Elevated Levels of CD4+CD25+FoxP3+ T Cells in Systemic Sclerosis Patients Contribute to the Secretion of IL-17 and Immunosuppression Dysfunction

Objective

Immune imbalance between regulatory T (Treg) and Th17 cells is a characteristic of systemic sclerosis (SSc). The functional heterogeneity among Treg can be elucidated by separating Treg into different subsets based on the expression of FoxP3 and CD45RA. The aim of this study was to investigate the role of Treg subsets in the immune imbalance in naïve SSc.

Methods

Peripheral blood mononuclear cells (PBMCs) of 31 SSc patients and 33 healthy controls were analyzed for the expression of CD4, CD25, CD45RA, CTLA-4, FoxP3, and IL-17 using flow cytometry. Treg immunesuppression capacity was measured in co-culture experiments. The expression of FoxP3, CTLA-4, IL-17A, and RORC mRNA was measured by real-time PCR.

Results

The frequency of CD4⁺CD25⁺FoxP3⁺ Treg cells was significantly elevated in patients with SSc (3.62±1.14 vs 1.97±0.75, p<0.001) with diminished immunosuppression capacity. In SSc, the proportion of FoxP3^highCD45RA⁻ activated Treg cells (aTreg) was decreased, the proportion of FoxP3^lowCD45RA⁻ T cells was increased, and the proportion of FoxP3^lowCD45RA⁺ resting Treg cells (rTreg) was decreased. The immune suppression capacity of aTreg and rTreg was diminished, while FoxP3^lowCD45RA⁻ T cells exhibited a lack of suppression capacity. The immune dysfunction of aTreg was accompanied by the abnormal expression of CTLA-4. Th17 cell numbers were elevated in SSc, FoxP3^lowCD45RA⁻ T cells produced IL-17, confirming their Th17 potential, which was consistent with the elevated levels of FoxP3⁺IL-17⁺ cells in SSc.

Conclusion

A decrease in aTreg levels, along with functional deficiency, and an increase in the proportion of FoxP3^lowCD45RA⁻ T cells, was the reason for the increase in dysfunctional Treg in SSc patients, potentially causing the immune imbalance between Treg and Th17 cells.     

Numerical defects in CD8CD28 T-suppressor lymphocyte population in patients with type 1 diabetes mellitus and multiple sclerosis

Type 1 diabetes mellitus (T1D) and multiple sclerosis (MS) are organ-specific autoimmune diseases leading to an attack of auto-aggressive lymphocytes against the pancreatic β-cells and central nervous system, respectively. Using four-colour flow cytometry, T-lymphocyte populations having an important function in autoimmune processes were analyzed. T-regulatory cells (Treg) CD4⁺CD25⁺CD127^low, T-suppressor cells (Ts) CD8⁺CD28⁻, activated helper CD4⁺CD25⁺CD127⁺ and cytotoxic CD8⁺CD25⁺ T-cells and also naive CD4⁺CD45RA⁺ and memory T-cells CD4⁺CD45RO⁺ were compared in the group of patients with T1D (n = 30), MS (n = 31) and in the group of healthy controls (n = 29). Significant differences in Ts cells, activated helper and cytotoxic cells and also memory T-cells were recognized in the group of T1D patients compared to healthy controls. Ts population was significantly lowered in MS patients as well. However, no significant differences were noticed in Treg population. The observed data demonstrate significant differences among patients with T1D and MS in comparison to healthy individuals.     

Impaired thymic export and apoptosis contribute to regulatory T-cell defects in patients with chronic heart failure

Objective

Animal studies suggest that regulatory T (T_reg) cells play a beneficial role in ventricular remodeling and our previous data have demonstrated defects of T_reg cells in patients with chronic heart failure (CHF). However, the mechanisms behind T_reg-cell defects remained unknown. We here sought to elucidate the mechanism of T_reg-cell defects in CHF patients.

