Search

Purpose:Adoptive T-cell therapy (ACT) of cancer, which involves the infusion of ex vivo–engineered tumor epitope reactive autologous T cells into the tumor-bearing host, is a potential treatment modality for cancer. However, the durable antitumor response following ACT is hampered either by loss of effector function or survival of the antitumor T cells. Therefore, strategies to improve the persistence and sustain the effector function of the antitumor T cells are of immense importance. Given the role of metabolism in determining the therapeutic efficacy of T cells, we hypothesize that inhibition of PIM kinases, a family of serine/threonine kinase that promote cell-cycle transition, cell growth, and regulate mTORC1 activity, can improve the potency of T cells in controlling tumor.Experimental Design:The role of PIM kinases in T cells was studied either by genetic ablation (PIM1−/−PIM2−/−PIM3−/−) or its pharmacologic inhibition (pan-PIM kinase inhibitor, PimKi). Murine melanoma B16 was established subcutaneously and treated by transferring tumor epitope gp100–reactive T cells along with treatment regimen that involved inhibiting PIM kinases, anti-PD1 or both.Results:With inhibition of PIM kinases, T cells had significant reduction in their uptake of glucose, and upregulated expression of memory-associated genes that inversely correlate with glycolysis. In addition, the expression of CD38, which negatively regulates the metabolic fitness of the T cells, was also reduced in PimKi-treated cells. Importantly, the efficacy of antitumor T-cell therapy was markedly improved by inhibiting PIM kinases in tumor-bearing mice receiving ACT, and further enhanced by adding anti-PD1 antibody to this combination.Conclusions:This study highlights the potential therapeutic significance of combinatorial strategies where ACT and inhibition of signaling kinase with checkpoint blockade could improve tumor control.

Supplementary Figures and Figure Legends

Purpose:Application of allogeneic hematopoietic cell transplantation (allo-HCT) for patients with hematologic disorders is limited by the development of GVHD. Separation of GVHD and graft-versus-leukemia (GVL) remains a great challenge in the field. We investigated the contribution of individual pathways involved in the complement cascade in GVH and GVL responses to identify specific targets by which to separate these two processes.Experimental Design:We used multiple preclinical murine and human-to-mouse xenograft models involving allo-HCT recipients lacking components of the alternative pathway (AP) or classical pathway (CP)/lectin pathway (LP) to dissect the role of each individual pathway in GVHD pathogenesis and the GVL effect. For translational purposes, we used the AP-specific complement inhibitor, CR2-fH, which localizes in injured target organs to allow specific blockade of complement activation at sites of inflammation.Results:Complement deposition was evident in intestines of mice and patients with GVHD. In a preclinical setting, ablation of the AP, but not the CP/LP, significantly improved GVHD outcomes. Complement activation through the AP in host hematopoietic cells, and specifically dendritic cells (DC), was required for GVHD progression. AP deficiency in recipients decreased donor T-cell migration and Th1/Th2 differentiation, while increasing the generation of regulatory T cells. This was because of decreased activation and stimulatory activity of recipient DCs in GVHD target organs. Treatment with CR2-fH effectively prevented GVHD while preserving GVL activity.Conclusions:This study highlights the AP as a new therapeutic target to prevent GVHD and tumor relapse after allo-HCT. Targeting the AP by CR2-fH represents a promising therapeutic approach for GVHD treatment.

