Phenotypic analysis of perennial airborne allergen‐specific CD4+ T cells in atopic and non‐atopic individuals

Background Accumulating evidence suggests that T cells play an important role in the pathogenesis of atopic dermatitis (AD); yet, little is known of the differentiation status of CD4⁺ T cells specific for common environmental allergens, such as the major cat allergen, Fel d 1. Objective To determine the frequency, differentiation phenotype and function of circulating Fel d 1‐specific CD4⁺ T cells in adult individuals with severe persistent AD in comparison with healthy controls. Methods Using HLA class II tetrameric complexes based on a HLA‐DPB1^*0401‐restricted Fel d 1 epitope, ex vivo and cultured T cell frequency and phenotype were analysed in individuals with AD and healthy controls. Cytokine secretion was measured by ex vivo and cultured IL‐4 and IFN‐γ ELISpots. Results Ex vivo Fel d 1‐specific DPB1^*0401‐restricted CD4⁺ T cells in both atopics and non‐atopics express high levels of CCR7, CD62L, CD27 and CD28, placing the cells largely within the central memory subgroup. However, the functional phenotype was distinct, with greater IL‐4 production from the cells derived from atopics, which correlated with disease severity. Conclusions and Clinical Relevance Circulating Fel d 1‐specific DPB1^*0401‐restricted CD4⁺ T cells in both atopic and non‐atopic donors maintain a central memory phenotype; however in atopics, the cells had greater Th2 effector function, compatible with a disease model of altered antigen delivery in atopic individuals. Cite this as: L. R. Crack, H. W. Chan, T. McPherson and G. S. Ogg, Clinical & Experimental Allergy, 2011 (41) 1555–1567.     

Expression of CCR8 is increased in asthma

Background Chemokines and their receptors could play key roles in the recruitment of T cells to the asthmatic lung. CCR8 is preferentially expressed on T‐helper type 2 cells, and is thought to play a role in the pathogenesis of human asthma. Objective Determine the expression of CCR8 on T cells in blood, bronchoalveolar lavage (BAL) and bronchial mucosa from asthmatics and normal subjects. Methods CCR8 expression in blood and BAL from asthma and normal subjects was studied using flow cytometry. CCR8 expression on IFN‐γ⁺ and IL‐4⁺/IL‐13⁺ blood and BAL T cells was studied following stimulation with Phorbol–Myristate–Acetate and Calcium Ionophore. Paraffin‐embedded bronchial biopsies were used to study CCR8 in bronchial epithelium. Results The percentage of CD3⁺ cells expressing CCR8 in the blood was higher in asthmatics (4.7±0.4%) compared with normal subjects (3.0±0.4%; P<0.01). There was an approximately sixfold enrichment of CCR8 on IL‐4⁺/IL‐13⁺ cells compared with IFN‐γ⁺ T cells (P<0.001) in both asthmatic and normal subjects in both blood and BAL. Significantly more BAL T cells expressed CCR8 in asthmatic (8.6±0.8%) compared with normal subjects (3.9±0.7%) (P<0.01). In paired blood‐BAL samples from asthmatics, significantly more CCR8+CD3⁺ T cells were present in BAL (9.0±0.9%) than in blood (5.6±0.9%; P<0.05). There were more CCR8‐positive cells in bronchial biopsies from asthmatic (93±11 cells/mm²) compared with normal subjects (30±16 cells/mm²) (P<0.05). The ligand CCL1 was increased in the BAL of asthmatics compared with normal subjects (35±6 vs. 12.9±7 pg/mL; P<0.05). Conclusion There may be a role for CCR8 in the recruitment of T cells to the lung in asthmatics. Cite this as: K. Mutalithas, C. Guillen, C. Raport, R. Kolbeck, D. Soler, C. E. Brightling, I. D. Pavord and A. J. Wardlaw, Clinical & Experimental Allergy, 2010 (40) 1175–1185.     

