Atopic asthma: differential activation phenotypes among memory T helper cells

Background T cells have been implicated in the pathogenesis of atopic asthma. We have previously shown that memory T helper cells (CD4⁺CD45RO⁺) are preferentially activated relative to naïve T helper cells (CD4⁺CD45RA⁺) after bronchial allergen challenge. However, specific T helper subpopulations that are activated in atopy and/or asthma remain undefined. Objective To determine the T helper subpopulations and activation phenotypes relevant to acute and stable asthma that may be common with or distinct from atopy. Methods Two groups of atopic asthmatics (ten acute and nine stable asthmatics) and two non‐asthmatic groups (14 non‐asthmatic atopics and eight normal non‐atopic controls) were analysed. Ten acute asthmatics were assessed in the emergency room during an acute episode (FEV₁ 43.6% ± 18.4). Nine stable asthmatics were assessed during a symptom‐free period (FEV₁ 85% ± 6). Using multiple colour flow cytometry we analysed T cell subpopulations and the expression of IL‐2‐receptor (IL‐2R) and MHC‐class II antigens (MHC II) on naïve and memory T helper cells in the peripheral blood of asthmatic and non‐asthmatic groups. Results Atopic asthmatics (acute and stable) had an increased percentage of memory T helper cells expressing IL‐2R compared with normal non‐atopics (mean SD 16.1 ± 6%, 12.4 ± 2% and 7.7 ± 1.8%, P < 0.05) but not compared with non‐asthmatic atopics (10 ± 3.5%). Naïve T helper cells had low expression of IL‐2R and MHC II in all four groups. MHC II antigen expression was increased in memory T helper cells of asthmatics (acute and stable) compared with normal non‐atopics (13.9 ± 7.5, 10.6 ± 5 and 4.9 ± 2.5, P < 0.05) but not compared with non‐asthmatic atopics (7.92 4). A novel finding was that IL‐2R and the MHC II molecules were mainly expressed in non‐overlapping populations and coexpression was found predominantly on memory T helper cells. Asthmatics (acute and stable) had higher proportion of double positive memory T helper cells (IL‐2R⁺MHC II⁺) compared with both non‐asthmatic groups (P < 0.05). Conclusions We demonstrate a differential expression of IL‐2R⁺ and MCH II⁺ on CD45RO⁺ T helper cells that would suggest that there are three subsets of activated memory T helper cells in asthmatics. Two non‐overlapping IL‐2R⁺ or MHC II⁺ CD45RO⁺ T helper cells and a third subpopulation of activated cells that coexpress IL‐2R and MHC II (double positives). This latter subpopulation is significantly higher in asthmatics (acute or stable) compared with both non‐asthmatic groups, suggesting a specific T helper activation phenotype distinct to atopic asthmatics as compared with atopic non‐asthmatics.     

TGF‐β interactions with IL‐1 family members trigger IL‐4‐independent IL‐9 production by mouse CD4+ T cells

TGF‐β and IL‐4 were recently shown to selectively upregulate IL‐9 production by naïve CD4⁺ T cells. We report here that TGF‐β interactions with IL‐1α, IL‐1β, IL‐18, and IL‐33 have equivalent IL‐9‐stimulating activities that function even in IL‐4‐deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T‐cell activation. Based on intracellular IL‐9 staining, all IL‐9‐producing cells were CD4⁺ and 80–90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL‐9, IL‐13 and IL‐17 were strongly stimulated by IL‐1 and either inhibited (IL‐13) or were unaffected (IL‐17) by addition of TGF‐β. IL‐9 and IL‐17 production also differed in their dependence on IL‐2 and regulation by IL‐1/IL‐23. As IL‐9 levels were much lower in Th2 and Th17 cultures, our results identify TGF‐β/IL‐1 and TGF‐β/IL‐4 as the main control points of IL‐9 synthesis.     

