Loss of IFN-gamma production by invariant NK T cells in advanced cancer

Invariant NK T cells express certain NK cell receptors and an invariant TCRalpha chain specific for the MHC class I-like CD1d protein. These invariant NK T cells can regulate diverse immune responses in mice, including antitumor responses, through mechanisms including rapid production of IL-4 and IFN-gamma, but their physiological functions remain uncertain. Invariant NK T cells were markedly decreased in peripheral blood from advanced prostate cancer patients, and their ex vivo expansion with a CD1d-presented lipid Ag (alpha-galactosylceramide) was diminished compared with healthy donors. Invariant NK T cells from healthy donors produced high levels of both IFN-gamma and IL-4. In contrast, whereas invariant NK T cells from prostate cancer patients also produced IL-4, they had diminished IFN-gamma production and a striking decrease in their IFN-gamma:IL-4 ratio. The IFN-gamma deficit was specific to the invariant NK T cells, as bulk T cells from prostate cancer patients produced normal levels of IFN-gamma and IL-4. These findings support an immunoregulatory function for invariant NK T cells in humans mediated by differential production of Th1 vs Th2 cytokines. They further indicate that antitumor responses may be suppressed by the marked Th2 bias of invariant NK T cells in advanced cancer patients.     

Differential regulation of IFN-gamma, TNF-alpha, and IL-10 production by CD4(+) alphabetaTCR+ T cells and vdelta2(+) gammadelta T cells in response to monocytes infected with Mycobacterium tuberculosis-H37Ra.

Mycobacterium tuberculosis bacilli readily activate CD4(+) and gammadelta T cells. CD4(+) and gammadelta T cells were compared for their ability to regulate IFN-gamma, TNF-alpha, and IL-10 production, cytokines with significant roles in the immune response to M. tuberculosis. PBMC from healthy tuberculin positive donors were stimulated with live M. tuberculosis-H37Ra. CD4(+) and gammadelta T cells were purified by negative selection and tested in response to autologous monocytes infected with M. tuberculosis. Both subsets produced equal amounts of secreted IFN-gamma. However, the precursor frequency of IFN-gamma secreting gammadelta T cells was half that of CD4(+) T cells, indicating that gammadelta T cells were more efficient producers of IFN-gamma than CD4(+) T cells. TNF-alpha production was markedly enhanced by addition of CD4(+) and gammadelta T cells to M. tuberculosis infected monocytes, and TNF-alpha was produced by both T cells and monocytes. No differences in TNF-alpha enhancement were noted between CD4(+) and gammadelta T cells. IL-10 production by M. tuberculosis infected monocytes was not modulated by CD4(+) or gammadelta T cells. Thus CD4(+) and gammadelta T cells had similar roles in differential regulation of IFN-gamma, TNF-alpha, and IL-10 secretion in response to M. tuberculosis infected monocytes. However, the interaction between T cells and infected monocytes differed for each cytokine. IFN-gamma production was dependent on antigen presentation and costimulators provided by monocytes. TNF-alpha levels were increased by addition of TNF-alpha produced by T cells and IL-10 production by monocytes was not modulated by CD4(+) or gammadelta T cells.     

Increased nonobese diabetic Th1:Th2 (IFN-gamma:IL-4) ratio is CD4+ T cell intrinsic and independent of APC genetic background

