PARP14 promotes the Warburg effect in hepatocellular carcinoma by inhibiting JNK1-dependent PKM2 phosphorylation and activation.

Most tumour cells use aerobic glycolysis (the Warburg effect) to support anabolic growth and evade apoptosis. Intriguingly, the molecular mechanisms that link the Warburg effect with the suppression of apoptosis are not well understood. In this study, using loss-of-function studies in vitro and in vivo, we show that the anti-apoptotic protein poly(ADP-ribose) polymerase (PARP)14 promotes aerobic glycolysis in human hepatocellular carcinoma (HCC) by maintaining low activity of the pyruvate kinase M2 isoform (PKM2), a key regulator of the Warburg effect. Notably, PARP14 is highly expressed in HCC primary tumours and associated with poor patient prognosis. Mechanistically, PARP14 inhibits the pro-apoptotic kinase JNK1, which results in the activation of PKM2 through phosphorylation of Thr365. Moreover, targeting PARP14 enhances the sensitization of HCC cells to anti-HCC agents. Our findings indicate that the PARP14-JNK1-PKM2 regulatory axis is an important determinant for the Warburg effect in tumour cells and provide a mechanistic link between apoptosis and metabolism.

Allospecific CD4(+) T cells retain effector function and are actively regulated by Treg cells in the context of transplantation tolerance.

Although donor-specific transfusion (DST) plus CD154 blockade represents a robust protocol for inducing transplantation tolerance, the underlying mechanisms are incompletely understood. In a murine T-cell adoptive transfer model, we have visualized alloantigen-specific, TCR-transgenic for H2-A(b) /H2-K(d) 54-68 epitope (TCR75) CD4(+) T cells with indirect allospecificity during the course of tolerance induction. Three main observations were made. First, although the majority of TCR75 CD4(+) T cells were deleted following DST plus CD154 blockade, the surviving TCR75 CD4(+) T cells were capable of making IL-2, upregulating CD44, and undergoing cell division, suggesting that they were functionally active. Indeed, residual TCR75 CD4(+) T cells reisolated from the primary recipients given DST plus CD154 blockade were fully capable of rejecting allografts upon secondary transfer. Second, in tolerant mice, TCR75 CD4(+) T cells were not induced to express Foxp3 in the graft-draining lymph node. TCR75 CD4(+) T cells were also absent in accepted graft tissues in which endogenous Treg cells were enriched. Finally, DST plus CD154 blockade resulted in an abortive expansion of TCR75 CD4(+) T cells, a process that required the presence of endogenous Treg cells. Collectively, surviving TCR75 CD4(+) T cells are immunocompetent but kept in check by an endogenous immunosuppressive network induced by DST plus CD154 blockade.

Intranasal peptide-induced tolerance and linked suppression: consequences of complement deficiency.

A role for complement, particularly the classical pathway, in the regulation of immune responses is well documented. Deficiencies in C1q or C4 predispose to autoimmunity, while deficiency in C3 affects the suppression of contact sensitization and generation of oral tolerance. Complement components including C3 have been shown to be required for both B-cell and T-cell priming. The mechanisms whereby complement can mediate these diverse regulatory effects are poorly understood. Our previous work, using the mouse minor histocompatibility (HY) model of skin graft rejection, showed that both C1q and C3 were required for the induction of tolerance following intranasal peptide administration. By comparing tolerance induction in wild-type C57BL/6 and C1q-, C3-, C4- and C5-deficient C57BL/6 female mice, we show here that the classical pathway components including C3 are required for tolerance induction, whereas C5 plays no role. C3-deficient mice failed to generate a functional regulatory T (Treg) -dendritic cell (DC) tolerogenic loop required for tolerance induction. This was related to the inability of C3-deficient DC to up-regulate the arginine-consuming enzyme, inducible nitric oxide synthase (Nos-2), in the presence of antigen-specific Treg cells and peptide, leading to reduced Treg cell generation. Our findings demonstrate that the classical pathway and C3 play a critical role in the peptide-mediated induction of tolerance to HY by modulating DC function.

Transitional-2 B cells acquire regulatory function during tolerance induction and contribute to allograft survival.