Lck regulates IL-10 expression in memory-like Th1 cells

The Src family kinase Lck is thought to facilitate Th2 differentiation; however, its role in Th1 cells has not been well explored. Using mice that lack Lck in mature T cells, we find that lck^−/− Th1 skewed cells have normal expression of T‐bet and produce IFN‐γ at WT levels. However, there is a 3‐fold increase in IL‐10 producing cells in the mutant cultures. These cells do not have elevated levels of IL‐4, GATA3, IL‐17 or Foxp3, indicating that they are not Th2, Th17, or Foxp3⁺ T regulatory cells (Treg). Nor do these cells behave in a similar manner as the type 1 Treg. Most of the IL‐10 in the lck^−/− Th1 cultures is derived from the memory/activated subset, as the cytokine profile from Th1 cultures established from purified CD62L⁺ (naïve) cells are similar to WT cells. Furthermore, this IL‐10 expression appears to be dependent on IL‐12 and correlates with elevated c‐Maf. These data highlight a previously unappreciated role for Lck in regulating IL‐10 in Th1 cells.     

Interleukin‐4 downregulates CD127 expression and activity on human thymocytes and mature CD8+ T cells

Signaling via the IL‐7 receptor complex (IL‐7Rα/CD127 and IL‐2Rγ/CD132) is required for T‐cell development and survival. Decreased CD127 expression has been associated with persistent viral infections (e.g. HIV, HCV) and cancer. Many IL‐2Rγ‐sharing (γ_C) cytokines decrease CD127 expression on CD4⁺ and CD8⁺ T cells in mice (IL‐2, IL‐4, IL‐7, IL‐15) and in humans (IL‐2, IL‐7), suggesting a common function. IL‐4 is of particular interest as it is upregulated in HIV infection and in thyroid and colon cancers. The role of IL‐4 in regulating CD127 expression and IL‐7 activity in human thymocytes and mature CD8⁺ T cells is unknown and was therefore investigated. IL‐4 decreased CD127 expression on all thymocyte subsets tested and only on naïve (CD45RA⁺) CD8⁺ T cells, without altering membrane‐bound CD127 mRNA expression. Pre‐treatment of thymocytes or CD8⁺ T cells with IL‐4 inhibited IL‐7‐mediated phosphorylation of STAT5 and decreased proliferation of CD8⁺ T cells. By downregulating CD127 expression and signaling on developing thymocytes and CD8⁺ T cells, IL‐4 is a potential contributor to impaired CD8⁺ T‐cell function in some anti‐viral and anti‐tumor responses. These findings are of particular consequence to diseases such as HIV, HCV, RSV, measles and cancer, in which CD127 expression is decreased, IL‐7 activity is impaired and IL‐4 concentrations are elevated.     

CXCL4 is a novel inducer of human Th17 cells and correlates with IL‐17 and IL‐22 in psoriatic arthritis

CXCL4 regulates multiple facets of the immune response and is highly upregulated in various Th17‐associated rheumatic diseases. However, whether CXCL4 plays a direct role in the induction of IL‐17 production by human CD4⁺ T cells is currently unclear. Here, we demonstrated that CXCL4 induced human CD4⁺ T cells to secrete IL‐17 that co‐expressed IFN‐γ and IL‐22, and differentiated naïve CD4⁺ T cells to become Th17‐cytokine producing cells. In a co‐culture system of human CD4⁺ T cells with monocytes or myeloid dendritic cells, CXCL4 induced IL‐17 production upon triggering by superantigen. Moreover, when monocyte‐derived dendritic cells were differentiated in the presence of CXCL4, they orchestrated increased levels of IL‐17, IFN‐γ, and proliferation by CD4⁺ T cells. Furthermore, the CXCL4 levels in synovial fluid from psoriatic arthritis patients strongly correlated with IL‐17 and IL‐22 levels. A similar response to CXCL4 of enhanced IL‐17 production by CD4⁺ T cells was also observed in patients with psoriatic arthritis. Altogether, we demonstrate that CXCL4 boosts pro‐inflammatory cytokine production especially IL‐17 by human CD4⁺ T cells, either by acting directly or indirectly via myeloid antigen presenting cells, implicating a role for CXCL4 in PsA pathology.     