Supplemental Materials

Purpose: Application of allogeneic hematopoietic cell transplantation (allo-HCT) for patients with hematologic disorders is limited by the development of GVHD. Separation of GVHD and graft-versus-leukemia (GVL) remains a great challenge in the field. We investigated the contribution of individual pathways involved in the complement cascade in GVH and GVL responses to identify specific targets by which to separate these two processes. Experimental Design: We used multiple preclinical murine and human-to-mouse xenograft models involving allo-HCT recipients lacking components of the alternative pathway (AP) or classical pathway (CP)/lectin pathway (LP) to dissect the role of each individual pathway in GVHD pathogenesis and the GVL effect. For translational purposes, we used the AP-specific complement inhibitor, CR2-fH, which localizes in injured target organs to allow specific blockade of complement activation at sites of inflammation. Results: Complement deposition was evident in intestines of mice and patients with GVHD. In a preclinical setting, ablation of the AP, but not the CP/LP, significantly improved GVHD outcomes. Complement activation through the AP in host hematopoietic cells, and specifically dendritic cells (DC), was required for GVHD progression. AP deficiency in recipients decreased donor T-cell migration and Th1/Th2 differentiation, while increasing the generation of regulatory T cells. This was because of decreased activation and stimulatory activity of recipient DCs in GVHD target organs. Treatment with CR2-fH effectively prevented GVHD while preserving GVL activity. Conclusions: This study highlights the AP as a new therapeutic target to prevent GVHD and tumor relapse after allo-HCT. Targeting the AP by CR2-fH represents a promising therapeutic approach for GVHD treatment.

Oxidative stress is elevated in the recipients of allogeneic hematopoietic transplantation (allo-HCT) and likely contributes to the development of graft-versus-host disease (GVHD). GVHD is characterized by activation, expansion, cytokine production and migration of alloreactive donor T cells, and remains a major cause of morbidity and mortality after allo-HCT. Hence, strategies to limit oxidative stress in GVHD are highly desirable. Thioredoxin1 (Trx1) counteracts oxidative stress by scavenging reactive oxygen species (ROS) and regulating other enzymes that metabolize H2O2. The present study sought to elucidate the role of Trx1 in the pathophysiology of GVHD. Using murine and xenograft models of allogeneic bone marrow transplantation (allo-BMT) and genetic (human Trx1-transgenic, Trx1-Tg) as well as pharmacologic (human recombinant Trx1, RTrx1) strategies; we found that Trx1-Tg donor T cells or administration of the recipients with RTrx1 significantly reduced GVHD severity. Mechanistically, we observed RTrx1 reduced ROS accumulation and cytokine production of mouse and human T cells in response to alloantigen stimulation in vitro. In allo-BMT settings, we found that Trx1-Tg or RTrx1 decreased downstream signaling molecules including NFκB activation and T-bet expression, and reduced proliferation, IFN-γ production and ROS accumulation in donor T cells within GVHD target organs. More importantly, administration of RTrx1 did not impair the graft-versus-leukemia (GVL) effect. Taken together, the current work provides a strong rationale and demonstrates feasibility to target the ROS pathway, which can be readily translated into clinic.

Adoptive transfer of tumor epitope–reactive T cells has emerged as a promising strategy to control tumor growth. However, chronically-stimulated T cells expanded for adoptive cell transfer are susceptible to cell death in an oxidative tumor microenvironment. Because oxidation of cell-surface thiols also alters protein functionality, we hypothesized that increasing the levels of thioredoxin (Trx), an antioxidant molecule facilitating reduction of proteins through cysteine thiol-disulfide exchange, in T cells will promote their sustained antitumor function. Using pre-melanosome protein (Pmel)–Trx1 transgenic mouse-derived splenic T cells, flow cytometry, and gene expression analysis, we observed here that higher Trx expression inversely correlated with reactive oxygen species and susceptibility to T-cell receptor restimulation or oxidation-mediated cell death. These Trx1-overexpressing T cells exhibited a cluster of differentiation 62L(hi) (CD62L(hi)) central memory-like phenotype with reduced glucose uptake (2-NBDG(lo)) and decreased effector function (interferon γ(lo)). Furthermore, culturing tumor-reactive T cells in the presence of recombinant Trx increased the dependence of T cells on mitochondrial metabolism and improved tumor control. We conclude that strategies for increasing the antioxidant capacity of antitumor T cells modulate their immunometabolic phenotype leading to improved immunotherapeutic control of established tumors.

