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.     

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.     

Activation‐induced CD137 is a fast assay for identification and multi‐parameter flow cytometric analysis of alloreactive T cells

Detection and isolation of viable alloreactive T cells at the single‐cell level requires a cell surface marker induced specifically upon T cell receptor activation. In this study, a member of the tumour necrosis factor receptor (TNFR)‐family, CD137 (4‐1BB) was investigated for its potential to identify the total pool of circulating alloreactive T cells. Optimal conditions for sensitive and specific detection of allogeneic‐induced CD137 expression on circulating T cells were established. Thereafter, CD137⁺ alloreactive T cells were phenotypically and functionally characterized by multi‐parameter flow cytometry. Alloantigen‐induced CD137 expression identified both alloreactive CD8⁺ T cells (mean ± standard error of the mean: 0·21 ± 0·07%) and alloreactive CD4⁺ T cells (0·21 ± 0·05%). CD137⁺ alloreactive T cells were detected in different T cell subsets, including naive T cells, but were found preferentially in CD28⁺ T cells and not in the terminally differentiated T cell subset. Upon allogeneic (re‐)stimulation, the cytokine‐producing as well as proliferative capacity of T cells resided mainly within the CD137‐expressing fraction. About 10% of the CD137⁺ alloreactive T cells produced any combination of interferon (IFN)‐γ, interleukin (IL)‐2 and TNF‐α. Polyfunctional alloreactive T cells, defined by multiple cytokine expression, were observed infrequently. In conclusion, activation‐induced CD137 expression is a fast assay allowing for detection and functional analysis of the total alloreactive T cell compartment at the single‐cell level by multi‐parameter flow cytometry.     

IFITM proteins drive type 2 T helper cell differentiation and exacerbate allergic airway inflammation

The interferon‐inducible transmembrane (Ifitm/Fragilis) genes encode homologous proteins that are induced by IFNs. Here, we show that IFITM proteins regulate murine CD4⁺ Th cell differentiation. Ifitm2 and Ifitm3 are expressed in wild‐type (WT) CD4⁺ T cells. On activation, Ifitm3 was downregulated and Ifitm2 was upregulated. Resting Ifitm‐family‐deficient CD4⁺ T cells had higher expression of Th1‐associated genes than WT and purified naive Ifitm‐family‐deficient CD4⁺ T cells differentiated more efficiently to Th1, whereas Th2 differentiation was inhibited. Ifitm‐family‐deficient mice, but not Ifitm3‐deficient mice, were less susceptible than WT to induction of allergic airways disease, with a weaker Th2 response and less severe disease and lower Il4 but higher Ifng expression and IL‐27 secretion. Thus, the Ifitm family is important in adaptive immunity, influencing Th1/Th2 polarization, and Th2 immunopathology.     

Relationship of CD146 expression to secretion of interleukin (IL)‐17, IL‐22 and interferon‐γ by CD4+ T cells in patients with inflammatory arthritis

Expression of the adhesion molecule, CD146/MCAM/MelCAM, on T cells has been associated with recent activation, memory subsets and T helper type 17 (Th17) effector function, and is elevated in inflammatory arthritis. Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA) and spondyloarthritides (SpA). Here, we compared the expression of CD146 on CD4⁺ T cells between healthy donors (HD) and patients with RA and SpA [ankylosing spondylitis (AS) or psoriatic arthritis (PsA)] and examined correlations with surface markers and cytokine secretion. Peripheral blood mononuclear cells (PBMC) were obtained from patients and controls, and synovial fluid mononuclear cells (SFMC) from patients. Cytokine production [elicited by phorbol myristate acetate (PMA)/ionomycin] and surface phenotypes were evaluated by flow cytometry. CD146⁺CD4⁺ and interleukin (IL)‐17⁺CD4⁺ T cell frequencies were increased in PBMC of PsA patients, compared with HD, and in SFMC compared with PBMC. CD146⁺CD4⁺ T cells were enriched for secretion of IL‐17 [alone or with IL‐22 or interferon (IFN)‐γ] and for some putative Th17‐associated surface markers (CD161 and CCR6), but not others (CD26 and IL‐23 receptor). CD4⁺ T cells producing IL‐22 or IFN‐γ without IL‐17 were also present in the CD146⁺ subset, although their enrichment was less marked. Moreover, a majority of cells secreting these cytokines lacked CD146. Thus, CD146 is not a sensitive or specific marker of Th17 cells, but rather correlates with heterogeneous cytokine secretion by subsets of CD4⁺ helper T cells.     

