An antagonist IL-15/Fc protein prevents costimulation blockade-resistant rejection.

IL-15 is a powerful T cell growth factor (TCGF) with particular importance for the maintenance of CD8(+) T cells. Because costimulation blockade does not result in universal tolerance, we hypothesized that "escape" from costimulation blockade might represent a CD8(+) and IL-15/IL-15R(+)-dependent process. For this analysis, we have used an IL-15 mutant/Fcgamma2a protein, a potentially cytolytic protein that is also a high-affinity receptor site specific antagonist for the IL-15Ralpha receptor protein, as a therapeutic agent. The IL-15-related fusion protein was used as monotherapy or in combination with CTLA4/Fc in murine islet allograft models. As monotherapies, CTLA4/Fc and an IL-15 mutant/Fcgamma2a were comparably effective in a semiallogeneic model system, and combined treatment with IL-15 mutant/Fcgamma2a plus CTLA4/Fc produced universal permanent engraftment. In a fully MHC-mismatched strain combination known to be refractory to costimulation blockade treatment, combined treatment with both fusion proteins proved to be highly effective; >70% of recipients were tolerized. The analysis revealed that the IL-15 mutant/Fc treatment confers partial protection from both CD4(+) and CD8(+) T cell graft infiltration. In rejections occurring despite CTLA4/Fc treatment, concomitant treatment with the IL-15 mutant/Fcgamma2a protein blocked a CD8(+) T cell-dominated rejection processes. This protection was linked to a blunted proliferative response of alloreactive T cells as well silencing of CTL-related gene expression events. Hence, we have demonstrated that targeting the IL-15/IL-15R pathway represents a new and potent strategy to prevent costimulation blockade-resistant CD8(+) T cell-driven rejection.     

CTLA4 signals are required to optimally induce allograft tolerance with combined donor-specific transfusion and anti-CD154 monoclonal antibody treatment

Sensitization to donor Ags is an enormous problem in clinical transplantation. In an islet allograft model, presensitization of recipients through donor-specific transfusion (DST) 4 wk before transplantation results in accelerated rejection. We demonstrate that combined DST with anti-CD154 (CD40L) therapy not only prevents the deleterious presensitization produced by pretransplant DST in the islet allograft model, it also induces broad alloantigen-specific tolerance and permits subsequent engraftment of donor islet or cardiac grafts without further treatment. In addition, our data strongly indicate that CTLA4-negative T cell signals are required to achieve prolonged engraftment of skin allograft or tolerance to islet allograft in recipients treated with a combination of pretransplant DST and anti-CD154 mAb. We provide direct evidence that a CD28-independent CTLA4 signal delivers a strong negative signal to CD4+ T cells that can block alloimmune MLR responses. In this study immune deviation into a Th2 (IL-4) response was associated with, but did not insure, graft tolerance, as the inopportune timing of B7 blockade with CTLA4/Ig therapy prevented uniform tolerance but did not prevent Th2-type immune deviation. While CTLA4-negative signals are necessary for tolerance induction, Th1 to Th2 immune deviation cannot be sufficient for tolerance induction. Combined pretransplant DST with anti-CD154 mAb treatment may be attractive for clinical deployment, and strategies aimed to selectively block CD28 without interrupting CTLA4/B7 interaction might prove highly effective in the induction of tolerance.     

The Ratio of Circulating Regulatory T Cells (Tregs)/Th17 Cells Is Associated with Acute Allograft Rejection in Liver Transplantation

CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) and Th17 cells are known to be involved in the alloreactive responses in organ transplantation, but little is known about the relationship between Tregs and Th17 cells in the context of liver alloresponse. Here, we investigated whether the circulating Tregs/Th17 ratio is associated with acute allograft rejection in liver transplantation. In present study, thirty-eight patients who received liver transplant were enrolled. The patients were divided into two groups: acute allograft rejection group (Gr-AR) (n = 16) and stable allograft liver function group (Gr-SF) (n = 22). The frequencies of circulating Tregs and circulating Th17 cells, as well as Tregs/Th17 ratio were determined using flow cytometry. The association between Tregs/Th17 ratio and acute allograft rejection was then analyzed. Our results showed that the frequency of circulating Tregs was significantly decreased, whereas the frequency of circulating Th17 cells was significantly increased in liver allograft recipients who developed acute rejection. Tregs/Th17 ratio had a negative correlation with liver damage indices and the score of rejection activity index (RAI) after liver transplantation. In addition, the percentages of CTLA-4⁺, HLA-DR⁺, Ki67⁺, and IL-10⁺ Tregs were higher in Gr-SF group than in Gr-AR group. Our results suggested that the ratio of circulating Tregs/Th17 cells is associated with acute allograft rejection, thus the ratio may serve as an alternative marker for the diagnosis of acute rejection.     

