Restricting Glutamine or Glutamine-Dependent Purine and Pyrimidine Syntheses Promotes Human T Cells with High FOXP3 Expression and Regulatory Properties.

T cell subsets differ in their metabolic requirements, and further insight into such differences might be harnessed to selectively promote regulatory T cells (Tregs) for therapies in autoimmunity and transplantation. We found that Gln restriction during human T cell activation favored CD4 T cells with high expression of the Treg transcription factor FOXP3. This resulted from shrinking numbers and reduced proliferation of activated FOXP3(lo/-)CD4 T cells while FOXP3(hi)CD4 T cell numbers increased. This gain was abolished by blocking Gln synthetase, an enzyme that responds to Gln and purine/pyrimidine deficiencies. The shift toward FOXP3(hi)CD4 T cells under Gln restriction was recapitulated with inhibitors of Gln-dependent pyrimidine and purine syntheses that together closely mimicked declining cell numbers and cell cycles, and by small interfering RNA knockdown of the respective rate-limiting Gln-consuming enzymes CAD and PPAT. FOXP3(hi)-enriched CD25(hi)CD4 T cells from these cultures inhibited proliferation, but they also produced effector cytokines, including IL-17A. The latter was largely confined to CTLA-4(hi)-expressing FOXP3(hi)-enriched CD25(hi)CD4 T cells that suppressed proliferation more weakly than did CTLA-4(lo/-)CD25(hi)FOXP3(hi)-enriched T cells. A causal link between high IL-17A production and impaired suppression of proliferation could not be demonstrated, however. Collectively, these results reveal a Gln synthetase-dependent increase and resilience of FOXP3(hi) cells under Gln restriction, and they demonstrate that impaired Gln-dependent nucleotide synthesis promotes FOXP3(hi) cells with regulator properties. It remains to be investigated to what extent the concomitant retention of IL-17A-producing CD4 T cells may limit the therapeutic potential of Tregs enriched through targeting these pathways in vivo.

Th17 cells, not IL-17+ γδ T cells, drive arthritic bone destruction in mice and humans.

The mechanism whereby IL-17 drives rheumatoid arthritis remains incompletely understood. We demonstrate that anti-IL-17 therapy in collagen-induced arthritis ameliorates bone damage by reducing the number of osteoclasts in joints. We found equal numbers of CD4(+) Th17 and IL-17 producing γδ T cells in the joints of arthritic mice, and in vitro, both populations similarly induced osteoclastogenesis. However, individual depletion and adoptive transfer studies revealed that in vivo, Th17 cells dominated with regard to bone destruction. Unlike γδ T cells, Th17 cells were found in apposition to tartrate-resistant acid phosphatase positive osteoclasts in subchondral areas of inflamed joints, a pattern reproduced in patient biopsies. This localization was caused by Ag-specific retention, because OVA-primed Th17 cells showed a γδ T cell-like diffuse distribution. Because IL-23, as produced by osteoclasts, enhanced T cell-mediated osteoclastogenesis, we propose that Ag-specific juxtaposition is key to foster the molecular cross talk of Th17 cells and osteoclasts, thus driving arthritic bone destruction.

Effects of the novel protein kinase C inhibitor AEB071 (Sotrastaurin) on rat cardiac allograft survival using single agent treatment or combination therapy with cyclosporine, everolimus or FTY720.

NVP-AEB071 (AEB, sotrastaurin), an oral inhibitor of protein kinase C (PKC), effectively blocks T-cell activation. The immunosuppressive effects of oral AEB were demonstrated in a rat local graft versus host (GvH) reaction and rat cardiac transplantation models. T-cell activation was suppressed by 95% in blood from AEB-treated rats, with a positive correlation between T-cell inhibition and AEB blood concentration. In GvH studies, AEB inhibited lymph node swelling dose-dependently (3-30 mg/kg). BN and DA cardiac allografts were acutely rejected within 6-10 days post-transplantation in untreated LEW rats. AEB at 10 and 30 mg/kg b.i.d. prolonged BN graft survival to a mean survival time of 15 and >28 days, and DA grafts to 6.5 and 17.5 days, respectively. In the DA to LEW model, combining a nonefficacious dose of AEB (10 mg/kg b.i.d.) with a nonefficacious dose of cyclosporine, everolimus or FTY720 led to prolonged median survival times (26 days, >68 days and >68 days, respectively). Pharmacokinetic monitoring excluded drug-drug interactions, suggesting synergy. In conclusion, these studies are the first to demonstrate that AEB prolongs rat heart allograft survival safely as monotherapy and in combination with nonefficacious doses of cyclosporine, everolimus or FTY720. Thus, AEB may have the potential to offer an alternative to calcineurin inhibitor-based therapies.

