Activation of the Notch signaling pathway disturbs the CD4+/CD8+, Th17/Treg balance in rats with experimental autoimmune uveitis

Objective and design

The present study aimed to investigate the relationship between the disturbed balance of CD4⁺/CD8⁺, Th17/Treg and the activation of the Notch signaling pathway in experimental autoimmune uveitis (EAU).

Methods

An EAU rat model was induced in Lewis rats, and pathology analysis was performed by hematoxylin and eosin (H&E) staining. CD4⁺, CD8⁺, Th17, and Treg levels in spleen, lymph nodes and eye tissues were determined by flow cytometry. Meanwhile, the expression of Notch1, DLL4, IL-10, and IL-17 was determined by quantitative polymerase chain reaction (Q-PCR) and enzyme-linked immunosorbent assay (ELISA). In addition, the inhibitory effect of N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT) on Th17 differentiation by Notch signaling in vitro was further investigated using T lymphocytes from EAU rats on day 12 post-immunization by flow cytometry.

Results

The pathological results showed that inflammatory cell infiltration occurred in ocular tissues in EAU rats. The CD4⁺/CD8⁺ and Th17/Treg ratios in EAU rats were apparently higher than those in normal control individuals. Q-PCR and ELISA analyses indicated the expression of Notch1, DLL4, IL-10, and IL-17 in EAU rats gradually increased on day 6 after immunization, peaked on day 12, and then gradually decreased. The dynamic trends in Notch1 and DLL4 expression in EAU rats were identical to those of CD4⁺/CD8⁺ and Th17/Treg levels. DAPT can significantly inhibit the activation of Notch signaling, decrease Th17 cell differentiation, and attenuate the level of the Th17 cell lineage, contributing to the balance of the Th17/Treg ratio.

Conclusion

The activation of the Notch signaling pathway can regulate Th17 and Treg cell differentiation, disrupt the CD4⁺/CD8⁺ and Th17/Treg balance, and aggravate the severity of EAU; inactivation of the Notch signaling pathway contributes to the CD4⁺/CD8⁺ and Th17/Treg balance in EAU rats. Our findings highlighted that the dynamic change in the CD4⁺/CD8⁺ and Th17/Treg ratio was consistent with the expression trend of Notch signaling in EAU rats, suggesting that Notch signaling may be a potentially important therapeutic target in clinical practice.

     

Increased Th17 cells are accompanied by FoxP3 Treg cell accumulation and correlated with psoriasis disease severity

Both T-helper 17 cells (Th17) and CD4⁺CD25⁺ regulatory T cells (Treg) play important roles in the pathogenesis of psoriasis. However, the relationship between Th17 and Treg cells and their dynamic variations in psoriasis remain unclear. In this study, we found that both Th17 and FoxP3⁺ Treg cells were increased in psoriasis patients both in the peripheral circulation and skin tissue lesions and were positively correlated with disease severity. The ratio of Th17 to Treg cells in skin tissue lesions was inversely correlated with PASI scores, while it was positively correlated with PASI scores in the circulation. IL-17 secretion by CD4⁺ T cells was not regulated by Treg cells, even though Treg cells exhibited significant inhibition on CD4⁺ T cells proliferation and IFN-γ production. These findings provide new information regarding the association between Th17 and Treg cells, which will further our understanding of the pathogenesis of psoriasis.     

Enrichment of regulatory T cells in invasive breast tumor correlates with the upregulation of IL‐17A expression and invasiveness of the tumor

