Costimulation of CD3/TcR complex with either integrin or nonintegrin ligands protects CD4+ allergen-specific T-cell clones from programmed cell death

An optimal stimulation of CD4⁺ cells in an immune response requires not only signals transduced via the TcR/CD3 complex, but also costimulatory signals delivered as a consequence of interactions between T-cell surface-associated costimulatory receptors and their counterparts on antigen-presenting cells ‘APC). The intercellular adhesion molecule-1 ‘ICAM-1, CD54) efficiently costimulates proliferation of resting, but not antigen-specific, T cells. In contrast, CD28 and CD2 support interleukin ‘IL)-2 synthesis and proliferation of antigen-specific T cells more efficiently than those of resting T cells. The molecular basis for this differential costimulation of T cells is poorly understood. Cypress-specific T-cell clones ‘TCC) were generated from four allergic subjects during in vivo seasonal exposure to the allergen. Purified cypress extract was produced directly from fresh collected pollen and incubated with the patients' mononuclear cells. Repeated allergen stimulation was performed in T-cell cultures supplemented with purified extract and autologous APC. The limiting-dilution technique was then adopted to generate allergen-specific TCC, which were also characterized by their cytokine secretion pattern as ThO ‘IL-4 plus interferon-gamma) or Th2 ‘IL-4). Costimulation-induced proliferation or apoptosis was measured by propidium iodide cytofluorometric assay. By cross-linking cypress-specific CD4⁺ and CD8⁺ T-cell clones with either anti-CD3 or anti-CD2, anti-CD28, and anti-CD54 monoclonal antibodies, we demonstrated that CD4+ clones ‘with ThO- or Th2-type cytokine production pattern) undergo programmed cell death only after anti-CD3 stimulation, whereas costimulation with either anti-CD54 or anti-CD28 protects target cells from apoptosis. The costimulation-induced protection from apoptotic death was associated with a significant rise in IL-4 secretion in both Th0 and Th2-type clones. In contrast, cypress-specific Th0 CD8⁺ clones were more susceptible to stimulation-induced apoptosis via either anti-CD3 or anti-CD2, alone or in combination with anti-CD54 or anti-CD28, thus displaying only slight but nonsignificant modifications in the pattern of IL-4 secretion. The death-promoting costimulatory effects were not observed with highly purified normal resting CD4⁺ or CD8⁺ lymphocytes. Taken together, these results suggest that TcR engagement by an allergen in the context of functionally active APC induces activation-dependent cell death of some, perhaps less specific, cells, and this may be an important homeostatic mechanism through which functional expansion of allergen-specific T cells is regulated during an ongoing immune response.     

CD8+ CD28− T cells in vertically HIV-infected children

To evaluate whether vertical HIV infection interferes with the expression of CD28 on T lymphocytes, 25 HIV-infected children and 29 seroreverted children born to HIV⁺ mothers were studied. The percentage of CD28⁻ cells among CD8⁺ T lymphocytes was higher in HIV-infected children than in controls (P < 0.001). In fact, in HIV-infected children, this percentage was elevated from the first year of life, while in healthy seroreverted children, the proportion of CD28⁻ cells among CD8⁺ cells rose progressively with age (r = 0.49; P = 0.008). In HIV⁺ children, the CD8⁺ CD28⁻, but not CD8⁺ CD28⁺ cell proportion was significantly correlated with immunological markers of disease progression, such as CD4⁺ cell loss (r = −0.65; P < 0.001) and the level of in vitro spontaneous lymphocyte apoptosis (r = 0.53; P = 0.03).     

CD28 activation promotes Th2 subset differentiation by human CD4+ cells

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4⁺ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-γ (IFN-γ) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.     

