Cell Receptor Variable Region Research Articles

Monoclonal antibodies derived from B cells in subjects with cystic fibrosis reduce Pseudomonas aeruginosa burden in mice.

Pseudomonas aeruginosa (PA) is an opportunistic, frequently multidrug-resistant pathogen that can cause severe infections in hospitalized patients. Antibodies against the PA virulence factor, PcrV, protect from death and disease in a variety of animal models. However, clinical trials of PcrV-binding antibody-based products have thus far failed to demonstrate benefit. Prior candidates were derivations of antibodies identified using protein-immunized animal systems and required extensive engineering to optimize binding and/or reduce immunogenicity. Of note, PA infections are common in people with cystic fibrosis (pwCF), who are generally believed to mount normal adaptive immune responses. Here we utilized a tetramer reagent to detect and isolate PcrV-specific B cells in pwCF and, via single-cell sorting and paired-chain sequencing, identified the B cell receptor (BCR) variable region sequences that confer PcrV-specificity. We derived multiple high affinity anti-PcrV monoclonal antibodies (mAbs) from PcrV-specific B cells across 3 donors, including mAbs that exhibit potent anti-PA activity in a murine pneumonia model. This robust strategy for mAb discovery expands what is known about PA-specific B cells in pwCF and yields novel mAbs with potential for future clinical use.

Antigen-Specific Single B Cell Sorting and Monoclonal Antibody Cloning in Guinea Pigs.

Here, we have established an antigen-specific single B cell sorting and monoclonal antibody (mAb) cloning platform for analyzing immunization- or viral infection-elicited antibody response at the clonal level in guinea pigs. We stained the peripheral blood mononuclear cells (PBMCs) from a guinea pig immunized with HIV-1 envelope glycoprotein trimer mimic (BG505 SOSIP), using anti-guinea pig IgG and IgM fluorochrome conjugates, along with fluorochrome-conjugated BG505 SOSIP trimer as antigen (Ag) probe to sort for Ag-specific IgGhi IgMlo B cells at single cell density. We then designed a set of guinea pig immunoglobulin (Ig) gene-specific primers to amplify cDNAs encoding B cell receptor variable regions [V(D)J segments] from the sorted Ag-specific B cells. B cell V(D)J sequences were verified by sequencing and annotated by IgBLAST, followed by cloning into Ig heavy- and light-chain expression vectors containing human IgG1 constant regions and co-transfection into 293F cells to reconstitute full-length antibodies in a guinea pig-human chimeric IgG1 format. Of 88 antigen-specific B cells isolated, we recovered 24 (27%) cells with native-paired heavy and light chains. Furthermore, 85% of the expressed recombinant mAbs bind positively to the antigen probe by enzyme-linked immunosorbent and/or BioLayer Interferometry assays, while five mAbs from four clonal lineages neutralize the HIV-1 tier 1 virus ZM109. In summary, by coupling Ag-specific single B cell sorting with gene-specific single cell RT-PCR, our method exhibits high efficiency and accuracy, which will facilitate future efforts in isolating mAbs and analyzing B cell responses to infections or immunizations in the guinea pig model.

Human γδ T cells: diverse orchestrators of the host response to influenza infection. (VIR5P.1145)

Abstract While often mild, influenza infection can occasionally progress to severe disease and even death. It is recognized that disease progression and outcome is often worse in the young and the old; however, the reason infection progresses in some individuals and not others is still unclear. Previous findings, reported by our laboratory, from a five-year longitudinal human study have shown that a definable inflammatory cytokine signature correlates with poor clinical outcome. Moreover, we have shown that this cytokine profile correlates with inflammatory monocyte numbers. Here we show a correlation between γδ T cell numbers and inflammatory monocyte numbers during infection within this clinical cohort. Further, we show that a subset of peripheral γδ T cells exhibit a surprisingly high T cell receptor (TCR) variable region gene diversity in infected individuals. Additionally, the CDR3 TCR sequences of this cellular subset show very high diversity, while retaining repetitive VJ gene junction-determined conserved motifs. The data presented here suggest that human peripheral γδ T cells are a more diverse cellular population than previously realized and are capable of playing a pivotal role in influencing the local immune environment during influenza infection with the potential to effect long-term outcome.

