CXCR4, but not CXCR3, drives CD8+ T‐cell entry into and migration through the murine bone marrow

The BM serves as a blood‐forming organ, but also supports the maintenance and immune surveillance function of many T cells. Yet, in contrast to other organs, little is known about the molecular mechanisms that drive T‐cell migration to and localization inside the BM. As BM accumulates many CXCR3‐expressing memory CD8⁺ T cells, we tested the involvement of this chemokine receptor, but found that CXCR3 is not required for BM entry. In contrast, we could demonstrate that CXCR4, which is highly expressed on both naive and memory CD8⁺ T cells in BM, is critically important for homing of all CD8⁺ T‐cell subsets to the BM in mice. Upon entry into the BM parenchyma, both naïve and memory CD8⁺ T cells locate close to sinusoidal vessels. Intravital imaging experiments revealed that CD8 T cells are surprisingly immobile and we found that they interact with ICAM‐1+VCAM‐1+BP‐1+ perivascular stromal cells. These cells are the major source of CXCL12, but also express key survival factors and maintenance cytokines IL‐7 and IL‐15. We therefore conclude that CXCR4 is not only crucial for entry of CD8⁺ T cells into the BM, but also controls their subsequent localization toward BM niches that support their survival.     

The regulation of regulation: interleukin‐10 increases CD4+ CD25+ regulatory T cells but impairs their immunosuppressive activity in murine models with schistosomiasis japonica or asthma

CD4⁺ CD25⁺ Foxp3⁺ regulatory T (Treg) cells play an important role in maintaining immune homeostasis. Interleukin‐10 (IL‐10), a cytokine with anti‐inflammatory capacities, also has a critical role in controlling immune responses. In addition, it is well known that production of IL‐10 is one of the suppression mechanisms of Treg cells. However, the action of IL‐10 on Treg cells themselves remains insufficiently understood. In this study, by using a Schistosoma japonicum‐infected murine model, we show that the elevated IL‐10 contributed to Treg cell induction but impaired their immunosuppressive function. Our investigations further suggest that this may relate to the up‐regulation of serum transforming growth factor (TGF‐β) level but the decrease in membrane‐bound TGF‐β of Treg cells by IL‐10 during S. japonicum infection. In addition, similar IL‐10‐mediated regulation on Treg cells was also confirmed in the murine model of asthma. In general, our findings identify a previously unrecognized opposing regulation of IL‐10 on Treg cells and provide a deep insight into the precise regulation in immune responses.     

Type I interferon potentiates T‐cell receptor mediated induction of IL‐10‐producing CD4+ T cells

Type I interferons (IFNs) have the dual ability to promote the development of the immune response and exert an anti‐inflammatory activity. We analyzed the integrated effect of IFN‐α, TCR signal strength, and CD28 costimulation on human CD4⁺ T‐cell differentiation into cell subsets producing the anti‐ and proinflammatory cytokines IL‐10 and IFN‐γ. We show that IFN‐α boosted TCR‐induced IL‐10 expression in activated peripheral CD45RA⁺CD4⁺ T cells and in whole blood cultures. The functional cooperation between TCR and IFN‐α efficiently occurred at low engagement of receptors. Moreover, IFN‐α rapidly cooperated with anti‐CD3 stimulation alone. IFN‐α, but not IL‐10, drove the early development of type I regulatory T cells that were mostly IL‐10⁺ Foxp3^? IFN‐γ^? and favored IL‐10 expression in a fraction of Foxp3⁺ T cells. Our data support a model in which IFN‐α costimulates TCR toward the production of IL‐10 whose level can be amplified via an autocrine feedback loop.     

Dermal IL‐17‐producing γδ T cells establish long‐lived memory in the skin

Conventional αβ T cells have the ability to form a long‐lasting resident memory T‐cell (T_RM) population in nonlymphoid tissues after encountering foreign antigen. Conversely, the concept of ‘innate memory’, where the ability of nonadaptive branches of the immune system to deliver a rapid, strengthened immune response upon reinfection or rechallenge, is just emerging. Using the αβ T‐cell‐independent Aldara psoriasis mouse model in combination with genetic fate‐mapping and reporter systems, we identified a subset of γδ T cells in mice that is capable of establishing a long‐lived memory population in the skin. IL‐17A/F‐producing Vγ4⁺Vδ4⁺ T cells populate and persist in the dermis for long periods of time after initial stimulation with Aldara. Experienced Vγ4⁺Vδ4⁺ cells show enhanced effector functions and mediate an exacerbated secondary inflammatory response. In addition to identifying a unique feature of γδ T cells during inflammation, our results have direct relevance to the human disease as this quasi‐innate memory provides a mechanistic insight into relapses and chronification of psoriasis.     

