PRMT5 Promotes Cyclin E1 and Cell Cycle Progression in CD4 Th1 Cells and Correlates With EAE Severity.

Multiple Sclerosis (MS) is a debilitating central nervous system disorder associated with inflammatory T cells. Activation and expansion of inflammatory T cells is thought to be behind MS relapses and influence disease severity. Protein arginine N-methyltransferase 5 (PRMT5) is a T cell activation-induced enzyme that symmetrically dimethylates proteins and promotes T cell proliferation. However, the mechanism behind PRMT5-mediated control of T cell proliferation and whether PRMT5 contributes to diseases severity is unclear. Here, we evaluated the role of PRMT5 on cyclin/cdk pairs and cell cycle progression, as well as PRMT5's link to disease severity in an animal model of relapsing-remitting MS. Treatment of T helper 1 (mTh1) cells with the selective PRMT5 inhibitor, HLCL65, arrested activation-induced T cell proliferation at the G1 stage of the cell cycle, suggesting PRMT5 promotes cell cycle progression in CD4+ T cells. The Cyclin E1/Cdk2 pair promoting G1/S progression was also decreased after PRMT5 inhibition, as was the phosphorylation of retinoblastoma. In the SJL mouse relapsing-remitting model of MS, the highest PRMT5 expression in central nervous system-infiltrating cells corresponded to peak and relapse timepoints. PRMT5 expression also positively correlated with increasing CD4 Th cell composition, disease severity and Cyclin E1 expression. These data indicate that PRMT5 promotes G1/S cell cycle progression and suggest that this effect influences disease severity and/or progression in the animal model of MS. Modulating PRMT5 levels may be useful for controlling T cell expansion in T cell-mediated diseases including MS.

Ubiquitin Activating Enzyme UBA6 Regulates Th1 and Tc1 Cell Differentiation.

Ubiquitination is a crucial mechanism in regulating the immune response, setting the balance between immunity and tolerance. Here, we investigated the function of a poorly understood alternative branch of the ubiquitin-activating E1 enzyme UBA6 in activating immune cells. UBA6 expression levels were elevated in T cells by toll-like receptor agonists and anti-CD3/28 antibody stimulation, but not in dendritic cells, macrophages, B cells, and natural killer cells. Additionally, we generated T cell-specific UBA6-deficient mice and found that UBA6-deficient CD4 and CD8 T cells elevated the production of interferon-gamma (IFN-γ). Moreover, the transfer of UBA6-deficient CD4 and CD8 T cells in RAG1-knockout mice exacerbated the development of multi-organ inflammation compared with control CD4 and CD8 T cell transfer. In human peripheral blood CD4 and CD8 T cells, basal levels of UBA6 in lupus patients presented much lower than those in healthy controls. Moreover, the IFN-γ production efficiency of CD4 and CD8 T cells was negatively correlated to UBA6 levels in patients with lupus. Finally, we found that the function of UBA6 was mediated by destabilization of IκBα degradation, thereby increasing NF-κB p65 activation in the T cells. Our study identifies UBA6 as a critical regulator of IFN-γ production in T cells by modulating the NF-κB p65 activation pathway.

Pyruvate kinase M2 is requisite for Th1 and Th17 differentiation.

Th1 and Th17 are important in the pathogenesis of autoimmune diseases and they depend on glycolysis as a source of energy. T cell antigen receptor signaling phosphorylates a serine/threonine kinase, calcium/calmodulin-dependent protein kinase IV (CaMK4), and promotes glycolysis. Based on these findings we hypothesized that CaMK4 promotes glycolysis. Camk4-deficient CD4+ T cells and cells treated with a CaMK4 inhibitor had less glycolysis compared with their counterparts. Pull-down of CaMK4 and mass spectrometry identified pyruvate kinase muscle isozyme (PKM), the final rate-limiting enzyme in glycolysis, as a binding partner. Coimmunoprecipitation and Western blotting showed that CaMK4 interacts directly with PKM2. Camk4-deficient CD4+ T cells displayed decreased pyruvate kinase activity. Silencing or pharmacological inhibition of PKM2 reduced glycolysis and in vitro differentiation to Th1 and Th17 cells, while PKM2 overexpression restored Th17 cell differentiation. Treatment with a PKM2 inhibitor ameliorated experimental autoimmune encephalomyelitis and CD4+ T cells treated with PKM2 inhibitor or Pkm2-shRNA caused limited disease activity in an adoptive cell transfer model of experimental autoimmune encephalomyelitis. Our data demonstrate that CaMK4 binds to PKM2 and promotes its activity, which is requisite for Th1 and Th17 differentiation in vitro and in vivo. PKM2 represents a therapeutic target for T cell-dependent autoimmune diseases.

