Post-Transplant Immunosuppression: Regulation of the Efflux of Allospecific Effector T Cells from Lymphoid Tissues

A functional sphingosine-1-phosphate (S1P) receptor antagonist specifically inhibited the egress of activated allospecific T cells from draining popliteal lymph nodes in alloantigen-sensitised mice. The level of S1P receptor 1 (S1PR1) mRNA was similarly reduced 1 and 3 days after mitogenic activation of T cells. However, the response of these cells to the S1PR1-specific agonist SEW2871 was only reduced on the first day after T cell activation with normal receptor-mediated Akt-phosphorylation restored by day 3. Longitudinal analysis of CD69 expression showed that almost all T cells expressed this antigen on days 1 and 3 after activation. However, the absolute level of cell-surface expression of CD69 peaked on undivided T cells and was then halved by each of the first 3 cycles of mitosis. CD69-specific small interfering RNA (siRNA) reduced the maximal level of CD69 expression by undivided, mitogen-stimulated T cells. These cells retained their capacity to phosphorylate Akt in response to stimulation with SEW2871. These data show that S1P receptors are involved in controlling the egress of activated T cells from lymph nodes, and that S1PR1 function is regulated by the level of T cell surface CD69. They suggest a potential for augmentation of this process to deplete alloreactive effector cells after organ transplantation.     

Th22 Cells as Well as Th17 Cells Expand Differentially in Patients with Early-Stage and Late-Stage Myelodysplastic Syndrome

Background

Immunological mechanisms are increasingly recognized in the progression of myelodysplastic syndrome (MDS). Early-stage MDS (E-MDS) is characterized by autoimmune-mediated myelosuppression whereas late-stage MDS (L-MDS) involves immune evasion, giving dysplastic cells growth potential to progress into acute myeloid leukemia. T-helper (Th) 22 is involved in the pathogenesis of inflammatory autoimmunity and tumorigenesis. The roles of Th22 cells in the pathophysiology of E-MDS and L-MDS remain unsettled.

Design and Methods

We studied 37 MDS patients (E-MDS, n = 17; L-MDS, n = 20) and 20 healthy controls to characterize their peripheral blood (PB), as well as 25 MDS patients and 10 healthy controls to characterize their bone marrow(BM). The expression of Interleukin-22 (IL-22), IL-17 or interferon gamma (IFN-γ) was examined in E-MDS, L-MDS patients and controls by flow cytometry. The mRNA expression levels of RAR-related orphan receptor C (RORC), IL-6, tumor necrosis factor alpha (TNF-α) and IL-23 in peripheral blood mononuclear cells (PBMCs) were determined by real-time quantitative polymerase chain reaction. The levels of IL-22 and IL-17 both in PB and BM plasma were examined by enzyme-linked immunosorbent assay.

Results

In E-MDS, peripheral Th17 cells were significantly elevated and correlated with peripheral Th22 cells compared with healthy controls and L-MDS. Significantly higher levels of peripheral Th22 expansion, mRNA expression of IL-6, TNF-α and lower level of RORC mRNA expression were observed in L-MDS compared with E-MDS. No statistical difference was found in IL-23 mRNA expression or plasma IL-22, IL-17 levels among E-MDS, L-MDS and controls.

Conclusions

Our data demonstrated that L-MDS cohort had increased frequencies of peripheral Th22 cells and higher mRNA expression levels of IL-6 and TNF-α, indicating that Th22 cells along with Th17 cells or not are involved in the dynamic immune responses of MDS.     

