The genomic structure of human V beta 6 T cell antigen receptor genes.

Six genomic clones were characterized containing members of the human V beta 6 subfamily of T cell antigen receptor genes. There were four major findings. (a) New V beta genes were discovered, including V beta 6.10, V beta 13.4, V beta 13.5, and V beta 5.5. (b) Members of the V beta 13, V beta 6, and V beta 5 subfamilies cluster together in the V beta locus and may have evolved through multiple duplication events of an ancestral cassette containing V beta 13-V beta 6-V beta 5 genes. These V beta subfamilies are used by an estimated one-third of T cells in humans and probably represent a highly useful component of the V beta repertoire. (c) The promoters of V beta 13, V beta 6, and V beta 5 genes contain conserved decamer motifs, but discrete differences were observed between promoters of different V beta subfamilies, raising the question of different transcriptional control depending on V beta subfamily usage. (d) The new V beta 6.10 gene is probably a pseudogene, which may have been inactivated due to retrotransposition of Alu elements into its promoter region, a mutation affecting a highly conserved cysteine residue or mutations of the 3' recombinase signal sequence.

Human malignant T cells capable of inducing an immunoglobulin class switch.

Evidence is presented for the existence of a "switch" T cell derived from a patient with mycosis fungoides/Sezary's syndrome. The serum immunoglobulin profile in this patient revealed high IgG and IgA but no detectable IgM. Peripheral blood mononuclear cells from this patient secreted only IgG and IgA in the presence of pokeweed mitogen. T cells (Trac) co-cultured with normal allogeneic non-T cells and pokeweed mitogen resulted in only IgG and IgA PFC, with little or no IgM secretion. There was no evidence of active suppression of IgM. Rather, these T cells appeared to induce an Ig class switch from IgM to IgG and IgA, when co-cultured with mu+ tonsillar B cells. Further evidence was obtained using mononuclear cells derived from a patient with immunodeficiency and hyper-IgM, a syndrome characterized by a lack of IgG and IgA secretion. The addition of Trac cells to either peripheral blood mononuclear cells or non-T cells from a patient with hyper-IgM syndrome resulted in new secretion of IgG, with a concomitant decrease in IgM secretion, whereas control T cells were not effective in inducing secretion of any isotype other than IgM. Isolated Tac+ T cells from Trac appear to be responsible for this effect.

Stimulation of a subset of normal resting T lymphocytes by a monoclonal antibody to a crossreactive determinant of the human T cell antigen receptor.

A previous study from this laboratory described a monoclonal antibody, S511, that reacted with the T cell antigen receptor on a human T cell leukemia and also on 1-2% of circulating T lymphocytes in all normal individuals tested. The data presented in the present study demonstrate that, when normal T lymphocytes are cultured with or without irradiated non-T cells in the presence of soluble S511 antibody, a concentration- and time-dependent proliferation of the S511-reactive population occurred. Proliferation indices as high as 184 times greater than control were observed, which represents a major stimulatory effect on the initially minor S511+ subset. When S511+ cells were studied for evidence of prior activation, they were shown to be unresponsive to interleukin 2 (IL-2) unless exposed to S511 antibody, and were shown to be in the G0/G1 phase of the cell cycle. Thus, the S511 antibody activated resting normal T cells in a manner analogous to specific antigen binding to the T cell antigen receptor. The leukemic S511 molecule has been shown previously to differ from most other antigen receptors in the mobility of the two chains at 43 and 38 kD and the neutral isoelectric point of each chain. Expansion of reactive normal cells by S511-Sepharose permitted the development of IL-2-dependent T cell lines enriched for S511-bearing cells. The antigen receptor molecules on one such polyclonal S511-enriched T cell line were immunoprecipitated with S511 antibody and shown to have comparable mobility to that present on the leukemic cells, but to possess a greater heterogeneity of mobility. Thus, the leukemic cells and normal cells express similar T cell receptor molecules. The differences in the S511 T cell antigen receptor molecule possibly relate to differences in glycosylation or polypeptide structure.

Clonal populations of T cells in normal elderly humans: the T cell equivalent to "benign monoclonal gammapathy".