Methods and Results

We performed flow cytometry analysis and demonstrated reduced numbers of peripheral blood CD4⁺CD25⁺FOXP3⁺CD45RO⁻CD45RA⁺ naïve T_reg (nT_reg) cells and CD4⁺CD25⁺FOXP3⁺CD45RO⁺CD45RA⁻ memory T_reg (mT_reg) cells in CHF patients as compared with non-CHF controls. Moreover, the nT_reg/mT_reg ratio (p<0.01), CD4⁺CD25⁺FOXP3⁺CD45RO⁻ CD45RA⁺CD31⁺ recent thymic emigrant T_reg cell (RTE-T_reg) frequency (p<0.01), and T-cell receptor excision circle levels in T_reg cells (p<0.01) were lower in CHF patients than in non-CHF controls. Combined annexin-V and 7-AAD staining showed that peripheral T_reg cells from CHF patients exhibited increased spontaneous apoptosis and were more prone to interleukin (IL)-2 deprivation- and CD95 ligand-mediated apoptosis than those from non-CHF individuals. Furthermore, analyses by both flow cytometry and real-time polymerase chain reaction showed that T_reg-cell frequency in the mediastinal lymph nodes or Foxp3 expression in hearts of CHF patients was no higher than that of the non-CHF controls.

Conclusion

Our data suggested that the T_reg-cell defects of CHF patients were likely caused by decreased thymic output of nascent T_reg cells and increased susceptibility to apoptosis in the periphery.     

Skewed Distribution of IL-7 Receptor-α-Expressing Effector Memory CD8+ T Cells with Distinct Functional Characteristics in Oral Squamous Cell Carcinoma

CD8⁺ T cells play important roles in anti-tumor immunity but distribution profile or functional characteristics of effector memory subsets during tumor progression are unclear. We found that, in oral squamous carcinoma patients, circulating CD8⁺ T cell pools skewed toward effector memory subsets with the distribution frequency of CCR7⁻CD45RA⁻CD8⁺ T cells and CCR7⁻ CD45RA⁺CD8⁺ T cells negatively correlated with each other. A significantly higher frequency of CD127^lo CCR7⁻CD45RA⁻CD8⁺ T cells or CCR7⁻CD45RA⁺CD8⁺ T cells among total CD8⁺ T cells was found in peripheral blood or tumor infiltrating lymphocytes, but not in regional lymph nodes. The CD127^hi CCR7⁻CD45RA⁻CD8⁺ T cells or CCR7⁻CD45RA⁺CD8⁺ T cells maintained significantly higher IFN-γ, IL-2 productivity and ex vivo proliferative capacity, while the CD127^lo CCR7⁻CD45RA⁻CD8⁺ T cells or CCR7⁻CD45RA⁺CD8⁺ T cells exhibited higher granzyme B productivity and susceptibility to activation induced cell death. A higher ratio of CCR7⁻CD45RA⁺CD8⁺ T cells to CCR7⁻CD45RA⁻CD8⁺ T cells was associated with advanced cancer staging and poor differentiation of tumor cells. Therefore, the CD127^lo CCR7⁻CD45RA⁻CD8⁺ T cells and CCR7⁻CD45RA⁺CD8⁺ T cells are functionally similar CD8⁺ T cell subsets which exhibit late differentiated effector phenotypes and the shift of peripheral CD8⁺ effector memory balance toward CCR7⁻CD45RA⁺CD8⁺ T cells is associated with OSCC progression.     

Intracerebral human regulatory T cells: analysis of CD4+ CD25+ FOXP3+ T cells in brain lesions and cerebrospinal fluid of multiple sclerosis patients

Impaired suppressive capacity of CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Treg) from peripheral blood of patients with multiple sclerosis (MS) has been reported by multiple laboratories. It is, however, currently unresolved whether Treg dysfunction in MS patients is limited to reduced control of peripheral T cell activation since most studies analyzed peripheral blood samples only. Here, we assessed early active MS lesions in brain biopsies obtained from 16 patients with MS by FOXP3 immunohistochemistry. In addition, we used six-color flow cytometry to determine numbers of Treg by analysis of FOXP3/CD127 expression in peripheral blood and cerebrospinal fluid (CSF) of 17 treatment-naïve MS patients as well as quantities of apoptosis sensitive CD45RO^hiCD95^hi cells in circulating and CSF Treg subsets. Absolute numbers of FOXP3⁺ and CD4⁺ cells were rather low in MS brain lesions and Treg were not detectable in 30% of MS biopsies despite the presence of CD4⁺ cell infiltrates. In contrast, Treg were detectable in all CSF samples and Treg with a CD45RO^hiCD95^hi phenotype previously shown to be highly apoptosis sensitive were found to be enriched in the CSF compared to peripheral blood of MS patients. We suggest a hypothetical model of intracerebral elimination of Treg by CD95L-mediated apoptosis within the MS lesion.     

