Regulation of IL-17 in human CCR6+ effector memory T cells

IL-17-secreting T cells represent a distinct CD4(+) effector T cell lineage (Th17) that appears to be essential in the pathogenesis of numerous inflammatory and autoimmune diseases. Although extensively studied in the murine system, human Th17 cells have not been well characterized. In this study, we identify CD4(+)CD45RO(+)CCR7(-)CCR6(+) effector memory T cells as the principal IL-17-secreting T cells. Human Th17 cells have a unique cytokine profile because the majority coexpress TNF-alpha but not IL-6 and a minor subset express IL-17 with IL-22 or IL-17 and IFN-gamma. We demonstrate that the cytokines that promote the differentiation of human naive T cells into IL-17-secreting cells regulate IL-17 production by memory T cells. IL-1beta alone or in association with IL-23 and IL-6 markedly increase IL-17(+) CCR6(+) memory T cells and induce IL-17 production in CCR6(-) memory T cells. We also show that T cell activation induces Foxp3 expression in T cells and that the balance between the percentage of Foxp3(+) and IL-17(+) T cells is inversely influenced by the cytokine environment. These studies suggest that the cytokine environment may play a critical role in the expansion of memory T cells in chronic autoimmune diseases.     

CCR4 participation in Th type 1 (mycobacterial) and Th type 2 (schistosomal) anamnestic pulmonary granulomatous responses

CCR4 is purported to be a Th type 2 (Th2) cell-biased receptor but its functional role is unclear. Recent studies suggest that chemokine receptor expression and function are more complex in vivo and raise doubts regarding restricted CCR4 expression by Th2 cells. To address these issues, we analyzed the role of CCR4 in highly polarized models of Th type 1 (Th1) and Th2 cell-mediated pulmonary granulomas, respectively, elicited by i.v. challenge of primed mice with either mycobacterial purified protein derivative or schistosomal egg Ag-coated beads. CCR4 agonists were expressed during both responses, correlating with a shift of CCR4+ CD4+ T cells from blood to lungs. CCL22 dominated in draining nodes during the Th1 response. Analysis of CD4+ effector T cells revealed CCR4 expression and CCR4-mediated chemotaxis by both IFN-gamma and IL-4 producers. Studies of CCR4 knockout (CCR4(-/-)) mice showed partial impairment of the local type-2 cytokine response and surprisingly strong impairment of the Th1 response with abrogated IFN-gamma production during secondary but not primary challenge. Adoptive transfer indicated CCR4(-/-)CD4+ Th1 cell function was defective but this could not be reconstituted with wild-type (CCR4(+/+)) CD4+ T cells indicating involvement of another CCR4+ population. Coculture of CCR4(+/+)CD4+ T cells and CCR4(-/-) dendritic cells revealed intact IL-2 but impaired IFN-gamma production, pointing to a role for CCR4+ dendritic cells in effector cell expression. Therefore, CCR4 is not Th2-restricted and was required for sustenance and expression of the Th1 effector/memory response to mycobacterial Ags.     

Human T cells that are able to produce IL-17 express the chemokine receptor CCR6

Some pathways of T cell differentiation are associated with characteristic patterns of chemokine receptor expression. A new lineage of effector/memory CD4+ T cells has been identified whose signature products are IL-17 cytokines and whose differentiation requires the nuclear receptor, RORgammat. These Th17 cells are critical effectors in mouse models of autoimmune disease. We have analyzed the association between chemokine receptor expression and IL-17 production for human T cells. Activating cord blood (naive) CD4+ T cells under conditions driving Th17 differentiation led to preferential induction of CCR6, CCR9, and CXCR6. Despite these data, we found no strong correlation between the production of IL-17 and expression of CCR9 or CXCR6. By contrast, our analyses revealed that virtually all IL-17-producing CD4+ T cells, either made in our in vitro cultures or found in peripheral blood, expressed CCR6, a receptor found on approximately 50% of CD4+ memory PBL. Compared with CD4+CD45RO+CCR6- cells, CD4+CD45RO+CCR6+ cells contained at least 100-fold more IL-17A mRNA and secreted 100-fold more IL-17 protein. The CCR6+ cells showed a similar enrichment in mRNA for RORgammat. CCR6 was likewise expressed on all IL-17-producing CD8+ PBL. CCR6 has been associated with the trafficking of T, B, and dendritic cells to epithelial sites, but has not been linked to a specific T cell phenotype. Our data reveal a fundamental feature of IL-17-producing human T cells and a novel role for CCR6, suggesting both new directions for investigating IL-17-related immune responses and possible targets for preventing inflammatory injury.     

