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To study antitumor immunity in patients with choroidal melanoma, T cells were generated from the peripheral blood of choroidal melanoma patients by mixed lymphocyte/tumor cell culture (MLTC). Because autologous tumors are generally unavailable, an allogeneic choroidal melanoma cell line, OCM-1, was used as the specific stimulus. Lymphocyte cultures from 27 patients were characterized by cell-surface phenotypes, patterns of reactivity towards cells of the melanocytic origin and T-cell-receptor gene usage. Antimelanoma reactivity was found in cell-sorter-purified CD4+ and CD8+ T cell subsets. To analyze this reactivity, sorter-purified CD4+ and CD8+ cells from a MLTC were cloned by limiting dilution in the presence of exogenous interleukin-2 and interleukin-4 as well as irradiated OCM-1. Under these conditions, CD4+ T cells did not proliferate, perhaps because of the absence of antigen-presenting cells. However, CD8+ grew vigorously and 29 cytolytic CD8+ T cell clones were isolated. On the basis of their pattern of lysis of OCM-1, a skin melanoma cell line M-7 and its autologous lymphoblastoid cell line LCL-7, the clones were categorized into three groups. Group 1, representing 52% of the clones, lysed all three target cells, and are alloreactive. However, since OCM-1 and M-7 did not share class I antigens, these clones recognized cross-reactive epitope(s) of the histocompatibility locus antigen (HLA) molecule. Group 2, constituting 28% of the clones, lysed both the ocular and skin melanoma cell lines but not LCL-7, and were apparently melanoma-specific. Unlike classical HLA-restricted cytolytic T lymphocytes, these T cells might mediate the lysis of melanoma cells via other ligands or a more degenerate type of HLA restriction. For the latter, the HLA-A2 and -A28 alleles would have to act interchangeably as the restriction element for shared melanoma-associated antigen(s). Group 3, representing only 10% of the T cell clones, was cytotoxic only to OCM-1, but not to M-7 or LCL-7. These clones may recognize antigens unique to ocular melanoma cells. Our data suggest that choroidal melanoma patients can recognize melanoma-associated antigens common to both ocular and cutaneous melanoma cells, and presumbly their autologous tumor. Thus, choroidal melanoma, like its skin counterpart, may be responsive to immunotherapeutic regimens such as active specific or adoptive cellular immunotherapy.

Human T-cell lymphotropic virus (HTLV-I) was recently reported to be etiologically associated with multiple sclerosis (MS). Genomic DNA from peripheral blood lymphocytes and brain plaques of patients with MS was analyzed for the presence of sequences homologous to the HTLV-I pol gene using the polymerase chain reaction and dot blot techniques. Comparison of DNA amplification patterns between patients with MS, and with control subjects who have other autoimmune conditions, with those in healthy control subjects and with an HTLV-I-infected cell line indicates that HTLV-I pol sequence is not present in the peripheral blood of patients with MS, and that the virus is not active in MS brain plaques.

Expression of T cell receptor (TCR) V alpha genes in tumor-infiltrating lymphocytes (TILs) within intraocular melanoma was studied. Primers for 18 different human TCR V alpha families were used to analyze TCR V alpha-C alpha gene rearrangements in TIL in these melanomas obtained at surgery. A limited number of TCR V alpha genes were expressed and rearranged in these tumors, and TILs expressing V alpha 7 were found in seven of eight of these uveal melanomas. TCR gene usage is also restricted in experimental autoimmune disease, in T cells within organs like skin and other epithelial tissues, and in the brain of patients with multiple sclerosis (MS). The restricted usage of TCR genes in TIL may indicate that a specific antigen in these melanomas is targeted.

Nonradioactive sequence-specific oligonucleotide (SSO) probes specific for the HLA-DP beta locus have been used in a simple dot-blot assay to DP beta-type samples amplified by the polymerase chain reaction (pcr) from Caucasoid (n = 24) and Japanese (n = 23) patients with multiple sclerosis (ms) as well as ethnically matched controls. In contrast to previous reports, no DP beta allele was found to be increased in either patient population. However, the results do show a dramatic difference in the allele frequencies between the two control populations, further emphasizing the need for ethnically matched controls in studies of HLA and disease.

