T cell receptor repertoire in BALB/c mice varies according to tissue type, sex, age, and hydrocortisone treatment.

Diversity in T cell recognition of antigens is determined by diverse usage of T cell receptor (TCR) repertoire. TCR repertoire analysis provides fundamental information for understanding T cell immune responses in the pathogenesis of various diseases. In the present study, we examined the TCR repertoire in various tissues in normal BALB/c mice. The TCR alpha chain variable region repertoires were consistent among the spleen, lymph nodes, and the thymus. The TCR beta chain variable region (TCRBV) repertoires were consistent between the spleen and lymph nodes, but different in the thymus. The TCR repertoires also differed in the lungs and the intestinal tract. The TCR repertoires were consistent between male and female mice, except for TCRBV15-1. TCR repertoire was almost similar in 3- and 7-week-old mice, except for TCRBV1-1, 8-3, and 14-1. The present findings suggest that the TCR repertoire of mice varies according to tissue type, sex and age. Additional analysis of the TCR repertoire, i.e., the effect of hydrocortisone (HC), was carried out. After the HC treatment, although the thymic T cells decreased to one-tenth, only a small fraction of CD4(+)CD8(+) T cells survived the treatment. Furthermore, the percentages of thymic T cells bearing TCRBV3-1, 5-1, 5-2, and 16-1 substantially decreased, but the percentage of cells bearing TCRBV12-1 did not decrease. The present findings suggest that the HC susceptibility of immature thymic T cells is different between TCR families.

Regulation of reactive oxygen species by Atm is essential for proper response to DNA double-strand breaks in lymphocytes.

The ataxia telangiectasia-mutated (ATM) gene plays a pivotal role in the maintenance of genomic stability. Although it has been recently shown that antioxidative agents inhibited lymphomagenesis in Atm(-/-) mice, the mechanisms remain unclear. In this study, we intensively investigated the roles of reactive oxygen species (ROS) in phenotypes of Atm(-/-) mice. Reduction of ROS by the antioxidant N-acetyl-l-cysteine (NAC) prevented the emergence of senescent phenotypes in Atm(-/-) mouse embryonic fibroblasts, hypersensitivity to total body irradiation, and thymic lymphomagenesis in Atm(-/-) mice. To understand the mechanisms for prevention of lymphomagenesis, we analyzed development of pretumor lymphocytes in Atm(-/-) mice. Impairment of Ig class switch recombination seen in Atm(-/-) mice was mitigated by NAC, indicating that ROS elevation leads to abnormal response to programmed double-strand breaks in vivo. Significantly, in vivo administration of NAC to Atm(-/-) mice restored normal T cell development and inhibited aberrant V(D)J recombination. We conclude that Atm-mediated ROS regulation is essential for proper DNA recombination, preventing immunodeficiency, and lymphomagenesis.

Quantitation of aberrant interlocus T-cell receptor rearrangements in mouse thymocytes and the effect of the herbicide 2,4-dichlorophenoxyacetic acid.

Small studies in human populations have suggested a correlation between the frequency of errors in antigen receptor gene assembly and lymphoid malignancy risk. In particular, agricultural workers exposed to pesticides have both an increased risk for lymphoma and an increased frequency of errors in antigen receptor gene assembly. In order to further investigate the potential of such errors to serve as a mechanistically based biomarker of lymphoid cancer risk, we have developed a sensitive PCR assay for quantifying errors of V(D)J recombination in the thymocytes of mice. This assay measures interlocus rearrangements between two T-cell receptor loci, V-gamma and J-beta, located on chromosomes 13 and 6, respectively. The baseline frequency in four strains of mice was determined at several ages (2-8 weeks of age) and was found to be stable at approximately 1.5 x 10(-5) per thymocyte. Strain AKR, which has a high susceptibility to T-cell lymphomas, did not show an elevated frequency of aberrant V(D)J events. We used this assay to examine the effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on the frequency of these events. Female B6C3F1 mice, 27 days of age, were exposed to 2,4-D by gavage at doses of 0, 3, 10, 30, and 100 mg/kg/day for 4 successive days and sacrificed on day 5. Thymus DNA was isolated and examined for illegitimate V(D)J recombination-mediated gene rearrangements. In addition, pregnant mice were exposed to 2,4-D and thymocytes from the offspring examined at 2 weeks of age. No significant increase in aberrant V(D)J rearrangements was found, indicating that under these conditions 2,4-D does not appear to effect this important mechanism of carcinogenesis.

Characterization of drug-specific T cells in phenobarbital-induced eruption.