In humans, tolerance to renal transplants has been associated with alterations in B-cell gene transcription and maintenance of the numbers of circulating transitional B cells. Here, we use a mouse model of transplantation tolerance to investigate the contribution of B cells to allograft survival. We demonstrate that transfer of B cells from mice rendered tolerant to MHC class I mismatched skin grafts can prolong graft survival in a dose-dependent and antigen-specific manner to a degree similar to that afforded by graft-specific regulatory T (Treg) cells. Tolerance in this model was associated with an increase in transitional-2 (T2) B cells. Only T2 B cells from tolerized mice, not naïve T2 nor alloantigen experienced T2, were capable of prolonging skin allograft survival, and suppressing T-cell activation. Tolerized T2 B cells expressed lower levels of CD86, increased TIM-1, and demonstrated a preferential survival in vivo. Furthermore, we demonstrate a synergistic effect between tolerized B cells and graft-specific Treg cells. IL-10 production by T2 B cells did not contribute to tolerance, as shown by transfer of B cells from IL-10(-/-) mice. These results suggest that T2 B cells in tolerant patients may include a population of regulatory B cells that directly inhibit graft rejection.

Enhanced and aberrant T cell trafficking following total body irradiation: a gateway to graft-versus-host disease?

Pre-transplant conditioning regimens play a major role in triggering graft-versus-host disease (GVHD). This study investigated the effect of irradiation on donor T cell trafficking to lymphoid and non-lymphoid tissues by comparing the migration of carboxy-fluorescein diacetate succinimidyl ester-labelled, naïve donor T lymphocytes in vivo in irradiated and non-irradiated syngeneic mice recipients. Recruitment of adoptively transferred naïve T cells to secondary lymphoid organs was increased in irradiated mice and naïve T cells also aberrantly localized to non-lymphoid tissues. Irradiation also induced aberrant effector memory T cell migration into lymph nodes and their localization to homing-privileged non-lymphoid sites, such as the gut. The presence of a minor histocompatibility mismatch further enhanced the aberrant accumulation of T cells in both lymphoid and non-lymphoid tissue, whilst their migratory pattern was not modified as compared to fully matched irradiated recipients. These effects correlated with decreased permeability of, and the secretion of chemotactic factors by the endothelium. Our findings are consistent with the possibility that excessive, dysregulated extravasation of T cells induced by irradiation promotes the development of GVHD.

Distinct in vivo CD8 and CD4 T cell responses against normal and malignant tissues.

Normal tissue and tumour grafts expressing the same alloantigens often elicit distinct immune responses whereby only normal tissue is rejected. To investigate the mechanisms that underlie these distinct outcomes, we compared the responses of adoptively transferred HY-specific conventional (CD8 and CD4) or regulatory T (Treg) cells in mice bearing HY-expressing tumour, syngeneic male skin graft or both. For local T cell priming, T cell re-circulation, graft localization and retention, skin grafts were more efficient than tumours. Skin grafts were also capable of differentiating CD4 T cells into functional Th1 cells. Donor T cell responses were inversely correlated with tumour progression. When skin graft and tumour transplants were performed sequentially, contemporary graft and tumour burden enhanced CD8 but reduced CD4 T cell responses causing accelerated skin-graft rejection without influencing tumour growth. Although both skin grafts and tumours were able to expand HY-specific Treg cells in draining lymph node (dLN), the proportion of tumour-infiltrating Treg cells was significantly higher than that within skin grafts, correlating with accelerated tumour growth. Moreover, there was a higher level of HY antigen presentation by host APC in tumour-dLN than in graft-dLN. Finally, tumour tissues expressed a significant higher level of IDO, TGFß, IL10 and Arginase I than skin grafts, indicating that malignant but not normal tissue represents a stronger immunosuppressive environment. These comparisons provide important insight into the in vivo mechanisms that conspire to compromise tumour-specific adaptive immunity and identify new targets for cancer immunotherapy.

Suppression of the allogeneic response by the anti-allergy drug N-(3,4-dimethoxycinnamonyl) anthranilic acid results from T-cell cycle arrest.