Cutting edge: blockade of the CD28/B7 costimulatory pathway inhibits intestinal allograft rejection mediated by CD4+ but not CD8+ T cells.

The effect of blocking the CD28/B7 costimulatory pathway on intestinal allograft rejection was examined in mice. Murine CTLA4Ig failed to prevent the rejection of allografts transplanted into wild-type or CD4 knockout (KO) mice but did inhibit allograft rejection by CD8 KO recipients. This effect was associated with decreased intragraft mRNA for IFN-gamma and TNF-alpha and increased mRNA for IL-4 and IL-5. This altered pattern of cytokine production was not observed in allografts from murine CTLA4Ig-treated CD4 KO mice. These data demonstrate that blockade of the CD28/B7 pathway has different effects on intestinal allograft rejection mediated by CD4+ and CD8+ T cells and suggest that these T cell subsets have different costimulatory requirements in vivo. The results also suggest that the inhibition of CD4+ T cell-mediated allograft rejection by CTLA4Ig may be related to down-regulation of Th1 cytokines and/or up-regulation of Th2 cytokines.     

Schistosoma mansoni infection alters co-stimulatory molecule expression and cell activation in asthma

Chronic schistosomiasis induces Th2/T regulatory responses which are able to down-modulate allergic inflammation and asthma. Because co-stimulatory molecules and IL-10 are essential for inducing tolerance, the aim of this study was to determine by flow cytometry, the expression of CD28, CTLA4, CD40L, CD80, CD86, HLA-DR, IL-10 and IL-10 receptor, by mononuclear cells from asthmatic individuals infected with Schistosoma mansoni and compare with non-infected individuals. Peripheral blood mononuclear cells were stained with fluorochrome conjugated antibodies for the expression of co-stimulatory molecules, and for intracellular CTLA4 and IL-10 expression. There was no significant difference in the frequency of T cells expressing CD28 between the two groups. However, the frequency of TCD4⁺ cells expressing CTLA4 and CD40L was higher in infected asthmatics. The frequency of monocytes expressing CD80 and CD86 did not differ between groups, while the expression of HLA-DR and IL-10 receptor was higher on monocytes of infected individuals. Furthermore, monocytes and CD4⁺CD25⁺ cells of infected individuals expressed higher levels of IL-10. We conclude that, besides alternatively-activated monocytes that are, together with CD4⁺CD25⁺ cells, important sources of IL-10, CTLA4 and CD40L expression may also participate in the down-modulation of inflammatory allergic response in S. mansoni-infected asthmatics.     

Combination of an Antigen-Specific Therapy and an Immunomodulatory Treatment to Simultaneous Block Recurrent Autoimmunity and Alloreactivity in Non-Obese Diabetic Mice