To determine whether T cells, like B cells, can become clonally expanded in normal individuals as a function of age, we compared the T cell V beta repertoire of cord blood to that of peripheral blood from normal donors over 65 yr of age. T cells from elderly subjects contained expanded subsets (greater than the mean+three standard deviations) of T cell receptor (TCR) V beta populations. These expanded subsets were observed primarily among CD8, but not CD4 cells, represented up to 37.5% of all CD8 cells, and were present in most elderly subjects. An expanded V beta 5.2/3 CD8 subset and a V beta 6.7a CD8 subset from separate donors were analyzed by reverse transcriptase-polymerase chain reaction, cloning and sequencing of the TCR beta chain VDJ junction. In both cases the expanded subsets were mono- or oligoclonal while control CD4 populations were polyclonal. Using two-color flow cytometry it was possible to identify the expanded V beta 6.7a subset as CD8+ CD28-CD11b+ cells. In three of five random old subjects similar expansions of V beta subsets were found specifically in the CD8+ CD28- subpopulation, an interesting subset of cytotoxic T lymphocytes, known to lack proliferative responses to TCR stimuli. It is common practice to use the demonstration of clonality as a diagnostic indicator for T cell lymphoma/leukemia. In view of the high frequency of expanded T clones of T cells in normal elderly subjects the diagnostic usefulness of this test should be reexamined.

A new HLA-linked T cell membrane molecule, related to the beta chain of the clonotypic receptor, is associated with T3.

The 38 kD molecule is noncovalently associated with beta 2 microglobulin (beta 2m)-free HLA heavy chain-like molecule, and thus forms a second heterodimer distinct from the clonotypic alpha/beta T cell receptor expressed by the same clone of leukemia cells. This second heterodimer (38 kD/HLA) is variably expressed and appears to be associated with the T3 molecule. We suggest, therefore, that it has a functional role in T cell activation.

Highly biased CDR3 usage in restricted sets of beta chain variable regions during viral superantigen 9 response.

Superantigens encoded by the mouse mammary tumor virus can stimulate a large proportion of T cells through interaction with germline-encoded regions of the T cell receptor beta chain like the hypervariable region 4 (HV4) loop. However, several lines of evidence suggest that somatically generated determinants in the CDR3 region might influence superantigen responses. We stimulated T cells from donors differing at the BV6S7 allele with vSAG9 to assess the nature and structure of the T cell receptor in amplified T cells and to evaluate the contribution of non-HV4 elements in vSAG recognition. This report demonstrates that vSAG9 stimulation caused the expansion of TCR BV6-expressing T cells, although to varying degrees depending on the BV6 subfamily. The BV6S7 subfamily was preferentially expanded in all donors, but in donors homozygous for the BV6S7*2 allele, a significant number of BV6S5 T cells were amplified and showed a highly biased beta chain junctional region (BJ) and CDR3 usage. As CDR3 regions are involved in major histocompatibility complex (MHC)-peptide interaction, such a selection is highly suggestive of an intimate MHC-TCR interaction and would imply that the topology of the MHC-vSAG-TCR complex is similar to the one occurring during conventional antigen recognition.

Mycoplasma superantigen is a CDR3-dependent ligand for the T cell antigen receptor.

Superantigens are defined as proteins that activate a large number of T cells through interaction with the Vbeta region of the T cell antigen receptor (TCR). Here we demonstrate that the superantigen produced by Mycoplasma arthritidis (MAM), unlike six bacterial superantigens tested, interacts not only with the Vbeta region but also with the CDR3 (third complementarity-determining region) of TCR-beta. Although MAM shares typical features with other superantigens, direct interaction with CDR3-beta is a feature of nominal peptide antigens situated in the antigen groove of major histocompatibility complex (MHC) molecules rather than superantigens. During peptide recognition, Vbeta and Valpha domains of the TCR form contacts with MHC and the complex is stabilized by CDR3-peptide interactions. Similarly, recognition of MAM is Vbeta-dependent and is apparently stabilized by direct contacts with the CDR3-beta region. Thus, MAM represents a new type of ligand for TCR, distinct from both conventional peptide antigens and other known superantigens.

Allelic variations in the human T cell receptor V beta 6.7 gene products.

