IL‐33 promotes DC development in BM culture by triggering GM‐CSF production

Short‐term DC cultures generated with GM‐CSF and other cytokines have markedly improved our ability to study the immunobiology of DC. Here, we tested 65 cytokines individually for their potential to promote the generation of CD11c⁺ cells in a murine BM culture system. In addition to several cytokines known to promote DC survival and/or growth, IL‐33 was found to augment DC development time‐ and dose‐dependently. Although the resulting CD11c⁺ cells generated in the presence of IL‐33 exhibited a typical dendritic morphology, they expressed MHC class II molecules only at modest levels, showed negligible responses to TLR ligands, produced no detectable IL‐12 p70, displayed PD‐L1 and PD‐L2 on the surface, and failed to activate immunologically naïve T cells efficiently. IL‐33‐induced expansion of CD11c⁺ cells was completely blocked by anti‐GM‐CSF mAb, and GM‐CSF mRNA and protein expression in BM culture was markedly elevated by added IL‐33, indicating that IL‐33 promotes in vitro DC generation indirectly by a GM‐CSF‐dependent manner. With regard to the cellular source, IL‐33‐dependent GM‐CSF production was observed exclusively within the CD45⁺/FcεRI⁺ BM population. Not only do our results reinforce the notion that GM‐CSF serves as a primary DC growth factor, but they also reveal a previously unrecognized mechanism supporting DC development.     

Pathogenic IL‐23 signaling is required to initiate GM‐CSF‐driven autoimmune myocarditis in mice

Using a mouse model of experimental autoimmune myocarditis (EAM), we showed for the first time that IL‐23 stimulation of CD4⁺ T cells is required only briefly at the initiation of GM‐CFS‐dependent cardiac autoimmunity. IL‐23 signal, acting as a switch, turns on pathogenicity of CD4⁺ T cells, and becomes dispensable once autoreactivity is established. Il23a^?/? mice failed to mount an efficient Th17 response to immunization, and were protected from myocarditis. However, remarkably, transient IL‐23 stimulation ex vivo fully restored pathogenicity in otherwise nonpathogenic CD4⁺ T cells raised from Il23a^?/? donors. Thus, IL‐23 may no longer be necessary to uphold inflammation in established autoimmune diseases. In addition, we demonstrated that IL‐23‐induced GM‐CSF mediates the pathogenicity of CD4⁺ T cells in EAM. The neutralization of GM‐CSF abrogated cardiac inflammation. However, sustained IL‐23 signaling is required to maintain IL‐17A production in CD4⁺ T cells. Despite inducing inflammation in Il23a^?/? recipients comparable to wild‐type (WT), autoreactive CD4⁺ T cells downregulated IL‐17A production without persistent IL‐23 signaling. This divergence on the controls of GM‐CSF‐dependent pathogenicity on one side and IL‐17A production on the other side may contribute to the discrepant efficacies of anti‐IL‐23 therapy in different autoimmune diseases.     

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.     

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.     

Different interleukin‐17‐secreting Toll‐like receptor+ T‐cell subsets are associated with disease activity in multiple sclerosis

Signalling through Toll‐like receptors (TLRs) may play a role in the pathogenesis of autoimmune diseases, such as multiple sclerosis (MS). In the present study, the expression of TLR‐2, ‐4 and ‐9 was significantly higher on CD4⁺ and CD8⁺ T‐cells from MS patients compared to healthy individuals. Following in‐vitro activation, the proportion of interleukin (IL)‐17⁺ and IL‐6⁺ CD4⁺ and CD8⁺ T‐cells was higher in the patients. In addition, the proportion of IFN‐γ‐secreting TLR⁺ CD8⁺ T‐cells was increased in MS patients. Among different IL‐17⁺ T‐cell phenotypes, the proportion of IL‐17⁺ TLR⁺ CD4⁺ and CD8⁺ T‐cells producing IFN‐γ or IL‐6 were positively associated with the number of active brain lesions and neurological disabilities. Interestingly, activation of purified CD4⁺ and CD8⁺ T‐cells with ligands for TLR‐2 (Pam3Csk4), TLR‐4 [lipopolysaccharide (LPS)] and TLR‐9 [oligodeoxynucleotide (ODN)] directly induced cytokine production in MS patients. Among the pathogen‐associated molecular patterns (PAMPs), Pam3Csk4 was more potent than other TLR ligands in inducing the production of all proinflammatory cytokines. Furthermore, IL‐6, IFN‐γ, IL‐17 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) levels produced by Pam3Csk4‐activated CD4⁺ cells were directly associated with disease activity. A similar correlation was observed with regard to IL‐17 levels released by Pam3Csk4‐stimulated CD8⁺ T‐cells and clinical parameters. In conclusion, our data suggest that the expansion of different T helper type 17 (Th17) phenotypes expressing TLR‐2, ‐4 and ‐9 is associated with MS disease activity, and reveals a preferential ability of TLR‐2 ligand in directly inducing the production of cytokines related to brains lesions and neurological disabilities.     