Glucocorticoids are a highly effective first-line treatment option for many inflammatory diseases, including asthma. Some patients develop a steroid-resistant condition, yet, the cellular and molecular mechanisms underlying steroid resistance remain largely unknown. In this study, we used a murine model of steroid-resistant airway inflammation and report that combining systemic dexamethasone and intranasal IL-27 is able to reverse the inflammation. Foxp3+ regulatory T cells (Tregs) were required during dexamethasone/IL-27 treatment of steroid-resistant allergic inflammation, and importantly, direct stimulation of Tregs via glucocorticoid or IL-27 receptors was essential. Mechanistically, IL-27 stimulation in Tregs enhanced expression of the agonistic glucocorticoid receptor-α isoform. Overexpression of inhibitory glucocorticoid receptor-β isoform in Tregs alone was sufficient to elicit steroid resistance in a steroid-sensitive allergic inflammation model. Taken together, our results demonstrate for the first time, to our knowledge, that Tregs are instrumental during steroid resistance and that manipulating steroid responsiveness in Tregs may represent a novel strategy to treat steroid refractory asthma.

RNA binding protein PCBP1 guards effector and antitumor T cell functions., Distinct lineages of T cells can act in response to various environmental cues to either drive or restrict immune-mediated pathology. Here, we identify the RNA binding protein, poly(C)-binding protein 1 (PCBP1) as an intracellular immune checkpoint that is up-regulated in activated T cells to prevent conversion of effector T (Teff) cells into regulatory T (Treg) cells, by restricting the expression of Teff cell–intrinsic Treg commitment programs. This was critical for stabilizing Teff cell functions and subverting immune-suppressive signals. T cell–specific deletion of Pcbp1 favored Treg cell differentiation, enlisted multiple inhibitory immune checkpoint molecules including PD-1, TIGIT, and VISTA on tumor-infiltrating lymphocytes, and blunted antitumor immunity. Our results demonstrate a critical role for PCBP1 as an intracellular immune checkpoint for maintaining Teff cell functions in cancer immunity.

Adoptive transfer of induced regulatory T cells (iTregs) can ameliorate graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT). CD4(+) iTregs can effectively prevent GVHD but impair the graft-versus-leukemia (GVL) effect, whereas CD8(+) iTregs preserve the GVL effect but have limited efficacy in GVHD control because of their instability under inflammatory conditions. Thus, we aimed to stabilize CD8(+) iTregs via treatment with vitamin C (Vit C) to improve their efficacy in controlling GVHD. We found that addition of Vit C significantly improved the stability of forkhead box P3 (Foxp3) expression in CD8(+) iTregs. Moreover, Vit C–treated CD8(+) iTregs exhibited high efficacy in attenuating acute and chronic GVHD. The mechanistic study revealed that addition of Vit C to CD8(+) iTreg culture markedly increased DNA demethylation in the conserved noncoding sequence 2 region and, hence, maintained higher Foxp3 expression levels compared with untreated controls. In acute GVHD, Vit C–treated CD8(+) iTregs were able to inhibit pathogenic T-cell expansion and differentiation while reducing thymus damage and B-cell activation in cGVHD. Importantly, in contrast to CD4(+) iTregs, Vit C–treated CD8(+) iTregs retained the ability to control tumor relapse. These results provide a strong rationale to use Vit C in the clinic to stabilize CD8(+) iTregs for the control of GVHD and preservation of GVL after allo-HCT.

Distinct lineages of T cells can act in response to various environmental cues to either drive or restrict immune-mediated pathology. Immunotherapy involving the block of cell surface immune checkpoints CTLA-4 and PD-1/PD-L1 has revolutionized treatment of several cancers. However, additional immune regulatory mechanisms remain to be discovered, and will facilitate development of next generation agents to overcome resistance to current therapies. Here, we identify the RNA-binding protein poly(C)-binding protein 1 (PCBP1) as an intracellular checkpoint that is upregulated in activated T cells to suppress expression of effector T cell-intrinsic regulatory T (Treg) cell-commitment programs. This is critical for stabilizing effector T cell functions and subverting immune-suppressive signals. T cell-specific deletion of Pcbp1 increased Treg development, enlisted multiple inhibitory checkpoint molecules including PD-1, TIGIT and VISTA on TILs, and thwarted anti-tumor immunity. Our results demonstrate a critical role for PCBP1 as an intracellular checkpoint for balancing effector versus regulatory T cells.