CD45 Isoform Profile Identifies Natural Killer (NK) Subsets with Differential Activity

The leucocyte-specific phosphatase CD45 is present in two main isoforms: the large CD45RA and the short CD45RO. We have recently shown that distinctive expression of these isoforms distinguishes natural killer (NK) populations. For example, co-expression of both isoforms identifies in vivo the anti tumor NK cells in hematological cancer patients. Here we show that low CD45 expression associates with less mature, CD56^bright, NK cells. Most NK cells in healthy human donors are CD45RA⁺CD45RO^-. The CD45RA^-RO⁺ phenotype, CD45RO cells, is extremely uncommon in B or NK cells, in contrast to T cells. However, healthy donors possess CD45RA^dimRO^- (CD45RA^dim cells), which show immature markers and are largely expanded in hematopoietic stem cell transplant patients. Blood borne cancer patients also have more CD45RA^dim cells that carry several features of immature NK cells. However, and in opposition to their association to NK cell progenitors, they do not proliferate and show low expression of the transferrin receptor protein 1/CD71, suggesting low metabolic activity. Moreover, CD45RA^dim cells properly respond to in vitro encounter with target cells by degranulating or gaining CD69 expression. In summary, they are quiescent NK cells, with low metabolic status that can, however, respond after encounter with target cells.     

Peripheral Blood T Cell Dynamics Predict Relapse in Multiple Sclerosis Patients on Fingolimod

Background

Fingolimod efficiently reduces multiple sclerosis (MS) relapse by inhibiting lymphocyte egress from lymph nodes through down-modulation of sphingosine 1-phosphate (S1P) receptors. We aimed to clarify the alterations in peripheral blood T cell subsets associated with MS relapse on fingolimod.

Methods/Principal Findings

Blood samples successively collected from 23 relapsing-remitting MS patients before and during fingolimod therapy (0.5 mg/day) for 12 months and 18 healthy controls (HCs) were analysed for T cell subsets by flow cytometry. In MS patients, the percentages of central memory T (CCR7⁺CD45RO⁺) cells (TCM) and naïve T (CCR7⁺CD45RO^-) cells decreased significantly, while those of effector memory T (CCR7^-CD45RA^-) and suppressor precursor T (CD28^-) cells increased in both CD4⁺T and CD8⁺T cells from 2 weeks to 12 months during fingolimod therapy. The percentages of regulatory T (CD4⁺CD25^highCD127^low) cells in CD4⁺T cells and CCR7^-CD45RA⁺T cells in CD8⁺T cells also increased significantly. Eight relapsed patients demonstrated greater percentages of CD4⁺TCM than 15 non-relapsed patients at 3 and 6 months (p=0.0051 and p=0.0088, respectively). The IL17-, IL9-, and IL4-producing CD4⁺T cell percentages were significantly higher at pre-treatment in MS patients compared with HCs (p<0.01 for all), while the IL17-producing CD4⁺T cell percentages tended to show a transient increase at 2 weeks of fingolimod therapy (p^corr=0.0834).

Conclusions

The CD4⁺TCM percentages at 2 weeks to 12 months during fingolimod therapy are related to relapse.     

Functional inactivation of EBV-specific T-lymphocytes in nasopharyngeal carcinoma: implications for tumor immunotherapy