Breast cancer is a leading cause of neoplasia‐associated death in women worldwide. Regulatory T (Treg) and Th17 cells are enriched within some tumors, but the role these cells play in invasive ductal carcinoma (IDC) of the breast is unknown. We show that CD25⁺CD4⁺ T cells from PBMCs and tumor express high levels of Foxp3, GITR, CTLA‐4, and CD103, indicating that tumor‐infiltrating Treg cells are functional and possibly recruited by CCL22. Additionally, we observed upregulation of Th17‐related molecules (IL‐17A, RORC, and CCR6) and IL‐17A produced by tumor‐infiltrating CD4⁺ and CD8⁺ T lymphocytes. The angiogenic factors CXCL8, MMP‐2, MMP‐9, and vascular endothelial growth factor detected within the tumor are possibly induced by IL‐17 and indicative of poor disease prognosis. Treg and Th17 cells were synchronically increased in IDC patients, with positive correlation between Foxp3, IL‐17A, and RORC expression, and associated with tumor aggressiveness. Therefore, Treg and Th17 cells can affect disease progression by Treg‐cell‐mediated suppression of the effector T‐cell response, as indicated by a decrease in the proliferation of T cells isolated from PBMCs of IDC patients and induction of angiogenic factors by IL‐17‐producing Th17. The understanding of regulation of the Treg/Th17 axis may result in novel perspectives for the control of invasive tumors.     

The effect of mammalian target of rapamycin inhibition on T helper type 17 and regulatory T cell differentiation in vitro and in vivo in kidney transplant recipients

Sirolimus (SRL) is a promising alternative to calcineurin inhibitors, such as tacrolimus (TAC), in kidney transplant recipients (KTRs), but the immunological benefits of conversion from calcineurin inhibitors to SRL are not fully investigated. In the present study, we evaluated the effect of conversion from TAC to SRL on the T helper type 17/regulatory T (Th17/Treg) axis in three separate studies. First, the effect of SRL on the Th17/Treg axis was evaluated in vitro using peripheral blood mononuclear cells (PBMCs). Second, the effect of conversion from TAC to SRL on the Th17/Treg axis was studied in KTRs. Finally, the effect of SRL on CD8⁺ Treg cells was evaluated. In vitro analysis of PBMCs isolated from KTRs showed that SRL suppressed Th17 cell differentiation but TAC did not. Conversion from TAC to SRL markedly decreased the number of effector memory CD8⁺ T cells and significantly increased the proportion of CD4⁺ and CD8⁺ Treg cells compared with TAC in KTRs. SRL treatment induced the CD8⁺ Treg cells, and these cells inhibited the proliferation of allogeneic CD4⁺ T cells and Th17 cells. In conclusion, conversion from TAC to SRL favourably regulates Th17 and Treg cell differentiation in KTRs. These findings provide a rationale for conversion from TAC to SRL in KTRs.     

In vivo anti-inflammatory activities of novel cytokine IL-38 in Murphy Roths Large (MRL)/lpr mice

The newly named interleukin (IL)-36 subfamily member IL-38 has been shown to exert anti-inflammatory activity. However, the in vivo immunomodulatory activity of IL-38 was poorly investigated in systemic lupus erythematosus (SLE). We have investigated the expression of CD4⁺IL-17⁺ Th17, CD4⁺IFN-γ⁺ Th1 and CD3⁺CD4⁻CD8⁻ double negative (DN) T cells and the related immunopathological mechanisms in female MRL/lpr mice model of spontaneous lupus-like disease, with or without IL-38 treatment. Intravenous administration of murine recombinant IL-38 into MRL/lpr mice can ameliorate the lupus-like clinical symptoms including proteinuria, leukocyteuria and skin lesions. A remission of histopathology characteristics of skin and nephritis was also observed upon IL-38 treatment. Accordingly, IL-38 receptor was expressed on the cell surface of both CD4⁺ Th and CD19⁺ B lymphocytes. The splenic Th17 and DN T lymphocytes, the average mRNA level of epigenetically regulated gene expression of Th17 cells, and serum concentrations of IL-17 and IL-22 were significantly decreased upon the treatment of IL-38 (all p < 0.05). The in vivo results suggest that IL-38 can ameliorate skin inflammation and nephritis in SLE mice probably via suppressing the formation of inflammatory cytokines such as IL-17 and IL-22, and pathogenic DN T cells. These findings may provide a biochemical basis for further investigation of the therapeutic mechanisms of IL-38 for the treatment of autoimmune-mediated inflammation.     