Graft-versus-host disease (GVHD) remains one of the major complications after allogeneic bone marrow transplantation (allo-BMT). Sirtuin-1 (Sirt-1) plays a crucial role in various biological processes including cellular senescence, metabolism, and inflammatory responses. Sirt-1 deacetylation regulates different transcription factors that are important for modulating immune responses. In the current study, we addressed the role of Sirt-1 in GVHD induction by employing Sirt-1 conditional knockout mice as well as a pharmacological Sirt-1 inhibitor. Using major histocompatibility complex (MHC)–mismatched and MHC-matched murine BMT models, we found that Sirt-1−/− T cells had a reduced ability to induce acute GVHD (aGVHD) via enhanced p53 acetylation. Sirt-1-deficient T cells also promoted induced regulatory T cell (iTreg) differentiation and inhibited interferon-γ production after allo-BMT. Sirt-1 deletion in iTregs increased Foxp3 stability and restrained iTreg conversion into pathogenic T cells. Furthermore, we found that administration with a Sirt-1 inhibitor, Ex-527, significantly improved recipient survival and clinical scores, with no signs of tumor relapse. These results indicate that Sirt-1 inhibition can attenuate GVHD while preserving the graft-versus-leukemia effect. Consistently, Sirt-1-deficient T cells also displayed a remarkably reduced ability to induce chronic GVHD (cGVHD). Mechanistic studies revealed that Sirt-1 deficiency in T cells enhanced splenic B-cell reconstitution and reduced follicular T helper cell development. Sirt-1 deficiency in T cells modulated donor B-cell responses reducing both B-cell activation and plasma cell differentiation. In addition, therapeutic Sirt-1 inhibition could both prevent cGVHD and reduce established cGVHD. In conclusion, Sirt-1 is a promising therapeutic target for the control of aGVHD and cGVHD pathogenesis and possesses high potential for clinical application.

CD8+ induced regulatory T cells (iTregs) have been identified to suppress alloreactive immune responses and expressed regulatory T cell (Treg) ontological markers as similar as CD4+ iTregs. However, adoptive transfer of CD8+ iTreg–based therapy is hampered by the instability of Treg specific-transcription factor, Foxp3. As CD8+ iTregs were previously demonstrated to possess superior tumor-killing ability to CD4+ iTregs, adoptive transfer of stabilized CD8+ iTregs would be a potential therapy to prevent tumor relapse during graft-versus-leukemia disease (GVHD) treatment. In the current study, we generated alloantigen reactive CD8+ iTregs from JAK2−/− T cells and adoptively transferred them to MHC-mismatched and haploidentical murine models of allogeneic bone marrow transplantation. JAK2−/− CD8+ iTregs not only attenuated GVHD but also preserved graft-versus-leukemia effect. Mechanistic analysis revealed that JAK2−/− CD8+ iTregs upregulated natural Treg marker (neuropilin-1), and augmented DNA demethylation of CNS2 region within Foxp3 gene. These properties licensed JAK2−/− CD8+ iTregs to retain high Foxp3 expression resulting in less conversion to type 1 CTLs; as a result, JAK2−/− CD8+ iTregs were able to maintain their suppressive and cytolytic function. Thus, our findings provide a strong rationale and means to stabilize CD8+ iTregs by targeting JAK2, and the stabilized CD8+ iTregs exhibit therapeutic potential for alleviating GVHD and preserving the graft-versus-leukemia effect.