Human CD8+ T Cells from TB Pleurisy Respond to Four Immunodominant Epitopes in Mtb CFP10 Restricted by HLA-B Alleles

CD8+ T cells are essential for host defense to Mycobacterium tuberculosis (Mtb) infection and identification of CD8+ T cell epitopes from Mtb is of importance for the development of effective peptide-based diagnostics and vaccines. We previously demonstrated that the secreted 10-KDa culture filtrate protein (CFP10) from Mtb is a potent CD8+ T cell antigen but the repertoire and dominance pattern of human CD8 epitopes for CFP10 remained poorly characterized. In the present study, we undertook to define immunodominant CD8 epitopes involved in CFP10 using a panel of CFP10-derived 13–15 amino acid (aa) peptides overlapping by 11 aa. Four peptides in CFP10 were observed to induce significant CD8+ T cell responses and we further determined the size of the epitopes involved in each individual peptide tested. Four 9 aa CD8 epitopes were finally identified and deleting a single amino acid from the N or C terminus of either peptide markedly reduced IFN-γ production, suggesting that they are minimum of CD8 epitopes. In the individuals tested, each epitope represented a single immunodominant response in CD8+ T cells. The epitope-specific CD8+ T cells displayed effector or effector memory phenotypes and could upregulate the expression of CD107a/b upon antigen stimulation. In addition, we found that epitope-specific CD8+ T cells shared biased usage of T cell receptor (TCR) variable region of β chain (Vβ) 12, 9, 7.2 or Vβ4 chains. As judged from HLA-typing results and using bioinformatics technology for prediction of MHC binding affinity, we found that the epitope-specific CD8+ T cells are all restricted by HLA-B alleles. Our findings suggest that the four epitopes in CFP10 recognized by CD8+ T cells might be of importance for the development of Mtb peptide-based vaccines and for improved diagnosis of TB in humans.

Plasticity in the Contribution of T Cell Receptor Variable Region Residues to Binding of Peptide–HLA-A2 Complexes

One hypothesis accounting for major histocompatibility complex (MHC) restriction by T cell receptors (TCRs) holds that there are several evolutionary conserved residues in TCR variable regions that contact MHC. While this “germline codon” hypothesis is supported by various lines of evidence, it has been difficult to test. The difficulty stems in part from the fact that TCRs exhibit low affinities for pep/MHC, thus limiting the range of binding energies that can be assigned to these key interactions using mutational analyses. To measure the magnitude of binding energies involved, here we used high-affinity TCRs engineered by mutagenesis of CDR3. The TCRs included a high-affinity, MART-1/HLA-A2-specific single-chain TCR and two other high-affinity TCRs that all contain the same Vα region and recognize the same MHC allele (HLA-A2), with different peptides and Vβ regions. Mutational analysis of residues in CDR1 and CDR2 of the three Vα2 regions showed the importance of the key germline codon residue Y51. However, two other proposed key residues showed significant differences among the TCRs in their relative contributions to binding. With the use of single-position, yeast-display libraries in two of the key residues, MART-1/HLA-A2 selections also revealed strong preferences for wild-type germline codon residues, but several alternative residues could also accommodate binding and, hence, MHC restriction. Thus, although a single residue (Y51) could account for a proportion of the energy associated with positive selection (i.e., MHC restriction), there is significant plasticity in requirements for particular side chains in CDR1 and CDR2 and in their relative binding contributions among different TCRs.

B‐cell tolerance to the B‐cell receptor variable regions

An enormous number of B cells with different B-cell receptors (BCRs) are continuously produced in the bone marrow. BCRs are further diversified during the germinal center reaction. Due to extensive recirculation, B cells with mutually binding BCR are likely to meet in lymphoid organs. We have addressed possible outcomes of such an encounter in vitro. B lymphoma cells were transfected with complementary BCR, one transfectant expressing an Idiotype⁺ (Id⁺) BCR and the other an anti-Id BCR. To exclude confounding effects of secreted Ig, the transfected B lymphoma cells only expressed membrane IgD. Coincubation of paired Id⁺/anti-Id lymphoma cells results in conjugate formation, signaling, activation of Caspase 3/7, and apoptosis of at least one of the two cells in the pair. Our data provide suggestive evidence for a mechanism whereby the B-cell compartment is partly purged of B cells with complementary BCRs.