Polymorphisms of human TCR gene products have been suggested by the description of a mAb, OT145, that identifies a subset of TCRs in some individuals but not in others (6). Here we demonstrate that this mAb detects a TCR allotype of the V beta 6.7 gene. Two allelic products of this V gene differ by two nonconservative amino acid substitutions. The mAb OT145 appears to react with V beta 6.7 a gene products ("+" allele), but not with V beta 6.7b gene products ("-" allele). This represents the first direct demonstration that TCR V gene allotypes exist and provides a possible explanation for immune responses under the control of TCR V genes and for disease associations with TCR V genes.

Helper T cell-dependent human B cell differentiation mediated by a mycoplasmal superantigen bridge.

Experimentally induced murine graft-vs.-host disease may be characterized by hypergammaglobulinemia, autoantibody formation, and immune complex-mediated organ system damage that mimics SLE. These autoimmune phenomena are mediated by abnormal Th-B cell cooperation, across MHC disparities, in which donor-derived allospecific Th cells recognize and interact with MHC class II antigens on the surface of recipient B cells. Microbial toxins, termed superantigens, which bind to MHC class II molecules and activate selected T cells based on TCR variable gene usage, may induce a similar form of Th-B cell interaction. In the present study, we generated and characterized human Th cell lines reactive with the Mycoplasma arthritidis superantigen (MAM). The essential observation is that resting human B cells bind MAM and present it to superantigen-reactive autologous or allogeneic Th cells, resulting in both Th cell activation and a consequent polyclonal Ig response by the superantigen-bearing B cells.

Detection of a gamma interferon-induced protein IP-10 in psoriatic plaques.

The pathologic features of psoriatic plaques are inflammation and increased epidermal turnover. IP-10, a cytokine the expression of which is induced by gamma-interferon, is a member of a family of soluble mediators with inflammatory and growth-promoting activities. IP-10 protein was detected in keratinocytes and the dermal infiltrate from active psoriatic plaques using an affinity-purified rabbit anti-IP-10 antibody in immunoperoxidase studies. Successful treatment of active plaques decreased IP-10 expression in plaques. These results were corroborated by Northern blot analysis with an IP-10 cDNA probe. We have previously detected activated T cells and HLA-DR keratinocytes in active psoriatic plaques. Since IP-10 is detected in delayed cellular immune responses, the present study further points to the role of ongoing cellular immune responses in the pathogenesis of psoriasis.

Detection of transforming growth factor alpha in normal, malignant, and hyperproliferative human keratinocytes.

Transforming growth factor alpha (TGF-alpha) is a 50-amino acid peptide, previously demonstrated only in transformed cell lines and human tumors, which is structurally homologous to epidermal growth factor (EGF). TGF-alpha expression in keratinocytes from normal individuals, patients with psoriasis, and patients with malignant skin diseases was investigated using an mAb raised against synthetic human TGF-alpha. mAb A1.5 reacted with TGF-alpha, but not EGF, in a sensitive ELISA. Keratinocytes in eight nodular basal cell carcinomas, one morpheic basal cell carcinoma, and one squamous cell carcinoma demonstrated intense membranous immunoperoxidase staining with mAb A1.5. Of even greater interest was the observation that the overlying normal epidermis, as well as the epidermis from five normal skin specimens, were stained by the mAb. Keratinocytes in plaques from 18 psoriasis patients were more intensely stained than those from normal skin. Cultured normal keratinocytes demonstrated membranous staining with mAb A1.5. Absorption of mAb A1.5 with synthetic human TGF-alpha completely removed the reactivity of mAb A1.5 with both basal cell tumors and normal epidermis. The demonstration of TGF-alpha in normal keratinocytes suggests that it plays a role in normal keratinocyte growth, wound healing, and in the pathogenesis of acanthosis.

T cell receptor V-segment frequencies in peripheral blood T cells correlate with human leukocyte antigen type.

We compared T cell receptor (TCR) V-segment frequencies in human leukocyte antigen (HLA) identical siblings to sibling pairs who differ at one or both HLA haplotypes using four V beta-specific and one V alpha-specific monoclonal antibody. In every one of nine families HLA-identical sibs had the most similar patterns of V-segment frequencies in their peripheral blood, whereas totally mismatched sibs were, in general, the most dissimilar; HLA haploidentical sibs tended to be intermediate between the two groups. The degree of similarity among HLA-identical sibs was comparable to that observed among three pairs of identical twins suggesting that HLA is the major genetic component influencing TCR V-segment frequency. Consistent with this observation, it was found that the frequency of T cells expressing particular V beta segments was skewed towards either CD4+ or CD8+ cells indicating that T cells expressing some V beta genes may be positively selected primarily by class I or class II major histocompatibility complex proteins. Finally, it was observed that individuals who express the HLA class I specificity, B38, tend to express high levels of V alpha 2.3+ cells among their CD8+ T cells. These observations represent definitive proof that human V-segment frequencies are profoundly influenced by the HLA complex.