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV) associated malignancy with high prevalence in Southern Chinese. In order to assess whether defects of EBV-specific immunity may contribute to the tumor, the phenotype and function of circulating T-cells and tumor infiltrating lymphocytes (TILs) were investigated in untreated NPC patients. Circulating naïve CD3⁺CD45RA⁺ and CD4⁺CD25⁻ cells were decreased, while activated CD4⁺CD25⁺ T-cells and CD3⁻CD16⁺ NK-cells were increased in patients compared to healthy donors. The frequency of T-cells recognizing seven HLA-A2 restricted epitopes in LMP1 and LMP2 was lower in the patients and remained low after stimulation with autologous EBV-carrying cells. TILs expanded in low doses of IL-2 exhibited an increase of CD3⁺CD4⁺, CD3⁺CD45RO⁺ and CD4⁺CD25⁺ cells and 2 to 5 fold higher frequency of LMP1 and LMP2 tetramer positive cells compared to peripheral blood. EBV-specific cytotoxicity could be reactivated from the blood of most patients, whereas the TILs lacked cytotoxic activity and failed to produce IFNγ upon specific stimulation. Thus, EBV-specific rejection responses appear to be functionally inactivated at the tumor site in NPC.     

CD8+ Treg Cells Associated with Decreasing Disease Activity after Intravenous Methylprednisolone Pulse Therapy in Lupus Nephritis with Heavy Proteinuria

We focus on the role of CD8⁺ Treg cell in Intravenous methyl-prednisolone (IVMP) pulse therapy in forty patients with active Class III/IV childhood lupus nephritis (LN) with heavy proteinuria. IVMP therapy for five days. From peripheral blood mononuclear cells (PBMCs) and renal tissues, we saw IVMP therapy definitely restoring both CD4⁺CD25⁺FoxP3⁺ and CD8⁺CD25⁺Foxp3⁺ Treg cell number plus greater expression with intracellular IL-10 and granzyme B in CD8⁺FoxP3⁺ Treg from PBMCs. IVMP-treated CD8⁺CD25⁺ Treg cells directly suppressed CD4⁺ T proliferation and induced CD4⁺CD45RO⁺ apoptosis. Histologically, CD4⁺FoxP3⁺ as well as CD8⁺FoxP3⁺ Treg cells appeared in renal tissue of LN patients before IVMP by double immunohistochemical stain. CD8⁺FoxP3⁺ Treg cells increased in 10 follow-up renal biopsy specimens after IVMP. Reverse correlation of serum anti-C1q antibody and FoxP3⁺ Treg cells in PBMNCs (r = −0.714, P<0.01). After IVMP, serum anti-C1q antibody decrease accompanied increase of CD4⁺FoxP3⁺ Treg cells. CD8⁺Treg cells reduced interferon-r response in PBMCs to major peptide autoepitopes from nucleosomes after IVMP therapy; siRNA of FoxP3 suppressed granzyme B expression while decreasing CD8⁺CD25⁺Treg-induced CD4⁺CD45RO⁺ apoptosis. Renal activity of LN by SLEDAI-2k in childhood LN was significantly higher than two weeks after IVMP (P<0.01). CD8⁺FoxP3⁺ Treg cells return in post-IVMP therapy and exert crucial immune modulatory effect to control autoimmune response in LN.

Trial Registration

DMR97-IRB-259     

Regulatory T Cell Responses to High-Dose Methylprednisolone in Active Systemic Lupus Erythematosus

Background/Purpose

A slight increase in the proportion of circulating regulatory T (Treg) cells has been reported in systemic lupus erythematosus (SLE) patients taking oral prednisone. The effects of intravenous (IV) high dose methylprednisolone (MP) on Tregs have not yet been described, especially in active SLE.

Methods

We prospectively analyzed the proportion of circulating CD4⁺ Treg cell subsets defined as follows: (1) naïve Treg (nTreg) FoxP3^lowCD45RA⁺ cells; (2) effector Treg (eTreg) FoxP3^highCD45RA⁻ cells; and (3) non-suppressive FoxP3^lowCD45RA⁻ cells (non-regulatory Foxp3^low T cells). Peripheral blood mononuclear cells of patients with active SLE were analyzed before the first infusion of IV high dose MP (day 0) and the following days (day 1, day 2, ±day 3 and ±day 8). The activity of SLE was assessed by the SLEDAI score.

