Longitudinal Analysis of T-Cell Receptor Variable β Chain Repertoire in Patients with Acute Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation

T-cell receptor variable β chain (TCRBV) repertoire spectratyping involves the estimation of CDR3 length distributions for monitoring T-cell receptor diversity and has proven useful for analyses of immune reconstitution and T-cell clonal expansions in graft-versus-host disease (GVHD) and graft-versus-leukemia after allogeneic stem cell transplantation. We performed a longitudinal spectratype analysis of 23 TCRBV families in 28 patients who underwent allogeneic T cell–depleted peripheral blood stem cell transplantation. Sixteen patients subsequently developed acute GVHD. We recently developed statistical methods that bring increased power and flexibility to spectratype analysis and allow us to analyze TCRBV repertoire development under appropriately complex statistical models. Applying these methods, we found that patients with acute GVHD demonstrated TCRBV repertoire development statistically distinct from that repertoire development in patients without GVHD. Specifically, GVHD patients showed spectratypes indicative of lower diversity and greater deviation from the spectratypes expected in healthy individuals at intermediate times. Most individual TCRBV subfamilies had spectratypes statistically distinguishable between GVHD and non-GVHD patients at 6 months after transplantation. These results suggest that the T-cell receptor repertoire perturbations associated with acute GVHD are widely spread throughout the TCRBV families.     

Selection of Distinct Vα/β T-Cell Receptor Families During In Vivo and In Vitro T-Cell Maturation

The experimental conditions influencing the use of Valphabeta TCR families were examined in lymph node (LN) cells from peptide-immunized C57BL/6 and Vbeta8.2 transgenic mice. Expanded proportions of Vbeta5, Vbeta8.2, Vbeta9, Vbeta12 and Vbeta14 positive cells and an association of Vbeta8.2 with Valpha11 was found in freshly harvested 8-day or 34-day immune LN cells. In contrast, peptide-specific T-cell lines generated in vitro from 8-day immune lymph node cells were found to be almost exclusively of the Valpha2/Vbeta12 family. However, T-cell lines originating from Vbeta8.2 transgenic mice did not show preferential Valpha usage. Anti-Vbeta8.2 antibody produced different effects: when added to cultures of LN cells from C57BL/6 or Vbeta8.2 transgenic strains, the peptide-induced proliferation was suppressed; however, following the injection of mice, subsequent in vitro proliferation and cytokine production induced by both peptide and Concanavalin A was suppressed in Vbeta8.2 transgenic, but much less in C57BL/6 mice. Hence, compensatory expansion of different Vbeta gene products occurred in vivo, but not under the employed in vitro conditions. In conclusion, these results suggest that the TCR family usage is influenced by the experimental conditions in which the T cells are selected and expanded and by the genetic potentials of the precursor pool.     

T-Cell Proliferation and Expression of MHC Class II Antigens

Two monoclonal antibodies to major histocompatibility complex (MHC) class II antigens, which in combination identify β chains encoded by the SB and DR loci, were used to investigate which of these gene- products were expressed at the cell surface of unstimulated T cells and at various stages of mitogen-induced T-cell maturation. In tests on blood lymphocytes from healthy donors 12% of Tcells expressed class II antigens, but only SB antigens were expressed. During activation of T lymphocytes, SB-coded antigens were expressed before DR antigens, and the kinetics of SB expression correlated with the proliferative response of T cells. These results and consideration of recent reports from other laboratories lead us to suggest that 58-coded class II antigens play a role in T-cell proliferation.     

The Ontogeny of T-Cell Antigens in the Thymus

T-cell-specific antigens were examined in tissue sections of thymus from 24 fetuses, infants, and children. OKT6 monoclonal antibodies faintly stained some thymocytes at 11 weeks of gestation, whereas OKT3, OKT4 and OKT8 did not stain the thymocytes before 14 weeks of gestation. OKT4, OKT6 and OKT8 preferentially stained the cortical thymocytes, in contrast to OKT3, which preferentially stained the medullary thymocytes. The fraction of OKT3- and OKT6-positive cells increased gradually during fetal life. At 24 weeks of gestation about half of the medullary thymocytes were stained by OKT3 and nearly all the cortical thymocytes were stained by OKT6. The fraction of cells stained with OKT4 and OKT8 was maximal at 14-20 weeks of gestation and decreased slightly with increasing age. The preference for either cortex or medulla of cells possessing the various antigens was less pronounced in fetal life than in infancy and childhood. An anti-T-cell antiserum produced in rabbit stained most thymocytes at 10 weeks of gestation. The staining intensity increased gradually until 17 weeks of gestation and then remained constant.     