IL‐10 interferes directly with TCR‐induced IFN‐γ but not IL‐17 production in memory T cells

IL‐10 is a potent immunoregulatory and anti‐inflammatory cytokine. However, therapeutic trials in chronic inflammation have been largely disappointing. It is well established that IL‐10 can inhibit Th1 and Th2 cytokine production via indirect effects on APC. Less data are available about the influence of IL‐10 on IL‐17 production, a cytokine which has been recently linked to chronic inflammation. Furthermore, there are only few reports about a direct effect of IL‐10 on T cells. We demonstrate here that IL‐10 can directly interfere with TCR‐induced IFN‐γ production in freshly isolated memory T cells in the absence of APC. This effect was independent of the previously described effects of IL‐10 on T cells, namely inhibition of IL‐2 production and inhibition of CD28 signaling. In contrast, IL‐10 did not affect anti‐CD3/anti‐CD28‐induced IL‐17 production from memory T cells even in the presence of APC. This might have implications for the interpretation of therapeutic trials in patients with chronic inflammation where Th17 cells contribute to pathogenesis.     

CD8+ T cells producing IL‐3 and IL‐5 in non‐IgE‐mediated eosinophilic diseases

The cytokines IL‐5, IL‐3, and GM‐CSF are crucial for eosinophil development, survival, and function. To better understand their role in non‐IgE‐mediated eosinophilic diseases, we investigated plasma levels of these cytokines as well as cytokine expression in peripheral blood T cells. While we did not find any evidence for an involvement of T‐cell‐derived GM‐CSF, some of these patients did show an increased proportion of IL‐5‐ or IL‐3‐producing CD4⁺ T cells. However, in a significant proportion of patients, IL‐5‐producing CD8⁺ T cells, so‐called Tc2 cells, which in healthy donors can only be detected at very low levels, were prominent. Furthermore, increased IL‐3 production by CD8⁺ T cells was also observed, strongly supporting the notion that CD8⁺ T cells, not just CD4⁺ T cells, must also be considered as a potential source of the cytokines promoting eosinophilia.     

IL‐17‐producing γδ T cells

IL‐17 is produced not only by CD4⁺ αβ T cells, but also CD8⁺ αβ T cells, NKT cells, and γδ T cells, plus some non‐T cells, including macrophages and neutrophils. The ability of IL‐17 to deploy neutrophils to sites of inflammation imparts this cytokine with a key role in diseases of several types. Surprisingly, γδ T cells are responsible for much of the IL‐17 produced in several disease models, particularly early on.     

Rheumatoid arthritis fibroblast‐like synoviocytes co‐cultured with PBMC increased peripheral CD4+CXCR5+ICOS+ T cell numbers

‘Circulating’ T follicular helper cells (Tfh), characterized by their surface phenotypes CD4⁺chemokine receptor 5 (CXCR5)⁺ inducible co‐stimulatory molecule (ICOS)⁺, have been identified as the CD4⁺ T cell subset specialized in supporting the activation, expansion and differentiation of B cells. Fibroblast‐like synoviocytes (FLS) are critical in promoting inflammation and cartilage destruction in rheumatoid arthritis (RA), and the interaction between FLS and T cells is considered to facilitate FLS activation and T cell recruitment. However, it remains unknown whether RA‐FLS co‐cultured with activated peripheral blood mononuclear cells (PBMC) has immunoregulatory effects on peripheral Tfh. In the present study, we co‐cultured RA‐FLS with or without anti‐CD3/CD28‐stimulated PBMC. The results showed that RA‐FLS co‐cultured with stimulated PBMC could increase the numbers of CD4⁺CXCR5⁺ICOS⁺ T cells of RA PBMC possibly via the production of interleukin (IL)‐6, a critical cytokine involved in the differentiation of Tfh cells. We also observed increased reactive oxygen species (ROS) levels in the co‐culture system of RA‐FLS and PBMC. The percentage of CD4⁺CXCR5⁺ICOS⁺ T cells was decreased when ROS production was inhibited by N‐acetyl‐L‐cysteine (NAC), a specific inhibitor which can decrease ROS production. In addition, we showed that the higher levels of tumour necrosis factor (TNF)‐α and IL‐1β in the co‐culture system and the blocking of TNF receptor 2 (TNF‐R2) and IL‐1β receptor (IL‐1βR) both decreased the numbers of CD4⁺CXCR5⁺ICOS⁺ T cells. Our study reveals a novel mechanistic insight into how the interaction of RA‐FLS and PBMC participates in the RA pathogenesis, and also provides support for the biologicals application for RA.     