Naïve donor responses to Schistosoma mansoni soluble egg antigens

Schistosome infection induces profound Th‐biasing and immune suppression. Although much has been examined in mice, few studies have examined responses of naïve humans to schistosome antigens. In this study, we examined the response of naïve human peripheral blood mononuclear cells (nPBMC) to stimulation with Schistosoma mansoni soluble egg antigen (SEA) using a priming in vitro (PIV) assay. We found that SEA induced a pronounced CD4⁺ T‐helper cell response based on cytokine secretion and phenotyping markers. SEA‐stimulated nPBMC (SEA cells) at day 7 post‐priming and after the first recall consisted predominantly of Th0‐like CD4⁺ T cells. Following the second recall, the majority of donor (10/12) responses were Th2‐like. The cell population consisted of approximately 64% CD4⁺, 17% CD8^+high, 12% CD19⁺, and 7% CD23⁺ cells. The CD4⁺ population also expressed HLA‐DR⁺, CD54⁺, CD45RO⁺ and CD25⁺ whereas the CD19⁺ cells expressed CD80 and CD86. Following priming, we detected high levels of IL‐6, IFN‐γ, IL‐12p40, IL‐10 and IL‐5. Upon restimulation, SEA cells secreted IL‐5 and high levels of IL‐10, typical of a Th2‐like response. The data presented herein shows that the majority of naïve donor dendritic cells, following stimulation with SEA, prime and clonally expand SEA‐specific T cells towards a Th2‐type response. However, two donors responded with an atypical response, producing IFN‐γ coincident with low levels of IL‐10. Whether this differential response was due to HLA or other genes was not determined but is currently under investigation.     

TGF‐β indirectly favors the development of human Th17 cells by inhibiting Th1 cells

Human Th17 clones and circulating Th17 cells showed lower susceptibility to the anti‐proliferative effect of TGF‐β than Th1 and Th2 clones or circulating Th1‐oriented T cells, respectively. Accordingly, human Th17 cells exhibited lower expression of clusterin, and higher Bcl‐2 expression and reduced apoptosis in the presence of TGF‐β, in comparison with Th1 cells. Umbilical cord blood naïve CD161⁺CD4⁺ T cells, which contain the precursors of human Th17 cells, differentiated into IL‐17A‐producing cells only in response to IL‐1β plus IL‐23, even in serum‐free cultures. TGF‐β had no effect on constitutive RORγt expression by umbilical cord blood CD161⁺ T cells but it increased the relative proportions of CD161⁺ T cells differentiating into Th17 cells in response to IL‐1β plus IL‐23, whereas under the same conditions it inhibited both T‐bet expression and Th1 development. These data suggest that TGF‐β is not critical for the differentiation of human Th17 cells, but indirectly favors their expansion because Th17 cells are poorly susceptible to its suppressive effects.     

Osteopontin promotes inflammation in patients with acute coronary syndrome through its activity on IL‐17 producing cells

Atherosclerosis is a progressive disease with a strong inflammatory component. Here we confirm the existence of a critical imbalance in the ratio of Th17 to Treg‐cell populations in peripheral CD4⁺ T cells from patients with acute coronary syndrome (ACS), which favors inflammation. This was concurrent with increased IL‐17 production from the CD4⁺CD45RA^?FOXP3^lo Treg‐cell subset, and elevated osteopontin (OPN) levels in serum from ACS patients. We demonstrate a direct effect of OPN in serum from ACS patients on increased IL‐17 production by CD4⁺CD45RA^?FOXP3^lo T cells, mediated through recruitment of the OPN receptors CD29 and CD44, and dependent on STAT3 and the nuclear hormone receptor retinoic‐acid‐related orphan receptor‐γt (RORγt) pathway, but not IL‐6 production. To our knowledge and beyond the disease context of ACS, this study constitutes the first demonstration of a critical role for OPN in the positive regulation of inflammation through increased IL‐17 production by CD4⁺CD45RA^?FOXP3^lo cells.     

Preferential production of interferon-γ by CD4+ T cells expressing the homing receptor integrin α4/β7