RT-PCR Topography of Chronic Psoriasis Skin Based on Analysis of T-Cell Receptor B Variable Region Gene Usage

Psoriasis is a hyperproliferative inflammatory disease and 70% of patients develop a chronic plaque form. The pathogenesis of psoriasis is not known but evidence exists that T cells play a crucial role. The T cell V-gene receptor repertoire from psoriasis skin (different layers) was compared with peripheral blood T cells by employing RNA polymerase chain reaction (PCR) amplification. T cell receptor ( TCR) BV 5.1, 11, 12, 13.1 and 16 were utilized to a significantly higher degree in areas close to the basal layers when compared to CD4+, CD8+ or unfractionated blood T cells from the same patients, whereas only BV11 and 13.1 genes of T cells from deeper layers of the dermis showed such a skewed usage. No biased usage of TCRBV genes was observed in superficial layers or in whole skin. Furthermore, T cell receptor junctional diversity analysed by high resolution gel electrophoresis showed skin psoriatic T cells to be poly- or oligoclonal. In conclusion, we show that TCRBV gene usage from different layers of psoriatic skin has a different pattern compared with the corresponding gene usage in circulating peripheral blood T cells. This pattern may implicate possible skin-associated antigen or superantigens activating a limited number of T cells in areas of skin close to basal layers, which in turn could promote keratinocyte proliferation.     

Non-MHC-Restricted T-Cell Interaction with B Cells: Role of the T-Cell Receptor

Non-specific antibody production usually accompanies the T-cell-regulated B-cell response. In this paper the mechanisms involved in non-MHC-restricted T-B-cell interaction were studied. As previously shown for NK cells, activated B cells induce IFN-gamma and TNF alpha production in non-MHC-restricted cytotoxic T lymphocytes (NrCTL). Using an in vitro model system, we demonstrate that direct cell-cell interactions are required to induce these cytokines in NrCTL. Receptor ligand systems involved are leucocyte function antigen-1/intercellular adhesion molecule-1 (LFA-1/ICAM-1)(CD11a, CD18/CD54), T11/LFA-3 (CD2/CD58), and the clonotypic T-cell receptor (TCR) structure NKTa of JT9/JT10 with its non-MHC-related target antigen TNKtar (4F2). Cytokine production can be induced by activating monoclonal antibodies against CD2R. Antibodies against the clonotypic TCR (NKTa) or CD3 had no cytokine-inducing effect on NrCTL cultured alone, but were able to retrieve the effect of blocking the target antigen on co-cultured B cells. We could further demonstrate that the inhibition of the TCR/target antigen interaction could be overcome by close cell-cell contact culture conditions. From these findings it is concluded that the role of the TCR in non-MHC-restricted cell-cell interaction is to facilitate LFA-1/ICAM-1-mediated effector target adhesion in a specific way rather than to mediate direct activating signals upon lymphokine production or cytotoxicity.     

Mechanism of T-Cell Activation by Mycobacterial Antigens in Inflammatory Synovitis

Earlier studies demonstrated enhanced proliferative responses to an acetone precipitable Mycobacterium tuberculosis (AP-MT) antigenic complex by T lymphocytes from the synovial fluid, compared with the peripheral blood, of patients with inflammatory synovitis, including rheumatoid arthritis. In contrast, decreased proliferation and interleukin 2 (IL-2) production in response to mitogens by synovial fluid lymphocytes from patients with rheumatoid arthritis has been demonstrated. In order to determine if IL-2 was produced in response to AP-MT, the peripheral blood and synovial fluid of patients with inflammatory arthritis were analysed by measuring proliferation and IL-2 production in response to AP-MT and tetanus toxoid. A reduction of IL-2 production relative to proliferation was observed in some, but not all, synovial fluids of patients who responded to the AP-MT. Nevertheless, antibodies to IL-2 as well as interleukin 4 (IL-4), significantly inhibited proliferation of synovial fluid lymphocytes by AP-MT. There was no inhibition by antibodies to interleukin 6 (IL-6). We conclude that AP-MT induced proliferation by synovial fluid lymphocytes is mediated by both IL-2 and IL-4.     