Phenobarbital has a high potential to elicit adverse reactions including severe skin eruptions and systemic involvements among the worldwide-prescribed drugs. Although phenobarbital hypersensitivity is thought to be mediated by T cells specific to the drug, its precise mechanism remains not fully elucidated. To characterize T cells reactive with phenobarbital, we generated drug-specific T cell clones and lines from PBMCs of patients with phenobarbital hypersensitivity showing various degrees of cutaneous and extracutaneous involvements. Although the TCR Vbeta repertoire and phenotype in the T cell clones/T cell lines were heterogeneous among the patients, Vbeta13.1(+) and Vbeta5.1(+) clones or lines were raised from the individuals examined who possessed different HLA haplotypes. Histopathological examination suggested that Vbeta5.1(+)CD8(+) T cells and Vbeta13.1(+) T cells played a role in cutaneous and extracutaneous involvements, respectively. A Vbeta13.1(+)CD4(+) clone was found to proliferate in response to the Ag with processing-impaired, fixed APCs. Most of the clones and lines belonged to the Th2 phenotype, producing IL-4 and IL-5 but not IFN-gamma upon phenobarbital stimulation. Clones/lines with Th1 or Th0 phenotypes also constituted minor populations. These observations clearly indicate the heterogeneity and a marked individual deviation of reactive T cell subsets among the patients in terms of CD4/8 phenotype, Vbeta repertoire, Ag recognition pattern, and cytokine production; and thus provide evidence whereby each pathogenic T cell subset contributes to special elements of clinical presentation.

T cell infiltration of the prostate induced by androgen withdrawal in patients with prostate cancer.

Manipulations capable of breaking host tolerance to induce tissue-specific T cell-mediated inflammation are of central importance to tumor immunotherapy and our understanding of autoimmunity. We demonstrate that androgen ablative therapy induces profuse T cell infiltration of benign glands and tumors in human prostates. T cell infiltration is readily apparent after 7-28 days of therapy and is comprised predominantly of a response by CD4+ T cells and comparatively fewer CD8+ T cells. Also, T cells within the treated prostate exhibit restricted TCR Vbeta gene usage, consistent with a local oligoclonal response. Recruitment/activation of antigen-presenting cells in treated prostate tissues may contribute to local T cell activation. The induction of T cell infiltration in prostate tissues treated with androgen ablation may have implications for the immunotherapeutic treatment of prostate cancer as well as other hormone-sensitive malignancies, including breast carcinoma.

Longitudinal analysis of T-cell receptor gene use by CD8(+) T cells in early human immunodeficiency virus infection in patients receiving highly active antiretroviral therapy.

The effects of early antiretroviral therapy on the peripheral CD8(+) T-cell population were assessed by sequentially determining the T-cell receptor (TCR) repertoire complexity in a cohort of 15 individuals recently diagnosed with human immunodeficiency virus infection. Analysis was based on quantitative TCR variable B gene (TCRBV) usage and complementary-determining region 3 length assessment. Repertories were assessed at baseline and at weeks 2, 4, 12, 24, and 72 after initiation of therapy. Early administration of highly active antiretroviral therapy has a positive effect on the preservation and homeostasis of the CD8(+) cell repertoire. Nevertheless, differences from average baseline and control TCR profiles and initial development of repertoire perturbations were observed. The findings suggest that additional therapeutic protocols will be required during primary infection to significantly prevent long-term erosion of the T-cell-mediated immune response.

Identification of pathogenic T cells in patients with beryllium-induced lung disease.

Chronic beryllium disease (CBD) is caused by beryllium exposure and is characterized by granulomatous inflammation with accumulation of CD4+ T cells in the lung. We analyzed TCR beta-chain and alpha-chain genes expressed by these CD4+ T cells. In the lungs of individual patients, as well as among four of five CBD patients studied, different oligoclonal expansions within the Vbeta3 subset were found to express homologous or even identical CDR3 amino acid sequences. These related expansions were specific for CBD patients, were compartmentalized to lung, and persisted at high frequency in patients with active disease. Limiting dilution cloning and analysis of coexpressed TCR alpha-chain genes confirmed that these TCRs were selectively expanded by a common Ag involving beryllium. Overall, homologous TCR beta- and alpha-chains showed identical V regions and invariant charged residues within the CDR3 but considerable variability in TCRJ usage. Remarkably, CBD patients expressing nearly identical TCRs did not share common HLA-DRB1 or DQ alleles. These results implicate particular CD4+ cells in the pathogenesis of CBD and provide insight into how beryllium is recognized in human disease.

