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.     

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.     

Monocyte-derived CD1a+ dendritic cells generated in two different culture systems: immunophenotypic and functional comparison

Previous studies demonstrated that CD1a⁺ dendritic cells (DCs) could not be prepared ex vivo without using fetal calf serum (FCS). Recently, we developed a method of using heparin to induce differentiation of human monocytes into CD1a⁺ DCs without using FCS. In order to determine the potential clinical applicability of heparin-induced CD1a⁺ DCs, we conducted this study to compare both types of CD1a⁺ DCs, immunophenotypically and functionally. Our results showed that the expression of CD1a on heparin-DCs was lower than that on FCS-DCs. Both types of DCs expressed similar levels of CD11c, HLA-DR, CD40, CD83, CD80 and CD86 before and after lipopolysaccharide stimulation. Immature heparin-DCs and FCS-DCs had similar phagocytic activities. Heparin-DCs consistently secreted higher interleukin-10 (IL-10) and lesser IL-12 than FCS-DCs after activation. Mature heparin-DCs were slightly more active than mature FCS-DCs in stimulating the proliferation of allogeneic CD4⁺ T cells. Both types of mature CD1a⁺ DCs primed the naïve CD4⁺ T cells to produce large amount of interferon-γ (IFN-γ). However, naïve CD4⁺ T cells stimulated with FCS-DCs produced more IFN-γ, while the naïve CD4⁺ T cells stimulated with heparin-DCs produced more IL-5. The results indicate that both types of CD1a⁺ DCs do not have identical function in the priming of CD4⁺ T cells and have minor difference in immunophenotypes.     

Single-cell analysis of lymphokine imbalance in asymptomatic HIV-1 infection: evidence for a major alteration within the CD8+ T cell subset

In this study we investigated at single-cell level by flow cytometry the potential of T cell cytokine production in asymptomatic HIV-1-infected subjects with > 200 CD4 counts and possible correlation with T helper cell depletion and viral load. Mitogen-stimulated peripheral blood mononuclear cells from 32 HIV-1⁺ patients and 16 healthy subjects were intracytoplasmically stained for IL-2, interferon-gamma (IFN-γ), IL-4 or IL-10, and the frequency of cytokine-producing cells was assessed in total T cells, CD4, CD8 and CD45RO subsets as well as in CD69⁺ CD3⁺ gated lymphocytes. HIV-1⁺ patients, irrespective of their degree of CD4 depletion, exhibited a major increase in IFN-γ⁺ CD8 T cells, largely due to CD28⁻ cells, as well as a decrease in the capacity of CD8 T cells to produce IL-2. Patients with > 500 CD4 counts showed a diminished frequency of IL-4 expression in CD4 T cells and a negative correlation was found between this parameter and the ex vivo CD4 counts in the 32 patients. Analysis of patients stratified according to viral load revealed a significantly higher proportion of IL-2-producing CD4 cells in the group with < 5000 RNA copies/ml. In short, using single-cell analysis and an antigen-presenting cell-independent stimulus, we have not been able to find any significant cytokine imbalances in the CD4 subset, suggesting that the well described T helper defects are not due to intrinsic alterations in the potential of CD4 T cells to produce cytokines. On the other hand, the major disturbances in the CD8 T lymphocytes agree with the marked activation and possible replicative senescence of CD8 T cells and emphasize the role of this subset in HIV immunopathogenesis.     

Expression of selectin-binding epitopes and cytokines by CD4+ T cells repopulating scid mice with colitis