Autoreactive CD4(+) T cells play a major role in the pathogenesis of autoimmune diabetes in nonobese diabetic (NOD) mice. We recently showed that the non-MHC genetic background controlled enhanced entry into the IFN-gamma pathway by NOD vs B6.G7 T cells. In this study, we demonstrate that increased IFN-gamma, decreased IL-4, and decreased IL-10 production in NOD T cells is CD4 T cell intrinsic. NOD CD4(+) T cells purified and stimulated with anti-CD3/anti-CD28 Abs generated greater IFN-gamma, less IL-4, and less IL-10 than B6.G7 CD4(+) T cells. The same results were obtained in purified NOD.H2(b) vs B6 CD4(+) T cells, demonstrating that the non-MHC NOD genetic background controlled the cytokine phenotype. Moreover, the increased IFN-gamma:IL-4 cytokine ratio was independent of the genetic background of APCs, since NOD CD4(+) T cells generated increased IFN-gamma and decreased IL-4 compared with B6.G7 CD4(+) T cells, regardless of whether they were stimulated with NOD or B6.G7 APCs. Cell cycle analysis showed that the cytokine differences were not due to cycle/proliferative differences between NOD and B6.G7, since stimulated CD4(+) T cells from both strains showed quantitatively identical entry into subsequent cell divisions (shown by CFSE staining), although NOD cells showed greater numbers of IFN-gamma-positive cells with each subsequent cell division. Moreover, 7-aminoactinomycin D and 5-bromo-2'-deoxyuridine analysis showed indistinguishable entry into G(0)/G(1), S, and G(2)/M phases of the cell cycle for both NOD and B6.G7 CD4(+) cells, with both strains generating IFN-gamma predominantly in the S phase. Therefore, the NOD cytokine effector phenotype is CD4(+) T cell intrinsic, genetically controlled, and independent of cell cycle machinery.     

Selective requirement for CD40-CD154 in drug-induced type 1 versus type 2 responses to trinitrophenyl-ovalbumin

CD154 is transiently expressed by activated T cells and interacts with CD40 on B cells, dendritic cells, macrophages, and monocytes. This costimulatory receptor-ligand couple seems decisive in Ag-driven immune responses but may be differentially involved in type 1 vs type 2 responses. We studied the importance of CD40-CD154 in both responses using the reporter Ag popliteal lymph node assay in which selectively acting drugs generate clearly polarized type 1 (streptozotocin) or type 2 (D-penicillamine, diphenylhydantoin) responses to a constant coinjected Ag in the same mouse strain. Treatment of mice with anti-CD154 reduced characteristic immunological parameters in type 2 responses (B and CD4(+) T cell proliferation, IgG1 and IgE Abs, and IL-4 secretion) and only slightly affected the type 1 response (small decrease in IFN-gamma production, influx of CD11c(+) and F4/80(+) cells, and prevention of architectural disruption of the lymph node, but no effect on IgG2a Ab and TNF-alpha secretion or B and CD4(+) T cell proliferation). The findings indicate that the CD40-CD154 costimulatory interaction is a prerequisite in drug-induced type 2 responses and is only marginally involved in type 1 responses. The observed expression patterns of CD80 and CD86 on different APC (B cells in type 2 and dendritic cells in type 1) may be responsible for this discrepancy.     

Experimental African trypanosomiasis: a subset of pathogenic, IFN-gamma-producing, MHC class II-restricted CD4+ T cells mediates early mortality in highly susceptible mice

Infections of highly susceptible BALB/c mice with virulent strains of Trypanosoma congolense or Trypanosoma brucei result in rapid death (8 days). We have previously shown that this mortality is IFN-gamma dependent. In this study we show that IFN-gamma is produced predominantly by CD3+Thy1.2+TCRbeta+CD4+ T cells shortly before the death of infected mice. Mortality may therefore be dependent on IFN-gamma-producing CD4+ T cells. Surprisingly, infected CD4+/+ and CD4-/- BALB/c mice have similar parasitemia and survival time. In infected CD4-/- mice, the production of both IFN-gamma and IL-10 is very low, suggesting that both cytokines are predominantly produced by CD4+ T cells and that the outcome of the disease might depend on the balance of their effects. Infected BALB/c mice partially depleted of CD4+ T cells or MHC class II function have lower parasitemia and survive significantly longer than infected normal BALB/c mice or infected BALB/c mice whose CD4+ T cells are fully depleted. Partial depletion of CD4+ T cells markedly reduces IFN-gamma secretion without a major effect on the production of IL-10 and parasite-specific IgG2a Abs. Based on our previous and current data, we conclude that a subset of a pathogenic, MHC class II-restricted CD4+ T cells (Tp cells), activated during the course of T. congolense infection, mediates early mortality in infected BALB/c mice via excessive synthesis of IFN-gamma. IFN-gamma, in turn, exerts its pathological effect by enhancing the cytokine release syndrome of the macrophage system activated by the phagocytosis of parasites. We speculate that IL-10-producing CD4+ T cells might counteract this effect.     