Phenotypic and functional characterization of CD4 T cells expressing killer Ig-like receptors

Killer Ig-like receptors (KIR) are commonly found on human NK cells, gammadelta T cells, and CD8 T cells. Although KIR(+) CD4 T cells are found in certain patients, their prevalence in healthy donors is controversial. We now provide definitive proof that such cells are present in most individuals, and report on their frequency, surface phenotype, cytokine profile, and Ag specificity. The number of KIR(+) CD4 T cells detected in peripheral blood increased with age. In contrast with regular KIR(-) CD4 T cells, the majority of KIR(+) CD4 T cells lacked surface expression of CD27, CD28, CCR4, and CCR7, but did express CD57 and 2B4. In addition, KIR were detected on approximately one-tenth of CD28(-) and CD57(+) memory CD4 T cells. In line with the absence of the Th2 marker CCR4, the KIR(+) CD4 cells produced mainly IFN-gamma and little IL-4, IL-10, or IL-17 upon TCR triggering. Furthermore, the KIR(+) population contained cells that responded to recall Ags in an HLA class II-restricted fashion. Together, our data indicate that KIR-expressing CD4 T cells are predominantly HLA class II-restricted effector memory Th1 cells, and that a significant, previously unrecognized fraction of effector memory Th1 cells expresses KIR.     

Dominant role for TL1A/DR3 pathway in IL-12 plus IL-18-induced IFN-gamma production by peripheral blood and mucosal CCR9+ T lymphocytes

The TNF-like cytokine TL1A augments IFN-gamma production by anti-CD3 plus anti-CD28 and IL-12/IL-18-stimulated peripheral blood (PB) T cells. However, only a small subset of PB T cells respond to TL1A stimulation with IFN-gamma production. PB CCR9+ T cells represent a small subset of circulating T cells with mucosal T cell characteristics and a Th1/Tr1 cytokine profile. In the current study, we show that TL1A enhanced IFN-gamma production by TCR- or CD2/CD28-stimulated CCR9(+)CD4+ PB T cells. However, TL1A had the most pronounced effect on augmenting IFN-gamma production by IL-12/IL-18-primed CCR9(+)CD4+ PB T cells. TL1A enhanced both the percentage and the mean fluorescence intensity of IFN-gamma in CCR9(+)CD4+ T cells as assessed by intracellular cytokine staining. IL-12 plus IL-18 up-regulated DR3 expression in CCR9(+)CD4+ T cells but had negligible effect on CCR9(-)CD4+ T cells. CCR9(+)CD4+ T cells isolated from the small intestine showed a 37- to 105-fold enhancement of IFN-gamma production when TL1A was added to the IL-12/IL18 cytokine combination. Cell membrane-expressed TL1A was preferentially expressed in CCR9(+)CD4+ PB T cells, and a blocking anti-TL1A mAb inhibited IFN-gamma production by cytokine-primed CCR9(+)CD4+ T cells by approximately 50%. Our data show that the TL1A/DR3 pathway plays a dominant role in the ultimate level of cytokine-induced IFN-gamma production by CCR9+ mucosal and gut-homing PB T cells and could play an important role in Th1-mediated intestinal diseases, such as Crohn's disease, where increased expression of IL-12, IL-18, TL1A, and DR3 converge in the inflamed intestinal mucosa.     

Stepwise differentiation of CD4 memory T cells defined by expression of CCR7 and CD27

