Therapeutic Blockade Research Articles

Identification of a subset of human natural killer cells expressing high levels of programmed death 1: A phenotypic and functional characterization

Background: PD-1 is an immunological checkpoint that limits immune responses by delivering potent inhibitory signals to T cells upon interaction with specific ligands expressed on tumor/virus-infected cells, thus contributing to immune escape mechanisms (1). Therapeutic PD-1 blockade has been shown to mediate tumor eradication with impressive clinical results. Little is known on the expression/function of PD-1 on human NK cells (2). Objective: To clarify whether human NK cells may express PD-1 and analyze their phenotypic/functional features. Methods: Multiparametric cytofluorimetric analysis of PD-1+ NK cells and their functional characterization by degranulation, cytokine production and proliferation assays. Results: We provide unequivocal evidence that PD-1 is highly expressed (PD-1bright) on a NK cell subset detectable in the peripheral blood of approximately one fourth of healthy individuals. These donors are always serologically positive for HCMV. PD-1 is expressed by CD56dim but not by CD56bright NK cells and is confined to fully mature NK cells characterized by the NKG2A-KIR+CD57+ phenotype. The proportions of PD-1bright NK cells were higher in the ascites of a cohort of ovarian-carcinoma patients suggesting their possible induction/expansion in tumor environments. Functional analysis revealed a reduced proliferative capability in response to cytokines, low degranulation and impaired cytokine production upon interaction with tumor targets. Conclusions: We have identified and characterized a novel subpopulation of human NK cells expressing high levels of PD-1. These cells have the phenotypic characteristics of fully mature NK cells and are increased in ovarian-carcinoma patients. They display low proliferative responses and impaired anti-tumor activity that can be partially restored by antibody-mediated disruption of PD-1/PD-L interaction.This work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro- (AIRC-) Special Project 5x1000 no. 9962 and AIRC-IG 2014 Id. 15704 (Alessandro Moretta), AIRC-IG 2014 Id. 15283 (Lorenzo Moretta), Progetto di Ricerca di Ateneo 2014 (Emanuela Marcenaro) and Olive Daniel laboratory is supported by the Fondation pour la Recherche Medicale (Equipe FRM DEQ20140329534).

Mobilization of CD8+ Central Memory T-Cells with Enhanced Reconstitution Potential in Mice By a Combination of G-CSF and GMI-1271-Mediated E-Selectin Blockade

T-cells are critical mediators of immune defense against pathogens and cancer. Adoptive T cell immunotherapy and T-cell engineering have promising clinical applications but T cell survival and exhaustion are current limitations. Central memory cells (TCM CD62L+ CCR7+) and their precursors, stem central memory T-cells (TSCM) possess the stem-like properties needed to reconstitute and prolong an effective immune response long-term. These cells have been shown to significantly improve therapeutic efficacy of adoptive T-cell therapy. The challenge remains to harvest good quality TCM-cells for these immunotherapy approaches.The bone marrow (BM) is the major reservoir of CD8+ TCM and their precursors. We have previously shown that E-selectin is expressed in the BM vasculature and drives activation and differentiation of hematopoietic stem cells during G-CSF induced mobilization to the blood. We find therapeutic blockade of E-selectin promotes HSC self-renewal and reconstitution in vivo. We now examine the impact of E-selectin blockade on CD8+ T cell mobilization from the bone marrow to the blood and hypothesize that E-selectin blockade may also dampen the activation/differentiation of this subset.First we administered a standard G-CSF regime (filgastim 250ug/kg/day for 3 days) to mice and then dosed some cohorts with GMI-1271 (40mg/kg BID) from 12 to 72 hours within this 3 day period. Administration of G-CSF alone results in a near complete disappearance of bone marrow resident CD8+ TCM cells, and their apparent migration (increase in numbers) to the blood, while CD8+ subsets in the lymph nodes and spleen were barely affected by G-CSF. Furthermore among T-cell subsets, CD8+ but not CD4+ TCM were specifically mobilized into the blood when GMI-1271 was co-administered for the last 12 to 24 hours of G-CSF. These findings are consistent with reports demonstrating the bone marrow to be a major reservoir for CD8+ but not CD4+ central memory T-cells.Administration of GMI-1271 caused a marked enhancement in mobilization into the blood of CD8+ TCM/SCM (CD62Lhi, CCR7+) cells over treatment with G-CSF alone (p<0.05). To determine the functional consequences of this skewed mobilization following GMI-1271 co-administration, 25 uL of mobilized blood was transplanted into irradiated congenic B6.SJL recipients together with 2x105 congenic BM cells to analyze long-term donor T-cell engraftment in the recipient mice. We found G-CSF mobilized donor blood did not contribute CD8+ TCM cells that can persist post-transplant (<0.5% at 20 weeks post-transplant). In contrast when donor mice were mobilized with G-CSF together with E-selectin blockade (GMI-1271), we found elevated levels of donor blood derived CD8+ T-cells demonstrating robust long-term CD8+ T-cell persistence / regeneration (5.3 ±3.2% of total recipient T-cells, p=0.04). This dramatic boost in donor CD8+ T-cell reconstitution in mobilized blood following GMI-1271 co-administration is likely to be due to the long-term persistence and in vivo amplification of CD8+ TCM cells from donor mobilized blood. Similar in vivo enhancing effects of GMI-1271 were also observed with other mobilizing agents such as combined CXCR4 and VLA-4 blockade and GM-CSF resulting in a significant 4.9-fold boost in donor CD8+ reconstitution with GMI-1271. Importantly, only 12 hours of E-selectin blockade was sufficient to achieve this boost in CD8+ TCM numbers in the blood following G-CSF.In a previous report we have shown that therapeutic blockade of E-selectin promotes HSC self-renewal in vivo. Thus, it is possible that E-selectin blockade boosts mobilization of CD8+ TCM/SCM with stem-like properties into the blood by loosening factors retaining CD8+ TCM/SCM in the bone marrow and/or blocking the E-selectin-mediated activation and differentiation of this T-cell subset.In summary, our studies identify E-selectin blockade as a novel target to improve harvesting of CD8+ TCM/SCM cells with stem-like properties. Blockade of this target with GMI-1271 significantly improves the in vivo reconstitution potential and regenerative properties of CD8+ T-cells from donor blood allowing a valuable source of desired T-cells for use in adoptive immunotherapy and T-cell engineering. DisclosuresWinkler:GlycoMimetics Inc: Research Funding. Barbier:GlycoMimetics Inc: Research Funding. Davies:GlycoMimetics Inc: Research Funding. Smith:GlycoMimetics, Inc.: Employment. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