Restoration of endogenous insulin production by islet transplantation is considered a curative option for patients with type 1 diabetes. However, recurrent autoimmunity and alloreactivity cause graft rejection hindering successful transplantation. Here we tested whether transplant tolerance to allogeneic islets could be achieved in non-obese diabetic (NOD) mice by simultaneously tackling autoimmunity via antigen-specific immunization, and alloreactivity via granulocyte colony stimulating factor (G-CSF) and rapamycin (RAPA) treatment. Immunization with insB_9-23 peptide alone or in combination with two islet peptides (IGRP_206-214 and GAD_524-543) in incomplete Freund’s adjuvant (IFA) were tested for promoting syngeneic pancreatic islet engraftment in spontaneously diabetic NOD mice. Treatment with G-CSF/RAPA alone or in combination with insB_9-23/IFA was examined for promoting allogeneic islet engraftment in the same mouse model. InsB_9-23/IFA immunization significantly prolonged syngeneic pancreatic islet survival in NOD mice by a mechanism that necessitated the presence of CD4⁺CD25⁺ T regulatory (Treg) cells, while combination of three islet epitopes was less efficacious in controlling recurrent autoimmunity. G-CSF/RAPA treatment was unable to reverse T1D or control recurrent autoimmunity but significantly prolonged islet allograft survival in NOD mice. Blockade of interleukin-10 (IL-10) during G-CSF/RAPA treatment resulted in allograft rejection suggesting that IL-10-producing cells were fundamental to achieve transplant tolerance. G-CSF/RAPA treatment combined with insB_9-23/IFA did not further increase the survival of allogeneic islets. Thus, insB_9-23/IFA immunization controls recurrent autoimmunity and G-CSF/RAPA treatment limits alloreactivity, however their combination does not further promote allogeneic pancreatic islet engraftment in NOD mice.     

IL-2 limits IL-12 enhanced lymphocyte proliferation during Leishmania amazonensis infection

C3H mice infected with Leishmania amazonensis develop persistent, localized lesions with high parasite loads. During infection, memory/effector CD44^hiCD4⁺ T cells proliferate and produce IL-2, but do not polarize to a known effector phenotype. Previous studies have demonstrated IL-12 is insufficient to skew these antigen-responsive T cells to a functional Th1 response. To determine the mechanism of this IL-12 unresponsiveness, we used an in vitro assay of repeated antigen activation. Memory/effector CD44^hiCD4⁺ T cells did not increase proliferation in response to either IL-2 or IL-12, although these cytokines upregulated CD25 expression. Neutralization of IL-2 enhanced CD4⁺ T cell proliferation in response to IL-12. This cross-regulation of IL-12 responsiveness by IL-2 was confirmed in vivo by treatment with anti-IL-2 antibodies and IL-12 during antigen challenge of previously infected mice. These results suggest that during chronic infection with L. amazonensis, IL-2 plays a dominant, immunosuppressive role independent of identifiable conventional T_reg cells.     

CD160Ig Fusion Protein Targets a Novel Costimulatory Pathway and Prolongs Allograft Survival

CD160 is a cell surface molecule expressed by most NK cells and approximately 50% of CD8⁺ cytotoxic T lymphocytes. Engagement of CD160 by MHC class-I directly triggers a costimulatory signal to TCR-induced proliferation, cytokine production and cytotoxic effector functions. The role of CD160 in alloimmunity is unknown. Using a newly generated CD160 fusion protein (CD160Ig) we examined the role of the novel costimulatory molecule CD160 in mediating CD4⁺ or CD8⁺ T cell driven allograft rejection. CD160Ig inhibits alloreactive CD8⁺ T cell proliferation and IFN-γ production in vitro, in particular in the absence of CD28 costimulation. Consequently CD160Ig prolongs fully mismatched cardiac allograft survival in CD4^−/−, CD28^−/− knockout and CTLA4Ig treated WT recipients, but not in WT or CD8^−/− knockout recipients. The prolonged cardiac allograft survival is associated with reduced alloreactive CD8⁺ T cell proliferation, effector/memory responses and alloreactive IFN-γ production. Thus, CD160 signaling is particularly important in CD28-independent effector/memory CD8⁺ alloreactive T cell activation in vivo and therefore may serve as a novel target for prevention of allograft rejection.     

Increase of Th17 Cell Phenotype in Kidney Transplant Recipients with Chronic Allograft Dysfunction