Level of human TCRBV3S1 (V beta 3) expression correlates with allelic polymorphism in the spacer region of the recombination signal sequence.

One of the causes of variations in the expressed human T cell receptor (TCR) BV (V beta) repertoire is genetic variation in the germline DNA. Herein evidence is provided that allelic polymorphism may affect recombination frequency for a specific V gene. Two alleles of the TCR BV3 differ only at a single nucleotide position (C/T) within the 23-bp spacer region of the recombination signal sequence. These alleles are associated with variable percentages of BV3 cells in the peripheral blood, as shown in families and in unrelated normal donors. Individuals homozygous for allele 2 have a mean of 8.1% BV3 cells, heterozygous individuals have a mean of 4.7% BV3 cells, and homozygotes for allele 1 have a mean of 1.2% BV3 cells in CD3+ CD4+ peripheral blood T cells. Since the correlation is tight in unrelated individuals and other genetic differences were not found in the vicinity of BV3, we suggest that the spacer region sequence itself modifies recombination efficiency. This allelic system provides an example of a novel mechanism by which cis-acting genetic elements may affect recombination in a natural in vivo system.

TCR binding differs for a bacterial superantigen (SEE) and a viral superantigen (Mtv-9).

Both superantigens (SAG) and many anti-TCR monoclonal antibodies (mAb) have specificity for the V beta region of the TCR encoded by TCRBV genes. For instance the bacterial SAG staphylococcal enterotoxin E (SEE), the retroviral SAG MTV-9 and the mAb OT145 each react with human T cells expressing BV6S7. This BV gene encodes two common alleles. We found that SEE and the mAb preferentially activate T cells expressing BV6S7*1 as opposed to BV6S7*2, but Mtv-9 activates T cells expressing either allele. Thus binding to the TCR differs between the two SAGs. A mutation in the TCR HVR-4 region of BV6S7*1 (G72E), where the two BV6S7 alleles differ, indicated that HVR-4 is a component of the binding site for SEE and for the mAb OT145. BV6S7*2 has a charged E72 which may result in electrostatic repulsion of SEE, as SEE contains a similarly acidic aspartic acid residue at a TCR interaction site (204D).

Human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus is a new transmissible virus that infects B cells.

Herpesviral DNA fragments isolated from AIDS-associated Kaposi's sarcoma (KS) tissue (KSHV-DNA) share homology with two lymphotropic oncogenic gamma-herpesviruses, Epstein-Barr virus and Herpesvirus saimiri, and are present in the lesions of more than 95% of HIV and non-HIV-associated forms of KS, AIDS-related body cavity-based lymphomas, and AIDS-related multicentric Castleman's disease. Here we show that BC-1, a KSHV-DNA-positive, body cavity-based lymphoma cell line, produces infective herpesviral particles carrying a linear 270-kb genome that specifically transmits KSHV-DNA to CD19+ B cells. Transmission of KSHV-DNA is dependent upon a biologically active, replicating virus, since it is blocked by UV irradiation and foscarnet, an inhibitor of viral DNA-polymerase. This study represents the first isolation and transmission of the human herpesvirus-8/KS-associated herpesvirus.

Characterization of human T cells reactive with the Mycoplasma arthritidis-derived superantigen (MAM): generation of a monoclonal antibody against V beta 17, the T cell receptor gene product expressed by a large fraction of MAM-reactive human T cells.