ACP-196 Is a Second Generation Inhibitor of Bruton Tyrosine Kinase (BTK) with Enhanced Target Specificity

Background: Signaling through the B-cell receptor (BCR) results in cell survival and proliferation, and this pathway is critical for the progression of many B-cell malignancies, such a chronic lymphocytic leukemia (CLL). BTK, a TEC family kinase, is essential to the BCR signaling cascade. The recent approval of the first-in-class BTK inhibitor for the treatment certain B-cell malignancies, ibrutinib, has validated BTK as a clinical target. ACP-196 is a novel, second generation inhibitor of BTK with an IC50of 3 nM against purified BTK and EC50 of 8 nM in a human whole-blood CD69 B-cell activation assay. In vivo, oral administration of ACP-196 inhibits BCR-induced CD69 expression in murine splenic lymphocytes with an ED50 of 1.3 mg/kg compared with 2.9 mg/kg for ibrutinib. ACP-196 also has improved target specificity over ibrutinib with 323-,94-, 19- and 9-fold selectivity over the other TEC kinase familymembers (ITK, TXK, BMX, and TEC, respectively) and no activity against EGFR (Covey AACR, 2015). TXK and ITK are critical for T-cell receptor activation and development. ITK is also necessary for natural killer (NK)-cell mediated antibody dependent cell cytotoxicity (ADCC), which is important for the efficacy of monoclonal antibody therapy, eg, obinutuzumab. Additionally, therapeutic blockade of EGFR signaling is employed for treatment of epithelial cancers and is associated with adverse events, such as diarrhea and skin rash. Here, we describe results from signaling and functional assays to further elucidate the differential effects of ACP-196 on primary CLL cells compared with T-cells, NK-cells and epithelial cells.Methods: CLL patient samples were evaluated for pathway activation in the presence and absence of ACP-196 using phospho-protein immunoblotting. Helper T-cell (Th) skewing assays were performed using purified naive CD4+ T-cells from C57BL/6, Itk knock out (KO) and Itk/Txk double KO mice in the presence ACP-196 or ibrutinib. CD8+ T-cell activity was assessed by cytolytic activity (CTL) assays using T-cells isolated from C57BL/6 mice. The CTL response was initiated with various effector:T-cell ratios using C57BL/6 T-cells and murine BALB/c lymphoblasts. To evaluate NK-cell ADCC function, standard chromium release assays were done using purified NK-cells isolated from healthy volunteer peripheral blood mononuclear cells and cultured with obinutuzumab-coated (10 µg/mL) CLL cells in the presence and absence of ACP-196. Immunoblotting of H460 lung cancer cells for phospho-EGFR was used to evaluate the effect of ACP-196 on EGFR signaling.Results: ACP-196 treatment of primary human CLL cells showed a dose-dependent (0.01μM-1μM) decrease in p-BTK (Y223), p-ERK (T202/Y204) and p-AKT (T308) and p-IκB-α (S32), without impact on total protein levels. These findings demonstrate ACP-196 inhibits autophosphorylation of BTK as well as down-stream effectors of BCR signaling.Phospho-protein immunoblotting of primary human T-cells and Jurkat cells showed that ACP-196 does not inhibit T-cell receptor signaling molecules downstream of ITK and TXK such as IκBα, NFAT, or JunB. In in vitro helper T cell skewing assays, ACP-196, unlike ibrutinib, showed no effect on differentiation of Th subsets (Th1/Th2/Th17) or regulatory T cells (Tregs). The effect of ibrutinib on Th and Treg differentiation was similar to that observed in cells from Itk KO and Itk/Txk KO mice. In addition, ACP-196 had no effect on CD8+ T-cell viability or cytotoxicity as compared with ibrutinib-treated CD8+ T cells (46% and 36% reduction, respectively).In the in vitro ADCC assay, ACP-196 had no effect on NK cell function (chromium release of 34.8% vs 39.6% for the vehicle control). Lastly, at clinically relevant concentrations ACP-196 did not attenuate autophosphorylation of EGFR in H460 lung epithelial cells demonstrating no effect of ACP-196 on EGFR signaling.Conclusions: ACP-196 selectively inhibits BTK and BCR signaling at pharmacologic concentrations without off-target effects on T-cells, NK-cells and lung epithelial cells. ACP-196 is currently in Phase 3 trials for the treatment of CLL (ClinicalTrials.gov NCT0247568 and NCT02477696).This work was supported by the NIH NCI R01CA197870 and Acerta Pharma. DisclosuresGulrajani:Acerta Pharma: Employment. Covey:Acerta Pharma BV: Employment, Equity Ownership, Patents & Royalties. Kaptein:Acerta Pharma: Employment. Van Lith:Acerta Pharma: Employment. Izumi:Acerta Pharma: Employment. Hamdy:Acerta Pharma BV: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Ulrich:Acerta Pharma: Employment. Lannutti:Acerta Pharma: Employment. Johnson:Acerta Pharma: Research Funding.