Previously we have shown that indoleamine 2,3-dioxygenase (IDO) and the tryptophan metabolite, 3-hydroxykynurenine (3HK) can prolong corneal allograft survival. IDO modulates the immune response by depletion of the essential amino acid tryptophan by breakdown to kynurenines, which themselves act directly on T lymphocytes. The tryptophan metabolite analogue N-(3,4-dimethoxycinnamonyl) anthranilic acid (DAA, 'Tranilast') shares the anthranilic acid core with 3HK. Systemic administration of DAA to mice receiving a fully MHC-mismatched allograft of cornea or skin resulted in significant delay in rejection (median survival of controls 12 days, 13 days for cornea and skin grafts, respectively, and of treated mice 24 days (P < 0.0001) and 17 days (P < 0.03), respectively). We provide evidence that DAA-induced suppression of the allogeneic response, in contrast to that induced by tryptophan metabolites, was a result of cell cycle arrest rather than T-cell death. Cell cycle arrest was mediated by up-regulation of the cell cycle-specific inhibitors p21 and p15, and associated with a significant reduction in interleukin-2 production, allowing us to characterize a novel mechanism for DAA-induced T-cell anergy. Currently licensed as an anti-allergy drug, the oral bioavailability and safe therapeutic profile of DAA make it a candidate for the prevention of rejection of transplanted cornea and other tissues.

The T-cell receptor is not hardwired to engage MHC ligands.

The bias of αß T cells for MHC ligands has been proposed to be intrinsic to the T-cell receptor (TCR). Equally, the CD4 and CD8 coreceptors contribute to ligand restriction by colocalizing Lck with the TCR when MHC ligands are engaged. To determine the importance of intrinsic ligand bias, the germ-line TCR complementarity determining regions were extensively diversified in vivo. We show that engagement with MHC ligands during thymocyte selection and peripheral T-cell activation imposes remarkably little constraint over TCR structure. Such versatility is more consistent with an opportunist, rather than a predetermined, mode of interface formation. This hypothesis was experimentally confirmed by expressing a hybrid TCR containing TCR-γ chain germ-line complementarity determining regions, which engaged efficiently with MHC ligands.

Mechanisms of endogenous MHC class II presentation by tumor cells.

Evaluation of: Tsuji T, Matsuzaki J, Caballero OL et al. Heat shock protein 90-mediated peptide-selective presentation of cytosolic tumor antigen for direct recognition of tumors by CD4(+) T cells. J. Immunol. 188, 3851-3858 (2012). In this study, Tsuji and colleagues investigated how tumor antigen NY-ESO-1 was processed by melanoma cells and subsequently presented on HLA class II for the recognition of NY-ESO-1-specific CD4(+) T cells. Using a combination of specific inhibitors and RNAi techniques, they found that tumor cells utilize a novel peptide selective antigen presentation pathway that requires both proteasome and endosomal protease-dependent processing, as well as heat-shock protein 90-dependent chaperoning. This newly described tumor-specific, endogenous MHC class II antigen presentation could have an impact on both antitumor or protumor T-cell responses.

Absence of Galectin-1 accelerates CD8⁺ T cell-mediated graft rejection.

Galectin-1 (Gal-1) is a member of a family of endogenous ß-galactose-binding proteins with a role in preventing autoimmune diseases and chronic inflammation. In this study, the involvement of Gal-1 in graft rejection was investigated by using Gal-1-deficient mice (Gal-1⁻/⁻). We demonstrate that in the absence of Gal-1, skin grafts are rejected earlier compared with those of WT mice, and that this is due to the role played by CD8⁺ T cells in graft rejection. The difference in graft survival observed between Gal-1⁻/⁻ and WT mice was explained by both an increase in the percentage of antigen-specific CD8+ T cells and by preferential secretion of IFN-γ and IL-17 by CD8⁺ T cells in Gal-1⁻/⁻ mice compared with WT mice. This study suggests that endogenous expression of Gal-1 contributes to graft survival. The results obtained from the use of mice deficient in Gal-1 also confirm a key role for CD8⁺ T cells in graft rejection.

Arginine depletion as a mechanism for the immune privilege of corneal allografts.

The cornea is an immune privileged tissue. Since arginase has been found to modulate T-cell function by depleting arginine, we investigated the expression of arginase in the cornea and its possible role in immune privilege using a murine transplant model. We found that both the endothelium and epithelium of murine corneas express functional arginase I, capable of down-regulating T-cell proliferation in an in vitro culture system. The administration of the specific arginase inhibitor N-hydroxy-nor-L-Arg to recipient mice resulted in an accelerated rejection of allogeneic C57BL/6 (B6) corneal grafts. In contrast, in vivo blockade of arginase activity had no effect in altering the course of rejection of primary skin grafts that express little, if any, arginase. In addition, the inhibition of arginase did not alter systemic T-cell proliferation. These data show that arginase is functional in the cornea and contributes to the immune privilege of the eye, and that modulation of arginase contributes to graft survival.

