Association between discordant immunological response to highly active anti‐retroviral therapy,regulatory T cell percentage,immune cell activation and very low‐level viraemia in HIV‐infected patients

The mechanisms sustaining the absence of complete immune recovery in HIV‐infected patients upon long‐term effective highly active anti‐retroviral therapy (HAART) remain elusive. Immune activation, regulatory T cells (T_regs) or very low‐level viraemia (VLLV) have been alternatively suspected, but rarely investigated simultaneously. We performed a cross‐sectional study in HIV‐infected aviraemic subjects (mean duration of HAART: 12 years) to concomitantly assess parameters associated independently with inadequate immunological response. Patients were classified as complete immunological responders (cIR, n = 48) and inadequate immunological responders (iIR, n = 39), depending on the CD4⁺ T cell count (> or < 500/mm³). Clinical and virological data (including very low‐level viraemia) were collected. In parallel, immunophenotyping of CD4⁺ lymphocytes, including T_reg subsets, and CD8⁺ T cells was performed. Percentages of activated CD4⁺ T cells, T_regs, effector T_regs and terminal effector T_regs were found to be significantly elevated in iIR. Neither the percentage of activated CD8⁺ T cells nor VLLV were found to be associated with iIR. In the multivariate analysis, nadir of CD4⁺ T cell count and percentage of T_regs were the only two parameters associated independently with iIR [odds ratio (OR) = 2·339, P = 0·001, and OR = 0·803, P = 0·041]. We present here the largest study investigating simultaneously the immune response to long‐term HAART, activation of CD4⁺ and CD8⁺ T cells, T_reg percentages and very low‐level viraemia. Causative interactions between T_regs and CD4⁺ T cells should now be explored prospectively in a large patients cohort.     

Increased levels of regulatory T cells (Tregs) in human immunodeficiency virus‐infected patients after 5 years of highly active anti‐retroviral therapy may be due to increased thymic production of naive Tregs

This study determines levels of regulatory T cells (T_regs), naive T_regs, immune activation and cytokine patterns in 15 adult human immunodeficiency virus (HIV)‐infected patients receiving prolonged highly active anti‐retroviral therapy (HAART) who have known thymic output, and explores if naive T_regs may represent recent thymic emigrant T_regs. HIV‐infected patients treated with HAART with a median of 1 and 5 years were compared with healthy controls. Percentages of T_regs (CD3⁺CD4⁺CD25⁺CD127^low), naive T_regs (CD3⁺CD4⁺CD25⁺CD45RA⁺) and activation markers (CD38⁺human leucocyte antigen D‐related) were determined by flow cytometry. Forkhead box P3 mRNA expression and T cell receptor excision circles (T_REC) content in CD4⁺ cells were determined by polymerase chain reaction and cytokines analysed with Luminex technology. Levels of T_regs were significantly higher in HIV‐infected patients compared with controls, both after 1 and 5 years of HAART (P < 0·001), despite fully suppressed HIV‐RNA and normalization of both CD4 counts, immune activation and cytokine patterns. Furthermore, levels of naive T_regs were elevated significantly in HIV‐infected patients (P < 0·001) and were associated with thymic output measured as the T_REC frequency in CD4⁺ cells (P = 0·038). In summary, T_reg levels in HIV‐infected patients are elevated even after 5 years of HAART. Increased thymic production of naive T_regs may contribute to higher T_reg levels in HIV‐infection.     

Interleukin‐2 treatment reverses effects of cAMP‐responsive element modulator α‐over‐expressing T cells in autoimmune‐prone mice

Systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), are often characterized by a failure of self‐tolerance and result in an uncontrolled activation of B cells and effector T cells. Interleukin (IL)‐2 critically maintains homeostasis of regulatory T cells (T_reg) and effector T cells in the periphery. Previously, we identified the cAMP‐responsive element modulator α (CREMα) as a major factor responsible for decreased IL‐2 production in T cells from SLE patients. Additionally, using a transgenic mouse that specifically over‐expresses CREMα in T cells (CD2CREMαtg), we provided in‐vivo evidence that CREMα indeed suppresses IL‐2 production. To analyse the effects of CREMα in an autoimmune prone mouse model we introduced a Fas mutation in the CD2CREMαtg mice (FVB/Fas^–/–CD2CREMαtg). Overexpression of CREMα strongly accelerated the lymphadenopathy and splenomegaly in the FVB/Fas^–/– mice. This was accompanied by a massive expansion of double‐negative (DN) T cells, enhanced numbers of interferon (IFN)‐γ‐producing T cells and reduced percentages of T_regs. Treatment of FVB/Fas^–/–CD2CREMαtg mice with IL‐2 restored the percentage of T_regs and reversed increased IFN‐γ production, but did not affect the number of DNTs. Our data indicate that CREMα contributes to the failure of tolerance in SLE by favouring effector T cells and decreasing regulatory T cells, partially mediated by repression of IL‐2 in vivo .     