In order to study the immunogenetics of myasthenia gravis (MG), we analysed the TCR and HLA-class II genes from Italian and Californian myasthenic patients. We investigated polymorphisms of the TCR using the full length cDNA probes pGA5 and the pT10 for the alpha and beta chains, respectively. The 6.3 kb and 2.0 kb polymorphic markers, revealed by the PssI enzyme and the alpha chain probe, were shown to be significantly associated with MG. Italian MG patients were HLA typed, and allele frequencies showed a significant association of DR3 and DQw2 with MG. The relative risk calculated for DR3 was 7.4. T-cell proliferative responses to peptides of the AchR alpha chain were also studied and no associations with TCR RFLP analysis or HLA-class II typing were observed.

The identification of activated T cells in the brain of individuals with multiple sclerosis (MS) indicates that these cells are critical in the pathogenesis of this disease. In an attempt to elucidate the nature of the lymphocytic infiltration, we used the polymerase chain reaction to amplify T-cell antigen receptor (TCR) V alpha sequences from transcripts derived from MS brain lesions. In each of three MS brains, only two to four rearranged TCR V alpha transcripts were detected. No V alpha transcripts could be found in control brains. Sequence analysis of transcripts encoded by the V alpha 12.1 region showed rearrangements to a limited number of J alpha region segments. These results imply that TCR V alpha gene expression in MS brain lesions is restricted.

The responses of peripheral blood human T lymphocytes supported by decidual antigen-presenting cells (DAPCs) to a variety of immunogenic stimuli were studied and compared to those of T cells supported by peripheral blood antigen-presenting cells (PAPCs). Antigen-presenting cells were isolated from early normal decidual tissue or peripheral blood by elution with ethylenediamine tetraacetic acid of cells that after Ficoll-Paque separation bear receptors for all have bound to fibronectin. DAPCs pulsed with soluble or particulate antigens induced proliferation of T cells with an efficiency equivalent to PAPCs. Decidual tissue APCs also showed the ability to stimulate auto- and alloreactivity. Treatment with anti-human lymphocyte antigen (HLA) class II antibody and ultraviolet radiation resulted in substantial inhibition of the accessory cell function of DAPCs as well as of PAPCs. Bromodeoxyuridine and light treatment of alloreactive T cells generated in vitro was used to demonstrate that DAPCs primed with a synthetic polypeptide antigen (T,G)-A-L can stimulate only HLA class II-compatible T lymphocytes.

The role of cAMP in lymphocyte proliferation was investigated in the response of a monoclonal T-cell population to a specific antigen and compared to the response to interleukin-2 (IL-2) and allogeneic cells. Myelin basic protein (MBP)-reactive and encephalitogenic T-cell clones were established from long-term lines derived from SJL/J (H-2s) mice. The clone 4b.14a recognizes the peptide sequence 89-101 of the MBP molecule in association with 1-As products of the major histocompatibility complex (MHC). Incubation of 4b.14a cells with syngeneic antigen-presenting cells, previously pulsed with the 89-101 synthetic peptide or with 80 U/ml of IL-2, or allogeneic H-2Ik cells, resulted in a significant increase in the accumulation of intracellular cAMP. This increase was preceded by a peak in membranal adenylate cyclase (AC) activity. Parallel time kinetics but significantly higher cAMP production and AC activity were observed when the cells were treated with pertussis toxin. At the same concentrations the toxin inhibits cellular proliferative responses, assayed by [3H]thymidine incorporation. Our results indicate the involvement of cAMP as a positive signal in the activation of the 4b.14a clone.

Antigen presenting cells were isolated from early human decidua or peripheral blood by elution with EDTA of cells that after Ficoll-Paque separation bear receptors for, and have bound to, fibronectin. Decidual antigen presenting cells (DAPCs) co-cultured with TNP-modified autologous T cells induced the development of cytotoxic anti-TNP T lymphocytes with an efficiency comparable to that of peripheral blood antigen presenting cells (PAPCs). Treatment with anti HLA-class II antibody plus complement and UV radiation resulted in substantial inhibition of the accessory cell function. The T cell mediated lysis of TNP-modified targets is restricted by the major histocompatibility complex. Our results show that HLA-class I molecules are the most prominent restriction elements. The relevance of these data to the immunological mechanisms operating at the feto-maternal interface is discussed.

