Coxsackievirus B4 infection of the mouse pancreas: the role of natural killer cells in the control of virus replication and resistance to infection.

The role of natural killer (NK) cells in the early immune response to a pancreatropic isolate of coxsackievirus B4 (CVB4) was investigated in a murine model of pancreatitis. Endogenous (background) NK cell activity in fresh spleen effector cells from eight mouse strains was compared with virus-augmented NK cell activity 4 days post-infection (p.i.). A significant virus-induced increase (P less than or equal to 0.003) in NK cell activity was seen in seven of eight infected mouse strains, when virus titres in the pancreas were beginning to fall. Lesions in the exocrine pancreas were least extensive in the three strains with the highest endogenous NK cell activity. In C3H/HeJ mice that had been depleted of NK cells prior to infection with a low virus concentration, resistance to infection of the pancreas was completely abolished; myocarditis was also observed in one of these animals. Thus, NK cells may limit virus replication in the pancreas and play a role in resistance in C3H/HeJ mice. Virus-specific neutralizing antibody was not detected in the serum until 5 to 6 days p.i. in most strains and did not appear to influence pancreatic virus titres. It may be significant that CVB4 infection did not induce the expression of major histocompatibility complex (MHC) class I molecules on target acinar cells. With certain tumour cells, an inverse relationship between MHC class I expression and susceptibility to NK cell-mediated lysis is well documented.

Is rheumatoid arthritis in Indians associated with HLA antigens sharing a DR beta 1 epitope?

HLA class II antigens were identified in a group of 44 patients with rheumatoid arthritis (RA) originating largely from the north or northeast of the Indian subcontinent and resident now in east London. Compared with 67 locally typed east London Asian controls, the prevalence of three HLA-DR antigens was raised in the patients: DR1 18.2% v 6.0% chi 2 = 3.99, DR4 20.5% v 11.9% chi 2 = 1.48, and DRw10 27.3% v 8.9% chi 2 = 6.56. These differences were also found when the patients with RA were compared with a larger control group of 110 northern Indians: DR1 18.2% v 7.2% chi 2 = 4.02, DR4 20.5% v 7.2% chi 2 = 5.56, and DRw10 27.3% v 8.1% chi 2 = 9.7. Twenty five (57%) of the patients expressed at least one of these antigens. All patients were also characterised for HLA-Dw types by mixed lymphocyte culture typing. The prevalence of the HLA-DR4 associated Dw types in the patients was: Dw4 2.3%, Dw10 0%, Dw14 11.4%, and Dw15 6.8%. The DR beta 1 chains of DR1 and DRw10 together with the Dw types of DR4 other than Dw10 share amino acid residues in a region of the third hypervariable region considered to be critical in antigen presentation. It is concluded that RA in Indians is associated with these HLA antigens, and data from this study support the hypothesis of a cross reactive epitope common to HLA specificities associated with RA.

Specificity of lymphocytotoxic autoantibodies (LCAbs) found in the serum of leprosy patients: class I MHC antigens.

Lymphocytotoxic autoantibodies (LCAbs) of the IgM class have been identified in patients with borderline tuberculoid (BT) and borderline lepromatous (BL) leprosy with Type I reactions (I) as well as lepromatous leprosy (LL) patients with erythema nodosum leprosum reactions (ENL). The observation that lymphocytotoxic activity (LCA) was reduced in the presence of platelets led us to determine whether LCAbs had specificities for Class I Major Histocompatibility Complex (MHC) determinants. Absorption of LCA positive sera with platelets, classically used to deplete Class I specific lymphocytotoxic antibodies, reduced LCA towards autologous as well as allogeneic target cells. This was true for LCA positive sera from all patient classifications (group BT in the autologous system, p less than 0.01; in all other patient groups, p less than 0.001). Introducing B-2m to cytotoxicity assays only marginally reduced LCA when added at high concentrations (5 mg/ml). An anti-Class I MHC antiserum which blocked the lytic activity. The data indicate that LCAbs while absorbed by platelets, are not specific for the Class I MHC antigens. The autoantigen recognized by these autoantibodies therefore remains to be identified.

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.

T-cell receptor beta-subunit gene polymorphism and autoimmune disease.