Purpose: Adoptive T-cell therapy (ACT) of cancer, which involves the infusion of ex vivo–engineered tumor epitope reactive autologous T cells into the tumor-bearing host, is a potential treatment modality for cancer. However, the durable antitumor response following ACT is hampered either by loss of effector function or survival of the antitumor T cells. Therefore, strategies to improve the persistence and sustain the effector function of the antitumor T cells are of immense importance. Given the role of metabolism in determining the therapeutic efficacy of T cells, we hypothesize that inhibition of PIM kinases, a family of serine/threonine kinase that promote cell-cycle transition, cell growth, and regulate mTORC1 activity, can improve the potency of T cells in controlling tumor. Experimental Design: The role of PIM kinases in T cells was studied either by genetic ablation (PIM1−/−PIM2−/−PIM3−/−) or its pharmacologic inhibition (pan-PIM kinase inhibitor, PimKi). Murine melanoma B16 was established subcutaneously and treated by transferring tumor epitope gp100–reactive T cells along with treatment regimen that involved inhibiting PIM kinases, anti-PD1 or both. Results: With inhibition of PIM kinases, T cells had significant reduction in their uptake of glucose, and upregulated expression of memory-associated genes that inversely correlate with glycolysis. In addition, the expression of CD38, which negatively regulates the metabolic fitness of the T cells, was also reduced in PimKi-treated cells. Importantly, the efficacy of antitumor T-cell therapy was markedly improved by inhibiting PIM kinases in tumor-bearing mice receiving ACT, and further enhanced by adding anti-PD1 antibody to this combination. Conclusions: This study highlights the potential therapeutic significance of combinatorial strategies where ACT and inhibition of signaling kinase with checkpoint blockade could improve tumor control.

Janus kinase 2 (JAK2) signal transduction is a critical mediator of the immune response. JAK2 is implicated in the onset of graft-versus-host disease (GVHD), which is a significant cause of transplant-related mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Transfer of JAK2-/- donor T cells to allogeneic recipients leads to attenuated GVHD yet maintains graft-versus-leukemia. Th1 differentiation among JAK2-/- T cells is significantly decreased compared with wild-type controls. Conversely, iTreg and Th2 polarization is significantly increased among JAK2-/- T cells. Pacritinib is a multikinase inhibitor with potent activity against JAK2. Pacritinib significantly reduces GVHD and xenogeneic skin graft rejection in distinct rodent models and maintains donor antitumor immunity. Moreover, pacritinib spares iTregs and polarizes Th2 responses as observed among JAK2-/- T cells. Collectively, these data clearly identify JAK2 as a therapeutic target to control donor alloreactivity and promote iTreg responses after allo-HCT or solid organ transplantation. As such, a phase I/II acute GVHD prevention trial combining pacritinib with standard immune suppression after allo-HCT is actively being investigated (https://clinicaltrials.gov/ct2/show/NCT02891603).

PIM kinase family members play a crucial role in promoting cell survival and proliferation via phosphorylation of their target substrates. In this study, we investigated the role of the PIM kinases with respect to T cell responses in transplantation and tumor immunity. We found that the PIM-2 isoform negatively regulated T cell responses to alloantigen, in contrast to the PIM-1 and PIM-3 isoforms, which acted as positive regulators. T cells deficient in PIM-2 demonstrated increased T cell differentiation toward Th1 subset, proliferation, and migration to target organs after allogeneic bone marrow transplantation, resulting in dramatically accelerated graft-versus-host disease (GVHD) severity. Restoration of PIM-2 expression markedly attenuated the pathogenicity of PIM-2-deficient T cells to induce GVHD. On the other hand, mice deficient in PIM-2 readily rejected syngeneic tumor, which was primarily dependent on CD8+ T cells. Furthermore, silencing PIM-2 in polyclonal or antigen-specific CD8+ T cells substantially enhanced their antitumor response in adoptive T cell immunotherapy. We conclude that PIM-2 kinase plays a prominent role in suppressing T cell responses, and provide a strong rationale to target PIM-2 for cancer immunotherapy.