The responses of human milk lymphocytes (MIL) to a variety of immunogenic stimuli were studied and compared to those of peripheral blood lymphocytes (PBL) from the milk donors. MIL showed a decreased proliferative response to mitogens and allogeneic leukocytes in vitro but displayed the ability to stimulate alloreactivity equivalent to PBL. Neither pretreatment with cell-free autologous milk nor co-cultured MIL were capable of suppressing the proliferative responses of PBL. Moreover, macrophages isolated from milk and pulsed with soluble antigen or allogeneic cells effectively induced proliferation by peripheral blood T cells whereas the response of milk nonadherent cells to antigen presented by peripheral macrophages was very low. MIL respond better to pathogenic enteric E. coli than PBL not as well as PBL to Yersinia enterocolitica. Treatment of MIL with monoclonal antibodies cytotoxic for T cells abolished their response to bacterial antigens. Application of an anti HLA class II antigen monoclonal antibody to mixed lymphocyte or lymphocyte-bacteria cultures resulted in substantial inhibition of the MIL response similarly to that of PBL. The relevance of these data to the immunological needs of the neonate are discussed.

Polymorphic markers in genes encoding that alpha chain of the human T-cell receptor (TcR) have been detected by Southern blot analysis in Pss I digests. Polymorphic bands were observed at 6.3 and 2.0 kilobases (kb) with frequencies of 0.30 and 0.44, respectively, in the general population. Using the polymerase chain reaction (PCR) method, we amplified selected sequences derived from the full-length TcR alpha cDNA probe. These PCR products were used as specific probes to demonstrate that the 6.3-kb polymorphic fragment hybridizes to the variable (V)-region probe and the 2.0-kb fragment hybridizes to the constant (C)-region probe. Segregation of the polymorphic bands was analyzed in family studies. To look for associations between these markers and autoimmune diseases, we have studied the restriction fragment length polymorphism distribution of the Pss I markers in patients with multiple sclerosis, myasthenia gravis, and Graves disease. Significant differences in the frequency of the polymorphic V alpha and C alpha markers were identified between patients and healthy individuals.

In order to investigate the HLA-D clusters associated with DR2 in Israeli Jews, 40 DR2 positive unrelated individuals were studied with a panel of DR2 associated homozygous typing cells (HTC's) which detect the lymphocyte defined specificities HLA-Dw2, Dw12, Dw9 and D-WJR. The results confirmed the existence of two distinct HLA-D clusters associated with the same serologically defined DR2. Of 40 individuals 22.5% (9/40) were Dw2 and 50% (20/40) were Dw12 carriers. Yet, no HLA-D specificity could be assigned to the remaining 11 DR2 positive individuals. In the present study we have defined a unique DR2-associated Dw specificity, HLA-D"AZH". The donor of the HTC was of Moroccan origin and an offspring of a first cousin marriage. This cell was not typeable with the known DR2-associated homozygous typing cells nor with other HTC's which define the well established HLA-Dw1 to Dw11 specificities. It was shown to segregate with DR2 positive HLA haplotypes in family analysis and in a population study, typed out 7 of 11 unrelated DR2 positive, Dw blank individuals, thus identifying a unique and new HLA-D cluster provisionally designated D"AZH".

Immunogenetic studies of a consanguineous family revealed discordance in the inheritance pattern of the HLA-D and HLA-DR antigens in one offspring. The findings suggest a recombination between the HLA-D and HLA-DR loci in one of the paternal chromosomes. Results on segregation of B-cell alloantigens. MLC reactivity, and glyoxalase isoenzyme determination map the DR gene between the HLA-B and D loci.