We have investigated the distribution of genotypes of a restriction fragment length polymorphism of the T-cell receptor beta-subunit gene in Caucasoid controls and patients with insulin-dependent diabetes mellitus, celiac disease, dermatitis herpetiformis, and idiopathic membranous nephropathy and also in South Indian controls and diabetics. We found no significant differences between the controls and patients with any disease in either ethnic group, a result which contrasts with previous reports of associations with both insulin-dependent diabetes mellitus and idiopathic membranous nephropathy. However, the most striking finding was a marked disparity between the genotype distribution in our Caucasoid control population and that previously reported by other investigators.

HLA DQ region gene polymorphism associated with primary IgA nephropathy.

IgA nephropathy (IgAN) has been associated with HLA-DR4. We have recently described two non-allelic Taq I DQ beta gene-associated fragments sized 2.0 kb (T2) and 6.0 kb (T6), which strongly associate with DR4. T2 represents a polymorphism of the DQ beta gene and has been redesignated DQw8 (10th International HLA Workshop). The origin of the T6 fragment has not been determined, but probably represents a polymorphism of either the DQ beta or DX beta gene. When present together T2 and T6 define a subgroup of DR4 subjects at high risk of developing autoimmune disease. We have, therefore, studied DQ beta gene polymorphisms in IgAN. The DR antigen distribution was similar in IgAN and normal controls. The T2+/T6+ phenotype was present in 49% patients with IgAN compared to 15% of controls [P less than 0.0001, chi 2 = 32.8, Cramer's V = 0.41; relative risk = 5.5 (range, 2.8-11.0)]. Seventy-two percent of DR4+ IgAN patients and 29% of DR4+ controls were T2+/T6+ (P = 0.007, chi 2 = 17.0). These findings confirm the hypothesis that disease susceptibility genes are important in IgAN, and suggest that the putative gene(s) are located within or near to the DQ subregion. Moreover, similar DQ beta gene associations have been found in IDDM and pemphigus vulgaris, pointing to a common immunogenetic mechanism predisposing to several autoimmune diseases.

Protection of newborn mice from graft versus host disease by maternal pre-immunization.

Alloimmunization of BALB/c (H-2d) female mice with allogeneic spleen cells from C57BL/6 (H-2b) or CBA/H (H-2k) mice protects BALB/c offspring from graft-versus-host disease (GVH-D) following neonatal intraperitoneal inoculation of high doses of spleen cells respectively of C57BL/6 or CBA/H strains of mice. The mice survived GVH-D over one year after the allogeneic inoculum 24-48 h after birth and they did not show any signs of GVH reaction nor splenomegaly. We show that this phenomenon is antibody mediated and affects the developing immune system of the foetus. Repeated immunization of virgin female BALB/c with anti-H-2b or anti-H-2k antisera (Ab1) can equally abrogate GVH-D in their newborn offspring challenged at 24-48 h after birth with allogeneic spleen cells of H-2b or H-2k phenotype. Our results demonstrate that protection from GVH-D is not specific to the immunizing strain and occurs when the neonatal mice are challenged with C57BL/6 or CBA/H spleen cells. There is thus crossreactivity of tolerance against H-2 specificities. In this study we also report on the in vitro cellular immune responses of the surviving GVH-resistant mice and demonstrate that these responses against both the challenge and third party lymphocytes are impaired.

Major histocompatibility complex (MHC) protein analysis by optimised two-dimensional electrophoretic methods.

Human histocompatibility molecules HLA-Class I and Class II (DR, DQ, DP) were analysed using three two-dimensional protocols: nonequilibrium pH gradient electrophoresis (NEPHGE), isoelectric focusing-acidic gradient (IEF-AG) and isoelectric focusing-basic gradient (IEF-BG). The three methods differ in their carrier ampholyte combinations and electrophoretic conditions. They provide different pH gradients and therefore different electrofocusing profiles. The NEPHGE protocol was adequate for separating proteins across a broad range of pI mobilities, i.e. 4.4 pH units between the acidic and the basic end. In contrast, the IEF-AG and the IEF-BG protocols gave a separation power across a narrow pH range, 1.9 and 1.7 pH units respectively. Thus, whereas the NEPHGE protocol provides a tool for a global major histocompatibility complex (MHC) antigen profile analysis, the IEF-AG and -BG allows one to investigate subcomponents of the individual MHC chains. For example, NEPHGE analysis of the HLA Class I heavy chain revealed a single spot. However, IEF-BG revealed the presence of six equidistantly spaced spots spanning a short pH gradient with identical molecular weight. Similar improved resolution was seen for the HLA-DR, DQ, and DP molecules. The IEF acidic gradient was adequate for separating the alpha chain; the IEF basic gradient gave better resolution of the beta chains. This data provides a baseline set of conditions for both analytical and preparative MHC protein studies prior to amino acid sequencing.