CD8+ lymphocyte phenotype and cytokine production in long-term non-progressor and in progressor patients with HIV-1 infection

In most HIV-1-infected patients, clinical and immunological progression develops within a few years. Few infected people, termed long-term non-progressors (LTNP), remain healthy and immunologically stable for a long time. The factors governing the maintenance of this condition are not well known, but it is conceivable that CD8⁺ lymphocytes, cells that play a central role in controlling in vitro HIV replication, may have a part in vivo in this process. The aim of this study was to characterize the phenotypic profile and the cytokine production of CD8⁺ cells in a group of LTNP patients who had stable CD4⁺ cell counts (>500/mm³) for at least 7 years. Their CD8⁺ absolute numbers were similar to a control group composed of HIV-1⁺ patients who have a progressive decline of their CD4⁺ cell counts. However, our multiparameter immunofluorescence studies show that a clinical and immunologically stable condition is associated with the presence of a CD28⁺, CD95 strongly positive CD8⁺ population, while disease progression is marked by the CD28⁻CD95⁺CD8⁺ subset. Purified CD8⁺ cells from LTNP retain their ability to produce IL-2, interferon-gamma (IFN-γ) and, to a lesser degree, to produce IL-10 and IL-4. In contrast, CD8⁺ cells from progressors are unable to secrete IL-2 and IL-10. Although CD8⁺ cytokine profile does not fit with the proposed T helper (Th)1/Th2 switch in progressive HIV infection, LTNP CD8⁺ T cells maintain their capacity to produce IL-2 and IL-10 (Th0-like), a pattern very similar to that observed in normal HIV healthy controls. We suggest that CD8⁺ cells expressing CD28, CD95 and having a Th0-like profile may be considered to be associated with long-term survival.     

Reversal of the inhibitory effects of HIV-gp120 on CD4+ T cells by stimulation through the CD28 pathway

The effects of exposure to HIV-gp120 on proliferation and cytokine production by T cell lines were investigated. T cell lines were generated by stimulation of peripheral blood mononuclear cells from several healthy donors with cross-linked anti-CD3 antibodies and IL-2. These T cell lines exhibited the characteristics of Th1 cells, producing IL-2 and interferon-gamma (IFN-γ), but not IL-4, on stimulation with anti-CD3 antibodies. In the presence of gp120, stimulation with anti-CD3 antibodies was inhibited in terms of both proliferative responses and the secretion of IL-2 and IFN-γ. Similar effects were observed when a T cell line was stimulated in the presence of a synthetic peptide representing the CD4-binding region of gp120. Neither gp120 nor the CD4-binding region peptide had any effect on IL-4 production by the T cell lines. Stimulation through the CD28 pathway partially restored both the proliferative effect and cytokine production by T cell lines in response to anti-CD3 antibodies in the presence of gp120. Anti-CD28 antibodies also partially restored cytokine production when purified CD4⁺ T cells from a T cell line were stimulated with anti-CD3 antibodies in the presence of gp120. Anti-gp120 antibodies partially or completely reversed the inhibitory effects of gp120 on T cell proliferation. These results indicate that stimulation through the CD28 pathway may restore defective CD4⁺ T cell responses in HIV-infected individuals.     

Superantigen activation of CD4+ and CD8+ T cells from HIV-infected subjects: role of costimulatory molecules and antigen-presenting cells (APC)

T cell receptor (TCR) triggering via superantigens induces decreased proliferative responses and increased apoptosis in T cells from HIV-infected patients compared with controls. Our aim was to delineate the role of intrinsic T cell defects, of APC dysfunction and of cytokines and costimulatory signal dysregulation in the deficient responses of CD4⁺and CD8⁺ T cells from HIV⁺ subjects to the superantigen Staphylococcus enterotoxin A (SEA). Proliferation and IL-2Rα up-regulation on SEA-stimulated CD4⁺and CD8⁺T cells in whole blood were reduced in HIV⁺ subjects with CD4 counts < 500, compared with controls. Neither addition of IL-2, IL-12 or phorbol myristate acetate (PMA) nor neutralization of endogenous IL-10, tumour necrosis factor-alpha (TNF-α), TNF-β or transforming growth factor-beta (TGF-β) could restore the decreased activation by SEA. Possible intrinsic T cell defects were studied by presenting SEA on HLA-DR-transfected Chinese hamster ovary (CHO) cells, co-expressing LFA3 and/or CD80, to purified T cells. In this system CD8⁺T cells from most HIV⁺ patients were hyporesponsive with regard to IL-2 production, IL-2Rα up-regulation and proliferation, whereas clearly reduced responses were only shown in CD4⁺T cells from AIDS patients. Similarly, apoptosis was increased in CD8⁺T cells from all patients, but only in CD4⁺T cells from AIDS patients. During HIV infection, the responses to TCR triggering through SEA are deficient in both T cell subsets. The intrinsic defect appears earlier during disease progression in purified CD8⁺T than in CD4⁺T cells, it occurs in conjunction with both CD2 and CD28 costimulation, and it is correlated with increased levels of apoptosis.     

