Different cytokine production profiles of γδ T cell clones: Relation to inflammatory arthritis

This study was performed to investigate whether γδ T cells could also be divided into subsets, identified by a cytokine profile, as described for αβ T helper (Th) cell subsets. Cytokine production was studied in 22 γδ T cell clones obtained from the synovial fluid and peripheral blood of one patient with inflammatory arthritis and compared to that of 26 αβ T cell clones of the same and different patients. Interferon-γ (IFN-γ) was produced by 18 (82%) and interleukin-4 (IL-4) by 17 (77%) out of 22 γδ T cell clones, respectively. In contrast, IL-10 was not produced, except at very low level in one case. The mean levels of IL-4 were lower for clones derived from synovial fluid. When considering the production of IFN-γ as an indicator of Th1 and that of IL-4 as an indicator of Th2, respectively, the most common pattern was a γδTh1-like pattern, with the combination of high levels of IFN-γ and low levels of IL-4. This pattern was found in Vδ1⁺ clones, all from synovial fluid. Additional patterns were also observed: a mixed, probably γδ Th0-like pattern with a more balanced production of both IFN-γ and IL-4; a γδTh1 pattern with the production of IFN-γ alone; a γδTh2 pattern with the production of IL-4 alone. These three patterns were also seen in blood γδ T cells which were all Vδ2, indicating that these patterns were independent of the γδ phenotype. γδ T cell clones produced lower levels of IFN-γ (p = 0.001) and higher levels of IL-4 than αβ clones (p < 0.02). These differences in cytokine production between αβ and γδ subsets and within these subsets may contribute to their respective role in chronic inflammation.     

Interleukin-12 but not interferon-γ production correlates with induction of T helper type-1 phenotype in murine candidiasis

By means of polymerase chain reaction-assisted mRNA amplification, we have monitored message levels of interleukin (IL)-12 in splenic macrophages and of interferon-γ (IFN-γ), IL-4, and IL-10 in CD4⁺ and CD8⁺ T cells using Candida albicans/host combinations that result either in a T helper type-1 (Th1)-associated self-limiting infection (“healer mice”) or in a Th2-associated progressive disease (“nonhealer mice”). The timing and pattern of message detection did not differ qualitatively by the expression of IFN-γ or IL-10 mRNA in CD4⁺ and CD8⁺ cells from healer (i.e. PCA-2 into CD2F1) vs. nonhealer (i.e. CA-6 into CD2F1 or PCA-2 into DBA/2) mice. In contrast, IL-4 mRNA was uniquely expressed by CD4⁺ cells from nonhealer animals. IL-12p40 was readily detected in macrophages from healer mice but was detected only early in infection in mice with progressive disease. Cytokine levels were measured in sera, and antigen-driven cytokine production by CD4⁺ and CD8⁺ cells was assessed in vitro, while IFN-γ-producing cells were enumerated in CD4^? CD8^? cell fractions. Overall, our results showed that (i) antigen-specific secretion of IFN-γ protein in vitro by CD4⁺ cells occurred only in healing infection; (ii) IL-4- and IL-10-producing CD4⁺ cells would expand in nonhealer mice in the face of high levels of circulating IFN-γ, likely released by CD4^? CD8^? lymphocytes; (iii) a finely regulated IFN-γ production correlated in the healer mice with IL-12 mRNA detection, and IL-12 was required in vitro for yeast-induced development of IFN-γ-producing CD4⁺ cells. Although the mutually exclusive production of IL-4/IL-10 and IFN-γ by early CD4⁺ cells may be the major discriminative factor of cure and noncure responses in candidiasis, IL-12 rather than IFN-γ production may be an indicator of Th1 differentiation.     