In vitro induction of immunological tolerance.

IL-2 was previously shown to induce cytotoxic effectors with a broad spectrum of target specificities in thymus and spleen cell cultures. This study was designed to show whether T cells activated by H-2 allogeneic cells in MLC or by syngeneic tumor cells in MLTC are also potential targets for these cytotoxic effectors. We found that thymocytes activated in vitro for 5 days by rIL-2 were capable of killing tumor cells as well as activated T cells. Thymocytes activated by IL-2 were accordingly utilized as a means of effecting clonal deletion of T cells activated by H-2 allogeneic target cells in MLC. To establish whether the unresponsiveness is specific. IL-2-activated thymocytes were added as third party cells to MLC and MLTC. The results showed that both T cells, proliferating in response to H-2 allogeneic cells, and CTL, reactive against syngeneic tumors or H-2 allogeneic cells, are eliminated from the T cell pool. Only alloreactive T cells are specifically eliminated in MLC by IL-2-activated thymocytes, as the remaining T cells are capable of proliferating and generating CTL in response to antigenically unrelated third party allogeneic cells. The possibility that unresponsiveness might be due to soluble factors was ruled out by studies performed with a diffusable "chamber insert" culture system. The results provide evidence that IL-2-activated thymocytes induce in vitro T cell tolerance.

Functional studies of H-2k-like epitopes on DTIC treated and untreated L1210 (H-2d) clones.

Following 'in vivo' treatment with 5-(3-3'-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC), murine leukemic cells acquire new antigenic specificities not detectable on parental cells and responsible for the rejection of the tumour by syngeneic hosts. 'In vivo' and 'in vitro' experiments pointed out an immunological cross reactivity between DTIC treated and untreated lines. Furthermore, specific CTLs raised against DTIC treated L1210 tumour cells (H-2d) were cytotoxic for H-2k target cells. The aim of this study is to investigate whether the H-2k cross reactivity displayed by L1210/DTIC is related to the drug treatment rather than due to an antigen already present in the parental line and maintained after treatment. Cloned cells from L1210, obtained by limiting dilution 'in vitro', were recloned 'in vivo' and then treated with DTIC. Syngeneic and allogeneic CTLs raised 'in vitro' against parental and treated clones showed lytic activity against H-2k target cells. Treated and untreated clones were then checked for the presence of H-2k-like determinants using monoclonal antibodies. One of these, HB-53 (IgG2bKkDk) was highly positive with all the clones tested in binding assay using iodinated Fab anti-mouse Ig, fluorescence and FACS analysis. Others displayed a low reactivity against both treated and untreated clones without significant differences. After neuraminidase treatment of two clones (D and D/DTIC), the H-100.5 (anti H-2Kk)-reactive epitope was dramatically exposed on the DTIC tumour cells but not on the parental clones. These data suggest that the H-2k cross reactivity is related to the presence of a TAA that is maintained after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological significance of beta hCG, HLA and other membrane antigen expression on bladder tumours and their relationship to tumour infiltrating lymphocytes (TIL).

Expression of beta human chorionic gonadotropin (beta hCG) by bladder tumours has been shown to be associated with increased metastases and resistance to treatment with radiotherapy and chemotherapy. Preliminary results from typing frozen tumours using monoclonal antibodies against HLA determinants show reduced or lost expression of one or more antigens in two thirds of patients studied with a trend for more malignant behaviour and inability to generate tumour infiltrating lymphocyte expression using Interleukin-2 in those patients whose tumours demonstrate loss. In this series beta hCG expression was only seen in a subgroup of those demonstrating loss of HLA antigen expression. Studies of beta hCG secreting bladder cancer cell lines showed that it was possible to induce class II HLA antigen expression with gamma Interferon, and that this treatment but not alpha Interferon reduced beta hCG production by the cell line.

Biochemical analysis of a novel H-2 class I-like glycoprotein expressed in five AKR-(Gross virus) derived spontaneous T cell leukemias.

