Absence of detectable HL-A antigens on cultured fibroblasts in progeria.

HL-A phenotypes were determined by a cytotoxicity assay on circulating lymphocytes of two boys with the premature aging syndrome, progeria. The antigenic phenotypes were not unusual inasmuch as they are frequently seen in the normal population. However, none of these antigens could be detected by the same assay on cultured skin fibroblasts from either individual, even when a significant mitotic potential remained before cessation of growth. Fibroblasts from normal donors were concordant with corresponding lymphocytes for HL-A antigens and maintained these antigens until mitotic division had virtually ceased. Absorption studies on fibroblasts with two HL-A2 antisera revealed that HL-A antigens are either absent or have a drastically reduced expression on progeric fibroblasts. The data are in accord with the concept of an immunological role in the pathogenesis of progeria.

Juvenile-onset diabetes HLA-A, -B, -C, and -DR alloantigens.

We studied the distribution of HLA-A, -B, and -C antigens in 94 juvenile-onset diabetic patients and of HLA-DR antigens in 62 of these patients. The frequencies for HLA-B15, -B40, and -Cw3 were significantly increased in the patient group as compared with the control group. With respect to the B-cell specificities, DRw4 was significantly increased in the patients. Analysis of the data to detect the possible presence of primary and secondary associations between HLA alleles and diabetogenic gene(s) indicated that DRw4 possessed a primary association with the diabetogenic gene(s). As a result, B15, B40, and Cw3 possessed secondary associations.

Cloning of a short HLA-DQ beta locus-specific cDNA probe: typing for DQw specificities.

A short HLA-DQ beta locus-specific (141 bp) probe was cloned from the full-length pII-beta-1 cDNA. Pst 1-digested genomic DNA from homozygous typing cell lines (HTC) was hybridized with this short DQ beta locus-specific, pDQ beta 141, probe. Restriction fragment length polymorphism (RFLP) patterns generated with this DQ beta locus-specific probe were compared with those obtained with the full-length (627 bp) DQ beta, pII-beta-1, probe. The results demonstrate that the RFLP patterns with the pDQ beta 141 probe were very simple, and no crossreacting DR beta and DX beta bands were observed. DQw1, 2, 3 and 4 specificities could each be identified by a single RFLP.

Polymorphism of three HLA-DR7 bearing major histocompatibility complex extended haplotypes.

We have studied the complexity of HLA class II region in DR7 bearing extended haplotypes by restriction fragment length polymorphism (RFLP). Genomic DNA from homozygous cell lines and from unrelated individuals was digested with a number of restriction endonucleases and probed with DR alpha, DR beta, DQ alpha and DQ beta cDNA probes. We detected RFLPs that distinguished subspecificities of DRA, DRB1, DQA1 and DQB1 chain genes. On the basis of polymorphism in these genes, three distinct types of DR7 bearing extended haplotypes could be identified: (1) B44 or Bw47 or B14, DR7a (DRA1, B1.1), DRw53a (DRA1,B4), DQw2 (DQA1.1, B1.1); (2) B13 or B40, DR7b (DRA1, B1.2), DRw53a (DRA1, B4), DQw2 (DQA1.1, B1.1); and (3) Bw57, DR7c (DRA2, B1.2), DRw53b (DRA2, B4), DQw9 (DQA1.2, B1.2). Available evidence indicates that independent examples belonging to a haplotype were similar in RFLP patterns, suggesting that most examples of an extended haplotype belonging to a subtype are similar. The results in the present study have important implications for immune function and disease susceptibility.

The influence of lymphocyte counts and disease progression on circulating and inducible anti-HIV-1 cytotoxic T-cell activity in HIV-1-infected subjects.