Cycloastragenol mediates activation and proliferation suppression in concanavalin A-induced mouse lymphocyte pan-activation model

Context: Cycloastragenol (CAG) is a molecule isolated from various species in the genus Astragalus. Although the regulatory activity of Astragalus on immune system has been investigated, the effect of CAG on activated lymphocytes is poorly understood.

Objective: We aimed to biologically address the possible anti-inflammation potential of CAG on concanavalin A (Con A)-mediated mouse lymphocyte pan-activation model.

Materials and methods: Mouse lymphocytes were obtained from spleens and subjected to Con A for 24?h. Herein, the cells were treated with different concentrations of CAG. Cell viability was assayed by MTT. Pretreated by CAG and stimulated by Con A, the expression of CD69 and CD25, Th1/Th2/Th17 cytokines, cell cycle, proliferation and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were analyzed by flow cytometry.

Results: The results declared that CAG significantly downregulated both CD69 and CD25 expressed on Con A activated CD3?+?T cells’ surface, as well as inhibiting proliferation of activated lymphocytes. In addition, CAG blocked the Con A-induced mitogenesis, exhibiting lymphocyte G₀/G₁-phase cell-cycle arrest with significant reduction of cells in S and G₂/M phases. Meanwhile, pretreated by CAG, a significant decline in [Ca²⁺]_i was observed. Furthermore, CAG significantly inhibited the production of Th1 cytokines IFN-γ, TNF, IL-2, Th2 cytokines IL-4, IL-6, IL-10 and Th17 cytokine IL-17?A on Con A-activated lymphocytes.

Conclusion: Our results reinforce that CAG has important anti-inflammatory activity by inhibiting lymphocytes activation, proliferation and cytokines expression, and shows, that this effect may be related to reduction of overall intracellular Ca²⁺ overload.     

Ribavirin modulates the conversion of human CD4+ CD25− T cell to CD4+ CD25+ FOXP3+ T cell via suppressing interleukin‐10‐producing regulatory T cell

Because regulatory T (Treg) cells play an important role in modulating the immune system response against both endogenous and exogenous antigens, their control is critical to establish immunotherapy against autoimmune disorders, chronic viral infections and tumours. Ribavirin (RBV), an antiviral reagent used with interferon, is known to polarize the T helper (Th) 1/2 cell balance toward Th1 cells. Although the immunoregulatory mechanisms of RBV are not fully understood, it has been expected that RBV would affect T reg cells to modulate the Th1/2 cell balance. To confirm this hypothesis, we investigated whether RBV modulates the inhibitory activity of human peripheral CD4⁺ CD25⁺ CD127⁻ T cells in vitro. CD4⁺ CD25⁺ CD127⁻ T cells pre-incubated with RBV lose their ability to inhibit the proliferation of CD4⁺ CD25⁻ T cells. Expression of Forkhead box P3 (FOXP3) in CD4⁺ CD25⁻ T cells was down-modulated when they were incubated with CD4⁺ CD25⁺ CD127⁻ T cells pre-incubated with RBV without down-modulating CD45RO on their surface. In addition, transwell assays and cytokine-neutralizing assays revealed that this effect depended mainly on the inhibition of interleukin-10 (IL-10) produced from CD4⁺ CD25⁺ CD127⁻ T cells. These results indicated that RBV might inhibit the conversion of CD4⁺ CD25⁻ FOXP3⁻ naive T cells into CD4⁺ CD25⁺ FOXP3⁺ adaptive Treg cells by down-modulating the IL-10-producing Treg 1 cells to prevent these effector T cells from entering anergy and to maintain Th1 cell activity. Taken together, our findings suggest that RBV would be useful for both elimination of long-term viral infections such as hepatitis C virus infection and for up-regulation of tumour-specific cellular immune responses to prevent carcinogenesis, especially hepatocellular carcinoma.     

