Brief Definitive Report: Human visceral leishmaniasis is not associated with expansion or accumulation of Foxp3+ CD4 cells in blood or spleen

Natural regulatory T cells (CD4⁺ CD25⁺ Foxp3⁺), natural regulatory T cells (nTreg), play an important role in the regulation of inflammatory immune responses. However, the immunosuppressive properties of nTreg may unfavourably affect the host’s ability to clear certain infections. In human visceral leishmaniasis (VL), reports on the frequency and function of nTreg are not conclusive. A limitation of our own previous studies that did not indicate a major role for Foxp3⁺ nTreg in VL pathogenesis was that Foxp3 was measured by mRNA expression alone, as other tools were not available at the time. We have in this study assessed CD4⁺CD25⁺Foxp3⁺ cells in splenic aspirates and peripheral blood mononuclear cells (PBMC) from an extensive series of patients with VL and endemic controls (EC) by flow cytometry (FACS). The results do not show increased frequencies of Foxp3⁺ cells in patient with VL pre‐ and post‐treatment, neither were they elevated when compared to PBMC of EC. We conclude that active VL is not associated with increased frequencies of peripheral Foxp3 Treg or accumulation at the site of infection.     

Suppressive activity and altered conventional phenotype markers/mediators of regulatory T cells in patients with self‐limiting hepatitis E

Hepatitis E virus (HEV) is a major cause of self‐limiting acute viral hepatitis in several developing countries. Elevated levels of peripheral CD4⁺CD25⁺Foxp3⁺, CD4⁺CD25⁻Foxp3⁺ and rise in IL‐10 in hepatitis E have been associated with the involvement of regulatory T cells (Treg). The functional role of the same is yet elusive. In the current study, we have assessed (i) Foxp3 expression by real‐time PCR and by flow cytometry, (ii) the levels of antigen‐specific IL‐10 and TGF‐β by ELISA, (iii) functional analysis of Treg cells and (iv) expression of Treg‐associated conventional phenotypes by flow cytometry in 54 acute patients, 44 recovered individuals from hepatitis E and in 33 healthy controls. Foxp3 mRNA elevation in the acute compared with recovered group and elevation in Foxp3⁺ cells in both patient groups were significantly elevated. The levels of IL‐10 and TGF‐β in the acute patients and TGF‐β in the recovered individuals were elevated. Significantly higher expression of CTLA‐4, PD1, GITR, CD95, CD103 and CD73 on Treg and T effector (Teff) cells was detected in the patient groups. Treg cells of acute patients and recovered individuals exhibited suppressive activity indicating that the Treg cells of hepatitis E patients are functional. The suppressive capacity of Treg cells in acute hepatitis E patients was significantly higher compared with the recovered individuals. Based on our findings, the suppressive functionality of these key markers associated with hepatitis E Treg function need further exploration to get a better understanding of the mechanisms of Treg‐mediated suppression.     

Idiotype-specific T lymphocytes in monoclonal gammopathies: evidence for the presence of CD4+ and CD8+ subsets

Tumour-specific CD4⁺ T helper (Th) and CD8⁺ T cytotoxic (Tc) cells may participate in the control and eradication of tumour cells. In the present study, idiotype-specific stimulation of CD4⁺ and CD8⁺ blood T cells from patients with monoclonal gammopathy of undetermined significance and patients with untreated multiple myeloma stage I was examined. Activation was measured in the CD4⁺ and CD8⁺ subsets enriched by magnetic microbeads as the incorporation of ³H-thymidine and the secretion of interferon (IFN)-γ, interleukin (IL)-2 and IL-4 by single cells using the enzyme-linked immunospot assay. Idiotype-specific T cells were found in four of seven patients. Stimulation was mainly confined to the CD4⁺ subset in three of the four responding patients. This type of response was major histocompatibility complex (MHC) class II restricted as it could be inhibited by monoclonal antibodies against MHC class II (HLA-DR), but not against class I (HLA-ABC) molecules. Idiotype-specific CD8⁺ T cells were also demonstrated in these patients although at a lower frequency. One patient showed a strong and dominating activation of CD8⁺ T cells which could be blocked by antibodies against HLA-ABC but not against HLA-DR. Idiotype-specific CD4⁺ or CD8⁺ T cells were mainly of the type-1 subsets as judged by their secretion of IFN-γ and IL-2. Thus, this study provides evidence for the presence of idiotype-specific and MHC-restricted CD4⁺ and CD8⁺ T cells of the type-1 subsets in patients with monoclonal gammopathies. Such T cells with the potential to control the growth of tumour B cells may be a suitable target for immunotherapeutic interventions in patients.     