Recruitment into the gut of CD4⁺ T cells and their activation in the colonic lamina propria (LP) are key events in the development of colitis in scid mice reconstituted with CD4⁺ T cells from immunocompetent, congenic donor mice. This study investigated the expression of cytokines and selectin-binding epitopes by CD4⁺ T cells repopulating different tissues of the adoptive scid host. Cells from the inflamed colonic LP of transplanted scid mice produced high amounts of IL-12, IFN-γ and TNF-α but only low amounts IL-4 and IL-10. Intracellular cytokine staining confirmed the presence of large numbers of IFN-γ- and TNF-α-producing effector CD4⁺ T cells in the colonic LP of scid mice with colitis but also in non-inflamed tissues [spleen (S), peritoneal cavity (PC) and mesenteric lymph nodes (mLN)] of the adoptive host. Cells from these tissues furthermore produced large amounts of IL-12. Ligands for endothelial selectins are involved in recruiting T cells into inflamed tissues. We have analyzed the expression of selectin-binding epitopes on CD4⁺ T cells repopulating different tissues of the adoptive scid host. We found that a large fraction of CD4⁺ T cells from inflamed colonic LP and from non-inflamed PC, mLN and S expressed high levels of P- and E-selectin-binding epitopes (P-L^hi) in transplanted scid mice, but not in congenic, immunocompetent control mice. Although P-L^hi CD4⁺ T cells were enriched in IFN-γ-producing subsets from most (but not all) tissues, we also found large numbers of in vivo generated P-L^lo CD4⁺ T cells producing pro-inflammatory cytokines. This was in contrast to in vitro generated Th1 CD4⁺ T blasts that were almost exclusively P-L^hi. In this mouse model, production of Th1-type pro-inflammatory cytokines and expression of surface epitopes binding endothelial selectins are hence strikingly up-regulated in CD4⁺ T cells residing in inflamed and non-inflamed tissues during the development of colitis.     

Manipulation of the superantigen-induced lymphokine response. Selective induction of interleukin-10 or interferon-γ synthesis in small resting CD4+ T cells

The production of several lymphokines by freshly isolated CD4⁺ T cells has been analyzed at the single-cell level, after stimulation with staphylococcal enterotoxin B (SEB). High frequencies of cells producing interleukin-2 (IL-2) and interferon-γ (IFN-γ) were induced, but very low frequencies of CD4⁺ T cells produced IL-4, IL-5 or IL-10 in response to SEB. Exogenously added IL-4 markedly altered the lymphokine profile induced during primary SEB stimulation. IFN-γ production was reduced, while a high fraction of cells contained IL-10 and IL-4 after activation in the presence of IL-4. We further demonstrate that IL-4 and IL-10 or IFN-y production was selectively induced in resting, high-density CD4⁺ T cells during primary stimulation, by SEB + IL-4 or SEB. Under conditions where both IL-10 and IFN-γ were produced, most cells contained only one of the two lymphokines.     

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.     

Protein kinase 2 (CK2) controls CD4+ T cell effector function in the pathogenesis of colitis

Crohn's disease (CD), one of the major forms of inflammatory bowel disease (IBD), is characterized by chronic inflammation of the gastrointestinal tract and associated with aberrant CD4⁺ T-helper type 1 (Th1) and Th17 responses. Protein kinase 2 (CK2) is a conserved serine–threonine kinase involved in signal transduction pathways, which regulate immune responses. CK2 promotes Th17 cell differentiation and suppresses the generation of Foxp3⁺ regulatory T cells. The function of CK2 in CD4⁺ T cells during the pathogenesis of CD is unknown. We utilized the T cell-induced colitis model, transferring CD45RB^hi-naive CD4⁺ T cells from CK2α^fl/fl controls and CK2α^fl/fldLck-Cre mice into Rag1^−/− mice. CD4⁺ T cells from CK2α^fl/fldLck-Cre mice failed to induce wasting disease and significant intestinal inflammation, which was associated with decreased interleukin-17A-positive (IL-17A⁺), interferon-γ-positive (IFN-γ⁺), and double-positive IL-17A⁺IFN-γ⁺ CD4⁺ T cells in the spleen and colon. We determined that CK2α regulates CD4⁺ T cell proliferation through a cell-intrinsic manner. CK2α is also important in controlling CD4⁺ T cell responses by regulating NFAT2, which is vital for T cell activation and proliferation. Our findings indicate that CK2α contributes to the pathogenesis of colitis by promoting CD4⁺ T cell proliferation and Th1 and Th17 responses, and that targeting CK2 may be a novel therapeutic treatment for patients with CD.     