T Cell Receptor Variable Regions in Diabetes Bind to Each Other, to Insulin, Glucagon or Insulin Receptor, and to Their Antibodies

Our objective is to elucidate the nature of the autoimmune disregulation in diabetes through the antigen speci- ficity of the T-cell receptor (TCR) sequences generated by patients with type 1 diabetes mellitus (T1DM). Previously we demonstrated that TCR from T1DM patients and NOD mice mimic insulin, glucagon and their receptors. We hypothesize that these TCR will bind to each other (as insulin and glucagon do to their receptors) and also be targets of anti-insulin and anti-glucagon antibodies. The hypervariable regions of multiple TCR from three patients were synthesized and their binding specificities determined using UV spectroscopy. ELISA was used to determine whether these TCR were recog- nized by anti-insulin and anti-glucagon antibodies. Each patient produced TCR that recognized insulin, glucagon and the insulin receptor (IR). These TCR also recognized each other as complementary (possibly idiotype-antiidiotype) pairs. In addition, each TCR peptide was recognized with nanomolar affinity as an antigen by an antibody against insulin, gluca- gon, and/or IR. Finally, each of the antibodies against insulin, glucagon and IR formed a complementary antibody (or idiotype-antiidiotype) pair with another antibody involved in the disease, again at nanomolar affinities. Every possible ex- pression of complementarity (or idiotype-antiidiotype cross-reactivity) involving TCRs and antibodies was manifested by each patient. Two interpretations of these observations are offered. One, following Marchelonis, is that TCR-antibody complementarity is a mechanism for down-regulating the autoimmune process to re-establish tolerance to self-antigens. A non-exclusive alternative is that the trigger for autoimmunity is antigenic complementarity, which results in the produc- tion of complementary TCR and antibodies that appear to have idiotype-antiidiotype relationships among themselves.

Observation of T cell receptor Vbeta gene usage in the peripheral blood of patients with chronic hepatitis B after being treated by Hejie Decoction

AIM: To date, few studies have been conducted to determine whether T cell receptor (TCR) variable regions (V)of βare involved in the clinical course of chronic hepatitis B. This study was performed to assess the TCR Vβusage and clinical course of CHB and clinical parameters. METHODS: Fluorescence Quantitative PCR (FQ-PCR) technique was used to detect the TCRVβ subfamilies gene expression in the peripheral blood of 3 patients with chronic hepatitis B before and after being treated by Hejie Decoction, and clinical parameters (ALT, AST, and viral load) were observed in the same time. RESULTS: The percentage of TCRVb gene usage was determined during acute exacerbation and the remission stages. Figure 1 illustrates the percentage of TCRVb usage in the acute exacerbation and the remission stages. Those whose expressions were increased during remission stages compared with that during acute exacerbation stages were TCRVβ1(t=7.00, P

Differential utilization of T cell receptor TCR alpha/TCR delta locus variable region gene segments is mediated by accessibility.

T cell receptor (TCR) variable region exons are assembled from germline V, (D), and J gene segments, each of which is flanked by recombination signal (RS) sequences that are composed of a conserved heptamer, a spacer of 12 or 23 bp, and a characteristic nonamer. V(D)J recombination only occurs between V, D, and J segments flanked by RS sequences that contain, respectively, 12(12-RS)- and 23(23-RS)-bp spacers (12/23 rule). Additional mechanisms can restrict joining of 12/23 RS matched segments beyond the 12/23 rule (B12/23). The TCRdelta locus is contained within the TCRalpha locus; TCRalpha variable region exons are encoded by TRAV and TRAJ segments and those of TCRdelta by TRDV, TRDD, and TRDJ segments. On the basis of the 12/23 rule, both TRAV and TRDV gene segments are compatible to rearrange with TRDD gene segments; however, TRAV-to-TRDD joins are not observed in vivo. Absence of TRAV-to-TRDD rearrangement might be explained either by B12/23 restriction or by differential accessibility of the TRDV versus TRAV gene segments for rearrangement to TRDD. We used in vitro substrate analysis to reveal that both TRAV and TRDV 23-RSs mediate rearrangements to the 5'TRDD1 12-RS, demonstrating that B12/23 restriction does not explain these rearrangement biases. However, targeted replacement of TRDD1 and its 12-RSs with TRAJ38 and its 12-RS showed that TRDV gene segments rearrange with the ectopic TRAJ38, whereas TRAV segments do not. Our results demonstrate that sorting of TRAV and TRDV gene segments is determined by differential locus accessibility during T cell development.

