are present within the peripheral blood CD4+CD25+ T cell compartment. Here, we describe an original method to purify human CD4+CD25+CD152+ T lymphocytes as living cells by forcing the exportation of CTLA‐4 molecules stored in intracellular vesicules at the cell surface. By doing so, we demonstrate that CD4+CD25+ T cells contain a smaller and more homogeneous population enriched in cells with in vitro regulatory activity. Moreover, we show that this enrichment in regulatory T cells is associated with an increased expression of Foxp3 and that CD4+CD25+CD152+ T lymphocytes display a much stronger suppressive activity in controlling in vitro proliferation of alloantigen‐specific T cells than CD4+CD25+CD152– T lymphocytes purified in parallel. Lastly, by purifying such cells expressing CTLA‐4, we demonstrate that indeed CTLA‐4 is involved in CD4+CD25+CD152+ T cell regulatory activity, while suppressive cytokines are not.

TAP2 GENE POLYMORPHISM CONTRIBUTES TO GENETIC SUSCEPTIBILITY TO MULTIPLE SCLEROSIS

MS is an autoimmune demyelinating disease that has been known to be associated with the HLA-DRB1*1501-DQA1*0102-DQB1*0602 haplotype. TAP1 and TAP2, two genes encoded within the MHC class II region between HLA-DP and -DQ loci, display genetic variability and are involved in the transport of antigenic peptides from the cytoplasm to the endoplasmic reticulum. Comparison of 116 MS patients with Caucasoid controls did not reveal any significant correlation between the previously described alleles of the TAP1 and TAP2 genes and MS. We report here an additional TAP2 dimorphism at codon 386, called I and J, corresponding to a silent mutation. An increased frequency of the J variant was observed in the patient population. The J mutation was not found in linkage disequilibrium with the HLA-DRB1*1501 allele and can be considered an additional genetic susceptibility marker of the disease.

Genetic interaction of CTLA-4 with HLA-DR15 in multiple sclerosis patients

Multiple sclerosis is a chronic inflammatory disease of the central nervous system with a genetic component. Until now, the more consistent association with the disease is found with the major histocompatibility complex, especially HLA‐DRB1*1501‐DQB1*0602 haplotype. In this report, we demonstrate the interaction of Cytotoxic T Lymphocyte‐associated antigen 4 (CTLA‐4 [CD152]) gene with DRB1*15 haplotype in multiple sclerosis genetic susceptibility. Our data were obtained from two European independent family‐based studies including 610 multiple sclerosis family trios. Ann Neurol 2003;54:119–122

Soluble human leucocyte antigen‐G molecules in peripheral blood haematopoietic stem cell transplantation: a specific role to prevent acute graft‐versus‐host disease and a link with regulatory T cells

Haematopoietic stem cell transplantation is often complicated by the life‐threatening graft‐versus‐host disease (GVHD) which consists of an allogeneic reaction of the graft cells against the host organs. The aim of this study was to investigate the putative involvement of soluble human leucocyte antigen (sHLA) class I molecules, and particularly sHLA‐G molecules, in the occurrence and/or prevention of acute GVHD (aGVHD) in allogeneic peripheral blood stem cell (PSC) transplantation. Whole sHLA class I molecules seem to be involved in aGVHD pathogenesis because detection of a high concentration of these molecules in the first month post allograft is correlated with aGVHD occurrence. Conversely, a high level of sHLA‐G molecules before and after allograft could indicate good prognosis in PSC allograft transplantation. sHLA‐G molecules seem to be involved in aGVHD prevention, not only because they are enriched in plasma of patients without aGVHD, but also because: (i) a positive correlation has been found between sHLA‐G level and CD4+ CD25+ CD152+ natural regulatory T cell (Treg) frequency in the blood of transplanted patients; and (ii) the presence of CD4+ CD25+ CD152+ natural Treg is correlated with increased sHLA‐G expression in in vitro mixed leucocyte reaction cultures. Altogether, these results support the immunomodulatory function of sHLA‐G molecules that might create a regulatory network together with the natural Treg to foster the induction of a tolerogenic environment and improve PSC transplantation favourable outcome.

Special Section:Future Trendsin Vaccination: Advances in cancer vaccine development

Traditionally, cancer vaccines have used whole tumour cells administered in adjuvant or infected with viruses to increase the immunogenicity of the cells. With the identification of tumour-associated and tumour-specific antigens (TAA, TSA), antigen and epitope-specific vaccines have been designed. Compared to tumour cell vaccines, antigen and epitope vaccines are more specific and easier to produce in large quantities but may display lower immunogenicity and lead to the in vivo selection of antigen or epitope-negative escape tumour variant cells. The optimal vaccine will elicit both humoral and cellular immunity in the patients as both parameters have been positively correlated with the induction of beneficial clinical responses. The choice of adjuvant, costimulation and delivery mode greatly determines the outcome of vaccinations and may favour the induction of T-cell responses of T helper (Th)1, Th2, or both Thl and Th2 types. Animal models of TAA vaccines must take into account the normal tissue expres...

Anti-idiotypic antibody and recombinant antigen vaccines in colorectal cancer patients

The colorectal carcinoma (CRC)-associated GA733 antigen (also known as CO17-1A, KS1-4, KSA or EpCAM) has been the target of a phase II/III randomized trial of passive immunotherapy with monoclonal antibody CO17-1A and phase I active immunotherapy trials with polyclonal anti-idiotypic antibodies mimicking the CO17-1A or GA733 epitope on the antigen. The CO17-1A antigen was molecularly cloned and the extracellular domain expressed in baculovirus (BV) GA733-2E. Whereas, anti-idiotypic antibody mimics a single epitope on the antigen, BV GA733-2E expresses multiple potentially immunogenic epitopes. In animals, the immunogenicity of BV GA733-2E in aluminum hydroxide was superior to that of anti-idiotype in the same adjuvant. Here, we compared the immunogenicity of anti-idiotypic antibody and GA733-2E antigen in CRC patients. These studies indicate that the antigen is superior to the anti-idiotype antibody in inducing humoral and cellular immunity in CRC patients.