Regulatory T‐cells in acute dengue viral infection

Although regulatory T‐cells (T_regs) have been shown to be expanded in acute dengue, their role in pathogenesis and their relationship to clinical disease severity and extent of viraemia have not been fully evaluated. The frequency of T_regs was assessed in 56 adult patients with acute dengue by determining the proportion of forkhead box protein 3 (FoxP3) expressing CD4⁺ CD25⁺T‐cells (FoxP3⁺ cells). Dengue virus (DENV) viral loads were measured by quantitative real‐time polymerase chain reaction (PCR) and DENV‐specific T‐cell responses were measured by ex‐vivo interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) assays to overlapping peptide pools of DENV‐NS3, NS1 and NS5. CD45RA and CCR4 were used to phenotype different subsets of T‐cells and their suppressive potential was assessed by their expression of cytotoxic T lymphocyte‐antigen 4 (CTLA‐4) and Fas. While the frequency of FoxP3⁺ cells in patients was significantly higher (P < 0·0001) when compared to healthy individuals, they did not show any relationship with clinical disease severity or the degree of viraemia. The frequency of FoxP3⁺ cells did not correlate with either ex‐vivo IFN‐γ DENV‐NS3‐, NS5‐ or NS1‐specific T‐cell responses. FoxP3⁺ cells of patients with acute dengue were predominantly CD45RA⁺ FoxP3^low, followed by CD45RA‐FoxP3^low, with only a small proportion of FoxP3⁺ cells being of the highly suppressive effector Treg subtype. Expression of CCR4 was also low in the majority of T‐cells, with only CCR4 only being expressed at high levels in the effector T_reg population. Therefore, although FoxP3⁺ cells are expanded in acute dengue, they predominantly consist of naive T_regs, with poor suppressive capacity.     

Infusion of ex‐vivo expanded human TCR‐αβ+ double‐negative regulatory T cells delays onset of xenogeneic graft‐versus‐host disease

Despite the demonstration of potent immunosuppressive function of T cell receptor (TCR)‐αβ⁺ double‐negative regulatory T cells (DN T_regs), scarce numbers and lack of effective expansion method limit their clinical applications. Here we describe an approach that allows for ～3500‐fold ex‐vivo expansion of human DN T_regs within 3 weeks with > 97% purity. Ex‐vivo‐expanded DN T_regs suppress proliferation of polyclonally stimulated autologous T and B cells in vitro through direct cell‐to‐cell contact. In vivo, we demonstrate for the first time that infusion of human DN T_regs delayed an onset of xenogeneic graft‐versus‐host disease (GVHD) significantly in a humanized mouse model. Furthermore, preincubation of ex‐vivo‐expanded DN T_regs with a mechanistic target of rapamycin (mTOR) inhibitor rapamycin enhanced their immune regulatory function further. Taken together, this study demonstrates that human DN T_regs can be expanded ex vivo to therapeutic numbers. The expanded DN T_regs can suppress proliferation of T and B cells and attenuate GVHD, highlighting the potential clinical use of DN T_regs to mitigate GVHD.     