GdX/UBL4A-knockout mice resist collagen-induced arthritis by balancing the population of Th1/Th17 and regulatory T cells.

Rheumatoid arthritis (RA) is an autoimmune disease associated with synovial hyperplasia and bone and cartilage destruction. T cells, notably T helper (Th)-1 and Th17 cells, play a critical role in the pathologic process of RA. However, it remains unclear how Th1 and Th17 cells are regulated during RA. In this study, we report that the small ubiquitin-like protein X-linked gene in the G6PD cluster at Xq28 (GdX) regulates the balance of Th17 and regulatory T (Treg) cells during collagen-induced arthritis (CIA). We discovered that the splenocytes of GdX-knockout (KO) mice were insensitive to T-cell stimulants. Correspondingly, GdX-KO mice showed alleviative Th1-mediated delayed-type hypersensitivity and were resistant to CIA compared with wild-type mice. GdX-KO mice showed fewer swollen paws, lower serum proinflammatory cytokine and anti-collagen IgG levels, and decreased synovial hyperplasia. Mechanistically, we observed that deletion of GdX decreased the transcription of proinflammatory cytokines and impaired the Th1 and Th17 differentiation but increased the Treg cell proliferation. Consistently, deletion of GdX decreased the transcription level of T-cell-specific T-box transcription factor and RAR-related orphan receptor-γ transcription factor but increased that of forkhead box P3 after being challenged with type-II collagen. These findings suggested that GdX functions as an important regulator of Th1 or Th17 and Treg cell balance during the inflammatory responses. Therefore, GdX may be a potential target for the therapy of RA.-Fu, Y., Liu, S., Wang, Y., Ren, F., Fan, X., Liang, J., Liu, C., Li, J., Ju, Y., Chang, Z. GdX/UBL4A-knockout mice resist collagen-induced arthritis by balancing the population of Th1/Th17 and regulatory T cells.

S-nitrosoglutathione reductase (GSNOR) inhibitor as an immune modulator in experimental autoimmune encephalomyelitis.

We previously reported that S-nitrosoglutathione (GSNO), an endogenous nitric oxide carrier, attenuated TH17-mediated immune responses in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Cellular GSNO homeostasis is regulated via its synthesis by reaction between nitric oxide and glutathione and its enzymatic catabolism by GSNO reductase (GSNOR). In this study, we evaluated potential of reversible inhibitor of GSNOR (N6022) in comparison with exogenous GSNO in immunopathogenesis of EAE. Daily treatment of EAE mice with N6022 or exogenous GSNO significantly attenuated the clinical disease of EAE, but N6022 treatment showed greater efficacy than GSNO. Both N6022 and exogenous GSNO treatments increased the spleen levels of GSNO, as documented by increased protein-associated S-nitrosothiols, and inhibited polarization and CNS effector function of proinflammatory TH17 cells while inducing the polarization and CNS effector function of anti-inflammatory CD4+ CD25+ FOXP3- regulatory T (Treg) cells. Moreover, N6022 further attenuated TH1 while inducing TH2 and CD4+ CD25+ FOXP3+ Treg in their polarization and CNS effector functions. Similar to GSNO, the N6022 treatment protected against the EAE disease induced demyelination. However, neither exogenous GSNO nor N6022 treatment did not cause significant systemic lymphopenic effect as compared to FTY720. Taken together, these data document that optimization of cellular GSNO homeostasis by GSNOR inhibitor (N6022) in NO metabolizing cells attenuates EAE disease via selective inhibition of pro-inflammatory subsets of CD4+ cells (TH1/TH17) while upregulating anti-inflammatory subsets of CD4+ cells (TH2/Treg) without causing lymphopenic effects and thus offers a potential treatment option for MS/EAE.

Aerobic glycolysis promotes T helper 1 cell differentiation through an epigenetic mechanism.