Deficiency of forkhead box P3 and cytotoxic T-lymphocyte-associated antigen-4 gene expressions and impaired suppressor function of CD4+CD25+ T cells in patients with autoimmune hepatitis

Aim:  Recently, forkhead box P3 (Foxp3), cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR), and CD28 were identified as the key molecules that control the development and activation of CD4+CD25+ regulatory T cells (T-reg). We investigated the expression pattern of these molecules on T-reg, and investigated the ability of T-reg to produce cytokines in patients with autoimmune hepatitis (AIH).
Methods:  Fifteen patients with AIH and nine healthy patients were included. To determine the frequency of T-reg, a two-color flow cytometry analysis was performed. T-reg were isolated using immunomagnetic beads, and the mRNA levels of Foxp3 , CTLA-4, GITR , and CD28 were quantified by real-time polymerase chain reaction (PCR). The ability of T-reg to produce interferon-γ, interleukin (IL)-10, transforming growth factor-β, and tumor necrosis factor-α after stimulation by OKT3 was evaluated by measuring the levels of mRNA in T-reg by real-time PCR.
Results:  The frequency of T-reg was increased in AIH. The mRNA levels of Foxp3 and CTLA-4 were significantly lower in AIH. The ability of T-reg to produce IL-10 was impaired in AIH.
Conclusion:  We speculate that the inferiority of the Foxp3 and CTLA-4 gene expressions on T-reg results in the impaired suppressor function of T-reg, and eventually in the breakdown of self-tolerance.     

Decreased CTLA4+ and Foxp3+ CD25highCD4+ Cells in Induced Sputum from Patients with Mild Atopic Asthma

BackgroundDetails of the comparisons between airway and peripheral blood regulatory T cells (Tregs) in patients with atopic asthma are still unclear. The objective of this study is to investigate the profiles of both airway and circulating Tregs in atopic asthma.MethodsWe measured the numbers of Tregs and eosinophils in induced sputum and peripheral blood in 28 patients with mild atopic asthma and compared these with numbers in 18 healthy controls. The frequency (%) of Tregs (surface CTLA4⁺, intracellular Foxp3⁺, and CTLA4⁺Foxp3⁺ on CD25^highCD4⁺ T cells) in sputum and blood was determined by intracellular 5-color flow cytometry. We also correlated the numbers with the level of airway hyperresponsiveness (AHR) in asthmatics.ResultsThe mean frequencies of cells expressing CTLA4⁺ (19.4 ± 2.1%, p = 0.075), Foxp3⁺ (16.4 ± 3.3%, p = 0.001), and CTLA4⁺Foxp3⁺ (7.0 ± 1.1%, p = 0.008) in induced sputum from asthmatics were significantly lower than controls (27.2 ± 3.7%, 37.4 ± 4.7%, and 18.2 ± 3.6%, respectively), whereas in peripheral blood, there was no inter-group difference in the frequencies of cells expressing CTLA4⁺ (7.1 ± 1.5% vs 5.7 ± 1.7%, p > 0.05), Foxp3⁺ (35.7 ± 3.2% vs 21.1 ± 3.9%, p > 0.05), and CTLA4⁺Foxp3⁺ (6.6 ± 1.5% vs 4.2 ± 1.0%, p > 0.05). Moreover, the frequency of CD25^highCD4⁺ cells expressing CTLA4⁺, but not Foxp3⁺, in induced sputum was associated with AHR (r = 0.60, p = 0.009) and airway eosinophilic inflammation (r = ? 0.60, p = 0.008) in asthmatics.ConclusionsAirway, but not circulating, Tregs are decreased in mild atopic asthmatics, and are negatively correlated to an increase of airway eosinophilic inflammation and AHR.     