Human decidual antigen presenting cells (DAPCs) exposed to fetal cells in vitro induce generation of suppressor T cells among a population of peripheral blood lymphocytes. Human lymphocyte antigen (HLA)-class II positive antigen presenting cells were isolated from early normal human decidual tissue and from peripheral blood (PAPCs) by adhering Ficoll-Paque separated cell suspensions to fibronectin. In contrast to PAPCs, DAPCs pulsed with fetal antigens induced a radio-sensitive, Leu 1,2-positive T suppressor cell population. A nylon wool adherent B cell population is required during the in vitro induction of the suppressor cells. These suppressor cells impair primary mitogen and mixed lymphocyte culture (MLC) responses, generation of anti-trinitrophenyl (TNP) cytotoxic T lymphocytes, and antibody response of autologous and allogeneic lymphocytes. Only intact viable embryonic cells can effectively confer upon DAPCs the ability to induce T suppressor cells. The T suppressor cell induction by DAPCs primed with fetal antigens is restricted by the major histocompatibility complex. Our results show that the HLA-DR molecules are the most prominent restriction elements.

The immunogenicity and reactogenicity of Bordetella pertussis vaccine are mediated in part by the S1 subunit of pertussis toxin (PT). To identify the immune epitopes in the S1 subunit of PT, synthetic peptides were prepared and tested for their capacity to induce antibodies in mice with different MHC genotypes. In BALB/c mice, peptides corresponding to sequences 1-17, 70-82 and 189-199 generate T cell proliferative responses, induce the production of antibodies capable of neutralization of the toxin in the Chinese hamster ovary-cell assay, and protect mice from a shock-like syndrome caused by alternate injections of BSA and PT. Protection and neutralization correlated with the ability of these peptides to elicit high anti-PT titers. Different B cell epitopes were detected in other inbred mouse strains. The antibody reactivity against synthetic peptides from two infants vaccinated with pertussis vaccine was tested. These infants had antibodies reactive to a variety of epitopes in the S1 subunit, including peptides 1-17, 70-82, 99-112, 135-145, and 189-199. Thus, it appears that there are multiple T and B cell epitopes in the S1 subunit of PT.

Polymorphic markers for the human T-cell receptor (TcR) genes are described. With a TcR beta-chain probe, polymorphic allelic fragments of 5.4 and 1.8 kb were detected in KpnI digests, and fragments of 12.5 and 11.5 kb were seen in the BglII digests. Polymorphism in alpha chain genes was observed in TaqI-digested DNA samples with bands at 10.2 and 6.2-2.1 kb. Mendelian codominant inheritance for all three polymorphisms was confirmed in family studies. The gene frequencies for these alleles were determined in a sample of 70 normal unrelated Caucasian individuals, and were shown to be in Hardy-Weinberg equilibrium. There were no significant differences in the frequency of these polymorphic alpha and beta alleles between patients with multiple sclerosis and patients with myasthenia gravis as compared to a panel of control healthy individuals.

Early mouse embryos at the two-cell stage were cultured in medium supplemented with sera from women with primary and secondary multiple spontaneous abortions and with long term unexplained infertility as compared to sera obtained from normal fertile women and pooled human male sera. On the basis of microscopic observation and uptake of 3H-thymidine we report a relationship between reproductive histories and the presence of a serum embryo inhibition factor in eight of ten sera samples from women with primary habitual abortions, six of ten sera from women with secondary habitual abortions, and ten of ten sera from women with unexplained infertility. This activity occurs independently of positive maternal antipaternal lymphocytotoxicity. Fractionation of serum samples by ammonium sulphate precipitation, resulted in removal of the embryo-inhibition factor with the IgG fraction in four of five primary habitual abortion cases and in two of five secondary habitual abortion patients, but not in the case of unexplained infertility. We propose that the appearance of such inhibition factor may be of relevance in the etiologies discussed in this paper and may possibly provide the basis for a new classification of idiopathic spontaneous habitual abortions, i.e. positive or negative for the embryo inhibition factor.

