Peter Stastny

LYMPHOKINES IN RHEUMATOID SYNOVITIS

In antigen-induced experimental arthritis of rabbits, a macrophage migration inhibitory factor was released from the inflamed synovial tissues. A migration inhibitory factor, blastogenic factor, and B-cell-stimulating factor were also found in human rheumatoid synovial fluids and culture supernatants of rheumatoid tissue explants. Joint fluids from patients with inflammatory conditions other than RA sometimes also displayed these activities. OA fluids were usually inactive. At present, little is known of the origin or role in vivo of the lymphokine-like activities observed in the joints of rheumatoid patients. In related experiments, injection of lymphokine-rich antigen-free lymphocyte supernatants into normal rabbit knee joints produced a synovitis characterized by lining layer hyperplasia and infiltration of the sublining layer by macrophages. The lymphocytic and plasmacytic components seen in active antigen-induced synovitis were absent. It seems likely that some of the changes observed in active chronic synovitis are mediated by soluble factors of the lymphokine variety.

Antigen-specific HLA-restricted human T-cell lines - II. A GAT-specific T-cell line restricted by a determinant carried by an HLA-DQ molecule

Human T-cell lines responsive to the polypeptide antigens GAT and (T, G)-A--L were developed. They were specific for the priming antigens and required the participation of accessory cells matched for HLA-linked determinants, as shown in family studies. In panel studies, the ability of accessory cells to present antigen was shown to be associated with HLA-D-region antigens. However, the specificity of these determinants did not fully correspond to any HLA antigens as currently defined. One GAT-specific subline, derived by limiting dilution, utilized a restriction determinant associated with, but distinct from, the DQw3 (MB3) allospecificity. Blocking studies with mouse monoclonal antibodies indicated that this restriction determinant was carried by HLA-DQ molecules. The epitopes recognized in these molecules appear to be distinct from the alloantigenic determinants currently defined by serology.

Cell-mediated cytotoxicity against HLA-D-region products expressed in monocytes and B lymphocytes - II. HLA-A- or B-antigen compatibility is not required for HLA-D-region directed cytotoxicity

When effector cells were produced by sensitization in vitro with HLA-A- and B-compatible but HLA-D/DR-incompatible stimulator cells, cytotoxicity apparently was directed against a product of genes closely associated withHLA-D. For effective killing to occur with effector cells against certain minor histocompatibility antigens it is necessary that the effector cells and the target cells be matched forHLA-A orB. We have, therefore, investigated whether sharing of HLA-A or B antigens is needed to obatin efficient lysis with effector cells primed againstHLA-D. Good killing was observed with effector cells against HLA-D/DRw3 and against HLA-D/DRw4 whether or not the HLA-A or B antigens were matched. The results indicated that compatibility atHLA-A orB is not required for cell-mediated cytotoxicity against a product of theHLA-D region. TheHLA-D region appears to code for products that behave as strong histocompatibility antigens that can by themselves cause cell-mediated killing.

Increase in Capillary Basement Membrane Width in Parents of Children with Type I Diabetes Mellitus: Association with HLA-DR4

Hereditary factors related to HLA antigens are known to play a role in determining risk for the development of type I diabetes. In the present investigation, asymptomatic first degree relatives of diabetic patients were investigated to determine whether any abnormalities could be associated with the HLA haplo-types. Muscle biopsies were performed to obtain measurements of the width of the capillary basement membranes in 16 type I diabetic children, 20 of their unaffected siblings, and 38 parents. Only two diabetic children had capillary basement membranes greater than 2000 Å. The mean width of the capillary basement membranes was not different in affected compared with unaffected siblings. In contrast, the capillary basement membranes in the parents were considerably larger, with 14 of the 38 parents (37%) having measurements greater than 2000 Å. Moreover, the width of the capillary basement membranes in the parents correlated with the presence of the antigen HLA-DR4. The mean capillary basement membrane width in DR4 positive parents was 2026 ± 350 Å; that of DR4 negative parents was 1642 ± 333 Å. The difference was highly significant (P < 0.001). There was no correlation of capillary basement membrane width with HLA-DR3. The results suggest that a risk factor for type I diabetes associated with HLA-DR4 was associated in parents of type I diabetic patients with an asymptomatic increase in capillary basement membrane width in the absence of any clinical evidence of diabetes. The possible role of these abnormalities in the pathogenesis of type I diabetes and of the vascular complications of the disease require further study.

Antigen presentation by adherent cells from human peripheral blood. Correlation between T-cell activation and expression of HLA-DQ and -DR antigens

The ability of cells with different amounts of HLA-DQ or -DR to support T-cell proliferation in response to foreign antigens was investigated. Adherent cells were stained with monoclonal antibodies and sorted in the fluorescence-activated cell sorter (FACS) into (a) DQ-positive and DQ-negative subsets, with monoclonal anti-DQ; or (b) subsets expressing different density of DR determinants. Expression of HLA-DQ correlated with increased density of DR. The subset of cells expressing detectable DQ and increased density of DR was found to be more efficient in presenting mumps or tetanus toxoid antigen to T cells than were the DQ-negative, low-DR density adherent cells. Similar results were obtained with primary cultures of T cells from blood and with cloned antigen-specific T-cell lines, restricted by a single DR-subregion specificity. Our results suggest that quantitative variation in DR/DQ molecules expressed on monocytes correlates with their ability to support T-cell responses to nominal antigens. It is not clear whether this is due to only class II antigen density on the surface of the accessory cells or whether other factors are involved.