To study the steps in the differentiation of human memory CD4 T cells, we characterized the functional and lineage relationships of three distinct memory CD4 subpopulations distinguished by their expression of the cysteine chemokine receptor CCR7 and the TNFR family member CD27. Using the combination of these phenotypic markers, three populations were defined: the CCR7+CD27+, the CCR7-CD27+, and the CCR7-CD27- population. In vitro stimulation led to a stepwise differentiation from naive to CCR7+CD27+ to CCR7-CD27+ to CCR7-CD27-. Telomere length in these subsets differed significantly (CCR7+CD27+ > CCR7-CD27+ > CCR7-CD27-), suggesting that these subsets constituted a differentiative pathway with progressive telomere shortening reflecting antecedent in vivo proliferation. The in vitro proliferative response of these populations declined, and their susceptibility to apoptosis increased progressively along this differentiation pathway. Cytokine secretion showed a differential functional capacity of these subsets. High production of IL-10 was only observed in CCR7+CD27+, whereas IFN-gamma was produced by CCR7-CD27+ and to a slightly lesser extent by CCR7-CD27- T cells. IL-4 secretion was predominantly conducted by CCR7-CD27- memory CD4 T cells. Thus, by using both CCR7 and CD27, distinct maturational stages of CD4 memory T cells with different functional activities were defined.     

Preferential HIV Infection of CCR6+ Th17 Cells Is Associated with Higher Levels of Virus Receptor Expression and Lack of CCR5 Ligands

Th17 cells are enriched in the gut mucosa and play a critical role in maintenance of the mucosal barrier and host defense against extracellular bacteria and fungal infections. During chronic human immunodeficiency virus (HIV) infection, Th17 cells were more depleted compared to Th1 cells, even when the patients had low or undetectable viremia. To investigate the differential effects of HIV infection on Th17 and Th1 cells, a culture system was used in which CCR6⁺ CD4⁺ T cells were sorted from healthy human peripheral blood and activated in the presence of interleukin 1β (IL-1β) and IL-23 to drive expansion of Th17 cells while maintaining Th1 cells. HIV infection of these cultures had minimal effects on Th1 cells but caused depletion of Th17 cells. Th17 loss correlated with greater levels of virus-infected cells and cell death. In identifying cellular factors contributing to higher susceptibility of Th17 cells to HIV, we compared Th17-enriched CCR6⁺ and Th17-depleted CCR6⁻ CD4 T cell cultures and noted that Th17-enriched CCR6⁺ cells expressed higher levels of α4β7 and bound HIV envelope in an α4β7-dependent manner. The cells also had greater expression of CD4 and CXCR4, but not CCR5, than CCR6⁻ cells. Moreover, unlike Th1 cells, Th17 cells produced little CCR5 ligand, and transfection with one of the CCR5 ligands, MIP-1β (CCL4), increased their resistance against HIV. These results indicate that features unique to Th17 cells, including higher expression of HIV receptors and lack of autocrine CCR5 ligands, are associated with enhanced permissiveness of these cells to HIV.     

CD127+CCR5+CD38+++ CD4+ Th1 effector cells are an early component of the primary immune response to vaccinia virus and precede development of interleukin-2+ memory CD4+ T cells

The stages of development of human antigen-specific CD4+ T cells responding to viral infection and their differentiation into long-term memory cells are not well understood. The inoculation of healthy adults with vaccinia virus presents an opportunity to study these events intensively. Between days 11 and 14 postinoculation, there was a peak of proliferating CCR5+CD38+++ CD4+ effector cells which contained the cytotoxic granule marker T-cell intracellular antigen 1 and included gamma interferon (IFN-gamma)-producing vaccinia virus-specific CD4+ T cells. The majority of these initial vaccinia virus-specific CD4+ T cells were CD127+ and produced interleukin-2 (IL-2) but not CTLA-4 in response to restimulation in vitro. Between days 14 and 21, there was a switch from IFN-gamma and IL-2 coexpression to IL-2 production only, coinciding with a resting phenotype and an increased in vitro proliferation response. The early CCR5+CD38+++ vaccinia virus-specific CD4+ T cells were similar to our previous observations of human immunodeficiency virus (HIV)-specific CD4+ T cells in primary HIV type 1 (HIV-1) infection, but the vaccinia virus-specific cells expressed much more CD127 and IL-2 than we previously found in their HIV-specific counterparts. The current study provides important information on the differentiation of IL-2+ vaccinia virus-specific memory cells, allowing further study of antiviral effector CD4+ T cells in healthy adults and their dysfunction in HIV-1 infection.     

Increased frequency of CCR4+ and CCR6+ memory T-cells including CCR7+CD45RAmed very early memory cells in granulomatosis with polyangiitis (Wegener's)

Introduction

Chemokine receptors play an important role in mediating the recruitment of T cells to inflammatory sites. Previously, small proportions of circulating Th1-type CCR5+ and Th2-type CCR3+ cells have been shown in granulomatosis with polyangiitis (GPA). Wondering to what extent CCR4 and CCR6 expression could also be implicated in T cell recruitment to inflamed sites in GPA, we investigated the expression of CCR4 and CCR6 on T cells and its association with T cell diversity and polarization.

