Blocking of IL-6 signaling pathway prevents CD4+ T cell-mediated colitis in a T(h)17-independent manner

Naive CD4(+) T cells rapidly proliferate to generate effector cells after encountering an antigen and small numbers survive as memory T cells in preparation for future immunological events. In the present work, adoptive transfer of naive CD4(+) T cells into RAG2(-/-) mice caused the generation of memory-type effector T cells including T(h)1, T(h)2, T(h)17 and regulatory T cells, and eventually induced T cell-dependent colitis. We found here that blocking of the IL-6R with a specific mAb remarkably inhibited the CD4(+) T cell-mediated colitis in parallel with the inhibition of T(h)17 cell generation. However, the transfer of naive CD4(+) T cells prepared from IL-17(-/-) mice still induced severe colitis. At the effector phase, the mAb significantly inhibited IL-17 but not IFN-gamma production. The blockade of IL-6 signaling enhanced the generation of IL-4- and IL-10-producing CD4(+) T cells, and inhibited up-regulation of tumor necrosis factor -alpha mRNA expression in the colon. These findings clearly demonstrated that IL-6 is a critical factor for the induction of colitis by expansion of naive CD4(+) T cells in RAG2(-/-) mice. Thus, the IL-6-mediated signaling pathway may be a significant therapeutic target in T cell-mediated autoimmune diseases.     

Nasal tolerance induces antigen-specific CD4+CD25- regulatory T cells that can transfer their regulatory capacity to naive CD4+ T cells

The mucosal immune system is uniquely adapted to elicit immune responses against pathogens but also to induce tolerogenic responses to harmless antigens. In mice, nasal application of ovalbumin (OVA) leads to suppression of both T(h)1 and T(h)2 responses. This tolerance can be transferred to naive mice by CD4(+) T(r) cells from the spleen. Using the allotypic Ly5 system, we were able to demonstrate in vivo that T(r) cells not only suppress naive CD4(+) T cells, but also induce them to differentiate into T(r) cells. The effector function of these mucosal T(r) cells is not restricted by cytokine polarization, since T(r) cells from T(h)1-tolerant mice can suppress a T(h)2 response and vice versa. Transfer of splenic CD4(+)CD25(+) and CD4(+)CD25(-) T cell subsets from OVA-tolerized mice revealed that both subsets were equally able to suppress a delayed-type hypersensitivity response in acceptor mice. In contrast to the CD25(-) T cell subset, the CD25(+) cells were not specific for the antigen used for tolerization. Together, these findings demonstrate a role for CD4(+)CD25(-) T(r) cells in mucosal tolerance, which suppresses CD4(+) T cells in an antigen-specific fashion, irrespective of initial T(h)1/T(h)2 skewing of the immune response. This offers a major advantage in the manipulation of mucosal tolerance for the treatment of highly cytokine-polarized disorders such as asthma and autoimmune diseases.     

Human blood CXCR5(+)CD4(+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion

Although a fraction of human blood memory CD4(+) T cells expresses chemokine (C-X-C motif) receptor 5 (CXCR5), their relationship to T follicular helper (Tfh) cells is not well established. Here we show that human blood CXCR5(+)CD4(+) T cells share functional properties with Tfh cells and appear to represent their circulating memory compartment. Blood CXCR5(+)CD4(+) T cells comprised three subsets: T helper 1 (Th1), Th2, and Th17 cells. Th2 and Th17 cells within CXCR5(+), but not within CXCR5(-), compartment efficiently induced naive B cells to produce immunoglobulins via interleukin-21 (IL-21). In contrast, Th1 cells from both CXCR5(+) and CXCR5(-) compartments lacked the capacity to help B cells. Patients with juvenile dermatomyositis, a systemic autoimmune disease, displayed a profound skewing of blood CXCR5(+) Th cell subsets toward Th2 and Th17 cells. Importantly, the skewing of subsets correlated with disease activity and frequency of blood plasmablasts. Collectively, our study suggests that an altered balance of Tfh cell subsets contributes to human autoimmunity.     

CD16+ and CD16- human blood monocyte subsets differentiate in vitro to dendritic cells with different abilities to stimulate CD4+ T cells.

