Martin R. Hodge

T Follicular helper cells express a distinctive transcriptional profile, reflecting their role as non-Th1/Th2 effector cells that provide help for B cells

Effector T cell responses have long been viewed in the context of the Th1/Th2 paradigm. Recently, a third major subset of nonpolarized effector T cells that provides help to B cells has been identified. These T cells, termed T follicular helper (TFH) cells, home to the B cell areas of secondary lymphoid tissue, through interactions mediated via the chemokine receptor CXCR5 and its ligand CXCL13. Affymetrix microarrays were used to identify transcription factors, cytokines, and cell surface molecules that underlie the differentiation pathways and functional properties of the TFH subset. The transcriptional profile of human CXCR5+ TFH cells was compared with that of Th1 and Th2 cells, which enabled the identification of numerous genes expressed preferentially by TFH cells, over the other effector subsets. Certain TFH genes were also expressed by B cells and thus appear to be particularly relevant for humoral immunity. Abs were used to confirm the expression of several factors. In particular, CD84 and CD200, the cytokine IL-21, and the transcription factor BCL6 were all strongly associated with TFH cells. Gene microarrays reveal a highly distinctive transcriptional profile for a third subset of effector T cells that differs markedly from Th1 and Th2 cells.

Unique subpopulations of CD56+ NK and NK-T peripheral blood lymphocytes identified by chemokine receptor expression repertoire.

CD56, an adhesion molecule closely related to neual cell adhesion molecule, is an immunophenotypic marker for several unique populations of PBLs. Although CD56+ cells derive from multiple lymphocyte lineages, they share a role in immunosurveillance and antitumor responses. We have studied the chemokine receptor expression patterns and functional migratory responses of three distinct CD56+ populations from human peripheral blood. NK-T cells were found to differ greatly from NK cells, and CD16+ NK cells from CD16− NK cells. CD16+ NK cells were the predominant population responding to IL-8 and fractalkine, whereas NK-T cells were the predominant population responding to the CCR5 ligand macrophage-inflammatory protein-1β. CD16− NK cells were the only CD56+ population that uniformly expressed trafficking molecules necessary for homing into secondary lymphoid organs through high endothelial venule. These findings describe a diverse population of cells that may have trafficking patterns entirely different from each other, and from other lymphocyte types.

Expression of the chemokine receptors CCR4, CCR5, and CXCR3 by human tissue-infiltrating lymphocytes.

Differential expression of adhesion molecules and chemokine receptors has been useful for identification of peripheral blood memory lymphocyte subsets with distinct tissue and microenvironmental tropisms. Expression of CCR4 by circulating memory CD4(+) lymphocytes is associated with cutaneous and other systemic populations while expression of CCR9 is associated with a small intestine-homing subset. CCR5 and CXCR3 are also expressed by discrete memory CD4(+) populations in blood, as well as by tissue-infiltrating lymphocytes from a number of sites. To characterize the similarities and differences among tissue-infiltrating lymphocytes, and to shed light on the specialization of lymphocyte subsets that mediate inflammation and immune surveillance in particular tissues, we have examined the expression of CCR4, CXCR3, and CCR5 on CD4(+) lymphocytes directly isolated from a wide variety of normal and inflamed tissues. Extra-lymphoid tissues contained only memory lymphocytes, many of which were activated (CD69(+)). As predicted by classical studies, skin lymphocytes were enriched in CLA expression whereas intestinal lymphocytes were enriched in alpha(4)beta(7) expression. CCR4 was expressed at high levels by skin-infiltrating lymphocytes, at lower levels by lung and synovial fluid lymphocytes, but never by intestinal lymphocytes. Only the high CCR4 levels characteristic of skin lymphocytes were associated with robust chemotactic and adhesive responses to TARC, consistent with a selective role for CCR4 in skin lymphocyte homing. In contrast, CXCR3 and CCR5 were present on the majority of lymphocytes from each non-lymphoid tissue examined, suggesting that these receptors are unlikely to determine tissue specificity, but rather, may play a wider role in tissue inflammation.

