Inhibitory and enhancing effects of IFN-gamma and IL-4 on SHIV(KU) replication in rhesus macaque macrophages: correlation between Th2 cytokines and productive infection in tissue macrophages during late-stage infection

HIV-1 is dual-tropic for CD4+ T lymphocytes and macrophages, but virus production in the macrophages becomes manifest only during late-stage infection, after CD4+ T cell functions are lost, and when opportunistic pathogens begin to flourish. In this study, the SHIV/macaque model of HIV pathogenesis was used to assess the role of cytokines in regulating virus replication in the two cell types. We injected complete Freund's adjuvant (CFA) intradermally into SHIV(KU)-infected macaques, and infused Schistosoma mansoni eggs into the liver and lungs of others. Tissues examined from these animals demonstrated that macrophages induced by CFA did not support viral replication while those induced by S. mansoni eggs had evidence of productive infection. RT-PCR analysis showed that both Th1 (IL-2 and IFN-gamma) and Th2 cytokines (IL-4 and IL-10) were present in the CFA lesions but only the Th2 cytokines were found in the S. mansoni lesions. Follow-up studies in macaque cell cultures showed that whereas IFN-gamma caused enhancement of virus replication in CD4+ T cells, it curtailed viral replication in infected macrophages. In contrast, IL-4 enhanced viral replication in infected macrophages. These studies strongly suggest that cytokines regulate the sequential phases of HIV replication in CD4 T cells and macrophages.     

High frequency of virus-specific interleukin-2-producing CD4(+) T cells and Th1 dominance during lymphocytic choriomeningitis virus infection

Analysis of C57BL/6 mice acutely infected with lymphocytic choriomeningitis virus (LCMV) by using intracellular cytokine staining revealed a high frequency (2 to 10%) of CD4(+) T cells secreting the Th1-associated cytokines interleukin-2 (IL-2), gamma interferon (IFN-gamma), and tumor necrosis factor alpha, with no concomitant increase in the frequency of CD4(+) T cells secreting the Th2-associated cytokines IL-4, IL-5, and IL-10 following stimulation with viral peptides. In LCMV-infected C57BL/6 CD8(-/-) mice, more than 20% of the CD4(+) T cells secreted IFN-gamma after viral peptide stimulation, whereas less than 1% of the CD4(+) T cells secreted IL-4 under these same conditions. Mice persistently infected with a high dose of LCMV clone 13 also generated a virtually exclusive Th1 response. Thus, LCMV induces a much more profound virus-specific CD4(+) T-cell response than previously recognized, and it is dramatically skewed to a Th1 phenotype.     

Effect of interferon-beta and atorvastatin on Th1/Th2 cytokines in multiple sclerosis

Beneficial effects by both interferon-beta and statin treatment in patients with multiple sclerosis (MS) may be linked to interference with the Th1/Th2 cytokine balance. We determined patterns of Th1/Th2 cytokines (interleukin (IL)-1beta, IL-2, IL-6, IL-12p70, tumor-necrosis factor (TNF)-alpha and interferon-gamma, and IL-4, IL-5 and IL-10, respectively) in the serum of patients with relapsing-remitting MS treated with 250microg interferon-beta 1b or with interferon-beta plus 40mg atorvastatin. In treatment na?ve patients with MS, a trend for lower TNF-alpha serum levels compared to controls was detected (P=0.08). Interferon-beta treatment increased TNF-alpha levels, while a trend for lowering of IL-5 serum levels was found (P=0.07). Addition of atorvastatin raised IL-12p70 serum levels (P<0.05). Mean levels of two Th2 cytokines (IL-4, IL-10) showed a non-significant increase after addition of atorvastatin. We conclude that interferon-beta and atorvastatin exert divergent action on Th1/Th2 serum cytokines levels in MS. Supplemental atorvastatin might promote a Th1-type response by raising IL-12p70. Further studies are required to support a Th2 cytokine shift by atorvastatin in patients with MS.     

Circulating Th1 and Th2 cytokines in patients with hepatitis C virus infection.