Dysregulation of melanocyte function by Th17-related cytokines: significance of Th17 cell infiltration in autoimmune vitiligo vulgaris

The aim of this study was to determine whether CD4(+) IL-17A(+) Th17 cells infiltrate vitiligo skin and to investigate whether the proinflammatory cytokines related to Th17 cell influence melanocyte enzymatic activity and cell fate. An immunohistochemical analysis showed Th17 cell infiltration in 21 of 23 vitiligo skin samples in addition to CD8(+) cells on the reticular dermis. An in vitro analysis showed that the expression of MITF and downstream genes was downregulated in melanocytes by treatment with interleukin (IL)-17A, IL-1β, IL-6, and tumor necrosis factor (TNF)-α. Treatment with these cytokines also induced morphological shrinking in melanocytes, resulting in decreased melanin production. In terms of local cytokine network in the skin, IL-17A dramatically induced IL-1β, IL-6, and TNF-α production in skin-resident cells such as keratinocytes and fibroblasts. Our results provide evidence of the influence of a complex Th17 cell-related cytokine environment in local depigmentation in addition to CD8(+) cell-mediated melanocyte destruction in autoimmune vitiligo.     

Stromal Cells Induce Th17 during Helicobacter pylori Infection and in the Gastric Tumor Microenvironment

Gastric cancer is associated with chronic inflammation and Helicobacter pylori infection. Th17 cells are CD4⁺ T cells associated with infections and inflammation; but their role and mechanism of induction during carcinogenesis is not understood. Gastric myofibroblasts/fibroblasts (GMF) are abundant class II MHC expressing cells that act as novel antigen presenting cells. Here we have demonstrated the accumulation of Th17 in H. pylori-infected human tissues and in the gastric tumor microenvironment. GMF isolated from human gastric cancer and H. pylori infected tissues co-cultured with CD4⁺ T cells induced substantially higher levels of Th17 than GMF from normal tissues in an IL-6, TGF-β, and IL-21 dependent manner. Th17 required interaction with class II MHC on GMF for activation and proliferation. These studies suggest that Th17 are induced during both H. pylori infection and gastric cancer in the inflammatory milieu of gastric stroma and may be an important link between inflammation and carcinogenesis.     

Treg细胞和Th17细胞在多发性骨髓瘤中的研究进展

多发性骨髓瘤(MM)系血液系统的恶性肿瘤,以老年人多见.目前治疗以化疗和自身干细胞移植为主,仍难以治愈.多发性骨髓瘤的进展涉及到一系列基因和骨髓微环境的改变,这些改变恰好促进了肿瘤细胞的生长并瓦解了局部的免疫反应.CD4+和CD8+T细胞在多发性骨髓瘤患者体内数量和功能的改变都已经阐明.Treg细胞和Th17细胞的平衡在维持多发性骨髓瘤患者的抗肿瘤免疫中起着至关重要的作用.Treg细胞负责维持机体对外来抗原和自身抗原的免疫耐受.Th17细胞主要参与机体抵抗真菌和寄生虫感染、炎症反应和自身免疫.多发性骨髓瘤患者体内TGF-β和IL-6高水平表达,并可直接或通过其他促炎因子影响Th17细胞的分化,进而调节机体的抗肿瘤免疫应答.因此我们针对Treg细胞和Th17细胞在多发性骨髓瘤中的研究进展进行阐述.     

Elevated Frequencies of Circulating Th22 Cell in Addition to Th17 Cell and Th17/Th1 Cell in Patients with Acute Coronary Syndrome

Background

Atherosclerosis is a chronic inflammatory disease mediated by immune cells. Th22 cells are CD4⁺ T cells that secret IL-22 but not IL-17 or IFN-γ and are implicated in the pathogenesis of inflammatory disease. The roles of Th22 cells in the pathophysiologic procedures of acute coronary syndrome (ACS) remain unclear. The purpose of this study is to investigate the profile of Th22, Th17 and Th17/Th1 cells in ACS patients, including unstable angina (UA) and acute myocardial infarction (AMI) patients.

Design and Methods

In this study, 26 AMI patients, 16 UA patients, 16 stable angina (SA) patients and 16 healthy controls were included. The frequencies of Th22, Th17 and Th17/Th1 cells in AMI, UA, SA patients and healthy controls were examined by flow cytometry. Plasma levels of IL-22, IL-17 and IFN-γ were measured by enzyme-linked immunosorbent assay (ELISA).