Increased resistance to CD4+CD25hi regulatory T cell‐mediated suppression in patients with type 1 diabetes

Type I diabetes (T1D) is a T cell‐mediated autoimmune disease characterized by loss of tolerance to islet autoantigens, leading to the destruction of insulin‐producing beta cells. Peripheral tolerance to self is maintained in health through several regulatory mechanisms, including a population of CD4⁺CD25^hi naturally occurring regulatory T cells (T_regs), defects in which could contribute to loss of self‐tolerance in patients with T1D. We have reported previously that near to T1D onset, patients demonstrate a reduced level of suppression by CD4⁺CD25^hi T_regs of autologous CD4⁺CD25^‐ responder cells. Here we demonstrate that this defective regulation is also present in subjects with long‐standing T1D (> 3 years duration; P = 0·009). No difference was observed in forkhead box P3 or CD127 expression on CD4⁺CD25^hi T cells in patients with T1D that could account for this loss of suppression. Cross‐over co‐culture assays demonstrate a relative resistance to CD4⁺CD25^hi T_reg‐mediated suppression within the CD4⁺CD25^‐ T cells in all patients tested (P = 0·002), while there appears to be heterogeneity in the functional ability of CD4⁺CD25^hi T_regs from patients. In conclusion, this work demonstrates that defective regulation is a feature of T1D regardless of disease duration and that an impaired ability of responder T cells to be suppressed contributes to this defect.     

Two separate effects contribute to regulatory T cell defect in systemic lupus erythematosus patients and their unaffected relatives

Forkhead box P3 (FoxP3)⁺ regulatory T cells (T_regs) are functionally deficient in systemic lupus erythematosus (SLE), characterized by reduced surface CD25 [the interleukin (IL)‐2 receptor alpha chain]. Low‐dose IL‐2 therapy is a promising current approach to correct this defect. To elucidate the origins of the SLE T_reg phenotype, we studied its role through developmentally defined regulatory T cell (T_reg) subsets in 45 SLE patients, 103 SLE‐unaffected first‐degree relatives and 61 unrelated healthy control subjects, and genetic association with the CD25‐encoding IL2RA locus. We identified two separate, uncorrelated effects contributing to T_reg CD25. (1) SLE patients and unaffected relatives remarkably shared CD25 reduction versus controls, particularly in the developmentally earliest CD4⁺FoxP3⁺CD45RO^–CD31⁺ recent thymic emigrant T_regs. This first component effect influenced the proportions of circulating CD4⁺FoxP3^highCD45RO⁺ activated T_regs. (2) In contrast, patients and unaffected relatives differed sharply in their activated T_reg CD25 state: while relatives as control subjects up‐regulated CD25 strongly in these cells during differentiation from naive T_regs, SLE patients specifically failed to do so. This CD25 up‐regulation depended upon IL2RA genetic variation and was related functionally to the proliferation of activated T_regs, but not to their circulating numbers. Both effects were found related to T cell IL‐2 production. Our results point to (1) a heritable, intrathymic mechanism responsible for reduced CD25 on early T_regs and decreased activation capacity in an extended risk population, which can be compensated by (2) functionally independent CD25 up‐regulation upon peripheral T_reg activation that is selectively deficient in patients. We expect that T_reg‐directed therapies can be monitored more effectively when taking this distinction into account.     

Immunological monitoring of extracorporeal photopheresis after heart transplantation

Extracorporeal photopheresis (ECP) has been used as a prophylactic and therapeutic option to avoid and treat rejection after heart transplantation (HTx). Tolerance‐inducing effects of ECP such as up‐regulation of regulatory T cells (T_regs) are known, but specific effects of ECP on regulatory T cell (T_reg) subsets and dendritic cells (DCs) are lacking. We analysed different subsets of T_regs and DCs as well as the immune balance status during ECP treatment after HTx. Blood samples were collected from HTx patients treated with ECP for prophylaxis (n = 9) or from patients with histologically proven acute cellular rejection (ACR) of grade ≥ 1B (n = 9), as well as from control HTx patients without ECP (HTxC; n = 7). Subsets of T_regs and DCs as well as different cytokine levels were analysed. Almost 80% of the HTx patients showed an effect to ECP treatment with an increase of T_regs and plasmacytoid DCs (pDCs). The percentage of pDCs before ECP treatment was significantly higher in patients with no ECP effect (26·3% ± 5·6%) compared to patients who showed an effect to ECP (9·8% ± 10·2%; P = 0·011). Analysis of functional subsets of CD4⁺CD25^highCD127^low T_regs showed that CD62L‐, CD120b‐ and CD147‐positive T_regs did not differ between the groups. CD39‐positive T_regs increased during ECP treatment compared to HTxC. ECP‐treated patients showed higher levels for T helper type 1 (Th1), Th2 and Th17 cytokines. Cytokine levels were higher in HTx patients with rejection before ECP treatment compared to patients with prophylactic ECP treatment. We recommend a monitoring strategy that includes the quantification and analysis of T_regs, pDCs and the immune balance status before and up to 12 months after starting ECP.     