The H-2 class I Ag profiles of five spontaneous AKR (H-2K) Gross virus leukemic cell lines were analyzed. A novel H-2 class I, "alloantigen"-like glycoprotein was immunoprecipitated and isolated from all the tumor cell lines using an H-2Dd-specific mAb 35-5-8. The novel Ag was also recognized in vitro by anti-H-2Dd-specific CTL. In addition, DNA from all the thymomas, but not the DNA from normal adult AKR thymic cells showed a transcribed gene detectable with an H-2Dd-specific oligonucleotide probe. The molecular profile of the novel antigen was further studied by two-dimensional gel electrophoresis and analyzed by a computer based image analyzer system and reverse-phase HPLC tryptic peptide mapping. Its molecular pattern was different from the syngeneic H-2Kk, H-2Dk, and the allogeneic H-2Dd gene products. The two-dimensional gel pattern of the novel H-2 class I molecule had a different overall structure reflected in isoelectric point, number, and distribution of polypeptide spots. The tryptic peptide map analysis showed six peaks exclusively identified with the novel Ag. The calculated degree of homology with the corresponding H-2Dd, H-Dk, and H-Kk peptides was 41, 56, and 51%, respectively. In addition, an unusual cell surface distribution of the novel Ag was observed in most of the leukemic lines. The removal of sialic acid residues by neuraminidase treatment facilitated the detection of the allodeterminants by anti-H-2Dd-specific mAb and CTL. Furthermore, we showed that in one AKR tumor line, 424, there is a close association of the novel Ag with the syngeneic class I molecules. Prior preclearance of the syngeneic class I molecules revealed the presence of the H-2Dd-like allospecificity. The genetic and molecular relationship between the expression of this novel class I-like glycoprotein and the recently sequenced Q5 gene is under current investigation.

Analysis of HLA antigens on germ cells in human semen.

Nucleated cells other than sperm (NCOS) were isolated from human semen by centrifugation on a Ficoll density gradient. Using tissue-specific monoclonal antibodies (mAb) greater than 99% of the NCOS were found to be sperm cell precursors (SpP). These cells were tested for the expression of class I and II (DR, DP and DQ) HLA antigens by using specific mAb. The anti-HLA class I and II and anti-beta 2-microglobulin mAb reacted with less than 1% of the NCOS. This was demonstrated by indirect immunofluorescence microscopy and fluorescence-activated cell sorter analysis. These results were similar to those obtained from testing germ cells in frozen sections of normal adult testis using the same panel of mAb. In mixed lymphocyte-NCOS cultures, the SpP failed to stimulate allogeneic lymphocytes even when different concentrations of cells were used. These results indicate little or no expression of HLA class I and II including the HLA-D (T cell-defined) determinant on the SpP, a phenomenon which could be of biological importance.

Are certain HLA haplotypes responsible for low testosterone levels in males?

The level of testosterone on 138 HLA typed healthy males and 71 male rheumatoid arthritis patients were analysed. HLA-B15 was associated with a lower mean serum testosterone level in males than any other tissue type. This was observed in both the normal and RA groups.

Serum lymphocytotoxic activity in leprosy.

Sera from 167 patients across the spectrum of leprosy and 46 endemic controls were screened for lymphocytotoxic activity (LCA). The Terasaki microdroplet lymphocytotoxicity assay was performed at 37 degrees C and 15 degrees C to test sera for LCA against a panel of lymphocytes from 50 donors which represented most known HLA-ABC antigens. Raised complement-dependent LCA at 15 degrees C was seen in leprosy patients with histories of erythema nodosum leprosum (ENL) or reversal/Type I (I) reactions. Eighty-six per cent of lepromatous (LL) patients with a history of ENL (n = 21, P less than 0.001), 83% of borderline lepromatous (BL) and 88% of borderline tuberculoid patients (BT) with a history of Type I reactions (n = 12, P less than 0.01 and n = 24, P less than 0.001 respectively) had LCA compared to 39% of endemic controls (n = 46). LCA was attributed to IgM on the basis of reduced activity when serum was treated with both dithiothreitol or absorbed with antiserum for IgM. Removal of immune complexes and rheumatoid factor did not influence LCA. LCA-positive sera reacted similarly with allogeneic lymphocytes from either healthy donors or leprosy patients. Moreover LCA-positive sera reacted with autologous lymphocytes. Specificities for HLA-ABC antigens were not identified. The potential role of these autoantibodies, manifested in leprosy patients with hypersensitivity reactions remains speculative.

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.