Immune activation in the context of HIV infection

Our objective was to study whether CD4⁺ or CD8⁺ T cells expressing particular T cell receptors (TCR) would accumulate in the lungs of patients with allergic asthma following allergen exposure. We thus analysed the TCR Vα and Vβ gene usage of CD4⁺ and CD8⁺lung and peripheral blood lymphocytes (PBL) of eight patients with allergic asthma before and 4 days after inhalation challenge with the relevant allergen. Lung cells obtained by bronchoalveolar lavage (BAL) and paired PBL samples were analysed by flow cytometry using a panel of anti-TCR V-specific monoclonal antibodies that encompass ≈ 50% of the T cell repertoire. Lung-limited T cell expansions were recorded in both the CD4⁺and the CD8⁺subsets. In BAL CD8⁺, out of a total of 126 analyses, the number of T cell expansions increased from two to 11 after challenge, some of them dramatic. In BAL CD4⁺the frequency of expansions was moderately increased already before challenge, but remained unchanged. A few expansions that tended to persist were noted in PBL CD8⁺. When analysing the overall change in TCR V gene usage the largest changes were also recorded in the BAL CD8⁺subset. Specific interactions between T cells and antigens may lead to an increased frequency of T cells using selected TCR V gene segments. In this study we demonstrate that following allergen bronchoprovocation in allergic asthmatic subjects, T cell expansions preferentially emerge in the lung CD8⁺T cell subset.     

Strict Adherence to a Common Rank Order of T-Cell Receptor Vβ Usage in Human Leucocyte Antigen Disparate Individuals

In order to investigate the T-cell receptor (TCR) Vβ usage in different T-cell subsets, the authors performed flow cytometric analyses using a large panel of TCR Vβ-specific monoclonal antibodies on CD4⁺, CD8⁺ CD28⁺ and CD8⁺ CD28^? T cells from 15 random blood donors, six umbilical cords and seven human leucocyte antigen (HLA) identical non-twin sibling pairs. The authors found that the proportion of T cells expressing each Vβ gene product was similar within CD4⁺ and CD8⁺ CD28⁺ T cells from all samples studied. For these T-cell subsets a rank order of Vβ usage could be identified which was adhered to by all donors. In contrast, within CD8⁺ CD28^? T cells a wide variation of Vβ usage was found between individuals, and no rank order correlation could be detected. Members of HLA identical sibling pairs were found to be no more similar in their usage of Vβ gene products than pairs of HLA disparate random blood donors. Groups of individuals sharing HLA antigens were no different from the groups not sharing such antigens in their usage of Vβ segments. The results suggest that HLA polymorphisms play no more than a minor part in determining TCR Vβ usage.     

A bias in the αβ 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 α/β (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, γδ⁺ T cells and natural killer cells were comparable to those in the control group.     

T-Cell Receptor Vβ Repertoire CDR3 Length Diversity Differs within CD45RA and CD45RO T-Cell Subsets in Healthy and Human Immunodeficiency Virus-Infected Children

The T-cell receptor (TCR) CDR3 length heterogeneity is formed during recombination of individual Vβ gene families. We hypothesized that CDR3 length diversity could be used to assess the fundamental differences within the TCR repertoire of CD45RA and CD45RO T-cell subpopulations. By using PCR-based spectratyping, nested primers for all 24 human Vβ families were developed to amplify CDR3 lengths in immunomagnetically selected CD45RA and CD45RO subsets within both CD4⁺ and CD8⁺ T-cell populations. Umbilical cord blood mononuclear cells or peripheral blood mononuclear cells obtained from healthy newborns, infants, and children, as well as human immunodeficiency virus (HIV)-infected children, were analyzed. All T-cell subsets from newborn and healthy children demonstrated a Gaussian distribution of CDR3 lengths in separated T-cell subsets. In contrast, HIV-infected children had a high proportion of predominant CDR3 lengths within both CD45RA and CD45RO T-cell subpopulations, most commonly in CD8⁺ CD45RO T cells. Sharp differences in clonal dominance and size distributions were observed when cells were separated into CD45RA or CD45RO subpopulations. These differences were not apparent in unfractionated CD4⁺ or CD8⁺ T cells from HIV-infected subjects. Sequence analysis of predominant CDR3 lengths revealed oligoclonal expansion within individual Vβ families. Analysis of the CDR3 length diversity within CD45RA and CD45RO T cells provides a more accurate measure of disturbances in the TCR repertoire than analysis of unfractionated CD4 and CD8 T cells.     