Functional deficits of pertussis-specific CD4(+) T cells in infants compared to adults following DTaP vaccination

Understanding the immune responses that explain why infants require multiple doses of pertussis vaccine to achieve protection against infection is a high priority. The objective of this study was to compare the function and phenotypes of antigen‐specific CD4⁺ T cells in adults (n = 12), compared to infants (n = 20), following vaccination with acellular pertussis (DTaP) vaccine. Peripheral blood mononuclear cells (PBMCs) were stimulated with pertussis toxoid (PT), pertactin (PRN) and filamentous haemagglutinin (FHA). Multi‐parameter flow cytometry was used to delineate CD4⁺ T cell populations and phenotypes producing interferon (IFN)‐γ, interleukin (IL)‐2, tumour necrosis factor (TNF)‐α and IL‐4. Based on surface CD69 expression, infants demonstrated activation of vaccine antigen‐specific CD4⁺ T cells similar to adults. However, among infants, Boolean combinations of gates suggested that type 1 (Th‐1) CD4⁺ T cell responses were confined largely to TNF‐α⁺IL‐2⁺IFN‐γ^‐ or TNF‐α⁺IL‐2^‐IFN‐γ^‐. A significantly lower percentage of polyfunctional T helper type 1 (Th1) responses (TNF‐α⁺IFN‐γ⁺IL‐2⁺) and type 2 (Th2) responses (IL‐4) were present in the infants compared to adults. Moreover, a significantly higher percentage of infants' functional CD4⁺ T cells were restricted to CD45RA^‐CCR7⁺CD27⁺ phenotype, consistent with early‐stage differentiated pertussis‐specific memory CD4⁺ T cells. We show for the first time that DTaP vaccination‐induced CD4⁺ T cells in infants are functionally and phenotypically dissimilar from those of adults.     

Bystander stimulation of activated CD4+ T cells of unrelated specificity following a booster vaccination with tetanus toxoid

Antigen‐specific CD4⁺ T cells are central to natural and vaccine‐induced immunity. An ongoing antigen‐specific T‐cell response can, however, influence surrounding T cells with unrelated antigen specificities. We previously observed this bystander effect in healthy human subjects following recall vaccination with tetanus toxoid (TT). Since this interplay could be important for maintenance of memory, we have moved to a mouse model for further analysis. We investigated whether boosting memory CD4⁺ T cells against TT in vivo would influence injected CD4⁺ TCR transgenic T cells (OT‐II) specific for an unrelated OVA peptide. If OT‐II cells were pre‐activated with OVA peptide in vitro, these cells showed a bystander proliferative response during the ongoing parallel TT‐specific response. Bystander proliferation was dependent on boosting of the TT‐specific memory response in the recipients, with no effect in naive mice. Bystander stimulation was also proportional to the strength of the TT‐specific memory T‐cell response. T cells activated in vitro displayed functional receptors for IL‐2 and IL‐7, suggesting these as potential mediators. This crosstalk between a stimulated CD4⁺ memory T‐cell response and CD4⁺ T cells activated by an unrelated antigen could be important in human subjects continually buffeted by environmental antigens.     

