Modification of murine T-cell cytotoxicity by preimmunization with M locus and H-2 incompatibilities.

The effect of preimmunization of CBA/H mice with M locus and H-2 incompatible lymphocytes was examined on the in vitro generation of cytotoxic cells to both types of targets. Preimmunization with M locus did not help to generate cytotoxic cells to M-locus targets; however, the anti-H-2 cytotoxicity of M-locus-preimmunized lymphocytes was suppressed between the 6th and 19th day after preimmunization. In contrast, preimmunization with H-2 incompatibility produced an amplified lytic anti-H-2 response. The possible mechanisms are discussed.

A new sensitive assay for antibody against cell surface antigens based on inhibition of cell-dependent antibody-mediated cytotoxicity. I. Specificity and sensitivity.

Inhibition of cell-dependent antibody-mediated cytotoxicity has been investigated as a new assay for antibody against cell surface antigens. The cytotoxicity system consisted of effector cells (normal mouse spleen cells), target cells ((61)Cr-labeled chicken erythrocytes), and antitarget cell antibody. Addition of antibody against cell surface antigens in the effector cell population regularly inhibited the cytotoxicity measured in this system. This cytotoxicity inhibition assay (CIA) detected antibody with a variety of specificities: anti-H-2, anti-Thy 1.2, anti-immunoglobulin, and antimouse bone marrow-derived lymphocyte antigen. When the inhibition by anti-H-2 sera was analyzed using effector cells from congenic mice, the activity was found to be directed against specificities mapping in the H-2K, H-2D, and I regions of the H-2 complex, correlating well with the specificities characterized by complement-dependent assays. A comparison between the sensitivity of the CIA and complement-dependent lysis revealed that the CIA was 2-11 times more sensitive for anti-H-2 antisera and 20-780 times more sensitive for certain antisera against subpopulations of the spleen cells (i.e., T cells or B cells). The CIA proved to be precise, sensitive, and reliable. It may become a very useful antibody assay in various species including man.

Human alloimmune sera against T cell subsets. Detection and influence on pokeweed mitogen-stimulated Ig production in vitro.

Sera from multiparous women contain antibodies against T cell subsets. Population and family studies of four anti-T cell-subset antibodies are given. Two of these reacted with part of the suppressor T cell fraction (T gamma) and two with part of the helper T cell fraction (T mu) cells. By mixing T gamma- and mu-enriched cell suspensions in different concentrations, preliminary evidence was obtained that the anti-T cell-subset sera recognized T cells that had different functions in pokeweed mitogen-stimulated cytoplasmic immunoglobulin synthesis.

DY determinants, possibly associated with novel class II molecules, stimulate autoreactive CD4+ T cells with suppressive activity.

A set of T cell clones (TCC) isolated from HLA-DR-, Dw-, DQ-matched allogeneic MLCs was found to proliferate autonomously when stimulated with cells carrying a wide range of class I or II specificities. This apparently unrestricted proliferation was relatively weak, and only low levels of IL-2 were present in the supernatants of stimulated cells. Autologous as well as allogeneic PBMC and B lymphoblastoid cell lines (B-LCL) were capable of stimulating such clones, which were also restimulated by suppressive, but not by helper, TCC. Moreover, such clones displayed the unusual property of autostimulation. mAb inhibition experiments suggested that class II- or class II-restricted antigens were involved in stimulation. Thus, certain "broad" mAbs (TU39, SG520) reacting with multiple locus products inhibited activation of these reagents, but none of those reacting more specifically with DR (TU34, TU37, L243, Q2/70, SG157), DQ (TU22, SPV-L3, Leu 10), or DP (B7/21), or mixtures of these mAbs, were able to do so. Evidence from sequential immunoprecipitation experiments suggested that mAb TU39 bound class II-like molecules other than DR, DQ, and DP on TCC and B-LCL, and it is therefore proposed that such putative novel class II-like molecules may carry the stimulating determinants for these autoreactive clones. DY-reactive clones lacked helper activity for B cells but mediated potent suppressive activity on T cell proliferative responses that was not restricted by the HLA type of the responding cells. Suppressive activity was induced in normal PBMC by such clones, as well as by independent suppressive clones, which was also inhibited only by mAb TU39. These findings lead to the proposal that DY-reactive autostimulatory cells may constitute a self-maintaining suppressive circuit, the level of activity of which would be regulated primarily by the availability of IL-2 in the microenvironment.