Allergen-specific IL-10-secreting type I T regulatory cells,but not CD4CD25Foxp3 T cells,are decreased in peripheral blood of patients with persistent allergic rhinitis

We investigate the frequencies of CD4⁺CD25⁺Foxp3⁺ T cells and allergen-specific IL-10⁺IL-4^-, IFN-γ⁺IL-4^-, IL-4⁺IFN-γ^-CD4⁺ T cells (which display characteristics of nTreg, Tr1-, Th1- and Th2- cells, respectively) in peripheral blood mononuclear cells (PBMCs) of patients with AR and of healthy individuals. In addition, we estimated the suppressive effect of CD4⁺CD25⁺ Treg cells and allergen-specific, IL-10-secreting cells from both two groups. The frequency of CD4⁺CD25⁺Foxp3⁺ T cells is similar in 43 AR patients compared with 38 healthy subjects. CD4⁺CD25^high cells retain suppressive activity on allergen-stimulated cell proliferation and cytokine production of Th1 but not Th2 cells in both groups. However, the frequency of allergen-specific IL-10⁺IL-4^-CD4⁺ T cells is reduced in AR patients, and correlates inversely to clinical symptom scores. Allergen-specific, IL-10-secreting cells potently suppressed D. pteronyssinus major allergen 1-stimulated cell proliferation and cytokine production (IFN-γ and IL-4) in healthy individuals. Altogether our data indicate that the number and function of CD4⁺CD25⁺ Treg cells from allergic patients are not impaired. However, the deficiency of allergen-specific Tr1 cells may play a role in the development of AR.     

Changes in T helper cell-related factors in patients with type 2 diabetes mellitus after empagliflozin therapy

The immune factors related to T helper (Th) 1 (T-bet, STAT1, and IFN-γ), Th17 (ROR-γt, STAT3, and IL-17), and Treg (FOXP3, STAT5, and IL-10) cells, and SOCS1/3 and the proliferation of Th cells were investigated in type 2 diabetes mellitus patients before (baseline) and after empagliflozin therapy.A total of 56 patients on metformin and gliclazide were separated into two groups: Group 1 did not receive empagliflozin (EMPA^?) and the Group 2 received 10 mg/day of empagliflozin for 6 months (EMPA⁺). The expressions of T-bet, ROR-γt, FOXP3, STAT1/3/5 and SOCS1/3 were evaluated in CD4⁺ T cells with real-time PCR. The production of IFN-γ, IL-17, and IL-10 from CD4⁺ T cells was measured using ELISA. The proliferation of Th cells was assessed with flow cytometry.Six months of empagliflozin therapy significantly reduced the expression of ROR-γt and increased FOXP3 and STAT5 expression, compared to baseline. Production of IL-17 decreased after empagliflozin treatment, while IL-10 was enhanced in the EMPA⁺ group. Oral administration of empagliflozin or the addition of empagliflozin to the cell cultures diminished the proliferation of Th cells.Empagliflozin showed anti-inflammatory effects on Th cells by decreasing Th17-related factors, reducing proliferation capacity, and increasing Treg cell properties.     

Infiltration of IL-17-Producing T Cells and Treg Cells in a Mouse Model of Smoke-Induced Emphysema

Chronic obstructive pulmonary disease (COPD) is a progressive and irreversible chronic inflammatory disease associated with the accumulation of activated T cells. To date, there is little information concerning the intrinsic association among Th17, Tc17, and regulatory T (Treg) cells in COPD. The objective of this study was to investigate the variation of lungs CD4⁺Foxp3⁺ Treg cells and IL-17-producing CD4 and CD8 (Th17 and Tc17) lymphocytes in mice with cigarette-induced emphysema. Groups of mice were exposed to cigarette smoke or room air. At weeks 12 and 24, mice were sacrificed to observe histological changes by HE stain. The frequencies of Th17 (CD4⁺IL-17⁺T), Tc17 (CD8⁺IL-17⁺T), and Treg (CD4⁺Foxp3⁺T) cells in lungs from these mice were analyzed by flow cytometry. The mRNA levels of orphan nuclear receptor ROR γt and Foxp3 were performed by real-time quantitative polymerase chain reaction. The protein levels of interleukin-17 (IL-17), IL-6, IL-10, and transforming growth factor-beta (TGF-β1) were measured by enzyme-linked immunosorbent assay. Cigarette smoke caused substantial enlargement of the air spaces accompanied by the destruction of the normal alveolar architecture and led to emphysema. The frequencies of Th17 and Tc17 cells, as well as the expressions of IL-6, IL-17, TGF-β1, and ROR γt were greater in the lungs of cigarette smoke (CS)-exposed mice, particularly in the 24-week CS-exposed mice. The frequencies of Treg cells and the expressions of IL-10 and Foxp3 were lower in CS-exposed mice compared to control group. More important, the frequencies of Tregs were negatively correlated with Th17 cells and with Tc17 cells. Interestingly, a significant portion of the cells that infiltrate the lungs was skewed towards a Tc17 phenotype. Our findings suggest the contribution of Th17, Tc17, and Treg cells in the pathogenesis of COPD. Rebalance of these cells will be helpful for developing and refining the new immunological therapies for COPD.     