Structural features of T cell receptor variable regions that enhance domain stability and enable expression as single-chain VαVβ fragments

The variable (V) domains of antibodies and T cell receptors (TCRs) share sequence homology and striking structural similarity. Single-chain antibody V domain constructs (scFv) are routinely expressed in a variety of heterologous systems, both for production of soluble protein as well as for in vitro engineering. In contrast, single-chain T cell receptor V domain constructs (scTCR) are prone to aggregation and misfolding and are refractory to display on phage or yeast in their wild-type form. However, through random mutagenesis and yeast display engineering, it has been possible to isolate scTCR mutants that are properly folded and displayed on the yeast surface. These displayed mutants can serve not only as a scaffold for further engineering but also as scTCR variants that exhibit favorable biophysical properties in Escherichia coli expression. Thus, a more comprehensive understanding of the V domain mutations that allowed display would be beneficial. Our goal here was to identify generalizable patterns of important mutations that can be applied to different TCRs. We compared five different scTCRs, four from mice and one from a human, for yeast surface display. Analysis of a collection of mutants revealed four distinct regions of TCR V domains that were most important for enabling surface expression: the Vα–Vβ interface, the HV4 of Vβ, and the region of the Vα and Vβ domains normally apposed against the constant (C) domains. Consistent with the role of the V–C interface in surface display, reconstitution of this interface, by including the constant domains of each chain, allowed V domain display and αβ chain association on the yeast surface, thus providing an alternative TCR scaffold. However, the surface levels of TCR achieved with engineered scTCR mutants were superior to that of the VαCα/VβCβ constructs. Therefore, we describe further optimization of the current strategy for surface display of the single-chain format in order to facilitate yeast display engineering of a broader range of scTCRs.

Primer sets for cloning the human repertoire of T cell Receptor Variable regions

BackgroundAmplification and cloning of naïve T cell Receptor (TR) repertoires or antigen-specific TR is crucial to shape immune response and to develop immuno-based therapies. TR variable (V) regions are encoded by several genes that recombine during T cell development. The cloning of expressed genes as large diverse libraries from natural sources relies upon the availability of primers able to amplify as many V genes as possible.ResultsHere, we present a list of primers computationally designed on all functional TR V and J genes listed in the IMGT®, the ImMunoGeneTics information system®. The list consists of unambiguous or degenerate primers suitable to theoretically amplify and clone the entire TR repertoire. We show that it is possible to selectively amplify and clone expressed TR V genes in one single RT-PCR step and from as little as 1000 cells.ConclusionThis new primer set will facilitate the creation of more diverse TR libraries than has been possible using currently available primer sets.

Predominance of T cell receptor V delta 3 in small bowel biopsies from coeliac disease patients.

Increased numbers of T cells bearing the gamma delta antigen receptor (gamma delta T cells) have been reported in small bowel biopsies of patients with latent, active or treated coeliac disease. We have studied jejunal biopsies from seven children with coeliac disease and 10 children with normal gut histology to characterize gamma delta T cell receptor (TCR) variable region gene subfamily expression in resident gamma delta T cells and compared the results with the findings in peripheral blood mononuclear cells (PBMC) obtained on the same day as the gut biopsy. Molecular analysis of RNA extracted from PBMC and biopsies was performed by reverse transcription and amplification with the polymerase chain reaction using primers specific for six TCR V delta families and four TCR V gamma families. We report, first, that a significantly increased number of gamma delta T cells expressing the TCR V delta 3 subfamily (P = 0.008) was observed in jejunal biopsies from children with coeliac disease, and second, that gamma delta T cell V region subfamily populations in gut differed from those seen in PBMC for both control and coeliac patients. Significantly reduced numbers of TCR V delta 2, V delta 3, V delta 5 (P < 0.01) and V gamma 2, V gamma 4 (P < 0.01) T cells were found in gut compared with PBMC. The difference in gamma delta T cell repertoire observed between gut and blood may reflect differences in the nature of the antigens usually encountered in these two compartments. The over-representation of TCR V delta 3 in patients with coeliac disease suggests a specific role for these cells in the induction or maintenance of the jejunal abnormality associated with this disease.