HLA class II alpha chain gene polymorphisms in patients with insulin-dependent diabetes mellitus, dermatitis herpetiformis, and celiac disease.

We have investigated DNA polymorphism of the class II alpha chain genes in HLA typed patients with insulin dependent diabetes mellitus (IDDM; n = 79), celiac disease (CD; n = 46), dermatitis herpetiformis (DH; n = 53), and controls (n = 86). Preferential allelic associations of HLA genes and gene products have thus been constructed for susceptibility to these diseases. DR alpha and DQ alpha gene polymorphisms indicated heterogeneity of HLA DR3, DRw6, and DR7, and HLA DR2 and DRw6, respectively. In DR7 positive CD patients a 3.8-kilobase (kb) DR alpha fragment, which correlated with DQw3, was found in only 11% of patients compared with 45% of corresponding controls (P less than 0.05). An increased frequency of a DX alpha genotype UU in all three diseases was found (IDDM 59%, DH 45%, CD 48%, compared to 21% in controls, P less than 0.001), which is not explained solely by the increased frequencies of DR3-DX alpha U. We therefore conclude part of the genetic susceptibility for these three conditions is encoded by genes within the DQ-DX subregion.

Identification of murine H-2Db histocompatibility antigens in cells transfected with cloned H-2 genes.

Clones of mouse L-cells transformed with 21 cosmids containing 15 major histocompatibility complex class I genes of C57BL10 (H-2b) sperm cell DNA were analyzed for the expression of their transfected H-2 and Qa/Tla genes. Three cosmids contained a single gene, mapping to the H-2D region. This gene encodes the H-2Db alloantigen: mouse L-cells transfected with cosmids containing this gene reacted with monoclonal antibodies and alloantisera specific for the H-2Db antigen and expressed a 46-kd H-2 heavy chain associated with beta 2-microglobulin in their cell membranes. Furthermore, these transfected cells were stimulators of, and targets for, anti-H-2Db cytotoxic T-lymphocytes. Eighteen cosmids contained 14 different genes mapping to the Qa and Tla regions. L-cells transfected with these genes did not express class I genes reacting with alloantisera or monoclonal antibodies against Qa2, Qa4 or TL differentiation antigens. In particular, the Qa2,3 gene of C57BL10 was not identified.

HLA-D typing of human lymphocytes using frozen and thawed spermatozoa.

A method is described for the preservation of haploid populations of human spermatozoa in liquid nitrogen for their subsequent use as stimulators in HLA-D (SL) lymphocyte typing. The typing results obtained using thawed spermatozoa correlated well with those obtained when using fresh sperm and also with other cellular typing methods.

Solubilisation effect of Nonidet P-40, triton X-100 and CHAPS in the detection of MHC-like glycoproteins.

We have analysed the differential solubilisation effect of three detergents on cell-membrane histocompatibility glycoproteins. Two nonionic detergents (Nonidet P-40 and Triton X-100) which are extensively used in the extraction of MHC proteins and a zwitterionic detergent (CHAPS) which is sulphobetaine derivative of cholic acid were used. An AKR (H-2k) derived spontaneous leukaemic cell line--424--was used as the experimental model. In this tumour cell line a class I-like antigen is expressed but not directly detected by cell-binding radioimmunoassay or immunoprecipitation from NP-40 or Triton X-100 solubilised glycoproteins. However, 46 kDa and 12 kDa bands consistent with the classical H-2 class I pattern were seen by SDS-PAGE after immunoprecipitation with the 34.5.8 anti-H-2Dd MoAb using CHAPS solubilised 424 glycoproteins. The H-2Dd-reactive molecule appears to be associated with at least one of the syngeneic class I specificities (H-2Kk, H-2Dk) and not accessible to react with the specific anti H-2Dd MoAb. The detergents NP-40 and Triton X-100 appear to be less efficient than CHAPS in breaking protein-protein interactions. This property of CHAPS permitted the adequate solubilisation of the novel antigen and its direct detection. The results of this study suggest that the alternative use of a non-denaturing zwitterionic detergent may contribute to the detection and characterisation of MHC-related, membrane-bound proteins of tumours and normal cells.