HIV infection and aging: enhanced Interferon- and Tumor Necrosis Factor-alpha production by the CD8+ CD28- T subset

Background  

T cells from HIV⁺ and aged individuals show parallels in terms of suppressed proliferative activity and interleukin-2 (I1-2) production and an increased number of CD8⁺ CD28^- T cells. In order to compare cytokine production from T cells from these two states, CD4⁺ and CD8⁺ T cells from HIV⁺ aged, and normal young donors (controls) were monitored for cytokine production by flow cytometry, quantitative PCR and ELISA upon activation by PMA and anti-CD3. In addition, the CD8⁺ T cell subsets CD28⁺ and CD28^- from the HIV⁺ and the aged groups were evaluated for cytokine production by flow cytometry, and compared with those from young controls.     

Increased expression of CD80, CD86 and CD70 on T cells from HIV-infected individuals upon activation in vitro: regulation by CD4+ T cells

T cells express CD28 and CD27 which transduce co-stimulatory signals after interaction with their ligands on antigen-presenting cells (APC). These ligands, CD80, CD86 and CD70, are also expressed to some extent on activated T cells. Here, we show that in human immunodeficiency virus (HIV)-infected individuals, CD28 and CD27 expression is decreased on CD8⁺ T cells. On the other hand, T cell stimulation in vitro induced high CD80, CD86 and CD70 expression on T cells from HIV-infected individuals. It appeared that an inverted CD4:CD8 T cell ratio could explain this enhanced expression of co-stimulatory ligands. Indeed, high expression levels of CD80, CD86 and CD70 were found on activated CD8⁺ T cells from HIV⁻ individuals cultured in the absence of CD4⁺ T cells. Addition of CD4⁺ T cells prevented this up-regulation. However, in HIV-infected individuals, addition of excess autologous or healthy control CD4⁺ T cells did not completely counteract up-regulation of co-stimulatory ligand expression on CD8⁺ T cells. Thus, to some extent, CD8⁺ T cells in HIV-infected individuals appeared to be refractory to CD4⁺ T cell-mediated regulation of ligand expression in vitro. Activated T cells from HIV-infected individuals and activated CD8⁺ T cells from healthy controls were able to act as accessory cells in CD3-induced T cell proliferation, which was dependent on cell-cell contact. Thus, we showed that T cells from HIV-infected individuals express enhanced levels of co-stimulatory ligands upon activation, which provides them with accessory cell properties. Enhanced stimulatory potential of these nonprofessional APC may contribute to persistently high levels of immune activation in HIV infection related to disease progression.     

Type 2 diabetes mellitus is associated with altered CD8+ T and natural killer cell function in pulmonary tuberculosis