Paeoniflorin regulates the function of human peripheral blood mononuclear cells stimulated by rhIL-1β by up-regulating Treg expression

Abstract

The aim of the present study was to investigate the effect of paeoniflorin (Pae) on recombinant human interleukin-1β (rhIL-1β)-stimulated human peripheral blood mononuclear cells (PBMCs) in vitro. PBMCs were collected by Ficoll density gradient centrifugation and were co-cultured with rhIL-1β for different time periods. The proliferation response was determined by a cell counting kit-8 (CCK-8) assay. The production of IL-17 and IL-10 was measured by enzyme-linked immunosorbent assay (ELISA). The percentage of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) was detected by flow cytometry analysis. These results indicated that rhIL-1β stimulation induced the proliferation of PBMCs in a concentration- and time-dependent manner; it also increased the level of IL-17 and decreased the level of IL-10 in a concentration-dependent manner. The flow cytometry analysis demonstrated that the stimulation of rhIL-1β significantly downregulated the percentage of CD4⁺CD25⁺Foxp3⁺ Treg in CD4⁺ T cells. However, administration of Pae significantly suppressed the proliferation response of rhIL-1β-induced PBMCs and regulated the secretion function of IL-17 and IL-10. Additional experiments demonstrated that Pae treatment significantly reduced rhIL-1β-induced decreases in PBMCs CD4⁺CD25⁺Foxp3⁺ subpopulation numbers. These results suggest that the anti-inflammatory action of Pae is attributable to its regulation of IL-17/IL-10 secretion and Treg expression.     

CD39 expression on Treg and Th17 cells is associated with metabolic factors in patients with type 2 diabetes

Th17 cells are involved in the pathogenesis of multiple inflammatory diseases such as type two diabetes (T2D). CD39⁺ Treg cells have been implicated as responsible for suppressing Th17 cells. The aim of this study was to evaluate the number and function of CD4⁺CD25^highCD39⁺ Treg and Th17 cells in peripheral blood mononuclear cells (PBMC) from T2D patients and healthy control subjects. The Th17 cells were detected in PBMC under culture with human anti-CD3/CD28 and PMA/ionomycin and the levels of IL-17 were assessed by ELISA and qPCR. The T2D patients with obesity showed significantly lower percentages of CD39⁺ Treg cells. A negative correlation between CD39⁺ Treg cells and weight, and body mass index was detected. In contrast, the low levels of CD4⁺IL-17⁺ cells in overweight and obese T2D patients showed a positive correlation with glucose and HbA1c. Additionally, we found a subpopulation of Th17 cells that express CD39 and were correlated with glucose and HbA1c. Our findings suggest that the expression of CD39 on Treg cells and also in CD4⁺IL-17⁺ cells from T2D patients is related to hyperglycemia as well as to overweight and obesity and therefore may participate as a modulator of the effector capacity of Th17 cells.     