An abnormal T cell repertoire in hypergammaglobulinaemic primary Sjogren’s syndrome

T cell antigen specificity is determined by the products of the genes which encode the variable regions of their receptors. Of the T cell receptor (TCR) variable region gene products examined, only V beta 6.7a TCR-positive lymphocytes were reduced in primary Sjögren's syndrome patients with IgG1 hypergammaglobulinaemia compared with an age-, sex- and HLA-matched control population. The levels of V beta 6.7a T cells were also significantly reduced when these patients were compared with an age- and sex-matched but HLA-unmatched control group and non-tissue typed normal people of both sexes. Since published studies show no such abnormality in rheumatoid arthritis, systemic lupus erythematosus or other autoimmune diseases, this abnormality may reflect a pathogenic process specific to primary Sjögren's syndrome.

Effect of Staphylococcal Enterotoxin A on the Distribution and Clonal Expansion of TCR Vβ Subfamilies and the Cytotoxicity of T Cells Stimulated by PML-RARα Peptid.

Bacterial superantigens (SAgs) are potent T cell activators. Superantigens stimulate some population of T cells, based on Sag binding to specific T cell receptor variable region β chains, a property that distinguishes SAgs from conventional T cell mitogens. The biologic properties of SAgs make them attractive for use in immunotherapy. However, the T cell stimulatory effects of SAgs cannot be confined to specific antigens, its important role might be to enhance the immune effect for tumor antigens. PML-RARα peptide is a specific antigen associated with acute promyelocytic leukemia, which can induce specific proliferation of T cells from healthy human donor in vitro. But the immunogenicity of PML-RARα peptide is weak based on our previous study. In order to investigate the differentiation and proliferation of T cells induced by PML-RARα peptide that combined with Staphyloccucal enterotoxins A(SEA), T cell phenotype, TCR Vβ repertoire usage, clonality and cytotoxicity of T cells were analyzed. Peripheral blood mononuclear cells from healthy donors were cultured with PML-RARα peptide and SEA for 20 days. After induction, the distribution and clonal expansion of TCR Vβ subfamilies of T cells were examined by RT-PCR and GeneScan. The cytotoxicity of T cells induced against NB4 cell lines were examined by Cell Counting Kit-8 (CCK-8). The CD4 and CD8 surface marker on the harvested CD3+ T cells were detected by flow cytometry (FCM). And cytokine levels of IFN-γ, IL-4 and IL-10 in the cultured supernatants were measured by Enzyme-Linked Immunosorbent Assay (ELISA) as well. The results showed that restricted expression of Vβ repertoire (11 Vβ subfamilies) was found, which were Vβ1, Vβ2, Vβ3, Vβ6, Vβ7, Vβ8, Vβ13, Vβ14, Vβ16, Vβ17, Vβ19 subfamilies. Clonal expanded T cells could be identified in some Vβ subfamilies, which were preferentially used in Vβ13 and Vβ14. The specific cytotoxicity of T cells induced by PML-RARα peptide with SEA was higher than that from PML-RARα peptide alone induction group against NB4 cells. The FCM assay showed that the activation of CD8+ T cells was in priority induced by PML-RARα peptide whether with SEA or not, while it came out to be an equal between CD4+ and CD8+ T cells induced only by SEA. And the IFN-γ level of Th1-type cytokine was higher than that of the Th2-type cytokines (IL-4 and IL-8) in both cultured supernatants of T cells induced by PML-RARα peptide with or without SEA. In conclusion, PML-RARα peptide can induce specific T cells with clonal expansions of TCR Vβ subfamilies. SEA could enhance the induction of the specific PML-RARα peptide. The T cells induced by combination of PML-RARα peptide and SEA have higher specific antileukemia effect.

Activation-Induced Cytidine Deaminase Acts as a Mutator in BCR-ABL1-Transformed Acute Lymphoblastic Leukemia Cells.