Stimulation of peripheral blood lymphocytes with Campylobacter jejuni generates a γδ T cell response in patients with Guillain–Barré syndrome

In three patients whose Guillain–Barré syndrome (GBS) was preceded by gastrointestinal infection due to Campylobacter jejuni, γδ T cells were generated from peripheral blood in response to in vitro stimulation with C. jejuni. In one of the patients, where a diagnostic sural nerve biopsy was performed, γδ T cells were also isolated following culture of the nerve tissue. Studies with healthy volunteers and C. jejuni gastroenteritis patients also showed preferential enrichment for γδ T cells in peripheral blood cells stimulated with C. jejuni, although the response was significantly lower than that seen in GBS patients. In two out of three GBS patients and all of the controls, γδ T cell receptor (TCR) gene usage was shown to be Vγ9/Vδ2⁺. In the GBS patient where nerve-infiltrating γδ T cells were isolated, these and C. jejuni-specific peripheral blood cells had similar TCR gene usage, predominantly consisting of Vγ5/Vδ1⁺ cells. Sequencing the Vδ1 products from nerve and peripheral blood showed similarities in CDR3 length, but the single Vδ1 sequence obtained from nerve was not identified in peripheral blood. These results suggest that the generation of γδ T cells is part of a normal immune response to C. jejuni, which, in patients with GBS, may contribute to the pathogenesis of their inflammatory neuropathy.     

CD4+ CD25high Foxp3+ regulatory T cells downregulate human Vδ2+ T-lymphocyte function triggered by anti-CD3 or phosphoantigen

Vδ2⁺ T cells, the major circulating T-cell receptor-γδ-positive (TCR-γδ⁺) T-cell subset in healthy adults, are involved in immunity against many microbial pathogens including Mycobacterium tuberculosis. Vδ2⁺ T cells recognize small phosphorylated metabolites (phosphoantigens), expand in response to whole M. tuberculosis bacilli, and complement the protective functions of CD4⁺ T cells. CD4⁺ CD25^high Foxp3⁺ T cells (Tregs) comprise 5–10% of circulating T cells and are increased in patients with active tuberculosis (TB). We investigated whether, in addition to their known role in suppressing TCR-αβ⁺ lymphocytes, Tregs suppress Vδ2⁺ T-cell function. We found that depletion of Tregs from peripheral blood mononuclear cells increased Vδ2⁺ T-cell expansion in response to M. tuberculosis (H37Ra) in tuberculin-skin-test-positive donors. We developed a suppression assay with fluorescence-activated cell sorting-purified Tregs and Vδ2⁺ T cells by coincubating the two cell types at a 1 : 1 ratio. The Tregs partially suppressed interferon-γ secretion by Vδ2⁺ T cells in response to anti-CD3 monoclonal antibody plus interleukin-2 (IL-2). In addition, Tregs downregulated the Vδ2⁺ T-cell interferon-γ responses induced by phosphoantigen (BrHPP) and IL-2. Under the latter conditions there was no TCR stimulus for Tregs and therefore IL-2 probably triggered suppressor activity. Addition of purified protein derivative (PPD) increased the suppression of Vδ2⁺ T cells, suggesting that PPD activated antigen-specific Tregs. Our study provides evidence that Tregs suppress both anti-CD3 and antigen-driven Vδ2⁺ T-cell activation. Antigen-specific Tregs may therefore contribute to the Vδ2⁺ T-cell functional deficiencies observed in TB.     