Vitamin D3 inhibits the proliferation of T helper cells,downregulate CD4+ T cell cytokines and upregulate inhibitory markers

1 α, 25-dihydroxyvitamin D3 (VitD3) has been suggested to have strong modulatory properties in the immune system. Researchers in the present study primarily aimed to understand the effect of VitD3 on human CD4⁺ T cell proliferation in antigen presenting cells (APCs) free condition in vitro. The effect of VitD3 on intracellular cytokine responses trend to Th1, Th2, Th17 and Th22 was evaluated using the flow cytometry. Moreover the effect of VitD3 on the expression of inhibitory markers such as PD1, PD-L1, and CTLA4 which are induced upon polyclonal T cell receptor (TCR) activation on CD4⁺ T cells, was assessed. We observed that the stimulation of CD4⁺ T cells with VitD3, suppressed proliferation capacity, enhanced the expression of PD1, PD-L1 and CTLA4 inhibitory markers on CD4⁺ T cells, and diminished the percentage of pro-inflammatory cytokines including, IFN-γ, IL-17, and IL-22 except IL-4 in CD4⁺ T cells. The data suggested a potential insight into the consideration of VitD3 in the prevention/control of pro-inflammatory immune response/autoimmune disorders.     

Low-dose IL-2 therapy restores imbalance between Th17 and regulatory T cells in patients with the dermatomyositis combined with EBV/CMV viremia

ObjectiveDermatomyositis (DM) is closely associated with infection, the levels of peripheral lymphocyte subpopulations are rarely studied in patients with DM combined with Epstein-Barr virus (EBV)/cytomegalovirus (CMV) infection. Here, we aimed to observe the level of lymphocyte subsets, especially Th17, regulatory T (Treg) cells in DM combined with EBV/CMV viremia, and explore the effects of short-term low-dose IL-2.Methods34 DM patients combined with EBV/CMV viremia (DM infection group), 31 DM patients without infection (DM non-infection group) and 20 healthy controls were entrolled in our study. In DM infection group, 13 patients received low-dose IL-2 at 0.50 Million IU/day for a five-day course on the basis of conventional treatment. All subjects had completed the decetion of the absolute numbers of lymphocytes subsets in peripheral blood by flow cytometry.ResultsThe infection group had significant decreases levels of total T, total B, NK, CD4 + T cells and CD4 + T subsets (Th1, Th2, Th17, Treg cells). Compare to the healthy controls, Th17 cells was significantly reduced in the infection group, but not in the non-infection group (P < 0.001 vs. P = 0.171). After low-dose IL-2 therapy, the levels of Treg (P = 0.001) cells and Th17 cells were significantly elevated, re-balancing the Th17 and Treg proportions.ConclusionsThe absolute numbers of Th17 and Treg cells in DM patients with EBV/CMV viremia is further reduced. In addition to Treg cells, a decrease in Th17 cells may be also a crucial feature. Low-dose IL-2 treatment may be beneficial and safe prospect immunomodulatory therapy to restores imbalance between Th17 and Treg cells for these patients. Low-dose IL-2 therapy may be a new prospect field with some challenges such as long-term immunoregulatory utility in various virus infection.     

Avian CD4CD25 regulatory T cells: Properties and therapeutic applications

Regulatory T cells (Tregs) are a subset of T cells that specialize in immune suppression. CD4⁺CD25⁺FoxP3⁺ T cells have been characterized as Tregs and extensively studied in mammals. In the absence of a putative FoxP3 ortholog in avians, CD4⁺CD25⁺ cells is characterized as Tregs in avians. Avian CD4⁺CD25⁺ cells produce high amounts of IL-10, TGF-β, CTLA-4, and LAG-3 mRNA; lack IL-2 mRNA; and suppress T cell proliferation in vitro through both contact-dependent and -independent pathways. Depleting avian CD4⁺CD25⁺ cells increases the proliferation of, IL-2 amount, and IFNγ mRNA amount of CD4⁺CD25⁻ cells. Avian CD4⁺CD25⁺ cells lose their suppressive properties immediately after inflammation and acquire supersuppressive properties once inflammation subsides. Although Treg activity could be beneficial to the host, Tregs simultaneously inhibit host immunity and cause persistent infections of certain pathogens. Therapy targeted toward alleviating Treg mediated immune suppression can improve host immunity against those persistent pathogens and benefit poultry production.     