The imbalance of T-helper (Th) lymphocyte cytokine production may play an important role in immunopathogenesis of persistent hepatitis C virus (HCV) infection. To know whether an imbalance between Th1 and Th2 cytokines is present in chronic HCV infection, serum levels of Th1 cytokines, interferon gamma (IFN-gamma) and interleukin (IL)-2, and Th2 cytokines, IL-4 and IL-10, were measured using enzyme-linked immunosorbent assay in this study. Eighteen individuals with chronic HCV infection, 11 healthy subjects as normal controls and 10 chronic HBV infected patients as disease controls were observed. The results showed that the levels of Th2 cytokines (IL-4 and IL-10) were significantly increased in chronic HCV infected patients compared with normal controls (IL-4: 30.49+/-17.55 vs. 14.94+/-13.73, pg/ml, P<0.025; IL-10: 50.30+/-19.59 vs. 17.87+/-9.49, pg/ml, P<0.001). Similarly, the levels of Th1 cytokine, IL-2, was also elevated in individuals with chronic HCV infection when compared with normal controls (IL-2: 118.53+/-95.23 vs. 61.57+/-28.70, pg/ml, P<0.05). However, Th1 cytokine IFN-gamma level was not significantly changed during HCV infection (IFN-gamma: 28.09+/-15.65 vs. 24.10+/-15.61, pg/ml, P>0.05). Furthermore, the elevated levels of Th2 cytokines are greater than Th1 cytokines in HCV infection. Thus, the study indicates that an enhanced Th2 responses are present during chronic HCV infection, which may partly be responsible for the persistence of HCV infection.     

Differential production of Th1- and Th2-derived cytokines does not determine the genetically controlled or vaccine-induced rate of cure in murine visceral leishmaniasis

Recent studies with models of cutaneous leishmaniasis have provoked much interest in the role of CD4+ T cell subsets in determining the outcome of infectious disease. In Leishmania major infections, cure vs progressive disease correlates with the expansion of Th1-like or Th2-like CD4+ populations, respectively. We have investigated whether similar responses are associated with the differential patterns of infection seen in models of visceral leishmaniasis, caused by L. donovani. Splenic lymphocytes from infected Lsh congenic C57BL/10 (Lshs;H-2b) and B10.L-Lshr (Lshr;H-2b) mice and MHC congenic non-curing B10.D2/n (Lshs;H-2d) mice were examined for the production of cytokines representative of these CD4+ populations (IL-2, IL-3, IL-4, IL-5, and IFN-gamma). In all three strains examined, there was no evidence for the production of Th2-restricted cytokines. In addition, levels of serum IgE were depressed during the early phase of infection, indicative of in vivo IFN-gamma production. In the non-curing B10.D2/n strain, late phase of infection was associated with the decreased ability to produce cytokines in response to Ag and not with the production of IL-4 or IL-5 in response to Ag or mitogen. Serum IgE levels were also not raised above levels seen in uninfected controls. C57BL/10 mice were vaccinated with SDS-PAGE fractionated amastigote Ag bound to nitrocellulose and cytokine levels determined at various times after infection. The protocol used for vaccination was able to induce significant modulation of the course of infection in this strain and it was clear that IFN-gamma production in vitro provided an excellent correlate of rate of cure. Occasional individuals produced low levels of IL-5 in culture in response to parasite Ag, but this did not correlate with disease progression. Together, these data suggest that over-expansion of Th2-type cells and production of their specific cytokines (IL-4 and IL-5) is not a contributing factor to the variable long term course of L. donovani infection in these strains of mice.     

Differential Th1 and Th2 cell responses in male and female BALB/c mice infected with coxsackievirus group B type 3.