Preclinical Analyses Support Clinical Investigation of Combined PD-L1 Blockade and Anti-CD19 CAR T Cell Therapy for the Treatment of NHL

In clinical studies, CD19-targeted CAR T cells have demonstrated encouraging clinical activity in adult and pediatric subjects with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (ALL) and B-cell non-Hodgkin lymphoma (NHL). In general, the anti-tumor activity of CAR T cells in NHL is less than that observed in ALL patients. Immunosuppressive aspects of the NHL tumor microenvironment may inhibit CAR T cell expansion, effector function and tumor killing capacity. One mechanism of suppression may be PD-L1 expression on tumor cells and on cells within the tumor microenvironment. PD-L1 may interact with the inhibitory receptor PD-1 expressed by T cells infiltrating the tumor and limit anti-tumor activity. Reported antitumor activity by nivolumab, an anti-PD-1 inhibitor, in patients with B-cell NHL supports this hypothesis. The data to be presented suggest that the PD-1/PD-L1 pathway could limit CAR T cell efficacy in patients with NHL.Presented data will show that PD-L1 is expressed on tumor cells and in the tumor microenvironment of a subset of NHL patients. In addition, CAR T cells upregulate PD-1 and, to a more limited extent, PD-L1 under certain manufacturing conditions. Co-culture of anti-CD19 CAR T cells with CD19-expressing target cells results in a rapid increase in expression of both PD-1 and PD-L1 on CAR T cells and expression levels are dependent upon the amount of available CAR target antigen. The upregulation of PD-1 and PD-L1 occurs on T cells transduced with CAR constructs containing either a CD28 or 4-1BB costimulatory signaling domain. Thus, the expression pattern of PD-1 on CAR T cells together with the presence of PD-L1 in NHL tumors suggests that this axis could play a role in the modulation of CAR T cell function in NHL.A series of in vitro studies were carried out to characterize the effects of PD-L1 blockade on the activity of anti-CD19 CAR T cells. Tumor B cell lines expressing CD19 and PD-L1 were cultured with anti-CD19 CAR T cells in the presence or absence of anti-PD-L1 blocking antibody. Dose-dependent effects of the blocking antibody on CAR T cell effector function, including cytokine production, proliferation, and cytotoxicity, will be discussed. Collectively, these results provide justification for clinical evaluation of combining therapeutic blockade of PD-L1 with anti-CD19 CAR T cells for the treatment of NHL. A Phase 1b study of anti-CD19 CAR T cells in combination with durvalumab (MEDI4736) for R/R B Cell NHL is planned. DisclosuresLarson:Juno Therapeutics: Employment. Ports:Juno Therapeutics: Employment. Jessup:Juno Therapeutics: Employment. Hay:MedImmune LLC: Employment. Stewart:AstraZeneca: Equity Ownership; MedImmune: Employment. Rebelatto:MedImmune/AstraZeneca: Employment, Equity Ownership. Hollingsworth:MedImmune: Employment. Odegard:Juno Therapeutics: Employment.

Expression of Novel Immune Checkpoint Molecules PVR and PVRL2 Confers a Negative Prognosis to Patients with Acute Myeloid Leukemia and Their Blockade Augments T-Cell Mediated Lysis of AML Cells Alone or in Combination with the BiTE® Antibody Construct AMG 330