Excessive production of IFN-gamma in patients with systemic lupus erythematosus and its contribution to induction of B lymphocyte stimulator/B cell-activating factor/TNF ligand superfamily-13B

Expression and immunological significance of IFN-gamma, a pivotal cytokine in murine lupus, have not been clearly demonstrated in human systemic lupus erythematosus (SLE). In the present study we investigated the expression of IFN-gamma in peripheral blood T cells from patients with SLE and its role in the production of the soluble B lymphocyte stimulator (sBLyS). Peripheral blood T cells from patients with SLE expressed significantly larger amounts of IFN-gamma in response to stimulation with anti-CD3 mAb plus anti-CD28 mAb than those from normal controls as shown by three analytical methods, including ELISA, flow cytometry, and quantitative RT-PCR. The ratio of IFN-gamma-producing T cells to effector memory T cells in CD3(+)CD4(+) and CD3(+)CD8(+) populations in patients with SLE was significantly higher than that of normal controls. The T-box expressed in T cells (T-bet) mRNA/GATA-binding protein-3 (GATA-3) mRNA ratio was significantly higher in patients with SLE than in normal controls. T cell culture supernatants from patients with SLE contained significantly higher sBLyS-inducing activity than normal controls; this was almost completely inhibited by the addition of anti-human IFN-gamma mAb. Percentages of BLyS-expressing peripheral blood monocytes in patients with SLE were significantly higher than those of normal controls. Monocytes from patients with SLE produced significantly larger amounts of sBLyS in response to IFN-gamma than those from normal controls. Taken together, these data strongly indicate that the overexpression of IFN-gamma in peripheral blood T cells contributes to the immunopathogenesis of SLE via the induction of sBLyS by monocytes/macrophages, which would promote B cell activation and maturation.     

Agrin signalling contributes to cell activation and is overexpressed in T lymphocytes from lupus patients

It is shown in this study that the heparan sulfate proteoglycan agrin is overexpressed in T cells isolated from patients with the autoimmune disease systemic lupus erythematosus (SLE). Freshly isolated CD4(+) and CD8(+) subpopulations both exhibited higher expression over healthy controls, which however, gradually declined when cells were cultured in vitro. Agrin expression was induced following in vitro activation of cells via their Ag receptor, or after treatment with IFN-alpha, a cytokine shown to be pathogenic in lupus. Furthermore, serum from SLE patients with active disease was able to induce agrin expression when added to T cells from healthy donors, an increase that was partially blocked by neutralizing anti-IFN-alpha Abs. Cross-linking agrin with mAbs resulted in rapid reorganization of the actin cytoskeleton, activation of the ERK MAPK cascade, and augmentation of anti-CD3-induced proliferation and IL-10 production, indicating that agrin is a functional receptor in T cells. These results demonstrate that agrin expression in human T cells is regulated by cell activation and IFN-alpha, and may have an important function during cell activation with potential implications for autoimmunity.     

Increased expression of SLAM receptors SLAMF3 and SLAMF6 in systemic lupus erythematosus T lymphocytes promotes Th17 differentiation

Altered T cell function in systemic lupus erythematosus (SLE) is determined by various molecular and cellular abnormalities, including increased IL-17 production. Recent evidence suggests a crucial role for signaling lymphocyte activation molecules (SLAMs) in the expression of autoimmunity. In this study, we demonstrate that SLAMF3 and SLAMF6 expression is increased on the surface of SLE T cells compared with normal cells. SLAM coengagement with CD3 under Th17 polarizing conditions results in increased IL-17 production. SLAMF3 and SLAMF6 T cell surface expression and IL-17 levels significantly correlate with disease activity in SLE patients. Both naive and memory CD4(+) T cells produce more IL-17 in response to SLAM costimulation as compared with CD28 costimulation. In naive CD4(+) cells, IL-17 production after CD28 costimulation peaks on day 3, whereas costimulation with anti-SLAMF3 and anti-SLAMF6 Abs results in a prolonged and yet increasing production during 6 d. Unlike costimulation with anti-CD28, SLAM costimulation requires the presence of the adaptor molecule SLAM-associated protein. Thus, engagement of SLAMF3 and SLAMF6 along with Ag-mediated CD3/TCR stimulation represents an important source of IL-17 production, and disruption of this interaction with decoy receptors or blocking Abs should mitigate disease expression in SLE and other autoimmune conditions.     