Human Th1 cells preferentially induce interleukin (IL)-1β while Th2 cells induce IL-1 receptor antagonist production upon cell/cell contact with monocytes

The role of human T cells in the induction and regulation, upon cell/cell contact, of inflammatory responses by monocytic cells was investigated. The production of interleukin (IL)-1β and IL-1 receptor antagonist (IL-1Ra) by the monocytic THP-1 cell line was measured upon contact with either Th1 or Th2 cell clones. CD4⁺ T cell clones specific for purified protein derivative of Mycobacterium tuberculosis, predominantly Th1 [high interferon (IFN)-γ and low IL-4 producers], or tetanus toxoid, predominantly Th2 (low IFN-γ and high IL-4 producers), were generated. Cell membranes from antigen-stimulated, but not from resting T cell clones induced dose-dependent cytokine production by THP-1 cells. Th1 clones induced higher levels of IL-1β production (484–806 pg/ml) than did Th2 clones (21–114 pg/ml). In contrast, Th1 clones induced lower levels of IL-1Ra (0.9–7.8 ng/ml) than did Th2 clones (7.0–49.6 ng/ml). Similar results were obtained when T cell clones were activated by cross-linked CD3 and CD28. IL-1β production by THP-1 cells correlated with IFN-γ production by T cell clones but was unaffected by IFN-γ neutralization. IL-1Ra production by THP-1 cells correlated with IL-4 production by T cells and was partially inhibited by IL-4 neutralization. These data indicate that activated Th1 and Th2 cells express different molecules on the cell surface able to induce distinct pro-inflammatory (IL-1β) or anti-inflammatory (IL-1Ra) responses in monocytes. This differential induction of molecules with opposite effects on inflammation stresses the functional heterogeneity in CD4⁺ T cells.     

Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response

Mice with homologous disruption of the gene coding for either the p35 subunit or the p40 subunit of interleukin-12 (IL-12) and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in resistance to infection and the differentiation of functional CD4⁺ T cell subsets in vivo. Wild-type 129/Sv/Ev mice are resistant to infection with L. major showing only small lesions which resolve spontaneously within a few weeks and develop a type 1 CD4⁺ T cell response. In contrast, mice lacking bioactive IL-12 (IL-12p35^?/? and IL-12p40^?/?) developed large, progressing lesions. Whereas resistant mice were able to mount a delayed-type hypersensitivity (DTH) response to Leishmania antigen, susceptible BALB/c mice as well as IL-12-deficient 129/Sv/Ev mice did not show any DTH reaction. To characterize the functional phenotype of CD4⁺ T cells triggered in infected wild-type mice and IL-12-deficient mice, the expression of mRNA for interferon-γ (IFN-γ) and interleukin-4 (IL-4) in purified CD4⁺ lymph node cells was analyzed. Wild-type 129/Sv/Ev mice showed high levels of mRNA for IFN-γ and low levels of mRNA for IL-4 which is indicative of a Th1 response. In contrast, IL-12- deficient mice and susceptible BALB/c mice developed a strong Th2 response with high levels of IL-4 mRNA and low levels of IFN-γ mRNA in CD4⁺ T cells. Similarly, lymph node cells from infected wild-type 129 mice produced predominantly IFN-γ in response to stimulation with Leishmania antigen in vitro whereas lymph node cells from IL-12-deficient mice and susceptible BALB/c mice produced preferentially IL-4. Taken together, these results confirm in vivo the importance of IL-12 in induction of Th1 responses and protective immunity against L. major.     

A shift to Th0 cytokine production by CD4+ cells in human longevity: Studies on two healthy centenarians

Centenarians, particularly healthy centenarians, constitute the example of successful aging and the study of their immune status can help to define the endpoint of the changes occurring throughout life. We characterized T cell clones (TCC) of two healthy centenarians, studying their phenotypes and production of representative Th1 and Th2 cytokines (IFN-γ and IL-4) and compared them with TCC obtained by three young normal subjects; in all 180 TCC were analyzed. In young donors, 35TCC were CD4⁺, 56 CD8⁺ and 2 were αβ⁴CD4⁻CD8⁻ (double negative). In centenarians, we obtained 46 CD4⁺ TCC, 38 CD8⁺, 2 CD4⁺CD8⁺ (double positive) and 1 γδ⁺ double negative. Of the young subjects' TCC, 71% produced IFN-γ but no IL-4 (Th1 pattern) and this prevalence decreased to 39% in TCC from the centenarians. The number of clones showing the opposite Th2 pattern was similar in young and aged donors (3 out of 93 TCC and 2 out of 87 TCC, respectively). The intermediate profile of TCC producing both IL-4 and IFN-γ (Th0) was found in 25.8% of clones from young people, but it almost doubled to 58.6% in centenarians. The analysis shows that the Th profiles of CD8⁺ TCC is nearly superimposable in the two groups, whereas a major shift from a Th1 to a Th0 pattern is presented by CD4⁺ TCC. The balance provided by a majority of CD4⁺ TCC showing a Th0 pattern may ensure both humoral and cell-mediated defences. In CD8⁺ TCC, however, a Th1 pattern still is present, possibly for efficient generation of cytotoxic responses. These findings should be extended by studying other centenarians and elderly subjects.     