Results

Seventeen patients were included. Following MP infusions, the median (range) percentage of eTregs significantly increased from 1.62% (0.53–8.43) at day 0 to 2.80% (0.83–14.60) at day 1 (p = 0.003 versus day 0), 4.64% (0.50–12.40) at day 2 (p = 0.06 versus day 1) and 7.50% (1.02–20.70) at day 3 (p = 0.008 versus day 2), and declined to baseline values at day 8. Expanding eTreg cells were actively proliferating, as they expressed Ki-67. The frequency of non-regulatory FoxP3^low T cells decreased from 6.39% (3.20–17.70) at day 0 to 4.74% (1.03–9.72) at day 2 (p = 0.005); nTreg frequency did not change. All patients clinically improved immediately after MP pulses. The absence of flare after one year of follow up was associated with a higher frequency of eTregs at day 2.

Conclusion

IV high dose MP induces a rapid, dramatic and transient increase in circulating regulatory T cells. This increase may participate in the preventive effect of MP on subsequent flares in SLE.     

Immunophenotypic Characterization of CD45RO+ and CD45RA+ T Cell Subsets in Peripheral Blood of Peripheral T Cell Lymphoma Patients

To study the distribution profile of CD45RO⁺ and CD45RA⁺ T cells in the peripheral blood of peripheral T cell lymphoma (PTCL) patients and its clinical significance. 27 patients with PTCL were enrolled in this study, together with 30 healthy individuals as the control group. Flow cytometry analysis was employed to examinate the differences in the distribution of CD45RO⁺ and CD45RA⁺ T cells in peripheral blood between two groups. In PTCL patient’s lymphnode tissues, the T cell population displayed diverse antigenic expression, with CD4⁺ T cells as the major subset. No B cell-related antigen was expressed. The percentage of CD4⁺/CD8⁺ and CD4⁺CD45RO⁺ T cells in patients’ peripheral blood were significantly lower than that in the control samples, while the percentage of CD4⁺CD45RA⁺, CD8⁺CD45RA⁺, and CD8⁺CD45RO⁺ T cells in patients’ peripheral blood were significantly higher than that in the control samples. The percentage of CD4⁺/CD8⁺, CD4⁺CD45RO⁺ cells in stage I/II PTCL patients’ peripheral blood were significantly higher than that in the samples from patients with stage III/IV PTCL. The percentage of CD4⁺CD45RA⁺, CD8⁺CD45RA⁺, and CD8⁺CD45RO⁺ T cells were notably lower than that in the samples from III/IV period PTCL patients. Both CD45RO⁺ and CD45RA⁺ T cells play important roles in the process of PTCL. The immunophenotypic profile from this study will help to develop the differential diagnosis and treatment of PTCL patients in the future, and improve the accuracy rate of diagnosis and to ameliorate the prognosis.     

Calcium Homeostasis Change in CD4<Superscript>+</Superscript> T Lymphocytes from Human Peripheral Blood during Differentiation <Emphasis Type="Italic">in vivo</Emphasis>

Resting naive CD4⁺CD45R0^?CD45RA⁺ T cells are sensitive to ionomycin. In contrast, resting CD4⁺CD45RA^?CD45R0⁺ memory T cells show resistance to this Ca²⁺ ionophore. In the present study, the ability of activated T lymphocytes to respond to ionomycin during the transition from naive precursors into memory T cells has been analyzed. Activated CD4⁺CD45RA⁺CD45R0⁺ T cells are always present both in human peripheral blood (HPB) and in the ionomycin-resistant (IR) fraction. Therefore, some activated T cells are resistant toward the Ca²⁺ ionophore. CD69 molecules are markers of the very early stage of T cell activation. However, CD4⁺CD69⁺ T cells have never been found in the IR fraction. Thus, the majority of CD4⁺ T lymphocytes at the early stage of activation are ionomycin-sensitive cells. The proportion of CD4⁺CD25⁺ T cells did not differ significantly in HPB and in the IR fraction. The presence of CD4⁺CD25⁺ T lymphocytes in the IR fraction reflects changes in the Ca²⁺-signaling pathway at this differentiation step of activated cells. Depending on the expression level of CD25 molecules, the population of CD4⁺CD25⁺ cells is divided in T-regulatory (CD25^high) and proliferating (CD25^low) subpopulations. The action of ionomycin results in a decrease in the portion of the CD4⁺CD25^low T-cells, but it leads to an increase in the proportion of the CD4⁺CD25^high T lymphocytes. Consequently, greater portion of CD4⁺CD25^high T lymphocytes and smaller portion of CD4⁺CD25^low T cells are IR cells. Expression of HLA-DR molecules can be used as the marker for the late activation step. The IR fraction is significantly rich in CD4⁺HLA-DR⁺ T lymphocytes in comparison to the blood of the same donor. The link between different differentiation steps of CD4⁺ T-lymphocytes and alterations in calcium ion homeostasis is discussed.     

