Lack of correlation between chemokine receptor and T(h)1/T(h)2 cytokine expression by individual memory T cells

Chemokine and chemokine receptor interactions may have important roles in leukocyte migration to specific immune reaction sites. Recently, it has been reported that CXC chemokine receptor (CXCR) 3 and CC chemokine receptor (CCR) 5 were preferentially expressed on T(h)1 cells, and CCR3 and CCR4 were preferentially expressed on T(h)2 cells. To investigate chemokine receptor expression by T(h) subsets in vivo, we analyzed cytokine (IL-2, IL-4 and IFN-gamma) and chemokine receptor (CXCR3, CXCR4, CCR3, CCR4 and CCR5) mRNA expression by individual peripheral CD4(+) memory T cells after short-term stimulation, employing a single-cell RT-PCR method. This ex vivo analysis shows that the frequencies of cells expressing chemokine receptor mRNA were not significantly different between T(h)1 and T(h)2 cells in normal peripheral blood. To assess a potential role of in vivo stimulation, we also analyzed unstimulated rheumatoid arthritis synovial CD4(+) memory T cells. CXCR3, CXCR4, CCR3 and CCR5 expression was detected by individual synovial T cells, but the frequencies of chemokine receptor mRNA were not clearly different between T(h)1 and non-T(h)1 cells defined by expression of IFN-gamma or lymphotoxin-alpha mRNA in all RA patients. These data suggest that chemokine receptor expression does not identify individual memory T cells producing T(h)-defining cytokines and therefore chemokine receptor expression cannot be a marker for T(h)1 or T(h)2 cells in vivo.     

Inadequate induction of suppressor of cytokine signaling-1 causes systemic autoimmune diseases

Suppressor of cytokine signaling (SOCS)-1 is a cytokine-inducible, negative regulatory molecule of Janus kinases (JAK) and its deficiency causes hyper-response to various cytokines. SOCS-1(-/-) mice spontaneously develop a fatal disease depending on aberrantly activated lymphocytes. Here, we show that partial restoration of SOCS-1 in lymphoid cells rescues SOCS-1(-/-) mice from the early-onset fatal disease, indicating that SOCS-1 expression in vivo is especially required in lymphocytes. However, SOCS-1 expression in these SOCS-1-restored mutant mice (E( micro )-SOCS-1(-/-) mice) was insufficient for proper down-regulation of its target signaling, and these mice spontaneously exhibit hyperactivation of lymphocytes, an increase in the levels of serum Ig and anti-DNA autoantibodies, and glomerulonephritis with glomerular IgG deposition. These phenotypes resemble those of murine systemic autoimmune diseases, models for systemic lupus erythematosus (SLE). Interestingly, similar phenotypes were also observed in adult female SOCS-1(+/-) mice, indicating that the autoimmune phenotypes of these mice can be ascribed primarily to the inadequate expression of SOCS-1. In addition, autoimmune phenotypes were not observed in SOCS-1(+/-)CD4(-/-) mice, suggesting that autoimmunity is dependent on hyper-activated CD4(+) T cells. Our findings also suggest that insufficient expression of SOCS-1 results in impaired function of CD25(+)CD4(+) regulatory T cells, which may contribute to aberrant activation of CD4(+) T cells. These findings suggest that dysfunction of SOCS-1 can be a pathogenic factor of systemic autoimmune diseases such as SLE.     

Increased antigen presentation and T(h)1 polarization in genetically histamine-free mice

Histamine is a well-known inflammatory mediator exerting various immunomodulatory effects and affecting the development of antigen-specific immune responses. Dendritic cells (DCs) are the most potent antigen-presenting cells specialized for capture, uptake, transport, processing and presentation of antigens to T cells. Using a genetically histamine-free [histidine decarboxylase knockout (HDC-/-)] mouse model, we examined the effects of histamine on DC-mediated antigen presentation. Applying an in vitro antigen presentation assay, we found that spleen DCs, derived from HDC-/- mice, display a higher efficiency in antigen presentation compared with wild-type cells. Flow cytometric characterization of DCs disclosed that this difference was not due to an altered distribution of DCs between or within the major functional sub-populations (assessed by CD11b and CD4 as myeloid and CD8alpha and DEC205 as lymphoid DC markers) or major changes in the co-stimulatory molecule profile (CD40, CD80, CD86). However, real-time PCR analysis of in vivo CFA-induced IL-12p35, IFNgamma, IL-10 and IL-4 expression showed that DCs matured in a histamine-free environment exhibit significantly elevated levels of IL-12p35 and IFNgamma mRNA. In vitro investigations confirmed that isolated DCs, developed in the absence of histamine, exhibit indeed a predominantly T(h)1-polarized cytokine pattern, as they show elevated levels of IFNgamma mRNA upon LPS stimulation. Similar difference was found at the protein level by ELISA, as well. Our study demonstrates that histamine interferes with antigen presentation and alters the cytokine profile of DCs.     