DX5+CD4+ T cells modulate CD4+ T‐cell response via inhibition of IL‐12 production by DCs

DX5⁺CD4⁺ T cells have been shown to dampen collagen‐induced arthritis and delayed‐type hypersensitivity reactions in mice. These cells are also potent modulators of T‐helper cell responses through direct effects on CD4⁺ T cells in an IL‐4 dependent manner. To further characterize this T‐cell population, we studied their effect on DCs and the potential consequences on T‐cell activation. Here, we show that mouse DX5⁺CD4⁺ T cells modulate DCs by robustly inhibiting IL‐12 production. This modulation is IL‐10 dependent and does not require cell contact. Furthermore, DX5⁺CD4⁺ T cells modulate the surface phenotype of LPS‐matured DCs. DCs modulated by DX5⁺CD4⁺ T‐cell supernatant express high levels of the co‐inhibitor molecules PDL‐1 and PDL‐2. OVA‐specific CD4⁺ T cells primed with DCs exposed to DX5⁺CD4⁺ T‐cell supernatant produce less IFN‐γ than CD4⁺ T cells primed by DCs exposed to either medium or DX5^?CD4⁺ T‐cell supernatant. The addition of IL‐12 to the co‐culture with DX5⁺ DCs restores IFN‐γ production. When IL‐10 present in the DX5⁺CD4⁺ T‐cell supernatant is blocked, DCs re‐establish their ability to produce IL‐12 and to efficiently prime CD4⁺ T cells. These data show that DX5⁺CD4⁺ T cells can indirectly affect the outcome of the T‐cell response by inducing DCs that have poor Th1 stimulatory function.     

Interleukin‐17 and interleukin‐22 promote tumor progression in human nonmelanoma skin cancer

Interleukin‐17 (IL‐17) and IL‐22 have been reported to play critical roles in autoimmunity and inflammation but information about their role in cancer is limited. In this study, we investigated the role of IL‐17 and IL‐22 in the progression of human skin basal‐cell carcinoma (BCC) and squamous‐cell carcinoma (SCC). We found that both tumor types are infiltrated with an high number of IL‐17⁺ and IL‐22⁺ T lymphocytes, as demonstrated by immunohistochemistry and by FACS analysis performed on peritumoral T‐cell lines isolated from skin biopsies. In vitro studies demonstrated that proliferation and migration of the BCC‐ and SCC‐cell lines M77015 and CAL27 were increased by IL‐17 and IL‐22. Moreover, IL‐17, alone or in combination with TNF‐α, was able to induce the production of two cytokines important for tumor progression, IL‐6 and IL‐8, in CAL27. We also showed that IL‐17 upregulated NF‐κB signaling, while IL‐22 activated the STAT3 pathway and the antiapoptotic AKT protein in M77015 and CAL27. Finally, in vivo experiments demonstrated that IL‐17 and IL‐22 enhanced tumor growth in nude mice injected with CAL27. Altogether, our findings indicate that high levels of IL‐22 and IL‐17 in the BCC and SCC microenvironment promote tumor progression.     

Interleukin‐17‐ and interleukin‐22‐secreting myelin‐specific CD4+ T cells resistant to corticoids are related with active brain lesions in multiple sclerosis patients

Multiple sclerosis (MS) is thought to be an autoimmune disorder. It is believed that immunological events in the early stages have great impact on the disease course. Therefore, we aimed to evaluate the cytokine profile of myelin basic protein (MBP)‐specific T cells from MS patients in the early phase of the disease and correlate it to clinical parameters, as well as to the effect of in vitro corticoid treatment. Peripheral T cells from MS patients were stimulated with MBP with our without hydrocortisone for 5 days. The cytokines level were determined by ELISA. The number of active brain lesions was determined by MRI scans, and the neurological disabilities were assessed by Expanded Disability Status Scale scores. Our results demonstrated that MS‐derived T cells responded to MBP by producing high levels of T helper type 1 (Th1) and Th17 cytokines. Although the production of interleukin‐6 (IL‐6), granulocyte–macrophage colony‐stimulating factor, IL‐17 and IL‐22 was less sensitive to hydrocortisone inhibition, only IL‐17 and IL‐22 levels correlated with active brain lesions. The ability of hydrocortisone to inhibit IL‐17 and IL‐22 production by MBP‐specific CD4⁺ T cells was inversely related to the number of active brain lesions. Finally, the production of both cytokines was significantly higher in cell cultures from Afrodescendant patients and it was less sensitive to hydrocortisone inhibition. In summary, our data suggest that IL‐17‐ and IL‐22‐secreting CD4⁺ T cells resistant to corticoids are associated with radiological activity of the MS in early stages of the disease, mainly among Afrodescendant patients who, normally, have worse prognosis.     