Evidence for increased expression of regulatory cytokine receptors interleukin-12R and interleukin-18R in common variable immunodeficiency

We investigated the expression of T helper (Th)1/Th2 regulatory cytokine receptors on lymphocytes from patients with common variable immunodeficiency (CVID), a disorder associated with raised Th1 cytokine production, comparing the results with those from healthy individuals and atopic asthmatics, the latter generally considered to have a Th2‐driven disease. We proposed that alterations in some of the relevant receptors might be related to the observed imbalances in Th1/Th2 cytokines. Cells from CVID patients showed an increase in the percentages of CD212 [interleukin (IL)‐12Rβ1] cells within the CD4⁺ CD45RA⁺ and CD8⁺ CD45RA⁺ subsets (24% and 41%, respectively), as compared to CD4⁺ CD45RA⁺ and CD8⁺ CD45RA⁺ in healthy subjects (6% and 23%, respectivey). Approximately 21% of the CD4⁺ CD45RA⁺ naïve cells expressed IL‐18Rα, compared with 11% in healthy subjects. In contrast, the cytokine‐receptor expression in asthmatics was similar to that of controls. In spite of the above differences, after 72 h of stimulation with anti‐CD3 and anti‐CD28, cytokine receptor up‐regulation was similar in all three groups, with up to 80% of both CD45RA⁺ and CD45RO⁺ lymphocytes expressing CD212 (IL‐12Rβ1) and IL‐18Rα. Approximately 50% of the ‘naïve’, and 25% of the ‘memory’ subpopulations up‐regulated IL‐12Rβ2. These findings provide further evidence of a polarization towards a Th1 immune response in CVID, the mechanism possibly involving up‐regulation of IL‐12‐mediated pathways.     

CRTH2 expression on T cells in asthma

Mast cell‐derived prostaglandin D₂ (PGD₂) is the major prostanoid found within the airway of asthmatics immediately following allergen challenge. PGD₂ has been shown to have chemokinetic effects on eosinophils and T helper type 2 (Th2) cells in vitro. This occurs through the interaction of PGD₂ with the G‐protein‐coupled chemokine receptor homologous molecule expressed on Th2 lymphocytes (CRTH2). The expression of CRTH2 has been shown to be highly selective for Th2 cells. Using flow cytometry we have studied the expression of CRTH2 on T cells in blood and bronchoalveolar lavage fluid in asthmatics and normal subjects. CRTH2 expression was confined to a small percentage of blood T cells in asthmatics (1·8% ± 0·2) and normal (1·6% ± 0·2) subjects. CRTH2 was enriched significantly on interleukin (IL)‐4⁺/IL‐13⁺ T cells compared to interferon (IFN)‐γ⁺ T cells (P < 0·001). There was a small population of CRTH2⁺ T cells in the bronchoalveolar lavage (BAL) of asthmatics (2·3% ± 0·6) and normal subjects (0·3% ± 0·1), and there was a significant difference between the two groups (P < 0·05). There were similar amounts of PGD₂ in the BAL of asthma and normal subjects. Within paired blood–BAL samples from the same subject there was no increase in CRTH2⁺ T cells in the BAL compared to blood in asthmatics. Enrichment of CRTH2 on IL‐4⁺ and IL‐13⁺ T cells compared to IFN‐γ⁺ T cells was also seen in BAL from asthmatics (P < 0·001). CRTH2 is expressed preferentially by IL‐4⁺/IL‐13⁺ T cells compared to IFN‐γ⁺ T cells. However, given their small numbers they are unlikely to have a significant involvement in the pathogenesis of asthma. CRTH2 antagonism may not diminish T cell accumulation in the asthmatic lung.     

Excessive CD4+ T cells co-expressing interleukin-17 and interferon-γ in patients with Behçet's disease