Functional heterogeneity among bone marrow-derived dendritic cells conditioned by T(h)1- and T(h)2-biasing cytokines for the generation of allogeneic cytotoxic T lymphocytes

Three distinct bone marrow (BM)-derived dendritic cells (BMDC) were expanded from BALB/c BM cells by culture with (i) granulocyte macrophage colony stimulating factor (GM-CSF) plus IL-3, (ii) GM-CSF, IL-3 plus T(h)1-biasing cytokines (IL-12 and IFN-gamma) or (iii) GM-CSF, IL-3 plus T(h)2-biasing cytokines (IL-4). All of these cells expressed the DC-specific marker CD11c, and were designated as BMDC0, BMDC1 and BMDC2 cells respectively. BMDC1 cells exhibited superior T cell-stimulating activity in allogeneic mixed lymphocyte culture (MLC), while BMDC2 showed inferior stimulating activity. Specifically, BMDC1, as compared with BMDC2, induced a higher frequency of IFN-gamma-producing CD8(+) T cells in MLC. Moreover, BMDC1, but not BMDC2, were strong inducers of H-2(d)-specific cytotoxic T lymphocytes (CTL) in MLC. BMDC0 always showed intermediate stimulatory activity; however, when BMDC0 were cultured with IFN-gamma, they differentiated into BMDC1-like stimulator cells concomitant with the up-regulation of both MHC antigens and co-stimulatory molecules. In contrast, BMDC2 were refractory to differentiation into superior stimulator cells by treatment with IFN-gamma, although this treatment enhanced MHC expression. These findings indicate that T(h)1- and T(h)2-biasing cytokines, in addition to their effect on T(h) cell differentiation, may play a critical role in the functional skewing of DC. These findings have important implications for the development of DC-based immunotherapies.     

Antigen-specific cellular hyporesponsiveness in a chronic human helminth infection is mediated by T(h)3/T(r)1-type cytokines IL-10 and transforming growth factor-beta but not by a T(h)1 to T(h)2 shift

Exposure to infective larvae of the filarial nematode Onchocerca volvulus (Ov) either results in patent infection (microfilaridermia) or it leads to a status called putative immunity, characterized by resistance to infection. Similar to other chronic helminth infections, there is a T cell proliferative hyporesponsiveness to Ov antigen (OvAg) by peripheral blood mononuclear cells (PBMC) from individuals with patent infection, i.e. generalized onchocerciasis (GEO), compared to PBMC from putatively immune (PI) individuals. In this study, mechanisms mediating this cellular hyporesponsiveness in GEO were investigated: the low proliferative response in PBMC from GEO individuals was associated with a lack of IL-4 production and significantly lower production of IL-5 compared to those from PI individuals, arguing against a general shift towards a T(h)2 response being the cause of hyporesponsiveness. In contrast, IL-10 and transforming growth factor (TGF)-beta, two cytokines associated with a T(h)3 response, seemed to mediate hyporesponsiveness: PBMC from individuals with GEO produced significantly more IL-10, and T cell proliferative hyporesponsiveness in this group could be reversed by the addition of anti-IL-10 and anti-TGF-beta antibodies. Hyporesponsiveness was specific for OvAg and not observed upon stimulation with related nematode antigens, arguing for a T cell-mediated, Ov-specific down-regulation. Ov-specific T cells could be cloned from GEO PBMC which have a unique cytokine profile (no IL-2 but high IL-10 and/or TGF-beta production), similar to the T cell subsets known to suppress ongoing inflammation (T(h)3 and T(r)1), indicating that this cell type which has not been found so far in infectious diseases may be involved in maintaining Ov-specific hyporesponsiveness.     

Antigen-specific T(h)1 cells as direct effectors of Propionibacterium acnes-primed lipopolysaccharide-induced hepatic injury

T(h)1 cells are cytotoxic effector cells that utilize Fas ligand (FasL) and tumor necrosis factor. The physiological roles of cytotoxic T(h)1 cells are considered to be immunoregulation by eliminating autoreactive lymphocytes or hyper-activated foreign antigen-specific lymphocytes. Their pathological roles, however, remain to be clarified. To investigate whether T(h)1 cells can destroy organs, we generated a Propionibacterium acnes-specific T(h)1 clone from C57BL/6 mice and tested whether the clone could serve as an effector in a P. acnes-primed lipopolysaccharide (LPS)-induced hepatic injury system, one of the septic shock models. B6SMN:C3H-FasL(gld) (B6-gld) mice, which were deficient in functional FasL, were resistant to P. acnes/LPS-induced hepatic shock. The T(h)1 clone rendered B6-gld mice sensitive to the hepatic shock after the i.v. transfer. The hepatic injury in the clone-transferred B6-gld mice, which was evaluated by both biochemical and histological examination, was inhibited by an anti-FasL mAb that we developed. These results suggested that bacterial antigen-specific T(h)1 cells like this clone can participate in organ destruction in vivo as one of the cytotoxic effectors and play a critical role in endotoxin-induced hepatic injury.     