This study was performed to determine the association of Th17 cell phenotype with chronic allograft dysfunction in kidney transplant recipients (KTRs). We compared the expression of Th17 cell phenotype in KTRs with chronic allograft dysfunction group (CAD, n = 52) with four control groups (long-term stable KTRs (LTS, n = 67), early stable KTRs (ES, n = 28), end stage renal disease (ESRD, n = 45), and healthy control (HC, n = 26). We also performed in vitro study using human proximal renal tubular epithelial cell line (HPRTEpiC) to evaluate the effect of IL-17 on human renal tubular epithelial cells. The CAD group showed increased percentage of Th17 cells out of CD4⁺ T cells and also increased proportion of IL-17 producing cells out of effector memory T cells or out of CCR4⁺CCR6⁺/CD4⁺ T cells compared to the LTS group and other control groups. Also, the serum level of IL-17, IL-33, and RAGE, and the expression of IL-1beta, RAGE, and HMGB1 mRNA showed an increase in the CAD group compared to the LTS group. In vitro study revealed that IL-17 increased production of IL-6 and IL-8 and up-regulated profibrotic gene expression such as ACTA-2 and CTGF in HPRTEpiC in a dose-dependent manner, which suggests that IL-17 has a role in the development of renal tubular cell injury. The results of our study may suggest that increase of Th17 cell phenotype could be a marker for the chronic allograft injury; hence there is a need to develop diagnostic and therapeutic tools targeting the Th17 cells pathway.     

Primary Murine CD4+ T Cells Fail to Acquire the Ability to Produce Effector Cytokines When Active Ras Is Present during Th1/Th2 Differentiation

Constitutive Ras signaling has been shown to augment IL-2 production, reverse anergy, and functionally replace many aspects of CD28 co-stimulation in CD4⁺ T cells. These data raise the possibility that introduction of active Ras into primary T cells might result in improved functionality in pathologic situations of T cell dysfunction, such as cancer or chronic viral infection. To test the biologic effects of active Ras in primary T cells, CD4⁺ T cells from Coxsackie-Adenovirus Receptor Transgenic mice were transduced with an adenovirus encoding active Ras. As expected, active Ras augmented IL-2 production in naive CD4⁺ T cells. However, when cells were cultured for 4 days under conditions to promote effector cell differentiation, active Ras inhibited the ability of CD4⁺ T cells to acquire a Th1 or Th2 effector cytokine profile. This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression. Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus. Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4⁺ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci. Alternative strategies to potentiate Ras pathway signaling in T cells in a more regulated fashion should be considered as a therapeutic approach to improve immune responses in vivo.     

Plasticity of Migrating CD1b+ and CD1b- Lymph Dendritic Cells in the Promotion of Th1, Th2 and Th17 in Response to Salmonella and Helminth Secretions

Dendritic cells (DCs) are pivotal in the development of specific T-cell responses to control pathogens, as they govern both the initiation and the polarization of adaptive immunity. To investigate the capacities of migrating DCs to respond to pathogens, we used physiologically generated lymph DCs (L-DCs). The flexible polarization of L-DCs was analysed in response to Salmonella or helminth secretions known to induce different T cell responses. Mature conventional CD1b⁺ L-DCs showed a predisposition to promote pro-inflammatory (IL-6), pro-Th1 (IL-12p40) and anti-inflammatory (IL-10) responses which were amplified by Salmonella, and limited to only IL-6 induction by helminth secretions. The other major population of L-DCs did not express the CD1b molecule and displayed phenotypic features of immaturity compared to CD1b⁺ L-DCs. Salmonella infection reduced the constitutive expression of TNF-α and IL-4 mRNA in CD1b^- L-DCs, whereas this expression was not affected by helminth secretions. The cytokine response of T cells promoted by L-DCs was analysed in T cell subsets after co-culture with Salmonella or helminth secretion-driven CD1b⁺ or CD1b^- L-DCs. T cells preferentially expressed the IL-17 gene, and to a lesser extent the IFN-γ and IL-10 genes, in response to Salmonella-driven CD1b⁺ L-DCs, whereas a preferential IL-10, IFN-γ and IL-17 gene expression was observed in response to Salmonella-driven CD1b^- L-DCs. In contrast, a predominant IL-4 and IL-13 gene expression by CD4⁺ and CD8⁺ T cells was observed after stimulation of CD1b⁺ and CD1b^- L-DCs with helminth secretions. These results show that mature conventional CD1b⁺ L-DCs maintain a flexible capacity to respond differently to pathogens, that the predisposition of CD1b^- L-DCs to promote a Th2 response can be oriented towards other Th responses, and finally that the modulation of migrating L-DCs responses is controlled more by the pathogen encountered than the L-DC subsets.     