Results

Th22, Th17 and Th17/Th1 cells were significantly increased in AMI and UA patients compared with SA patients and healthy controls. Moreover, plasma IL-22 level was significantly elevated in AMI and UA patients. In addition, Th22 cells correlated positively with IL-22 as well as Th17 cells in AMI and UA patients.

Conclusion

Our findings showed increased frequencies of both Th22 and Th17 cells in ACS patients, which suggest that Th22 and Th17 cells may play a potential role in plaque destabilization and the development of ACS.     

Frequency of Th17 CD4+ T Cells in Early Rheumatoid Arthritis: A Marker of Anti-CCP Seropositivity

Objective

To examine the frequency and phenotype of Th17 cells in the peripheral blood of early RA (eRA) patients.

Methods

CD4+ T cells were isolated from the peripheral blood of 33 eRA patients, 20 established RA patients and 53 healthy controls (HC), and from the synovial fluid of 20 established RA patients (RASF), by ficoll-hypaque gradient and magnetical negative selection. After polyclonal stimulation, the frequency of Th17 and Th1 cells was determined by flow cytometry and concentrations of IL-17, IFN-γ, TNF-α and IL-10 were measured by ELISA in cell-free supernatants.

Results

When all of our eRA patients were analyzed together, a significantly lower percentage of circulating Th17 cells and a lower CD4-derived IL-17 secretion were observed in comparison with HC. However, after stratifying by anti-CCP antibody status, circulating Th17 cells were decreased in anti-CCP(+) but not in anti-CCP(-)-eRA. All Th17 cells were CD45RO+CD45RA- and CCR6+. Dual Th17/Th1 cells were also exclusively decreased in anti-CCP(+)-eRA. Circulating Th17 and Th17/Th1 cells were negatively correlated with anti-CCP titres. When anti-CCP(+)-eRA patients were retested one year after initiating treatment with oral methotrexate, their circulating Th17 frequency was no longer different from HC. Of note, the percentage of circulating Th1 cells and the secretion of CD4-derived IFN-γ, TNF-α and IL-10 were not different between eRA patients and HC. In established RA patients, circulating Th17 and T17/Th1 cell frequencies were comparable to HC. In RASF, both Th17 and Th1 cells were increased when compared with blood of eRA patients, established RA patients and HC.

Conclusion

Decreased circulating Th17 levels in eRA seem to be a marker of anti-CCP seropositivity, and return to levels observed in healthy controls after treatment with methotrexate.     

Expansion of Pathogen-Specific Mono- and Multifunctional Th1 and Th17 Cells in Multi-Focal Tuberculous Lymphadenitis

Background

Th1 and Th17 responses are known to play an important role in immunity to pulmonary tuberculosis (PTB), although little is known about their role in extrapulmonary forms of tuberculosis (TB).

Methods

To identify the role of Th1, Th17, and Th22 cells in multi-focal TB lymphadenitis (TBL), we examined mycobacteria–specific immune responses in the whole blood of individuals with PTB (n = 20) and compared them with those with TBL (n = 25).

Results

Elevated frequencies of CD4⁺ T cells expressing IFN- γ, TNF-α, and IL-2 were present in individuals with TBL compared with those with PTB at baseline and in response to ESAT-6 and CFP-10. Similarly, increased frequencies of CD4⁺ T cells expressing IL-17A, IL-17F, and IFN-γ were also present in individuals with TBL at baseline and following ESAT-6 and CFP-10 stimulation although no significant difference in frequency of Th22 cells was observed. Finally, frequencies of Th1 (but not Th17) cells exhibited a significantly negative correlation with natural regulatory T cell frequencies at baseline.

Conclusions

Multi-focal TB lymphadenitis is therefore characterized by elevated frequencies of Th1 and Th17 cells, indicating that Th1 and Th17 responses in TB disease are probably correlates of disease severity rather than of protective immunity.     