OBJECTIVE: To evaluate specific anti-HIV cytotoxic T-lymphocyte (CTL) activity in relation to basic clinical and laboratory parameters used to follow HIV infection. METHODS: Lymphocytes from HIV-1-infected subjects with different clinical and immunologic features of HIV infection were tested for circulating and inducible anti-HIV CTL activity using autologous B-lymphoblastoid cells infected with recombinant vaccinia viruses expressing the HIV gag, pol and env genes as targets. Anti-HIV CTL were induced by stimulation with HIV-infected autologous lymphoblasts in vitro. RESULTS: We detected circulating anti-HIV CTL in asymptomatic subjects exclusively and found a significant association (P < 0.01) between CD8+ lymphocyte counts > or = 900/microliters blood and detectable levels of circulating anti-HIV CTL. Subjects with circulating anti-HIV CTL also had a higher mean CD8+ lymphocyte count than those without detectable circulating activity (P < 0.001), but there was no significant difference in mean CD4+ lymphocyte count. CD8+ human histocompatibility leukocyte antigen (HLA) class I-restricted anti-HIV CTL were induced in all HIV-infected subjects tested following stimulation with HIV-infected autologous lymphoblasts in vitro. In subjects without detectable circulating anti-HIV CTL, circulating HLA-DR+ cells contributed to anti-HIV CTL activity induced by stimulation with HIV or concanavalin A in vitro. CONCLUSIONS: Circulating anti-HIV CTL activity is associated with CD8+ lymphocyte counts > or = 900/microliters. Anti-HIV CTL retain proliferative and functional capacity following in vitro stimulation with HIV and interleukin-2 through all stages of HIV infection. Persistent inducible anti-HIV CTL activity in subjects with advanced HIV disease and without circulating CTL suggests impaired activation and/or proliferation of the CTL in vivo.

Human Y-box transcription factors: sequences of two new YB-1 alleles.

The complete nucleotide (nt) sequences of two new alleles of the human Y-box-binding protein are described. These alleles show over 93% nt similarity to published sequences of YB-1.

Association of renal failure with Lewis incompatibility after allogeneic bone marrow transplantation.

The cause of the renal failure that occurs in approximately 20 percent of patients following allogeneic bone marrow transplantation is poorly understood. A patient is described in whom acute renal failure occurred one week after allogeneic bone marrow transplantation. The onset of the renal failure was associated with the demonstration of anti-Lewis antibodies in the patient's serum, which could only have been derived from donor lymphocytes. Recovery of renal function coincided with the disappearance of the Lewis antibody. It is postulated that Lewis incompatibility between graft and host tissue may have contributed to the renal failure in this patient and that incompatibility associated with determinants present on renal cells may account for other instances of acute renal failure following allogeneic bone marrow transplantation.

Effect of blood transfusions from different H-2 donors on immune responses in mice.

We studied the effect of blood transfusions (BT) from different H-2 donors on the induction of suppressor cells (SC) and of MLC inhibitory activity in serum in a drug-unmodified mouse model. Balb/c (H-2d) mice were transfused at weekly intervals with whole blood from donors of three strains using two transfusion protocols. In protocol I, blood was transfused first from C3H/HeJ (C3H) (H-2k), then C57Bl (H-2b), and then SJL (H-2s) strain mice, and in protocol II the order of blood donors was reversed. Spleen cells and serum samples were obtained from the transfused mice one and two weeks after the last BT. In both transfusion protocols, the kinetics of responses of cells from recipient transfused mice to cells from the blood donors in MLC were similar to those of cells from nontransfused mice. The peak responses of cells from transfused mice were consistently lower than those of cells from nontransfused mice. In cell-mixing experiments, radiosensitive SC capable of inhibiting responses of Balb/c mice to cells from all three blood donors in MLC could be demonstrated one week after the last transfusion in both protocols. Two weeks after the last BT, SC were demonstrable only against the first (C3H) blood donor in protocol I, and against all three blood donors in protocol II. Serum obtained one week after transfusion in protocol I inhibited responses of Balb/c mice to stimulator lymphocytes from all three blood donors in MLC. Serum obtained two weeks after BT, however, inhibited responses of recipient mice only to the first blood donor. In contrast, in protocol II, serum obtained both one and two weeks after BT did not cause inhibition of responses of cells from Balb/c mice to blood donor cells in MLC. Similar results were obtained when Balb/c mice were transfused at weekly intervals with whole blood from either C3H or from SJL mice. The data suggest that the induction of SC and/or MLC-inhibitory activity in the serum after BT is dependent on the H-2 type of the first blood donor.