Human Th1 responses driven by IL-12 are associated with enhanced expression of CD40 ligand

IL- 12 is the prominent inducer of Th1 responses in humans and in the mouse. CD40 ligand (CD40L) plays important roles in regulation of immune responses, including T cell-dependent activation of B cells and cytokine production by monocytes and dendritic cells. The present study examined the influences of IL-12 on the CD40L expression of activated human CD4⁺ T cells. IL-12 enhanced CD40L expression on CD4⁺ T cells stimulated with immobilized anti-CD3 in the complete absence of accessory cells, whereas IL-4 and IL-10 decreased it. Exogenous interferon-gamma (IFN-γ) did not increase CD40L expression on immobilized anti-CD3 stimulated CD4⁺ T cells at any time up to 168 h of culture. The IL-12-induced enhancement of CD40L expression on anti-CD3 activated CD4⁺ T cells was not influenced in the presence of a metalloproteinase inhibitor KB8301, which up-regulated CD40L expression by preventing the processing of membrane-bound CD40L, or B cells, which down-regulated CD40L expression by receptor-mediated endocytosis. These results indicate that IL-12 enhances the CD40L expression of activated CD4⁺ T cells independently of the IFN-γ production. The data thus suggest that Th1 responses induced by IL-12 might play an important role in the regulation of humoral immune responses through up-regulated CD40L expression.     

In Vivo CD3+CD25+ Lymphocyte Subpopulation Is Down-regulated Without Increased Serum-Soluble Interleukin-2 Receptor (sIL-2R) by Gonadotropin Releasing Hormone Agonist (GnRH-a)

PROBLEM: To test further whether the suppression of the CD3⁺CD25⁺ lymphocyte sub-population by gonadotropin-releasing hormone agonist (GnRH-a) is related to the change in levels of cytokines and soluble interleukin-2 receptor (sIL-2R). METHOD: Twenty-seven infertile patients were enrolled under the long protocol of GnRH-a agonist (buserelin acetate) and superovulation with gonadotropin from our IVF-ET program. Peripheral B cells, NK cells, CD4⁺ and CD8⁺ T cells and the expression of CD69, CD25, HLA-DR, and CD71 antigens on the T cells were serially examined by dual-color flow cytometry. Serum levels of cytokines and sIL-2R were measured. RESULTS: The B cells, NK cells, T cells, CD4⁺, CD8⁺ T cells, CD3⁺DR⁺, and CD3⁺CD71⁺ lymphocyte subpopulations were not changed after the use of GnRH-a. The CD25⁺ T cell subpopulation was significantly down-regulated, but the CD69⁺ T cell subpopulation was increased when the GnRH-a was used for approximately 2 wk. The serum levels of interleukin-lp (IL-1β), interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-γ (IFN-γ), and sIL-2R were not changed. CONCLUSION: The GnRH-a had a transiently suppressive effect on CD25⁺ T cells, but a stimulatory effect on CD69⁺ T cells. However, the serum level of cytokines or sIL-2R did not change. These immunological modulations seems to be the result of interaction between GnRH-a and estrogen.     

Opposite role for interleukin-4 and interferon-γ on CD30 and lymphocyte activation gene-3 (LAG-3) expression by activated naive T cells

Polarized human type 1 and type 2 T helper cells not only produce different sets of cytokines, but they also preferentially express certain activation markers, such as lymphocyte activation gene-3 (LAG-3) and CD30, respectively. In this study we have examined the LAG-3 and CD30 expression in relation to the lineage commitment of human naive CD4⁺ T cells, as assessed at the single-cell level of committed T cells. Purified CD45RA⁺ umbilical cord blood T lymphocytes were activated with phytohemagglutinin and interleukin (IL)-2 in the absence or presence of interleukin IL-4 or IL-12 and assessed for CD30 and LAG-3 expression, as well as for intracellular cytokine synthesis. Significant numbers of CD30⁺ cells were only found in CD4⁺ and CD8⁺ T lymphocytes of cultures primed with IL-4, which developed into cells able to produce IL-4 and IL-13 in addition to interferon (IFN)-γ. By contrast, LAG-3 expression was strongly up-regulated in CD4⁺ and CD8⁺ T cells from cultures primed with IL-12, which developed into high numbers of IFN-γ producers. The addition of a neutralizing anti-IFN-γ antibody to IL-12-primed CD4⁺ T cell cultures virtually abolished the development of LAG-3-expressing CD4⁺ T cells. Taken together, these data suggest that CD30 expression is dependent on the presence of IL-4, whereas LAG-3 expression is dependent on the production of IFN-γ during the lineage commitment of human naive T cells.     