Type 2 diabetes mellitus (DM) is associated with expanded frequencies of mycobacterial antigen-specific CD4⁺ T helper type 1 (Th1) and Th17 cells in individuals with active pulmonary tuberculosis (TB). No data are available on the role of CD8⁺ T and natural killer (NK) cells in TB with coincident DM. To identify the role of CD8⁺ T and NK cells in pulmonary TB with diabetes, we examined mycobacteria-specific immune responses in the whole blood of individuals with TB and DM (TB-DM) and compared them with those without DM (TB-NDM). We found that TB-DM is characterized by elevated frequencies of mycobacterial antigen-stimulated CD8⁺ T cells expressing type 1 [interferon-γ and interleukin-2 (IL-2)] and type 17 (IL-17F) cytokines. We also found that TB-DM is characterized by expanded frequencies of TB antigen-stimulated NK cells expressing type 1 (tumour necrosis factor-α) and type 17 (IL-17A and IL-17F) cytokines. In contrast, CD8⁺ T cells were associated with significantly diminished expression of the cytotoxic markers perforin, granzyme B and CD107a both at baseline and following antigen or anti-CD3 stimulation, while NK cells were associated with significantly decreased antigen-stimulated expression of CD107a only. This was not associated with alterations in CD8⁺ T-cell or NK cell numbers or subset distribution. Therefore, our data suggest that pulmonary TB complicated with type 2 DM is associated with an altered repertoire of cytokine-producing and cytotoxic molecule-expressing CD8⁺ T and NK cells, possibly contributing to increased pathology.     

Changes to the cytokine microenvironment in the genital tract mucosa of HIV+ women

As previous studies have indicated that genital tract mucosal T cell function may be impaired in HIV infection, we investigated the T cell cytokine mRNA in the genital tract mucosa of HIV-infected women to determine if there are alterations in the cytokine profile which may explain the T cell impairment. The in situ hybridization technique was used to investigate the T helper-1 (Th1: IL-2, interferon-gamma (IFN-γ)) and Th2 cytokine (IL-4, IL-5, IL-10) mRNA profile in cervical biopsies from 10 HIV⁺ and 10 HIV^? subjects. Cervical intraepithelial neoplasia (CIN) and genital infection had previously been excluded and the distribution of immunocompetent cells within the cervical mucosa was known for each subject. Non-parametric tests were used to compare the optical density (OD) of cytokine mRNA in the HIV⁺ and HIV^? groups. Comparisons were also made between peripheral CD4 lymphocyte counts, cervical CD4/CD8 T lymphocyte ratios and cytokine mRNA OD in HIV⁺ subjects. The HIV⁺ women had significantly higher mRNA OD for the Th2 cytokines IL-4, IL-5 and IL-10 than HIV^? women. There was also significantly lower IL-2 mRNA OD in the former group. HIV⁺ women had lower IFN-γ mRNA than HIV^? women, but the difference was not statistically significant. There was no correlation between cytokine mRNA OD and peripheral CD4 count or cervical CD4/CD8 ratio. The predominance of Th2 cytokines, which are immuno-inhibitory, in the cervical mucosa of HIV⁺ women may underlie the impaired cytotoxic potential observed in the CD8⁺ T lymphocytes and may contribute to the susceptibility of HIV-infected women to recurrent genital tract infections and cervical neoplasia.     

Association of IL-12+ DC with High CD3+CD4-DR+ Lymphocyte Counts in Long-term HIV-infected Hemophilia Patients With Clinically Stable Disease

We investigated dendritic cell (DC) subsets as well as cellular and humoral immune parameters in long-term HIV-infected hemophilia patients with clinically stable disease. DC subsets were determined by their function to produce either IL-10 or IL-12. CD11c⁺CD83⁺CD40⁺IL-10⁺ and CD11c⁺CD83⁺CD40⁺IL-12⁺ DC were studied in freshly obtained blood samples of 28 HIV⁺ and 15 HIV⁻ patients and 39 healthy controls using four-color flow cytometry, and were analyzed in relation to blood lymphocyte subpopulation counts, proportions of IgG-coated CD4⁺ blood lymphocytes, neopterin, and HIV-1 viral load in the plasma, and in vitro responses of patient lymphocytes to mitogens. Proportions and ratios of IL-10⁺ DC and IL-12⁺ DC were similar in HIV⁺ and HIV⁻ patients and healthy controls. Whereas IL-12⁺ DC in HIV⁺ patients were associated with high CD3⁺CD4^-DR⁺ lymphocyte counts, IL-10⁺ DC were associated with the proportion of IgG-coated CD4⁺ blood lymphocytes. These data suggest that long-term HIV-infected hemophilia patients with clinically stable disease have normal levels of functional IL-10⁺ DC and IL-12⁺ DC that might be involved in halting the progression of disease.     