Hyaluronan binding identifies the most proliferative activated and memory T cells

CD44 is expressed on T cells where its ability to bind hyaluronan is tightly regulated. Here, we investigated when T cells bind hyaluronan during an immune response. We found that naïve, murine T cells do not bind fluoresceinated hyaluronan but are induced to bind upon antigen‐induced T‐cell activation in vitro and in vivo. Hyaluronan binding occurred on proliferating T cells and the percentage of hyaluronan‐binding cells correlated with the strength of the activation stimulus. A small percentage of hyaluronan‐binding cells persisted after in vitro activation and had a memory phenotype (CD122⁺CD44^hi). This hyaluronan‐binding population increased after culture with IL‐7 or IL‐15 and proliferated more rapidly than nonbinding cells. In vivo, approximately 20–30% of antigen‐specific OT‐I CD8⁺ memory T cells in the spleen and BM bound hyaluronan. Hyaluronan binding identified memory cells that proliferated faster in IL‐7 and IL‐15, and enriched for CD62L⁺ central memory cells. In vivo homeostatic proliferation induced hyaluronan binding on a small percentage of the most rapidly dividing cells after several cell divisions. This study demonstrates that hyaluronan binding is induced upon antigen‐induced T‐cell activation and occurs on a percentage of the most proliferative activated and memory T cells.     

Functional exhaustion of CD4+ T cells induced by co‐stimulatory signals from myeloid leukaemia cells

To cope with immune responses, tumour cells implement elaborate strategies such as adaptive resistance and induction of T‐cell exhaustion. T‐cell exhaustion has been identified as a state of hyporesponsiveness that arises under continuous antigenic stimulus. Nevertheless, contribution of co‐stimulatory molecules to T‐cell exhaustion in cancer remains to be better defined. This study explores the role of myeloid leukaemia‐derived co‐stimulatory signals on CD4⁺ T helper (Th) cell exhaustion, which may limit anti‐tumour immunity. Here, CD86 and inducible T‐cell co‐stimulator ligand (ICOS‐LG) co‐stimulatory molecules that are found on myeloid leukaemia cells supported Th cell activation and proliferation. However, under continuous stimulation, T cells co‐cultured with leukaemia cells, but not with peripheral blood monocytes, became functionally exhausted. These in vitro‐generated exhausted Th cells were defined by up‐regulation of programmed cell death 1 (PD‐1), cytotoxic T‐lymphocyte antigen 4 (CTLA‐4), lymphocyte activation gene 3 (LAG3) and T‐cell immunoglobulin and mucin domain‐containing protein 3 (TIM‐3) inhibitory receptors. They were reluctant to proliferate upon re‐stimulation and produced reduced amounts of interleukin‐2 (IL‐2), tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ). Nonetheless, IL‐2 supplementation restored the proliferation capacity of the exhausted Th cells. When the co‐stimulation supplied by the myeloid leukaemia cells were blocked, the amount of exhausted Th cells was significantly decreased. Moreover, in the bone marrow aspirates from patients with acute myeloid leukaemia (AML) or myelodysplastic syndrome (MDS), a subpopulation of Th cells expressing PD‐1, TIM‐3 and/or LAG3 was identified together with CD86⁺ and/or ICOS‐LG⁺ myeloid blasts. Collectively, co‐stimulatory signals derived from myeloid leukaemia cells possess the capacity to facilitate functional exhaustion in Th cells.     

CD4+ T helper 2 cells--microbial triggers, differentiation requirements and effector functions

Over the past 10 years we have made great strides in our understanding of T helper cell differentiation, expansion and effector functions. Within the context of T helper type 2 (Th2) cell development, novel innate‐like cells with the capacity to secrete large amounts of interleukin‐5 (IL‐5), IL‐13 and IL‐9 as well as IL‐4‐producing and antigen‐processing basophils have (re)‐emerged onto the type 2 scene. To what extent these new players influence αβ⁺ CD4⁺ Th2 cell differentiation is discussed throughout this appraisal of the current literature. We highlight the unique features of Th2 cell development, highlighting the three necessary signals, T‐cell receptor ligation, co‐stimulation and cytokine receptor ligation. Finally, putting these into context, microbial and allergenic properties that trigger Th2 cell differentiation and how these influence Th2 effector function are discussed and questioned.     