Methods

Multicolor flow cytometry was used to analyze CCR4, CCR6, and intracellular cytokine expression of T cells from whole blood of GPA-patients (n = 26) and healthy controls (n = 20). CCR7 and CD45RA were included for phenotypic characterization.

Results

We found a significant increase in the percentages of circulating CCR4+ and CCR6+ cells within the total CD4+ T cell population in GPA. In contrast, there was no difference in the percentages of CD8+CCR4+ and CD8+CCR6+ T cells between GPA and healthy controls. CCR4 and CCR6 expression was largely confined to central (T_CM) and effector memory T cells (T_EM, T_EMRA). A significant increase in the frequency of CCR4+ and CCR6+ T_EMRA and CCR6+ T_CM was shown in GPA. Of note, we could dissect CCR4 and CCR6 expressing CCR7+CD45RA^med very early memory T cells (T_VEM) from genuine CCR7+CD45RA^high naïve T cells lacking CCR4 and CCR6 expression for peripheral tissue-migration within the CCR7+CD45RA+ compartment. The frequencies of CCR4+ and CCR6+ T_VEM were also significantly increased in GPA. An increased percentage of IL-17+ and IL-22+ cells was detected in the CCR6+ cell subsets and IL-4+ cells in the CRR4+ cell subset when compared with CD4+ cells lacking CCR4 and CCR6 expression.

Conclusions

Increased frequencies of circulating CCR4+ and CCR6+ memory T cell subsets including hitherto unreported T_VEM suggest persistent T cell activation with the accumulation of CCR4+ and CCR6+ cells in GPA. CCR4 and CCR6 could be involved in the recruitment of T cells including cytokine-producing subsets to inflamed sites in GPA.     

Functional expression of the chemokine receptor CCR5 on virus epitope-specific memory and effector CD8+ T cells

Because the chemokine receptor CCR5 is expressed on Th1 CD4(+) cells, it is important to investigate the expression and function of this receptor on other T cells involved in Th1 immune responses, such as Ag-specific CD8(+) T cells, which to date have been only partially characterized. Therefore, we analyzed the expression and function of CCR5 on virus-specific CD8+ T cells identified by HLA class I tetramers. Multicolor flow cytometry analysis demonstrated that CCR5 is expressed on memory (CD28+CD45RA-) and effector (CD28-CD45RA- and CD28-CD45RA+) CD8+ T cells but not on naive (CD28+CD45RA+) CD8+ T cells. CCR5 expression was much lower on two effector CD8+ T cells than on memory CD8+ T cells. Analysis of CCR7 and CCR5 expression on the different types of CD8+ T cells showed that memory CD8+ T cells have three phenotypic subsets, CCR5+CCR7-, CCR5+CCR7+, and CCR5-CCR7+, while naive and effector CD8+ T cells have CCR5-CCR7+ and CCR5+CCR7- phenotypes, respectively. These results suggest the following sequence for differentiation of memory CD8+ T cells: CCR5-CCR7+-->CCR5+CCR7+-->CCR5+CCR7-. CCR5+CD8+ T cells effectively migrated in response to RANTES, suggesting that CCR5 plays a critical role in the migration of Ag-specific effector and differentiated memory CD8+ T cells to inflammatory tissues and secondary lymphoid tissues. This is in contrast to CCR7, which functions as a homing receptor in migration of naive and memory CD8+ T cells to secondary lymphoid tissues.     