Maintaining adequate regulatory T cell (Treg) and effector T cell balance is critical for efficient regulation of tolerance and immune response, respectively. There is increasing evidence that in addition to regulating RNA fate, RNA-binding proteins (RBPs) may also control gene expression and determine the fate of immune cells. Here by studying mice in which the RBP Pcbp1 gene was disrupted in T cells, we define PCBP1 as a global regulator of immune activity that stabilizes immune responses, while repressing the signature programs of Tregs. PCBP1 expression was upregulated in response to T cell activation, but functionally inhibited by TGF-β-mediated phosphorylation. Loss of PCBP1 expression in T cells was associated with dysregulated peripheral homeostasis and impaired effector functions in knockout mice. Indeed, PCBP1-deficient T cells produced less IFN-γ and TNF-α upon stimulation with PMA, plus ionomycin, compared to wild-type littermates. Strikingly, lack of PCBP1 constrained the pathogenicity of effector T cells by conferring resistance to acute graft-vs-host disease in mice. Mechanistically, assessment of the genome-wide function of PCBP1 revealed that it limits the expression of genes associated with commitment to the Treg lineage. Consequently, its absence resulted in a marked increase in the frequency of Foxp3+Treg cells. Together, our findings identify PCBP1 as a distinct regulator of effector T cell functions and the formation of Treg cells in order to control the balance between tolerance and immunity.

Graft-versus-host disease (GvHD) remains a leading cause of non-relapse mortality after allogeneic hematopoietic cell transplantation (allo-HCT). The imbalance between T effector (Teffs) and regulatory cells (Tregs) is a defined characteristic of GvHD. Therefore, promoting Tregs for controlling GvHD has drawn an intense interest in preclinical and clinical studies of allo-HCT. Although CD4 Tregs (CD4+Foxp3+) have been shown to effectively prevent GvHD, they impaired graft-versus-leukemia (GVL) effect. Our lab has previously demonstrated that CD8 iTregs (CD8+Foxp3+) not only suppressed allogeneic T-cell responses, but also possessed the GVL activity themselves. However, instability of Foxp3 in CD8 iTregs is a major obstacle to translate this Treg population into clinic application. Vitamin C has been reported to promote Foxp3 stability by accelerating its demethylation. In the current study, we asked whether vitamin C could stabilize CD8 iTregs and enhance their therapeutic potential in controlling GvHD. Addition of vitamin C in the culture significantly increased the generation of allo-reactive CD8 iTregs in vitro (Fig. 1A). These CD8 iTregs generated with vitamin C had strikingly increased demethylation on Foxp3 CpGs (Fig. 1B) and superior suppressive activity (data not shown) compared to control-treated CD8 iTregs. We further evaluated the capability of vitamin C-treated CD8 iTregs in preventing GvHD in allo-HCT. Using a MHC-mismatched murine BMT model, vitamin C-treated CD8 iTregs could significantly alleviate GvHD reflected by a marked reduction of recipient mortality (Fig. 1C) and clinical scores (data not shown). To determine the Foxp3 stability in vivo, we analyzed the transferred CD8 iTregs and allogeneic Teffs in recipient spleens 7 days after allo-HCT. Vitamin C-treated CD8 iTregs (Ly5.2+) remarkably suppressed Teff (Ly5.1+) expansion and maintained higher Foxp3 expression compared to the control iTregs (Fig. 1D). To address a critical question whether vitamin C-treated CD8 iTregs can preserve the GVL activity, we performed a haploidentical transplant with a leukemia relapse model, and found that vitamin C-treated CD8 iTregs not only attenuated GvHD severity, but also prevented recipients from tumor relapse (Fig. 1E). In conclusion, our studies provide a rationale and mean to use stabilized CD8 iTregs with vitamin C for controlling GvHD and leukemia relapse in the clinic. Disclosures No relevant conflicts of interest to declare.