Preferential expression of T(h)2-type chemokine and its receptor in atopic dermatitis

Lesional skin of patients with atopic dermatitis (AD) is histologically characterized by hypertrophy of the skin, and the infiltration of a large number of eosinophils and T cells into the dermis. Recent studies have indicated that Th2 cells play a crucial role in the pathogenesis of AD skin. Chemokines and their receptors are implicated in the development of symptoms of various skin diseases such as AD and psoriasis vulgaris (psoriasis). We have examined the in situ expression of a typical Th2-type chemokine, thymus- and activation-regulated chemokine (TARC), and its receptor (CCR4) using immunohistochemical techniques. TARC was found to be highly expressed in the basal epidermis of the lesional skin of AD patients and only slightly in the non-lesional skin. On the other hand, no positive cells were seen in the lesional skin of psoriasis. Consistently, CCR4+ cells were present predominantly in the lesional skin of AD patients, but not in the non-lesional skin. In contrast, in the lesional skin of psoriasis patients, cells positive for CCR5, which is expressed on Th1 cells, were abundantly present. Interestingly, psoralen plus ultraviolet A therapy reduced the number of CCR4+ cells in the AD skin lesions. These results suggest that Th2-type cytokines such as TARC are involved in the pathogenesis of skin lesions in AD patients through the preferential recruitment of Th2 cells.     

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.     

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.     

Endotoxin can induce MyD88-deficient dendritic cells to support T(h)2 cell differentiation

Toll-like receptor (TLR) signaling activates dendritic cells (DC) to secrete proinflammatory cytokines and up-regulate co-stimulatory molecule expression, thereby linking innate and adaptive immunity. A TLR-associated adapter protein, MyD88, is essential for cytokine production induced by TLR. However, in response to a TLR4 ligand, lipopolysaccharide (LPS), MyD88-deficient (MyD88(-/-)) DC can up-regulate co-stimulatory molecule expression and enhance their T cell stimulatory activity, indicating that the MyD88-independent pathway through TLR4 can induce some features of DC maturation. In this study, we have further characterized function of LPS-stimulated, MyD88(-/-) DC. In response to LPS, wild-type DC could enhance their ability to induce IFN-gamma production in allogeneic mixed lymphocyte reaction (alloMLR). In contrast, in response to LPS, MyD88(-/-) DC augmented their ability to induce IL-4 instead of IFN-gamma in alloMLR. Impaired production of T(h)1-inducing cytokines in MyD88(-/-) DC cannot fully account for their increased T(h)2 cell-supporting ability, because absence of T(h)1-inducing cytokines in DC caused impairment of IFN-gamma, but did not lead to augmentation of IL-4 production in alloMLR. In vivo experiments with adjuvants also revealed T(h)2-skewed immune responses in MyD88(-/-) mice. These results demonstrate that the MyD88-independent pathway through TLR4 can confer on DC the ability to support T(h)2 immune responses.     

Early in vitro priming of distinct T(h) cell subsets determines polarized growth of visceralizing Leishmania in macrophages

An in vitro priming system of murine naive splenocytes was established to investigate early immune responses to LEISHMANIA: chagasi, the agent of visceral leishmaniasis in the New World. Priming of splenocytes from resistant C3H and CBA or susceptible BALB and B10 mice with L. chagasi resulted in blast transformation and in proliferating parasite-specific CD4(+) T cells secreting a differential complement of cytokines (IFN-gamma and low IL-10 levels for resistant T cells; IFN-gamma, IL-4 and high IL-10 levels for susceptible T cells). After priming, intracellular parasite load was much higher in susceptible than in resistant-type splenocyte cultures. On the other hand, infection of purified splenic macrophages from either resistant or susceptible mice with live L. chagasi promastigotes, resulted in comparable parasite loads. Moreover, when early CD4(+) T cell priming in splenocyte cultures was disrupted with anti-CD4 mAb, polarized parasite growth was abolished, becoming comparable in resistant and susceptible cultures. Neutralizing IL-4 activity during splenocyte priming did not affect the final parasite load in susceptible cultures. However, neutralizing IL-10 activity markedly decreased parasite load in susceptible, but not in resistant splenic macrophages. These results suggest that IL-10 plays an important role in L. chagasi infection in susceptible hosts. The results also indicate that innate control of growth of a visceralizing LEISHMANIA: in splenic macrophages results from the ability to activate different CD4(+) T cell subsets.     