Aerobic glycolysis (the Warburg effect) is a metabolic hallmark of activated T cells and has been implicated in augmenting effector T cell responses, including expression of the proinflammatory cytokine interferon-γ (IFN-γ), via 3' untranslated region (3'UTR)-mediated mechanisms. Here, we show that lactate dehydrogenase A (LDHA) is induced in activated T cells to support aerobic glycolysis but promotes IFN-γ expression independently of its 3'UTR. Instead, LDHA maintains high concentrations of acetyl-coenzyme A to enhance histone acetylation and transcription of Ifng Ablation of LDHA in T cells protects mice from immunopathology triggered by excessive IFN-γ expression or deficiency of regulatory T cells. These findings reveal an epigenetic mechanism by which aerobic glycolysis promotes effector T cell differentiation and suggest that LDHA may be targeted therapeutically in autoinflammatory diseases.

Oxygen Sensing by T Cells Establishes an Immunologically Tolerant Metastatic Niche.

Cancer cells must evade immune responses at distant sites to establish metastases. The lung is a frequent site for metastasis. We hypothesized that lung-specific immunoregulatory mechanisms create an immunologically permissive environment for tumor colonization. We found that T-cell-intrinsic expression of the oxygen-sensing prolyl-hydroxylase (PHD) proteins is required to maintain local tolerance against innocuous antigens in the lung but powerfully licenses colonization by circulating tumor cells. PHD proteins limit pulmonary type helper (Th)-1 responses, promote CD4(+)-regulatory T (Treg) cell induction, and restrain CD8(+) T cell effector function. Tumor colonization is accompanied by PHD-protein-dependent induction of pulmonary Treg cells and suppression of IFN-γ-dependent tumor clearance. T-cell-intrinsic deletion or pharmacological inhibition of PHD proteins limits tumor colonization of the lung and improves the efficacy of adoptive cell transfer immunotherapy. Collectively, PHD proteins function in T cells to coordinate distinct immunoregulatory programs within the lung that are permissive to cancer metastasis. PAPERCLIP.

Immunoprotective responses of T helper type 1 stimulatory protein-S-adenosyl-L-homocysteine hydrolase against experimental visceral leishmaniasis.

It is well known that a patient in clinical remission of visceral leishmaniasis (VL) remains immune to reinfection, which provides a rationale for the feasibility of a vaccine against this deadly disease. In earlier studies, observation of significant cellular responses in treated Leishmania patients as well as in hamsters against leishmanial antigens from different fractions led to its further proteomic characterization, wherein S-adenosyl-L-homocysteine hydrolase (AdoHcy) was identified as a helper type 1 (Th1) stimulatory protein. The present study includes immunological characterization of this protein, its cellular responses [lymphoproliferation, nitric oxide (NO) production and cytokine responses] in treated Leishmania-infected hamsters and patients as well as prophylactic efficacy against Leishmania challenge in hamsters and the immune responses generated thereof. Significantly higher cellular responses were noticed against recombinant L. donovani S-adenosyl-L-homocysteine hydrolase (rLdAdoHcy) compared to soluble L. donovani antigen in treated samples. Moreover, stimulation of peripheral blood mononuclear cells with rLdAdoHcy up-regulated the levels of interferon (IFN)-γ, interleukin (IL)-12 and down-regulated IL-10. Furthermore, vaccination with rLdAdoHcy generated perceptible delayed-type hypersensitivity response and exerted considerably good prophylactic efficacy (∼70% inhibition) against L. donovani challenge. The efficacy was confirmed by the increased expression levels of inducible NO synthase and Th1-type cytokines, IFN-γ and IL-12 and down-regulation of IL-4, IL-10 and transforming growth factor (TGF)-ß. The results indicate the potentiality of rLdAdoHcy protein as a suitable vaccine candidate against VL.

The methylcytosine dioxygenase Tet2 promotes DNA demethylation and activation of cytokine gene expression in T cells.