Experimental protocols for cancer immunotherapy include the utilization of autologous monocyte-derived dendritic cells (moDC) pulsed with tumor antigens. However, disease can alter the characteristics of monocyte precursors and some patients have increased numbers (up to 40%) of the minor CD16(+) monocyte subpopulation, which in healthy individuals represent 10% of blood monocytes. At the present, the capacity of CD16(+) monocytes to differentiate into DC has not been evaluated. Here, we investigated the ability of CD16(+) monocytes cultured with granulocyte- macrophage colony-stimulating factor, IL-4 and tumor necrosis factor-alpha to generate DC in vitro, and we compared them to DC derived from regular CD16(-) monocytes. Both monocyte subsets gave rise to cells with DC characteristics. They internalized soluble and particulate antigens similarly, and both were able to stimulate T cell proliferation in autologous and allogeneic cultures. Nevertheless, CD16(+) moDC expressed higher levels of CD86, CD11a and CD11c, and showed lower expression of CD1a and CD32 compared to CD16(-) moDC. Lipopolysaccharide-stimulated CD16(-) moDC expressed increased levels of IL-12 p40 mRNA and secreted greater amounts of IL-12 p70 than CD16(+) moDC, whereas levels of transforming growth factor-beta1 mRNA were higher on CD16(+) moDC. Moreover, CD4(+) T cells stimulated with CD16(+) moDC secreted increased amounts of IL-4 compared to those stimulated by CD16(-) moDC. These data demonstrate that both moDC are not equivalent, suggesting either that they reach different stages of maturation during the culture or that the starting monocytes belong to cell lineages with distinct differentiation capabilities.     

Differential representations of memory T cell subsets are characteristic of polarized immunity in leprosy and atopic diseases.

We identified functionally polarized subsets of CD4 memory T cells on the basis of the expression of CD11a, CD45RA and CD62L. Within the several phenotypically distinct subsets of CD4 memory cells are two that, upon stimulation, produce primarily IL-4 (MT(2), CD45RA(-)CD62L(+)CD11a(dim)) or primarily IFN-gamma (MT(1), CD45RA(-)CD62L(-)CD11a(bright)). In addition, four other phenotypically distinct subsets of CD4 cells have unique cytokine profiles. To determine the clinical relevance of the representation of these cell types, we analyzed blood from patients with the chronic diseases leprosy and atopy. These diseases are characterized as immunologically polarized, since T cell responses in affected individuals are often strongly biased towards T(h)1 (dominated by IFN-gamma production) or T(h)2 (IL-4 production). We show here that this polarization reflects homeostatic or differentiation mechanisms affecting the representation of the functionally distinct subsets of memory CD4 T cells, MT(1) and MT(2). Significantly, the representation of the MT(1) and MT(2) subsets differs dramatically between subjects with tuberculoid leprosy (a T(h)1 disease), or lepromatous leprosy or atopic disease (T(h)2 diseases). However, there was no difference in the cytokine profiles of these or any of the other finely resolved CD4 subsets, when compared between individuals across all disease states. Thus, it is the representation of these subsets in peripheral blood that is diagnostic of the polarized state of the immune system.     

A co-stimulatory molecule on activated T cells,H4/ICOS,delivers specific signals in T(h) cells and regulates their responses

We examined the co-stimulatory activity of H4/ICOS on murine activated CD4(+) T cells and found that the cross-linking of H4/ICOS enhanced their proliferation, in addition to raising IFN-gamma, IL-4 and IL-10 production to levels comparable to those induced by CD28. However, IL-2 production was only marginally co-stimulated by H4/ICOS. This distinct pattern of lymphokine production appears to be induced by a specific intracellular signaling event. Compared with CD28, H4/ICOS dominantly elicited the Akt pathway via phosphatidylinositol 3-kinase. In addition, mitogen-activated protein kinase family kinases were activated in different ways by CD28 and H4/ICOS. The strong phosphorylation of p46 c-Jun N-terminal kinase was observed upon CD28 co-stimulation, but was less potently induced by H4/ICOS. The strain diversity in the induction of H4/ICOS was recognized. The expression of H4/ICOS on BALB/c activated CD4(+) T cells was >6-fold higher compared with C57BL/6 activated CD4(+) T cells. Furthermore, BALB/c activated CD4(+) T cells exhibited more T(h)2-deviated lymphokine production as compared with C57BL/6 activated CD4(+) T cells and signaling through H4/ICOS during the primary stimulation of naive CD4(+) T cells promoted the generation of T(h)2 cells. Thus, the difference in H4/ICOS expression on activated CD4(+) T cells, which is regulated among the mouse strains, may also regulate the polarization of T(h) cells.     

The initial response of CD4+ IL-4-producing cells

Naive CD4(+) T cells were reported to produce small amounts of IL-4 in vitro, which are implicated to be sufficient to initiate T(h)2 response in vivo. However, IL-4-producing naive CD4(+) T cells are difficult to study in vivo because they are present in low numbers shortly after the first antigen exposure. Here, we used IL-4/green fluorescence protein (GFP) reporter mice (G4 mice) to track the initial response of CD4(+) IL-4-producing cells. We first established a flow cytometry method to estimate the number of GFP(+) cells. We demonstrated the effectiveness of this method by showing that the responding CD4(+)GFP(+) cells exhibited an activated phenotype, possessed the capacity to express IL-5 and IL-13, but not IFN-gamma mRNA, and showed enhanced levels of GATA3 and c-maf mRNA expression. More importantly, we showed that the cell lines derived from FACS-sorted CD4(+)GFP(+) cells were antigen specific. By using this newly established method, we showed that the majority of responding GFP(+) cells were CD4(+) T cells. Our study provides direct ex vivo evidence to show that a small percent of CD4(+) T cells that have no previous experience of antigenic stimulation might produce IL-4 to initiate T(h)2 response.     