Acute lymphoblastic leukemia (ALL) cells are derived from B and T cell precursors and typically carry rearranged immunglobulin (Ig) or T cell receptor (TCR) variable (V) region genes devoid of somatic mutations. The Philadelphia chromsome (Ph) encoding the oncogenic BCR-ABL1 kinase defines a subset of ALL with a particularly unfavorable prognosis. Here we show that oncogenic BCR-ABL1 kinase activity induces aberrant somatic hypermutation in Ph-positive ALL cells. Under physiological conditions, somatic hypermutation is restricted to mature germinal center B cells and depends on expression of the DNA-deaminating enzyme AID. Comparing Ph-positive and Ph-negative ALL cells, AID expression was found in 24 of 28 Ph-positive but only 3 of 80 Ph-negative ALLs. As shown by RT-PCR and Western blot, expression of AID in Ph-positive ALL cells reached similar levels as in germinal center B cells.Forced expression of BCR-ABL1 in Ph-negative ALL cells and usage of the BCR-ABL1-kinase inhibitor STI571 revealed that BCR-ABL1 kinase activity is required and sufficient to induce aberrant expression of AID in Ph-positive ALL. Consistent with aberrant AID expression in Ph-positive ALL, Ig VH region genes were mutated in most Ph-positive but unmutated in Ph-negative cases. Of note, also non-Ig genes including BCL6 and MYC harbored somatic mutations in Ph-positive but not Ph-negative ALL cells. Likewise, Ph-positive T cell lineage ALL cells express AID and carry somatically mutated TCRβ V region genes. As demonstrated by ligation-mediated PCR, AID introduced DNA-single-strand breaks also within the tumor suppressor gene CDKN2B in Ph-positive ALL cells, which was sensitive to both inhibition of BCR-ABL1 kinase activity and silencing of AID expression by RNA interference. These findings identify AID as a BCR-ABL1-induced mutator in Ph-positive ALL cells, which may be relevant with respect to the particularly unfavorable prognosis of this leukemia subset.

A bias in the alphabeta T cell receptor variable region gene usage in Takayasu's arteritis.

Takayasu's arteritis (TA) is a chronic large vessel vasculitis with a predilection for the aortic arch and its branches. T lymphocytes may be important in the pathogenesis, as they have been found to infiltrate the vascular lesions. To elucidate further the role of T cells in the disease, we studied circulating CD4+ and CD8+ T cells, expression of the activation marker (HLA-DR), marker for naive (CD45RA) and primed (CD45RO) cells and the different variable alpha/beta (AV/BV) gene segments on them. The TCR AV/BV repertoire was studied using a panel of 15 T cell receptor (TCR) V-specific MoAbs by flow cytometry in 18 patients and 23 age- and sex-matched controls. Patients had a higher percentage of AV12S1 (P < 0.05), BV6S7 (P < 0.05) and BV9 (P < 0.001)-bearing CD4+ cells. Patients also had a higher frequency of expansions, i.e. of T cell populations with an abnormally high TCR AV/BV gene usage. In patients' CD4+ subset of cells, there were 22 expansions out of 231 analyses (9.5%), whereas in controls, four were expanded out of 310 analyses (1%) (P < 0.001). For CD8+ cells, the frequency of expansions was 32 in 231 analyses (14%) in patients and nine out of 304 analyses in controls (3%) (P < 0.01). In addition, there was a correlation between CD4+ expansions and disease activity; nine out of 10 patients with active disease in comparison with two out of eight patients with inactive disease (P < 0.01) had an expansion. Some of the expanded populations in patients were phenotypically characterized and observed to be HLA-DR+, CD28+, CD45RA+ and CD45RO+, with a greater proportion being CD45RO+. Patients had a higher percentage of expression of HLA-DR on both CD4+ and CD8+ T cells (P < 0.01). The percentages of naive and primed CD4+ and CD8+ T cells, gammadelta+ T cells and natural killer cells were comparable to those in the control group.

Combination antiretroviral therapy results in a rapid increase in T cell receptor variable region beta repertoire diversity within CD45RA CD8 T cells in human immunodeficiency virus-infected children.

Human immunodeficiency virus (HIV) type 1 disrupts the T cell receptor (TCR) variable region (V) beta repertoire in CD8 T cells by impairing thymic capacity and skewing postthymic cellular maturation. The TCR repertoire was examined using spectratyping of CDR3 length diversity within CD45RA and CD45RO CD8 T cells in HIV-infected and healthy children. In healthy children, CDR3 lengths displayed Gaussian distribution in both CD45RA and CD45RO subsets. Vbeta families in HIV-infected children displayed a large proportion of perturbations in both subsets. High virus load and advanced immunosuppression correlated with increased perturbations within CD45RA but not CD45RO CD8 T cells. After therapy and virus suppression, there was rapid reestablishment of Gaussian distributions in CD45RA cells. HIV-1-induced disruption of TCR diversity within CD45RA CD8 T cells correlates with disease progression. Suppression of viral replication by treatment results in the rapid correction of TCR diversity in this CD8 subset because of emergence of new T cells from the thymus.