SGK1 Governs the Reciprocal Development of Th17 and Regulatory T Cells

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The Metalloprotease ADAM12 Regulates the Effector Function of Human Th17 Cells

A key modulator of immune homeostasis, TGFβ has an important role in the differentiation of regulatory T cells (Tregs) and IL-17-secreting T cells (Th17). How TGFβ regulates these functionally opposing T cell subsets is not well understood. We determined that an ADAM family metalloprotease called ADAM12 is specifically and highly expressed in both Tregs and CCR6+ Th17 cells. ADAM12 is induced in vitro upon differentiation of naïve T cells to Th17 cells or IL-17-secreting Tregs. Remarkably, silencing ADAM12 expression in CCR6+ memory T cells enhances the production of Th17 cytokines, similar to suppressing TGFβ signaling. Further, ADAM12 knockdown in naïve human T cells polarized towards Th17/Treg cells, or ectopically expressing RORC, greatly enhances IL-17-secreting cell differentiation, more potently then inhibiting TGFβ signals. Together, our findings reveal a novel regulatory role for ADAM12 in Th17 cell differentiation or function and may have implications in regulating their aberrant responses during immune pathologies.     

Linking Power Doppler Ultrasound to the Presence of Th17 Cells in the Rheumatoid Arthritis Joint

Background

Power Doppler ultrasound (PDUS) is increasingly used to assess synovitis in Rheumatoid Arthritis (RA). Prior studies have shown correlations between PDUS scores and vessel counts, but relationships with T cell immunopathology have not been described.

Methodology/Principal Findings

PBMC were isolated from healthy controls (HC) or RA patients and stimulated ex vivo with PMA and ionomycin for 3 hours in the presence of Golgistop. Paired synovial fluid (SF) or synovial tissue (ST) were analysed where available. Intracellular expression of IL-17, IFNγ, and TNFα by CD4+ T cells was determined by flow cytometry. Synovial blood flow was evaluated by PDUS signal at the knees, wrists and metacarpophalangeal joints of RA patients. Serum, SF and fibroblast culture supernatant levels of vascular endothelial growth factor-A (VEGF-A) were measured by ELISA. The frequency of IL17+IFNγ-CD4+ T cells (Th17 cells) was significantly elevated in peripheral blood (PB) from RA patients vs. HC (median (IQR) 0.5 (0.28–1.59)% vs. 0.32 (0.21–0.54)%, p = 0.005). Th17 cells were further enriched (mean 6.6-fold increase) in RA SF relative to RA PB. Patients with active disease had a higher percentage of IL-17+ T cells in ST than patients in remission, suggesting a possible role for Th17 cells in active synovitis in RA. Indeed, the percentage of Th17 cells, but not Th1, in SF positively correlated with CRP (r = 0.51, p = 0.04) and local PDUS-defined synovitis (r = 0.61, p = 0.002). Furthermore, patients with high levels of IL-17+CD4+ T cells in SF had increased levels of the angiogenic factor VEGF-A in SF. Finally, IL-17, but not IFNγ, increased VEGF-A production by RA synovial fibroblasts in vitro.

Conclusions/Significance

Our data demonstrate a link between the presence of pro-inflammatory Th17 cells in SF and local PDUS scores, and offer a novel immunological explanation for the observation that rapid joint damage progression occurs in patients with persistent positive PDUS signal.     