Excessive T helper type 1 (Th1) cell activity has been reported in Behçet's disease (BD). Recently, association of Th17 cells with certain autoimmune diseases was reported, and we thus investigated circulating Th17 cells in BD. CD4⁺CD45RO^– (naive) T cells were cultured with Th0‐, Th1‐, Th2‐ and Th17‐related cytokines and antibodies, and their mRNA was studied by real‐time polymerase chain reaction (PCR). When naive CD4⁺ T cells were cultured with Th1‐ and Th17‐related cytokines, interferon (IFN)‐γ mRNA and interleukin (IL)‐17 mRNA were up‐regulated, respectively, in BD patients. Naive CD4⁺ T cells cultured in a Th17 cell‐inducing condition expressed IL‐23 receptor (IL‐23R) mRNA excessively. IL‐17 mRNA expression was induced only when naive CD4⁺T cells were cultured in the presence of IL‐23. CD4⁺ T cells cultured with Th17 cytokines expressed excessive RAR‐related orphan receptor C (RORC) mRNA. Using intracellular cytokine staining, we found that CD45RO⁺(memory) CD4⁺ T cells producing IL‐17 and IFN‐γ simultaneously were increased significantly. Memory CD4⁺ T cells producing IFN‐γ but not IL‐17 decreased profoundly in BD patients. CD4⁺ T cells producing IL‐17 and IFN‐γ simultaneously were found in BD skin lesions. Collectively, we found excessive CD4⁺ T cells producing IL‐17 and IFN‐γ (Th1/Th17) cells in patients with BD, and possible involvement of IL‐23/IL‐23R pathway for the appearance of excessive Th1/Th17 cells.     

Secretion of the eosinophil-active cytokines interleukin-5, granulocyte/macrophage colonystimulating factor and interleukin-3 by bronchoalveolar lavage CD4+ and CD8+ T cell lines in atopics asthmatics,and atopic and nonatopic controls

Specific eosinophil accumulation and activation within the asthmatic bronchial mucosa are thought to occur at least partly through the actions of cytokines, including interleukin (IL)-5, IL-3 and granulocyte/macrophage colonystimulating factor (GM-CSF). Although mRNA encoding some of these cytokines has been demonstrated in bronchoalveolar lavage (BAL) fluid cells and bronchial biopsies from asthmatics, it has yet to be established whether these cells produce the translated products and whether expression is associated with CD4⁺ T helper or CD8⁺ cytotoxic T cells. We addressed this problem by raising polyclonal CD4⁺ and CD8⁺ T cell lines from the BAL fluid of six atopic asthmatics, five atopic non-asthmatics and seven non-atopic non-asthmatic controls. BAL fluid cells obtained at fiberoptic bronchoscopy were depleted of adherent cells, and then T lymphocytes expanded by stimulation with monoclonal anti-CD3 antibody and recombinant human IL-2. When lymphocytes had expanded to sufficient numbers, CD4⁺ and CD8⁺ cells were separated by positive selection with magnetic beads coated with anti-CD4 or anti-CD8 monoclonal antibodies and further expanded. Cytokine secretion by standardized cell numbers was measured by enzyme-linked immunosorbent assays. BAL CD4⁺ T cell lines from the asthmatics secreted significantly elevated quantities of both IL-5 and GM-CSF as compared with lines from the atopic and non-atopic controls (p = 0.023–0.003). In contrast, IL-3 secretion did not significantly differ between the groups. In some subjects, CD8⁺ T cell lines also secreted significant quantities of these cytokines and there was a trend for IL-5 secretion by these cells to be higher in asthmatics than non-atopic controls (p = 0.035). These data are consistent with the hypothesis that activated T lymphocytes from asthmatics, particularly of the CD4⁺ subset, are predisposed to release elevated quantities of cytokines relevant to the accumulation and activation of eosinophils.     

Inhaled allergen-driven CD1c up-regulation and enhanced antigen uptake by activated human respiratory-tract dendritic cells in atopic asthma