Modulation of T cell homeostasis and alloreactivity under continuous FTY720 exposure.

The immunomodulator FTY720 inhibits lymph node (LN) and thymic egress, thereby constraining T cell circulation and reducing peripheral T cell numbers. Here, we analyzed in mouse models the as yet scarcely characterized impact of long-term (up to 6 months) FTY720 exposure on T cell homeostasis and possible consequences for alloreactivity. In green fluorescent protein (GFP) hemopoietic chimeras, the turnover of (initially GFP(-)) peripheral T cell pools was markedly delayed under FTY720, while normal homeostatic differences between CD4 and CD8 T cell sub-populations were retained or amplified further. Homeostatic proliferation was enhanced, and within shrinking T cell pools, the proportions of effector memory phenotype CD4 T cells (CD4T(PEM)) increased in spleens and LNs and of central memory phenotype CD8 T cells (CD8T(PCM)) in LNs. By contrast, the fractions of CD8T(PEM) and CD4T(PCM) remained stably small under FTY720. The enrichment for CD4T(PEM) and CD8T(PCM) correlated with larger proportions of IFNgamma-producing T cells upon nonspecific but not allospecific stimulation. Splenic CD4 T cells from FTY720-treated mice proliferated more strongly upon transfer to semi-allogeneic hosts. However, heart allograft survival was not compromised in FTY720 pre-treated recipients. It correlated with reduced intra-graft CD8 T cells, and the longest surviving transplants contained the highest numbers of CD4 T cells. Thus, continuous FTY720 exposure reveals differential homeostatic responses by memory phenotype CD4 and CD8 T cell sub-populations, and it may enhance alloreactive CD4 T cell proliferation and tissue infiltration without accelerating allograft rejection.

Differential promotion of hematopoietic chimerism and inhibition of alloreactive T cell proliferation by combinations of anti-CD40Ligand, anti-LFA-1, everolimus, and deoxyspergualin.

Allogeneic bone marrow (BM) engraftment for chimerism and transplantation tolerance may be promoted by combinations of costimulation blocking biologics and small molecular weight inhibitors. We showed previously in a mouse model that anti-CD40Ligand (anti-CD40L, CD154) combined with anti-LFA-1 or everolimus (40-O-(2-hydroxyethyl)-rapamycin) resulted in stable chimerism in almost all BM recipients, whereas anti-LFA-1 plus everolimus conferred approximately 50% chimerism stability. Here, we investigated whether this lower incidence could be increased with deoxyspergualin (DSG) in place of or in addition to everolimus. However, DSG and everolimus were similarly synergistic with costimulation blockade for stable hematopoietic chimerism. This correlated with allospecific T cell depletion and inhibition of acute but not chronic skin allograft rejection. Different treatments were also compared for their inhibition of alloreactive T cell proliferation in vivo. While anti-CD40L did not impair T cell proliferation, anti-LFA-1 reduced both CD4 and CD8 T cell proliferation, and combining anti-LFA-1 with everolimus or DSG had an additive inhibitory effect on CD4 T cell proliferation. Thus, despite their strong inhibition of alloreactive T cell proliferation, combinations of anti-LFA-1 with everolimus or DSG did not reach the unique potency of anti-CD40L-based combinations to support stable hematopoietic chimerism in this system.

Partial deficiency of sphingosine-1-phosphate lyase confers protection in experimental autoimmune encephalomyelitis.