Initiation of ART during Early Acute HIV Infection Preserves Mucosal Th17 Function and Reverses HIV-Related Immune Activation

Mucosal Th17 cells play an important role in maintaining gut epithelium integrity and thus prevent microbial translocation. Chronic HIV infection is characterized by mucosal Th17 cell depletion, microbial translocation and subsequent immune-activation, which remain elevated despite antiretroviral therapy (ART) correlating with increased mortality. However, when Th17 depletion occurs following HIV infection is unknown. We analyzed mucosal Th17 cells in 42 acute HIV infection (AHI) subjects (Fiebig (F) stage I-V) with a median duration of infection of 16 days and the short-term impact of early initiation of ART. Th17 cells were defined as IL-17+ CD4+ T cells and their function was assessed by the co-expression of IL-22, IL-2 and IFNγ. While intact during FI/II, depletion of mucosal Th17 cell numbers and function was observed during FIII correlating with local and systemic markers of immune-activation. ART initiated at FI/II prevented loss of Th17 cell numbers and function, while initiation at FIII restored Th17 cell numbers but not their polyfunctionality. Furthermore, early initiation of ART in FI/II fully reversed the initially observed mucosal and systemic immune-activation. In contrast, patients treated later during AHI maintained elevated mucosal and systemic CD8+ T-cell activation post initiation of ART. These data support a loss of Th17 cells at early stages of acute HIV infection, and highlight that studies of ART initiation during early AHI should be further explored to assess the underlying mechanism of mucosal Th17 function preservation.     

Expansion in CD39+ CD4+ Immunoregulatory T Cells and Rarity of Th17 Cells in HTLV-1 Infected Patients Is Associated with Neurological Complications

HTLV-1 infection is associated with several inflammatory disorders, including the neurodegenerative condition HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is unclear why a minority of infected subjects develops HAM/TSP. CD4⁺ T cells are the main target of infection and play a pivotal role in regulating immunity to HTLV and are hypothesized to participate in the pathogenesis of HAM/TSP. The CD39 ectonucleotidase receptor is expressed on CD4⁺ T cells and based on co-expression with CD25, marks T cells with distinct regulatory (CD39⁺CD25⁺) and effector (CD39⁺CD25⁻) function. Here, we investigated the expression of CD39 on CD4⁺ T cells from a cohort of HAM/TSP patients, HTLV-1 asymptomatic carriers (AC), and matched uninfected controls. The frequency of CD39⁺ CD4⁺ T cells was increased in HTLV-1 infected patients, regardless of clinical status. More importantly, the proportion of the immunostimulatory CD39⁺CD25⁻ CD4⁺ T-cell subset was significantly elevated in HAM/TSP patients as compared to AC and phenotypically had lower levels of the immunoinhibitory receptor, PD-1. We saw no difference in the frequency of CD39⁺CD25⁺ regulatory (Treg) cells between AC and HAM/TSP patients. However, these cells transition from being anergic to displaying a polyfunctional cytokine response following HTLV-1 infection. CD39⁻CD25⁺ T cell subsets predominantly secreted the inflammatory cytokine IL-17. We found that HAM/TSP patients had significantly fewer numbers of IL-17 secreting CD4⁺ T cells compared to uninfected controls. Taken together, we show that the expression of CD39 is upregulated on CD4⁺ T cells HAM/TSP patients. This upregulation may play a role in the development of the proinflammatory milieu through pathways both distinct and separate among the different CD39 T cell subsets. CD39 upregulation may therefore serve as a surrogate diagnostic marker of progression and could potentially be a target for interventions to reduce the development of HAM/TSP.     

Development of a unique epigenetic signature during in vivo Th17 differentiation

Activated naive CD4⁺ T cells are highly plastic cells that can differentiate into various T helper (Th) cell fates characterized by the expression of effector cytokines like IFN-γ (Th1), IL-4 (Th2) or IL-17A (Th17). Although previous studies have demonstrated that epigenetic mechanisms including DNA demethylation can stabilize effector cytokine expression, a comprehensive analysis of the changes in the DNA methylation pattern during differentiation of naive T cells into Th cell subsets is lacking. Hence, we here performed a genome-wide methylome analysis of ex vivo isolated naive CD4⁺ T cells, Th1 and Th17 cells. We could demonstrate that naive CD4⁺ T cells share more demethylated regions with Th17 cells when compared to Th1 cells, and that overall Th17 cells display the highest number of demethylated regions, findings which are in line with the previously reported plasticity of Th17 cells. We could identify seven regions located in Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 showing pronounced demethylation selectively in ex vivo isolated Th17 cells when compared to other ex vivo isolated Th cell subsets and in vitro generated Th17 cells, suggesting that this unique epigenetic signature allows identifying and functionally characterizing in vivo generated Th17 cells.     