Background:T-cell activity is regulated by immune checkpoints to maintain the sensitive balance of co-stimulatory and inhibitory immune signals. Therapeutic blockade of checkpoint molecules on tumor or T cells such as CTLA-4 or PD-L1 has shown clinical success in several tumor types including Hodgkin´s disease. Furthermore, Blinatumomab, a bispecific T-cell engager (BiTE antibody construct) directing cytotoxic T cells to CD19 positive leukemic cells has been approved for treatment of acute lymphoblastic leukemia. The CD33 specific BiTE antibody construct AMG 330 has been developed for the therapy of acute myeloid leukemia (AML) and will be evaluated in phase I studies shortly. In our current study, we investigated the therapeutic utility of blockade of the novel checkpoint proteins PVR (poliovirus receptor) and PVRL2 (poliovirus receptor-related 2) alone and in combination with AMG 330 in AML.Methods and results:Samples from 140 treatment naive patients with newly diagnosed AML (AMLSG 07-04, NCT00151242) were analyzed by RT-qPCR for expression of the immune checkpoint molecules PVR, PVRL2 and Galectin-9 (Gal-9). Expression was correlated with patient demographics (age, karyotype, FLT3 mutation status) and clinical survival data by multivariate cox regression. The majority of patients showed mRNA expression of PVR (94%), PVRL2 (95%) and Gal-9 (92%). In a multivariate stepwise cox regression for overall survival, an unfavorable karyotype, high PVR and high Gal-9 expression were identified as independent prognostic markers (p<0.001, HR: 2.10, CI 1.39-3.15 for the karyotype; p=0.001, HR: 1.64, CI 1.21-2.21 for PVR and p<0.001, HR: 0.67, CI 0.54-0.84 for Gal-9). Due to a high correlation between PVR and PVRL2 (Pearson's rho=0.827, p<0.001), PVRL2 was removed during the stepwise process. Nevertheless, if PVR was excluded from the multivariate cox regression, PVRL2 remained as significant term in the stepwise procedure in addition to the karyotype and Gal-9 (p=0.003, HR: 1.58, CI 1.17-2.13 for PVRL2).In a second, independent patient cohort containing microarray-based gene expression and clinical data of 291 AML patients (Verhaak et.al., Haematologica 2009;94) a high PVR and PVRL2 expression in contrast to expression of CD80, CD86 or PD-L1 was associated with poor overall survival (log-rank test p=0.003 and p=0.032, respectively).In in vitro killing assays the therapeutic effect of PVR and PVRL2 blockade was studied by FACS using 7-AAD staining. AML cell lines MV4-11, Kasumi-1 and Molm-13 were preincubated with blocking antibodies against PVR, PVRL2 or both and co-cultured for 24h with peripheral blood mononuclear cells (PBMCs) of healthy donors in the presence or absence of AMG 330.In the absence of AMG 330, the cell kill of MV4-11 increased from 12.6±4.7% (control) to 33.0±8.8% (PVR), to 40.4±10.4% (PVRL2) and to 56.0±12.0% (both PVR + PVRL2). In the presence of suboptimal concentration of AMG 330 (0.1 ng/ml) MV4-11 cell lysis was 29.4±9.0% (AMG 330 alone), 49.7±12.6% (AMG 330 + PVR), 57.9±11.3% (AMG 330 + PVRL2) and 70.0±9.8% (AMG 330 + PVR + PVRL2; n=4, p<0.05 for all comparisons). Comparable results were found for Kasumi-1 and Molm-13 with blockade of both checkpoint inhibitors being the most effective treatment, although additive effects of antibodies against PVR and PVRL2 could not be verified in all cases (data not shown). To confirm specificity of the approach and to exclude effects caused by antibody dependent cellular cytotoxicity (ADCC), PVR and PVRL2 double knockouts of the cell line MV4-11 were generated by CRISPR/Cas-9. Significantly increased killing was observed in PVR and PVRL2 double knockout cells compared to wild-type cells (40.5±8.1 % vs. 25.9±9.1; n=3, p<0.001). Further experiments using an irrelevant antibody against CD117 or Fcγ receptor blockade by purified IgG antibodies excluded ADCC confirming the functional relevance of PVR/PVRL2 blockade.Conclusion:The expression of immune checkpoint ligands PVR and PVRL2 confers a negative prognosis to AML patients possibly due to immune evasion. We could further show that the killing of AML cells by PBMCs could be augmented by blockade of these novel checkpoint inhibitors. Furthermore, addition of PVR and/or PVRL2 blocking antibodies to AMG 330 could enhance cytotoxicity. Therefore, blockade of PVR and PVRL2 represents a promising target for the treatment of AML. DisclosuresKischel:Amgen Research (Munich) GmbH: Employment. Stienen:Amgen Research (Munich) GmbH: Employment. Friedrich:Amgen Research (Munich) GmbH: Employment. Lutteropp:Amgen Research (Munich) GmbH: Employment. Nagorsen:Amgen: Employment, Equity Ownership, Patents & Royalties: Inventor on blinatumomab-related patent.