Role of γδ T lymphocytes in the development of Behçet's disease

Phenotypic and functional properties of γδ T cells, which play an important role in mucocutaneous immunity, were examined to elucidate whether immunological abnormality in Behc¸et's disease may be related to a specific T cell population. We found that CD45RA⁺Vγ9⁺Vδ2⁺γδ T cells, which constitute a minor population of γδ T cells in healthy individuals, were increased in number in Behc¸et's disease irrespective of disease activity. This CD45RA⁺ subset of γδ T cells in the active, but not inactive, phase of this disease expressed IL-2Rβ and HLA-DR, suggesting that they are activated in vivo in active Behc¸et's disease. In addition, the CD45RA⁺γδ T cells produced extreme amounts of tumour necrosis factor and contained perforin granules. These data indicate that a phenotypically distinct subset of γδ T cells, CD45RA⁺CD45RO⁻Vγ9⁺Vδ2⁺, may contribute to immunological abnormalities which may lead to complexity of pathophysiology in Behc¸et's disease.     

Peripheral blood T cell expansions in patients with Behc¸et's disease

Behc¸et's disease (BD) is a chronic multisystemic inflammatory disorder characterized mainly by recurrent oral and genital aphthous ulcerations and uveitis. Etiology and pathogenesis of BD remain unknown. T cell receptor (TCR) Vα/Vβ gene product expression as well as Jβ gene segment expression in peripheral blood of BD patients were analysed to investigate the possible role of T lymphocytes in the etiopathogenesis of BD. Flow cytometry with 12 TCR V-specific MoAbs was used for TCRV analyses. Jβ gene segment usage by T cell populations expressing certain Vβs was determined by polymerase chain reaction (PCR) technique with Vβ- and Cβ-specific primers, Southern blotting of PCR products, and subsequent hybridization with radiolabelled Jβ gene segment-specific probes. Although 13 of the 23 BD patients exhibited increases in expression of one or more TCR V-gene products, only expansions among the CD4⁺ T cell subset were significantly more frequent in BD patients (7/23) compared with healthy controls (0/15) (P = 0.019). Six out of eight cases followed for up to 20 months had at least one expansion correlated with disease activity. A strict preference for particular Jβ gene segments implicating clonality was apparent in all analysed T cell expansions and correlated well with disease activity. These results suggest a possible involvement of antigen-specific T lymphocytes in the pathogenesis of BD.     

Developmental regulation of p53-dependent radiation-induced thymocyte apoptosis in mice

The production of T cell receptor αβ⁺ (TCRαβ⁺) T lymphocytes in the thymus is a tightly regulated process that can be monitored by the regulated expression of several surface molecules, including CD4, CD8, cKit, CD25 and the TCR itself, after TCR genes have been assembled from discrete V, D (for TCR-β) and J gene segments by a site-directed genetic recombination. Thymocyte differentiation is the result of a delicate balance between cell death and survival: developing thymocytes die unless they receive a positive signal to proceed to the next stage. This equilibrium is altered in response to various physiological or physical stresses such as ionizing radiation, which induces a massive p53-dependent apoptosis of CD4⁺CD8⁺ double-positive (DP) thymocytes. Interestingly, these cells are actively rearranging their TCR-α chain genes. To unravel an eventual link between V(D)J recombination activity and thymocyte radio-sensitivity, we analysed the dynamics of thymocyte apoptosis and regeneration following exposure of wild-type and p53-deficient mice to different doses of γ-radiation. p53-dependent radio-sensitivity was already found to be high in immature CD4⁻CD8⁻ (double-negative, DN) cKit⁺CD25⁺ thymocytes, where TCR-β gene rearrangement is initiated. However, TCR-αβ⁻CD8⁺ immature single-positive thymocytes, an actively cycling intermediate population between the DN and DP stages, are the most radio-sensitive cells in the thymus, even though their apoptosis is only partially p53-dependent. Within the DP population, TCR-αβ⁺ thymocytes that completed TCR-α gene recombination are more radio-resistant than their TCR-αβ⁻ progenitors. Finally, we found no correlation between p53 activation and thymocyte sensitivity to radiation-induced apoptosis.     