HLA Antigens in 16 Families with Xeroderma Pigmentosum

Xeroderma pigmentosum is an autosomal recessive disease. HLA-A and -B typing was performed on peripheral blood lymphocytes and platelets. Sixteen Tunisian families were typed with 37 patients and 108 relatives. Genetic transmission of the disease and of the HLA system seemed to be independent in this study. Comparison of HLA gene frequencies between (unrelated) parents of patients and a control population showed no difference, proving that there is no clear association in populations between deleterious XP genes and a particular HLA gene. However, an excess of identical HLA among pairs of diseased siblings would suggest that the disease is polymorphic and a form of the XP could be linked to HLA.     

MaGiK Method of T-Cell Receptor Repertoire Analysis

T-cell receptor diversity enables the cellular immune response to recognize a broad range of viral and other pathogenic agents. An increasingly common method of characterizing T-cell receptor diversity and usage in response to antigenic challenges involves the identification of clonal expansions by PCR amplification of the CDR3 region of distinct TCRVβ families. Though clonal expansions often appear evident upon visual inspection of the results, a systematic method is needed for the valid enumeration of these expansions. Here, we describe a novel analysis method, termed the MaGiK method, for systematically identifying and enumerating clonal T-cell expansions and for applying the results to investigations of the T-cell receptor repertoire.     

Differential Patterns of T-Cell Receptor BV-Specific Activation of T Cells by gp120 from Different HIV Strains

Studies by several groups have suggested that HIV infection in vivo results in a BV-specific alteration of the TCR repertoire and that this might play a role in the pathogenesis of AIDS. Our earlier studies demonstrated tbat both a crude extract of HIV₄₅₁ as well as purified gp160 from HIV₄₅₁ could specifically activate, in vitro , T cells expressing a common set of TCRBV segments (TCRBV3, 12, 14, 15, and sometimes BV17 and 20) in individuals of disparate HLA type. Furthermore, purified gp120 from HIV₄₅₁ was shown to have a similar ability to activate T cells, although with a slightly diiferent TCRBV-specific pattern. In order to determine whether gp120 from other HIV strains could similarly activate T cells in a TCRBV-specific pattern, PBMC from HIV seronegative individuals of disparate HLA type were stimulated with gp120 from three strains of HIV (451, IIIB, and MN). The authors found that gp120 from all three strains activate T cells bearing TCRBV2 and BV3 in nearly every individual. T cells expressing other BV segments are also activated, but this is more variable and appears to be unique to each individual. Furthermore, gp120₄₅₁ and gp120 from HIV_IIIB and HIV_MNdiffer in their ability to activate T cells expressing these other TCRBV segments. These observations suggest that variation in the structure of gp120 and in the genetic and/or environmental background of the individual play an important role in determining which TCRBV segments are'triggered' by gp120. Furthermore, these observations may have important implications for the rate of disease progression in HIV-infected individuals.     

Analysis of the Conservation of T Cell Receptor Alpha and Beta Chain Variable Regions Gene in pp65 Peptide-Specific HLA-A＊0201-Restricted CD8^＋ T Cells

Many viral epitope specific T cell receptors （TCRs） in MHC-matched individuals have been demonstrated to involve conserved amino acid motifs in β chain complementarity-determining region 3 （CDR3）. However, it is not sure whether the conserved motifs can also be found in TCR β chain. In previous studies, we developed a modified method to enlarge the percentage of cytomegalovirus （CMV） pp65 peptide-specific CD8^＋ T cells in PBMC by continuous peptide stimulation in vitro, which provides sufficient number of specific T cells for detection. In this study, we further analyzed the restrictive usage of TCR Vα and Vβ gene families and investigated the CDR3 gene sequence of pp65 peptide-specific CD8β T cells. Analysis of CDR3 spectratypes suggested a restricted usage of TCR α chain AV8, AV12, AV21, AV31 families and TCR βchain BV3, BV14, BV21, BV23, BVll families in donor CD8^＋ T cells stimulated by pp65 peptide. The sequences of these T cells involved similar sequence （TX） G （X） A in CDR3 region of TCR α chain and L （XT） G （X） A in TCR β chain.