Cooperative Activity of BRAF F595L and Mutant HRAS in Histiocytic Sarcoma Provides New Insights into Oncogenic BRAF Signaling

Activating BRAF mutations, in particular V600E/K, drive many cancers, including a substantial proportion of systemic histiocytic disorders, and mutant BRAF-selective inhibitors are promising therapeutics for these diseases. Activating BRAF alleles are considered mutually exclusive with mutations in RAS family members, whereas inactivating BRAF mutations in the D(594)F(595)G(596) motif can coexist with oncogenic RAS and cooperate via transactivation of wildtype RAF proteins and paradoxical MEK/ERK activation. Due to the increasing use of global approaches to tumor genomic profiling, many non-V600 BRAF mutations are being detected whose functional consequences and therapeutic actionability are often unknown. We used several in vitro experimental systems, including Braf-deficient murine embryonic fibroblasts expressing a regulatable HRAS oncogene, to determine the biochemical properties and cellular effects of a largely uncharacterized mutation, F595L, in the DFG motif of the BRAF activation segment that was identified by clinical exome sequencing in a patient with histiocytic sarcoma and multiorgan involvement and also occurs as somatic alteration in colorectal adenoma or carcinoma, non-small cell lung cancer, cholangiocarcinoma, urothelial cancer, melanoma, and neuroblastoma and as germline mutation in cardio-facio-cutaneous syndrome. In addition, we investigated the interaction between BRAF F595L and a concomitant HRAS Q61R allele, which was present in the same tumor cell clone and occurs as acquired alteration in multiple tumor types and as inherited variant in Costello syndrome. Unlike previously described DFG motif mutants, BRAF F595L is a gain-of-function variant with intermediate activity towards MEK that, in sharp contrast to BRAF V600E, requires an intact dimer interface for downstream signaling. Furthermore, BRAF F595L does not act paradoxically, but nevertheless cooperates with mutant HRAS to induce maximal activity of the MEK-ERK signaling pathway. Of immediate clinical relevance, BRAF F595L shows divergent responses to the mutant BRAF-selective inhibitors vemurafenib and dabrafenib, whereas signaling driven by BRAF F595L with and without mutant HRAS is efficiently blocked by the pan-RAF inhibitors sorafenib and AZ628 and the MEK inhibitor trametinib. Consistent with this, sorafenib treatment led to abrogation of aberrant MEK/ERK signaling in the index case with histiocytic sarcoma driven by BRAF F595L and HRAS Q61R. Mutation data from patients and cell lines, representing 18 different tumor entities, show that BRAF F595L as well as other BRAF mutants with intermediate signaling activity coincide with mutant RAS in at least 40% and 23% of cases, respectively. These data define a distinct class of activating BRAF mutations that cooperate with oncogenic RAS in a non-paradoxical fashion, extend the spectrum of patients with systemic histiocytoses and other malignancies who are candidates for therapeutic blockade of the RAF-MEK-ERK pathway, and underscore the value of comprehensive genomic profiling for uncovering the vulnerabilities of individual tumors. DisclosuresOff Label Use: Administration of sorafenib in a patient with histiocytic sarcoma.

Tracking Effector T Cell Dynamics and Immune Inhibitory Receptors in Patients with Acute Myeloid Leukemia (AML)

Background: Targeting specific immune inhibitory receptors (iRs) with monoclonal antibodies has led to paradigm-shifting treatment practices in a variety of solid cancers. These advances were in part driven by tremendous progress in phenotypic and functional characterization of altered iR expression patterns and memory T cell differentiation states such as exhaustion and senescence. Effector T cell dynamics and iR expression patterns in AML patients (pts) at diagnosis and after induction chemotherapy are not well understood and, if deciphered, are poised to be critically important for optimal integration of therapeutic blockade of various iRs in the clinic.Methods: We analyzed T cell dynamics and iR expression in peripheral blood (PB, n=45) and bone marrow (BM, n=38) cells from 49 pts (median age 60, range 21-76) with newly diagnosed AML. After induction, 36 (73%) pts entered complete remission (CR) whereas 13 (27%) were non responders (NR). Samples were collected at diagnosis, upon recovery after induction and following consolidation/salvage chemotherapy. Using multi-parameter flow cytometry, we characterized the differentiation status (CD45RA, CCR7), and the expression of co-stimulatory receptors (CD27, CD28) and iRs. Our gating strategy excluded NK T cells (CD3+ CD56+) from downstream analyses. Co-expression of iRs was analyzed in combination of 3 (2B4, BTLA, TIM3) or 4 (KLRG-1, CD57, PD-1 and CD160) markers. As a control, we used PB (n=41)/BM (n=16) lymphocytes from healthy controls (HC). Percentage of cells expressing specific markers were log transformed and modeled with mixed-effect linear regression models. Differential response outcomes over time were tested with interaction terms. Co-expression of multiple iRs was also analyzed with SPICE software version 5.3.Results: At diagnosis, AML pts showed significantly lower median frequency of CD8+ naïve (CD45RA+ CCR7+) T cells in PB, but higher frequencies of terminal differentiated effector memory (TEMRA; CD45RA+ CCR7-) and phenotypically senescent CD8+ CD27- CD28- CD57+ T cells. Significantly higher percentages of PB CD4+ and CD8+ T cells were found to express PD1 and 2B4 compared to HC. Additionally, the frequency of PB CD8+ T cells co-expressing 2-4 iRs was significantly higher in the PB of AML pts (Fig.1). Surprisingly, in contrast to PB, the only significant finding in the BM of AML pts at diagnosis was increased frequency of CD8+ CD27- CD28- CD57+ T cells (p<0.001) compared to HC.At the time of hematopoietic recovery from chemotherapy, TEMRAs and CD8+ CD27- CD28- CD57+ T cells significantly decreased in PB and BM of AML pts, compared to pretreatment levels. However, if analyzed by response, frequencies of these populations declined only in CR pts (p<0.001), but remained unchanged in NR pts. Analysis of iR co-expression in relation to the response to chemotherapy and time revealed that the frequency of CD8+ T cells co-expressing multiple IRs decreases in CR pts but increases in NR pts. These response-associated changes in iR co-expression were observed only in BM while, in PB, the iR co-expression profile remained unchanged irrespective of response.Finally, we assessed the effect of diverse pre-treatment factors on T cell composition at AML diagnosis. We found that older age was associated with increased frequency of CD8+ T cells expressing the iR marker KLRG-1 and the senescent T cell phenotype CD8+ CD27- CD28- CD57+ (p<0.001) but age did not affect iR co-expression on T cells in either PB or BM. CMV seropositivity was associated with increased CD8+ TEMRAs in PB and CD8+ T cells co-expressing multiple iRs (mostly Tim3 and 2B4) in both compartments. The effect of sex, cytogenetic risk group, or ELN category was insignificant.Conclusion: Our study provides critical insights into T cell differentiation and iR expression at diagnosis and during the course of treatment in pts with AML. We have identified several dominant expression patterns suggesting that iR signatures are consistent with immune recognition of AML and their role in sculpting the effector T cell responses directed against AML cell populations. However, data need to be interpreted in the context of the anatomical compartment and non-inheritable variables such as CMV and age. While ongoing work is focused on the deciphering significance of IRs expression for the interpretation of T cell functionality, our data support the rationale for therapeutic blocking of iRs in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 12135: Toll-like Receptor Signalling Mediates C-kit+ Cell Migration in vivo Following Ischemia-reperfusion Injury via the Myd88 Pathway