Human recombinant interferon-β influences T helper subset differentiation by regulating cytokine secretion pattern and expression of homing receptors

Type I interferons (IFN) are important regulators of both innate and acquired immunity. We have used an in vitro system of human CD4⁺ T cell differentiation to determine how IFN-β influences development of T helper (Th) subsets and homing receptor expression. IFN-β promoted differentiation of CD4⁺ T cells that produce low levels of both IFN-γ and lymphotoxin compared to interleukin (IL)-12-derived Th1 CD4⁺ T cells. IFN-β inhibited production of Th2 cytokines (IL-5 and IL-13) and augmented IL-12-mediated IL-10 secretion. In addition, IFN-β significantly enhanced L-selectin expression on CD4⁺ T cells and synergized with IL-12 to induce expression of cutaneous lymphocyte-associated antigen (CLA). This Th1 L-selectin⁺, CLA⁺ phenotype is characteristic of T cells found in normal human skin and suggests a role for type I IFN in the regulation of Th subset differentiation and tissue-specific homing receptors.     

Development in vitro of human CD4+ thymocytes into functionally mature Th2 cells. Exogenous interleukin-12 is required for priming thymocytes to produce both Th1 cytokines and interleukin-10

Fresh postnatal thymocyte cell suspensions were directly cloned under limiting dilution conditions with either phytohemagglutinin or toxic shock syndrome toxin-1 (TSST-1), a bacterial superantigen. Cultures contained allogenic irradiated feeder cells and interleukin (IL)-2, in the absence or presence of exogenous IL-4, interferon (IFN)-γ or IL-12. The resulting CD4⁺ T cell clones generated under these different experimental conditions were then analyzed for their ability to produce IL-2, IL-4, IL-5, IL-10, IFN-γ and tumor necrosis factor (TNF)-β in response to stimulation with phorbol 12-myristate 13-acetate (PMA)+anti-CD3 monoclonal antibody or PMA + ionomycin. Different from T cell clones generated from peripheral blood, virtually all CD4⁺ T cell clones generated from human thymocytes produced high concentrations of IL-2, IL-4 and IL-5, but no IFN-γ, TNF-β or IL-10. Moreover, after activation, these clones expressed on their surface membrane both CD30 and CD40 ligand, but not the product of lymphocyte activation gene (LAG)-3, and provided strong helper activity for IgE synthesis by allogeneic B cells. The Th2 cytokine pattern could not be modified by the addition of IFN-γ. However, upon addition of exogenous IL-12, the resulting CD4⁺ thymocyte clones produced TNF-β, IFN-γ, and IL-10 in addition to IL-4 and IL-5. These results suggest that CD4⁺ human thymocytes have the potential to develop into cells producing the Th2 cytokines IL-4 and IL-5, whereas the ability to produce both Th1 cytokines and IL-10 is acquired only after priming with IL-12.     