Immunomagnetic isolation of CD4+CD25+FoxP3+ natural T regulatory lymphocytes for clinical applications

Although CD4⁺/CD25⁺ T regulatory cells (T_regs) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell‐separation strategy complying with good manufacturing practice guidelines. We isolated T_regs from standard leukapheresis products using double‐negative selection (anti‐CD8 and anti‐CD19 monoclonal antibodies) followed by positive selection (anti‐CD25 monoclonal antibody). The final cell fraction (CD4⁺/CD25⁺) showed a mean purity of 93·6% ± 1·1. Recovery efficiency was 81·52% ± 7·4. The CD4⁺/CD25^+bright cells were 28·4% ± 6·8. The CD4⁺/CD25⁺ fraction contained a mean of 51·9% ± 15·1 FoxP3 cells and a mean of 18·9% ± 11·5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4⁺CD25⁺FoxP3⁺ cells were in line with flow cytometric results. In Vβ spectratyping the complexity scores of CD4⁺/CD25⁺ cells and CD4⁺/CD25^‐ cells were not significantly different, indicating that T_regs had a broad T cell receptor repertoire. The inhibition assay showed that CD4⁺/CD25⁺ cells inhibited CD4⁺/CD25^‐ cells in a dose‐dependent manner (mean inhibition percentages: 72·4 ± 8·9 [ratio of T responder (T_resp) to T_regs, 1:2]; 60·8% ± 20·5 (ratio of T_resp to T_regs, 1:1); 25·6 ± 19·6 (ratio of T_resp to T_regs, 1:0·1)). Our study shows that negative/positive T_reg selection, performed using the CliniMACS device and reagents, enriches significantly CD4⁺CD25⁺FoxP3⁺ cells endowed with immunosuppressive capacities. The CD4⁺CD25⁺FoxP3⁺ population is a source of natural T_reg cells that are depleted of CD8⁺ and CD4⁺/CD25^‐ reacting clones which are potentially responsible for triggering graft‐versus‐host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft‐versus‐tumour effect.     

An increased CD25‐positive intestinal regulatory T lymphocyte population is dependent upon Cox‐2 activity in the Apcmin/+ model

Only mismatch repair (MMR)‐deficient colorectal cancer (CRC) appears to respond well to programmed death (PD)‐1 inhibition at the present time. Emerging evidence suggests a role for micro‐environmental factors such as CD25⁺ cells modulating response to PD‐1 inhibition. In the Apc^Min/+ model of familial adenomatous polyposis (MMR‐proficient CRC), increased Cyclooxygenase‐2 (Cox‐2) expression by cells which include alternatively activated mononuclear phagocytes promotes intestinal tumorigenesis by mechanisms which may include immune suppression. To gain insight into this, we compared regulatory T cell (T_reg) populations between Apc^Min/+ and wild‐type mice prior to and after the phase of increased intestinal Cox‐2‐dependent prostaglandin E₂ (PGE₂) production. There was no difference in systemic T_reg function or numbers between Apc^Min/+ and wild‐type mice. However, increased numbers of small intestinal CD25⁺ T_regs were observed with increased Cox‐2 activity in the absence of any difference in the expression of Tgf‐β or Tslp between Apc^Min/+ and wild‐type mice. Cox‐2 inhibitor therapy (Celecoxib) reversed the increase in Apc^Min/+ intestinal CD25⁺ T_reg numbers, without decreasing numbers of CD25⁺ systemic T_regs. Forkhead box protein 3 (FoxP3⁺) and Cox‐2⁺ cells were co‐localized to the interstitium of adenomas of Apc^min/+ mice. These results suggest selective dependence of an ‘activated T_reg’ phenotype on paracrine Cox‐2 activity in Apc^Min/+ small intestine. For therapeutic potential, further studies are required to evaluate the relevance of these findings to human cancer as well as the functional significance of CD25⁺ intestinal T_regs in cancer.     