B7 2 (CD86) is essential for the development of IL-4-producing T cells

The CD28/CTLA-4 ligands, B7–1 (CD80) and B7–2 (CD86),provide a co-stimulatory signal necessary for optimal T cellactivation. We have examined the effect of blocking B7–1and B7–2 in an in vitro system using ovalbumin-specificT cells from ß TCR-transgenic mice. This system allowedus to examine the interaction of B7 co-stimulators on physiologicantigen-presenting cells (APC) with antigen-specific T helperprecursor (T_hp) cells. We report that blocking T_hp/B7–1or B7–2 interactions in a primary response differentiallyaffects the cytokine profile observed in a secondary stimulation,even in the absence of additional anti-B7 antibody. Engagementof B7–2 in the primary stimulation was found to be essentialfor production of the T_h2 cytokine, IL-4, but not the T_h1 cytokines,IL-2 and IFN-, in a secondary stimulation. Conversely, inclusionof the anti-B7–1 mAb in cultures using highly purifiednaive T cells increased levels of IL-4 and significantly depressedlevels of IFN-, upon re-stimulation. The effect of the anti-B7–2mAb in reducing IL-4 production could be overcome by the additionof recombinant IL-4 in the primary stimulation. The effectsof the anti-B7–2 mAb appear to be due to blocking andnot cross-linking, as F(ab) fragments mimicked the intact antibody.Taken together, our data demonstrate that the interaction betweenThp and B7–2 favors the development of T_h2 cells.     

Cytokine profile and T cell adhesiveness to endothelial selectins: in vivo induction by a myasthenogenic T cell epitope and immunomodulation by a dual altered peptide ligand

Myasthenia gravis (MG) is a T cell-regulated antibody-mediated autoimmune disease. Immunization with two myasthenogenic peptides, p195-212 and p259-271, that are sequences of the human acetylcholine receptor alpha subunit was shown to induce experimental autoimmune MG (EAMG)-associated immune responses. A peptide composed of the two altered peptide ligands (APL) of the myasthenogenic peptides (designated as dual APL) inhibited, in vitro and in vivo, those responses. The objectives of this study were to examine (i) whether in vivo T cell activation by p259-271 affects the cytokine profile and the T cell migration ability, and (ii) whether the latter are immunomodulated by in vivo administration of the dual APL. Our results showed that immunization of mice with p259-271 enriched the population of lymph node and spleen cells with subsets of T cells with strong adhesiveness towards E- and P-selectins. This enrichment was associated with an acquisition of a T(h)1-type cytokine profile. Treatment of the immunized mice with the dual APL interfered with both the migratory potential of the autoreactive T cells, and the production of the T(h)1-type cytokines IL-2 and IFN-gamma (known to play a pathogenic role in MG and EAMG). T cells derived from APL-treated mice acquired a T(h)3-type cytokine profile, characterized by the secretion of the immunosuppresive cytokine transforming growth factor-ss. Thus, our results suggest that T cell selectin ligands and T cell-derived cytokines are involved in the induction and immunomodulation of EAMG- and MG-associated T cell responses.     

Development of an assay to measure in vivo cytokine production in the mouse.

The short in vivo lifespan of many cytokines can make measurement of in vivo cytokine production difficult. A method was developed to measure in vivo IL-4 and IFN-gamma production that eliminates this problem. Mice are injected with a biotin-labeled neutralizing IgG anti-IL-4 or anti-IFN-gamma mAb and bled 2-24 h later. Secreted cytokine is captured by the biotin-labeled mAb to produce a complex that has a relatively long in vivo half-life and consequently accumulates in serum. Serum concentrations of the complex are determined by ELISA, using wells coated with an antibody to a second epitope on the same cytokine to capture the complex. This technique is specific and increases sensitivity of detection of secreted IL-4 at least 1000-fold. The amount of cytokine measured is directly proportional to the amount produced and relatively independent of the site of cytokine production. Furthermore, because mice are injected with small quantities of biotin-labeled anti-cytokine mAb, which sample, rather than neutralize, all secreted cytokines, cytokine-dependent responses are not inhibited. The in vivo half-lives of the cytokine-anti-cytokine mAb complexes are sufficiently short to allow cytokine production to be measured every 2-3 days in the same mice. Thus, use of this assay provides a practical and relatively simple and inexpensive way to measure ongoing in vivo cytokine production. Furthermore, the techniques that have been developed to measure in vivo production of IL-4 and IFN-gamma can be applied to in vivo measurement of other molecules that have a short in vivo lifespan, including other cytokines.     

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.     

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.