Introduction: Toll-like-receptor (TLR) mediated signalling has been shown to regulate tissue damage following cardiac ischemia-reperfusion (I-R) injury. Whether progenitor cell based cardiac regeneration might also depend on TLR-signaling has not been described conclusively. As sufficient progenitor cell migration represents a crucial step of cell-based therapy protocols, we aimed at analyzing the migratory capacity of c-kit+ bone marrow stem cells in vivo following I-R injury as well as the impact of endothelial TLR's and related signalling pathways (MyD88,TRIF)on c-kit+ cell migration. Methods: Murine c-kit+ stem cells were isolated from the bone marrow. In vivo migration of fluorescently labeled c-kit+ following different extents of local I-R injury was visualized by intravital fluorescence microscopy, utilizing the cremaster muscle model in male mice.C-kit+ cell migration was quantified by analyzing early (endothelial rolling) and late (firm endothelial adhesion) c-kit+ cell interaction with the local endothelium. The role of TLR-2 and TLR-4, as well as MyD88 and TRIF on c-kit+ migration following I-R-injury was analyzed by applying specific knock-out models. Results: A sequence of 15 min ischemia followed by 15 min reperfusion induced relevant interactions of c-kit+ cells with the microvascular endothelium(Firm endothelial adhesion:5.6±1.3 cells/mm2 in control vs. 30.2±10.1 cells/mm2 in IR, p&lt;0.05). Prolonged I-R periods did not result in an additional gain of c-kit+ cell-endothelial cell interaction. Knock-out of TLR-2 and TLR-4 diminished I-R induced c-kit+ - endothelial cell interaction.Subsequent knock-out of downstream adaptor proteins resulted in a significantly decreased firm endothelial c-kit+ cell adhesion only for MyD88 but not for TRIF. Conclusions: Artificially applied c-kit+ cells are capable to interact with the target organ endothelium following I-R injury. C-kit+ cell-endothelial cell interaction following I-R injury depends on TLR associated signaling, with a special relevance being demonstrated here for the MyD88 pathway. Our findings suggest, that therapeutic blockade of TLR signaling following I-R injury for anti-inflammatory purposes might interfere with progenitor cell based therapy protocols.

CD47 blockade as another immune checkpoint therapy for cancer.

The role of CD47—often expressed on tumor cells—as a 'don't eat me' signal that inhibits macrophage phagocytosis is well established. But new work reveals a major role for other immune cell types—T cells and dendritic cells—in the anti-tumor effects of therapeutic CD47 blockade.

ID: 159: IFN-dependent macrophage-epithelial crosstalk induces and sustains lung injury in viral pneumonia

Influenza A viruses (IAV) may cause lung injury and acute respiratory distress syndrome (ARDS) characterized by apoptotic injury to the alveolar epithelium and accumulation of excessive fluid (edema) in the alveolar airspaces resulting in loss of barrier function, respiratory failure, and death. We recently demonstrated that inflammatory macrophages recruited from the circulation into the alveoli substantially contribute to influenza-induced lung epithelial injury by IFN-dependent expression of pro-apoptotic factors such as TNF-related apoptosis-inducing ligand (TRAIL). Following severe injury, clearance of excess edema fluid, driven by the alveolar epithelial Na, K-ATPase, is crucial for survival of patients with ARDS. We demonstrate that, apart from causing direct injury to the delicate alveolar barrier, macrophage mediators significantly impair resolution of inflammation in terms of edema clearance from the infected lungs. Using ex vivo and in vivo infection models including transgenic mice and adoptive cell transfer strategies, we identify a paracrine cell communication network between alveolar epithelial cells and alveolar macrophages leading to decreased alveolar epithelial Na, K-ATPase plasma membrane abundance and inhibition of alveolar fluid clearance. Our data furthermore reveal that therapeutic blockade of this cellular cross-talk improves edema resolution which is of clinical significance to patients with IAV-induced lung injury.