Active CD4+ helper T cells directly stimulate CD8+ cytotoxic T lymphocyte responses in wild-type and MHC II gene knockout C57BL/6 mice and transgenic RIP-mOVA mice expressing islet β-cell ovalbumin antigen leading to diabetes

CD4⁺ helper T (Th) cells play crucial role in priming, expansion and survival of CD8⁺ cytotoxic T lymphocytes (CTLs). However, how CD4⁺ Th cell's help is delivered to CD8⁺ T cells in vivo is still unclear. We previously demonstrated that CD4⁺ Th cells can acquire ovalbumin (OVA) peptide/major histocompatibility complex (pMHC I) and costimulatory CD80 by OVA-pulsed DC (DC_OVA) stimulation, and then stimulate OVA-specific CD8⁺ CTL responses in C57BL/6 mice. In this study, we further investigated CD4⁺ Th cell's effect on stimulation of CD8 CTL responses in major histocompatibility complex (MHC II) gene knockout (KO) mice and transgenic rat insulin promoter (RIP)-mOVA mice with moderate expression of self OVA by using CD4⁺ Th cells or Th cells with various gene deficiency. We demonstrated that the in vitro DC_OVA-activated CD4⁺ Th cells (3 × 10⁶ cells/mouse) can directly stimulate OVA-specific CD8⁺ T-cell responses in wild-type C57BL/6 mice and MHC II gene KO mice lacking CD4⁺ T cells. A large amount of CD4⁺ Th cells (12 × 10⁶ cells/mouse) can even overcome OVA-specific immune tolerance in transgenic RIP-mOVA mice, leading to CD8⁺ CTL-mediated mouse pancreatic islet destruction and diabetes. The stimulatory effect of CD4⁺ Th cells is mediated by its IL-2 secretion and CD40L and CD80 costimulations, and is specifically delivered to OVA-specific CD8⁺ T cells in vivo via its acquired pMHC I complexes. Therefore, the above elucidated principles for CD4⁺ Th cells will have substantial implications in autoimmunity and antitumor immunity, and regulatory T-cell-dependent immune suppression.     

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.     

Anomalies of the CD8+ T cell pool in haemochromatosis: HLA-A3-linked expansions of CD8+CD28− T cells

The present study consists of a phenotypic and functional characterization of peripheral blood T lymphocytes in a group of 21 patients with hereditary haemochromatosis (HH), an MHC class I-linked genetic disease resulting in iron overload, and a group of 30 healthy individuals, both HLA-phenotyped. The HH patients studied showed an increased percentage of CD8⁺ CD28⁻ T cells with a corresponding reduction in the percentage of CD8⁺ CD28⁺ T cells in peripheral blood relative to healthy blood donors. No anomalies of CD28 expression were found in the CD4⁺ subset. The presence of the HLA-A3 antigen but not age accounted for these imbalances. Thus, an apparent failure of the CD8⁺ CD28⁺ T cell population ‘to expand’, coinciding with an ‘expansion’ of CD8⁺ CD28⁻ T cells in peripheral blood of HLA-A3⁺ but not HLA-A3⁻ HH patients was observed when compared with the respective HLA-A3-matched control group. A significantly higher percentage of HLA-DR⁺ but not CD45RO⁺ cells was also found within the peripheral CD8⁺ T cell subset in HH patients relative to controls. Phytohaemagglutinin (PHA) stimulation of peripheral blood mononuclear cells (PBMC) for 5 days showed: (i) that CD8⁺ CD28⁺ T cells both in controls and HH were able to expand in vitro; (ii) that CD8⁺ CD28⁻ T cells decreased markedly after activation in controls but not in HH patients. Moreover, functional studies showed that CD8⁺ cytotoxic T lymphocytes (CTL) from HH patients exhibited a diminished cytotoxic activity (approx. two-fold) in standard ⁵¹Cr-release assays when compared with CD8⁺ CTL from healthy controls. The present results provide additional evidence for the existence of phenotypic and functional anomalies of the peripheral CD8⁺ T cell pool that may underlie the clinical heterogeneity of this iron overload disease. They are of particular relevance given the recent discovery of a novel mutated MHC class I-like gene in HH.     