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.     

CD8+ MAIT cells infiltrate into the CNS and alterations in their blood frequencies correlate with IL‐18 serum levels in multiple sclerosis

Recent findings indicate a pathogenic involvement of IL‐17‐producing CD8⁺ T cells in multiple sclerosis (MS). IL‐17 production has been attributed to a subset of CD8⁺ T cells that belong to the mucosal‐associated invariant T (MAIT) cell population. Here, we report a reduction of CD8⁺ MAIT cells in the blood of MS patients compared with healthy individuals, which significantly correlated with IL‐18 serum levels in MS patients. In vitro stimulation of peripheral blood mononuclear cells from healthy individuals and MS patients with IL‐18 specifically activated CD8⁺ MAIT cells. Moreover, IL‐18 together with T‐cell receptor stimulation induced, specifically on CD8⁺ MAIT cells, an upregulation of the integrin very late antigen‐4 that is essential for the infiltration of CD8⁺ T cells into the CNS. Notably, we were able to identify CD8⁺ MAIT cells in MS brain lesions by immunohistochemistry while they were almost absent in the cerebrospinal fluid (CSF). In summary, our findings indicate that an IL‐18–driven activation of CD8⁺ MAIT cells contributes to their CNS infiltration in MS, in turn leading to reduced CD8⁺ MAIT‐cell frequencies in the blood. Therefore, CD8⁺ MAIT cells seem to play a role in the innate arm of immunopathology in MS.     

EAE mediated by a non‐IFN‐γ/non‐IL‐17 pathway

Previous studies have shown that EAE can be elicited by the adoptive transfer of either IFN‐γ‐producing (Th1) or IL‐17‐producing (Th17) myelin‐specific CD4⁺ T‐cell lines. Paradoxically, mice deficient in either IFN‐γ or IL‐17 remain susceptible to EAE following immunization with myelin antigens in CFA. These observations raise questions about the redundancy of IFN‐γ and IL‐17 in autoimmune demyelinating disease mediated by a diverse, polyclonal population of autoreactive T cells. In this study, we show that an atypical form of EAE, induced in C57BL/6 mice by the adoptive transfer of IFN‐γ‐deficient effector T cells, required IL‐17 signaling for the development of brainstem infiltrates. In contrast, classical EAE, characterized by predominant spinal cord inflammation, occurred in the combined absence of IFN‐γ and IL‐17 signaling, but was dependent on GM‐CSF and CXCR2. Our findings contribute to a growing body of data, indicating that individual cytokines vary in their importance across different models of CNS autoimmunity.     

IL‐12‐polarized Th1 cells produce GM‐CSF and induce EAE independent of IL‐23

CD4⁺ T‐helper (Th) cells reactive against myelin antigens mediate the mouse model experimental autoimmune encephalomyelitis (EAE) and have been implicated in the pathogenesis of multiple sclerosis (MS). It is currently debated whether encephalitogenic Th cells are heterogeneous or arise from a single lineage. In the current study, we challenge the dogma that stimulation with the monokine IL‐23 is universally required for the acquisition of pathogenic properties by myelin‐reactive T cells. We show that IL‐12‐modulated Th1 cells readily produce IFN‐γ and GM‐CSF in the CNS of mice and induce a severe form of EAE via an IL‐23‐independent pathway. Th1‐mediated EAE is characterized by monocyte‐rich CNS infiltrates, elicits a strong proinflammatory cytokine response in the CNS, and is partially CCR2 dependent. Conversely, IL‐23‐modulated, stable Th17 cells induce EAE with a relatively mild course via an IL‐12‐independent pathway. These data provide definitive evidence that autoimmune disease can be driven by distinct CD4⁺ T‐helper‐cell subsets and polarizing factors.     