Expression of the type 2 receptor for cysteinyl leukotrienes (CysLT2R) by human mast cells: Functional distinction from CysLT1R

Cysteinyl leukotrienes (cysLTs) mediate vascular leakage and bronchoconstriction through the smooth muscle-associated CysLT type 1 receptor (CysLT1R), one of at least two loosely homologous cysLT-binding G protein-coupled receptors. We previously reported that CysLT1R is expressed by cultured human mast cells (hMCs), and that priming these cells with IL-4 enhances their sensitivity to calcium flux and cytokine generation in response to cys-LTs and the nucleotide ligand, uridine diphosphate (UDP), without increasing their surface expression of CysLT1R. We now report that hMCs express the type 2 receptor for cysLTs (CysLT2R) as well, and that the amount of surface CysLT2R protein increases in response to priming with IL-4. The selective function of CysLT2R was evident based on uninhibited IL-8 secretion by IL-4-primed hMCs stimulated with cys-LTs or UDP in the presence of the selective CysLT1R antagonist MK571. MK571 did inhibit IL-5 generation, calcium flux, and phosphorylation of extracellular signal-regulated kinase. IL-8 secretion was inhibited by pertussis toxin and a selective p38 kinase inhibitor, SB203580. The CysLT2 response may permit the cys-LTs and nucleotides generated in infection and tissue injury to elicit IL-8 generation by hMCs, potentially leading to neutrophilic infiltration, a characteristic of aerosol challenge-induced late-phase responses and of sudden death associated with asthma.

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-γ 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-γ-producing cells. Moreover, memory CCR9+CD4+ lymphocytes respond to CD2 stimulation with proliferation and IFN-γ/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.

CCR8 Expression Identifies CD4 Memory T Cells Enriched for FOXP3+ Regulatory and Th2 Effector Lymphocytes

CD4+ Th2 cells are important regulators of allergic inflammation. CCR8 is thought to play a role in Th2-mediated responses, however, expression of CCR8 in peripheral blood has not been fully characterized. Using a fluorescent form of the ligand selective for CCR8 (F-CCL1), we identified the leukocytes expressing CCR8 in human, monkey, and mouse peripheral blood. CCR8 expression is primarily restricted to a subset of human CD4 memory T lymphocytes (15%). Approximately 40% of CCR8+CD4+ T cells express Th2 cytokines IL-4 or IL-13 while 13% express the Th1 cytokine IFN-γ. In fact, 50% of all Th2, but only 5% of Th1, cells express CCR8. Upon anti-CD3/anti-CD28 mAb-mediated activation, CCR8+CD4+ T cells secrete 3- to 7-fold higher levels of IL-4, IL-5, IL-9, and IL-13 and 10- to 20-fold lower levels of IFN-γ or IL-17, compared with CCR8−CD4+ memory T cells. Two-thirds of CCR8+CD4 T cells express cutaneous lymphocyte-associated Ag while the majority lack gut-homing receptors. CCR8+CD4+ cells express CCR7 and CD62L and are present in spleen and lymph nodes of mice. Approximately 25% of CCR8+CD4 T cells express CD25high while 20% of CCR8+CD4+ express the T regulatory cell transcription factor FOXP3 accounting for 60% of all FOXP3-expressing CD4+ T cells. In conclusion, CCR8 marks a diverse subset of CD4 memory T cells enriched for T regulatory and Th2 cells which have the potential for recruitment into sites of allergic inflammation where they could participate in the induction and regulation of the allergic response.

CCR4 in human allergen-induced late responses in the skin and lung

We studied the regulation of CCR4 expression in peripheral blood and in human models of cutaneous and pulmonary allergen challenge. CCR4 expression was detectable on freshly isolated CD4+ lymphocytes and in CD4+ and CD8+ T cell lines derived from blood of atopic donors. Numbers of CCR4+ cells were up‐regulated in T cell lines expanded in the presence of IL‐4. CCR4 mRNA was absent at baseline in normal subjects in lung and skin, but present at baseline in the lung of some atopics. Baseline expression of CCR4 mRNA and protein was higher in lung vs. skin, but allergen‐induced increases in CCR4 mRNA+ cells were observed in both organs. CCR4 protein+ cells were present at higher levels after allergen challenge in atopics compared to normal subjects. CCR4 may be important in the recruitment of T lymphocytes at sites of allergic inflammation, in a non‐organ‐specific manner.