Intracellular cytokine production by human CD4+ and CD8+ T cells from normal and immunodeficient donors using directly conjugated anti-cytokine antibodies and three-colour flow cytometry

Using three-colour flow cytometry, we have measured intracellular IL-2, interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) induced in human CD4⁺ and CD8⁺ T cells from normal donors and patients with common variable immunodeficiency (CVID). Since a new range of directly FITC-conjugated anti-cytokine antibodies was used, conditions were optimized for the concentration of antibody, for cell permeabilization and fixation, and for the time of exposure to monensin to retain the cytokines within the cell. Kinetics of intracellular cytokine production were measured for up to 20h in culture with phorbol myristate acetate (PMA) and ionomycin, or with phytohaemagglutinin (PHA). Kinetic studies of activation with PMA and ionomycin show that a higher proportion of normal CD4⁺ cells can make IL-2 than the other two cytokines, and that there are more TNF-α-positive CD4⁺ cells than cells with IFN-γ. For normal CD8⁺ cells the highest production of cytokine is of IFN-γ (up to 50% of the cells) especially at longer times (10–20h) of stimulation. For CD8⁺ cells, IL-2-positive cells exceed those with TNF-α. The other mitogenic stimulus used (PHA) was grossly inferior to PMA and ionomycin in its ability to induce intracellular cytokines. The time of exposure to monensin was also examined. Its continuous presence in the cultures (up to a maximum of 20h) increased the detection of IL-2-positive cells without apparently reducing the percentage of cytokine-positive CD4⁺ or CD8⁺ cells. Finally, using optimal conditions, we compared cytokine production in cells from patients with the disease CVID and showed normal cellular levels of ability to produce IL-2 and TNF-α but significantly raised levels of production of IFN-γ in both CD4⁺ and CD8⁺ lymphocytes. This suggests that the pathology of this disease may involve an excessive Th1-type response.     

CD4 T Cell Clones Producing both Interferon-γ and Interleukin-10 Predominate in Bronchoalveolar Lavages of Active Pulmonary Tuberculosis Patients

The pattern of cytokine production in T cell clones derived from bronchoalveolar lavages (BAL) of active pulmonary tuberculosis (TB) patients was analyzed in clones obtained by limiting dilution procedures which expand with high efficiency either total T lymphocytes, independently of their antigen-recognition specificity, or Mycobacterium tuberculosis-specific T cells. BAL-derived clones, representative of CD4⁺ cells from five patients with active TB, produced significantly higher amounts of IFN-γ than BAL-derived CD4⁺ clones from three inactive TB donors or four controls (with unrelated, noninfectious pathology). Average IL-4 and IL-10 production did not differ significantly in the three groups. Although these data suggest a predominant Th1 response to M. tuberculosis infection in the lungs, the majority of BAL-derived CD4⁺ clones produced both IFN-γ and IL-10 and the percentage of clones with this pattern of cytokine production was significantly higher in clones derived from BAL of active TB patients than from controls. Only rare clones derived from peripheral blood (PB)-derived CD45RO⁺ CD4⁺ T cells of both patients (nine cases) and controls (four cases) produced both IFN-γ and IL-10; instead, the IL-10-producing clones derived from PB T cells most often also produced IL-4, displaying a typical Th2 phenotype. Higher average amounts of IFN-γ and IL-10 were produced by BAL-derived CD8⁺ clones of four active TB patients than of four controls, although the frequency of CD8⁺ clones producing both IFN-γ and IL-10 was lower than that of CD4⁺ clones. The M. tuberculosis-specific BAL-derived T cell clones from three active TB patients were almost exclusively CD4⁺ and produced consistently high levels of IFN-γ often in association with IL-10, but very rarely with IL-4. Unlike the BAL-derived clones, the M. tuberculosis-specific clones derived from PB CD45RO⁺ CD4⁺ T cells of three different active TB patients and two healthy donors showed large individual variability in cytokine production as well as in the proportion of CD4⁺, CD8⁺, or TCR γ/δ⁺ clones. These results indicate the predominance of CD4⁺ T cells producing both the proinflammatory cytokine IFN-γ and the anti-inflammatory cytokine IL-10 in BAL of patients with active TB.     