Enhanced high‐mobility group box 1 (HMGB1) modulates regulatory T cells (Treg)/T helper 17 (Th17) balance via toll‐like receptor (TLR)‐4‐interleukin (IL)‐6 pathway in patients with chronic hepatitis B

High‐mobility group box 1 (HMGB1) proteins are substantially up‐regulated in acute and chronic hepatitis. However, the immunopathogenic role of HMGB1 in patients with chronic hepatitis B (CHB) has not been elucidated. In this study, using a cohort of 36 CHB patients, we demonstrated a crucial role for HMGB1 to modulate balance between regulatory T (Treg) and T helper 17 (Th17) cells via the toll‐like receptor (TLR)‐4‐interleukin (IL)‐6 pathway. Serum HMGB1 levels were dramatically higher in CHB patients and increased along with liver injury, inflammation and fibrosis. Notably, HMGB1 increased along with decreased Treg/Th17 cells ratios in the periphery or intrahepatic microenvironment, which provides a clue for HMGB1 to favour Th17 responses whereas inhibit Treg responses. For in vitro studies, serum pools were constructed with serum from CHB patients at an advanced stage, whereas peripheral blood mononuclear cells (PBMC) pools were constructed with cells from those at an early stage. CHB‐serum significantly enhanced retinoic acid‐related orphan receptor‐γt (RORγt), whereas they inhibited forkhead box P3 (Foxp3) expression in CHB‐PBMC, which could be reversed by blocking of HMGB1, TLR4, or IL‐6. Besides, recombinant HMGB1 (rHMGB1) dose‐dependently up‐regulated RORγt whereas down‐regulated Foxp3 expression in CHB‐PBMC, and meanwhile, rHMGB1 enhanced TLR4 and IL‐6 expression in CHB‐PBMC. Moreover, the axis of HMGB1–TLR4‐IL‐6–Treg/Th17 required noncontact interactions between CD4 and non‐CD4 cells. In addition, rHMGB1 down‐regulated anti‐inflammatory proteins on CD4⁺CD25⁺ cells whereas up‐regulated pro‐inflammatory cytokines in CD4⁺CD25⁻ cells. In summary, enriched HMGB1 in CHB patients shifts Treg/Th17 balance to Th17 dominance via the TLR4‐IL‐6 pathway, which exacerbates liver injury and inflammation.     

Ascaris suum infection modulates inflammation: Implication of CD4+ CD25high Foxp3+ T cells and IL‐10

Helminth infections have the ability to modulate host's immune response through mechanisms that allow the chronic persistence of the worms in the host. Here, we investigated the mechanisms involved on the suppressive effect of Ascaris suum infection using a murine experimental model of LPS‐induced inflammation. We found that infection with A. suum markedly inhibited leucocyte influx induced by LPS into air pouches, suppressed secretion of pro‐inflammatory cytokines (IL‐1β, TNF‐α and IL‐6) and induced high levels of IL‐10 and TGF‐β. Augmented frequency of CD4⁺ CD25^high Foxp3⁺ T cells was observed in the mesenteric lymph nodes of infected mice. Adoptive transfer of purified CD4⁺ CD25⁺ T cells to recipient uninfected mice demonstrated that these cells were able to induce a suppressive effect in the LPS‐induced inflammation in air pouch model. In addition, adoptive transfer of CD4⁺ CD25⁺ T cells derived from IL‐10 knockout mice suggests that this suppressive effect of A. suum infection involves IL‐10 cytokine. In conclusion, our results demonstrated that A. suum experimental infection was capable of suppressing LPS‐induced inflammation by mechanisms, which seem to be dependent on responses of CD4⁺ CD25⁺ T cells and secretion of IL‐10 cytokine.     