Differential susceptibility to staphylococcal superantigen (SsAg)-induced apoptosis of CD4+ T cells from atopic dermatitis patients and healthy subjects: the inhibitory effect of IL-4 on SsAg-induced apoptosis

This study had two aims: 1) to determine whether there are differences between atopic dermatitis (AD) patients and healthy subjects in staphylococcal superantigen (SsAg)-induced CD4(+) T cell activation, cytokine production, chemokine receptor expression, and apoptosis; and 2) to investigate the effect of IL-4 on SsAg-induced apoptosis. By using immunofluorescence and annexin V staining, we analyzed PBMC with or without staphylococcal enterotoxin B (SEB) stimulation in the presence or absence of rIL-4 or anti-IL-4-neutralizing Abs in 15 healthy subjects and 27 AD patients. We found that SEB preferentially induced production of Th1 cytokine in SEB-reactive (TCRVbeta3(+) or Vbeta12(+) or Vbeta17(+)) CD4(+) T cells from healthy subjects and Th2 cytokine in those from AD patients. SEB induced up-regulation of CXCR3(+) cells in SEB-reactive CD4(+) T cells from healthy subjects and CCR4(+) cells in those from AD patients. SEB-reactive CD4(+) T cells from AD patients were more resistant to SEB-induced apoptosis than those from healthy subjects. There was no significant difference between AD and healthy subjects in SEB-induced activation of CD4(+) T cells. CXCR3(+) CD4(+) T cells were more susceptible to SEB-induced apoptosis than CCR4(+) CD4(+) T cells in healthy subjects. Exogenously added IL-4 inhibited SEB-induced apoptosis of SEB-reactive CD4(+) and CXCR3(+) CD4(+) T cells but not of CCR4(+) CD4(+) T cells in healthy subjects. Inhibition of endogenous IL-4 increased SEB-induced apoptosis of SEB-reactive CD4(+) T cells from AD patients. These results might provide new clues to the mechanism that SsAgs contribute to the persistence and exacerbation of allergic skin inflammation in AD.     

Chimeras of labile toxin one and cholera toxin retain mucosal adjuvanticity and direct Th cell subsets via their B subunit

Native cholera toxin (nCT) and the heat-labile toxin 1 (nLT) of enterotoxigenic Escherichia coli are AB5-type enterotoxins. Both nCT and nLT are effective adjuvants that promote mucosal and systemic immunity to protein Ags given by either oral or nasal routes. Previous studies have shown that nCT as mucosal adjuvant requires IL-4 and induces CD4-positive (CD4+) Th2-type responses, while nLT up-regulates Th1 cell production of IFN-gamma and IL-4-independent Th2-type responses. To address the relative importance of the A or B subunits in CD4+ Th cell subset responses, chimeras of CT-A/LT-B and LT-A/CT-B were constructed. Mice nasally immunized with CT-A/LT-B or LT-A/CT-B and the weak immunogen OVA developed OVA-specific, plasma IgG Abs titers similar to those induced by either nCT or nLT. Both CT-A/LT-B and LT-A/CT-B promoted secretory IgA anti-OVA Ab, which established their retention of mucosal adjuvant activity. The CT-A/LT-B chimera, like nLT, induced OVA-specific mucosal and peripheral CD4+ T cells secreting IFN-gamma and IL-4-independent Th2-type responses, with plasma IgG2a anti-OVA Abs. Further, LT-A/CT-B, like nCT, promoted plasma IgG1 more than IgG2a and IgE Abs with OVA-specific CD4+ Th2 cells secreting high levels of IL-4, but not IFN-gamma. The LT-A/CT-B chimera and nCT, but not the CT-A/LT-B chimera or nLT, suppressed IL-12R expression and IFN-gamma production by activated T cells. Our results show that the B subunits of enterotoxin adjuvants regulate IL-12R expression and subsequent Th cell subset responses.     