Blood-transfusion-induced suppression of cytotoxic T lymphocyte responses in mice.

B6AF1 (H-2KbkDbd) mice were transfused weekly with 0.1 ml of whole blood from DBA/2 (H-2d) mice. One week after each transfusion, spleen and serum samples were collected. Blood transfusions did not induce blood donor alloantigen-specific cytotoxic T lymphocytes (CTL) in spleens of B6AF1 mice. When spleen cells from transfused mice were sensitized to alloantigens in mixed lymphocyte culture in vitro, it was observed that 1-3 transfusions induced suppression of blood donor-specific CTL activity. No suppression of CTL activity was found after 4 transfusions. The cell-mixing experiments demonstrated that the suppression of CTL activity following initial 2 blood transfusions was due to the presence of suppressor cells. The presence of antibodies in sera of transfused B6AF1 mice capable of inhibiting CTL was investigated using the CTL-inhibition test. In these experiments, cytotoxic T lymphoblasts generated in MLC in vitro by culturing normal B6AF1 spleen cells with x-irradiated DBA/2 cells were treated with serum before testing them for cytotoxicity. The antibodies capable of inhibiting CTL responses were demonstrable in sera from transfused mice. Three and four BT sera caused significant inhibition of CTL responses. The CTL-inhibitory antibodies were specific for effector cells of the B6AF1 mice and for target cells of the blood donor DBA/2 mice. These results suggest that the inhibition of CTL responses is caused by antibodies directed against the recognition sites on effector T lymphocytes. The data from this study, therefore, demonstrate that BT cause suppression of the recipient's CTL responses against alloantigens present on the blood donor, and that this suppression is mediated by suppressor cells after the initial 1 to 2 transfusions and by antibodies directed against the CTL antigen-specific receptors after subsequent transfusions.

MLC-inhibiting antibodies in mice after blood transfusions.

Balb/c (H-2d) mice were transfused weekly with 0.1 ml of whole blood from C3H/HeJ (C3H) (H-2k) mice. One and two weeks after each transfusion, mice were bled and the sera were collected and pooled. Serum samples from transfused mice were absorbed with erythrocytes and spleen cells from C3H mice, and then heat-inactivated. The presence of antiidiotypic antibodies in these sera was investigated using the mixed lymphocyte culture (MLC) inhibition test, in which spleen cells from normal Balb/c mice were tested for proliferative responses to x-irradiated C3H stimulator cells in the presence of sera from transfused mice. Sera obtained from transfused mice caused significant inhibition of responses in MLC. This inhibition in MLC was specific for stimulator cells from the blood donor (C3H), and little or no inhibition was observed with stimulator cells from third-party SJL mice. In addition, the inhibitory effect in MLC was specific for responder cells from the recipient Balb/c mice and no inhibition was observed with responder cells from blood donor C3H mice. These results suggest that blood transfusions induce antiidiotypic antibodies that can block the T cell antigen-specific receptors and cause inhibition of responses of the recipient mice to blood donor alloantigens in MLC. Thus in this strain combination, disparate for the entire H-2 region, antiidiotypic antibodies developed after 1 blood transfusion. These findings are in contrast to our earlier published results in which antiidiotypic antibodies developed after 3 transfusions in recipient-blood donor combination incompatible for the K and I regions. These data suggest that the development of antiidiotypic antibodies may be related to the level of histoincompatibility at the H-2 complex between recipient and the blood donor.

Induction of antibodies by blood transfusions capable of inhibiting responses in MLC.