Quantification of Regulatory T Cells in Septic Patients by Real-Time PCR–Based Methylation Assay and Flow Cytometry

During sepsis, a relative increase of regulatory T (Treg) cells has been reported. Its persistence is associated with lymphocyte anergy, immunoparalysis and a poor prognosis. Currently, an exact quantification of human Treg cells based on protein expression of marker molecules is ambiguous, as these molecules are expressed also by activated non-regulatory T cells. Furthermore, no firm criteria for flow cytometer gate settings exist so far. Recently, a specific DNA methylation pattern within FOXP3-TSDR has been reported that allows distinguishing Treg and non-regulatory T cells, independent of their activation status. Using this epigenetic marker, we established a single-tube real-time PCR based methylation assay (QAMA) for relative quantification of Treg cells. Validation was performed on defined ratios of methylated and unmethylated target sequence and on mixtures of Treg and non-regulatory T cells. DNA-methylation was measured in CD4⁺ T cells isolated from blood samples of 30 septic patients and 30 healthy subjects and compared with results of Treg cell quantification by flow cytometry based on CD4⁺ CD25^hiCD127^low measurement. In septic patients both methods showed an increased ratio of Treg cells to all CD4⁺ T cells. In healthy individuals, the results obtained by both methods were clearly positively correlated. However, the correlation between both methods in septic patients was only weak. We showed that quantification of Treg cells by QAMA detects CD4⁺ T cells with unmethylated FOXP3-TSDR, hidden in the CD25^med/low fraction of flow cytometry. Given that unmethylated FOXP3-TSDR is the most specific feature of Treg cells to date, our assay precisely quantifies Treg cells, as it additionally detects those committed Treg cells, hidden in the CD25^med/low fraction of CD4⁺ cells. Furthermore, QAMA is a reliable method, which is easier to standardize among laboratories and can thus improve reproducibility of Treg cell quantification.     

Heterogeneity of CD4 and CD8+ memory T cells in localized and generalized Wegener's granulomatosis

Memory T cells display phenotypic heterogeneity. Surface antigens previously regarded as exclusive markers of naive T cells, such as L-selectin (CD62L), can also be detected on some memory T cells. Moreover, a fraction of CD45RO⁺ (positive for the short human isoform of CD45) memory T cells reverts to the CD45RA⁺ (positive for the long human isoform of CD45) phenotype. We analyzed patients with biopsy-proven localized Wegener's granulomatosis (WG) (n = 5), generalized WG (n = 16) and age- and sex-matched healthy controls (n = 13) to further characterize memory T cells in WG. The cell-surface expression of CD45RO, CD45RA, CD62L, CCR3, CCR5 and CXCR3 was determined on blood-derived T cells by four-color flow cytometric analysis. The fractions of CCR5⁺ and CCR3⁺ cells within the CD4⁺CD45RO⁺ and CD8⁺CD45RO⁺ memory T cell populations were significantly expanded in localized and generalized WG. The mean percentage of Th1-type CCR5 expression was higher in localized WG. Upregulated CCR5 and CCR3 expression could also be detected on a fraction of CD45RA⁺ T cells. CD62L expression was seen on approximately half of the memory T cell populations expressing chemokine receptors. This study demonstrates for the first time that expression of the inducible inflammatory chemokine receptors CCR5 and CCR3 on CD45RO⁺ memory T cells, as well as on CD45RA⁺ T cells ('revertants'), contributes to phenotypic heterogeneity in an autoimmune disease, namely WG. Upregulated CCR5 and CCR3 expression suggests that the cells belong to the effector memory T cell population. CCR5 and CCR3 expression on CD4⁺ and CD8⁺ memory T cells indicates a potential to respond to chemotactic gradients and might be important in T cell migration contributing to granuloma formation and vasculitis in WG.     