Human CCR4+ CCR6+ Th17 cells suppress autologous CD8+ T cell responses

The role of Th17 cells in cancer patients remains unclear and controversial. In this study, we have analyzed the phenotype of in vitro primed Th17 cells and further characterized their function on the basis of CCR4 and CCR6 expression. We show a novel function for a subset of IL-17-secreting CD4(+) T cells, namely, CCR4(+)CCR6(+)Th17 cells. When cultured together, CCR4(+)CCR6(+)Th17 cells suppressed the lytic function, proliferation, and cytokine secretion of both Ag-specific and CD3/CD28/CD2-stimulated autologous CD8(+) T cells. In contrast, CCR4(-)CCR6(+) CD4(+) T cells, which also secrete IL-17, did not affect the CD8(+) T cells. Suppression of CD8(+) T cells by CCR4(+)CCR6(+)Th17 cells was partially dependent on TGF-β, because neutralization of TGF-β in cocultures reversed their suppressor function. In addition, we also found an increase in the frequency of CCR4(+)CCR6(+), but not CCR4(-)CCR6(+) Th17 cells in peripheral blood of hepatocellular carcinoma patients. Our study not only underlies the importance of analysis of subsets within Th17 cells to understand their function, but also suggests Th17 cells as yet another immune evasion mechanism in hepatocellular carcinoma. This has important implications when studying the mechanisms of carcinogenesis, as well as designing effective immunotherapy protocols for patients with cancer.     

Coexpression of CCR5 and IL-2 in human genital but not blood T cells: implications for the ontogeny of the CCR5+ Th1 phenotype.

Memory T cells that home to inflamed tissues typically express the beta-chemokine receptor CCR5 and exhibit a Th1 cytokine profile. The migration of these cells into the genital tract following antigenic exposure has particular relevance to acquisition of HIV-1 infection, because CCR5 functions as the coreceptor for most sexually transmitted HIV-1 strains. We recently established methodology to purify and culture mononuclear cells from the female reproductive tract, and here we analyzed the phenotype, CCR5 expression, and cytokine production of cervicovaginal T cells in up to 16 donors. The proportion of mucosal T cells expressing CCR5 was markedly expanded as compared with peripheral blood (mean 88% vs 24% in 13 donors), but the receptor density on individual CCR5+ T cells was only slightly increased (mean 5837 vs 4191 MEPE (molecules of equivalent PE) units in 6 of 7 donors). Intracellular costaining for IL-2, IFN-gamma, IL-4, and IL-5 revealed a Th1-type pattern in cervical T cells, with significantly higher percentages of IL-2- and IFN-gamma-producing T cells in the mucosa than in blood (mean 67% vs 29%). Coexpression of surface CCR5 with intracellular IL-2 and IFN-gamma was observed only among T cells in the mucosa, but not among those in circulation. Thus, we postulate that T cell homing to the genital mucosa leads to differentiation into the combined CCR5+ Th1 phenotype. Moreover, the predominance of CCR5+ Th1-type T cells in normal cervical mucosa provides targets accessible for the efficient transmission of macrophage-tropic HIV-1 variants in women following sexual exposure.     

Chemokine receptor CCR4 on CD4+ T cells in juvenile rheumatoid arthritis synovial fluid defines a subset of cells with increased IL-4:IFN-gamma mRNA ratios

To understand the mechanisms that promote recruitment and survival of T cells within the pediatric inflamed joint, we have studied the expression of CCR4 and CCR5 on synovial fluid T cells and matched peripheral blood samples from juvenile rheumatoid arthritis (JRA) patients using three-color flow cytometric analysis. Thymus- and activation-regulated chemokine and macrophage-derived chemokine, ligands for CCR4, were measured by ELISA in JRA synovial fluid, JRA plasma, adult rheumatoid arthritis synovial fluid, and normal plasma. IL-4 and IFN-gamma mRNA production was assessed in CD4+/CCR4+ and CD4+/CCR4(-) cell subsets. We found accumulations of both CCR4+ and CCR5+ T cells in JRA synovial fluids and a correlation for increased numbers of CCR4+ T cells in samples collected early in the disease process. Thymus- and activation-regulated chemokine was detected in JRA synovial fluid and plasma samples, but not in adult rheumatoid arthritis synovial fluid or control plasma. Macrophage-derived chemokine was present in all samples. CD4+/CCR4+ synovial lymphocytes produced more IL-4 and less IFN-gamma than CD4+/CCR4(-) cells. These findings suggest that CCR4+ T cells in the JRA joint may function early in disease in an anti-inflammatory capacity through the production of type 2 cytokines and may play a role in determining disease phenotype.     