B6AF1 (H-2KbkDbd) mice were transfused weekly with 0.1 ml of whole blood from DBA/2 (H-2d) mice. One week after each transfusion, the mice were bled and the sera were collected and pooled. The presence of antiidiotypic antibodies in these sera was investigated using the mixed-lymphocyte culture (MLC) inhibition test, in which spleen cells from normal B6AF1 mice were treated with sera from transfused B6AF1 mice, washed, and then tested for their responses to DBA/2 stimulator cells. The sera collected following 3 and 4 transfusions caused a significant inhibition of responses in MLC, but sera obtained after 1 and 2 transfusions caused little or no inhibition. This inhibition was specific for stimulator cells from the blood donor (DBA/2) and was not observed against third-party SJL (H-2s) stimulator cells. In addition, the suppressive effect in MLC was specific for responder cells from the recipient B6AF1 mice, and no suppression was observed with responder cells from C3H (H-2k) and SJL mice. Treatment of DBA/2 stimulator cells with the serum caused no inhibition in MLC. The MLC inhibitory activity of the serum decreased gradually from the first to the third week following 4 transfusions, although a significant inhibition was still demonstrable after 3 weeks. These findings suggest that multiple blood transfusions induce antiidiotypic antibodies that can block the T cell antigen-specific receptors and cause suppression of the recipient's responses against the donor's alloantigens in MLC.

Induction of antiidiotypic antibodies by blood transfusions. Characterization of T cell alloantigen-specific receptors by sera from transfused mice.

Balb/c (H-2d) mice were transfused weekly with 0.1 ml of whole blood from C3H (H-2k) mice. One week after 3 blood transfusions (BT), the mice were bled and the sera collected and pooled. The 3BT serum was absorbed twice with C3H lymphocytes and IgG isolated by ion-exchange chromatography. Balb/c anti-C3H, Balb/c anti-Balb/c, and Balb/c anti-SJL (H-2s) lymphocytes were generated in the mixed lymphocyte cultures and metabolically labeled with 35S-methionine. Cell lysates were prepared from labeled lymphocytes and precleared by absorption with normal mouse serum. Immunoprecipitation was carried out by 3BT-IgG and NMS-IgG. 3BT-IgG specifically precipitated 7 molecules (30K, 60K, 72K, 86K, 92K, 97K, 145K) from Balb/c anti-C3H lymphocytes. In contrast, 3BT-IgG did not precipitate these molecules from Balb/c anti-Balb/c or from Balb/c anti-SJL lymphocytes. The data suggest that BT induces antibodies directed against the blood donor alloantigen-specific receptors on recipient's T lymphocytes.

Inhibition of injury induced thromboatherosclerotic lesions by anti-platelet serum in rabbits.

We have previously shown that repeated or continuous intimal injury caused by an indwelling aortic catheter causes a variety of lesions in rabbits maintained on a diet unsupplemented by lipid. These include fatty streaks, lipid-free fibrous plaques and lipid-rich raised thromboatherosclerotic plaques. Whether lipid-rich raised lesions are a result of injury or co-existing thrombosis or both is not clear. The present experiment was designed to answer this question. Anti-platelet serum (APS) to washed sonicated rabbit platelets was raised in sheep. PE 60 polyethylene catheters were placed in the aortas of 35 rabbits by way of a femoral artery. The animals were randomly divided into 2 groups. The experimental group (17 rabbits) received an intravascular injection of 1.0 ml of APS followed 8 hours later by a subcutaneous injection of 0.5 ml. Thereafter, 0.5 ml APS was given subcutaneously each day for 13 additional days. The control group (18 rabbits) received no APS. Platelet counts were done prior to surgery, at 5 minutes following surgery, at 4 days, 8 days and just prior to killing. Extent of lesions was estimated by photographing the opened aortas, projecting the photographs on cardboard, cutting out the areas occupied by the different lesions and weighing the cardboard. The mean weight of raised lesions in the control group was 6 to 7 times greater than in the experimental groups. Statistical analysis of this difference based on Welsh's "t" test for unequal variances was highly significant (P less than 0.001). Platelet counts in the experimental groups varied from 0 to 20,000 at 14 days. In animals with platelet counts less than or equal to 1,000 mm3 raised lesions were completely prevented. In a second experiment the effect of APS was compared with normal sheep serum (NSS). A similarly significant inhibition of raised lesions occurred in the APS group. The extent of lesions in the NSS control was similar to that in the No-APS group of the first experiment. These findings indicate that thrombosis is more important than injury in the development of lipid-rich raised lesions.