TRAF2 regulates peripheral CD8+ T‐cell and NKT‐cell homeostasis by modulating sensitivity to IL‐15

In this study, a critical and novel role for TNF receptor (TNFR) associated factor 2 (TRAF2) is elucidated for peripheral CD8⁺ T‐cell and NKT‐cell homeostasis. Mice deficient in TRAF2 only in their T cells (TRAF2TKO) show ∼40% reduction in effector memory and ∼50% reduction in naïve CD8⁺ T‐cell subsets. IL‐15‐dependent populations were reduced further, as TRAF2TKO mice displayed a marked ∼70% reduction in central memory CD8⁺CD44^hiCD122⁺ T cells and ∼80% decrease in NKT cells. TRAF2TKO CD8⁺CD44^hi T cells exhibited impaired dose‐dependent proliferation to exogenous IL‐15. In contrast, TRAF2TKO CD8⁺ T cells proliferated normally to anti‐CD3 and TRAF2TKO CD8⁺CD44^hi T cells exhibited normal proliferation to exogenous IL‐2. TRAF2TKO CD8⁺ T cells expressed normal levels of IL‐15‐associated receptors and possessed functional IL‐15‐mediated STAT5 phosphorylation, however TRAF2 deletion caused increased AKT activation. Loss of CD8⁺CD44^hiCD122⁺ and NKT cells was mechanistically linked to an inability to respond to IL‐15. The reduced CD8⁺CD44^hiCD122⁺ T‐cell and NKT‐cell populations in TRAF2TKO mice were rescued in the presence of high dose IL‐15 by IL‐15/IL‐15Rα complex administration. These studies demonstrate a critical role for TRAF2 in the maintenance of peripheral CD8⁺ CD44^hiCD122⁺ T‐cell and NKT‐cell homeostasis by modulating sensitivity to T‐cell intrinsic growth factors such as IL‐15.     

Both IFN‐γ and IL‐17 are required for the development of severe autoimmune gastritis

IL‐17, produced by a distinct lineage of CD4⁺ helper T (Th) cells termed Th17 cells, induces the production of pro‐inflammatory cytokines from resident cells and it has been demonstrated that over‐expression of IL‐17 plays a crucial role in the onset of several auto‐immune diseases. Here we examined the role of IL‐17 in the pathogenesis of autoimmune gastritis, a disease that was previously believed to be mediated by IFN‐γ. Significantly higher levels of IL‐17 and IFN‐γ were found in the stomachs and stomach‐draining lymph nodes of mice with severe autoimmune gastritis. Unlike IL‐17, which was produced solely by CD4⁺ T cells in gastritic mice, the majority of IFN‐γ‐producing cells were CD8⁺ T cells. However, CD8⁺ T cells alone were not able to induce autoimmune gastritis. T cells that were deficient in IL‐17 or IFN‐γ production were able to induce autoimmune gastritis but to a much lower extent compared with the disease induced by wild‐type T cells. These data demonstrate that production of neither IL‐17 nor IFN‐γ by effector T cells is essential for the initiation of autoimmune gastritis, but suggest that both are required for the disease to progress to the late pathogenic stage that includes significant tissue disruption.     

Phosphorylated STAT3 and PD‐1 regulate IL‐17 production and IL‐23 receptor expression in Mycobacterium tuberculosis infection