Allergen-specific IL-10-secreting type I T regulatory cells,but not CD4CD25Foxp3 T cells,are decreased in peripheral blood of patients with persistent allergic rhinitis

We investigate the frequencies of CD4⁺CD25⁺Foxp3⁺ T cells and allergen-specific IL-10⁺IL-4^-, IFN-γ⁺IL-4^-, IL-4⁺IFN-γ^-CD4⁺ T cells (which display characteristics of nTreg, Tr1-, Th1- and Th2- cells, respectively) in peripheral blood mononuclear cells (PBMCs) of patients with AR and of healthy individuals. In addition, we estimated the suppressive effect of CD4⁺CD25⁺ Treg cells and allergen-specific, IL-10-secreting cells from both two groups. The frequency of CD4⁺CD25⁺Foxp3⁺ T cells is similar in 43 AR patients compared with 38 healthy subjects. CD4⁺CD25^high cells retain suppressive activity on allergen-stimulated cell proliferation and cytokine production of Th1 but not Th2 cells in both groups. However, the frequency of allergen-specific IL-10⁺IL-4^-CD4⁺ T cells is reduced in AR patients, and correlates inversely to clinical symptom scores. Allergen-specific, IL-10-secreting cells potently suppressed D. pteronyssinus major allergen 1-stimulated cell proliferation and cytokine production (IFN-γ and IL-4) in healthy individuals. Altogether our data indicate that the number and function of CD4⁺CD25⁺ Treg cells from allergic patients are not impaired. However, the deficiency of allergen-specific Tr1 cells may play a role in the development of AR.     

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.     

FOXP3 expression and frequency of regulatory T cells in healed individuals from Leishmania major infection and the asymptomatic cases

Two groups of residents in an endemic area of Leishmania major infection in Iran with positive leishmanin skin tests who were either asymptomatic or had healed cutaneous leishmaniasis lesions were compared with respect to their T helper responses. The percentages of regulatory T cells (T_reg; CD4⁺CD25^high FoxP3⁺) from the peripheral blood and CD4⁺ T cells producing intracellular cytokines (IL-4, IL-10, IL-17 and IFN-γ) from the stimulated PBMCs were evaluated by flow cytometry and the expressions of RORC and FOXP3 genes were quantified by real-time RT-PCR. T responder (CD4⁺CD25⁻) and T_reg-enriched (CD4⁺CD25⁺) cells were isolated magnetically and the suppressive capacity of the latter and the cytokines (IFN-γ, TGF-β and IL-10) secreted from them were evaluated by in vitro assays. The results showed that the frequency of T_reg in the studied groups were similar and T_reg from both groups exhibited high yet similar suppressive capacities while significantly higher levels of FOXP3 expression was observed in the asymptomatic group. Taken together, similar frequency and suppressiveness of T_reg combined with high ratios of IFN-γ/IL-10 producing CD4⁺ T cells were common in both groups; however the members of the asymptomatic group appeared to require higher expression of FOXP3 to maintain their immunity to re-infection.     

Vitamin D3 inhibits the proliferation of T helper cells,downregulate CD4+ T cell cytokines and upregulate inhibitory markers

1 α, 25-dihydroxyvitamin D3 (VitD3) has been suggested to have strong modulatory properties in the immune system. Researchers in the present study primarily aimed to understand the effect of VitD3 on human CD4⁺ T cell proliferation in antigen presenting cells (APCs) free condition in vitro. The effect of VitD3 on intracellular cytokine responses trend to Th1, Th2, Th17 and Th22 was evaluated using the flow cytometry. Moreover the effect of VitD3 on the expression of inhibitory markers such as PD1, PD-L1, and CTLA4 which are induced upon polyclonal T cell receptor (TCR) activation on CD4⁺ T cells, was assessed. We observed that the stimulation of CD4⁺ T cells with VitD3, suppressed proliferation capacity, enhanced the expression of PD1, PD-L1 and CTLA4 inhibitory markers on CD4⁺ T cells, and diminished the percentage of pro-inflammatory cytokines including, IFN-γ, IL-17, and IL-22 except IL-4 in CD4⁺ T cells. The data suggested a potential insight into the consideration of VitD3 in the prevention/control of pro-inflammatory immune response/autoimmune disorders.