Male and female BALB/c mice differ dramatically in susceptibility to myocarditis subsequent to coxsackievirus B3 (CVB3) infection. CVB3 infection of male mice results in substantial inflammatory cell infiltration of the myocardium, and virus-immune lymphocytes from these animals give predominantly a Th1 cell phenotypic response, as determined by predominant immunoglobulin G2a isotypic antibody production and elevated numbers of gamma interferon and interleukin-2 (IL-2)-producing CD4+ T lymphocytes. Females infected with the same virus give predominantly a Th2 cell phenotypic response, as determined by preferential immunoglobulin G1 antibody isotypic responses and increased precursor frequencies of IL-4- and IL-5-producing CD4+ T cells. Treatment of females with testosterone or males with estradiol prior to infection alters subsequent Th subset differentiation, suggesting that the sex-associated hormones have either a direct or indirect effect on CD4+ lymphocyte responses in this model. Treatment of females with 0.1 mg of monoclonal antibody to IL-4 reduces precursor frequencies of IL-4-producing CD4+ T cells and increases frequencies of gamma interferon-producing cells. This treatment also enhances myocardial inflammation, indicating a correlation between Th1-like cell responses and pathogenicity in CVB3 infection. The Th2-like cell may regulate Th1 cell activation. Adoptive transfer of T lymphocytes from CVB3-infected female mice into male animals suppresses the development of myocarditis in the recipients. Treatment of the female donors with monoclonal antibodies to either CD3, CD4, or IL-4 molecules abrogates suppression.     

Interleukin-4 therapy of psoriasis induces Th2 responses and improves human autoimmune disease

Selective skewing of autoreactive interferon-gamma (IFN-gamma)-producing T helper cells (Th1) toward an interleukin-4 (IL-4)-producing (Th2) phenotype can in experimental animals alleviate autoimmune disease without inducing general immunosuppression. In a prospective dose escalation study, we assessed treatment with human IL-4 (rhuIL-4) in 20 patients with severe psoriasis. The therapy was well tolerated, and within six weeks all patients showed decreased clinical scores and 15 improved more than 68%. Stable reduction of clinical scores was significantly better at 0.2-0.5 microg rhuIL-4 than at < or =0.1 microg rhuIL-4 (P = 0.009). In psoriatic lesions, treatment with 0.2-0.5 microg/kg rhuIL-4 reduced the concentrations of IL-8 and IL-19, two cytokines directly involved in psoriasis; the number of chemokine receptor CCR5+ Th1 cells; and the IFN-gamma/IL-4 ratio. In the circulation, 0.2-0.5 microg/kg rhuIL-4 increased the number of IL-4+CD4+ T cells two- to three-fold. Thus, IL-4 therapy can induce Th2 differentiation in human CD4+ T cells and has promise as a potential treatment for psoriasis, a prototypic Th1-associated autoimmune disease.     

Th1 cytokines in autoimmune thyroiditis

This study was performed on a lot of 51 patients and intends to correlate the autoimmune thyroiditis to the synthesis of Th1 cytokines and to the activation of T lymphocytes. We find out that CD25, an activation marker of T lymphocytes, is significantly increased in these patients. We also find out that certain cytokine serum levels are increased (IL-2, TNF-alpha, IFN-gamma). These cytokines correspond to the secretor profile of the Th1 subset. Mononuclear cell culture supernatants showed an increased level of IL-2 and TNF-alpha in samples stimulated with ConA in comparison to unstimulated samples from the same patient, suggesting the existence of an expansioned Th1 and CD8+ cytotoxic population.     

Modulation of Th1/Th2 cytokines and inflammatory mediators by hydroxychavicol in adjuvant induced arthritic tissues

The present study was undertaken to investigate the anti-arthritic activity of hydroxychavicol (HC) a major phenolic compound isolated from the aqueous extract leaves of plant Piper betle (Piperaceae). The compound showed significant lowering of pro-inflammatory (Th1) cytokine levels in arthritic paw tissue homogenate supernatant viz. IL-2, IFN-γ, and TNF-α with maximum inhibition at higher dose levels of 2 and 4 mg/kg p.o. and enhanced the production of anti-inflammatory (Th2) cytokines IL-4 and IL-5 estimated by cytometric bead array immunoassay. Cytometric bead array uses the sensitivity of amplified fluorescence detection by flowcytometer to measure soluble analytes in a particle based immune assay. This assay can accurately quantitate five cytokines in a 50-μl sample volume. The T-helper (Th1) deviated cells produce detectable level of tumor necrosis factor (TNF-α), interleukin-2 (IL-2), and interferon-gamma (IFN-γ), while the Th2 deviated cells produce significant amount of interleukin-4 (IL-4) and interleukin-5 (IL-5). HC at graded doses also significantly decreased the expression of IL-1β, PGE₂, LTB₄, and nitric oxide levels showing significant inhibition of these parameters. Elevated levels of CD4⁺ T cell specific interferon-gamma (IFN-γ) in splenocytes of arthritic animals was also inhibited in treated animals. The oral LD₀ in both mice and rats was more than 1000 mg/kg.     