Differential production of IL-10 by T cells and monocytes of HIV-infected individuals: association of IL-10 production with CD28-mediated immune responsiveness

Immune unresponsiveness in HIV-1 infection can result from impaired signals delivered by the costimulatory CD28-B7 pathway and the altered production of immunoregulatory cytokines, in particular IL-10, whose production is altered in HIV-1 infection. In this study we investigate IL-10 regulation in T cells and monocytes from HIV⁺ individuals, and its association with CD28-mediated T cell proliferation. IL-10 production as analysed in T cell- and monocyte-depleted peripheral blood mononuclear cells (PBMC), and by intracellular staining at the single-cell level, reveals a defect in IL-10 production by CD4⁺ and CD8⁺ T cells, whereas monocytes constitute the major IL-10-producing cell type. To investigate the impact of IL-10 on immune responsiveness, CD28-mediated proliferative responses in HIV⁺ individuals were correlated with PHA-induced IL-10 production. CD4⁺ T cells expressed CD28, yet exhibited markedly reduced CD28-mediated cell proliferation. This CD28-mediated CD4⁺ T cell proliferation was found to be inversely associated with the levels of PHA-induced IL-10 production and could be restored, at least in part, by anti-IL-10 antibodies. These results suggest that IL-10 production is differentially regulated in T cells and monocytes of HIV⁺ individuals, and that IL-10 may have a role in inducing immune unresponsiveness by modulating the CD28-B7 pathway.     

Association of T cell dysfunction with the presence of IgG autoantibodies on CD4+ lymphocytes in haemophilia patients; results of a 10-year study