Therapeutic efficacy of PD-L1 blockade in a breast cancer model is enhanced by cellular vaccines expressing B7-1 and glycolipid-anchored IL-12

Immunotherapeutic approaches have emerged as promising strategies to treat various cancers, including breast cancer. A single approach, however, is unlikely to effectively combat the complex, immune evasive strategies found within the tumor microenvironment, thus novel, effective combination treatments must be explored. In this study, we investigated the efficacy of a combination therapy consisting of PD-L1 immune checkpoint blockade and whole cell vaccination in a HER-2 positive mouse model of breast cancer. We demonstrate that tumorigenicity is completely abrogated when adjuvanted with immune stimulatory molecules (ISMs) B7-1 and a cell-surface anchored (GPI) form of IL-12 or GM-CSF. Irradiated cellular vaccines expressing the combination of adjuvants B7-1 and GPI-IL-12 completely inhibited tumor formation which was correlative with robust HER-2 specific CTL activity. However, in a therapeutic setting, both cellular vaccination and PD-L1 blockade induced only 10–20% tumor regression when administered alone but resulted in 50% tumor regression as a combination therapy. This protection was significantly hindered following CD4 or CD8 depletion indicating the essential role played by cellular immunity. Collectively, these pre-clinical studies provide a strong rationale for further investigation into the efficacy of combination therapy with tumor cell vaccines adjuvanted with membrane-anchored ISMs along with PD-L1 blockade for the treatment of breast cancer.

IL-22 regulates lymphoid chemokine production and assembly of tertiary lymphoid organs

The series of events leading to tertiary lymphoid organ (TLO) formation in mucosal organs following tissue damage remain unclear. Using a virus-induced model of autoantibody formation in the salivary glands of adult mice, we demonstrate that IL-22 provides a mechanistic link between mucosal infection, B-cell recruitment, and humoral autoimmunity. IL-22 receptor engagement is necessary and sufficient to promote differential expression of chemokine (C-X-C motif) ligand 12 and chemokine (C-X-C motif) ligand 13 in epithelial and fibroblastic stromal cells that, in turn, is pivotal for B-cell recruitment and organization of the TLOs. Accordingly, genetic and therapeutic blockade of IL-22 impairs and reverses TLO formation and autoantibody production. Our work highlights a critical role for IL-22 in TLO-induced pathology and provides a rationale for the use of IL-22-blocking agents in B-cell-mediated autoimmune conditions.

Axial spondyloarthritis.

The term axial spondyloarthritis covers both non-radiographic disease and radiographic disease (also known as ankylosing spondylitis). Some studies have been performed to investigate the prevalence of axial spondyloarthritis, although most are limited to patients with radiographic disease. A strong genetic association has been shown between axial spondyloarthritis and human leukocyte antigen-B27 (HLA-B27), but the pathogenetic role of HLA-B27 has not yet been clarified. Tumour necrosis factor (TNF), IL-17, IL-23 and downstream pathways also seem to be important - based on the good results of therapies directed against these molecules - but their exact role in the inflammatory process is also not yet clear. Elucidating the interaction between osteoproliferation and inflammation will be crucial for the prevention of long-term structural damage of the bone. The development of new criteria for classification, diagnosis and screening of patients with axial spondyloarthritis will enable earlier intervention for this chronic inflammatory disease. MRI has become an important tool for the early detection of axial spondyloarthritis. NSAIDs and TNF blockers are effective therapies, including in the early non-radiographic stage. Therapeutic blockade of IL-17 or IL-23 seems to be a promising new treatment option. Tools for measuring quality of life in axial spondyloarthritis have become relevant to assess the impact that the disease has on patients. These diagnostic and therapeutic advances will continue to change the management of axial spondyloarthritis, and new insights into the disease pathogenesis will hopefully accelerate this process. For an illustrated summary of this Primer, visit: http://go.nature.com/51b1af.

Polymicrobial Sepsis Increases Susceptibility to Chronic Viral Infection and Exacerbates CD8+ T Cell Exhaustion.