Expansion of CD8+CD57+ T cells after allogeneic BMT is related with a low incidence of relapse and with cytomegalovirus infection

Summary. Peripheral blood lymphocytes of 46 recipients of lymphocyte-depleted bone marrow allografts were pheno-typically analysed over a period of 1 year. We investigated the repopulation of lymphocyte subpopulations and their relation with clinical parameters such as graft-versus-host disease (GVHD), graft-versus-leukaemia and cytomegalovirus (CMV) infection. The number of repopulated T cells varied strongly between the blood samples of the recipients. In 45% of the recipients the number of T cells recovered to or above normal levels within 3 months after bone marrow transplantation (BMT), whereas the other recipients remained below normal up to 1 year after BMT. In recipients with a high repopulation, the CD8⁺ T-cell subset contributed more to this high repopulation than the CD4⁺ T-cell subset. We showed that the majority of T cells of these recipients expressed the a/3 T-cell receptor, CD8, CD57 and CDllb. HLA-DR was also highly expressed reflecting the activation stage of T cells in these recipients. BMT recipients with a high repopulation of CD8⁺ T cells showed a lower incidence of leukaemic relapse than recipients with a low repopulation. The 3-year probability of relapse was 19% versus 64% (P=O03), respectively. The relative high number of CD8⁺ T cells at 3 months after BMT was not associated with the incidence of GVHD. In contrast, occurrence of CMV infection after BMT was significantly higher in these recipients. Our results indicate that CD8⁺ T cells, predominantly CD57⁺, of BMT recipients with an expansion of these cells represent an in vivo activated cell population. This CD8⁺ T-cell population may consist partially of cytotoxic cells with anti-leukaemic activity as suggested by a low relapse rate. The signal for the strong expansion of these CD8+CD57+ T cells after BMT is still unclear, but association with CMV infection suggests that viral antigens are involved.     

The role of CD4+ and CD8+ T cells in protective immunity to the murine nematode parasite Trichuris muris

The role ofCD4⁺ and CD8⁺ T cells in protective immunity to Trichuris muris was studied in CD4⁺ or CD8⁺ or both CD4⁺ and CD8⁺ T cell-depleted BALB/c mice. To assess in vivo depletion of T-cell subsets, CD4⁺ and CD8⁺ T cells in the Peyer's patches, the mesenteric lymph nodes (MLN), and the spleens of mice treated with T cell-specific monoclonal antibodies (MoAbs) were analysed by FACS. CD4⁺ T cells were selectively depleted in mice injected with anti-CD4 MoAb i.p. and injection of anti-CD8 MoAb resulted in selective depletion ofCD8⁺ T cells. The expulsion ofl. muris was inhibited in CD4⁺ T cell-depleted mice and numerous worms were detected in the large intestine on days 14 and 21 after infection, although no suppression of protective immunity to T. muris was observed in CD8⁺ T cell-depleted mice. Moreover, there was no difference in suppression of protective immunity to T. muris between CD4⁺ T cell-depleted and both CD4⁺ and CD8⁺ T cell-depleted mice. These results indicate that CD4⁺ T cells play a central role in protective immunity to T. muris infection.     

CCL17 and CCL22 chemokines within tumor microenvironment are related to infiltration of regulatory T cells in esophageal squamous cell carcinoma

It has been reported that an increased population of regulatory T cells (T‐regs) is one of the reasons for impaired anti‐tumor immunity. We investigated the frequency of Foxp3⁺ T‐regs in tumor‐infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs) of patients with esophageal squamous cell carcinoma (ESCC). Furthermore, in order to elucidate the mechanisms behind T‐regs accumulation within tumors, we evaluated the relationship between CCL17 or CCL22 expression and the frequency of Foxp3⁺ T‐regs. CD4⁺CD25⁺Foxp3⁺ T‐regs as a percentage of CD4⁺ cells were counted by flow cytometry. The frequency of CCL17⁺ or CCL22⁺ cells among CD14⁺ cells in tumors was also evaluated by flow cytometry. Moreover, an in vitro migration assay using T‐regs derived from ESCC was performed in the presence of CCL17 or CCL22. The frequency of Foxp3⁺ T‐regs in TILs was significantly higher than that in the normal esophageal mucosa (24.6 ± 10.0 vs 7.1 ± 5.9%, P < 0.01). The frequency of Foxp3⁺ T‐regs in PBLs of ESCC patients was significantly higher than that in normal healthy donors (7.0 ± 4.2 vs 2.5 ± 1.0%, P < 0.01). Furthermore, the frequency of CCL17⁺ or CCL22⁺ cells among CD14⁺ cells within tumors was significantly higher than that of normal esophageal mucosa, and there was a significant correlation between the frequency of CCL17⁺ or CCL22⁺ cells and Foxp3⁺ T‐regs in TILs. In addition, the in vitro migration assay indicated that T‐regs were significantly induced to migrate by CCL17 or CCL22. In conclusion, CCL17 and CCL22 within the tumor are related to the increased population of Foxp3⁺ T‐regs in ESCC.     

