The biologic importance of conserved major histocompatibility complex class II motifs in primates.

Phylogenetic comparisons of polymorphic second-exon sequences of MHC class II DRB genes showed that equivalents of the HLA-DRB1*03 alleles are present in various nonhuman primate species such as chimpanzees, gorillas, and rhesus macaques. These alleles must root from ancestral structure(s) that were once present in a progenitor species that lived about 35 million years ago. Due to accumulation of genetic variation, however, sequences that cluster into a lineage are generally unique to a species. To investigate the biologic importance of such conservation and variation, the peptide-binding capacity of various Mhc-DRB1*03 lineage members was studied. Primate Mhc-DRB1*03 lineage members successfully binding the p3-13 peptide of the 65-kD heat-shock protein of Mycobacterium tuberculosis/leprae share a motif that maps to the floor of the peptide-binding site. Apart from that, some rhesus macaque MHC class-II-positive cells were able to present the p3-13 peptide to HLA-DR17-restricted T cells whereas cells obtained from great ape species failed to do so. Therefore, these studies open ways to understand which MHC polymorphisms have been maintained in evolution and which MHC residues are essential for peptide binding and T-cell recognition.

Effects of inoculation with attenuated autologous T cells in patients with rheumatoid arthritis.

Injection of attenuated autoimmune T cells, T cell vaccination, has been used successfully in the prevention and treatment of experimental animal autoimmune disease. In order to determine whether such a procedure might be applied in rheumatoid arthritis (RA), a phase I study was conducted in thirteen RA patients with a mean disease duration of 12.8 years. All patients received a subcutaneous injection of attenuated autologous T lymphocytes from a CD4 positive clone (n = 4) or line (n = 9) isolated from synovial tissue (n = 3) or synovial fluid (n = 10). No toxic side effects were observed. On the average the patients showed a slight decrease in disease activity which was most marked at 8 weeks after the injection. Specific immune reactivity against the injected T cells was not detected, with the possible exception of one patient who was vaccinated with a clone selected in vitro with antigen and whose disease had begun one year earlier. In this patient a clear decrease in disease activity occurred, which was associated with a decrease in mitogen-induced proliferation of her peripheral blood mononuclear cells and in titres of serum rheumatoid factors. The results of this study show that inoculation of RA patients with autologous T cells is technically feasible and non-toxic, and may be associated with clinical and immunological effects. The data suggest that the potential of T cell vaccination should be further explored in diseases with defined antigen reactivity.

Activated synovial T cell clones from a patient with rheumatoid arthritis induce proliferation of autologous peripheral blood-derived T cells.

In order to investigate cellular interactions involved in the development of human autoimmune disease, a synovial fluid-derived T cell clone reactive with mycobacterial antigens, termed k38, was employed as a stimulus for autologous peripheral blood mononuclear cells (PBMC). Stimulator cells were used either activated with immobilized OKT3 mAb or in a resting state. Activated k38 cells triggered PBMC to proliferate. A T cell line prepared by coculturing autologous PBMC with irradiated activated k38 cells proliferated upon stimulation with activated k38 cells in the presence of PBMC as a source of accessory cells, as did T cell clones that were subsequently isolated from this line. Blocking studies revealed that proliferation of the anti-k38 line and anti-k38 clones in response to stimulation with clone k38 could be inhibited by monoclonal antibodies against a variety of cellular determinants including HLA class I and LFA-1 beta. It was demonstrated that the antigen reactivity of clone k38 was modulated by the presence of anti-k38 clones. These data provide a model for understanding the cellular interactions that may take place in vivo in the evolution of the chronic synovial inflammatory process.

HLA class-II-restricted Mycobacterium leprae-reactive T-cell clones from leprosy patients established with a minimal requirement for autologous mononuclear cells.