Background Dendritic cells (DC) mediate inflammation in rodent models of allergic airway disease, but the role played by human respiratory‐tract DC (hRTDC) in atopic asthma remains poorly defined. Recent data suggest that CD1 antigen presentation by hRTDC may contribute to asthma pathogenesis. Objective To investigate the influence of hRTDC on the balance between atopy and allergic asthma in human subjects and to determine whether CD1 expression by hRTDC is modulated during asthmatic inflammation. Methods Sputum cells were induced from steroid‐naïve, allergen‐challenged and allergen‐naïve subjects (atopic asthmatics, atopic non‐asthmatics and non‐atopic controls). hRTDC were identified using monoclonal antibody labelling and analysis by flow cytometry. Results hRTDC stained HLA‐DR⁺ (negative for markers of other cell lineages) were predominantly myeloid and comprised ∼0.5% of viable sputum cells. Sputum cells were potent stimulators of allogeneic CD4⁺ naïve T cells and enrichment/depletion experiments correlated stimulatory potency with DC numbers. Sputum contained cells that exhibited typical dendritic morphology when analysed by electron microscopy. Myeloid hRTDC were endocytically active, but uptake of FITC‐dextran was enhanced in cells from asthmatics (P<0.001). Despite their increased endocytic capacity, asthmatic myeloid hRTDC appeared mature and expressed increased levels of maturation markers (P<0.05–P<0.001), CD1c, CD1d and langerin (P<0.05). CD1c expression by asthmatic myeloid hRTDC was enhanced upon in vivo allergen challenge (three to ninefold within 24 h; P<0.05). CD11c⁻CD123^high hRTDC were only detected in asthmatic sputum and were increased in number following allergen challenge. Conclusion Despite limited cell numbers, it proved possible to analyse human RTDC in induced sputum, providing evidence that increased antigen uptake and enhanced CD1 presentation by activated hRTDC may contribute to allergic airway disease. CD1 presentation by hRTDC in atopic asthma may therefore constitute a novel target for future intervention strategies.     

IL‐34‐ and M‐CSF‐induced macrophages switch memory T cells into Th17 cells via membrane IL‐1α

Macrophages orchestrate the immune response via the polarization of CD4⁺ T helper (Th) cells. Different subsets of macrophages with distinct phenotypes, and sometimes opposite functions, have been described. M‐CSF and IL‐34 induce the differentiation of monocytes into IL‐10^high IL‐12^low immunoregulatory macrophages, which are similar to tumor‐associated macrophages (TAMs) in ovarian cancer. In this study, we evaluated the capacity of human macrophages induced in the presence of M‐CSF (M‐CSF macrophages) or IL‐34 (IL‐34 macrophages) and ovarian cancer TAMs to modulate the phenotype of human CD4⁺ T cells. Taken together, our results show that M‐CSF‐, IL‐34 macrophages, and TAMs switch non‐Th17 committed memory CD4⁺ T cells into conventional CCR4⁺ CCR6⁺ CD161⁺ Th17 cells, expressing or not IFN‐gamma. Contrary, the pro‐inflammatory GM‐CSF macrophages promote Th1 cells. The polarization of memory T cells into Th17 cells is mediated via membrane IL‐1α (mIL‐1α), which is constitutively expressed by M‐CSF‐, IL‐34 macrophages, and TAMs. This study elucidates a new mechanism that allows macrophages to maintain locally restrained and smoldering inflammation, which is required in angiogenesis and metastasis.     

Characteristics of Schistosoma japonicum infection induced IFN‐γ and IL‐4 co‐expressing plasticity Th cells

Schistosoma japonicum infection can induce granulomatous inflammation and cause tissue damage in the mouse liver. The cytokine secretion profile of T helper (Th) cells depends on both the nature of the activating stimulus and the local microenvironment (e.g. cytokines and other soluble factors). In the present study, we found an accumulation of large numbers of IFN‐γ⁺ IL‐4⁺ CD4⁺ T cells in mouse livers. This IFN‐γ⁺ IL‐4⁺ cell population increased from 0·68 ± 0·57% in uninfected mice to 7·05 ± 3·0% by week 4 following infection and to 9·6 ± 5·28% by week 6, before decreasing to 6·3 ± 5·9% by week 8 in CD4 T cells. Moreover, IFN‐γ⁺ IL‐4⁺ Th cells were also found in mouse spleen and mesenteric lymph nodes 6 weeks after infection. The majority of the IFN‐γ⁺ IL‐4⁺ Th cells were thought to be related to a state of immune activation, and some were memory T cells. Moreover, we found that these S. japonicum infection‐induced IFN‐γ⁺ IL‐4⁺ cells could express interleukin‐2 (IL‐2), IL‐9, IL‐17 and high IL‐10 levels at 6 weeks after S. japonicum infection. Taken together, our data suggest the existence of a population of IFN‐γ⁺ IL‐4⁺ plasticity effector/memory Th cells following S. japonicum infection in C57BL/6 mice.     