BACKGROUND: Sphingosine-1-phosphate (S1P) regulates the egress of T cells from lymphoid organs; levels of S1P in the tissues are controlled by S1P lyase (Sgpl1). Hence, Sgpl1 offers a target to block T cell-dependent inflammatory processes. However, the involvement of Sgpl1 in models of disease has not been fully elucidated yet, since Sgpl1 KO mice have a short life-span. METHODOLOGY: We generated inducible Sgpl1 KO mice featuring partial reduction of Sgpl1 activity and analyzed them with respect to sphingolipid levels, T-cell distribution, and response in models of inflammation. PRINCIPAL FINDINGS: The partially Sgpl1 deficient mice are viable but feature profound reduction of peripheral T cells, similar to the constitutive KO mice. While thymic T cell development in these mice appears normal, mature T cells are retained in thymus and lymph nodes, leading to reduced T cell numbers in spleen and blood, with a skewing towards increased proportions of memory T cells and T regulatory cells. The therapeutic relevance of Sgpl1 is demonstrated by the fact that the inducible KO mice are protected in experimental autoimmune encephalomyelitis (EAE). T cell immigration into the CNS was found to be profoundly reduced. Since S1P levels in the brain of the animals are unchanged, we conclude that protection in EAE is due to the peripheral effect on T cells, leading to reduced CNS immigration, rather than on local effects in the CNS. SIGNIFICANCE: The data suggest Sgpl1 as a novel therapeutic target for the treatment of multiple sclerosis.

Vav1 GEF activity is required for T cell mediated allograft rejection.

The GDP exchange factor (GEF) Vav1 is a central signal transducer downstream of the T cell receptor and has been identified as a key factor for T cell activation in the context of allograft rejection. Vav1 has been shown to transduce signals both dependent and independent of its GEF function. The most promising approach to disrupt Vav1 activity by pharmacological inhibition would be to target its GEF function. However, the contribution of Vav1 GEF activity for allogeneic T cell activation has not been clarified yet. To address this question, we used knock-in mice bearing a mutated Vav1 with disrupted GEF activity but intact GEF-independent functions. T cells from these mice showed strongly reduced proliferation and activation in response to allogeneic stimulation. Furthermore, lack of Vav1 GEF activity strongly abrogated the in vivo expansion of T cells in a systemic graft-versus-host model. In a cardiac transplantation model, mice with disrupted Vav1 GEF activity show prolonged allograft survival. These findings demonstrate a strong requirement for Vav1 GEF activity for allogeneic T cell activation and graft rejection suggesting that disruption of Vav1 GEF activity alone is sufficient to induce significant immunosuppression.

PKC theta cooperates with PKC alpha in alloimmune responses of T cells in vivo.

The physiological roles of PKC alpha and PKC theta were defined in T cell immune functions downstream of the antigen receptor. To investigate the hypothesis that both PKC isotypes may have overlapping functions, we generated mice lacking both genes. We find that PKC alpha(-/-)/theta(-/-) animals have additive T cell response defects in comparison to animals carrying single mutations in these genes. Our studies demonstrate that the activities of PKC alpha and PKC theta converge to regulate both IL-2 cytokine responses and T cell intrinsic alloreactivity in vivo. Mechanistically, this PKC alpha/theta crosstalk primarily affects the NFAT transactivation pathway in T lymphocytes, as observed by decreased phosphorylation of Ser-9 on GSK3 beta, reduced nuclear translocation and DNA binding of NFAT in isolated PKC alpha(-/-)/theta(-/-) CD3(+) T cells. This additive defect proved to be of physiological relevance, because PKC alpha(-/-)/theta(-/-) mice demonstrated significantly prolonged allograft survival in heart transplantation experiments, whereas both PKC alpha(-/-) and PKC theta(-/-) mice showed only minimal graft prolongation when compared to wild type controls. While PKC theta appears to be the rate-limiting PKC isotype mediating T lymphocyte activation, we here provide genetic evidence that PKC alpha and PKC theta have overlapping functions in alloimmunoreactivity in vivo and both PKC theta and PKC alpha isotypes must be targeted to prevent organ allograft rejection.

Precaecal and postileal metabolism of P, Ca and N in pigs as affected by different carbohydrate sources fed at low level of P intake.