Aging and human CD4 regulatory T cells

Alterations in immunity that occur with aging likely contribute to the development of infection, malignancy and inflammatory diseases. Naturally occurring CD4⁺ regulatory T cells (Treg) expressing high levels of CD25 and forkhead box P3 (FOXP3) are essential for regulating immune responses. Here we investigated the effect of aging on the number, phenotypes and function of CD4⁺ Treg in humans. The frequency and phenotypic characteristics of CD4⁺, FOXP3⁺ T cells as well as their capacity to suppress inflammatory cytokine production and proliferation of CD4⁺, CD25⁻ T cells (target cells) were comparable in young (age ≤40) and elderly (age ≥65) individuals. However, when CD4⁺, FOXP3⁺ Treg and CD4⁺, CD25⁻ T cells were co-cultured at a ratio of 1:1, the production of anti-inflammatory cytokine IL-10 from CD4⁺, CD25⁻ T cells was more potently suppressed in the elderly than in the young. This finding was not due to changes in CTLA-4 expression or apoptosis of CD4⁺, FOXP3⁺ Treg and CD4⁺, CD25⁻ T cells. Taken together, our observations suggest that aging may affect the capacity of CD4⁺, FOXP3⁺ T cells in regulating IL-10 production from target CD4⁺ T cells in humans although their other cellular characteristics remain unchanged.     

The immune responses of central and effector memory BCG-specific CD4 T cells in BCG-vaccinated PPD donors were modulated by Treg cells

Most of the studies evaluating the function of tuberculosis (TB)-specific T cells were only based on the ability to produce cytokines, which may not fully reflect the function of T cells. In the present study, we confirmed that Bacille Calmette Guerin (BCG) could significantly induce cytokine production by CD4⁺ T cells from BCG-vaccinated PPD⁺ donors. In addition, CD4⁺ T cells were activated, divided and proliferated in response to BCG stimulation. Phenotypic analysis showed that IFN-γ⁺CD4⁺ T cells displayed CD45RA⁻CCR7^+/−CD62L⁻, indicating that these CD4⁺ T cells were central and effector memory cells. The analysis of cytokine profiles demonstrated that most of BCG-specific BrdU⁺CD4⁺ T cells produced Th1 cytokines in response to polyclonal stimulation. In addition, we found that regulatory T cells (Treg) suppressed BCG-induced proliferation and IFN-γ production by memory CD4⁺ T cells. The suppressive effects of Treg on BCG-specific responses of CD4⁺ T cells could be partially reversed by blocking the production of IL-10. Taken together, our results demonstrated that functional central and effector memory BCG-specific CD4⁺ T cells could be detected based on the activation, proliferation and division of these cells, and modulated by Treg in PBMCs from BCG-vaccinated PPD⁺ donors.     

Quantitative and functional analysis of CD69+ NKG2D+ T regulatory cells in healthy subjects

T regulatory (Treg) cells have a key role in immune homeostasis and the pathogenesis of chronic inflammatory and autoimmune diseases. CD69 is an early leukocyte activation molecule that under steady state conditions is detected in a small proportion of lymphocytes in peripheral blood and lymphoid tissues. Although it has been reported that a subset of CD69⁺ T cells behaves as Treg lymphocytes, the possible relationship between CD69⁺ Treg cells and CD4⁺NKG2D⁺ T lymphocytes, which also exert immunosuppressive activity, has not been explored. In this study, we analyzed the expression of CD69 and NKG2D by T lymphocytes from the peripheral blood of twenty-five healthy subjects by multi-parametric flow cytometry analysis, and their suppressive activity by an assay of inhibition of lymphocyte activation (CD40L expression) and proliferation (carboxyfluorescein partition assay). We found a very small percentage of CD4⁺CD69⁺NKG2D⁺ T cells (median 0.002%, Q₁–Q₃, 0.001–0.004%), which also expressed TGF-β (Latency Associated Peptide or LAP) and IL-10, in all samples analyzed. These cells exerted an important in vitro suppressive effect on both activation and proliferation of T effector cells. Our data suggest that at very small numbers, CD4⁺CD69⁺NKG2D⁺ lymphocytes seem to exert a relevant functional immune-regulatory role in healthy subjects.