γδ T lymphocyte responses to HIV

Natural immunity may be involved in controlling viral spread in hosts infected with HIV. A panel of γδ T cell receptor-positive lymphocyte clones was isolated from the peripheral blood of healthy HIV⁻ donors and tested for anti-HIV cytotoxic responses. Twelve of 30 (40%) Vγ9⁺Vδ2⁺ T cell clones, but none of seven Vδ1⁺ T cell clones, displayed lytic activity against HIV-infected cells. The Vγ9⁺/Vδ2⁺ clones cytotoxic for HIV-infected cells also lysed Daudi cells. However, not all Vγ9⁺/Vδ2⁺ clones which lysed Daudi targets had the capacity to lyse HIV-infected cells. Some of the γδ T cell clones were also investigated for potential proliferative responses to HIV-infected cells. One Vγ9⁺/Vδ2⁺ T cell clone (ME8-7) and one Vδ1⁺ T cell clone (ME18-2) demonstrated proliferative responses toward HIV-infected cells. Another Vγ9⁺/Vδ2⁺ clone (VM39) proliferated in response to cell-free HIV. Taken together, these results provide direct evidence of anti-HIV γδ T cell responses in healthy, HIV⁻ persons.     

HLA-DQ ASSOCIATIONS AND T-CELL RECEPTOR V-GENE USAGE IN PERIPHERAL BLOOD OF SWEDISH MYASTHENIA GRAVIS PATIENTS

To characterize better the functional aspects of the HLA class II associations with myasthenia gravis (MG), T-cell receptor (TCR) Vα/β elements were studied in peripheral blood in 29 Swedish MG patients. HLA typing had previously been done using polymerase chain reaction with sequence-specific primers (PCR-SSP) or combined with sequence-specific oligonucleotide probes (PCR-SSO). The TCR V gene expression was determined by fluorescence-activated cell sorter (FACS) analysis using 12 monoclonal antibodies (mAb) that detected 30–40% of CD4⁺ and CD8⁺ T cells. No correlation between HLA-DQ genotype and TCR V elements could be found, nor was any restricted V gene usage seen. Fourteen (48%) of the patients had T cells showing signs of abnormal expansion in peripheral blood. There was an increased expression of TCR V gene elements in CD8⁺ T cells in patients (13/29) compared with CD4⁺ T cells in patients (5/29) (P < 0.05) and in unthymectomized patients compared with controls (14/56) (P < 0.005). TCR V gene expression was also increased in the CD8⁺ population in unthymectomized (7/8) compared with thymectomized patients (6/21) (P < 0.01). There was an increased expression in both CD4⁺ and CD8⁺ populations in unthymectomized patients (7/8, 88%), compared with thymectomized patients (7/21) (P < 0.05). We conclude that the abnormal T-cell expansion in peripheral blood could be a reflection of non-specific pathogenic processes in the muscle and thymus.     

Peripheral CD25 positive T lymphocytes with biased T cell receptor Vβ gene usage in autoimmune endogenous posterior uveitis

Aims—To determine T cell receptor (TCR) Vβ gene usage in peripheral blood T lymphocytes of patients with endogenous posterior uveitis (EPU). If biased TCR variable (V) gene usage occurs in this autoimmune disease, it should be detectable in immune activated peripheral blood T cells in vivo.     