Recent studies indicate that T helper type 1 (Th1) and 2 (Th2) lymphocytes differ in their expression of molecules that control T‐cell migration, including adhesion molecules and chemokine receptors. We investigated the relationship between cytokine production and expression of the homing receptor integrin α₄/β₇ on T cells. We began by analysing cytokine production by human CD4⁺ CD45RA^– memory/effector T cells following brief (4 hr) stimulation with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin. α₄/ CD4⁺ T cells were more likely to produce the Th1 cytokine interferon‐γ (IFN‐γ) than were α₄/β₇^? CD4⁺ T cells in all six subjects studied. In contrast, production of the Th2 cytokine interleukin‐4 (IL‐4) was similar on α₄/ and α₄/β₇^? CD4⁺ T cells. In addition, we found that human CD4⁺ CD45RA^– T cells that adhered to the α₄/β₇ ligand mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1) had a greater capacity to produce IFN‐γ than did non‐adherent cells, suggesting that the association between α₄/β₇ expression and IFN‐γ production has functional significance. These results suggested that primary activation under Th1‐promoting conditions might favour expression of α₄/β₇. We directly examined this possibility, and found that naïve murine CD4⁺ T cells activated under Th1‐promoting conditions expressed higher levels of α₄/β₇ compared to cells activated under Th2‐promoting conditions. The association between α₄/β₇ expression and IFN‐γ production by CD4⁺ T cells may help to determine the cytokine balance when MAdCAM‐1 is expressed at sites of inflammation in the intestine or elsewhere.     

Expression of IL‐15RA or an IL‐15/IL‐15RA fusion on CD8+ T cells modifies adoptively transferred T‐cell function in cis

IL‐15 and IL‐15 receptor alpha (IL‐15RA) play a significant role in multiple aspects of T‐cell biology. However, given the evidence that IL‐15RA can present IL‐15 in trans, the functional capacity of IL‐15RA expressed on CD8⁺ T cells to modify IL‐15 functions in cis is currently unclear. In the current study, we explore the functional consequences of IL‐15RA, expression on T cells using a novel method to transfect naive CD8⁺ T cells. We observed that RNA nucleofection led to highly efficient, non‐toxic, and rapid manipulation of protein expression levels in unstimulated CD8⁺ T cells. We found that transfection of unstimulated CD8⁺ T cells with IL‐15RA RNA led to enhanced viability of CD8⁺ T cells in response to IL‐15. Transfection with IL‐15RA enhanced IL‐15‐mediated phosphorylation of STAT5 and also promoted IL‐15‐mediated proliferation in vivo of adoptively transferred naïve CD8⁺ T cells. We demonstrated that IL‐15RA can present IL‐15 via cis‐presentation on CD8⁺ T cells. Finally, we showed that transfection with a chimeric construct linking IL‐15 to IL‐15RA cell autonomously enhances the viability and proliferation of primary CD8⁺ T cells and cytotoxic potential of antigen‐specific CD8⁺ T cells. The clinical implications of the current study are discussed.     

Human rhinoviruses induce IL‐35‐producing Treg via induction of B7‐H1 (CD274) and sialoadhesin (CD169) on DC

IL‐35 is a heterodimer of EBV‐induced gene 3 and of the p35 subunit of IL‐12, and recently identified as an inhibitory cytokine produced by natural Treg in mice, but not in humans. Here we demonstrate that DC activated by human rhinoviruses (R‐DC) induce IL‐35 production and release, as well as a suppressor function in CD4⁺ and CD8⁺ T cells derived from human peripheral blood but not in naïve T cells from cord blood. The induction of IL‐35‐producing T cells by R‐DC was FOXP3‐independent, but blocking of B7‐H1 (CD274) and sialoadhesin (CD169) on R‐DC with mAb against both receptors prevented the induction of IL‐35. Thus, the combinatorial signal delivered by R‐DC to T cells via B7‐H1 and sialoadhesin is crucial for the induction of human IL‐35⁺ Treg. These results demonstrate a novel pathway and its components for the induction of immune‐inhibitory T cells.     

Changes in thymic function in HIV-positive patients treated with highly active antiretroviral therapy and interleukin-2

Despite its potent antiviral activity, highly active antiretroviral therapy (HAART) only exerts a marginal effect on CD4⁺ T‐cell regeneration in HIV‐infected subjects. Combination therapies aimed at boosting T‐cell activity and maturation may provide an important contribution to the restoration of immune function. Here, we report the results obtained by a two‐year follow‐up of a cohort of HIV‐infected patients treated with a combination of HAART and interleukin‐2 (IL‐2). In these patients, in addition to a series of quantitative virological and immunological parameters, we investigated T‐cell regeneration by an immunophenotypic assay monitoring CD4⁺ naïve T cells, and by analysis of thymic function, through the quantification of the excision DNA products of T‐cell receptor rearrangement (TRECs) in lymphocytes. Compared with HAART alone, we found that the IL‐2 combination therapy was equally effective in reducing the levels of viremia and marginally more effective in decreasing proviral DNA load. Strikingly, the IL‐2 combination produced a marked increase in the number of CD4⁺ T cells bearing a naïve phenotype (CD45RA⁺, CD62L⁺), which was apparent for over 96 weeks after therapy. To assess whether these cells were the product of improved T‐cell generation, we exploited a competitive quantitative molecular assay to quantify TRECs in peripheral blood lymphocytes. Surprisingly, we found that the levels of these molecules were unchanged in these patients. These findings indicate that improved thymic function does not account for the early rise of CD4 naïve cells in HIV‐positive patients treated with IL‐2, and suggest that alternative mechanisms of T‐cell maturation and differentiation are responsible for this event.     