The Characteristics of Th1/Th2 Cytokine Receptors on Monocytes in Untreated Patients of Long Term Nonprogressor or Chronic HIV Infection

Monocytes/macrophages play crucial roles in immunity to microorganisms and are one of the important targets for human immunodeficiency virus (HIV) infection. The phenotypes and function of monocytes in HIV-infected patients were poorly determined. We herein detected the expression of Th1/Th2 cytokine receptors on monocyte subsets in the untreated HIV-infected patients of either long term nonprogressor (LTNP) or chronic infection (CHI). CD14+CD16- monocytes were significantly increased and CD14+CD16+ monocytes were reduced in patients of LTNP or CHI compared with healthy control. IL-6R expression on CD14+CD16- monocytes were decreased in patients of LTNP or CHI, whereas IL-4R and IL-10R expression on both CD14+CD16- and CD14+CD16+ monocyte subsets were increased in patients with LTNP or CHI, as determined by flow cytometry and real time PCR assays. The decreased IL-6R expression and enhanced IL-4R and IL-10R expression were also observed on CD4+ T cells of these patients, indicating that these changes in monocytes are not cell-specific. CD14+CD16- monocytes of HIV-infected patients produced less TNF-α and IL-1β but identical levels of IL-6, and IL-12 as the control after IFN-γ/LPS stimulation. However, in the presence of IL-4 or IL10, CD14+CD16- monocytes of HIV-infected patients produced more TNF-α, IL-6, IL-12 or Il-1β after IFN-γ/LPS stimulation than the healthy control, supporting the impaired IL-4R and IL-10R signal pathways in patients with LTNP and CHI. Therefore, our present study offered the basic information for the Th1/Th2 cytokine receptor expression and function on monocyte subsets in untreated HIV-infected individuals.     

Multi-way flow cytometric immune monitoring of antiretroviral therapy in HIV-infected children

In 40 HIV-infected children, 8-10 years old, belonging to the category A state of infection, the following flow-cytometric parameters were scored: percentage levels of different blood lymphocytes; surface expression of some activation and memory markers in CD4+ cells; switch to Th1 or Th2 of in vitro -stimulated CD4+ cell, tested by intracellular production of interleukin-2 or interleukin-4. Each investigation was carried out both before and 3 months after antiretroviral therapy (AZT and ddC). Some post-therapy changes concerning blood lymphocyte percentages were noticed, not only within CD4+ subpopulation, but also within CD8+, HLA-DR+/CD3 (T-activated) and CD16+CD56+ cells, respectively. On the other hand, following antiretroviral treatment, in HIV- originated CD4+ fresh cells, an improvement of pre-therapy increased values of surface activation (CD69, CD25) markers on memory (CD45RO+) cells, as well as of pre-therapy reduced rate of switching to Th1, revealed by intracellular interleukin-2 synthesis, was found. The significance of data obtained in the multi-way immune monitoring of antiretroviral therapy, in pediatric AIDS, as an additional investigation panel, is discussed.     

原发性肝癌患者Th1/Th2 亚群研究*

目的:探讨原发性肝癌患者血清中淋巴细胞亚群中Th1/Th2的变化,为分析肝癌的发生发展状症和临床治疗提供免疫学指标。方法:应用放射免疫分析及酶联免疫分析法(ELISA),测定46例肝癌患者,及43例正常对照组进行比较。以IL-2、INF-γ和TNF-α水平代表Th1型细胞因子,以IL-4,IL-6、IL-8、IL-10的水平代表Th2型细胞因子。结果:肝癌患者IL-2、TNF-γ、IL-6的水平明显低正常对照组,P<0.01。IL-4、IL-8、IL-10、TNF-α的水平明显高于正常对照组,P<0.01。结论:肝癌患者体内存在Th1/Th2细胞因子失衡,其中Th1亚群功能抑制,Th2亚群功能亢进,其与肿瘤在宿主体内生长密切相关。通过纠正这些免疫失调将成为肝癌治疗的重要手段。     

Salivary gland extract from Ixodes ricinus tick polarizes the cytokine profile toward Th2 and suppresses proliferation of T lymphocytes in human PBMC culture.