PIM kinases, a family of serine/threonine kinases, play an important role in regulating cell survival, cell proliferation, transcription activation and protein translation. Of the three isoforms, PIM-1, PIM-2 and PIM-3, some of them are overly expressed in several hematological malignancies as well as solid tumors. Thus, these kinases have been targeted clinically in several cancer studies. However, the roles of PIM kinases in T cells from previous studies are inconclusive and the functions of each isoform in these cells are yet to be investigated. In our study, we focused on the role of PIM-2 kinase in T cell responses to allo-antigen and tumor using pre-clinical models. Using allogeneic bone marrow transplantation (allo-BMT) models, we transferred allogeneic T cells isolated from mice deficient for Pim-2 (Pim-2-/-, FVB background) in comparison to WT and PIM1/3double deficient mice (PIM1/3-/-) into lethally irradiated Balb/C recipients. Our data revealed that upon allogeneic stimulation, PIM-2-/- T cells induced accelerated graft-versus-host-disease (GVHD) compared to WT or PIM1/3-/- T cells (Fig.1A). In addition, PIM-2-/- donor T cells were highly activated with significantly increased proliferation and IFN-γ production in recipient spleen and GVHD target organs. Donor PIM-2-/- T cells also expressed high levels of chemokine receptor, CXCR3 and a4b7 integrin (gut homing receptor) that correlated with increased capacity of donor T-cell migration to GVHD target organs such as liver and gut. In graft-versus-leukemia (GVL) study, PIM-2-/- donor T cells had roughly 4-fold higher ability to mediate GVL effect to WT and PIM1/3-/- T cells (Fig.1B). Negative regulation of T-cell responses to allo-antigens by PIM-2 was independently verified by restoring PIM-2 expression on PIM-2-/- T cells or inhibiting PIM kinases in PIM1/3-/- T cells. Altogether, the data from allo-BMT studies suggested that PIM-2 isoform functions as a negative regulator in T cell allo-response in contrast to PIM-1 and PIM-3 kinases. We then focused on evaluating T-cell immunity against syngeneic tumor. By administering tumor (TS-1 breast tumor in FVB background) into WT and PIM-2-/- mice either subcutaneously or intravenously, we observed that PIM-2-deficient mice efficiently control tumor growth in sharp contrast to WT mice (Fig. 1C). Mechanistic study revealed that tumor rejection in PIM-2-/- recipient relies on CD8+ tumor infiltrating lymphocytes (TILs) with a significant up-regulation of IFN-g, TNF-a and Fas-L expression. Lower infiltration of regulatory T cells and down regulation of PD-1 expression on T cells recovered from tumor site were also observed in PIM-2 deficient mice. Moreover, the tumor growth rate in PIM-2-/- mice after T-cell depletion with antibody was similar to that of WT mice. To further exclude the effect of other immune cells involved in tumor regression, we adoptively transferred WT and PIM-2-/- CD8+ T cells into WT mice with pre-established TS-1 tumor. CD8+ T cells deficient for PIM-2 was more efficient in controlling of tumor growth than WT or PIM1/3-/- CD8+ T cells. These data indicate that inhibition of PIM-2 in T cells display superior anti-tumor activity. In conclusion, PIM-2 kinase possesses a distinct role in anti-tumor immunity compared to the other two isoforms and it represents a potential target to enhance anti-tumor activity of adoptive T-cell immunotherapy. Disclosures No relevant conflicts of interest to declare.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapy for many hematological malignancies due to its potent graft-versus-leukemia (GVL) effect; yet its clinical application is limited by the development of graft-versus-host-disease (GVHD). Currently, the separation of GVHD and GVL is a great challenge in the field. Complement can be activated via three different pathways: the classical (CP), lectin (LP) and alternative pathway (AP). The AP can spontaneously activate, but it also serves as an amplification loop for the other pathways, and hence plays a central role in the development of autoimmune diseases such inflammatory bowel disease, which shares certain pathological similarities with intestinal GVHD. Complement activation has been implicated in GVHD development, yet how complement regulates GVHD is not known. In current study, deposition of complement activation product C3d occurs within GVHD target organs (Fig. 1A), suggesting that local complement activation