Recognition of class II molecules by human T cells. I. Analysis of epitopes of DR and DQ molecules in a DRw11, DRw52, DQw3 haplotype.

The HLA-D region of individuals with the DRw11, w52, DQw3 haplotype encodes multiple molecular products of three distinct subregions, DR, DP, and DQ. Since each molecule can carry multiple stimulatory epitopes, the repertoire of allogeneic T-cell responses to determinants of this haplotype can be quite large. In the present experiments, alloreactive cloned T-cell lines recognized six distinct epitopes associated with DRw11, DRw52, DQw3 haplotypes. Panel studies established that three epitopes were DRwll-like and three were DRw52-like. Blocking with monoclonal antibodies showed that two DRw11-like epitopes were carried by DR-subregion products and one DRwll-like epitope was carried by DQ-subregion molecules. DRw52-like epitopes were detected on separate DR subregion-encoded molecules. One of them carried both DRwl1-and DRw52-like epitopes, the other carried two of the DRw52-like epitopes. These epitopes, which represent functional units that trigger T-cell responses, can be detected at the present time only with the methods used in this report. Conventional allogeneic T-cell responses represent the summation of responses to multiple epitopes encoded by different D-subregion genes.

Cytofluorometric analysis of major histocompatibility antigens on human monocytes using monoclonal antibodies

The expression of HLA-A, B, C, and DR antigens was investigated on monocyte preparations by flow cytometry using various monoclonal antibodies. Essentially all human monocytes, either freshly isolated or after culture for several days, were stained for HLA-A, B, C, and DR antigens. When monocytes were incubated with Con-A-stimulated lymphocyte supernatants, an increase in HLA-A, B, C, and DR staining was observed. No increase was noted when two other monoclonal antibodies against non-HLA-related monocyte antigens (63D3 and 61D3) were studied under the same culture conditions. These results indicate that soluble factor(s) present in Con-A-stimulated lymphocyte supernatants modulate the expression of the major histocompatibility antigens on the surface of human monocytes.

Allostimulating cells in man. Quantitative variation in the expression of HLA-DR and HLA-DQ molecules influences T-cell activation

The adherent cells of the blood, which are powerful stimulators of the mixed lymphocyte reaction (MLR), contain subpopulations that differ in the quantity of class II major histocompatibility complex (MHC) antigens on their surface. Most of the adherent cells express HLA-DP and DR antigens, although in varying amounts (Nufiez et al. 1984). In contrast, detectable amounts of HLA-DQ products are found in less than half of the adherent cells (Gonwa et al. 1983, Nufiez et al. 1984, Chen et al. 1984). In the present report, experiments were performed to investigate the relationship between expression of class II MHC antigens on adherent cells and their ability to stimulate in the MLR.

Different specificities of an HLA-DRw6 haplotype detected by alloreactive T lymphocytes

DRw6 has been difficult to define serologically. In the present experiments we have developed T cell lines in order to characterize the components of a DRw6 haplotype. This was accomplished by priming T cells with allogeneic mononuclear cells mismatched for DRw6, Dw6, and MT2. Subsequently, three sublines with distinct reactivity patterns were derived by limiting dilution. The specificities detected by these sublines included: (a) a specificity found on a subset of cells positive for DRw6 which was inhibited by monoclonal antibodies against DS(DC), the human homologue of the murine IA-encoded molecules, (b) another DRw6-associated specificity blocked by an MT2-like antibody, and (c) an MT2-like specificity blocked by monoclonal antibodies reactive with a different MT2-associated determinant. These results show that more than one IE-like, as well as the DS/DC (IA-like) molecules, carry distinctive antigenic epitopes that can be recognized by allogeneic T cells. Primed T cell lines may be useful for a better definition of certain haplotypes which are at present difficult to characterize with serological reagents alone.

Polymorphisms of the Human T-Cell Receptor α and β Chain Genes and Their Relationship to Insulin Dependent Diabetes Mellitus

The association of certain HLA-D alleles, with insulin-dependent diabetes mellitus (IDDM) is well known. However, several analyses have suggested that a second locus, unlinked to the HLA complex may be involved in this disease. The availability of genetic probes for the T-cell receptor (TCR) genes has provided an opportunity to test the hypothesis that the second locus might be one or more of the TCR genes. The human TCR is a heterodimer consisting of two glycosylated polypeptide chains, α and β (1–3). Restriction fragment length polymorphisms (RFLP) of both human genes have been described (4–11). Previously, we described an association between one of these RFLPs and IDDM (12). This report extends that analysis by examining over 150 additional patients for the associated marker and additional TCR polymorphisms. These studies document an association between the TCRβ chain polymorphism and IDDM and provide yet an additional risk factor particularly among individuals who are DR3/X (where X ≠ 4).