Tick salivary gland extract (SGE) was previously shown to inhibit murine T cell proliferation. In mice, SGE has an inhibitory effect on Th1 and a stimulatory effect on Th2 cytokine elaboration. In the present study, tick-mediated immunomodulation of human T cell proliferation and cytokine elaboration was analyzed using human peripheral blood mononuclear cells (PBMCs) stimulated with concanavalin A (Con A) or lipopolysaccharide (LPS). Using flow cytometry, tick saliva-induced changes were investigated in human mononuclear cell subpopulations. SGE from Ixodes ricinus dose-dependently inhibited human T cell proliferation. This finding supports the flow cytometry data, showing that the percentage of Con A-activated HLA-DR-CD3+ T lymphocytes and CD4+ CD8+ double-positive T cells decreased after SGE treatment. SGE significantly inhibited the in vitro production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) secreted by Th1 lymphocytes. In contrast, the elaboration of IL-4, IL-6, and IL-10 secreted by Th2 lymphocytes was significantly stimulated by I. ricinus SGE. Similarly, the production of both IL-1alpha and IL-1beta was significantly stimulated after SGE treatment. These data indicate that the tick-induced immunomodulatory events in humans are similar to those previously described in a murine model.     

Exogenous nitric oxide inhibits experimental autoimmune uveoretinitis development in Lewis rats by modulation of the Th1-dependent immune response.

Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease of posterior uvea that closely resembles a human disease. The uveitogenic effector T cell has a Th1-like phenotype [high interferon-gamma (IFN-gamma), low interleukin-4 (IL-4)], and genetic susceptibility to EAU that is associated with an elevated Th1 response. Suppression of CD4+ Th1 cells for the treatment of autoimmune disease is an attractive potential therapeutic approach. Nitric oxide (NO) has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. In this study, we investigated the potential role of NO as an immunoregulator to alter Th1/Th2 cytokine production, as well as to inhibit the interphotoreceptor retinoid binding protein (IRBP)-induced EAU, a CD4+ Th1 cell-mediated autoimmune disease. Injection of IRBP (100 microg) into two footpads resulted in severe EAU. The beginning peak of the disease was days 12 to 15 after immunization. Oral treatment with molsidomine (MSDM), a NO donor, began 24 h before IRBP immunization to the end of the experiments, which resulted in a significant inhibition of the disease by clinical and histopathological criteria. When MSDM was administered until day 21, a complete reduction of incidence and severity of EAU was observed. To investigate the cytokine alterations from Th1 to Th2 cytokines by MSDM, the cytokines were assayed in a culture medium of IRBP-stimulated inguinal lymphocytes. IRBP-immunized rats secreted a high concentration of IFN-gamma and a low concentration of IL-10. In contrast, MSDM treatment enhanced IL-10 secretion and tended to decrease IFN-gamma secretion. In conclusion, we show that the administration of NO suppresses EAU by altering the Th1/Th2 balance of inflammatory immune responses. We suggest that NO may be useful in the therapeutic control of autoimmune uveitis.     

A standardized root extract of Withania somnifera and its major constituent withanolide-A elicit humoral and cell-mediated immune responses by up regulation of Th1-dominant polarization in BALB/c mice