While all known microbial superantigens are mitogenic for human peripheral blood lymphocytes (PBL), the functional response induced by Mycoplasma arthritidis-derived superantigen (MAM) is unique in that MAM stimulation of PBL consistently results in T cell-dependent B cell activation characterized by polyclonal IgM and IgG production. These immunostimulatory effects of MAM on the humoral arm of the human immune system warranted a more precise characterization of MAM-reactive human T cells. Using an uncloned MAM reactive human T cell line as immunogen, we have generated a monoclonal antibody (mAb) (termed C1) specific for the T cell receptor V beta gene expressed by the major fraction of MAM-reactive human T cells, V beta 17. In addition, a V beta 17- MAM-reactive T cell population exists, assessed by MAM, induced T cell proliferation and cytotoxic T cell activity. mAb C1 will be useful in characterizing the functional properties of V beta 17+ T cells and their potential role in autoimmune disease.

T cell antiidiotypic antibodies reveal differences between two human leukemias.

Two different human T cell leukemias were compared, using antiidiotype-like murine monoclonal antibodies. In each case these antibodies immunoprecipitated disulfide-linked heterodimer molecules from their respective leukemic cells. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the two idiotype-bearing molecules a major difference in molecular weight was observed, which could be attributed to a similar difference in size of the heavily iodinated chain of either heterodimer. The lightly iodinated chains of both molecules co-migrated at 43 Kd, but appeared to have different isoelectric points on two-dimensional gel analysis. The possibility that these two different heterodimers correspond to different classes of the putative T cell receptor for antigen is discussed. Assays of proliferation of the leukemic cells using Sepharose-bound antiidiotype-like monoclonal antibody showed that one of the leukemic cell types proliferated readily in response to its antiidiotypic antibody. This proliferation was not associated with measurable production of IL-2 and appeared to be a direct effect of the antiidiotypic antibody, which may mimic antigen in its interaction with the T cell receptor for antigen. The other leukemic cell type did not respond to Sepharose-bound antiidiotypic antibody and was generally unresponsive to lymphokines and mitogens. It is possible that the two leukemic cell types represent different stages of T cell differentiation.

V beta-specific stimulation of human T cells by staphylococcal toxins.

The staphylococcal toxins are responsible for a number of diseases in man and other animals. Many of them have also long been known to be powerful T cell stimulants. They do not, however, stimulate all T cells. On the contrary, each toxin reacts with human T cells bearing particular V beta sequences as part of their receptors for major histocompatibility complex protein-associated antigen. The specificity of these toxins for V beta s puts them in the recently described class of superantigens and may account for the differential sensitivity of different individuals to the toxic effects of these proteins.

Monoclonal antibodies with specificity for hairy cell leukemia cells.

Hairy cell leukemia is a well described clinical entity, but the cell of origin for this leukemic cell and its function are still unknown. There are no totally specific markers for this cell, although tartrate-resistant acid phosphatase staining has been used extensively as a diagnostic test. This study describes three monoclonal murine antibodies with variable specificity for hairy cells. Antibody 1 was highly specific for hairy cells and was not found to react with normal or leukemic cells in this limited study. It did not react with the cells of all patients. It also did not react with all of the hairy cells of some of the positive cases. Antibodies 2 and 3 reacted with virtually all hairy cells but not with normal peripheral blood cells. However, reactions were obtained with certain leukemic myelomonoblasts and some activated B cells. The most obvious use for these three antibodies is for diagnostic purposes. They should also be helpful reagents to investigate the origin of the leukemic hairy cell. The possibility that antibody 1 detects a tumor-specific antigen is discussed.

T cell antigen receptor V gene usage. Increases in V beta 8+ T cells in Crohn's disease.

Crohn's disease represents part of a spectrum of inflammatory bowel diseases characterized by immune regulatory defects and genetic predisposition. T cell antigen receptor V gene usage by T lymphocytes was investigated using four MAbs specific for various V gene products. One MAb (Ti3a), reactive with V beta 8 gene products, detected increased numbers of T cells in a subset of Crohn's disease patients as compared with normal controls and ulcerative colitis patients. In family studies there was no apparent inherited predisposition to the use of V beta 8 genes, and there was no association between a restriction fragment length polymorphism of the V beta 8.1 gene and Crohn's disease. The V beta 8+ T cells were concentrated in the mesenteric lymph nodes draining the inflammatory lesions and belonged to both the CD4+ and CD8+ T cell subsets. In contrast, lamina propria and intraepithelial T cells were not enriched in V beta 8+ T cells, suggesting that these cells were participating in the afferent limb of a gut-associated immune response. The expanded V beta 8+ T cells in Crohn's disease appear to result from an immune response to an as yet unknown antigen.