Relationship between FOXP3 positive populations and cytokine production in systemic lupus erythematosus

In this work we studied CD4⁺FOXP3⁺ populations in systemic lupus erythematosus (SLE) and the relationship with Th cytokine production. We found an increment in CD25^?FOXP3⁺ population in SLE associated with CD4⁺ downregulation and disease progression. CD25^low cells were also upregulated and showed increased percentages of FOXP3⁺ and CD127^?/low cells, supporting the activated status of SLE lymphocytes. Despite the normal levels of CD25^highFOXP3⁺ cells, the negative correlations observed in controls with the frequency of IFNγ, TNFα and IL-10 secreting cells were disrupted in patients, supporting a defective Treg function. Also, CD25^high cells showed an altered balance in the production of these cytokines. In addition, CD25^highFOXP3⁺ cells correlated directly with IL-17A and IL-8 but not with TGFβ in SLE. The increased proportion of IL-17⁺ cells among the CD25^high subset and the positive correlation between IL-17 levels and Treg cells suggest a trans-differentiation of Treg into Th17 cells in SLE.     

IL-33 Induces IL-9 Production in Human CD4+ T Cells and Basophils

IL-33, an IL-1 family member and ligand for the IL-1 receptor-related protein ST2, has been associated with induction of Th2 cytokines such as IL-4, IL-5, and IL-13. Here, we report that IL-33 can initiate IL-9 protein secretion in vitro in human CD4+ T cells and basophils isolated from peripheral blood. TGF-β has been described as a critical factor for IL-9 induction in Th2 cells; however, we found that TGF-β also induces co-production of IL-9 in purified, naïve (>99%) CD4⁺CD45RA⁺CD45RO⁻CD25⁻ T cells differentiated towards a Th1 profile. Subsequently, it was demonstrated that TGF-β is important, although not an absolute requirement, for IL-9 production in CD4+ T cells. IL-9 production by purified (>95%) human basophils, cultured for 24 h with IL-3 or IL-33, was found, with a strong synergy between the two, likely to be explained by the IL-3 upregulated ST2 expression. Collectively, these data indicate that barrier functioning cells are important for the regulation of IL-9 production by immune cells in inflamed tissue.     

Adaptive immune neuroprotection in G93A-SOD1 amyotrophic lateral sclerosis mice

Background

Innate neuroimmune dysfunction is a pathobiological feature of amyotrophic lateral sclerosis (ALS). However, links, if any, between disease and adaptive immunity are poorly understood. Thus, the role of T cell immunity in disease was investigated in human G93A superoxide dismutase 1 (SOD1) transgenic (Tg) mice and subsequently in ALS patients.

Methods and Findings

Quantitative and qualitative immune deficits in lymphoid cell and T cell function were seen in G93A-SOD1 Tg mice. Spleens of Tg animals showed reductions in size, weight, lymphocyte numbers, and morphological deficits at terminal stages of disease compared to their wild-type (Wt) littermates. Spleen sizes and weights of pre-symptomatic Tg mice were unchanged, but deficits were readily seen in T cell proliferation coincident with increased annexin-V associated apoptosis and necrosis of lymphocytes. These lymphoid deficits paralleled failure of Copolymer-1 (COP-1) immunization to affect longevity. In addition, among CD4⁺ T cells in ALS patients, levels of CD45RA⁺ (naïve) T cells were diminished, while CD45RO⁺ (memory) T cells were increased compared to age-matched caregivers. In attempts to correct mutant SOD1 associated immune deficits, we reconstituted SOD1 Tg mice with unfractionated naïve lymphocytes or anti-CD3 activated CD4⁺CD25⁺ T regulatory cells (Treg) or CD4⁺CD25⁻ T effector cells (Teff) from Wt donor mice. While naive lymphocytes failed to enhance survival, both polyclonal-activated Treg and Teff subsets delayed loss of motor function and extended survival; however, only Treg delayed neurological symptom onset, whereas Teff increased latency between disease onset and entry into late stage.

Conclusions

A profound and progressive immunodeficiency is operative in G93A-SOD1 mice and is linked to T cell dysfunction and the failure to elicit COP-1 neuroprotective immune responses. In preliminary studies T cell deficits were also observed in human ALS. These findings, taken together, suggest caution in ascribing vaccination outcomes when these animal models of human ALS are used for study. Nonetheless, the abilities to improve neurological function and life expectancy in G93A-SOD1 Tg mice by reconstitution with activated T cells do provide opportunities for therapeutic intervention.     