Three different carbohydrate sources (cellulose on wood basis, maize starch and apple pectin) in combination with a low phosphorus (P) basal diet were fed to eight pigs fitted with single ileum-T-cannulas in a balance trial. The effects on net disappearance of dry matter, N, P and Ca as well as phytate (IP6) degradation in the intestine were studied. A basal diet and three diets supplemented with 25% of one carbohydrate source were applied in three experimental periods following an incomplete Latin Square design. Animals were housed individually in balance crates. After 15 days of adaptation to the diets, five days of total excreta collection were followed by two days of spot sampling of ileal digesta. The results showed a significantly decreasing effect of cellulose on the precaecal, postileal and total tract digestibility of DM, N and P. Starch reduced precaecal IP6 degradation and P digestibility. The addition of pectin negatively affected the precaecal digestibility of DM, N and Ca as well as the total tract digestibility of P and Ca. Postileally, cellulose and pectin caused a net secretion while starch induced a net absorption of P.

Strongly reduced alloreactivity and long-term survival times of cardiac allografts in Vav1- and Vav1/Vav2-knockout mice.

Vav proteins mediate T- and B-cell activation by functioning as GTP/GDP exchange factors for small GTPases. We have studied the role of Vav1 and Vav2 in allogeneic T-cell activation, antibody responses and allograft rejection. Alloantigen-induced proliferation of T cells from Vav1- and Vav1/Vav2-knockout (ko) mice was decreased by >90% in a mixed lymphocyte reaction. In whole-blood cultures, Vav deficiency led to markedly impaired T- and B-cell activation. Expansion of Vav1- or Vav1/Vav2-ko T cells (C57BL/6) was reduced after transfer into severe combined immune deficiency/beige recipient mice (BALB/c). After priming with 2,4-dinitrophenyl (DNP)-keyhole limpet hemocyanin, T-cell-dependent anti-DNP IgM and IgG antibody levels were normal in Vav1-ko mice but undetectable in Vav1/Vav2-ko mice. The median survival time of BALB/c cardiac allografts transplanted into C57BL/6 Vav1-ko mice (n = 13) or Vav1/Vav2-ko mice (n = 5) was >100 and >77 days, compared with 8-9 days in the corresponding wild-type mice. Vav1/Vav2-ko mice with <100 days graft survival developed bacterial skin infections and were prematurely killed with beating cardiac allograft. Long-term surviving transplants of single and double ko mice showed mild cellular interstitial rejection and mild to severe vascular remodeling. In conclusion, our studies show for the first time that the absence of Vav1 and Vav1/Vav2 in ko mice strongly reduces alloreactivity and results in long-term allograft survival, whereas antibody responses were only affected in Vav1/Vav2 ko mice.

Combinations of anti-LFA-1, everolimus, anti-CD40 ligand, and allogeneic bone marrow induce central transplantation tolerance through hemopoietic chimerism, including protection from chronic heart allograft rejection.

Central transplantation tolerance through hemopoietic chimerism initially requires inhibition of allogeneic stem cell or bone marrow (BM) rejection, as previously achieved in murine models by combinations of T cell costimulation blockade. We have evaluated LFA-1 blockade as part of regimens to support mixed hemopoietic chimerism development upon fully allogeneic BALB/c BM transfer to nonirradiated busulfan-treated B6 recipient mice. Combining anti-LFA-1 with anti-CD40 ligand (CD40L) induced high incidences and levels of stable multilineage hemopoietic chimerism comparable to chimerism achieved with anti-CD40L and everolimus (40-O-(2-hydroxyethyl)-rapamycin) under conditions where neither Ab alone was effective. The combination of anti-LFA-1 with everolimus also resulted in high levels of chimerism, albeit with a lower incidence of stability. Inhibition of acute allograft rejection critically depended on chimerism stability, even if maintained at very low levels around 1%, as was the case for some recipients without busulfan conditioning. Chimerism stability correlated with a significant donor BM-dependent loss of host-derived Vbeta11(+) T cells 3 mo after BM transplantation (Tx). Combinations of anti-CD40L with anti-LFA-1 or everolimus also prevented acute rejection of skin allografts transplanted before established chimerism, albeit not independently of allospecific BMTx. All skin and heart allografts transplanted to stable chimeras 3 and 5 mo after BMTx, respectively, were protected from acute rejection. Moreover, this included prevention of heart allograft vascular intimal thickening ("chronic rejection").