We studied the factors that regulate IL‐23 receptor expression and IL‐17 production in human tuberculosis infection. Mycobacterium tuberculosis (M. tb)‐stimulated CD4⁺ T cells from tuberculosis patients secreted less IL‐17 than did CD4⁺ T cells from healthy tuberculin reactors (PPD⁺). M. tb‐cultured monocytes from tuberculosis patients and PPD⁺ donors expressed equal amounts of IL‐23p19 mRNA and protein, suggesting that reduced IL‐23 production is not responsible for decreased IL‐17 production by tuberculosis patients. Freshly isolated and M. tb‐stimulated CD4⁺ T cells from tuberculosis patients had reduced IL‐23 receptor and phosphorylated STAT3 (pSTAT3) expression, compared with cells from PPD⁺ donors. STAT3 siRNA reduced IL‐23 receptor expression and IL‐17 production by CD4⁺ T cells from PPD⁺ donors. Tuberculosis patients had increased numbers of PD‐1⁺ T cells compared with healthy PPD⁺ individuals. Anti‐PD‐1 antibody enhanced pSTAT3 and IL‐23R expression and IL‐17 production by M. tb‐cultured CD4⁺ T cells of tuberculosis patients. Anti‐tuberculosis therapy decreased PD‐1 expression, increased IL‐17 and IFN‐γ production and pSTAT3 and IL‐23R expression. These findings demonstrate that increased PD‐1 expression and decreased pSTAT3 expression reduce IL‐23 receptor expression and IL‐17 production by CD4⁺ T cells of tuberculosis patients.     

Marked enhancement of antigen‐specific T‐cell responses by IL‐7‐fused nonlytic,but not lytic,Fc as a genetic adjuvant

IL‐7 plays a crucial role in the homeostatic proliferation, differentiation and survival of T cells, as well as in the survival and proliferation of precursor B cells. Here, we demonstrated that utilizing nonlytic Fc‐fused IL‐7 (IL‐7‐Fc^m) as a genetic adjuvant significantly enhanced not only CD4⁺ but also CD8⁺ T‐cell responses by E7 DNA immunization, in addition to improving protection against TC‐1‐induced tumors in comparison to IL‐7 alone. Similar results were obtained in OT‐1 adoptive transfer experiments with OVA DNA injection, suggesting independence from antigenic nature and experimental conditions. In particular, the increased frequency of CD8⁺ T cells was mainly due to enhanced T‐cell proliferation in T‐cell priming, and not to decreased cellular apoptosis. Interestingly, the enhanced adjuvant effect was not seen in the co‐delivery of lytic Fc‐fused IL‐7 (IL‐7‐Fc) which increases T‐cell apoptosis as well as T‐cell proliferation, suggesting that the T‐cell proliferative effect may be neutralized by T‐cell apoptosis. Thus, our findings suggest that nonlytic Fc, in contrast to lytic Fc, fusion to cytokines may provide an insight in designing a potent genetic adjuvant for inducing CD4⁺ and CD8⁺ T‐cell responses.     

The transduction pattern of IL‐12‐encoding lentiviral vectors shapes the immunological outcome

In situ modification of antigen‐presenting cells garnered interest in cancer immunotherapy. Therefore, we developed APC‐targeted lentiviral vectors (LVs). Unexpectedly, these LVs were inferior vaccines to broad tropism LVs. Since IL‐12 is a potent mediator of antitumor immunity, we evaluated whether this proinflammatory cytokine could enhance antitumor immunity of an APC‐targeted LV‐based vaccine. Therefore, we compared subcutaneous administration of broad tropism LVs (VSV‐G‐LV) with APC‐targeted LVs (DC2.1‐LV)‐encoding enhanced GFP and ovalbumin, or IL‐12 and ovalbumin in mice. We show that codelivery of IL‐12 by VSV‐G‐LVs or DC2.1‐LVs augments CD4⁺ or CD8⁺ T‐cell proliferation, respectively. Furthermore, we demonstrate that codelivery of IL‐12 enhances the CD4⁺ T_H1 profile irrespective of its delivery mode, while an increase in cytotoxic and therapeutic CD8⁺ T cells was only induced upon VSV‐G‐LV injection. While codelivery of IL‐12 by DC2.1‐LVs did not enhance CD8⁺ T‐cell performance, it increased expression of inhibitory checkpoint markers Lag3, Tim3, and PD‐1. Finally, the discrepancy between CD4⁺ T‐cell stimulation with and without functional CD8⁺ T‐cell stimulation by VSV‐G‐ and DC2.1‐LVs is partly explained by the observation that IL‐12 relieves CD8⁺ T cells from CD4⁺ T‐cell help, implying that a T_H1 profile is of minor importance for antitumor immunotherapy if IL‐12 is exogenously delivered.     

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.