T cell response in murine Leishmanial mexicana amazonensis infection: production of interferon-γby CD8+ cells

The immune response to Leishmania major has been the subject of many investigations. However, Leishmania includes many species with different clinical manifestations. In this report, we studied the Tcell response to L. mexicana amazonensis, a New World species, in a murine model. We found that, similar to L. major, an Old World species, resistant C57BL/6 mice produced a high level of IFN-γ and a low level of IL-4. Conversely, susceptible BALB/c mice produced a much lower level of IFN-γ and higher level of IL-4. Although IFN-γ is one of the important lymphokines that mediate macrophage activation and thus the destruction of the intracellular parasites, which lymphocyte subsets are producing the IFN-γ is still a controversy. Much evidence including the isolation of protective, IFN-γ-producing, CD4⁺ cell lines have confirmed the participation of CD4⁺ Thl cells unequivocally. However, both CD4⁺ and CD8⁺ cells produced IFN-γ. Recently, an increasing body of evidence has appeared suggesting that CD8⁺ cells also play a role in the resolution of murine L. major infection. We found that in the L. m. amazonensis model, when CD8⁺ lymphocytes from resistant C57BL/6 mice were eliminated by anti-CD8 antibody and complement-mediated lysis, the IFN-γ production was reduced by 77%. This indicated that CD8⁺ cells produced a significant amount of the IFN-γ. However, our results also indicate that IFN-γ production by CD8⁺ cells was dependent on CD4⁺ cells.     

Effects of interferon-α on cytokine profile,T cell receptor repertoire and peptide reactivity of human allergen-specific T cells

A large panel of T cell clones (TCC) specific for the recombinant form of Poa pratensis allergen (rKBG7.2 or Poa p9) were established from the peripheral blood of a grass pollen-sensitive donor in the absence or presence of recombinant interferon-α (IFN-α) in bulk culture and their pattern of cytokine secretion, peptide reactivity and TCR Vβ repertoire was examined. The majority of allergen-specific TCC derived in absence of IFN-α produced high amounts of interleukin-4 (IL-4) and IL-5 but not IFN-γ (Th2 cells), while most of TCC derived in presence of IFN-α produced IFN-γ but not, or limited amounts of, IL-4 and IL-5 (Th1 or Th0 cells). Of 24 TCC established in the presence of IFN-α, 22 were able to recognize a single allergen peptide, p26, while none of the clones established in the absence of IFN-α showed a similar specificity. The majority of both clones expressed the Vβ2 element regardless of whether they were established in the presence of IFN-α, but the presence of IFN-α favored the expansion of Vβ2⁺, Vβ17⁺ and Vβ22⁺ Poa p9-specific T cells, whereas in the absence of IFN-α, other TCR Vβ-bearing T cells (Vβ5, Vβ6.7 and Vβ14) were expanded in addition to Vβ2⁺ T cells. None of Vβ2⁺ clones established in the absence of IFN-α reacted with p26, whereas all the Vβ2⁺ clones established in its presence responded to this peptide. IFN-α also shifted the TCR Vβ repertoire of both Poa p9- and Lolium perenne group 1 (Lol p1)-specific T cell lines generated from the same patient and from a different grass-sensitive individual. These data demonstrate that IFN-α modulates the development of allergen-specific T cells in vitro, and suggest that IFN-α may represent an useful tool for novel immunotherapeutic approaches in allergic disorders.     

Antigen-stimulated IL-4, IL-13 and IFN-γ production by human T cells at a single-cell level

To understand the intricate balance and the coordinate expression of the Th1 and Th2 cytokines following a natural mode of T cell triggering, antigen-stimulated IL-4, IL-13 and IFN-γ production was studied in primary peripheral blood mononuclear cell cultures at a single-cell level. Cells from filariasis patients who respond to parasite antigen by producing not only IFN-γ but also IL-4 and IL-13 were stimulated with Brugia malayi adult worm antigen and analyzed for co-expression of cytokines by intracellular staining. IL-4 and IL-13 were frequently co-expressed (54 % of IL-4⁺ cells stained for IL-13 and 29 % of IL-13⁺ cells expressed IL-4 at all time points), whereas IFN-γ expression was totally segregated from both IL-4 and IL-13. These data indicate that in human peripheral T cells the co-expression of the dominant Th1 and Th2 cytokines within a single cell is a rare event and that IL-13 is clearly more frequently associated with a Th2 than a Th1 type response in primary T cell cultures.     