IL-2 is required for the activation of memory CD8+ T cells via antigen cross-presentation

Dendritic cells (DCs) are capable of capturing exogenous Ag for the generation of MHC class I/peptide complexes. For efficient activation of memory CD8(+) T cells to occur via a cross-presentation pathway, DCs must receive helper signals from CD4(+) T cells. Using an in vitro system that reflects physiologic recall memory responses, we have evaluated signals that influence helper-dependent cross-priming, while focusing on the source and cellular target of such effector molecules. Concerning the interaction between CD4(+) T cells and DCs, we tested the hypothesis that CD40 engagement on DCs is critical for IL-12p70 (IL-12) production and subsequent stimulation of IFN-gamma release by CD8(+) T cells. Although CD40 engagement on DCs, or addition of exogenous IL-12 are both sufficient to overcome the lack of help, neither is essential. We next evaluated cytokines and chemokines produced during CD4(+) T cell/DC cross talk and observed high levels of IL-2 produced within the first 18-24 h of Ag-specific T cell engagement. Functional studies using blocking Abs to CD25 completely abrogated IFN-gamma production by the CD8(+) T cells. Although required, addition of exogenous IL-2 did not itself confer signals sufficient to overcome the lack of CD4(+) T cell help. Thus, these data support a combined role for Ag-specific, cognate interactions at the CD4(+) T cell/DC as well as the DC/CD8(+) T cell interface, with the helper effect mediated by soluble noncognate signals.     

Cytokine production by Vgamma(+)-T-cell subsets is an important factor determining CD4(+)-Th-cell phenotype and susceptibility of BALB/c mice to coxsackievirus B3-induced myocarditis

Two coxsackievirus B3 (CVB3) variants (H3 and H310A1) differ by a single amino acid mutation in the VP2 capsid protein. H3 induces severe myocarditis in BALB/c mice, but H310A1 is amyocarditic. Infection with H3, but not H310A1, preferentially activates Vgamma4 Vdelta4 cells, which are strongly positive for gamma interferon (IFN-gamma), whereas Vgamma1 Vdelta4 cells are increased in both H3 and H310A1 virus-infected animals. Depletion of Vgamma1(+) cells using monoclonal anti-Vgamma1 antibody enhanced myocarditis and CD4(+)-, IFN-gamma(+)-cell responses in both H3- and H310A1-infected mice yet decreased the CD4(+)-, IL-4(+)-cell response. Depleting Vgamma4(+) cells suppressed myocarditis and reduced CD4(+) IFN-gamma(+) cells but increased CD4(+) IL-4(+) T cells. The role of cytokine production by Vgamma1(+) and Vgamma4(+) T cells was investigated by adoptively transferring these cells isolated from H3-infected BALB/c Stat4 knockout (Stat4ko) (defective in IFN-gamma expression) or BALB/c Stat6ko (defective in IL-4 expression) mice into H3 virus-infected wild-type BALB/c recipients. Vgamma4 and Vgamma1(+) T cells from Stat4ko mice expressed IL-4 but no or minimal IFN-gamma, whereas these cell populations derived from Stat6ko mice expressed IFN-gamma but no IL-4. Stat4ko Vgamma1(+) cells (IL-4(+)) suppress myocarditis. Stat6ko Vgamma1(+) cells (IFN-gamma(+)) were not inhibitory. Stat6ko Vgamma4(+) cells (IFN-gamma(+)) significantly enhanced myocarditis. Stat4ko Vgamma4(+) cells (IL-4(+)) neither inhibited nor enhanced disease. These results show that distinct gammadelta-T-cell subsets control myocarditis susceptibility and bias the CD4(+)-Th-cell response. The cytokines produced by the Vgamma subpopulation have a significant influence on the CD4(+)-Th-cell phenotype.     