Cancer vaccination reprograms regulatory T cells into helper CD4 T cells to promote antitumor CD8 T-cell responses.

Evaluation of: Sharma MD, Hou DY, Baban B et al. : Reprogrammed Foxp3(+) regulatory T cells provide essential help to support cross-presentation and CD8(+) T cell priming in naive mice. Immunity 33, 942-954 (2010). It has been recognized that natural CD4(+)Foxp3(+) Tregs could display a phenotypic and functional plasticity in an inflammatory microenvironment. Following the loss of key transcription factor, Foxp3 and core inhibitory molecules associated with suppression, Tregs are reprogrammed into proinflammatory effector cells in vivo. However, the biological significance of this conversion is elusive. Sharma et al. demonstrate that in response to vaccines containing antigens, IFA and CpG, a large proportion of Tregs are dedifferentiated into Th1-like effector cells, which coexpress CD40L - a key molecule for CD8 help by licensing dendritic cells. Under certain experimental conditions, these reprogrammed Tregs are absolutely essential in helping the differentiation of CD8 T cells primed by antigen cross-presentation pathways. Treg conversion is diminished by tumor-induced indoleamine 2,3-dioxygenase in tumor-bearing mice, and blockade of indoleamine 2,3-dioxygenase activity in vivo is able to rescue Treg reprogramming. Collectively, in response to signaling from innate immune cells, Tregs are rapidly reprogrammed into Th1-like effector cells, which are also capable of providing timely help for antigen-specific CD8 T cells in the early phase of activation, when the traditional cognate help from conventional CD4 T cells has not yet became available.

Molecular mechanisms of induction of antigen-specific allograft tolerance by intranasal peptide administration.

We have previously shown that intranasal (i.n.) administration of a single MHC class II-restricted HY peptide to female mice induces tolerance to up to five additional epitopes expressed on test male grafts, a phenomenon known as linked suppression. In this study, we investigated the molecular mechanisms involved both in the induction phase following peptide administration and during linked suppression after grafting. We report that following initial i.n. administration, peptide is widely disseminated and is presented by functionally immature dendritic cells. These fail to cause optimal stimulation of the responding HY-specific CD4(+) T cells that express genes characteristic of regulatory T cells. Following i.n. peptide plus LPS administration, causing immunization, HY-specific CD4(+) T cells express genes characteristic of activated T cells. We further find that following male skin grafting, HY-specific CD8(+) T cells from peptide-treated tolerant mice display both quantitative and qualitative differences compared with similar cells from untreated mice that reject their grafts. In tolerant mice there are fewer HY-specific CD8(+) cells and they express several genes characteristic of exhausted T cells. Furthermore, associated with specific chemokine receptor and integrin expression, HY-specific CD8(+) T cells show more limited migration from the graft draining lymph node into other tissues.

Functional plasticity of antigen-specific regulatory T cells in context of tumor.

Although polyclonal regulatory T cells (Tregs) that once expressed Foxp3 (ex-Tregs) derived from Foxp3(+) Tregs have been described in homeostatic and autoimmune settings, little is known regarding the influence of the tumor environment on ex-Treg development. After adoptive transfer of HY-specific green Tregs (peripheral or thymic) to Rag2(-/-) B6 female mice bearing syngeneic HY-expressing MB49 tumors, a significant fraction rapidly lost expression of Foxp3. On the second transfer to a Rag2(-/-) B6 male environment, these ex-Tregs expanded strongly, whereas Tregs that maintained expression of Foxp3 expression did not. Both FACS and quantitative real-time-PCR analysis revealed that ex-Tregs upregulated genes characteristic of a Th1 effector-memory phenotype including IFN-γ and downregulated a panel of Treg-specific genes. Peripheral HY-specific green Tregs were adoptively transferred to Rag2(-/-) B6 male mice, to dissect the factors regulating ex-Treg differentiation. Development of ex-Tregs was more efficient in the mesenteric lymph node (mLN) than peripheral lymph node environment, correlating with a much greater level of IL-6 mRNA in mLN. In addition, the preferential development of ex-Tregs in mLN was significantly impaired by cotransfer of HY-specific naive CD4 T cells. Collectively, our study not only demonstrates the plasticity of Ag-specific Tregs in the context of the tumor environment, but also defines key molecular and cellular events that modulate ex-Treg differentiation.