Inhibition of T cell and promotion of natural killer cell development by the dominant negative helix loop helix factor Id3.

Bipotential T/natural killer (NK) progenitor cells are present in the human thymus. Despite their bipotential capacity, these progenitors develop predominantly to T cells in the thymus. The mechanisms controlling this developmental choice are unknown. Here we present evidence that a member(s) of the family of basic helix loop helix (bHLH) transcription factors determines lineage specification of NK/T cell progenitors. The natural dominant negative HLH factor Id3, which blocks transcriptional activity of a number of known bHLH factors, was expressed in CD34+ progenitor cells by retrovirus-mediated gene transfer. Constitutive expression of Id3 completely blocks development of CD34+ cells into T cells in a fetal thymic organ culture (FTOC). In contrast, development into NK cells in an FTOC is enhanced. Thus, the activity of a bHLH transcription factor is necessary for T lineage differentiation of bipotential precursors, in the absence of which a default pathway leading to NK cell development is chosen. Our results identify a molecular switch for lineage specification in early lymphoid precursors of humans.

Selective usage of defined TCRBV genes in response to filarial antigens.

The characterization of T cell reactivities that are prone to down-modulation by filarial parasites is central to understanding how these nematodes can survive for long periods of time within their human host and to design appropriate immunoprophylactic measures. In the present study, TCRBV gene usage was analyzed in response to filarial antigens by PCR using a panel of TCRBV gene segment family-specific oligonucleotide primers. Analysis of individuals highly responsive to Brugia malayi adult worm antigen (BmA) (n = 4) indicated that following stimulation with BmA a maximum of four TCRBV gene families were over-represented in each subject. Those were TCRBV2, 9, 19 and 23 in subject 1; TCRBV8, 9 and 16 in subject 2; TCRBV2, 8, 9 and 11 in subject 3; and TCRBV13 and 23 in subject 4. The analysis of one subject who was unresponsive to BmA before but regained responsiveness after diethylcarbamazine treatment revealed that there was no overexpression of a particular TCRBV gene family before chemotherapy, whereas after chemotherapy three TCRBV gene families (TCRBV8, 16 and 19) were found to be overexpressed. Complementarity determining region 3 size analysis of a selection of the overexpressed TCRBV genes displayed oligoclonality in some of the observed expansions. Together these observations show that limited T cell subpopulations are clonally amplified in BmA-stimulated peripheral blood mononuclear cells of filarial responder subjects, possibly driven by a restricted number of antigens.

Interleukin 7 induces TCR gene rearrangement in adult marrow-resident murine precursor T cells.

Rearrangement of the T cell antigen receptor genes is a complex, highly regulated process. To gain a better understanding of the extracellular factors involved in the regulation of TCR beta and gamma gene rearrangement in adult murine bone marrow-resident precursor T cells, several cytokines were tested for their ability to induce gene recombination. A selected population of C58/J bone marrow cells (Thy 1(low), CD3, CD8, B220) that is enriched for pre-T cell activity was propagated in vitro in medium supplemented with IL-3 and mast cell growth factor (MGF, also referred to as stem cell factor, Steele factor and c-kit ligand). These cytokines were required for the maintenance of pre-T cell activity in culture, but had no effect on TCR gene expression. Several additional cytokines were added to the culture medium. Of all those tested, only IL-7 induced complete rearrangement of the TCR gamma locus. Complete rearrangement of the TCR beta locus was not induced under any of the culture conditions analysed here. The bone marrow cells cultured in IL-3, MGF and IL-7 did not begin to express mature T cell proteins and maintained their in vivo progenitor potential. Furthermore, IL-7 cultured bone marrow cells were capable of differentiation in vivo into all phenotypic subpopulations of T cells, without an apparent bias toward the gammadelta lineage. The data presented here suggest that TCR gamma gene rearrangement in adult pre-T cells is regulated by IL-7, but that the TCR beta locus requires additional or alternative signals for the induction of complete rearrangement.

Stimulation of immune reconstitution by interleukin-7 after syngeneic bone marrow transplantation in mice.