Activation profile of Toll‐like receptors of peripheral blood lymphocytes in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease associated with aberrant activation of T and B lymphocytes for the production of inflammatory cytokines and autoreactive antibodies. Animal studies of SLE have indicated that Toll‐like receptors (TLR) are important in the pathogenesis of murine lupus. In the present clinical study, differential protein expressions of TLR‐1–9 of monocytes and different lymphocyte subsets from patients with SLE and normal control subjects were determined by flow cytometry. Results showed that the expression of intracellular TLRs (TLR‐3, ‐8, ‐9) and extracellular TLRs (TLR‐1, ‐2, ‐4, ‐5, ‐6) were elevated in monocytes, CD4⁺ T lymphocytes, CD8⁺ T lymphocytes and B lymphocytes of SLE patients compared to control subjects (all P < 0·001). Moreover, cell surface expression of TLR‐4 on CD4⁺ T lymphocytes and CD8⁺ T lymphocytes, and TLR‐6 on B lymphocytes, were correlated positively with SLE disease activity index (SLEDAI) (TLR‐4 on CD4⁺ T lymphocytes and CD8⁺ T lymphocytes: r = 0·536, P = 0·04; r = 0·713, P = 0·003; TLR‐6 in B lymphocytes: r = 0·572, P = 0·026). In concordance with the above results, there is an observable increased relative induction (%) of inflammatory cytokine interleukin (IL)‐1β, IL‐6, IL‐10 and IL‐12, chemokines CCL2, CXCL8, CCL5 and CXCL10 from peripheral blood mononuclear cells (PBMC) upon differential stimulation by PolyIC (TLR‐3 ligand), lipopolysaccharide (TLR‐4 ligand), peptidoglycan (TLR‐2 ligand), flagellin (TLR‐5 ligand), R837 (TLR‐7 ligand) and CpG DNA (TLR‐9 ligand) in SLE patients compared to controls. These results suggest that the innate immune response for extracellular pathogens and self‐originated DNA plays immunopathological roles via TLR activation in SLE.     

Programmed death (PD)‐1 molecule and its ligand PD‐L1 distribution among memory CD4 and CD8 T cell subsets in human immunodeficiency virus‐1‐infected individuals

Human immunodeficiency virus (HIV)‐1 causes T cell anergy and affects T cell maturation. Various mechanisms are responsible for impaired anti‐HIV‐1‐specific responses: programmed death (PD)‐1 molecule and its ligand PD‐L1 are negative regulators of T cell activity and their expression is increased during HIV‐1 infection. This study examines correlations between T cell maturation, expression of PD‐1 and PD‐L1, and the effects of their blockade. Peripheral blood mononuclear cells (PBMC) from 24 HIV‐1⁺ and 17 uninfected individuals were phenotyped for PD‐1 and PD‐L1 expression on CD4⁺ and CD8⁺ T cell subsets. The effect of PD‐1 and PD‐L1 blockade on proliferation and interferon (IFN)‐γ production was tested on eight HIV‐1⁺ patients. Naive (CCR7⁺CD45RA⁺) CD8⁺ T cells were reduced in HIV‐1 aviraemic (P = 0·0065) and viraemic patients (P = 0·0130); CD8 T effector memory subsets [CCR7^‐CD45RA^–(T_EM)] were increased in HIV‐1⁺ aviraemic (P = 0·0122) and viraemic (P = 0·0023) individuals versus controls. PD‐1 expression was increased in CD4 naive (P = 0·0496), central memory [CCR7⁺CD45RA^– (T_CM); P = 0·0116], T_EM (P = 0·0037) and CD8 naive T cells (P = 0·0133) of aviraemic HIV‐1⁺versus controls. PD‐L1 was increased in CD4 T_EMRA (CCR7^‐CD45RA⁺, P = 0·0119), CD8 T_EM (P = 0·0494) and CD8 T_EMRA (P = 0·0282) of aviraemic HIV‐1⁺versus controls. PD‐1 blockade increased HIV‐1‐specific proliferative responses in one of eight patients, whereas PD‐L1 blockade restored responses in four of eight patients, but did not increase IFN‐γ‐production. Alteration of T cell subsets, accompanied by increased PD‐1 and PD‐L1 expression in HIV‐1 infection contributes to anergy and impaired anti‐HIV‐1‐specific responses which are not rescued when PD‐1 is blocked, in contrast to when PD‐L1 is blocked, due possibly to an ability to bind to receptors other than PD‐1.     