Acute stimulation generates Tim‐3‐expressing T helper type 1 CD4 T cells that persist in vivo and show enhanced effector function

T‐cell immunoglobulin and mucin domain 3 (Tim‐3) is a surface receptor expressed by T helper type 1 (Th1) effector CD4 T cells, which are critical for defence against intracellular pathogens and have been implicated in autoimmune disease. Previous studies showed that Tim‐3 expression makes Th1 cells more susceptible to apoptosis and also marks functionally impaired T cells that arise due to chronic stimulation. However, other studies suggested that Tim‐3‐expressing Th1 cells do not always have these properties. To further define the relationship between Tim‐3 and Th1 cell function, we analysed the characteristics of Th1 cells that expressed Tim‐3 in response to brief stimulation in vitro or an acute viral infection in vivo. As expected, cultured CD4 T cells began expressing Tim‐3 during Th1 differentiation and secondary stimulation generated Tim‐3^? and Tim‐3⁺ fractions that were separated and further analysed. When injected into naive mice, Tim‐3⁺ cells down‐regulated Tim‐3 and survived equally well compared with Tim‐3^‐ cells. Further, Tim‐3^? and Tim‐3⁺ Th1 cells had similar functional responses when transferred into naive mice that were subsequently infected with lymphocytic choriomeningitis virus (LCMV). Cultured Th1 cells that expressed Tim‐3 following T‐cell receptor stimulation had a greater capacity to express signature Th1 cytokines than their Tim‐3^? counterparts and showed differential expression of genes that regulate CD4 T‐cell function. Consistent with these findings, Tim‐3⁺ Th1 cells generated in response to LCMV infection displayed augmented effector function relative to Tim‐3^? cells. These results suggest that Tim‐3 expression by Th1 cells responding to acute stimulation can mark cells that are functionally competent and have an augmented ability to produce cytokines.     

MicroRNA‐155 may be involved in the pathogenesis of atopic dermatitis by modulating the differentiation and function of T helper type 17 (Th17) cells

Our aims were to identify the differential expression of microRNA (miR)‐155, as well as to explore the possible regulatory effects of miR‐155 on the differentiation and function of T helper type 17 (Th17) cells in atopic dermatitis (AD). The Th17 cell percentage and expression levels of miR‐155, retinoic acid‐related orphan receptor (ROR)γt, interleukin (IL)‐17 and suppressor of cytokine signalling‐1 (SOCS1) in peripheral CD4⁺ T cells, plasma and skin specimens were detected and compared in AD patients and healthy subjects. A miR‐155 mimic and an inhibitor were transfected separately into AD CD4⁺ T cells to confirm the in‐vivo data. The Th17 cell percentage, miR‐155 expression, RORγt mRNA expression, IL‐17 mRNA expression and plasma concentration were increased significantly in AD patients compared with healthy subjects. Conversely, SOCS1 mRNA expression and plasma concentration were decreased significantly. Similar results were detected in cultured CD4⁺ T cells transfected with the miR‐155 mimic compared with a miR‐155 inhibitor or a negative control. Additionally, there was a sequential decrease in miR‐155 expression, as well as RORγt and IL‐17 mRNA expression, but an increase in SOCS1 mRNA expression, from AD lesional skin and perilesional skin to normal skin. Positive correlations were found between miR‐155 expression and AD severity, Th17 cell percentage, RORγt mRNA expression and IL‐17 mRNA expression and plasma concentration, while negative correlations were observed between miR‐155 expression and SOCS1 mRNA expression and plasma concentration in AD peripheral circulation and skin lesions. In conclusion, miR‐155 is over‐expressed and may be involved in AD pathogenesis by modulating the differentiation and function of Th17 cells.     