Successful outcome of autologous bone marrow transplantation (BMT) is severely handicapped by susceptibility to infection and by a high rate of relapse. While quantitative aspects of the immune system generally return to normal within the first 3-4 months after BMT, the recovery of qualitative immune functions is prolonged. Since interleukin-7 (IL-7) has growth-promoting and differentiating effects on pre-B cells and immature thymocytes, its role in the recovery of immune functions was investigated in BALB/c mice after syngeneic BMT (sBMT). After sBMT, mice treated with human recombinant IL-7 (rIL-7) showed an 11.9-fold increase in thymic cellularity associated with an enhanced response to a mitogenic stimulus compared with the controls. rIL-7 significantly increased RAG-1 expression and promoted V beta 8(D)J gene rearrangement of the T cell receptor in the thymus. Further, the cytokine boosted survival after challenge with influenza virus following sBMT. The finding that rIL-7 induces differentiation and proliferation of immature thymocytes and counteracts post-BMT immune deficiency makes it a promising medium for clinical application in BMT patients.

Block of T lymphocyte differentiation by activation of the cAMP-dependent signal transduction pathway.

Differentiation of T lymphocytes is a complex and finely tuned process. Here we show that treatment of mouse fetal thymus organ cultures with agents activating the cAMP-dependent signalling pathway results in the block of thymocyte differentiation. This is due to severe impairment of maturation beyond the CD4-/CD8- stage. In addition, rearrangements at the TCR alpha gene locus, but not at the TCR beta locus, are completely inhibited. The cAMP effect is reversible and is restricted to TCR alpha beta+ cells. cAMP acts both by triggering apoptosis and by inducing cell-cycle block in thymocytes. Thus, activation of the cAMP pathway provides a mechanism to modulate thymic function for hormones and ligands whose receptors are coupled to adenylate cyclase.

Granular lymphocyte leukemia with pure red cell aplasia: usefulness of gene analysis in assessing therapeutic effect.

A patient with granular lymphocyte leukemia (GLL) of the CD3+, CD4-, CD8+ phenotype accompanied by pure red cell aplasia (PRCA) is described. Surface marker analysis, nonmajor histocompatibility complex (MHC)-restricted cytotoxicity assay, gene analysis, and in vitro colony assay were performed on the granular lymphocytes before and after treatment. Cyclophosphamide therapy was highly effective, and after remission clonal granular lymphocytes were no longer identified by T-cell antigen receptor (TCR) gene analysis or surface marker analysis. Lymphocytes obtained after remission did not exhibit elevated levels of non-MHC-restricted cytotoxicity, nor did they demonstrate a suppressive effect on erythroid colony formation. TCR gene analysis proved to be a sensitive parameter for evaluating the residual malignant granular lymphocytes. Gene analysis will be useful both for timing the discontinuation of treatment and for the early detection of relapse. Various factors possibly related to the development of PRCA in this patient were investigated and their significance is discussed.

Recombinant gamma-interferon has activity in chronic myeloid leukemia.

We demonstrated the clinical effectiveness of recombinant interferon-gamma (rIFN gamma) (Biogen) in 18 patients with Philadelphia-positive chronic myeloid leukemia. Sequential cytogenetic studies and molecular analyses of the breakpoint cluster region and for immunoglobulin and T cell rearrangements were performed every 3-4 months. In 13 patients who received treatment for a minimum of 3 months, the majority were treated with 1.5 mg/m2, t.i.w., i.v. Nonhematologic effects--particularly chills, rigors, myalgia, fatigue, headaches, and nausea--were significant. Complete or partial hematologic responses were observed in six patients, two of whom had approximately 20% normal metaphases after an average of 74 weeks of treatment. However, reversion to 100% Ph+ cells occurred 30 weeks later. In these two patients, in whom normal metaphases were found, no changes were observed in the presence of rearrangements of the breakpoint cluster region. In addition, the marrows remained hypercellular, and the leukocyte alkaline phosphatase score and B12 levels remained abnormal. No immunoglobulin or T cell beta-chain gene rearrangements were found. These data indicate the clinical effectiveness of rIFN gamma in some patients with chronic myeloid leukemia, although the fundamental nature of the disease is unaltered by this form of treatment.

Induction by interleukin-2 of oligoclonal expansion of cultured tumor-infiltrating lymphocytes.

To better understand the modulatory effects of interleukin-2 (IL-2) on lymphocyte proliferation, we examined the clonality of the in vitro T-cell response by Southern blot hybridization. Tumor-infiltrating lymphocytes (TILs) grown in the presence of IL-2 for 15-26 days had detectable T-cell receptor beta-chain gene rearrangements, which indicated oligoclonal enhancement in culture in four of nine TIL samples. In contrast, none of 11 uncultured TIL samples had detectable gene rearrangements. Lack of detection in at least three of the five negative, cultured TIL samples could be explained by increased numbers of natural killer cells. We hypothesize that the oligoclonal expansion noted results from the enhanced response of immune-primed T cells to IL-2.