In susceptible mice,Leishmania major induce very rapid interleukin-4 production by CD4+ T cells which are NK1.1−

Susceptibility of BALB/c mice to infection with Leishmania major is associated with a T helper type 2 (Th2) response. Since interleukin-4 (IL-4) is critically required early for Th2 cell development, the kinetics of IL-4 mRNA expression was compared in susceptible and resistant mice during the first days of infection. In contrast to resistant mice, susceptible mice exhibited a peak of IL-4 mRNA in their spleens 90 min after i.v. injection of parasites and in lymph nodes 16 h after s.c. injection. IL-12 and interferon-γ (IFN-γ) down-regulated this early peak of IL-4 mRNA; the effect of IL-12 was IFN-γ dependent. Treatment of resistant C57BL/6 mice with anti-IFN-γ allowed the expression of this early IL-4 response to L. major. The increased IL-4 mRNA expression occurred in Vβ8, 7, 2^? CD4⁺ cells in BALB/c mice and NK1.1^? CD4⁺ cells in anti-IFN-γ treated C57BL/6 mice. These results show that the NK1.1⁺ CD4⁺ cells, responsible for the rapid burst of IL-4 production after i.v. injection of anti-CD3, do not contribute to the early IL-4 response to L. major.     

Defective lymphokine production by most CD8+ and CD4+ tumor-specific T cell clones derived from human melanoma-infiltrating lymphocytes in response to autologous tumor cells in vitro

Human melanomas are infiltrated by tumor-reactive T lymphocytes. However, the ability of these cells to elicit a specific anti-tumor response in vivo remains to be established. Because lymphokine production is critical for T cell functions, we have analyzed the capacity of melanoma-specific tumor-infiltrating lymphocyte (TIL) clones to produce major lymphokines: interleukin-2 (IL-2), interferon-γ (IFN-γ) and interleukin-4 (IL-4), as well as tumor necrosis factor (TNF), in response to direct antigen presentation by autologous and allogeneic tumor cells. We report here that, upon stimulation by autologous melanoma cells, all TIL clones secreted TNF but only a few of them produced significant amounts of IL-2, IL-4 or IFN-γ. Nonetheless, all these clones consistently produced two or three of these last lymphokines upon stimulation with phorbol myristate acetate and calcium ionophore, as well as IL-2 upon CD3 stimulation, showing the existence of three lymphokine profiles among them: Th1, Th0 and a profile characterized by IL-2 and IL-4, but not IFN-γ secretion. Stimulation of TIL clones by allogeneic melanoma lines sharing the appropriate HLA-peptide complexes revealed that defective IL-2 production seemed to be a constant feature for some clones, while it was, for other clones, dependent on the antigen-presenting tumor cells. For this last type of clone, we further showed that defective IL-2 induction resulted from an LFA-3 defect of some melanoma cells or from distinct yet undefined defects of other melanoma lines. Our data suggest that defective lymphokine secretion may be an essential component of the in vivo failure of melanoma-reactive TIL to control tumor development. Interestingly both CD4⁺ and CD8⁺ TIL clones from one patient were fully activated by the autologous melanoma cells in vitro, supporting a potential role of such TIL in spontaneous or induced tumor rejection.     

Reduced IL-4 and interferon-gamma (IFN-γ) expression by CD4 T cells in patients with chronic lymphocytic leukaemia

CD7 co-expression by CD4 T cells has been reported to be higher in the Th1 compared with the Th2 functional subset. Clinical immunodeficiency and immune dysregulation are more prevalent in the advanced stages of B cell chronic lymphocytic leukaemia (B-CLL). To analyse this further 25 patients with B-CLL and 11 healthy subjects were examined for cell surface CD7 and intracellular IFN-γ and IL-4 expression in the peripheral blood CD4⁺ T helper cell population. Significantly decreased CD7, IFN-γ and IL-4 expression was observed in the patients with B-CLL (P < 0.001). While CD7 negativity and IL-4 expression were more frequent in the later stages of the disease, this did not attain statistical significance. These results suggest a possible explanation for the reduced cellular and humoral immunity in B-CLL.     