Prevalence and Clinical Relevance of T-Helper Cells,Th17 and Th1, in Hepatitis B Virus-Related Hepatocellular Carcinoma

Background and Aims

An immune imbalance in the cytokine profile exerts a profound influence on the progression of hepatitis B virus (HBV) infections and hepatocellular carcinoma (HCC). The present study evaluated the immune status of T helper (Th) 17 and Th1 cells in patients with HBV-related and non-HBV-related HCC.

Methods

We randomly enrolled 150 patients with HCC. Blood samples and tissue samples were obtained. The distributions and phenotypic features of Th17 and Th1 cells were determined by flow cytometry and/or immunohistochemistry.

Results

Compared to corresponding non-tumor regions, the levels of Th17 and Th1 cells were significantly increased in tumors of patients with HCC (P<0.001). The intratumoral densities of IL-17-producing cells and IFN-γ-producing cells were associated with overall survival (OS, P = 0.001) and disease-free survival (DFS, P = 0.001) of patients with HCC. The ratio of Th17 to Th1 in HBV-related HCC was higher than in non-HBV-related HCC. A multivariate Cox analysis revealed that the Th17 to Th1 ratio was an independent prognostic factor for OS (HR = 2.651, P = 0.007) and DFS (HR = 2.456, P = 0.002).

Conclusions

HBV infections can lead to an imbalance in immune status in patients with HCC. An elevated Th17 to Th1 ratio may promote tumor progression. The Th17 to Th1 ratio could serve as a potential prognostic marker for scoring the severity of HCC.     

CD161 Expression Defines a Th1/Th17 Polyfunctional Subset of Resident Memory T Lymphocytes in Bronchoalveolar Cells

Alveolar resident memory T cells (T_RM) comprise a currently uncharacterized mixture of cell subpopulations. The CD3⁺CD161⁺ T cell subpopulation resides in the liver, intestine and skin, but it has the capacity for tissue migration; however, the presence of resident CD3⁺CD161⁺ T cells in the bronchoalveolar space under normal conditions has not been reported. Bronchoalveolar cells (BACs) from healthy volunteers were evaluated and found that 8.6% (range 2.5%-21%) of these cells were CD3⁺ T lymphocytes. Within the CD3⁺ population, 4.6% of the cells (2.1–11.3) expressed CD161 on the cell surface, and 74.2% of the CD161⁺CD3⁺ T cells expressed CD45RO. The number of CD3⁺CD161⁺ T cells was significantly lower in the bronchoalveolar space than in the blood (4.6% of BACs vs 8.4% of peripheral blood mononuclear cells (PBMCs); P<0.05). We also found that 2.17% of CD4⁺ T lymphocytes and 1.52% of CD8⁺ T lymphocytes expressed CD161. Twenty-two percent of the alveolar CD3⁺CD161⁺ T lymphocytes produced cytokines upon stimulation by PMA plus ionomycin, and significantly more interferon gamma (IFN-γ) was produced compared with other cytokines (P = 0.05). Most alveolar CD3⁺CD161⁺ T cells produced interleukin-17 (IL-17) and IFN-γ simultaneously, and the percentage of these cells was significantly higher than the percentage of CD3⁺CD161⁻ T cells. Moreover, the percentage of alveolar CD3⁺CD161⁺ T lymphocytes that produced IFN-γ/IL-17 was significantly higher than those in the peripheral blood (p<0.05). In conclusion, Th1/Th17-CD3⁺CD161⁺ T_RM could contribute to compartment-specific immune responses in the lung.