Immune Responses of Breast Cancer Patients to Mutated Epidermal Growth Factor Receptor (EGF-RvIII, ΔEGF-R, and de2–7 EGF-R)

Mutated epidermal growth factor receptor (EGF-RvIII, ΔEGF-R, and de2–7 EGF-R) is the result of an 801-bp deletion within the extracellular domain of wild-type EGF-R and is expressed by breast carcinomas, but not by normal breast tissues. EGF-RvIII is expressed both on the surface and in the cytoplasm of tumor cells. Thus, EGF-RvIII is a potential tumor-specific target for both Abs and T cells. However, it is not known whether breast cancer patients can raise immune responses to EGF-RvIII expressed by their tumors. The demonstration of EGF-RvIII-specific immune responses in patients would suggest that immunization of patients with EGF-RvIII vaccines is feasible, because these vaccines may boost a pre-existing immune response. We have evaluated humoral and cellular immune responses to EGF-RvIII in 16 breast cancer patients and three healthy donors. Seven of 16 patients developed EGF-RvIII-specific Abs that bound to isolated EGF-RvIII protein or the protein expressed by EGF-RvIII-transfected mouse fibroblasts. The Abs that bound to EGF-RvIII did not bind to wild-type EGF-R, and anti-EGF-RvIII Abs were not found in the sera of healthy donors. Three patients had EGF-RvIII peptide-specific lymphoproliferative responses, and two of these patients also had humoral immune responses. Humoral and cellular immune responses correlated with EGF-RvIII expression by patients’ tumors in most cases. These studies demonstrate that breast cancer patients specifically recognize EGF-RvIII with an overall immune response rate of 50%, suggesting that patients may benefit from vaccination against EGF-RvIII, boosting pre-existing immune responses.

Induction of cellular immunity by anti-idiotypic antibodies mimicking GD2 ganglioside

Abstract. Gangliosides are potentially useful targets for tumor destruction by antibodies. However, the role of gangliosides in T cell-mediated immunity to tumors is unclear. We produced three murine monoclonal anti-idiotypic antibodies (Ab2) against a monoclonal antibody (Ab1) that binds strongly to melanoma-associated GD2 ganglioside and weakly to GD3 ganglioside. All three Ab2 induced anti-anti-idiotypic antibodies (Ab3) with Ab1-like binding specificity to tumor cells and antigen in rabbits. The Ab3 specifically bound to GD2+ tumor cells and isolated GD2, and shared idiotopes with the Ab1. Two of the three Ab2 induced GD2-specific delayed-type hypersensitivity responses in BALB/c and C57BL/6 mice, but not in C57BL/6/CD4–/– mice. Peripheral blood mononuclear cells (PBMC) from a melanoma patient proliferated specifically in response to in vitro stimulation with Ab2. Proliferation was accompanied by Th1-type cytokine production. Our studies demonstrate the induction of ganglioside-specific T cell-dependent immunity by Ab2 in mice. These T cells showed specific reactivity to ganglioside expressed by tumor cells.

Colorectal Cancer Vaccines: Antiidiotypic Antibody, Recombinant Protein, and Viral Vector

Abstract: The colorectal cancer antigen GA733 (also termed CO17‐1A, KSI‐4, Ep‐CAM, KSA) has proved to be a useful target in passive immunotherapy with monoclonal antibody and in active immunotherapy with antiidiotypic antibodies in cancer patients. The GA733 antigen was molecularly cloned and expressed in baculovirus (BV), adenovirus (AV), and vaccinia virus (VV). Recombinant BV‐, VV‐, and AV‐GA733 induced antigen‐specific cytotoxic antibodies and proliferative and delayed‐type hypersensitive lymphocytes. However, only the AV recombinant induced antigen‐specific cytolytic T lymphocytes and regression of established tumors. Cured mice were protected against challenge with antigen‐negative tumors, indicating antigen spreading of immune responses. In a model of active immunotherapy against the murine homologue of the human GA733 antigen, murine epithelial glycoprotein (mEGP), BV‐derived mEGP protein in various adjuvants did not protect mice against a challenge with mEGP‐positive tumors. AV mEGP, only when combined with interleukin‐2, significantly inhibited growth of established mEGP‐positive tumors. This is in contrast to the same vaccine expressing the human antigen that was effective without interleukin‐2. AV GA733, in combination with interleukin‐2, is a candidate vaccine for colorectal cancer patients.

Genetic analysis of multiple sclerosis in Europeans: French data

We report the results of a genome-wide screen for linkage disequilibrium (LD) in multiple sclerosis (MS) performed on 200 cases, 200 controls and 200 case-parent trios from France employing pooled DNA methodology. A total of 3510 microsatellite markers supplied through the GAMES collaborative were analysed and ranked according to their evidence for association. The most promising 117 markers were then followed up in a two-step validation process. In the first step, additional PCR of the DNA pools was performed in order to refine the ranking order. In the second step, markers were genotyped in individual cases and parents from the trio families. Seven markers showing nominally significant allele frequency differences between affected and unaffected emerged-D6S265, D12S1064, TNFa, D7S1824, D14S1426, D14S605 and D21S2051. These potential associations will require confirmation in further studies.