Role of CD4(+)CD25(+) T regulatory cells in IL-2-induced vascular leak

T regulatory cells (CD4(+)CD25(+)) play an important role in the regulation of the immune response. However, little is known about the ability of T regulatory cells to regulate endothelial cell (EC) damage following activation of lymphocytes with IL-2. Therefore, in the current study, we examined the role of T regulatory cells and the subsequent T(h)1/T(h)2 bias in IL-2-mediated EC injury using the well-characterized C57BL/6 (T(h)1-biased) and BALB/c (T(h)2-biased) models. Following IL-2 treatment, BALB/c mice were less susceptible to IL-2-induced vascular leak syndrome (VLS) compared with C57BL/6 mice. Splenocytes from BALB/c mice displayed less cytotoxicity against ECs compared with those from C57BL/6 mice. Interestingly, BALB/c mice had significantly higher numbers of CD4(+)CD25(+) T regulatory cells, which proliferated more profoundly following IL-2 treatment, compared with CD4(+)CD25(+) T regulatory cells from C57BL/6 mice. In addition, T regulatory cells from naive BALB/c mice were more potent suppressors of anti-CD3 mAb-stimulated proliferation of T cells than similar cells from C57BL/6 mice. Depletion of T regulatory cells in both BALB/c and C57BL/6 mice led to a significant increase in IL-2-induced VLS. Together, the results from this study suggest that CD4(+)CD25(+) T regulatory cells play an important role in the regulation of IL-2-induced EC injury.     

Clustering of immunoreceptor tyrosine-based activation motif-containing signalling subunits in CD4(+) T cells is an optimal signal for IFN-gamma production,but not for the production of IL-4

CD4(+) T cells with pre-defined MHC-unrestricted specificity to type II collagen (CII) were engineered for cell-based anti-inflammatory gene therapy of autoimmune arthritis. To this end, recombinant chimeric immunoreceptors, C2gamma or C2zeta, were expressed in primary mouse keyhole limpet hemocyanin (KLH)-specific T(h)1 and T(h)2 cells using retrovirus vector-based somatic cell gene transfer. The ectodomain of these tyrosine-based activation motif (ITAM)-containing immunoreceptors is a single-chain IgG variable domain of an anti-CII mAb. The engineered cells might arrest migration when they encounter CII in articular cartilage. Up to 19 and 55% of transduced CD4(+) T cells expressed respectively C2gamma and C2zeta. The expression of C2gamma or C2zeta on the surface of CD4(+) T cells was down-regulated upon binding CII, and cells activated in such a way proliferated, up-regulated CD25 expression and produced cytokines. Comparison of cytokine levels normalized by the number of producer cells revealed that C2gamma and C2zeta were as potent as TCR in the induction of IFN-gamma, but induced lower levels of IL-4. It appears that the reason why CD4(+) T cells stimulated through C2gamma and C2zeta produce low levels of IL-4 is a lack of integration between co-stimulatory signals required for the optimal production of this cytokine and the ITAM-dependent signals generated by the immunoreceptors. The significance of these data for the development of anti-inflammatory gene therapy based on CD4(+) T cells targeted to a tissue-specific protein is discussed.     

IL-27 suppresses RANKL expression in CD4+ T cells in part through STAT3

The receptor activator of NF-κB ligand (RANKL), which is expressed by not only osteoblasts but also activated T cells, plays an important role in bone-destructive diseases such as rheumatoid arthritis. IL-27, a member of the IL-6/IL-12 family cytokines, activates STAT1 and STAT3, promotes early helper T (Th)1 differentiation and generation of IL-10-producing type 1 regulatory T (Tr1) cells, and suppresses the production of inflammatory cytokines and inhibits Th2 differentiation. In addition, IL-27 was recently demonstrated to not only inhibit Th17 differentiation but also directly act on osteoclast precursor cells and suppress RANKL-mediated osteoclastogenesis through STAT1-dependent inhibition of c-Fos, leading to amelioration of the inflammatory bone destruction. In the present study, we investigated the effect of IL-27 on the expression of RANKL in CD4(+) T cells. We found that IL-27 greatly inhibits cell surface expression of RANKL on naive CD4(+) T cells activated by T cell receptor ligation and secretion of its soluble RANKL as well. The inhibitory effect was mediated in part by STAT3 but not by STAT1 or IL-10. In contrast, in differentiated Th17 cells, IL-27 much less efficiently inhibited the RANKL expression after restimulation. Taken together, these results indicate that IL-27 greatly inhibits primary RANKL expression in CD4(+) T cells, which could contribute to the suppressive effects of IL-27 on the inflammatory bone destruction.     