Peptide transport in human lymphoblastoid and tumor cells: effect of transporter associated with antigen presentation (TAP) polymorphism.

CD8+ T-cells recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. These peptides bind to MHC class I molecules in the endoplasmic reticulum (ER) lumen. Antigenic peptides are translocated from the cytosol to the lumen of ER by transporter associated with antigen presentation (TAP) proteins. In this study, it is shown that TAP1 polymorphism influences the peptide substrate specificity in human B-lymphoblastoid and tumor cell lines. TAP1A and 1C alleles specifically enhance translocation of model peptides containing basic C-terminal amino acid residue. However, TAP1B allele does not show specificity for the peptide C-terminus. Human basophilic leukemia (Ku812), and hepatocellular carcinoma (PLC/PRF/5) cells express TAP1 molecules and exhibit TAP-mediated allele-specific peptide uptake after gamma-interferon (gamma-IFN) treatment. Ku812 cells express TAP1A and preferentially take up antigenic peptides with a basic C-terminus, however, PLC/PRF/5 cells with the TAP1B allele take up low but equivalent levels of peptides regardless of basic, acidic, or hydrophobic C-termini. Moreover, TAP2 polymorphisms have no influence on the peptide translocation in normal or tumor cell lines. In addition, Daudi, a beta2-microglobulin (beta2m) deficient human Burkitt lymphoma, cell line also showed TAP-dependent peptide uptake. Taken together, these results suggest that human TAP1 but not TAP2 polymorphisms influence the antigenic peptide transport and that this transport is independent of beta2m in this system.

Genetics of rheumatoid arthritis (RA): two separate regions in the major histocompatibility complex contribute to susceptibility to RA.

We analyzed HLA-DR antigens and microsatellite Bat2 alleles in 97 adult caucasian patients with classical seropositive rheumatoid arthritis (RA) and 95 normal healthy controls. The results demonstrate that the prevalence of microsatellite Bat2 138 allele was significantly higher in RA-susceptibility DRB1 QKRAA/QRRAA epitope-negative patients as compared with normal controls. Analysis of the data suggested that Bat2 138 allele has primary association with RA-susceptibility in QKRAA/QRRAA epitope-negative patients. The Bat2 138 allele thus provides an additional risk in RA-susceptibility. In addition, microsatellite Bat2 138 allele showed a highly significant positive association with microsatellite D6S273 138 allele, which has similar (identical) association with RA development in DRB1 QKRAA/QRRAA epitope-negative patients. The present data demonstrate that DRB1 QKRAA/QRRAA epitope and microsatellite Bat2 138/D6S273 138 alleles more completely define the risk for development of RA. The results in the present study therefore suggest that two regions in MHC, class II (DRB1) and class III (Bat2 and D6S273 in HSP70-Bat2 region), contribute to susceptibility to RA.

Markedly decreased expression of TAP1 and LMP2 genes in HLA class I-deficient human tumor cell lines.

HLA class I antigens of the human major histocompatibility complex play an important role in immune response. These molecules present foreign antigenic peptides to cytotoxic T lymphocytes and thereby play a role in the immune surveillance of cells infected with virus or other intracellular pathogens or altered by malignant transformation. A marked deficiency or lack of expression of these antigens has been reported in a variety of human neoplasms. In the present study, we examined the expression of class I alpha chain, beta 2-microglobulin, TAP (TAP1 and TAP2) and LMP (LMP2 and LMP7) genes in a number of human tumor cell lines including small-cell lung carcinoma, hepatocellular carcinoma, colon adenocarcinoma and basophilic leukaemia. These cell lines were deficient in expression of both class I alpha chain and beta 2-microglobulin gene products. In addition, these cell lines lacked the products of MHC-encoded proteasome subunit LMP2 as well as the putative peptide transporter TAP1 genes. In contrast, TAP2 and LMP7 genes were expressed in these cell lines. Treatment of cells with gamma-IFN markedly enhanced the expression of class I alpha chain, beta 2-microglobulin, TAP1 and LMP2 genes with a concomitant increase in cell-surface expression of class I molecules. The upregulation of TAP1 and LMP2 expression is associated with increased class I expression, suggesting that endogenous antigens, e.g. tumor antigens, could be presented by class I molecules following treatment of tumor cells with gamma-IFN.