Epigenetic regulation of lineage-specific genes is important for the differentiation and function of T cells. Ten-eleven translocation (Tet) proteins catalyze 5-methylcytosine (5 mC) conversion to 5-hydroxymethylcytosine (5 hmC) to mediate DNA demethylation. However, the roles of Tet proteins in the immune response are unknown. Here, we characterized the genome-wide distribution of 5 hmC in CD4(+) T cells and found that 5 hmC marks putative regulatory elements in signature genes associated with effector cell differentiation. Moreover, Tet2 protein was recruited to 5 hmC-containing regions, dependent on lineage-specific transcription factors. Deletion of Tet2 in T cells decreased their cytokine expression, associated with reduced p300 recruitment. In vivo, Tet2 plays a critical role in the control of cytokine gene expression in autoimmune disease. Collectively, our findings suggest that Tet2 promotes DNA demethylation and activation of cytokine gene expression in T cells.

Differential Effects of Calcineurin and Mammalian Target of Rapamycin Inhibitors on Alloreactive Th1, Th17, and Regulatory T Cells.

BACKGROUND: Previously, we had reported the role of tacrolimus (TAC) versus sirolimus (SRL) on the generation of regulatory T cells (Tregs) in primary MLR assays with SRL, demonstrating a uniquely supportive effect. However, the mechanisms associated with their actions on alloreactive human T cells are not fully understood. Therefore, we tested whether TAC and SRL differentially affect already alloactivated human CD4 T-cell subsets. METHODS: Alloreactive CD4CD45RA/CD45RO T cells generated in 9-day MLR were cocultured with anti-CD3 and autologous antigen presenting cells plus interleukin (IL)-2 in presence of TAC, SRL, or both, and the Tregs generated after another 5 to 6 days were phenotypically, molecularly, and functionally characterized. RESULTS: Tacrolimus significantly and SRL modestly inhibited interferon (IFN)-γ (Th1) and IL-17 (Th17)-producing cells. At clinical therapeutic concentrations, SRL, however, significantly increased forkhead/winged helix transcription factor P3 (FOXP3) Tregs, whereas TAC inhibited this T-cell population dose dependently and significantly. When used in combination, TAC and SRL had additive effects on inhibition of IFN-γ- and IL-17-producing cells. This was in contrast to the ability of SRL to reverse TAC-mediated inhibition of FOXP3-expressing cells. Proinflammatory cytokines (IL-1ß, IL-6, and tumor necrosis factor-α) added to cultures caused significant decrease in FOXP3 Tregs that was again reversed by SRL. Sirolimus-derived Tregs were phenotypically normal, anergic to allostimulation, and suppressed proliferation of allogeneic effector T-cells. CONCLUSIONS: Thus, although TAC inhibits all alloreactive T cells, SRL promotes the differentiation and expansion of donor-specific Tregs without secondary reprogramming to IFN-γFOXP3 and IL-17FOXP3 Treg subsets. These results, although performed in an artificial in vitro model, add clinically applicable information on how these agents affect T-cell subpopulations.

Treg cells require the phosphatase PTEN to restrain TH1 and TFH cell responses.

The interplay between effector T cells and regulatory T cells (Treg cells) is crucial for adaptive immunity, but how Treg cells control diverse effector responses is elusive. We found that the phosphatase PTEN links Treg cell stability to repression of type 1 helper T cell (TH1 cell) and follicular helper T cell (TFH cell) responses. Depletion of PTEN in Treg cells resulted in excessive TFH cell and germinal center responses and spontaneous inflammatory disease. These defects were considerably blocked by deletion of interferon-γ, indicating coordinated control of TH1 and TFH responses. Mechanistically, PTEN maintained Treg cell stability and metabolic balance between glycolysis and mitochondrial fitness. Moreover, PTEN deficiency upregulates activity of the metabolic checkpoint kinase complex mTORC2 and the serine-threonine kinase Akt, and loss of this activity restores functioning of PTEN-deficient Treg cells. Our studies establish a PTEN-mTORC2 axis that maintains Treg cell stability and coordinates Treg cell-mediated control of effector responses.

Critical role of histone demethylase Jmjd3 in the regulation of CD4+ T-cell differentiation.