Single-cell analysis by RT-PCR reveals differential expression of multiple type 1 and 2 cytokine genes among cells within polarized CD4+ T cell populations.

RT-PCR was used to examine the expression of IFN-gamma, IL-2, IL-4, IL-5, IL-6 and IL-10 mRNAs by single murine CD4+ T cells activated either in a strongly type 1-polarized mixed lymphocyte reaction (MLR) or in the type 2-polarized response to immunization with keyhole limpet hemocyanin (KLH) in alum. The frequencies of expression of each cytokine differed markedly between the two responses, consistent with their polarization at the population level. However, most cells expressed only none to three of the six cytokines assayed, few displayed the canonical type 1 profile and none in either response expressed a full type 2 or type 0 profile. A significant fraction of cells co-expressed IFN-gamma with IL-4 and/or other type 2 cytokines at frequencies that suggested that most of these genes were independently regulated. Collectively, these single-cell expression patterns indicate that polarization at the population level can mask substantial intercellular heterogeneity, and show directly that multiple type 1 and 2 cytokines can be expressed simultaneously in an individual T cell.     

Origins of CD4(+) effector and central memory T cells

Lineage-committed effector CD4(+) T cells are generated at the peak of the primary response and are followed by heterogeneous populations of central and effector memory cells. Here we review the evidence that T helper type 1 (T(H)1) effector cells survive the contraction phase of the primary response and become effector memory cells. We discuss the applicability of this idea to the T(H)2 cell, T(H)17 helper T cell, follicular helper T cell (T(FH) cell) and induced regulatory T cell lineages. We also discuss how central memory cells are formed, with an emphasis on the role of B cells in this process.     

Frequencies of CD4+ T cells reactive with Plasmodium chabaudi chabaudi: distinct response kinetics for cells with Th1 and Th2 characteristics during infection

The functional heterogeneity of the CD4⁺ T cell response toPlasmodium chabaudi has been evaluated. Using a limiting dilutionassay system and a variety of assays to detect -interferon (IFN-),interleukln-2 (IL-2), IL-3, and T helper (T_h) cells for malaria-specificantibody production, the precursor frequencies of P. chabaudl-reactiveT cells have been calculated. The patterns of lymphokines producedby individual microcultures of the limiting dilution assay generallysupported the Idea of two functionally distinct CD4⁺ subsets:one which produces IFN- and IL-2 (T_h1) and one which Is an effectivehelper cell for antibody production (T_h2) However, it couldnot be determined whether the overiapping functions observedin some cultures represented T cells which could produce allfactors or separate clones which were developing In the samewells. During the first 14 days of an erythrocytic Infectionof P. chabaudi the predominant T cell response was of the T_h1-tupe.The frequency of these cells decreased after 14 days. By 3 weeksafter Infection the CD4⁺ T cell response was characterized byT_h2 cells, as defined by their ability to act as helper cellsin the production of malaria-specific antibody. These data supportthe hypothesis that early clearance of P. chabaudi may be antibody-Independentbut that the final clearance mechanism coincides with the appearanceof helper cells and antibody.     

Sialyl lewisx antigen-expressing human CD4+ T and CD8+ T cells as initial immune responders in memory phenotype subsets

Cytokine production by memory T cells in secondary immune responses has a critical role in host defenses. Previously, we had demonstrated that a unique antigen composed of sialyl lewis(x) (sLe(x)) was expressed on CD45RO(+) memory-phenotype subsets of human T cells. Here, we found that the sLe(x) antigen was up-regulated on CD45RA(+) na?ve human CD4(+) T and CD8(+) T cells by TCR stimulation. In addition, sLe(x) antigen-expressing CD4(+) T and CD8(+) T cells in human PBMCs were activated immediately by cytokine stimulations composed of IL-2 plus IL-12 or IL-15 in an antigen-independent manner. Moreover, the sLe(x)-positive human CD8(+) T cells significantly enhanced reverse antibody-dependent cellular cytotoxicity compared with a sLe(x)-negative population. These findings clearly indicate that sLe(x) antigen-expressing memory phenotype CD4(+) T and CD8(+) T cells contribute to early-stage immunity by providing a source of IFN-gamma and cytotoxicity, suggesting that they would be a key immunomodulator in host defenses.