Analytical studies of the binding parameters describing the interaction of HLA-DR epitopes with a specific monomorphic monoclonal antibody.

In this paper we present an analytical study of the binding parameters related to the interactions of the HLA-DR-specific monoclonal antibody (L243) and its reacting epitope as expressed on the cell surface of seven Epstein-Barr virus-transformed B cell lines. Scatchard, Sips and Langmuir equations were used to plot and analyse the data obtained from each reaction. A single affinity constant (K) value was derived at low and high concentrations of free antibody for each antibody-cell interaction tested and was of the order of 10(7) M-1. Similar heterogeneity indices (a) (close to 1.0) and K values were obtained for most of the cells. These results suggest that the reacting HLA-DR epitopes are homogeneously distributed and equally accessible to the antibody on all the cells tested. The average number of epitopes per cell was 3.4 X 10(6), SD 0.5 X 10(6) and were similar for all the cell lines. The analytical and experimental model presented here can be useful for studying quantitative and qualitative variations in the expression of MHC epitopes in oncogenesis and disease associations.

New mixed lymphocyte microculture test harvested by a simple device developed for this purpose.

The methodology of the conventional mixed lymphocyte culture test has been simplified and miniaturized by developing a micromethod employing Terasaki trays with a final culture volume of 20 mul containing between 10,000 and 20,000 responding and stimulating cells each/culture. This represents one-tenth the cell concentration of the conventional mixed lymphocyte culture. For this new test, a special microharvester has been designed and developed. The test allows accurate and reproducible results giving a high level of discrimination between the different levels of response.

Generation of suppressor cells in mice immunized with M locus-incompatible lymphocytes.

Alloimmunization of mice with M locus-incompatible lymphocytes resulted in the generation of suppressor cells in the immunized host. Lymph node cells from such alloimmunized mice suppressed the in vitro cytotoxic response of normal cells to H-2 alloantigens. The suppression generated was greater than could be accounted for by dilution of the prekiller cell population with cells possibly devoid of cytotoxic potential from M locus preimmunized mice. Using M locus pseudocongenic mice, the suppressive effect was shown to be largely attributable to M locus determinants; restimulation of suppressor cells in culture with the specific M locus was required for suppression of effector cell generation. The in vivo effect of suppressor cells was tested in a graft-versus-host reaction; injection of M locus preimmunized cells into footpads of F1 hybrid mice suppressed the popliteal lymph node enlargement compared with lymph node size after injection of control preimmunized cells. Although the suppressive effect is mainly attributable to M locus determinants, incompatibility for the DBA/2 antigen may add to the suppression. The study of inhibitory effects on T cell cytotoxicity because of serologically undetectable lymphocyte-activating determinants (Mls) could lead to the better understanding of suppressive mechanisms which may allow the growth of syngeneic tumours.

Non-major histocompatibility system immunogenetic influences in rat renal allograft survival.

H-1-incompatible rat renal allograft survival rates are strongly influenced by the non-major histocompatibility system (MHS) genes. The same H-1 (MHS) incompatibility tested on four different non-MHS backgrounds gave widely differing results ranging from 11 to 60% fractional survival between the best and the poorest (P less than 0.01) at 105 days. These results can best be explained if non-H-1 and intra-MHS amplifiers and/or suppressors are invoked.

Effects of prepro-vasoactive intestinal peptide-derived peptides on the murine immune response.