The effects of graded doses of a chemically standardized aqueous alcoholic (1:1) root extract (AGB) of Withania somnifera on the immune system of SRBC immunized BALB/c mice were investigated. Mice were administrated AGB orally for 15 days. AGB stimulated cell mediated immunity, IgM and IgG titers reaching peak value with 30 mg/kg b.wt. Flow cytometric analysis of lymphocyte surface markers of T cells (CD3(+), CD4(+) and CD8(+)) and B cells (CD19(+)) indicated prominent enhancement in proliferation and differentiation of lymphocytes. The extract selectively, induced type 1 immunity because it guided enhanced expression of T helper cells (Th)1 cytokines interferon (IFN)-gamma and interleukin (IL)-2 while Th2 cytokine IL-4 observed a moderate decline. Confirmation of Th1 polarization was obtained from augmented levels of IgG2a over IgG1 in the blood sera of AGB treated groups. Withanolide-A, a major constituent of AGB appeared responsible for Th1 skewing effect of the extract as it significantly increased the levels of Th1 cytokines, decreased moderately IL-4 and significantly restored the selective dexamethasone inhibition of Th1 cytokines in mouse splenocytes cultures in vitro. In addition, AGB also strongly activated macrophage functions ex vivo and in vitro indicated by enhanced secretion of nitrite, IL-12 and TNF-alpha. In contrast IL-10 remained unchanged again suggesting that AGB critically influenced Th1 profile of the cytokines. The studies suggested that AGB supports predominantly Th1 immunity with increase in macrophage functions. The standardized root extract of no toxicological consequences might therefore, find useful applications against the intracellular pathogens and in the management of immune suppressed diseases.     

Kinetics of intracytoplasmic Th1 and Th2 cytokine production assessed by flow cytometry following in vitro activation of peripheral blood mononuclear cells

BACKGROUND: Standard cytokine detection methods are unable to determine which cells are the producing cells. We report on the extent and under which conditions the multilabeling capability of flow cytometry (FCM) can bring new advances into the field. METHODS: Five different cytokines, interleukin-2 (IL-2), -4, -5, -10 and interferon-gamma (IFN-gamma), were assessed simultaneously under five ex vivo stimulation conditions in peripheral blood mononuclear cells from five healthy volunteers in a 5-day kinetic study. A second group of 35 volunteers was assessed for IFN-gamma and IL-2 production. RESULTS: This study showed that (a) intracytoplasmic cytokines were almost undetectable within unstimulated cells, (b) intracytoplasmic cytokines were detected only in CD69(+) T lymphocytes, and (c) intracytoplasmic IL-2 and IFN-gamma were dramatically upregulated after stimulation with phorbol myristate acetate (PMA)-ionomycin in a biphasic response or with PMA-phytohemagglutinin (one major peak only at 18 h) but to a lesser extent with other stimuli such as monoclonal antibodies. Th2 cytokines were detected at a later time point and at lower levels. PMA/ionomycin stimulation after 4 h and 18 h of culture in 35 other volunteers individualized several subgroups according to the frequency of IFN-gamma- or IL-2-producing cells--IFN-gamma delayed producers (n = 10/35), IFN-gamma low producers (n = 8/35), and IL-2 delayed producers (n = 16/35)--as opposed to IFN-gamma or IL-2 normal producers. CONCLUSIONS: FCM appears to be a good tool to examine cell cytokine status in pathology (allergy, autoimmune disease, etc.) provided that optimal stimulation conditions and multiple time-point cultures are used. It also seems to be a relevant method to define new Th subsets further.     

CD4+ subset regulation in viral infection. Preferential activation of Th2 cells during progression of retrovirus-induced immunodeficiency in mice.

Progressive lymphoproliferation and increasingly severe immunodeficiency are prominent features of a syndrome, designated mouse AIDS, which develops in susceptible strains of mice infected with the mixture of murine leukemia viruses, termed LP-BM5. Development of splenomegaly and lymphadenopathy, caused primarily by increases in B cell immunoblasts, requires the presence of CD4+ T cells and is assumed to be mediated by lymphokines produced by these cells inasmuch as progression of disease is markedly inhibited by treatment of infected mice with cyclosporin A. Studies of spleen cells from infected mice revealed spontaneous production of cytokines (IFN-gamma, IL-2, IL-4, IL-5, and IL-10) characteristic of Th0 (or a mixture of Th1 and Th2) T helper cells at 1 wk after infection. At later times, IFN-gamma and IL-2, characteristic products of Th1 helper clones, were expressed poorly, either spontaneously or after stimulation of cells with Con A. In contrast, IL-4, IL-5, IL-6, and IL-10, cytokines typically synthesized by Th2 cells, were produced in response to Con A or spontaneously through 18 wk post-infection. Increased serum IgE levels and enhanced IL-10 mRNA expression were consistent with expression of Th2 cytokines at biologically significant levels in vivo. Selective depletion of T cell subsets before stimulation with Con A showed that CD4+ T cells were the primary source of IL-2, IL-4, IL-10, and, to a lesser extent, IFN-gamma in spleens and lymph nodes of normal or infected mice. These results suggest that persistent activation of CD4+ T cells with the lymphokine profile of Th2 helper clones is responsible for chronic B cell stimulation, down-regulation of Th1 cytokines, and impaired CD8+ T cell function in mouse AIDS. This provides the first demonstration that, like many parasitic infections, viruses encoding potent antigenic stimuli can markedly affect the balance of Th subset expression.     