Cytokine profile of protective anti-Trichinella spiralis CD4+ OX22− and non-protective CD4+ OX22+ thoracic duct cells in rats: secretion of IL-4 alone does not determine protective capacity

We analysed the cytokine profile of a T cell subset (CD4⁺ CD45 RC⁻) that confers protection against Trichinella spiralis infection in rats. These CD4⁺ cells are generated in the gut and appear in the thoracic duct lymph within 72 h after infection. Cytokine mRNA levels for IL-2, IL-3, IL-4, IL-5, IL-10 and IFN-γ and functional cytokine secretion for IL-4, IL-5, IFN-γ, TNF-α and mast cell differentiation activity were tested in vitro following stimulation with T. spiralis antigens. Compared to a non-protective T cell population (CD4⁺ CD45 RC⁺ or CD8⁺), also isolated from the same thoracic lymph, no significant differences were observed in the levels of mRNA for IL-2, IL-3, IL-4, IL-5, IL-10 or IFN-γ in the protective CD4⁺ CD45 RC⁻ cells. However, analysis of the cytokine activities in culture supernatant of these T cell subsets following 24 h stimulation in vitro with T. spiralis antigens showed that significant IL-4 and IL-5 activity but little IFN-γ or TNF-α was secreted by the protective CD4⁺ CD45 RC⁻ cells. Whereas the non-protective CD4⁺ CD45 RC⁺ cells secreted significant levels of IL-4, IFN-γ, mast cell differentiating activity and TNF-α but little IL-5 activity. Nonprotective CD8⁺ cells were found to secrete IL-4 but not IL-5. Production of IL-4 was essentially equal for both protective and non-protective T cell subsets. These findings suggest that the presence or absence of IFN-γ secretion, rather than IL-4 alone, determines whether a T cell subset has protective activity against T. spiralis infection in rats.     

Rapamycin combined with donor immature dendritic cells promotes liver allograft survival in association with CD4(+) CD25(+) Foxp3(+) regulatory T cell expansion

Aim: To determine whether donor immature dendritic cells (imDCs) combined with a short postoperative course of rapamycin (Rapa) has the ability to expand the CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells and prolong liver allograft survival. Methods: Orthotopic liver transplantation (OLT) was performed from Lewis rats to Brown Norway recipients. Three days before transplantation, animals were injected intravenously with 2 × 10⁶ donor bone marrow‐derived imDCs. Recipient rats (the combined treated group) also received Rapa for 7 d after liver transplantation. Additional groups received either imDCs alone, Rapa alone, or saline alone. Every six recipients from each group were killed at 14 days, 28 days after OLT. The changes of CD4⁺CD25⁺Foxp3⁺ Treg cells in peripheral blood and spleen, histological changes of liver grafts, and serum cytokine levels were investigated. The other six recipients were left in each group to observe the animal survival. Results: Donor imDCs followed by a short postoperative course of Rapa induced long‐term allograft survival. The percentage of CD4⁺CD25⁺Foxp3⁺ Treg cells in CD4⁺ T cells in the combination treatment group were significantly higher compared with the acute rejection group. Moreover, within the CD4⁺CD25⁺ T cell population the combination treatment recipients maintained a higher incidence of Foxp3⁺ T cells compared with the other groups. Despite the lower serum levels of interleukin (IL)‐2, IL‐12, and interferon‐γ in the combined treated group, the cytokine levels in the combined treated group at 7 days after OLT was nearly twice that at 3 days after OLT but decreased significantly compared with the other groups at 28 days after OLT. Serum IL‐10 level in the combined treated group was higher than the other groups. Conclusions: A single imDC infusion followed by a short postoperative course of Rapa prolongs liver allograft survival and enhances the expansion of Treg cells. This optimal protocol may be a promising administration protocol for the peritransplant tolerance induction.     