Epigenetic factors have been implicated in the regulation of CD4(+) T-cell differentiation. Jmjd3 plays a role in many biological processes, but its in vivo function in T-cell differentiation remains unknown. Here we report that Jmjd3 ablation promotes CD4(+) T-cell differentiation into Th2 and Th17 cells in the small intestine and colon, and inhibits T-cell differentiation into Th1 cells under different cytokine-polarizing conditions and in a Th1-dependent colitis model. Jmjd3 deficiency also restrains the plasticity of the conversion of Th2, Th17 or Treg cells to Th1 cells. The skewing of T-cell differentiation is concomitant with changes in the expression of key transcription factors and cytokines. H3K27me3 and H3K4me3 levels in Jmjd3-deficient cells are correlated with altered gene expression through interactions with specific transcription factors. Our results identify Jmjd3 as an epigenetic factor in T-cell differentiation via changes in histone methylation and target gene expression.

Deoxyhypusine synthase promotes differentiation and proliferation of T helper type 1 (Th1) cells in autoimmune diabetes.

In type 1 diabetes, cytokines arising from immune cells cause islet ß cell dysfunction even before overt hyperglycemia. Deoxyhypusine synthase catalyzes the crucial hypusine modification of the factor eIF5A, which promotes the translation of a subset of mRNAs involved in cytokine responses. Here, we tested the hypothesis that deoxyhypusine synthase and, secondarily, hypusinated eIF5A contribute to the pathogenesis of type 1 diabetes using the non-obese diabetic (NOD) mouse model. Pre-diabetic NOD mice that received injections of the deoxyhypusine inhibitor N1-guanyl-1,7-diaminoheptane (GC7) demonstrated significantly improved glucose tolerance, more robust insulin secretion, and reduced insulitis compared with control animals. Analysis of tissues from treated mice revealed selective reductions in diabetogenic T helper type 1 (Th1) cells in the pancreatic lymph nodes, a primary site of antigen presentation. Isolated mouse CD90.2(+) splenocytes stimulated in vitro with anti-CD3/anti-CD28 and IL-2 to mimic autoimmune T cell activation exhibited proliferation and differentiation of CD4(+) T cell subsets (Th1, Th17, and Treg), but those treated with the deoxyhypusine synthase inhibitor GC7 showed a dose-dependent block in T cell proliferation with selective reduction in Th1 cells, similar to that observed in NOD mice. Inhibition of deoxyhypusine synthase blocked post-transcriptional expression of CD25, the high affinity IL-2 receptor α chain. Our results suggest a previously unrecognized role for deoxyhypusine synthase in promoting T cell proliferation and differentiation via regulation of CD25. Inhibition of deoxyhypusine synthase may provide a strategy for reducing diabetogenic Th1 cells and preserving ß cell function in type 1 diabetes.

AMPKα1 deficiency amplifies proinflammatory myeloid APC activity and CD40 signaling.

AMPK is a serine/threonine kinase that regulates energy homeostasis and metabolic stress in eukaryotes. Previous work from our laboratory, as well as by others, has provided evidence that AMPKα1 acts as a negative regulator of TLR-induced inflammatory function. Herein, we demonstrate that AMPKα1-deficient macrophages and DCs exhibit heightened inflammatory function and an enhanced capacity for antigen presentation favoring the promotion of Th1 and Th17 responses. Macrophages and DCs generated from AMPKα1-deficient mice produced higher levels of proinflammatory cytokines and decreased production of the anti-inflammatory cytokine IL-10 in response to TLR and CD40 stimulation as compared with WT cells. In assays of antigen presentation, AMPKα1 deficiency in the myeloid APC and T cell populations contributed to enhanced IL-17 and IFN-γ production. Focusing on the CD154-CD40 interaction, we found that CD40 stimulation resulted in increased phosphorylation of ERK1/2, p38, and NF-κB p65 and decreased activation of the anti-inflammatory Akt -GSK3ß-CREB pathway in DCs deficient for AMPKα1. Our data demonstrate that AMPKα1 serves to attenuate LPS and CD40-mediated proinflammatory activity of myeloid APCs and that AMPKα1 activity in both APC and T cells contributes to T cell functional polarization during antigen presentation.

Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes.