IL‐33‐activated murine mast cells control the dichotomy between RORγt+ and Helios+ Tregs via the MK2/3‐mediated IL‐6 production in vitro

In mast cells, IL‐33 typically induces the activation of NF‐κB, which results in the production of cytokines such as IL‐6 and IL‐2. Here, we demonstrate that the IL‐33‐induced IL‐6 production in murine mast cells and the formation of RORγt⁺ T_regs essentially depends on the MAPKAPs, MK2, and MK3 (MK2/3) downstream of MyD88. In contrast to this, the IL‐33‐induced and MyD88‐dependent IL‐2 production in mast cells contributes to the maintenance of Helios⁺ T_regs. Thereby, the IL‐33‐induced IL‐2 response and, thus, the maintenance of Helios⁺ T_regs are limited by an IL‐6‐mediated autocrine negative feedback stimulation acting on mast cells. Collectively, we present MK2/3 in IL‐33‐activated mast cells as a signaling node, which controls the dichotomy between RORγt⁺ T_reg and Helios⁺ T_reg in vitro.     

Differentiation of ICOS+ and ICOS− recent thymic emigrant regulatory T cells (RTE Tregs) during normal pregnancy,pre‐eclampsia and HELLP syndrome

Two different subsets of naturally occurring regulatory T cells (nT_regs), defined by their expression of the inducible co‐stimulatory (ICOS) molecule, are produced by the human thymus. To examine the differentiation of ICOS⁺ and ICOS^?CD45RA⁺CD31⁺ recent thymic emigrant (RTE) T_regs during normal pregnancy and in the presence of pre‐eclampsia or haemolysis elevated liver enzymes low platelet (HELLP)‐syndrome, we used six‐colour flow cytometric analysis to determine the changes in the composition of the ICOS⁺ and ICOS^? T_reg pools with CD45RA⁺CD31⁺ RTE T_regs, CD45RA⁺CD31^? mature naive (MN) T_regs, CD45RA^?CD31⁺ and CD45RA^?CD31^? memory T_regs. With the beginning of pregnancy until term, we observed a strong differentiation of both ICOS⁺ and ICOS^?CD45RA⁺CD31⁺ RTE, but not CD45RA⁺CD31^? MN T_regs, into CD45RA^?CD31^? memory T_regs. At the end of pregnancy, the onset of spontaneous term labour was associated with a significant breakdown of ICOS⁺CD45RA^?CD31^? memory T_regs. However, in the presence of pre‐eclampsia, there was a significantly increased differentiation of ICOS⁺ and ICOS^?CD45RA⁺CD31⁺ RTE T_regs into CD45RA^?CD31⁺ memory T_regs, wherein the lacking differentiation into CD45RA^?CD31^? memory T_regs was partially replaced by the increased differentiation of ICOS⁺ and ICOS^?CD45RA⁺CD31^? MN T_regs into CD45RA^?CD31^? memory T_regs. In patients with HELLP syndrome, this alternatively increased differentiation of CD45RA^?CD31^? MN T_regs seemed to be exaggerated, and presumably restored the suppressive activity of magnetically isolated ICOS⁺ and ICOS^? T_regs, which were shown to be significantly less suppressive in pre‐eclampsia patients, but not in HELLP syndrome patients. Hence, our findings propose that the regular differentiation of both ICOS⁺ and ICOS^?CD45RA⁺CD31⁺ RTE T_regs ensures a healthy pregnancy course, while their disturbed differentiation is associated with the occurrence of pre‐eclampsia and HELLP syndrome.     