CD4+CD25hiCD127low Regulatory T Cells Are Increased in Oral Squamous Cell Carcinoma Patients

Regulatory T cells (Tregs), a subset of CD4⁺ T cells plays a pivotal role in regulating the immune system. An increase in Treg numbers enables cancer progression by dampening the immune system and allowing tumor cells to evade immune detection and destruction. An increase in Treg numbers and expression of inhibitory cytokines including TGF-β and IL-10 are mechanisms by which Tregs exert their immune suppressive function. However, the presence of Tregs and inhibitory cytokines in oral cancer patients is still unclear. In this study, the presence of circulating Tregs in 39 oral cancer patients and 24 healthy donors was examined by studying the presence of the CD4⁺CD25^hiCD127^low cell population in their peripheral blood mononuclear cells using flow cytometry. Serum levels of TGF-β and IL-10 were measured by ELISA. T cell subsets of OSCC patients were found to differ significantly from healthy donors where a decrease in CD8⁺ cytotoxic T cells and an increase in Tregs (CD4⁺CD25^hiCD127^low) were observed. Further, the ratio of CD8⁺ T cells/Tregs was also decreased in patients compared to healthy donors. The presence of Tregs was accompanied by a decrease in IL-10 but not TGF-β secretion in OSCC patients when compared to donors; in addition, the analysis also revealed that an increased presence of Tregs was accompanied by better patient survival. Amongst OSCC patients, smokers had significantly higher levels of TGF-β. It is apparent that the immune system is compromised in OSCC patients and the characterization of the Treg subpopulation could form a basis for improving our understanding of the perturbations in the immune system that occur during OSCC tumorigenesis.     

Mtb-Specific CD27(low) CD4 T Cells as Markers of Lung Tissue Destruction during Pulmonary Tuberculosis in Humans

Background

Effector CD4 T cells represent a key component of the host’s anti-tuberculosis immune defense. Successful differentiation and functioning of effector lymphocytes protects the host against severe M. tuberculosis (Mtb) infection. On the other hand, effector T cell differentiation depends on disease severity/activity, as T cell responses are driven by antigenic and inflammatory stimuli released during infection. Thus, tuberculosis (TB) progression and the degree of effector CD4 T cell differentiation are interrelated, but the relationships are complex and not well understood. We have analyzed an association between the degree of Mtb-specific CD4 T cell differentiation and severity/activity of pulmonary TB infection.

Methodology/Principal Findings

The degree of CD4 T cell differentiation was assessed by measuring the percentages of highly differentiated CD27^low cells within a population of Mtb- specific CD4 T lymphocytes (“CD27^lowIFN-γ⁺” cells). The percentages of CD27^lowIFN-γ+ cells were low in healthy donors (median, 33.1%) and TB contacts (21.8%) but increased in TB patients (47.3%, p<0.0005). Within the group of patients, the percentages of CD27^lowIFN-γ⁺ cells were uniformly high in the lungs (>76%), but varied in blood (12–92%). The major correlate for the accumulation of CD27^lowIFN-γ⁺ cells in blood was lung destruction (r = 0.65, p = 2.7×10⁻⁷⁾. A cutoff of 47% of CD27^lowIFN-γ⁺ cells discriminated patients with high and low degree of lung destruction (sensitivity 89%, specificity 74%); a decline in CD27^lowIFN-γ⁺cells following TB therapy correlated with repair and/or reduction of lung destruction (p<0.01).

Conclusions

Highly differentiated CD27^low Mtb-specific (CD27^lowIFN-γ⁺) CD4 T cells accumulate in the lungs and circulate in the blood of patients with active pulmonary TB. Accumulation of CD27^lowIFN-γ⁺ cells in the blood is associated with lung destruction. The findings indicate that there is no deficiency in CD4 T cell differentiation during TB; evaluation of CD27^lowIFN-γ⁺ cells provides a valuable means to assess TB activity, lung destruction, and tissue repair following TB therapy.