contributes to GVHD pathogenesis. To functionally dissect the roles of each complement pathway, we used mice deficient in fB (no AP) or C1q/MBL (no CP or LP) as recipients in MHC-mismatched murine models of GVHD. GVHD severity and mortality was significantly reduced in fB, but not C1q/MLB, deficient recipients compared to wild type (WT) counterparts, suggesting a crucial role for the AP in GVHD (Fig. 1B). Furthermore, using chimeric mice with a fB-deficiency specifically in the hematopoietic compartment, we demonstrated that the AP in hematopoietic cells is required for GVHD development, but not for the GVL effect (Fig. 1D). For translational purposes, we evaluated GVHD and GVL responses using CR2-fH, an AP-specific protein inhibitor (Fig. 1C). CR2-fH consists of the N-terminus of mouse complement factor H, which contains the AP-inhibitory domain, linked to complement receptor 2 (CR2) targeting fragment that binds the complement activation product C3d. CR2-fH has been shown to specifically co-localize to sites of injury and C3d deposition. In recipients harboring C1498 acute myeloid leukemia, systemic treatment of recipients with CR2-fH significantly ameliorated GVHD with no associated tumor relapse (Fig. 1D), suggesting CR2-fH does not impact GVL activity. Mechanistic studies revealed that host AP deficiency significantly impacted the phenotype of immune cells in GVHD target organs rather than in lymphoid organs. Complement deposition was decreased in GVHD target organs of AP-deficient recipients. Gut integrity was significantly preserved in fB-/- compared to WT recipients (Fig. 1E). Activation and maturation of host DCs in the gut, lung, and thymus were also significantly decreased in fB-/- recipients, while GVHD-protective CD8+DCs were increased. In contrast, AP deficiency did not affect the phenotype of recipient DCs in the lymphoid organs. The numbers of donor T cells and particularly CD103+CD8+ T cells, a cell subset that critically mediates intestinal GVHD, were decreased in the gut of fB-/- recipients, which was associated with reduced expression of the gut homing chemokine CCR9 on donor T cells. Donor iTregs were increased in the liver of fB-/- recipients (Fig. 1F), while Th1 and Tc1 cells were significantly decreased; lower cytolytic activity was also seen in these Tc1 cells. Donor Th2 and Tc1 were diminished in the lung while thymic integrity was significantly improved in fB-/- recipients. We observed no differences in differentiation or function of donor T cells in lymphoid organs (Fig. 1G). These studies further indicate AP activation within target organs is important for GVHD pathogenesis. Taken together, the current data identifies a central role for the AP in GVHD and validates the AP as a therapeutic target for the control of GVHD. We provide evidence that local complement generation in GVHD target organs is important for GVHD development. Since site-specific AP inhibition is effective at controlling GVHD while preserving GVL activity, this study identifies a novel strategy to reduce GVHD and spare GVL effects. Because site-specific complement inhibition has been shown to reduce potentially dangerous side effects and since a humanized version of CR2-fH (TT30) has been shown to be safe and nonimmunogenic in humans, the current findings have a high potential for clinical translation. Disclosures No relevant conflicts of interest to declare.

Multiplex PCR (mPCR) was established for the simultaneous detection of clarithromycin (CLR) resistance and species identification of Mycobacterium avium complex (MAC). mPCR was tested on 218 MAC clinical isolates. CLR-resistance was detected by mPCR in 31 of 35 isolates identified by a microdilution method. Of the remaining 187 susceptible isolates identified by mPCR, 183 isolates had MICor = 8 microg/ml (susceptible), 3 with MIC of 16 (intermediate resistant) and 1 with MIC ofor = 32 microg/ml (resistant). Comparing with the PCR-restriction enzyme analysis, mPCR concordantly identified 185 isolates either as being M. avium or M. intracellulare, whereas one isolate was misidentified and 32 isolates could not be identified. Comparing with reference methods, the mPCR showed the sensitivity, specificity, positive predictive and negative predictive value of 89, 100, 100, and 98% for detection of CLR resistance; 92, 98, 99, and 78% for identification of M. avium; and 57, 100, 100, and 89% for identification of M. intracellulare, respectively.