Impairment of CD8+ T suppressor cell function in patients with active systemic lupus erythematosus

Alteration of T cell suppression function has been recognized in patients with systemic lupus erythematosus (SLE). Recently, CD8(+) T suppressor lymphocytes (CD8(+) Ts) have been generated in vitro by incubating purified CD8(+) T cells with IL-2 and GM-CSF. Using this method, we generated CD8(+) Ts from patients affected by SLE. No major differences were found in the CD8(+) Ts phenotype between SLE patients and healthy subjects. CD8(+) Ts from SLE patients with active disease did not inhibit the anti-CD3 mAb-induced proliferation of autologous PBMC, whereas CD8(+) Ts from SLE patients in remission exerted an inhibitory activity comparable to normal subjects. The inhibitory effect of CD8(+) Ts cells was neither mediated by cytotoxic activity nor by apoptosis induction. Two cytokines, IFN-gamma and IL-6, were found to be responsible for the function of CD8(+) TS: In fact, counteraction of CD8(+) Ts suppression activity was obtained by blocking IFN-gamma with a specific Ab or by inhibiting CD8(+) Ts-mediated IL-6 secretion by an antisense oligonucleotide. Interestingly, CD8(+) Ts from SLE patients showed a peculiar cytokine pattern characterized by an impaired secretion of IL-6 and an increased secretion of IL-12. Thus, it appears that an altered balance between inhibitory (IL-6) and stimulatory (IL-12) cytokines might be responsible for the functional impairment of CD8(+) Ts in SLE patients.     

Hepatitis C virus-specific Th17 cells are suppressed by virus-induced TGF-beta

IL-17-secreting T (Th17) cells play a protective role in certain bacterial infections, but they are major mediators of inflammation and are pathogenic in organ-specific autoimmune diseases. However, human Th17 cells appear to be resistant to suppression by CD4(+)CD25(+)FoxP3(+) regulatory T cells, suggesting that they may be regulated by alternative mechanisms. Herein we show that IL-10 and TGF-beta suppressed IL-17 production by anti-CD3-stimulated PBMC from normal individuals. TGF-beta also suppressed IL-17 production by purified CD4(+) T cells, whereas the inhibitory effect of IL-10 on IL-17 production appears to be mediated predominantly by its effect on APC. An examination of patients infected with hepatitis C virus (HCV) demonstrated that Ag-specific Th17 cells are induced during infection and that these cells are regulated by IL-10 and TGF-beta. PBMC from HCV Ab-positive donors secreted IL-17, IFN-gamma, IL-10, and TGF-beta in response to stimulation with the HCV nonstructural protein 4 (NS4). Furthermore, NS4 induced innate TGF-beta and IL-10 expression by monocytes from normal donors and at higher levels from HCV-infected patients. Neutralization of TGF-beta, and to a lesser extent IL-10, significantly enhanced NS4-specific IL-17 and IFN-gamma production by T cells from HCV-infected donors. Our findings suggest that both HCV-specific Th1 and Th17 cells are suppressed by NS4-induced production of the innate anti-inflammatory cytokines IL-10 and TGF-beta. This may represent a novel immune subversion mechanism by the virus to evade host-protective immune responses. Our findings also suggest that TGF-beta and IL-10 play important roles in constraining the function of Th17 cells in general.     