Positive and Negative Selection in Transgenic Mice Expressing a T-Cell Receptor Specific for Influenza Nucleoprotein and Endogenous Superantigen

A transgenic mouse was generated expressing on most (>80%) of thymocytes and peripheral T cells a T-cell receptor isolated from a cytotoxic T-cell clone (F5). This clone is CD8⁺ and recognizes αα366-374 of the nucleoprotein (NP 366-374) of influenza virus (A/NT/60/68), in the context of Class ,MHC D^b (Townsend et al., 1986). The receptor utilizes the Vβ11 and Vα4 gene segments for the β chain and α chain, respectively (Palmer et al., 1989). The usage of Vβ11 makes this TcR reactive to Class II IE molecules and an endogenous ligand recently identified as a product of the endogenous mammary tumour viruses (Mtv) 8, 9, and 11 (Dyson et al., 1991). Here we report the development of F5 transgenic T cells and their function in mice of the appropriate MHC (C57BL/10 H-2^b, IE^-) or in mice expressing Class II MHC IE (e.g., CBA/Ca H-2^k and BALB/c H-2^d) and the endogenous Mtv ligands. Positive selection of CD8⁺ T cells expressing the Vβ11 is seen in C57BL/10 transgenic mice (H-2^b). Peripheral T cells from these mice are capable of killing target cells in an antigen-dependent manner after a period of ^{in vitro} culture with IL-2. In the presence of Class II MHC IE molecules and the endogenous Mtv ligand, most of the single-positive cells carrying the transgenic T-cell receptor are absent in the thymus. Unexpectedly, CD8⁺ peripheral T-cells in these (H-2^k or H-2^d) F5 mice are predominantly Vβ11 positive and also have the capacity to kill targets in an antigen-dependent manner. This is true even following backcrossing of the F5 TcR transgene to H-2^d scid/scid mice, in which functional rearrangement of endogenous TcR alpha- and beta-chain genes is impaired.     

Abnormal T cell receptor V gene usage in myasthenia gravis: prevalence and characterization of expanded T cell populations

The usage of T cell receptor (TCR) Vα/Vβ chains on cells from 38 patients with myasthenia gravis (MG) was determined by flow cytometry. There was a decreased number of cells expressing Vβ2 in CD8⁺ and Vβ3 in CD4⁺ cells in patients compared with healthy individuals. Abnormal expansions of T cells using particular TCR Vα/Vβ gene products were found in 18/38 patients. A significantly higher usage of Vβ13 was observed but there was no restriction with regard to other TCR Vα/Vβ. Expanded cells belonging to both CD4⁺ and CD8⁺ were present in MG patients while restricted to the CD8⁺ population in healthy individuals. To elucidate the role of the expanded populations, we studied characteristics of the expanded and non-expanded T cells from MG patients who had persistent T cell expansions over more than 2 years. The cells were analysed with regard to phenotype, cytokine secretion, cytokine mRNA expression and reactivity with the autoantigen, the acetylcholine receptor. The characteristics of the expanded populations in MG clearly differed from those found in healthy individuals. More cells in the CD4⁺ expanded populations expressed HLA-DR and there was also a tendency for higher expression of CD25, CD28 and CD57. The number of cells spontaneously secreting cytokines was higher in the expanded populations. A dominant Th1-type cytokine secretion and mRNA expression was noted. Autoantigen-reactive CD4⁺ T cells were largely restricted to the expanded populations.     

Defective integration of activating signals derived from the T cell receptor (TCR) and costimulatory molecules in both CD4+ and CD8+ T lymphocytes of common variable immunodeficiency (CVID) patients

CVID is characterized by hypogammaglobulinaemia and impaired antibody production. Previous studies demonstrated defects at the T cell level. In the present study the response of purified CD4⁺ and CD8⁺ T lymphocytes to stimulation with anti-TCR monoclonal antibody (the first signal) in combination with anti-CD4 or anti-CD8, anti-CD2 and anti-CD28 MoAbs (the costimulatory signals) was investigated. Both CD4⁺ and CD8⁺ T cells from the patients showed significantly reduced IL-2 release following stimulation via TCR and costimulation via CD4 or CD8 and CD2, respectively. However, normal IL-2 production following TCR plus phorbol myristate acetate (PMA) costimulation and normal expression of an early activation marker, CD69, after TCR + CD28 stimulation indicated that TCR was able to transduce a signal. Furthermore, both IL-2 and IL-4 release were impaired in CD4⁺ lymphocytes following TCR + CD28 stimulation. In addition, stimulation via TCR + CD28 resulted in significantly decreased expression of CD40 ligand in the patients. These results suggest that the integration of activating signals derived from the TCR and costimulatory molecules is defective in CVID patients; the defect is not confined to costimulation via a single molecule, or restricted to cells producing Thl-type cytokines such as IL-2, and is expressed in both CD4⁺ and CD8⁺T cell subsets.     