Pollen‐derived low‐molecular weight factors inhibit 6‐sulfo LacNAc+ dendritic cells' capacity to induce T‐helper type 1 responses

Background Evidence is accumulating that the pollen exsudate contains an array of non‐allergenic, pro‐inflammatory and immunomodulatory substances acting on the innate and adaptive immune system. In this context, pollen‐associated E₁‐phytoprostanes (PPE₁) were shown to licence human monocyte‐derived dendritic cells for T‐helper type 2 (Th2) polarization of naïve T cells. Objective This study aims at analysing the impact of pollen‐associated lipid mediators on cytokine secretion and maturation of 6‐sulfo LacNAc⁺ dendritic cells (slanDCs), the most abundant native dendritic cell (DC) in human peripheral blood, and further dissecting the biologically active substance(s) within aqueous pollen extracts. Results Aqueous birch pollen extracts dose‐dependently inhibited the lipopolysaccharide (LPS)‐induced IL‐12 p70 production, while the levels of IL‐6 remained unaffected. PPE₁ inhibited secretion of both IL‐12 p70 and IL‐6. Aqueous pollen extracts, but not PPE₁ or F₁‐phytoprostanes significantly reduced the LPS‐induced surface expression of the maturation markers CD80, CD83, CD40 and CCR‐7, an effect that was independent of proteins and that was still present in a 3 kDa cut‐off fraction of the pollen extract. These effects were observed irrespective of the atopy status of the donors. Finally, slanDCs exposed to aqueous pollen extracts were impaired in eliciting an IFN‐γ response in naïve CD4⁺ T cells. Conclusion Our data show that slanDCs, a subset of human blood DCs with constitutively high potency to induce Th1 responses, are susceptible to the Th2 polarizing effect of low molecular weight, non‐protein factors derived from pollen. Cite this as: S. Gilles, D. Jacoby, C. Blume, M. J. Mueller, T. Jakob, H. Behrendt, K. Schaekel and C. Traidl‐Hoffmann, Clinical & Experimental Allergy, 2010 (40) 269–278.     

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.     

T cell response to viral antigens in adults and children with common variable immunodeficiency and specific antibody deficiency

Several T cell abnormalities have been described in common variable immunodeficiency (CVID), a B cell disorder of mainly unknown origin. A subset of CVID patients suffers from frequent reactivations of herpes viruses. We studied T cell function in CVID [and in a subset of paediatric patients with specific antibody deficiency (SAD)] by measuring T cell proliferation and cytokine production in response to herpes virus‐antigens in paediatric CVID patients (n = 9) and paediatric SAD patients (n = 5), in adult CVID patients (n = 14) and in healthy controls. Paediatric CVID patients, but not SAD patients, displayed moderately increased CD8⁺ T cell proliferation in response to cytomegalovirus, human herpes virus type 6B (HHV6‐B) and herpes simplex virus compared to controls. CD8⁺ T cell responses in adult CVID patients tended to be increased in response to cytomegalovirus and herpes simplex virus. In response to stimulation with herpes virus antigens, the proinflammatory cytokines interleukin (IL)‐1β, IL‐6, tumour necrosis factor (TNF)‐α and interferon inducible protein (IP)‐10 were produced. Overall, no major differences were detected in cytokine production upon stimulation between patients and controls, although higher IL‐10 and IL‐12 production was detected in paediatric patients. In conclusion, cellular immunity against herpes virus antigens appears undisturbed in CVID patients, although defects in subpopulations of CVID patients cannot be excluded.