Selective activation of peripheral blood T cell subsets by endotoxin infusion in healthy human subjects corresponds to differential chemokine activation

Although activation of human innate immunity after endotoxin administration is well established, in vivo endotoxin effects on human T cell responses are not well understood. Most naive human T cells do not express receptors for LPS, but can respond to endotoxin-induced mediators such as chemokines. In this study, we characterized the in vivo response of peripheral human T cell subsets to endotoxin infusion by assessing alterations in isolated T cells expressing different phenotypes, intracellular cytokines, and systemic chemokines concentration, which may influence these indirect T cell responses. Endotoxin administration to healthy subjects produced T cell activation as confirmed by a 20% increase in intracellular IL-2, as well as increased CD28 and IL-2R alpha-chain (CD25) expression. Endotoxin induced indirect activation of T cells was highly selective among the T cell subpopulations. Increased IL-2 production (36.0 +/- 3.7 to 53.2 +/- 4.1) vs decreased IFN-gamma production (33.8 +/- 4.2 to 19.1 +/- 3.2) indicated selective Th1 activation. Th2 produced IL-13 was minimally increased. Differentially altered chemokine receptor expression also indicated selective T cell subset activation and migration. CXCR3+ and CCR5+ expressing Th1 cells were decreased (CXCR3 44.6 +/- 3.2 to 33.3 +/- 4.6 and CCR5 24.8 +/- 2.3 to 12 +/- 1.4), whereas plasma levels of their chemokine ligands IFN-gamma-inducible protein 10 and MIP-1alpha were increased (61.4 +/- 13.9 to 1103.7 +/- 274.5 and 22.8 +/- 6.2 to 55.7 +/- 9.5, respectively). In contrast, CCR4+ and CCR3 (Th2) proportions increased or remained unchanged whereas their ligands, eotaxin and the thymus and activation-regulated chemokine TARC, were unchanged. The data indicate selective activation among Th1 subpopulations, as well as differential Th1/Th2 activation, which is consistent with a selective induction of Th1 and Th2 chemokine ligands.     

Human effector memory CD4+ T cells directly recognize allogeneic endothelial cells in vitro and in vivo

The frequency of circulating alloreactive human memory T cells correlates with allograft rejection. Memory T cells may be divided into effector memory (T(EM)) and central memory (T(CM)) cell subsets, but their specific roles in allograft rejection are unknown. We report that CD4+ T(EM) (CD45RO+ CCR7- CD62L-) can be adoptively transferred readily into C.B-17 SCID/bg mice and mediate the destruction of human endothelial cells (EC) in vascularized human skin grafts allogeneic to the T cell donor. In contrast, CD4+ T(CM) (CD45RO+ CCR7+ CD62L+) are inefficiently transferred and do not mediate EC injury. In vitro, CD4+ T(EM) secrete more IFN-gamma within 48 h in response to allogeneic ECs than do T(CM). In contrast, T(EM) and T(CM) secrete comparable amounts of IFN-gamma in response to allogeneic monocytes (Mo). In the same cultures, both T(EM) and T(CM) produce IL-2 and proliferate in response to IFN-gamma-treated allogeneic human EC or Mo, but T(CM) respond more vigorously in both assays. Blockade of LFA-3 strongly inhibits both IL-2 and IFN-gamma secretion by CD4+ T(EM) cultured with allogeneic EC but only minimally inhibits responses to allogeneic Mo. Blockade of CD80 and CD86 strongly inhibits IL-2 but not IFN-gamma production by in response to allogeneic EC or Mo. Transduction of EC to express B7-2 enhances allogeneic T(EM) production of IL-2 but not IFN-gamma. We conclude that human CD4+ T(EM) directly recognize and respond to allogeneic EC in vitro by secreting IFN-gamma and that this response depends on CD2 but not CD28. Consistent with EC activation of effector functions, human CD4+ T(EM) can mediate allogeneic EC injury in vivo.     

CCR8 is expressed by antigen-elicited, IL-10-producing CD4+CD25+ T cells, which regulate Th2-mediated granuloma formation in mice