Depletion of CD4+CD25+ regulatory T cells enhances natural killer T cell‐mediated anti‐tumour immunity in a murine mammary breast cancer model

Both invariant natural killer T (NK T) cells and CD4⁺CD25⁺ T regulatory cells (T_regs) regulate the immune system to maintain homeostasis. In a tumour setting, NK T cells activated by α‐galactosylceramide (α‐GalCer) execute anti‐tumour activity by secreting cytokines. By contrast, T_regs intrinsically suppress antigen‐specific immune responses and are often found to be elevated in tumour patients. In this study, we have shown that T_regs regulate NK T cell function negatively in vitro, suggesting a direct interaction between these cell types. In a murine mammary tumour model, we demonstrated that administration of either α‐GalCer or anti‐CD25 antibody alone markedly suppressed tumour formation and pulmonary metastasis, and resulted in an increase in the survival rate up to 44% (from a baseline of 0%). When treatments were combined, depletion of T_regs boosted the anti‐tumour effect of α‐GalCer, and the survival rate jumped to 85%. Our results imply a potential application of combining T_reg cell depletion with α‐GalCer to stimulate NK T cells for cancer therapy.     

Characteristics of Schistosoma japonicum infection induced IFN‐γ and IL‐4 co‐expressing plasticity Th cells

Schistosoma japonicum infection can induce granulomatous inflammation and cause tissue damage in the mouse liver. The cytokine secretion profile of T helper (Th) cells depends on both the nature of the activating stimulus and the local microenvironment (e.g. cytokines and other soluble factors). In the present study, we found an accumulation of large numbers of IFN‐γ⁺ IL‐4⁺ CD4⁺ T cells in mouse livers. This IFN‐γ⁺ IL‐4⁺ cell population increased from 0·68 ± 0·57% in uninfected mice to 7·05 ± 3·0% by week 4 following infection and to 9·6 ± 5·28% by week 6, before decreasing to 6·3 ± 5·9% by week 8 in CD4 T cells. Moreover, IFN‐γ⁺ IL‐4⁺ Th cells were also found in mouse spleen and mesenteric lymph nodes 6 weeks after infection. The majority of the IFN‐γ⁺ IL‐4⁺ Th cells were thought to be related to a state of immune activation, and some were memory T cells. Moreover, we found that these S. japonicum infection‐induced IFN‐γ⁺ IL‐4⁺ cells could express interleukin‐2 (IL‐2), IL‐9, IL‐17 and high IL‐10 levels at 6 weeks after S. japonicum infection. Taken together, our data suggest the existence of a population of IFN‐γ⁺ IL‐4⁺ plasticity effector/memory Th cells following S. japonicum infection in C57BL/6 mice.     

Associations of B cell‐activating factor (BAFF) and anti‐BAFF autoantibodies with disease activity in multi‐ethnic Asian systemic lupus erythematosus patients in Singapore

To measure the levels of B cell‐activating factor (BAFF) and endogenous anti‐BAFF autoantibodies in a cohort of multi‐ethnic Asian systemic lupus erythematosus (SLE) patients in Singapore, to determine their correlation with disease activity. Serum samples from 121 SLE patients and 24 age‐ and sex‐matched healthy controls were assayed for BAFF and anti‐BAFF immunoglobulin (Ig)G antibody levels by enzyme‐linked immunosorbent assay (ELISA). The lowest reliable detection limit for anti‐BAFF‐IgG antibody levels was defined as 2 standard deviations (s.d.) from blank. Correlation of serum BAFF and anti‐BAFF IgG levels with disease activity [scored by SLE Activity Measure revised (SLAM‐R)], and disease manifestations were determined in these 121 patients. SLE patients had elevated BAFF levels compared to controls; mean 820 ± 40 pg/ml and 152 pg ± 45/ml, respectively [mean ± standard error of the mean (s.e.m.), P < 0·01], which were correlated positively with anti‐dsDNA antibody levels (r = 0·253, P < 0·03), and SLAM‐R scores (r = 0·627, P < 0·01). In addition, SLE patients had significantly higher levels of anti‐BAFF IgG, which were correlated negatively with disease activity (r = –0·436, P < 0·01), levels of anti‐dsDNA antibody (r = –0·347, P < 0·02) and BAFF (r = –0·459, P < 0·01). The majority of patients in this multi‐ethnic Asian SLE cohort had elevated levels of BAFF and anti‐BAFF antibodies. Anti‐BAFF autoantibody levels correlated negatively with clinical disease activity, anti‐dsDNA and BAFF levels, suggesting that they may be disease‐modifying. Our results provide further information about the complexity of BAFF pathophysiology in different SLE disease populations and phenotypes, and suggest that studies of the influence of anti‐cytokine antibodies in different SLE populations will be required when selecting patients for trials using targeted anti‐cytokine therapies.