Mechanisms of modulation of experimental autoimmune encephalomyelitis by chronic Trichinella spiralis infection in Dark Agouti rats

Trichinella spiralis is a helminth that provokes Th2 and anti‐inflammatory type responses in an infected host. Our previous studies using Dark Agouti (DA) rats indicated that T. spiralis infection reduced experimental autoimmune encephalomyelitis (EAE) severity in rats. The aim of this study was to analyse the mechanisms underlying EAE suppression driven by T. spiralis infection. Reduced clinical and histological manifestations of the disease were accompanied by increased IL‐4 and IL‐10 production and decreased IFN‐γ and IL‐17 production in draining lymph node cells. This indicates that T. spiralis infection successfully maintains a Th2 cytokine bias regardless of EAE induction. High IL‐10 signifies parasite‐induced anti‐inflammatory and/or regulatory cell responses. Transfer of splenic T cell‐enriched population of cells from T. spiralis‐infected rats into EAE immunized rats caused amelioration of EAE and in some cases protection from disease development. This population of cells contained higher proportion of CD4⁺ CD25⁺ Foxp3⁺ regulatory cells and produced high level of IL‐10 when compared with uninfected rats.     

Hepatic compartmentalization of exhausted and regulatory cells in HIV/HCV‐coinfected patients

Accelerated intrahepatic hepatitis C virus (HCV) pathogenesis is likely the result of dysregulation within both the innate and adaptive immune compartments, but the exact contribution of peripheral blood and liver lymphocyte subsets remains unclear. Prolonged activation and expansion of immunoregulatory cells have been thought to play a role. We determined immune cell subset frequency in contemporaneous liver and peripheral blood samples from chronic HCV‐infected and HIV/HCV‐coinfected individuals. Peripheral blood mononuclear cells (PBMC) and biopsy‐derived liver‐infiltrating lymphocytes from 26 HIV/HCV‐coinfected, 10 chronic HCV‐infected and 10 HIV‐infected individuals were assessed for various subsets of T and B lymphocytes, dendritic cell, natural killer (NK) cell and NK T‐cell frequency by flow cytometry. CD8⁺ T cells expressing the exhaustion marker PD‐1 were increased in HCV‐infected individuals compared with uninfected individuals (P = 0.02), and HIV coinfection enhanced this effect (P = 0.005). In the liver, regulatory CD4⁺CD25⁺Foxp3⁺ T cells, as well as CD4⁺CD25⁺PD1⁺ T cells, were more frequent in HIV/HCV‐coinfected than in HCV‐monoinfected samples (P < 0.001). HCV was associated with increased regulatory T cells, PD‐1⁺ T cells and decreased memory B cells, regardless of HIV infection (P ≤ 0.005 for all). Low CD8⁺ expression was observed only in PD‐1⁺CD8⁺ T cells from HCV‐infected individuals and healthy controls (P = 0.002) and was associated with enhanced expansion of exhausted CD8⁺ T cells when exposed in vitro to PHA or CMV peptides. In conclusion, in HIV/HCV coinfection, ongoing HCV replication is associated with increased regulatory and exhausted T cells in the periphery and liver that may impact control of HCV. Simultaneous characterization of liver and peripheral blood highlights the disproportionate intrahepatic compartmentalization of immunoregulatory T cells, which may contribute to establishment of chronicity and hepatic fibrogenesis in HIV coinfection.     