Preferential infiltration of interleukin‐4‐producing CXCR4+ T cells in the lesional muscle but not skin of patients with dermatomyositis

Dermatomyositis (DM) and polymyositis (PM) are collectively termed autoimmune myopathy. To investigate the difference between muscle‐ and skin‐infiltrating T cells and to address their role for myopathy, we characterized T cells that were directly expanded from the tissues. Enrolled into this study were 25 patients with DM and three patients with PM. Muscle and skin biopsied specimens were immersed in cRPMI medium supplemented with interleukin (IL)‐2 and anti‐CD3/CD28 antibody‐conjugated microbeads. The expanded cells were subjected to flow cytometry to examine their phenotypes. We analysed the cytokine concentration in the culture supernatants from the expanded T cells and the frequencies of cytokine‐bearing cells by intracellular staining. There was non‐biased in‐vitro expansion of tissue‐infiltrating CD4⁺ and CD8⁺ T cells from the muscle and skin specimens. The majority of expanded T cells were chemokine receptor (CCR) type 7^–CD45RO⁺ effecter memory cells with various T cell receptor (TCR) Vβs. The skin‐derived but not muscle‐derived T cells expressed cutaneous lymphocyte antigen (CLA) and CCR10 and secreted large amounts of IL‐17A, suggesting that T helper type 17 (Th17) cells may have a crucial role in the development of skin lesions. Notably, the frequency of IL‐4‐producing chemokine (C‐X‐C motif) receptor (CXCR)4⁺ Th2 cells was significantly higher in the muscle‐derived cells and correlated inversely with the serum creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) levels. stromal‐derived factor (SDF)‐1/CXCL12, a ligand for CXCR4, was expressed at a high level in the vascular endothelial cells between muscular fasciculi. Our study suggests that T cell populations in the muscle and skin are different, and the Th2 cell infiltrate in the muscle is associated with the low severity of myositis in DM.     

Predominance of activated,clonally expanded T helper type 17 cells within the CD4+ T cell population in psoriatic lesions

Recent evidence points to the T helper type 17 (Th17) subset as key in the pathogenesis of psoriasis, but cells of this type in lesions remain to be fully characterized. Here we isolated, enumerated, functionally tested and clonotyped the CD4⁺ Th cell population ex vivo from lesional biopsies and paired peripheral blood samples from psoriasis patients. Th17 cells were over‐represented dramatically in lesions from all patients, representing 49–93% of CD4⁺ Th cells compared with 3–18% in blood. Most lesional Th17 cells produced interleukin (IL)‐17A ex vivo without further stimulation and expressed the CD45RO⁺ phenotype characteristic of activated or memory cells. There was no increase in ‘natural’ [CD25^hiforkhead box protein 3 (FoxP3⁺)] regulatory T cells in lesions versus peripheral blood, but there was enrichment of ‘induced’ IL‐10⁺ regulatory T cell numbers in biopsies from some patients. The lesional Th17 cells exhibited a bias in T cell receptor Vβ chain usage, suggestive of specific expansion by antigen. The therapeutic challenge is to overcome the dominance of overwhelming numbers of such antigen‐specific Th17 cells in psoriatic lesions.     

Stress‐activated dendritic cells interact with CD4+ T cells to elicit homeostatic memory

Evidence is presented that thermal or oxidizing stress‐activated DC interact with CD4⁺ T cells to induce and maintain a TCR‐independent homeostatic memory circuit. Stress‐activated DC expressed endogenous intra‐cellular and cell surface HSP70. The NF‐κB signalling pathway was activated and led to the expression of membrane‐associated IL‐15 molecules. These interacted with the IL‐15 receptor complex on CD4⁺ T cells, thus activating the Jak3 and STAT5 phosphorylation signalling pathway to induce CD40 ligand expression, T‐cell proliferation and IFN‐γ production. CD40 ligand on CD4⁺ T cells in turn re‐activated CD40 molecules on DC, inducing DC maturation and IL‐15 expression thereby maintaining the feedback circuit. The proliferating CD4⁺ T cells were characterized as CD45RA^? CD62L⁺ central memory cells, which underwent homeostatic proliferation. The circuit is independent of antigen and MHC‐class‐II‐TCR interaction as demonstrated by resistance to TCR inhibition by ZAP70 inhibitor or MHC‐class II antibodies. These findings suggest that stress can activate a DC‐CD4⁺ T‐cell interacting circuit, which may be responsible for maintaining a homeostatic antigen‐independent memory.     