Potentiation of Th17 cytokines in aging process contributes to the development of colitis

Th17 cells, which produce IL-17 and IL-22, promote autoimmunity in mice and have been implicated in the pathogenesis of autoimmune/inflammatory diseases in humans. However, the Th17 immune response in the aging process is still not clear. In the present study, we found that the induction of IL-17-produing CD4⁺ T cells was significantly increased in aged individuals compared with young healthy ones. The mRNA expression of IL-17, IL-17F, IL-22, and RORC2 was also significantly increased in aged people. Similar to humans, Th17 cells as well as mRNAs encoding IL-17, IL-22 and RORγt were dramatically elevated in naïve T cells from aged mouse compared to young ones. In addition, CD44 positive IL-17-producing CD4⁺ T cells were significantly higher in aged mice, suggesting that memory T cells are an important source of IL-17 production. Furthermore, the percentage of IL-17-produing CD4⁺ T cells generated in co-culture with dendritic cells from either aged or young mice did not show significant differences, suggesting that dendritic cells do not play a primary role in the elevation of Th17 cytokines in aged mouse cells. Importantly, transfer of CD4⁺CD45Rb^hi cells from aged mice induced more severe colitis in RAG^−/− mice compared to cells from young mice, Taken together, these results suggest that Th17 immune responses are elevated in aging humans and mice and may contribute to the increased development of inflammatory disorders in the elderly.     

Regulatory T cells induced by B cells: a novel subpopulation of regulatory T cells

Regulatory T cells play a crucial role in the homeostasis of the immune response. In addition to CD4⁺Foxp3⁺ regulatory T cells, several subsets of Foxp3^- regulatory T cells, such as T helper 3 (Th3) cells and type 1 regulatory T (Tr1) cells, have been described in mice and human. Accumulating evidence shows that naïve B cells contribute to tolerance and are able to promote regulatory T cell differentiation. Naïve B cells can convert CD4⁺CD25^- T cells into CD25⁺Foxp3^- regulatory T cells, named Treg-of-B cells by our group. Treg-of-B cells express LAG3, ICOS, GITR, OX40, PD1, and CTLA4 and secrete IL-10. Intriguingly, B-T cell-cell contact but not IL-10 is essential for Treg-of-B cells induction. Moreover, Treg-of-B cells possess both IL-10-dependent and IL-10-independent inhibitory functions. Treg-of-B cells exert suppressive activities in antigen-specific and non-antigen-specific manners in vitro and in vivo. Here, we review the phenotype and function of Foxp3⁺ regulatory T cells, Th3 cells, Tr1 cells, and Treg-of-B cells.     

Th17 and Th17-stimulated CD8<Superscript>+</Superscript> T cells play a distinct role in Th17-induced preventive and therapeutic antitumor immunity

CD4⁺ Th17 cells induce antitumor immunity leading to the eradication of established tumors. However, the mechanism of antitumour immunity and CTL activation by Th17 cells and the distinct role of Th17 and Th17-activated CTLs in antitumor immunity are still elusive. In this study, we generated ovalbumin (OVA)-specific Th17 cells by cultivating OVA-pulsed dendritic cells with CD4⁺ T cells derived from transgenic OTII mice in the presence of IL-6, IL-23, TGF-β, and anti-IFN-γ antibody. We demonstrated that Th17 cells acquired major histocompatibility complex/peptide (pMHC)-I and expressed RORγt, IL-17, and IL-2. Th17 cells did not have any direct in vitro tumor cell–killing activity. However, Th17 cells were able to stimulate CD8⁺ CTL responses via IL-2 and pMHC I, but not IL-17 signaling, which play a major role in Th17-induced preventive immunity against OVA-expressing B16 melanoma. Th17 cells stimulated the expression of CCL2 and CCL20 in lung tumor microenvironments promoting the recruitment of various inflammatory leukocytes (DCs, CD4⁺, and CD8⁺ T cells) stimulating more pronounced therapeutic immunity for early-stage (5-day lung metastases or 3 mm, s.c.) tumor than for well-established (6 mm, s.c.) tumor. The therapeutic effect of Th17 cells is associated with IL-17 and is mediated by Th17-stimulated CD8⁺ CTLs and other inflammatory leukocytes recruited into B16 melanoma via Th17-stimulated CCL20 chemoattraction. Taken together, our data elucidate a distinct role of Th17 and Th17-stimulated CD8⁺ CTLs in the induction of preventive and therapeutic antitumor immunity, which may greatly impact the development of Th17-based cancer immunotherapy.     