Given its critical role in T-cell signaling, interleukin-2-inducible kinase (ITK) is an appealing therapeutic target that can contribute to the pathogenesis of certain infectious, autoimmune, and neoplastic diseases. Ablation of ITK subverts Th2 immunity, thereby potentiating Th1-based immune responses. While small-molecule ITK inhibitors have been identified, none have demonstrated clinical utility. Ibrutinib is a confirmed irreversible inhibitor of Bruton tyrosine kinase (BTK) with outstanding clinical activity and tolerability in B-cell malignancies. Significant homology between BTK and ITK alongside in silico docking studies support ibrutinib as an immunomodulatory inhibitor of both ITK and BTK. Our comprehensive molecular and phenotypic analysis confirms ITK as an irreversible T-cell target of ibrutinib. Using ibrutinib clinical trial samples along with well-characterized neoplastic (chronic lymphocytic leukemia), parasitic infection (Leishmania major), and infectious disease (Listeria monocytogenes) models, we establish ibrutinib as a clinically relevant and physiologically potent ITK inhibitor with broad therapeutic utility. This trial was registered at www.clinicaltrials.gov as #NCT01105247 and #NCT01217749.

Indoleamine 2,3-dioxygenase is differentially expressed by different white blood cell populations of rhesus macaques (Macaca mulatta).

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) is involved in immune processes such as transplant and fetal rejection, autoimmunity, cancer, and infection; however, its expression in rhesus macaques has not been fully addressed. METHODS: Indoleamine 2,3-dioxygenase mRNA and protein in the white blood cells (WBCs) of Chinese rhesus macaques were examined by RT-PCR, western blotting, real-time RT-PCR, and flow cytometry. RESULTS: Both IDO protein and mRNA could be readily detected in WBCs or peripheral blood mononuclear cells (PBMCs) of normal rhesus macaques. IDO+ cell frequency was the highest among CD14(+) mononuclear cells, followed by CD56(+) cells and DCs. No difference in the frequency of IDO+ cells between CD4(+) and CD8(+) T cells; however, Th17 cells have higher frequency of IDO+ cells than Th1 cells, with Th2 cells the lowest. Toll-like receptor (TLR) stimulation significantly increased IDO protein level in CD14(+) , CD56(+) , CD1c(+) , CD11c(+) , and CD123(+) myeloid cells. CONCLUSION: Rhesus macaques express IDO differentially in their leukocyte subsets and are suitable for IDO-related pathophysiological studies.

CD26-mediated co-stimulation in human CD8(+) T cells provokes effector function via pro-inflammatory cytokine production.

CD26 is an activation marker of human CD4(+) T cells, and is associated with T-cell signal transduction processes as a co-stimulatory molecule. We have previously demonstrated that high CD26 cell surface expression on CD4(+) T cells is correlated with the production of T helper type 1 cytokines, whereas CD26(+) T helper cells stimulate antibody synthesis in B cells. Although the cellular and molecular mechanisms involved in CD26-mediated CD4(+) T-cell activation have been extensively evaluated by our group and others, the role of CD26 in CD8(+) T cells has not been clearly elucidated. In the present study, we examine the effector function of CD8(+) T cells via CD26-mediated co-stimulation in comparison with CD28-mediated co-stimulation. We found that CD26(high)  CD8(+) T cells belong to the early effector memory T-cell subset, and that CD26-mediated co-stimulation of CD8(+) T cells exerts a cytotoxic effect preferentially via granzyme B, tumour necrosis factor-α, interferon-γ and Fas ligand. The effector function associated with CD26-mediated co-stimulation is enhanced compared with that obtained through CD28-mediated co-stimulation, suggesting that the CD26 co-stimulation pathway in CD8(+) T cells is distinct from the CD28 co-stimulation pathway. Targeting CD26 in CD8(+) T cells therefore has the potential to be useful in studies of immune responses to new vaccine candidates as well as innovative therapy for immune-mediated diseases.

Proviral integration site for Moloney murine leukemia virus (PIM) kinases promote human T helper 1 cell differentiation.

The differentiation of human primary T helper 1 (Th1) cells from naïve precursor cells is regulated by a complex, interrelated signaling network. The identification of factors regulating the early steps of Th1 cell polarization can provide important insight in the development of therapeutics for many inflammatory and autoimmune diseases. The serine/threonine-specific proviral integration site for Moloney murine leukemia virus (PIM) kinases PIM1 and PIM2 have been implicated in the cytokine-dependent proliferation and survival of lymphocytes. We have established that the third member of this family, PIM3, is also expressed in human primary Th cells and identified a new function for the entire PIM kinase family in T lymphocytes. Although PIM kinases are expressed more in Th1 than Th2 cells, we demonstrate here that these kinases positively influence Th1 cell differentiation. Our RNA interference results from human primary Th cells also suggest that PIM kinases promote the production of IFNγ, the hallmark cytokine produced by Th1 cells. Consistent with this, they also seem to be important for the up-regulation of the critical Th1-driving factor, T box expressed in T cells (T-BET), and the IL-12/STAT4 signaling pathway during the early Th1 differentiation process. In summary, we have identified PIM kinases as new regulators of human primary Th1 cell differentiation, thus providing new insights into the mechanisms controlling the selective development of human Th cell subsets.