Cytolytic mechanisms and T-cell receptor Vβ usage by ex vivo generated Epstein–Barr virus-specific cytotoxic T lymphocytes

Ex-vivo-generated Epstein–Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) have been used for cellular adoptive immunotherapy of EBV-associated lymphomas. Here we investigated the phenotypes, cytolytic mechanisms, polyfunctionality and T-cell receptor (TCR) usage in growing and established CTL, generated by weekly stimulation with an EBV-transformed autologous lymphoblastoid cell line (LCL). Our results showed that phenotypically mature CTL developed within the first 4 weeks of culture, with an increase in CD45RO and CD69, and a decrease in CD45RA, CD62L, CD27 and CD28 expression. Spectratyping analysis of the variable β-chain of the TCR revealed that TCR repertoire remained diverse during the course of culture. Cytotoxicity of CTL was significantly inhibited by concanamycin A (P < 0·0001) and ethylene glycol-bis tetraacetic acid (P < 0·0001), indicating that a calcium and perforin-mediated exocytosis pathway with the release of granzyme B was the principal cytotoxic mechanism. The CTL mainly produced interferon-γ (IFN-γ) or tumour necrosis factor-α (TNF-α) upon restimulation with autologous LCL, although there were some polyfunctional cells producing IFN-γ and TNF-α. Granzyme B, perforin and Fas ligand were detected in CD8⁺ and CD4⁺ cells in all CTL; however, a greater proportion of CD8⁺ than CD4⁺ T cells expressed granzyme B (P < 0·0001) and more granzyme B was detected in CD8⁺ T cells than in CD4⁺ T cells (P = 0·001). This difference was not observed with Fas ligand or perforin expression. Our results provide insight into the basic characteristics of ex-vivo-generated CTL.     

Proliferation of distinct human T cell subsets in response to live, killed or soluble extracts of Mycobacterium tuberculosisand Myco. avium

The proliferative responses of distinct cell subsets from healthy, bacille Calmette–Guérin (BCG)-vaccinated blood donors were assessed after in vitro stimulation with live or UV-killed Mycobacterium tuberculosis and Myco. avium or with soluble extracts obtained from either mycobacterial species. Proliferation of cell subsets was evaluated by flow cytometric determination of 5-bromo-2′-deoxy-uridine incorporation into DNA and simultaneous identification of surface phenotypic markers. In the presence of monocytes, the response to whole (live or killed) bacteria was characterized by a predominant proliferation of CD4⁺ αβ⁺ T cells and, to a lesser extent, of CD8⁺ αβ⁺ T cells. Proliferation of CD8⁺ αβ⁺ T cells was primarily elicited by live rather than killed bacilli (P < 0.05). Conversely, when soluble bacterial extracts were used as stimulators, a preferential proliferation of γδ⁺ T cells, expressing predominantly Vγ9⁺ and Vδ2⁺ T cell receptor chains, was recorded. Moreover, when monocyte-depleted cell populations were directly cultured with live bacteria, a marked proportion of CD3⁻CD16⁺ (natural killer (NK)) cells was detected among the responding cells. Although both αβ, γδ and NK cells have been previously shown to react with mycobacteria in vitro, their relative contributions to the response have been difficult to assess. Using a flow cytometric technique which allows direct identification of proliferating cells within complex cell populations, our study demonstrates significant differences in the ability of various mycobacterial antigen preparations to elicit proliferation of distinct cell subsets.