Interleukin-15 modulates interferon-gamma and beta-chemokine production in patients with HIV infection: implications for immune-based therapy

Interleukin (IL)-15 is a cytokine that has lymphocyte stimulatory activity similar to that of IL-2, and plays important immunoregulatory functions during HIV disease. To evaluate the role of IL-15 in HIV infection the following patients were studied: 18 antiretroviral-naive patients with advanced disease; 19 patients with continuous viral suppression and immunological response after 48-120 weeks of highly active antiretroviral therapy (HAART); and 12 patients with evidence of virological and immunological HAART treatment failure. Nineteen healthy blood donors were included as controls. The production of IL-15 by human peripheral blood monocytes stimulated with lipopolysaccharide and Mycobacterium avium complex, the priming effect of IL-15 on IFN-gamma production from purified CD4(+) and CD8(+) T cells, and the ability of IL-15 to stimulate the beta-chemokine release from purified CD4(+) and CD8(+) T cells were analyzed. In the present work IL-15 production by human peripheral blood monocytes was significantly increased in HIV-infected patients with long-term virological and immunological response to HAART. IL-15 enhanced the in vitro priming of CD4(+) and CD8(+) T cells for IFN-gamma production, also in patients receiving HAART. Finally, IL-15 had positive effects on RANTES, MIP-1alpha, and MIP-1beta release by CD4(+) and CD8(+) T cells. In conclusion IL-15 could affect the immune response of HIV-infected patients by augmenting and/or modulating IFN-gamma production and beta-chemokine release. These data about functional properties of IL-15 could provide new implications for immune-based therapies in HIV infection.     

A clonal culture system demonstrates that IL-4 induces a subpopulation of noncytolytic T cells with low CD8, perforin, and granzyme expression

Immune deviation of cytolytic T cell function, induced by type 2 cytokines like IL-4, is an attractive concept to explain failure of the immune system in some diseases. However, this concept is challenged by previous conflicting results on whether type 2 cytokine-producing CD8(+) T cells are cytolytic. Therefore, we have analyzed the relationship between cytolytic activity and cytokine production among large numbers of primary CD8(+) T cell clones. Single murine CD8(+) T cells of naive phenotype were activated at high efficiency with immobilized Abs to CD3, CD8, and CD11a in the presence of IL-2 (neutral conditions) or IL-2, IL-4, and anti-IFN-gamma Ab (type 2-polarizing conditions) for 8-9 days. Under neutral conditions, most clones produced IFN-gamma without IL-4 and were cytolytic. Under type 2-polarizing conditions, most clones produced IFN-gamma and IL-4 but displayed variable cytolytic activity and CD8 expression. Separation on the basis of surface CD8 levels revealed that, compared with CD8(high) cells from the same cultures, CD8(low) cells were poorly cytolytic and expressed low levels of perforin mRNA and protein and granzyme A, B, and C mRNA. A similar, smaller population of noncytolytic CD8(low) cells was identified among CD8(+) T cells activated in mixed lymphocyte reaction with IL-4. Variable efficiency of generation of the noncytolytic cells may account for the differing results of earlier studies. We conclude that IL-4 promotes the development of a noncytolytic CD8(low) T cell phenotype that might be important in tumor- or pathogen-induced immune deviation.     

Allergic inflammatory response to short ragweed allergenic extract in HLA-DQ transgenic mice lacking CD4 gene.

To investigate the role of HLA-DQ molecules and/or CD4(+) T cells in the pathogenesis of allergic asthma, we generated HLA-DQ6 and HLA-DQ8 transgenic mice lacking endogenous class II (Abeta(null)) and CD4 genes and challenged them intranasally with short ragweed allergenic extract (SRW). We found that DQ6/CD4(null) mice developed a strong eosinophilic infiltration into the bronchoalveolar lavage and lung tissue, while DQ8/CD4(null) mice were normal. However, neither cytokines nor eosinophil peroxidase in the bronchoalveolar lavage of DQ6/CD4(null) mice was found. In addition, the airway reactivity to methacholine was elevated moderately in DQ6/CD4(null) mice compared with the high response in DQ/CD4(+) counterparts and was only partially augmented by CD4(+) T cell transfer. The DQ6/CD4(null) mice showed Th1/Th2-type cytokines and SRW-specific Abs in the immune sera in contrast to a direct Th2 response observed in DQ6/CD4(+) mice. The proliferative response of spleen mononuclear cells and peribronchial lymph node cells demonstrated that the response to SRW in DQ6/CD4(null) mice was mediated by HLA-DQ-restricted CD4(-)CD8(-)NK1.1(-) T cells. FACS analysis of PBMC and spleen mononuclear cells demonstrated an expansion of double-negative (DN) CD4(-)CD8(-)TCRalphabeta(+) T cells in SRW-treated DQ6/CD4(null) mice. These cells produced IL-4, IL-5, IL-13, and IFN-gamma when stimulated with immobilized anti-CD3. IL-5 ELISPOT assay revealed that DN T cells were the cellular origin of IL-5 in allergen-challenged DQ6/CD4(null) mice. Our study shows a role for HLA-DQ-restricted CD4(+) and DN T cells in the allergic response.     