HLA-DQ and TAP2 genes in patients with insulin-dependent diabetes mellitus.

We examined the prevalence of HLA-DRB1, DQB1, DQA1 and TAP2 genes in children with insulin-dependent diabetes mellitus (type 1 diabetes). These HLA and TAP2 alleles were identified by dot-blot analysis of polymerase chain reaction (PCR)-amplified genomic DNA with sequence-specific oligonucleotide probes. The results show that those DQB1 alleles, which carry non-aspartic acid at position 57, in conjunction with DQA1 alleles carrying arginine at position 52, are strongly associated with susceptibility to type 1 diabetes. The prevalence of the TAP2* 0201 allele in diabetic patients was significantly lower than that in normal controls. Analysis of the data suggests that DQ alleles have the primary association with type 1 diabetes and that the association of TAP2 alleles with the disease is secondary.

Quantitative studies of alloantigen-reactive human lymphocytes in primary and secondary MLC.

The number of alloantigen-reactive cells in human peripheral blood was estimated by a limiting dilution analysis. In MLC combinations between allogeneic unrelated donors, the frequency of alloantigen-reactive cells ranged between 1:241 to 1:486. The frequency of alloantigen-reactive cells to the specific donor was increased six- to eight-fold after priming in MLC. The results demonstrate that specific "memory" cells are enriched in long-term MLC. In limiting dilution experiments between HLA-identical siblings, the frequency of alloantigen-reactive cells ranged from 1:1160 to 1:1740. The data point to the existence of a lymphocyte-defined antigen system controlled by a genetic region that is not linked to HLA. The results suggest that the lymphocyte clones that are able to react to non-HLA antigens probably consist of a small number of lymphocytes. Finally, the response of these clones of cells to non-HLA antigens was observed only under conditions where responder cells were limiting.

Role of blood transfusions on the induction of antibodies against recognition sites on T lymphocytes in renal transplant patients.

We have tested sera from 23 renal allograft recipients to study the effects of blood transfusions on the induction of antibodies directed against recognition sites on T lymphocytes. The results demonstrate that antibodies capable of inhibiting responses in MLC could be induced by blood transfusion. This inhibition in MLC is observed by treatment of responder lymphocytes with serum plus rabbit complement and is mediated by IgG antibodies. Also, the inhibitory effect is specific for certain responder cells and is not mediated by antibodies against common surface antigens of either the responder or the stimulator lymphocytes. The antibodies inhibiting proliferative responses in MLC against antigens present on the kidney donor were demonstrable in renal transplant recipients with functional allografts, but not in patients who had rejected the graft. The data suggest that antibodies directed against recognition sites on T lymphocytes could be induced by blood transfusions and these antibodies may be associated with prolonged graft survival.

In vitro cell-mediated cytotoxicity to the male specific (H-Y) antigen in man.

We have studied the role of HLA antigens in restricting specificity of the cytotoxic lymphocytes (CTL). CTL's developed between female responder/male stimulator combinations, were tested for H-Y antigen killing in cell-mediated lympholysis. Two CTL's demonstrated HLA-restricted H-Y cytotoxicity. In both instances, the responders are married parous females and both are positive for HLA-A2. These CTL's lysed target cells from donors who are either positive for the sensitizing HLA antigen or who are HLA-A2-positive males. On the other hand, one CTL where the HLA-A2-positive responder is not a parous female did not show HLA-restricted H-Y cytotoxicity. Also, CTL's where responders do not carry HLA-A2 showed no H-Y cytotoxicity. The data suggest that pregnancy(ies) is sufficient in itself to induce HLA-restricted H-Y cytotoxicity and that it can be recalled by in vitro stimulation with lymphocytes from an unrelated male donor. Also, in these studies HLA-restricted H-Y cytotoxicity was obtained only with targets that shared HLA-A2 with the effectors.