We have studied the effects of prepro-vasoactive intestinal polypeptide-derived peptides on lectin-induced lymphocyte proliferation and natural killer cell (NK) activity in cells from murine spleen, mesenteric lymph nodes, Peyer's patches, thymus and peripheral blood mononuclear lymphocytes (PBL). These peptides (vasoactive intestinal peptide (VIP), peptide histidine methionine (PHM-27) and peptide histidine valine (PHV-42)) showed differential effects in their immune response in a dose- and tissue-dependent manner. All peptides significantly decreased DNA synthesis in spleen (range: 45-30%), lymph nodes (range: 30-0%), Peyer's patches (range: 30-4%) and PBL (range: 30-16%). In these tissues there was no significant difference in their potency. In the thymus, however, PHM-27 (range: 27-15%) was significantly more potent (p less than 0.001) in inhibiting DNA synthesis than either VIP (range: 6-0%) or PHV-42 (range: 20-8%). The modulatory effects on NK activity by these peptides also showed an inhibitory effect. The order of potency was: VIP (range: 40-27%), PHV-42 (range: 22-11%) and PHM-27 (range: 20-8%). The presence of VIP inhibitor [( D-p-chloro-Phe6,Leu17]-VIP) at 10(-8) M in both functional assays caused a significant antagonism of the effects of VIP but not PHM-27 or PHV-42. Our results suggest the existence on lymphocytes of different receptors for prepro-VIP-derived peptides, and that they may be considered as important immunoregulatory molecules. Their mechanism of interaction, however, is not clearly understood.

Persistence of donor-specific class II antigens in allografted human heart two years after transplantation.

It has been suggested that one of the mechanisms of action of cyclosporin is by abrogation of major histocompatibility complex class II expression. We have tested this hypothesis by following the expression of DR7, a polymorphic determinant of the class II DR locus in cardiac biopsies from 12 heart or heart-lung recipients who were themselves DR7 negative but whose donors were DR7 positive. All patients received cyclosporine and azathioprine immunosuppression. Immunoperoxidase and immunofluorescent techniques were used. The DR7 determinant was found on interstitial structures on donor heart at all times studied, including at 2 years after transplantation. Double immunofluorescent labeling of donor heart before transplantation revealed that more than 60% of the DR7 was on endothelial cells. At later times the proportion of DR7 on endothelial cells increased, but even at 1 year some DR7 was found on interstitial structures not of endothelial origin. The significance of these findings to mechanisms of long-term immunosuppression is discussed.

Ontogenic and functional implications of the differential expression of HLA-DQ antigens on leukemic cells.

We have examined the HLA class II antigenic profiles on different types of leukemic cells and have attempted to relate these findings to the normal differentiation pathways of the cells from which they have arisen. Monoclonal antibodies reacting with the different HLA class II determinants, HLA-DR, DRw52(MT), and DQ, were used to study the expression of these antigens on Epstein-Barr virus transformed cell lines, chronic lymphocytic leukemic cells, acute lymphoblastic leukemic blasts, acute myeloblastic leukemic blasts, and established leukemic cell lines by indirect immunofluorescence binding and immunoprecipitations. The results showed that whereas the HLA-DR and HLA-DRw52(MT2) antigens are normally expressed on the majority of the cells tested, there is a different expression of the HLA-DQ antigens on acute leukemic blasts, chronic lymphocytic leukemic cells, and leukemic cell lines indicating that the DQ molecules may be differentiation antigens preferentially expressed on mature cells. Furthermore, when the pre-B cell leukemic line NALM 6 was induced to differentiate with phorbol ester (TPA), normal expression of the HLA-DQ antigen was obtained after 5 days of culture. The absence of HLA-DQ antigens from the acute leukemic blasts suggests that these immature cells "froze" in the early stages of cell differentiation. We discuss these findings in relation to the role of these HLA class II antigens in cell differentiation and the immune response.

Di-allelic alloantigenic systems on subsets of T cells.

Sera obtained from multiparous women and some of other origin contain antibodies which react with antigens on T cell subsets. These antibodies recognize two distinct diallelic systems, one of which is mainly present on T gamma cells while the other is present on T mu cells. The sera that reacted with the T gamma cells formed a pattern consistent with that of a diallelic system which we have called TCA system with alleles TCA 1 and TCA 2; the sera which reacted with the T mu cells formed a pattern consistent with another diallelic system, independent from TCA, which we have designated the TCB system, with alleles TCB 1 and TCB 2.

HLA-Dw specificity assignments are independent of HLA-DQ, HLA-DR, and other class II specificities and define a biologically important segregant series which strongly activates a functionally distinct T cell subset.