Concurrent allorecognition has a limited impact on posttransplant vaccination.

Transplantation of allogeneic hematopoietic stem cells with or without immunocompetent lymphocytes has proved a successful strategy in the treatment of hematological malignancies. We have recently shown that this approach can also cure mouse prostate cancer, provided that it is combined with tumor-specific vaccination. Whether the response to alloantigens acts by providing helper function to enhance vaccine-specific responses or in other ways impinges on vaccine immunogenicity remains to be clarified, and this question is of clinical relevance. In this study, we have addressed this issue by comparing the immunogenicity of dendritic cells pulsed with a peptide derived from a tumor/viral model Ag in recipients of donor cells either syngeneic to the host or differing for either Y-encoded or multiple minor H antigens. We report that vaccination elicits comparable proliferation and differentiation of peptide-specific CD8(+) T cells despite concurrent expansion and differentiation of minor H antigen-specific IFN-γ effector T cells. Depletion of alloreactive CD4(+) T cells reduced alloreactivity but not vaccine-induced CD8(+) T cell priming, suggesting that alloresponses do not provide helper functions in peripheral lymphoid tissues. Vaccine-mediated T cell priming was also preserved in the case of multiple minor H antigen disparities, prone to graft-versus-host disease. Thus, in the context of nonmyeloablative allotransplantation aimed at restoring an effective tumor-specific T cell repertoire, minor H antigen-specific T cells do not interfere with vaccine-induced T cell priming, supporting the notion that posttransplant vaccination is a valuable strategy to boost tumor and pathogen-specific protective immunity.

Ig gene-like molecule CD31 plays a nonredundant role in the regulation of T-cell immunity and tolerance.

CD31 is an Ig-like molecule expressed by leukocytes and endothelial cells with an established role in the regulation of leukocyte trafficking. Despite genetic deletion of CD31 being associated with exacerbation of T cell-mediated autoimmunity, the contribution of this molecule to T-cell responses is largely unknown. Here we report that tumor and allograft rejection are significantly enhanced in CD31-deficient mice, which are also resistant to tolerance induction. We propose that these effects are dependent on an as yet unrecognized role for CD31-mediated homophilic interactions between T cells and antigen-presenting cells (APCs) during priming. We show that loss of CD31 interactions leads to enhanced primary clonal expansion, increased killing capacity, and diminished regulatory functions by T cells. Immunomodulation by CD31 signals correlates with a partial inhibition of proximal T-cell receptor (TCR) signaling, specifically Zap-70 phosphorylation. However, CD31-deficient mice do not develop autoimmunity due to increased T-cell death following activation, and we show that CD31 triggering induces Erk-mediated prosurvival activity in T cells either in conjunction with TCR signaling or autonomously. We conclude that CD31 functions as a nonredundant comodulator of T-cell responses, which specializes in sizing the ensuing immune response by setting the threshold for T-cell activation and tolerance, while preventing memory T-cell death.

Nonhematopoietic antigen blocks memory programming of alloreactive CD8+ T cells and drives their eventual exhaustion in mouse models of bone marrow transplantation.

Allogeneic blood or BM transplantation (BMT) is the most commonly applied form of adoptive cellular therapy for cancer. In this context, the ability of donor T cells to respond to recipient antigens is coopted to generate graft-versus-tumor (GVT) responses. The major reason for treatment failure is tumor recurrence, which is linked to the eventual loss of functional, host-specific CTLs. In this study, we have explored the role of recipient antigen expression by nonhematopoietic cells in the failure to sustain effective CTL immunity. Using clinically relevant models, we found that nonhematopoietic antigen severely disrupts the formation of donor CD8+ T cell memory at 2 distinct levels that operate in the early and late phases of the response. First, initial and direct encounters between donor CD8+ T cells and nonhematopoietic cells blocked the programming of memory precursors essential for establishing recall immunity. Second, surviving CD8+ T cells became functionally exhausted with heightened expression of the coinhibitory receptor programmed death-1 (PD-1). These 2 factors acted together to induce even more profound failure in long-term immunosurveillance. Crucially, the functions of exhausted CD8+ T cells could be partially restored by late in vivo blockade of the interaction between PD-1 and its ligand, PD-L1, without induction of graft-versus-host disease, suggestive of a potential clinical strategy to prevent or treat relapse following allogeneic BMT.