Increased intratumoral IL-22-producing CD4+ T cells and Th22 cells correlate with gastric cancer progression and predict poor patient survival

IL-22-producing CD4⁺ T cells (IL-22⁺CD4⁺ T cells) and Th22 cells (IL-22⁺IL-17^?IFN-γ^?CD4⁺ T cells) represent newly discovered T-cell subsets, but their nature, regulation, and clinical relevance in gastric cancer (GC) are presently unknown. In our study, the frequency of IL-22⁺CD4⁺ T cells in tumor tissues from 76 GC patients was significantly higher than that in tumor-draining lymph nodes, non-tumor, and peritumoral tissues. Most intratumoral IL-22⁺CD4⁺ T cells co-expressed IL-17 and IFN-γ and showed a memory phenotype. Locally enriched IL-22⁺CD4⁺ T cells positively correlated with increased CD14⁺ monocytes and IL-6 and IL-23 detection ex vivo, and in vitro IL-6 and IL-23 induced the polarization of IL-22⁺CD4⁺ T cells in a dose-dependent manner and the polarized IL-22⁺CD4⁺ T cells co-expressed of IL-17 and IFN-γ. Moreover, IL-22⁺CD4⁺ T-cell subsets (IL-22⁺IL-17⁺CD4⁺, IL-22⁺IL-17^?CD4⁺, IL-22⁺IFN-γ⁺CD4⁺, IL-22⁺IFN-γ^?CD4⁺, and IL-22⁺IL-17⁺IFN-γ⁺CD4⁺ T cells), and Th22 cells were also increased in tumors. Furthermore, higher intratumoral IL-22⁺CD4⁺ T-cell percentage and Th22-cell percentage were found in patients with tumor-node-metastasis stage advanced and predicted reduced overall survival. In conclusion, our data indicate that IL-22⁺CD4⁺ T cells and Th22 cells are likely important in establishing the tumor microenvironment for GC; increased intratumoral IL-22⁺CD4⁺ T cells and Th22 cells are associated with tumor progression and predict poorer patient survival, suggesting that tumor-infiltrating IL-22⁺CD4⁺ T cells and Th22 cells may be suitable therapeutic targets in patients with GC.     

Islet allograft rejection in nonobese diabetic mice involves the common gamma-chain and CD28/CD154-dependent and -independent mechanisms

Once nonobese diabetic (NOD) mice become diabetic, they are highly resistant to islet transplantation. The precise mechanism of such resistance remains largely unknown. In the present study we tested the hypothesis that islet allograft survival in the diabetic NOD mouse is determined by the interplay of diverse islet-specific T cell subsets whose activation is regulated by CD28/CD154 costimulatory signals and the common gamma-chain (gammac; a shared signaling element by receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21). We found that common gammac blockade is remarkably effective in blocking the onset and the ongoing autoimmune diabetes, whereas CD28/CD154 blockade has no effect in suppressing the ongoing diabetes. However, CD28/CD154 blockade completely blocks the alloimmune-mediated islet rejection. Also, a subset of memory-like T cells in the NOD mice is resistant to CD28/CD154 blockade, but is sensitive to the common gammac blockade. Nonetheless, neither common gammac blockade nor CD28/CD154 blockade can prevent islet allograft rejection in diabetic NOD mice. Treatment of diabetic NOD recipients with CD28/CD154 blockade plus gammac blockade markedly prolongs islet allograft survival compared with the controls. However, allograft tolerance is not achieved, and all CTLA-4Ig-, anti-CD154-, and anti-gammac-treated diabetic NOD mice eventually rejected the islet allografts. We concluded that the effector mechanisms in diabetic NOD hosts are inherently complex, and rejection in this model involves CD28/CD154/gammac-dependent and -independent mechanisms.