CCR8 was initially described as a Th2 cell-restricted receptor, but this has not been fully tested in vivo. The present study used ex vivo and in vivo approaches to examine the distribution and functional significance of CCR8 among CD4+ T cells. Populations of cytokine-secreting CD4+ T cells were generated in primed mice with Th1 or Th2 cell-mediated pulmonary granulomas, respectively elicited by i.v. challenge with either Mycobacteria bovis purified protein derivative- or Schistosoma mansoni egg Ag (SEA)-coated beads. Cytokine-producing CD4+ T cells were isolated from Ag-stimulated draining lymph node cultures by positive selection. Quantitative analysis of cytokine mRNA indicated enriched populations of IFN-gamma-, IL-4-, and IL-10-producing cells. Analysis of chemokine receptor mRNA indicated that IL-10+ cells selectively expressed CCR8 in the SEA bead-elicited type 2 response. The IL-10+CCR8+ populations were CD25+ and CD44+ but lacked enhanced Foxp3 expression. Adoptive transfer to naive recipients indicated that IL-10+ T cells alone could not transfer type 2 inflammation. Analysis of SEA bead-challenged CCR8-/- mice indicated significantly impaired IL-10 production as well as reductions in granuloma eosinophils. Adoptive transfer of CD4+CCR8+/+ T cells corrected cytokine and inflammation defects, but the granuloma eosinophil recruitment defect persisted when donor cells were depleted of IL-10+ cells. Accordingly, local IL-10 production correlated with CCR8 ligand (CCL1) expression and the appearance of CCR8+ cells in granulomatous lungs. Thus, IL-10-producing, CCR8+CD4+CD25+CD44+ T cells are generated during SEA challenge, which augment the Th2-mediated eosinophil-rich response to the parasite Ags.     

Human Th17 cells share major trafficking receptors with both polarized effector T cells and FOXP3+ regulatory T cells

It is a question of interest whether Th17 cells express trafficking receptors unique to this Th cell lineage and migrate specifically to certain tissue sites. We found several Th17 cell subsets at different developing stages in a human secondary lymphoid organ (tonsils) and adult, but not in neonatal, blood. These Th17 cell subsets include a novel in vivo-stimulated tonsil IL17+ T cell subset detected without any artificial stimulation in vitro. We investigated in depth the trafficking receptor phenotype of the Th17 cell subsets in tonsils and adult blood. The developing Th17 cells in tonsils highly expressed both Th1- (CCR2, CXCR3, CCR5, and CXCR6) and Th2-associated (CCR4) trafficking receptors. Moreover, Th17 cells share major non-lymphoid tissue trafficking receptors, such as CCR4, CCR5, CCR6, CXCR3, and CXCR6, with FOXP3+ T regulatory cells. In addition, many Th17 cells express homeostatic chemokine receptors (CD62L, CCR6, CCR7, CXCR4, and CXCR5) implicated in T cell migration to and within lymphoid tissues. Expression of CCR6 and CCR4 by some Th17 cells is not a feature unique to Th17 cells but shared with FOXP3+ T cells. Interestingly, the IL17+IFN-gamma+ Th17 cells have the features of both IL17-IFN-gamma+ Th1 and IL17+IFN-gamma- Th17 cells in expression of trafficking receptors. Taken together, our results revealed that Th17 cells are highly heterogeneous, in terms of trafficking receptors, and programmed to share major trafficking receptors with other T cell lineages. These findings have important implications in their distribution in the human body in relation to other regulatory T cell subsets.     

In vivo differentiated cytokine-producing CD4(+) T cells express functional CCR7

Chemokines and their receptors fulfill specialized roles in inflammation and under homeostatic conditions. CCR7 and its ligands, CCL19 and CCL21, are involved in lymphocyte recirculation through secondary lymphoid organs and additionally navigate lymphocytes into distinct tissue compartments. The role of CCR7 in the migration of polarized T effector/memory cell subsets in vivo is still poorly understood. We therefore analyzed murine and human CD4(+) cytokine-producing cells developed in vivo for their chemotactic reactivity to CCR7 ligands. The responses of cells producing cytokines, such as IFN-gamma, IL-4, and IL-10, as well as of subsets defined by memory or activation markers were comparable to that of naive CD4(+) cells, with slightly lower reactivity in cells expressing IL-10 or CD69. This indicates that CCR7 ligands are able to attract naive as well as the vast majority of activated and effector/memory T cell stages. Chemotactic reactivity of these cells toward CCL21 was absent in CCR7-deficient cells, proving that effector cells do not use alternative receptors for this chemokine. Th1 cells generated from CCR7(-/-) mice failed to enter lymph nodes and Peyer's patches, but did enter a site of inflammation. These findings indicate that CD4(+) cells producing effector cytokines upon stimulation retain the capacity to recirculate through lymphoid tissues via CCR7.