Several lines of evidence indicate that HLA-Dw, as defined by HTC typing, is not the result of the combined stimulatory effect of HLA-DR and DQ. Therefore, responder cells do not have to share HLA-DQ antigens with the stimulator HTCs to give a typing response. The common HLA-DR-DQ associations observed in HTCs correspond to different patterns of linkage disequilibrium in different populations. HLA-DQ and HLA-Dw are functionally heterogeneous. Although HLA-DQ molecules may play a role in primary stimulation, this role is distinct from that of Dw determinants which have strong lymphocyte activating properties. The role of the HLA-DQ determinants on the other hand, is one of modulating the total T cell response by controlling the proliferation of suppressor and cytotoxic cells. The primary MLC response is the result of the proliferative effect of HLA-Dw, DR, DP, and other associated determinants, in conjunction with a modulatory effect of DQ molecules. However, HLA-Dw (as detected by HTC typing) are DR associated determinants which are immunodominant in primary MLR. The genes of the HLA-DR subregion have been named DR by the WHO nomenclature committee. This subregion encodes the HLA-DR specificities and the DRw52 and DRw53 determinants. Unfortunately this nomenclature does not take into account the need to define the genetic basis of the HLA-Dw determinants--whether they are encoded by separate genes within the HLA-DR subregion or whether they are encoded by as yet unspecified genes in the HLA class II region in linkage disequilibrium with HLA-DR DRw52/53. There are at least three and possibly four beta chain genes in the HLA-DR subregion, all in strong linkage disequilibrium with each other. Some of these are expressed in most haplotypes while others are not; some behave as pseudogenes in some haplotypes and in others, all the genes are expressed. All the genes of the class II region have not been fully characterized. HLA-Dw determinants may be specified by one or more of these genes. When more information becomes available, the genetic and molecular basis of the HLA-Dw series as well as the functional heterogeneity and antigenic strength of the various class II determinants will be better understood.

A new HLA Bw16 subtype defined in both Negroid and Saudi Arabian populations.

Serological identification of a new HLA-Bw16 subtype, B39B, was made by the analysis of reaction patterns of many alloantisera and one monoclonal antibody. The B39B pattern of reactivity was shown to be distinct from HLA-Bw38, Bw39, and 8w57. Cytotoxicity testing before and after absorption suggests that the B39B specificity belongs to the HLA-B7 cross-reactive group. The B39B was clearly demonstrated in two families. This antigen was detected in Negroids and Saudi Arabian Caucasoids but not in a large panel of British Caucasoids.

HLA-DR alpha, -DX alpha, and DR beta III gene association studies in DR3 individuals.

In this study we have examined the results of probing with synthetic oligomers at the DR beta III locus, together with restriction fragment length polymorphisms defined by BglII digestion and a cDNA DR alpha probe, and Taq 1 digestion and a genomic DQ alpha probe. We have demonstrated heterogeneity of the human leukocyte antigen DR3 and close association of the DR alpha, DR beta III, and DX alpha genes. Two DR3-related preferential allelic associations have been identified, which may prove useful in family analysis as well as for investigations of DR3-related diseases.

HLA class III haplotypes in multicase rheumatoid arthritis families.

The class III complement proteins (C2, BF, C4A, and C4B) were studied in 57 multicase rheumatoid arthritis (RA) families. When the gene frequencies for RA probands were compared to a normal control panel (162 haplotypes), a significantly higher frequency of the rare variant C4B*3 was observed (p less than 0.05). No significant differences were seen for the other C2, BF, C4A, or C4B alleles. The most common haplotype found in the probands was HLA-Cw5,B44,C2*C,BF*S,C4A*3,C4B*3,DR4, occurring with a frequency of 0.088. Haplotypes containing HLA-DR4 and Bw62 were found to carry either C4A*3,C4B*3; C4A*3,C4B*1; or C4A*4,C4B*2. When only haplotypes containing DR4 were compared between probands and controls, the frequency of the C4B*3-bearing haplotype remained higher in the probands. It is concluded that Bw62,C4A*3,C4B*3DR4 is a haplotype which is especially associated with RA. The low frequency in the RA population of this haplotype indicates that C4B*3 has a minor role in overall RA susceptibility.