Generation of regulatory dendritic cells and CD4+Foxp3+ T cells by probiotics administration suppresses immune disorders

The beneficial effects of probiotics have been described in many diseases, but the mechanism by which they modulate the immune system is poorly understood. In this study, we identified a mixture of probiotics that up-regulates CD4⁺Foxp3⁺ regulatory T cells (Tregs). Administration of the probiotics mixture induced both T-cell and B-cell hyporesponsiveness and down-regulated T helper (Th) 1, Th2, and Th17 cytokines without apoptosis induction. It also induced generation of CD4⁺Foxp3⁺ Tregs from the CD4⁺CD25⁻ population and increased the suppressor activity of naturally occurring CD4⁺CD25⁺ Tregs. Conversion of T cells into Foxp3⁺ Tregs is directly mediated by regulatory dendritic cells (rDCs) that express high levels of IL-10, TGF-β, COX-2, and indoleamine 2,3-dioxygenase. Administration of probiotics had therapeutical effects in experimental inflammatory bowel disease, atopic dermatitis, and rheumatoid arthritis. The therapeutical effect of the probiotics is associated with enrichment of CD4⁺Foxp3⁺ Tregs in the inflamed regions. Collectively, the administration of probiotics that enhance the generation of rDCs and Tregs represents an applicable treatment of inflammatory immune disorders.     

Inhibition of Plasmodium falciparum growth in vitro by CD4+ and CD8+ T cells from non-exposed donors

T cells from most adult non-exposed donors, which express a memory phenotype (CD45RO+), can respond by proliferation to P. falciparum asexual stages in vitro. Such cells may have arisen from exposure to environmental organisms. To address the efficacy of such cells in eliminating parasites and investigate the mechanisms involved, we have used an in vitro assay where parasite growth can be precisely monitored in the presence of different cell preparations. Unfractionated peripheral blood mononuclear cells (PBMC) from both malaria-exposed and non-exposed donors inhibited parasite growth by up to 62% in a two day assay. Purified T cells in the presence of adherent cells had a similar effect, but purified T cells alone or adherent cells alone had minimal effect. Antigens released at the time of schizont rupture were maximally effective in stimulating interferon-γ (IFNγ) production. Neutralizing antibodies to IFNγ showed a partial reduction of growth inhibition in some individuals tested suggesting that different mechanisms may be operative. Neutralizing antibody to TNFα had a partial effect in combination with anti-IFNγ. Antibodies to IL-1 and IL-4 had no effect. T cell fractionation experiments showed that while purified CD4⁺ T cells from some donors produced IFNγ and inhibited parasite growth, purified CD8⁺ T cells could inhibit parasite growth to a greater extent without production of detectable IFNγ. Four parasitised red blood cell clones (CD4⁺, TCRαβ⁺, IFNγ producing) derived from non-exposed donors inhibited parasite growth to comparable levels, but one clone showed low production of IFNγ whilst the other three produced high levels. Our data show that T cells from non-exposed donors have the potential to eliminate malaria parasites via non-IFNγ dependent mechanisms. Such mechanisms may contribute to a degree of innate resistance to malaria in vivo, and may be able to be targeted by malaria vaccine programs.     

CD8+/CD57+ cells and apoptosis suppress T-cell functions in multiple myeloma

The aim of this study was to evaluate the role of CD8⁺/CD57⁺ lymphocytes in the immune dysregulation of multiple myeloma (MM). Cytofluorimetry of peripheral blood lymphocytes (PBL) purified from 39 MM patients showed an inverse relationship between the percentage of CD8⁺/CD57⁺ cells and CD4/CD8 ratio. Analysis of their activation antigens revealed that they were prevalently HLA-DR⁺ and Fas⁺. Removal of CD8⁺/CD57⁺ cells from MM PBL significantly improved cell proliferation and pokeweed mitogen (PWM)-induced polyclonal Ig production in vitro, whereas the addition of supernatants from patients' CD8⁺/CD57⁺ cell cultures to normal PBL suppressed both the PWM-driven Ig synthesis and the proliferative rate of stimulated PBL, supporting the contention that CD8⁺/CD57⁺ cells release in vitro an inhibitory factor that is directly involved in T-cell regulatory function. However, since the proliferative recovery of PWM- and phytohaemagglutinin (PHA)-stimulated MM PBL in the absence of CD8⁺/CD57⁺ lymphocytes was only partial, a dysregulated activation-induced apoptosis was anticipated. In fact, patients' PBL displayed an increased susceptibility to apoptosis and this was significantly enhanced after PWM and, even more, after PHA stimulation. Analysis of CD57 antigen expression on apoptotic or viable cells demonstrated a substantial defect of apoptosis in the CD8⁺/CD57⁺ population. Our results indicate that both the immunosuppressive effect of CD8⁺/CD57⁺ cells and the enhanced susceptibility to apoptosis of PBL could be involved in the pathogenesis of the immunodeficiency observed in this disease.     