Single cell analysis reveals that IL-4 receptor/Stat6 signaling is not required for the in vivo or in vitro development of CD4+ lymphocytes with a Th2 cytokine profile

The concept that IL-4 is the primary signal for Th2 lymphocyte differentiation has recently been put in doubt by studies in which the production of Th2-associated cytokines was detected in mice deficient in IL-4 synthesis or IL-4R triggering. In this study, we formally demonstrate by single cell analysis that CD4+ lymphocytes with a classical Th2 phenotype (IL-4+, IL-5+, IFN-gamma-, IL-2-) develop in significant numbers in helminth-infected mice deficient in either IL-4R alpha-chain or Stat6. While an expanded population of Th1 (IL-4-, IL-5-, IFN-gamma+, IL-2+) lymphocytes was observed in the same animals, surprisingly, cells with a mixed Th0 cytokine pattern were rare. The cytokine production phenotypes of the Th1 and Th2 subpopulations generated in infected Stat6-deficient mice were unaffected by in vitro neutralization of endogenous IL-4 or IFN-gamma. Nevertheless, while addition of exogenous rIL-12 resulted in transitory IFN-gamma production by Th2 lymphocytes from both wild-type and Stat6-deficient mice, IL-4 synthesis was preserved in the former, but temporarily ablated in the latter cells. Importantly, IL-4+ IFN-gamma- and IL-4- IFN-gamma+ populations similar to those arising in helminth-infected Stat6-deficient mice could also be generated in vitro by repetitive polyclonal stimulation of CD4+CD62Lhigh lymphocytes from uninfected mice of the same strain. Together, the results of these single cell analysis experiments demonstrate that IL-4R/Stat6 signaling, while influencing the final frequency of Th2 lymphocytes, is not essential for Th2 cell development, and suggest that this pathway has a previously unrecognized function in stabilizing Th2 populations once they have emerged.     

CC chemokine receptor 9 expression defines a subset of peripheral blood lymphocytes with mucosal T cell phenotype and Th1 or T-regulatory 1 cytokine profile

The chemokine receptor CCR9 is expressed on most small intestinal lamina propria and intraepithelial lymphocytes and on a small subset of peripheral blood lymphocytes. CCR9-expressing lymphocytes may play an important role in small bowel immunity and inflammation. We studied the phenotype and functional characteristics of CCR9(+) lymphocytes in blood from normal donors. A subset of CCR9(+) T cells have a phenotype of activated cells and constitutively express the costimulatory molecules CD40L and OX-40. In contrast to CCR9(-), CCR9(+)CD4(+) peripheral blood T cells proliferate to anti-CD3 or anti-CD2 stimulation and produce high levels of IFN-gamma and IL-10. IL-10-producing cells were exclusively detected within the CCR9(+) subset of CD4(+) T cells by intracellular staining and were distinct from IL-2- and IFN-gamma-producing cells. Moreover, memory CCR9(+)CD4(+) lymphocytes respond to CD2 stimulation with proliferation and IFN-gamma/IL-10 production, whereas memory CCR9(-)CD4(+) cells were unresponsive. In addition, memory CCR9(+)CD4(+) T cells support Ig production by cocultured CD19(+) B cells in the absence of prior T cell activation or addition of exogenous cytokines. Our data show that the memory subset of circulating CCR9(+)CD4(+) T cells has characteristics of mucosal T lymphocytes and contains cells with either Th1 or T-regulatory 1 cytokine profiles. Studies on the cytokine profile and Ag specificity of this cell subset could provide important insight into small intestinal immune-mediated diseases and oral tolerance in humans.