Diversion of CD4+ T cell development from regulatory T helper to effector T helper cells alters the contact hypersensitivity response

Cutaneous sensitization to reactive haptens and subsequent challenge results in a T cell-mediated response, contact hypersensitivity (CHS). Recent results from this laboratory have indicated that hapten sensitization induces two populations of reactive T cells: CD8⁺ T cells producing interferon (IFN)-γ which mediate the response and CD4⁺ T cells producing interleukin (IL)-4 and IL-10 which negatively regulate the magnitude and duration of the response. Since CD4⁺ T cell development to either IFN-γ- (Th1) or IL-4/IL-10- (Th2)-producing cells is dependent upon the cytokine environment during antigen priming, we hypothesized that CD4⁺ T cell induction in a Th1-promoting environment would not only alter the CD4⁺ T cell cytokine-producing phenotype but also the course of the CHS response. Administration of the Th1-promoting cytokine IL-12 during hapten sensitization resulted in a CHS response of greater magnitude following challenge and extended the duration of the response. In hapten-sensitized mice depleted of CD8⁺ T cells, treatment with IL-12 induced effector CD4⁺ T cells. Histological examination of challenged ear tissue from these mice indicated minimal edema and an acute mononuclear cell infiltration more typical of classical delayed-type hypersensitivity than CHS. Hapten-primed CD4⁺ T cells from IL-12 treated, sensitized mice produced IFN-γ, but not IL-4 in response to T cell receptor-mediated stimulation. Use of neutralizing anti-IFN-γ antibody indicated that IL-12 not only directly promoted Th1 development but also indirectly inhibited Th2 development through stimulation of IFN-γ production at the time of hapten sensitization. Overall, these results demonstrate that diversion of CD4⁺ T cell development to Th1 effector cells rather than to Th2 cells alters the efferent nature of CHS and removes a primary regulatory mechanism of the immune response.     

T lymphocytes from the normal human peritoneum contain high frequencies of Th2-type CD8+ T cells

The majority of peritoneal T lymphocytes have been shown to be CD8⁺ and to co-express CDw60. Expression of CDw60 characterizes CD8 T cells capable of secreting interleukin (IL)-4 and supporting IgG production by B cells. We analyzed at the clonal level the functional cytokine profile of CD8⁺ T lymphocytes from the normal human peritoneum. While the majority of the clones produced interferon (IFN)-γ and exhibited high alloantigen-specific cytolytic activity, some clones secreted IL-4 and IL-5 but no detectable IFN-γ. These Th2-type CD8⁺ T cell clones provided substantial B cell help for IgG and IgA synthesis and exhibited reduced cytolytic activity. Our results suggest that distinct subsets of CD8⁺ T cell may occur in different immune compartments.     

IL-4-producing NK T cells are biased towards IFN-γ production by IL-12. Influence of the microenvironment on the functional capacities of NK T cells

NK T cells are an unusual T lymphocyte subset capable of promptly producing several cytokines after stimulation, in particular IL-4, thus suggesting their influence in Th2 lineage commitment. In this study we demonstrate that, according to the cytokines present in the micro environment, NK T lymphocytes can preferentially produce either IL-4 or IFN-γ. In agreement with our previous reports showing that their IL-4-producing capacity is strikingly dependent on IL-7, CD4⁻ CD8⁻ TCRα β⁺ NK T lymphocytes, obtained after expansion with IL-1 plus granulocyte-macrophage colony-stimulating factor, produced almost undetectable amounts of IL-4 or IFN-γ in response to TCR/CD3 cross-linking. However, the capacity of these T cells to produce IFN-γ is strikingly enhanced when IL-12 is added either during their expansion or the anti-CD3 stimulation, while IL-4 secretion is always absent. A similar effect of IL-12 on IFN-γ production was observed when NK T lymphocytes were obtained after expansion with IL-7. It is noteworthy that whatever cytokines are used for their expansion, IL-12 stimulation, in the absence of TCR/CD3 cross-linking, promotes consistent IFN-γ secretion by NK T cells without detectable IL-4 production. Experiments in vivo demonstrated a significant up-regulation of the capacity of NK T cells to produce IFN-γ after anti-CD3 mAb injection when mice were previously treated with IL-12. In conclusion, we provide evidence that the functional capacities of NK T cells, which ultimately will determine their physiological roles, are strikingly dependent on the cytokines present in their microenvironment.