CD4+Foxp3+ T cells,interleukin-35 (IL-35) and IL-10 in systemic lupus erythematosus patients: Relation to disease activity

Aim of the workTo evaluate three subtypes of CD4⁺Foxp3⁺ T cells, interleukin-35 (IL-35) and IL-10 in systemic lupus eryhtematosus (SLE) patients and study their relation to disease activity.Patients and methodsFifty SLE patients were included and divided according to the SLE disease activity index (SLEDAI) into 2 equal groups with activity or in remission. Twenty healthy subjects were included as controls. All subjects underwent flow cytometric analysis of CD4, CD25, CXCR5 and Foxp3 expression on T cells. Serum IL-35 and IL-10 levels were measured by ELISA.ResultsPatients were 46 females and 4 males with a mean age of 38.0 ± 10.0 years, disease duration of 9.2 ± 6.0 years. The mean SLEDAI was 6.8 ± 3.7 in active ones. SLE patients especially those with activity had significantly reduced percents of CD4⁺CD25⁺Foxp3⁺ and CD4⁺CXCR5⁺Foxp3⁺ T cells, but increased percents of CD4⁺CD25⁻Foxp3⁺ T cells. This was accompanied by significant higher levels of serum IL-35 and IL-10 (p < 0.0001). The SLEDAI in active patients significantly correlated with CD4⁺CD25⁻Foxp3⁺ T cell percent, serum IL-35 and IL-10 levels (p < 0.05) and inversely with the CD4⁺CD25⁺Foxp3⁺ and CD4⁺CXCR5⁺Foxp3⁺ T cell percents (p < 0.05). At cut-off values of 3.29% for CD4⁺CD25⁺Foxp3⁺ T cell, 7.62% for CD4⁺CD25⁻Foxp3⁺ T cell, 1.77% for CD4⁺CXCR5⁺Foxp3⁺ T cell, 22.04 pg/ml for IL-35 level and 30.51 pg/ml for serum IL-10 level were found to be highly sensitive and specific for detecting lupus activity.ConclusionCD4⁺Foxp3⁺ T cells, IL-35 and IL-10 showed high sensitivity and specificity for detecting SLE activity and may be considered as potentially promising therapeutic targets.     

A commensal symbiotic factor derived from Bacteroides fragilis promotes human CD39+Foxp3+ T cells and Treg function

Polysaccharide A (PSA) derived from the human commensal Bacteroides fragilis is a symbiosis factor that stimulates immunologic development within mammalian hosts. PSA rebalances skewed systemic T helper responses and promotes T regulatory cells (T_regs). However, PSA-mediated induction of Foxp3 in humans has not been reported. In mice, PSA-generated Foxp3⁺ T_regs dampen Th17 activity thereby facilitating bacterial intestinal colonization while the increased presence and function of these regulatory cells may guard against pathological organ-specific inflammation in hosts. We herein demonstrate that PSA induces expression of Foxp3 along with CD39 among naïve CD4 T cells in vitro while promoting IL-10 secretion. PSA-activated dendritic cells are essential for the mediation of this regulatory response. When cultured with isolated Foxp3⁺ T_regs, PSA enriched Foxp3 expression, enhanced the frequency of CD39⁺HLA-DR⁺ cells, and increased suppressive function as measured by decreased TNFα expression by LPS-stimulated monocytes. Our findings are the first to demonstrate in vitro induction of human CD4⁺Foxp3⁺ T cells and enhanced suppressive function of circulating Foxp3⁺ T_regs by a human commensal bacterial symbiotic factor. Use of PSA for the treatment of human autoimmune diseases, in particular multiple sclerosis and inflammatory bowel disease, may represent a new paradigm in the approach to treating autoimmune disease.