HIV induces progressive dysfunction followed by numerical depletion of CD4⁺ lymphocytes. IgG autoantibodies and gp120-containing immune complexes have been implicated in the pathogenesis of AIDS. We carried out a longitudinal study in 19 HIV^– and 72 HIV⁺ haemophilia patients over a 10-year period in order to investigate a possible relationship between the occurrence of autoantibodies and CD4⁺ lymphocyte changes. IgM, IgG, C3d and gp120 on the surface of CD4⁺ lymphocytes were determined in heparinized whole blood with flow cytometry and double-fluorescence. The in vitro response of autoantibody-coated cells was tested in cell cultures with concanavalin A (Con A), phytohaemagglutinin (PHA), pokeweed mitogen (PWM), anti-CD3 MoAb or pooled allogeneic stimulator cells (MLC). After a 10-year follow up, 12 of 71 HIV⁺ and 16 of 19 HIV^– haemophilia patients showed no evidence of immunoglobulins on circulating CD4⁺ lymphocytes. HIV^– haemophilia patients without autoantibodies had CD4⁺and CD8⁺ cell counts in the normal range (957 ± 642/μl and 636 ± 405/μl) and normal T cell responses in vitro (mean relative response (RR) ≥ 0.7). In contrast, HIV⁺ haemophilia patients showed immunological abnormalities which were associated with the autoantibody and immune complex load of CD4⁺ blood lymphocytes. HIV⁺ patients without autoantibodies had a mean CD4⁺ lymphocyte count of 372 ± 274/μl, a mean CD8⁺ lymphocyte count of 737 ± 435/μl, and normal T lymphocyte stimulation in vitro (mean RR ≥ 0.7). HIV⁺ patients with complement-fixing IgM on CD4⁺ lymphocytes had somewhat lower CD4⁺ (255 ± 246/μl, P= NS) and CD8⁺ (706 ± 468/μl, P= NS) lymphocyte numbers, and also normal T lymphocyte stimulation (mean RR ≥ 0.7) in vitro. However, patients with complement-fixing IgG autoantibodies showed a strong decrease of CD4⁺ (150 ± 146/μl, P< 0.02) and CD8⁺ (360 ± 300/μl, P< 0.02) lymphocytes and impaired CD4⁺ lymphocyte stimulation in vitro with a mean RR of 0.5 ± 0.5 for Con A (P= NS), 0.7 ± 0.8 for PHA (P< 0.03), 0.4 ± 0.4 for PWM (P= NS), 0.8 ± 1.2 for anti-CD3 MoAb (P< 0.04) and 0.7 ± 1.0 for pooled allogeneic stimulator cells (P= 0.05). Patients with gp120-containing immune complexes on CD4⁺ blood lymphocytes demonstrated strongly decreased CD4⁺(25 ± 35/μl, P< 0.0001) and CD8⁺ (213 ± 212/μl, P< 0.006) lymphocyte counts as well as strongly impaired T lymphocyte responses in vitro upon stimulation with PHA (RR 0.2 ± 0.1, P < 0.02), PWM (RR 0.2 ± 0.2, P= 0.05), anti-CD3 MoAb (RR 0.1 ± 0.1, P< 0.04), and allogeneic stimulator cells (RR 0.2 ± 0.1, P< 0.02). These data led us to speculate that autoantibody formation against CD4⁺ lymphocytes is an important mechanism in the pathogenesis of AIDS. We hypothesize that autoantibodies against circulating CD4⁺ lymphocytes inhibit CD4⁺ cell function, especially the release of cytokines, and induce CD4⁺ cell depletion. The reduction and dysfunction of CD4⁺ lymphocytes may be responsible for the CD8⁺ cell depletion observed in HIV⁺ patients.     

Interleukin-13 is produced by activated human CD45RA+ and CD45R0+ T cells: modulation by interleukin-4 and interleukin-12

Interleukin (IL)-13 is a cytokine originally identified as a product of activated T cells. Little is known, however, about IL-13 production by human T cells and its modulation by other cytokines. Here, we show that IL-13 is produced by activated human CD4⁺ and CD8⁺ CD45R0⁺ memory T cells and CD4⁺ and CD8⁺ CD45RA⁺ naive T cells. In contrast, IL-4, which shares many biological activities with IL-13, is only produced by CD45R0⁺ T cells following activation. Analysis of intracellular cytokine production by single CD45RA⁺ and CD45R0⁺ T cells indicated that IL-13 continued to be produced for more than 24 h after stimulation, whereas IL-4 could not be detected after 24 h. These data were confirmed by measurement of specific mRNA and suggest that IL-13, unlike IL-4, but like interferon-γ (IFN-γ), is a cytokine with long-lasting kinetics. The majority of human CD45R0⁺ T cells produced IL-4 and IL-13 simultaneously. In contrast, IFN-γ protein was generally not co-expressed with IL-4 or IL-13. IL-4 added to primary cultures of highly purified peripheral blood T cells activated by the combination of anti-CD3+anti-CD28 mAb enhanced IL-13 production by CD45RA⁺ and to a lesser extent by CD45R0⁺ T cells. Under these conditions, however, IL-12 inhibited IL-13 production by CD45RA⁺ T cells and to a lesser extent by CD45R0⁺ T cells in a dose-dependent fashion. These inhibiting effects were not related to enhanced IFN-γ production induced by IL-12, since IFN-γ by itself did not affect IL-13 production. Collectively, our data indicate that IL-13 is produced by peripheral blood T cells which also produce IL-4, but not IFN-γ, and by naive CD45RA⁺ T cells which, in contrast, fail to produce IL-4. These observations, together with the long-lasting production of IL-13, suggest that IL-13 may have IL-4-like functions in situations where T cell-derived IL-4 is still absent or where its production has already been down-regulated.