Effect of tyrosine hydroxylase gene silencing in CD4+ T lymphocytes on differentiation and function of helper T cells.

OBJECTIVES: We explored effect of gene silencing of tyrosine hydroxylase (TH), a rate-limiting enzyme for synthesis of catecholamines (CAs), in CD4+ T cells on differentiation and function of helper T (Th) cells to provide more evidence for functional significance of lymphocyte-derived CAs. METHODS: CD4+ T lymphocytes were isolated and purified from the mesenteric lymph nodes of mice. Recombinant TH miRNA expression vector (pcDNA6.2-GW/EmGFPmiR-TH) was constructed and transfected into concanavalin A (Con A)-activated CD4+ T lymphocytes using nucleofection technology. After incubated for 48 h, these cells were detected for TH gene and protein expression and CA content. Simultaneously, percentage of interferon-γ (IFN-γ)- and interleukin-4 (IL-4)-producing cells and levels of IL-2, IFN-γ, tumor necrosis factor (TNF), IL-4 and IL-5 in culture supernatants of Con A-stimulated CD4+ T cells were examined by flow cytometric analysis. RESULTS: CD4+ T lymphocytes with TH RNAi expressed less TH mRNA and protein and synthesized less CAs including norepinephrine, epinephrine and dopamine than control cells with mock transfection. The silencing of TH gene in CD4+ T lymphocytes reduced percentage of IL-4-producing cells and elevated ratio of IFN-γ-producing cells to IL-4-producing cells, although it did not alter proportion of IFN-γ-producing cells. The Th1 cytokines, IL-2, IFN-γ and TNF, were increased, but the Th2 cytokines, IL-4 and IL-5, were decreased in the culture supernatants of Con A-stimulated CD4+ T lymphocytes that were transfected with TH miRNA. CONCLUSION: TH gene silencing attenuates TH expression and CA synthesis in CD4+ T lymphocytes and promotes polarization of differentiation and function towards Th1 cells.

CD5-dependent CK2 activation pathway regulates threshold for T cell anergy.

CD5 activates casein kinase 2 (CK2), a serine/threonine kinase that constitutively associates with the CK2-binding domain at the end of its cytoplasmic tail. To determine the physiological significance of CD5-dependent CK2 activation in T cells, we generated a knock-in mouse that expresses a CD5 protein containing a microdeletion with selective inability to interact with CK2 (CD5ΔCK2BD). The levels of CD5 on developing and mature T cell populations from CD5ΔCK2BD mice and CD5 wild-type (WT) mice were similar. The thymus of CD5ΔCK2BD mice contained fewer double-positive thymocytes than did that of both CD5WT and CD5 knockout (KO) mice, although the numbers of all other immature and mature T cell populations were unaltered. CD5ΔCK2BD T cells hypoproliferated and exhibited enhanced activation-induced cell death when stimulated with anti-CD3 or cognate peptide in comparison with CD5WT T cells. We also found that functional CD5-dependent CK2 signaling was necessary for efficient differentiation of naive CD4+ T cells into Th2 and Th17 cells, but not Th1 cells. We previously showed that experimental autoimmune encephalomyelitis (EAE) in CD5KO mice was less severe and delayed in onset than in CD5WT mice. Remarkably, CD5ΔCK2BD mice recapitulated both EAE severity and disease onset of CD5KO mice. Increasing the immunization dose of myelin oligodendrocyte glycoprotein 35-55 peptide, a model that mimics high-dose tolerance, led to decreased severity of EAE in CD5WT mice but not in CD5KO or CD5ΔCK2BD mice. This property was recapitulated in in vitro restimulation assays. These results demonstrate that CD5-CK2 signaling sets the threshold for T cell responsiveness and is necessary for efficient generation of Th2 and Th17 cells.