Basophils inhibit proliferation of CD4+ T cells in autologous and allogeneic mixed lymphocyte reactions and limit disease activity in a murine model of graft versus host disease

Basophils are known to modulate the phenotype of CD4⁺ T cells and to enhance T helper type 2 responses in vitro and in vivo. In this study, we demonstrate that murine basophils inhibit proliferation of CD4⁺ T cells in autologous and allogeneic mixed lymphocyte reactions. The inhibition is independent of Fas and MHC class II, but dependent on activation of basophils with subsequent release of interleukin‐4 (IL‐4) and IL‐6. The inhibitory effect of basophils on T‐cell proliferation can be blocked with antibodies against IL‐4 and IL‐6 and is absent in IL‐4/IL‐6 double‐deficient mice. In addition, we show that basophils and IL‐4 have beneficial effects on disease activity in a murine model of acute graft‐versus‐host disease (GvHD). When basophils were depleted with the antibody MAR‐1 before induction of GvHD, weight loss, GvHD score, mortality and plasma tumour necrosis factor levels were increased while injection of IL‐4 improved GvHD. Basophil‐depleted mice with GvHD also have increased numbers of CD4⁺ T cells in the mesenteric lymph nodes. Our data show for the first time that basophils suppress autologous and allogeneic CD4⁺ T‐cell proliferation in an IL‐4‐dependent manner.     

Positive selection of self‐antigen‐specific CD8+ T cells by hematopoietic cells

In contrast to thymic epithelial cells, which induce the positive selection of conventional CD8⁺ T cells, hematopoietic cells (HCs) select innate CD8⁺ T cells whose Ag specificity is not fully understood. Here we show that CD8⁺ T cells expressing an H‐Y Ag‐specific Tg TCR were able to develop in mice in which only HCs expressed MHC class I, when HCs also expressed the H‐Y Ag. These HC‐selected self‐specific CD8⁺ T cells resemble innate CD8⁺ T cells in WT mice in terms of the expression of memory markers and effector functions, but are phenotypically distinct from the thymus‐independent CD8⁺ T‐cell population. The peripheral maintenance of H‐Y‐specific CD8⁺ T cells required presentation of the self‐Ag and IL‐15 on HCs. HC‐selected CD8⁺ T cells in mice lacking the Tg TCR also showed these features. Furthermore, by using MHC class I tetramers with a male Ag peptide, we found that self‐Ag‐specific CD8⁺ T cells in TCR non‐Tg mice could develop via HC‐induced positive selection, supporting results obtained from H‐Y TCR Tg mice. These findings indicate the presence of self‐specific CD8⁺ T cells that are positively selected by HCs in the peripheral T‐cell repertoire.     

IL‐7 is superior to IL‐2 for ex vivo expansion of tumour‐specific CD4+ T cells

It is well established that tumours hinder both natural and vaccine‐induced tumour‐specific CD4⁺ T‐cell responses. Adoptive T‐cell therapy has the potential to circumvent functional tolerance and enhance anti‐tumour protective responses. While protocols suitable for the expansion of cytotoxic CD8⁺ T cells are currently available, data on tumour‐specific CD4⁺ T cells remain scarce. We report here that CD4⁺ T cells sensitized to tumour‐associated Ag in vivo, proliferate in vitro in response to IL‐7 without the need for exogenous Ag stimulation and accumulate several folds while preserving a memory‐like phenotype. Both cell proliferation and survival accounts for the outgrowth of tumour‐sensitized T cells among other memory and naive lymphocytes following exposure to IL‐7. Also IL‐2, previously used to expand anti‐tumour CTL, promotes tumour‐specific CD4⁺ T‐cell accumulation. However, IL‐7 is superior to IL‐2 at preserving lymphocyte viability, in vitro and in vivo, maintaining those properties, that are required by helper CD4⁺ T cells to confer therapeutic efficacy upon transplantation in tumour‐bearing hosts. Together our data support a unique role for IL‐7 in retrieving memory‐like CD4⁺ T cells suitable for adoptive T‐cell therapy.