Dysregulation of circulating CD4+CXCR5+ T cells in type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is characterized by a chronic low‐grade inflammatory state. Follicular helper T cells (Tfh) play critical roles in inducing B‐cell activation and producing various cytokines, whereas circulating CD4+CXCR5+ T cells (CTfh) may act as a counterpart to measure Tfh cell disorders. In this study, we investigated whether Tfh could be involved in the development of T2DM by assessing CTfh in peripheral blood. CTfh and it subtypes were determined by measuring CD3, CD4, CXCR5, CXCR3, and CCR6 in 68 T2DM patients and 60 healthy controls using flow cytometry. Results showed that proportion of CTfh in the peripheral CD4+ T cells was significantly increased in T2DM patients (8.5 ± 0.5%) than in controls (4.5 ± 0.3%) (p < 0.001). Further study revealed that the balance of CTfh subtypes was greatly dysregulated, in which percentage of Th17 subtype was significantly increased in patients. Investigating the correlation between CTfh and risk factors of T2DM demonstrated that proportion of CTfh were significantly elevated in patients with body mass index (BMI) over 24.0 (p = 0.005). Interestingly, patients with abdominal obesity had further increase in CTfh than those without abdominal obesity. This study suggests the involvement of CTfh in T2DM, especially in T2DM‐related obesity.     

CD4+CXCR3+ T cells and plasmacytoid dendritic cells drive accelerated atherosclerosis associated with systemic lupus erythematosus

Cardiovascular disease due to accelerated atherosclerosis is the leading cause of death in patients with systemic lupus erythematosus (SLE). Noteworthy, accelerated atherosclerosis in SLE patients appears to be independant of classical Framingham risk factors. This suggests that aggravated atherosclerosis in SLE patients may be a result of increased inflammation and altered immune responses. However, the mechanisms that mediate the acceleration of atherosclerosis in SLE remain elusive. Based on experimental data which includes both humans (SLE patients and control subjects) and rodents (ApoE−/− mice), we herein propose a multi-step model in which the immune dysfunction associated with SLE (i.e. high level of IFN-α production by TLR 9-stimulated pDCs) is associated with, first, an increased frequency of circulating pro inflammatory CD4+CXCR3+ T cells; second, an increased production of CXCR3 ligands by endothelial cells; third, an increased recruitment of pro-inflammatory CD4+CXCR3+ T cells into the arterial wall, and fourth, the development of atherosclerosis. In showing how SLE may promote accelerated atherosclerosis, our model also points to hypotheses for potential interventions, such as pDCs-targeted therapy, that might be studied in the future.     

Zinc signals promote IL‐2‐dependent proliferation of T cells

Zinc signals, i.e. a change of the intracellular concentration of free zinc ions in response to receptor stimulation, are involved in signal transduction in several immune cells. Here, the role of zinc signals in T‐cell activation by IL‐2 was investigated in the murine cytotoxic T‐cell line CTLL‐2 and in primary human T cells. Measurements with the fluorescent dyes FluoZin‐3 and Zinquin showed that zinc is released from lysosomes into the cytosol in response to stimulation of the IL‐2‐receptor. Activation of the ERK‐pathway was blocked by chelation of free zinc with N,N,N′,N′‐tetrakis‐2(pyridyl‐methyl)ethylenediamine, whereas zinc was not required for STAT5 phosphorylation. In addition, the key signaling molecules MEK and ERK were activated in response to elevated free intracellular zinc, induced by incubation with zinc and the ionophore pyrithione. Downstream of ERK activation, ERK‐specific gene expression of c‐fos and IL‐2‐induced proliferation was found to depend on zinc. Further experiments indicated that inhibition of MEK and ERK‐dephosphorylating protein phosphatases is the molecular mechanism for the influence of zinc on this pathway. In conclusion, an increase of cytoplasmic free zinc is required for IL‐2‐induced ERK signaling and proliferation of T cells.