This report describes an effective method for the cloning of Mycobacterium leprae-reactive T lymphocytes with Epstein-Barr-virus transformed autologous B cells as antigen-presenting cells. The two advantages of this method are that it drastically reduces the number of autologous peripheral blood mononuclear cells (less than 10(7) cells) needed to obtain and propagate these T-cell clones (TLC), and that it enables us to expand individual TLC to large numbers of cells (greater than 10(8)). Thus the major obstacles for the cloning of T lymphocytes--especially important with regard to patients--are bypassed. Thus far, TLC from three leprosy patients have been established. These TLC are HLA class II restricted in their M. leprae-directed response. A marked enhancement in antigen responsiveness was observed after further expansion of several TLC, some of which turned from nonresponder into responder TLC. Four tested TLC display strikingly different antigen recognition patterns when tested against a number of other mycobacterial antigens; one TLC so far recognizes only M. leprae antigens.

Epstein-Barr virus-transformed B cell lines present M. leprae antigens to T cells.

We have investigated whether or not Epstein-Barr virus-transformed lymphoblastoid B cell lines (EBV-BLCL) are able to present Mycobacterium leprae (M. leprae) to antigen-reactive T cell lines and clones. Such EBV-BLCL would provide us with a homogeneous and unlimited source of antigen-presenting cells. Antigen-triggered proliferation of T cells has been studied with co-cultures either with autologous or allogeneic irradiated EBV-BLCL. Our results show that EBV-BLCL are able to present M. leprae as efficiently as peripheral blood mononuclear cells, and that they also function in an HLA-DR-restricted fashion. Apart from their possible in vivo relevance, these results have important practical implications, in particular for the generation and study of M. leprae-reactive T-cell clones.

Role of HLA class II products in proliferative T-lymphocyte responses to PPD. Evidence of a regulatory influence associated with MB1.

HLA class II determinants were analysed for their role in monocyte-T-cell interactions in the proliferative response to purified protein derivative (PPD). Allogeneic cell combinations of monocytes and T cells were tested with a range of suboptimal PPD concentrations. For each cell combination tested, a summary measure to characterize the antigen-induced response curve was calculated by means of regression analysis of the response to the dose of PPD added, thus obviating a further need to correct for mixed lymphocyte reactivity. In 200 distinct monocyte-T-cell combinations, HLA-DR appeared to be the major restricting element. Additionally, a marginal influence of HLA-MB or -MT matching was observed and no evidence of HLA-SB-restricted responses. Remarkably, the HLA-MB1 specificity was significantly associated with low responsiveness in DR sharing monocyte-T-cell combinations, indicating a modulating role of MB1 in DR-restricted T-cell responses.

Low T lymphocyte responsiveness to Mycobacterium leprae antigens in association with HLA-DR3.

The type of leprosy which develops after infection with Mycobacterium leprae is influenced by the presence or absence of HLA-DR3, as has been demonstrated in an ethnic group originating from Surinam. In the present study we investigated in this same ethnic group the role of HLA-DR, and of HLA-DR3 in particular, in monocyte-T cell interactions during leprosy specific proliferative responses in vitro. HLA-DR3 heterozygous T cells from tuberculoid leprosy patients were cultured with antigen and either HLA-DR3 positive or HLA-DR3 negative homozygous HLA-DR compatible allogeneic monocytes as antigen presenting cells (APCs). T cell proliferative responses with DR3 homozygous monocytes as APCs, were observed to be decreased as compared to T cell proliferative responses with DR3 negative homozygous monocytes as APCs. Furthermore, although the leprosy specific monocyte-T cell interactions were shown to be restricted for HLA-DR, in the anti-MLW-1 response HLA-DR3 appeared to function as a restricting element poorly or not at all. These observations may imply, that an in vitro correlate has been found for an (HLA associated) genetic control of leprosy in vivo.

An estimation of the recombination fraction between the MLC locus and the FOUR locus.

Thirty-nine families with 167 children were typed for HL-A and studied in the MLC test. Two maternal recombinations between the FOUR and the MLC locus in two different families were found. An estimate of the recombination fraction between the FOUR and the MLC locus was calculated.