Distinct and overlapping effector functions of expanded human CD4+, CD8α+ and CD4-CD8α- invariant natural killer T cells

CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+), CD8α(+) and CD4(-)CD8α(-) double-negative (DN) subsets. CD4(+) iNKT cells expanded more readily than CD8α(+) and DN iNKT cells upon mitogen stimulation. CD8α(+) and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+) cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4(+) and CD8α(+) fractions, respectively. Only CD4(+) iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α(+), DN or CD4(+) iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.     

Activation pathways implicate anti-HLA-DP and anti-LFA-1 antibodies as lead candidates for intervention in chronic berylliosis

CD4(+) T cells play a key role in granulomatous inflammation in the lung of patients with chronic beryllium disease. The goal of this study was to characterize activation pathways of beryllium-responsive bronchoalveolar lavage (BAL) CD4(+) T cells from chronic beryllium disease patients to identify possible therapeutic interventional strategies. Our results demonstrate that in the presence of APCs, beryllium induced strong proliferation responses of BAL CD4(+) T cells, production of superoptimal concentrations of secreted proinflammatory cytokines, IFN-gamma, TNF-alpha,and IL-2, and up-regulation of numerous T cell surface markers that would promote T-T Ag presentation. Ab blocking experiments revealed that anti-HLA-DP or anti-LFA-1 Ab strongly reduced proliferation responses and cytokine secretion by BAL CD4(+) T cells. In contrast, anti-HLA-DR or anti-OX40 ligand Ab mainly affected beryllium-induced proliferation responses with little impact on cytokines other than IL-2, thus implying that nonproliferating BAL CD4(+) T cells may still contribute to inflammation. Blockade with CTLA4-Ig had a minimal effect on proliferation and cytokine responses, confirming that activation was independent of B7/CD28 costimulation. These results indicate a prominent role for HLA-DP and LFA-1 in BAL CD4(+) T cell activation and further suggest that specific Abs to these molecules could serve as a possible therapy for chronic beryllium disease.     

B cells from patients with Graves' disease aberrantly express the IGF-1 receptor: implications for disease pathogenesis

Graves' disease (GD) is an autoimmune process involving the thyroid and connective tissues in the orbit and pretibial skin. Activating anti-thyrotropin receptor Abs are responsible for hyperthyroidism in GD. However, neither these autoAbs nor the receptor they are directed against have been convincingly implicated in the connective tissue manifestations. Insulin-like growth factor-1 receptor (IGF-1R)-bearing fibroblasts overpopulate connective tissues in GD and when ligated with IgGs from these patients, express the T cell chemoattractants, IL-16, and RANTES. Disproportionately large fractions of peripheral blood T cells also express IGF-1R in patients with GD and may account, at least in part, for expansion of IGF-1R(+) memory T cells. We now report a similarly skewed B cell population exhibiting the IGF-1R(+) phenotype from the blood, orbit, and bone marrow of patients with GD. This expression profile exhibits durability in culture and is maintained or increased with CpG activation. Moreover, IGF-1R(+) B cells produce pathogenic Abs against the thyrotropin receptor. In lymphocytes from patients with GD, IGF-1 enhanced IgG production (p < 0.05) and increased B cell expansion (p < 0.02) in vitro while those from control donors failed to respond. These findings suggest a potentially important role for IGF-1R display by B lymphocytes in patients with GD in supporting their expansion and abnormal Ig production.