Interleukin-7 activates human naive CD4+ cells and primes for interleukin-4 production

Interleukin (IL)-4 is considered to be essential for T helper (Th)2 cell development, yet in areas of primary T cell activation, CD4⁺ cells are its only source. This implies that other signals must drive the initial expression of IL-4 production. The role of CD28 co-stimulation in Th2 subset development has been described. However, in mice deficient for CD28, Th2 responses are diminished, but not abrogated. Cytokines produced within the lymphoid tissue, e.g. IL-7, may be important in the primary activation of naive CD4⁺ cells. We have found that human naive CD4⁺ cells purified from umbilical cord blood express the IL-7 receptor and respond vigorously to IL-7 during primary stimulation. Naive CD4⁺ cells grown in IL-4, in the presence or absence of IL-2, fail to produce Th2 cytokines upon restimulation. In contrast, IL-7 induces development of a population of T cells that produce large amounts of IL-4. Growth in IL-7 also increases IL-2-induced production of interferon (IFN)-γ and IL-10 production. IL-7-induced IL-4 production is not inhibited by neutralizing antibodies to IL-4 on its receptor. This implies that IL-7 acts directly to induce Th2 subset development and not by up-regulating either production of IL-4 during culture or expression of the IL-4 receptor. Moreover, IL-7 potentiates the effects of CD28 co-stimulation on both naive CD4⁺ cell proliferation and subsequent IL-4 production. Following primary stimulation, CD4⁺ cells lose expression of the IL-7 receptor, resulting in IL-7 unresponsiveness. This work reveals a novel role for IL-7 in the primary activation of CD4⁺ cells. We propose that in conjunction with CD28 co-stimulation, IL-7 induces the initial expression of IL-4 production and that IL-4 acts subsequently to expand Th2 cytokine-producing cells at the appropriate anatomical site.     

Antigen‐specific over‐expression of human cartilage glycoprotein 39 on CD4+CD25+ forkhead box protein 3+ regulatory T cells in the generation of glucose‐6‐phosphate isomerase‐induced arthritis

Human cartilage gp‐39 (HC gp‐39) is a well‐known autoantigen in rheumatoid arthritis (RA). However, the exact localization, fluctuation and function of HC gp‐39 in RA are unknown. Therefore, using a glucose‐6‐phosphate isomerase (GPI)‐induced model of arthritis, we investigated these aspects of HC gp‐39 in arthritis. The rise in serum HC gp‐39 levels was detected on the early phase of GPI‐induced arthritis (day 7) and the HC gp‐39 mRNA was increased significantly on splenic CD4⁺T cells on day7, but not on CD11b⁺cells. Moreover, to identify the characterization of HC gp‐39⁺CD4⁺T cells, we assessed the analysis of T helper (Th) subsets. As a result, HC gp‐39 was expressed dominantly in CD4⁺CD25⁺ forkhead box protein 3 (FoxP3)⁺ refulatory T cells (T_reg), but not in Th1, Th2 or Th17 cells. Furthermore, to investigate the effect of HC gp‐39 to CD4⁺T cells, T cell proliferation assay and cytokine production from CD4⁺T cells using recombinant HC gp‐39 was assessed. We found that GPI‐specific T cell proliferation and interferon (IFN)‐γ or interleukin (IL)‐17 production were clearly suppressed by addition of recombinant HC gp‐39. Antigen‐specific over‐expression of HC gp‐39 in splenic CD4⁺CD25⁺ FoxP3⁺ T_reg cells occurs in the induction phase of GPI‐induced arthritis, and addition of recombinant HC gp‐39 suppresses antigen‐specific T‐cell proliferation and cytokine production, suggesting that HC gp‐39 in CD4⁺ T cells might play a regulatory role in arthritis.