Class I human leukocyte antigens (HLA-A, -B) and class II (HLA-DR) antigens were determined in 60 and 30 carefully selected couples with multiple abortions, respectively. The study group was compared with fertile couples with no history of abortion and with a control group consisting of randomly matched women and men from our laboratory cell panel. No significant deviation from the calculated control mating frequencies was observed in the group with habitual abortions. When the study and control couples were grouped by ethnic origin into Ashkenazim and non-Ashkenazim, the frequencies of shared HLA-A, -B, and -DR antigens were similar in both groups. These results do not confirm the observations of greater HLA compatibility between partners of aborting couples reported by other laboratories. Moreover, the results of an informative family in which the woman, after three consecutive spontaneous abortions, conceived and bore a healthy male infant genotypically HLA-identical to his mother are presented. Taken together, these results challenge the concept that compatibility in determinants of the major histocompatibility complex have a major role in habitual abortions.

We have endeavored to ascertain the possible existence of deviant recognition of antigens controlled by the major histocompatibility complex by lymphocytes originating in couples with a history of multiple spontaneous abortions of unknown cause. Human leukocyte antigen (HLA) typing, as well as one-way mixed lymphocyte culture (MLC) tests, were performed on 33 couples with multiple spontaneous abortions and subsequently compared to 30 fertile couples with no abortion history. No significant differences in the frequency of HLA were detected between the fertile and infertile groups. The frequencies of shared HLA-A and -B antigens among members of the couples were similar in both groups. In 70% of the cases studied, the husbands revealed a depressed cellular response to their respective wives; whereas reactivity to cells from other women proved normal. However, the response of allogeneic controls to these infertile women was normal. This implies that women with multiple abortions are not poor stimulators. The specific one-way decrease in the MLC reactivity appeared to be mediated by female-derived suppressor mechanisms and may be considered as one of the causes of the interrupted development of pregnancy in vivo.

Class I human leukocyte antigens (HLA-A, -B) and class II (HLA-DR) antigens were determined in 18 carefully selected couples suffering from unexplained infertility and subsequently compared with 30 normal fertile couples with no history of secondary sterility and with a control group consisting of randomly matched women and men from our laboratory cell panel. No significant differences in the frequencies of HLA antigens were detected between infertile and control groups. The frequency of shared HLA-A, -B, and -DR antigens among members of the couples was similar in all the groups. Finally, the one-way mixed lymphocyte culture showed normal reactivity of both infertile parental pairs in all combinations tested.

The S1 subunit of Pertussis toxin (PT) is responsible for the reactogenicity and in part the immunogenicity of Bordetella pertussis vaccine. The critical residues associated with the immunomodulatory effects of PT were located around Glu140 in the S1 subunit. In man, T cell responses to PT are directed at S1 peptides distinct from Glu140. Two such epitopes, p64-75 and p151-161, are immunogenic in a panel of individuals covering a wide range of HLA genotypes. The response to PT peptides is HLA class II restricted. The response to p64-75 is blocked by an anti-HLA-DQ mAb, while that to p151-161 is blocked by an anti-HLA-DR mAb. These findings may allow for the development of a B. pertussis vaccine free from reactogenicity.

The expression of human leukocyte antigens (HLA) on highly purified human ejaculated sperm cells was studied using the sensitive enzyme-linked immunosorbent assay (ELISA) technique and a wide panel of monoclonal antibodies to class I and class II HLA. In addition, the stimulatory capacity of these cells was tested in mixed cultures of lymphocytes and spermatozoa, and the levels of RNA homologous to the HLA class I and class II genes were determined. The results obtained using the ELISA indicate that the class I and class II HLA serologically defined antigens are weakly expressed on the cell surface of the mature spermatozoa. Highly purified sperm cells consistently stimulated heterologous lymphocytes but not when HLA-DR compatibility was observed between stimulator and responder. The proliferative response of lymphocytes induced by sperm cells was lower than the response obtained in a lymphocyte-lymphocyte combination, though the kinetics of the response were similar in both cases. In addition, it was found that spermatozoa contained RNA species homologous to HLA class II DR beta and DQ beta genes sequences but not to HLA class I sequences. The levels of these RNA species were significantly reduced after interferon stimulation. Lymphocytes that served as positive control were found to contain RNA complementary to both HLA class I and class II genes.