Coordinated Involvement of Mast Cells and T Cells in Allergic Mucosal Inflammation: Critical Role of the CC Chemokine Ligand 1:CCR8 Axis

CCL1 is the predominant chemokine secreted from IgE-activated human and mouse mast cells in vitro, colocalizes to mast cells in lung biopsies, and is elevated in asthmatic airways. CCR8, the receptor for CCL1, is expressed by ∼70% of CD4+ T lymphocytes recruited to the asthmatic airways, and the number of CCR8-expressing cells is increased 3-fold in the airways of asthmatic subjects compared with normal volunteers. In vivo, CCL1 expression in the lung is reduced in mast cell-deficient mice after aeroallergen provocation. Neutralization of CCL1 or CCR8 deficiency results in reduced mucosal lung inflammation, airway hyperresponsiveness, and mucus hypersecretion to a similar degree as detected in mast cell-deficient mice. Adenoviral delivery of CCL1 to the lungs of mast cell-deficient mice restores airway hyperresponsiveness, lung inflammation, and mucus hypersecretion to the degree observed in wild-type mice. The consequences of CCR8 deficiency, including a marked reduction in Th2 cytokine levels, are comparable with those observed by depletion of CD4+ T lymphocytes. Thus, mast cell-derived CCL1- and CCR8-expressing CD4+ effector T lymphocytes play an essential role in orchestrating lung mucosal inflammatory responses.

Role of Regulator of G Protein Signaling 16 in Inflammation- Induced T Lymphocyte Migration and Activation

Chemokine-induced T lymphocyte recruitment to the lung is critical for allergic inflammation, but chemokine signaling pathways are incompletely understood. Regulator of G protein signaling (RGS)16, a GTPase accelerator (GTPase-activating protein) for Gα subunits, attenuates signaling by chemokine receptors in T lymphocytes, suggesting a role in the regulation of lymphocyte trafficking. To explore the role of RGS16 in T lymphocyte-dependent immune responses in a whole-organism model, we generated transgenic (Tg) mice expressing RGS16 in CD4+ and CD8+ cells. rgs16 Tg T lymphocytes migrated to CC chemokine ligand 21 or CC chemokine ligand 12 injection sites in the peritoneum, but not to CXC chemokine ligand 12. In a Th2-dependent model of allergic pulmonary inflammation, CD4+ lymphocytes bearing CCR3, CCR5, and CXCR4 trafficked in reduced numbers to the lung after acute inhalation challenge with allergen (OVA). In contrast, spleens of sensitized and challenged Tg mice contained increased numbers of CD4+CCR3+ cells producing more Th2-type cytokines (IL-4, IL-5, and IL-13), which were associated with increased airway hyperreactivity. Migration of Tg lymphocytes to the lung parenchyma after adoptive transfer was significantly reduced compared with wild-type lymphocytes. Naive lymphocytes displayed normal CCR3 and CXCR4 expression and cytokine responses, and compartmentation in secondary lymphoid organs was normal without allergen challenge. These results suggest that RGS16 may regulate T lymphocyte activation in response to inflammatory stimuli and migration induced by CXCR4, CCR3, and CCR5, but not CCR2 or CCR7.

CCR4 blockade does not inhibit allergic airways inflammation

The CC chemokine receptor 4 (CCR4) shows selectivity for the recruitment of memory T cell subsets, including those of the T helper cell type 2 (Th2) phenotype. In humans, CCR4+ T cells are recruited to the asthmatic lung in response to allergen challenge; however, the contribution of this pathway to allergic disease remains uncertain. We therefore investigated the role of CCR4 in allergic airways inflammation in the guinea pig. Blockade of CCR4 with a specific antibody resulted in only minor changes in numbers of CCR4+ Th cells in the bronchoalveolar lavage fluid of allergen‐challenged guinea pigs and failed to inhibit the generation of eotaxin/CC chemokine ligand (CCL)11 or macrophage‐derived chemokine/CCL22 or the recruitment of inflammatory leukocytes to the lung. These data suggest that although CCR4 was originally proposed as a marker of Th2 status, antigen‐specific Th2 cells are recruited to the lung predominantly by other pathways. This study casts doubts on the validity of CCR4 as a therapeutic target in the treatment of asthma.