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.     

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.     

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.     

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.     

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.     

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.     

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.     

Reversal of experimental allergic encephalomyelitis with non-mitogenic, non-depleting anti-CD3 mAb therapy with a preferential effect on T(h)1 cells that is augmented by IL-4

This study examined whether therapy with a non-mitogenic, non-activating anti-CD3 mAb (G4.18) alone, or in combination with the T(h)2 cytokines, could inhibit induction or facilitate recovery from experimental allergic encephalomyelitis (EAE) in Lewis rats. G4.18, but not rIL-4, rIL-5 or anti-IL-4 mAb, reduced the severity and accelerated recovery from active EAE. A combination of rIL-4 with G4.18 was more effective than G4.18 alone. The infiltrate of CD4(+) and CD8(+) T cells, B cells, dendritic cells, and macrophages in the brain stem was less with combined G4.18 and IL-4 than G4.18 therapy or no treatment. Residual cells had preferential sparing of T(r)1 cytokines IL-5 and transforming growth factor-beta with loss of T(h)1 markers IL-2, IFN-gamma and IL-12Rbeta2, and the T(h)2 cytokine IL-4 as well as macrophage cytokines IL-10 and tumor necrosis factor-alpha. Lymph nodes draining the site of immunization had less mRNA for T(h)1 cytokines, but T(h)2 and T(r)1 cytokine expression was spared. Treatment with G4.18, rIL-4 or rIL-5 from the time of immunization had no effect on the course of active EAE. MRC OX-81, a mAb that blocks IL-4, delayed onset by 2 days, but had no effect on severity of active EAE. G4.18 also inhibited the ability of activated T cells from rats with active EAE to transfer passive EAE. This study demonstrated that T cell-mediated inflammation was rapidly reversed by a non-activating anti-CD3 mAb that blocked effector T(h)1 cells, and spared cells expressing T(h)2 and T(r)1 cytokines.     

A regulatory role for suppressor of cytokine signaling-1 in T(h) polarization in vivo

Suppressor of cytokine signaling (SOCS)-1 is an inhibitory molecule for JAK, and its deficiency in mice leads to lymphocyte-dependent multi-organ disease and perinatal death. Crossing of SOCS-1(-/-) mice on an IFN-gamma(-/-), STAT1(-/-) and STAT6(-/-) background revealed that the fatal disease of SOCS-1(-/-) mice is also dependent on IFN-gamma/STAT1 and IL-4/STAT6 signaling pathways. Since IFN-gamma and IL-4 are representative T(h)1 and T(h)2 cytokines respectively, here we investigated the role of SOCS-1 in T(h) differentiation. Freshly isolated SOCS-1(-/-) CD4(+) T cells stimulated with anti-CD3 rapidly produced larger amounts of IFN-gamma and IL-4 than control cells, suggesting that these mutant T cells had already differentiated into T(h)1 and T(h)2 cells in vivo. In addition, SOCS-1(+/-) CD4(+) T cells cultured in vitro produced significantly larger amounts of IFN-gamma and IL-4 than SOCS-1(+/+) cells. Similarly, SOCS-1(+/-) CD4(+) T cells produced more IFN-gamma and IL-4 than SOCS-1(+/+) cells after infection with Listeria monocytogenes and Nippostrongyrus braziliensis respectively. Since IL-12-induced STAT4 and IL-4-induced STAT6 activation is sustained in SOCS-1(-/-) T cells, the enhanced T(h) functions in SOCS-1(-/-) and SOCS-1(+/-) mice appear to be due to the enhanced effects of these cytokines. These results suggest that SOCS-1 plays a regulatory role in both T(h)1 and T(h)2 polarizations.     

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.     

Immunologic immaturity, but high IL-4 productivity, of murine neonatal thymic CD4 single-positive T cells in the last stage of maturation

To determine the levels of maturation and differentiation ofmurine CD4 single-positive (SP) T cells, we compared the secondaryresponses of staphylococcal enterotoxin A (SEA)-induced neonatalthymic, adult thymic and adult splenic CD4 SP T cell blastsprepared from whole or heat-stable antigen^low CD4 SP T cells.Proliferative responses upon re-stimulation with SEA were strongin adult splenic CD4 SP T cell blasts, but quite weak in neonatalthymic and adult thymic CD4 SP T cell blasts. SEA-induced IL-2production was weaker in neonatal thymic blasts than in theadult splenic CD4 SP T cell blasts. In contrast, SEA-inducedIL-4 production was high in neonatal thymic CD4 SP T cell blasts,and low in adult splenic and thymic CD4 SP T cell blasts. Expressionof GATA-3, that directs production of IL-4 in T cells, examinedat protein and mRNA levels, was higher in neonatal thymic cellsthan in adult thymic and splenic cells. These results suggestthat neonatal and adult thymic CD4 SP T cells in the final stageof maturation are relatively immature compared with adult splenicCD4 SP T cells. The cytokine production profile of neonatalthymic CD4 SP T cells suggests that they are inclined towardsa T_h2 response.     

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.     

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.     

Mycobacterium tuberculosis in the adjuvant modulates the balance of Th immune response to self-antigen of the CNS without influencing a "core" repertoire of specific T cells

In the present study, we use modified CDR3 beta-chain spectratyping (immunoscope) to dissect the effect of Mycobacterium tuberculosis (MT)-derived proteins on individual PLP139-151-specific cells in the SJL mouse strain. In this model, the immunoscope technique allows the characterization of a public TCR that involves rearrangement of Vbeta10 and Jbeta1.1 and a semi-private TCR characterized by rearrangement of Vbeta4 and Jbeta1.6. Both rearrangements are specific for PLP139-151 and sequences of the CDR3 region of the two beta-chains show a conserved motif for the public rearrangement and related but more variable sequences for the semi-private rearrangement. MT-derived proteins promote increase of IFN-gamma-secreting cells. However, we observe that the presence and amount of MT used during immunization have no effect on the frequency of usage, polarization and in vivo expansion of cells carrying the studied rearrangements. Rather, the strong Th1-promoting effect of adjuvant is possibly due to recruitment toward Th1 of a wider spectrum of TCR repertoires. Therefore, instead of having a comprehensive effect on the entire repertoire, MT modulates the immune response by affecting a subset of antigen-specific T cells whose polarization can be adapted to the environment. This step establishes the final balance between Th1 and Th2 and may be essential for the enhancement or protection of disease.