The roles of antigen-specificity, responsiveness to transforming growth factor-ß and antigen-presenting cell subsets in tumour-induced expansion of regulatory T cells.

In this study we investigated the impact of several factors on the expansion of natural regulatory T (nTreg) cells by tumours, including antigen specificity, transforming growth factor-ß (TGF-ß) signalling and the antigen-presenting cell subsets responsible for expansion. We found that antigen non-specific expansion of nTreg cells is tumour cell line-dependent. Although both antigen-specific and non-specific pathways can contribute to expansion, the migration of activated nTreg cells to tumour tissues is strictly antigen-dependent. Intact TGF-ß signalling on nTreg cells is also essential for tumour-induced expansion. Finally, for stimulation of resting antigen-specific CD4 T cells, CD11c(+) cells purified from tumour-draining lymph nodes were more potent than CD11b(+) cells, suggesting that dendritic cells are the key antigen-presenting cell subset involved in cross-presentation of tumour antigens. This study not only provides an in vivo system in which cross-talk between nTreg cells and tumours can be explored but also reveals novel aspects of tumour immune evasion.

Ligand dimensions are important in controlling NK-cell responses.

Size-dependent protein segregation at the cell-cell contact interface has been suggested to be critical for regulation of lymphocyte function. We investigated the role of ligand dimensions in regulation of mouse NK-cell activation and inhibition. Elongated forms of H60a, a mouse NKG2D ligand, were generated and expressed stably in the RMA cell line. RMA cells expressing the normal size H60a were lysed efficiently by both freshly isolated and IL-2 stimulated C57BL/6 mouse-derived NK cells; however the level of lysis decreased as the H60a ligand size increased. Importantly, H60a elongation did not affect NKG2D binding, as determined by soluble NKG2D tetramer staining, and by examining NK-cell target cell conjugate formation. CHO cells are efficient at activating NK cells from C57BL/6 mice, and expression of a single chain form of H-2K(b), a ligand for the mouse inhibitory receptor Ly49C, strongly inhibited such activation of Ly49C/I positive NK cells. Elongation of H-2K(b) resulted in decreased inhibition of both lysis and IFN-gamma production by NK cells. These results establish that small ligand dimensions are important for both NK-cell activation and inhibition, and suggest that there are shared features between the mechanisms of receptor triggering on different types of lymphocytes.

Depletion of regulatory T cells by anti-GITR mAb as a novel mechanism for cancer immunotherapy.

In vitro, engagement of GITR on Treg cells by the agonistic anti-GITR mAb, DTA-1, appears to abrogate their suppressive function. The consequence of in vivo engagement of GITR by DTA-1 is, however, less clear. In this study, we show that Treg cells isolated from DTA-1-treated mice were as potent as those from untreated mice in suppressing conventional CD4 T cells in vitro, indicating that in vivo GITR ligation does not disable Treg cells. Treatment of Foxp3/GFP knock-in mice with DTA-1 led to a selective reduction of circulating Treg cells, suggesting that DTA-1 is a depleting mAb which preferentially targets Treg cells. In tumour-bearing mice, DTA-1-mediated depletion of Treg cells was most marked in tumours but not in tumour-draining lymph node. These features were confirmed in an adoptive transfer model using tumour antigen-specific Treg cells. Interestingly, Treg cells detected in tumour tissues expressed much higher levels of GITR than those in tumour-draining lymph nodes, indicating that the efficiency of depletion might be correlated with the level of GITR expression. Finally, in vivo labelling of GITR in naive or tumour-bearing mice demonstrated that Treg cells constitutively expressed higher levels of GITR than conventional T cells, independent of location and activation state, consistent with the preferential in vivo depletion of Tregs by DTA-1. Thus, depletion of Treg cells represents a previously unrecognised in vivo activity of DTA-1 which has important implications for the application of anti-GITR antibodies in cancer immunotherapy.