Analysis of T Cell Receptor Variable Regions and Complementarity Determining Region 3 of Infiltrating T Lymphocytes in the Liver of Patients with Chronic Type B Hepatitis

Objectives: T cell receptor (TCR) variable regions and complementarity determining regions 3 (CDR3) of infiltrating T cells in the livers of patients with chronic hepatitis B were examined in detail. Methods: TCR usage was examined by the inverse polymerase chain reaction in 2 hepatitis B e antigen-positive patients, one at an early stage and the other at an advanced stage. Results: In 61 productively rearranged clones bearing TCRα genes in the liver of the patient with early stage chronic hepatitis B, the Vα 7.2 gene segment was most frequently used (16.4%), while the other Vα gene segments were used less frequently. All but one of the clones with the Vα7.2 gene were joined with the Jα33 gene, and the CDR3 regions of these clones were the same length and similar in amino acid sequence. In contrast, in 68 productively rearranged clones from the liver of the patient with advanced stage disease, all Vα gene segments were used less frequently, and combinations of the Vα and Jα gene segments varied. Conclusions: Our data suggest a relatively restricted usage of TCR in the liver in early stage chronic hepatitis B.

Characterization of a Novel Staphylococcal Enterotoxin-like Superantigen, a Member of the Group V Subfamily of Pyrogenic Toxins

Staphylococcus aureus is an important human pathogen, causing a variety of diseases. Major virulence factors of this organism include staphylococcal enterotoxins (SEs) that cause food poisoning and toxic shock syndrome. Our study identified a novel enterotoxin-like protein that is a member of the new subfamily (group V) of pyrogenic toxin superantigens (PTSAgs) and examined its biochemical and immunobiological properties. The gene encoding the SE-like protein is directly 5' of another recently identified PTSAg, SEK. The SE-like protein had a molecular weight of 26000 and an experimentally determined isoelectric point between 7.5 and 8.0. We demonstrated that the PTSAg had many of the biological activities associated with SEs, including superantigenicity, pyrogenicity, and ability to enhance endotoxin shock, but lacked both lethality in rabbits when administered in subcutaneous miniosmotic pumps and emetic activity in monkeys. Recombinant protein stimulated human CD4 and CD8 T cells in a T cell receptor variable region, beta chain (TCRVbeta) specific manner. T cells bearing TCRVbeta 2, 5.1, and 21.3 were significantly stimulated.

Expression of T cell receptor variable region families by bone marrow gammadelta T cells in patients with IgA nephropathy.

IgA nephropathy (IgAN) is characterized by mesangial deposition of polymeric IgA (pIgA). Abnormalities of the IgA system include reduced mucosal and increased bone marrow (BM) pIgA production. Gammadelta T cells are regulators of mucosal IgA production and oral tolerance. We have described previously a deficiency of gammadelta T cells expressing Vgamma3 and Vdelta3 from the duodenal mucosa in IgAN. Since pIgA production is displaced to the BM, we have now studied BM gammadelta T cells in IgAN. Peripheral blood and BM aspirates were obtained from 14 patients with IgAN and 15 controls. Expression of TCR gamma and delta V region families was analysed by semiquantitative RT-PCR, and CDR3 spectratyping of Vgamma1-4 and Vdelta3 genes was performed. We found no difference between IgAN and controls in the V region usage of blood gammadelta T cells. However, in the BM of patients with IgAN, there was significantly reduced expression of the V region families Vgamma3 and Vdelta3, with the decrease in Vdelta3 being particularly striking. CDR3 spectratyping showed no abnormalities in blood or BM samples. Vgamma3 and Vdelta3 are underexpressed in the duodenum and the BM in IgAN. The combination of imbalanced mucosal and systemic pIgA production with deficient expression of gammadelta T cells using Vgamma3 and Vdelta3 in both sites may imply a role for these gammadelta T cells in the normal regulation of IgA immune responses, and in the complex immunopathogenesis of IgAN.