Patients who survive sepsis display suppressed immune functions, often manifested as an increased susceptibility to secondary infections. Recently, using a cecal-ligation and puncture (CLP) model of sepsis, we showed that sepsis induces substantial and long-lasting changes in the available naive CD8(+) T cell repertoire affecting the capacity of the host to respond to newly encountered acute infections. However, the extent to which sepsis changes the host susceptibility to chronic infection and affects CD8(+) T cell responses is currently unknown. In this study, we demonstrate that inbred and outbred mice recovering from a septic event are more susceptible to lymphocytic choriomeningitis virus (LCMV) clone-13 infection exhibited by mortality and viral burden. Primary virus-specific CD8(+) T cells in LCMV clone-13-infected septic mice displayed exacerbated CD8(+) T cell exhaustion illustrated by increased inhibitory molecule expression (e.g., programmed cell death 1, lymphocyte-activation gene 3, and 2B4) and diminished Ag-driven cytokine production (e.g., IFN-γ, TNF-α) compared with similarly infected sham-treated mice. Importantly, therapeutic inhibitory molecule dual blockade (anti-PD-L1 and anti-lymphocyte-activation gene 3) increased the number of circulating LCMV-specific CD8(+) T cells, and improved CD8(+) T cell function and pathogen control in chronically infected septic mice. Together, these results illustrate that polymicrobial sepsis compromises the overall health of the host leading to increased vulnerability to chronic infection and exacerbated CD8(+) T cell exhaustion. Collectively, our findings suggest that septic survivors may be more susceptible and at greater risk for developing exhaustible CD8(+) T cells upon encountering a subsequent chronic infection.

From T cell “exhaustion” to anti-cancer immunity

The immune system has the potential to protect from malignant diseases for extended periods of time. Unfortunately, spontaneous immune responses are often inefficient. Significant effort is required to develop reliable, broadly applicable immunotherapies for cancer patients. A major innovation was transplantation with hematopoietic stem cells from genetically distinct donors for patients with hematologic malignancies. In this setting, donor T cells induce long-term remission by keeping cancer cells in check through powerful allogeneic graft-versus-leukemia effects. More recently, a long awaited breakthrough for patients with solid tissue cancers was achieved, by means of therapeutic blockade of T cell inhibitory receptors. In untreated cancer patients, T cells are dysfunctional and remain in a state of T cell “exhaustion”. Nonetheless, they often retain a high potential for successful defense against cancer, indicating that many T cells are not entirely and irreversibly exhausted but can be mobilized to become highly functional. Novel antibody therapies that block inhibitory receptors can lead to strong activation of anti-tumor T cells, mediating clinically significant anti-cancer immunity for many years. Here we review these new treatments and the current knowledge on tumor antigen-specific T cells.

Inhibitory Receptors Beyond T Cell Exhaustion.

Inhibitory receptors (iRs) are frequently associated with “T cell exhaustion”. However, the expression of iRs is also dependent on T cell differentiation and activation. Therapeutic blockade of various iRs, also referred to as “checkpoint blockade”, is showing ­unprecedented results in the treatment of cancer patients. Consequently, the clinical potential in this field is broad, calling for increased research efforts and rapid refinements in the understanding of iR function. In this review, we provide an overview on the significance of iR expression for the interpretation of T cell functionality. We summarize how iRs have been strongly associated with “T cell exhaustion” and illustrate the parallel evidence on the importance of T cell differentiation and activation for the expression of iRs. The differentiation subsets of CD8 T cells (naïve, effector, and memory cells) show broad and inherent differences in iR expression, while activation leads to strong upregulation of iRs. Therefore, changes in iR expression during an immune response are often concomitant with T cell differentiation and activation. Sustained expression of iRs in chronic infection and in the tumor microenvironment likely reflects a specialized T cell differentiation. In these situations of prolonged antigen exposure and chronic inflammation, T cells are “downtuned” in order to limit tissue damage. Furthermore, we review the novel “checkpoint blockade” treatments and the potential of iRs as biomarkers. Finally, we provide recommendations for the immune monitoring of patients to interpret iR expression data combined with parameters of activation and differentiation of T cells.

MDM2 beyond cancer: podoptosis, development, inflammation, and tissue regeneration.

Murine double minute (MDM)-2 is an intracellular molecule with diverse biological functions. It was first described to limit p53-mediated cell cycle arrest and apoptosis, hence, gain of function mutations are associated with malignancies. This generated a rationale for MDM2 being a potential therapeutic target in cancer therapy. Meanwhile, several additional functions and pathogenic roles of MDM2 have been identified that either enforce therapeutic MDM2 blockade or raise caution about potential side effects. MDM2 is also required for organ development and tissue homeostasis because unopposed p53 activation leads to p53-overactivation-dependent cell death, referred to as podoptosis. Podoptosis is caspase-independent and, therefore, different from apoptosis. The mitogenic role of MDM2 is also needed for wound healing upon tissue injury, while MDM2 inhibition impairs re-epithelialization upon epithelial damage. In addition, MDM2 has p53-independent transcription factor-like effects in nuclear factor-kappa beta (NFκB) activation. Therefore, MDM2 promotes tissue inflammation and MDM2 inhibition has potent anti-inflammatory effects in tissue injury. Here we review the biology of MDM2 in the context of tissue development, homeostasis, and injury and discuss how the divergent roles of MDM2 could be used for certain therapeutic purposes. MDM2 blockade had mostly anti-inflammatory and anti-mitotic effects that can be of additive therapeutic efficacy in inflammatory and hyperproliferative disorders such as certain cancers or lymphoproliferative autoimmunity, such as systemic lupus erythematosus or crescentic glomerulonephritis.

Predictive factors for immunotherapy in melanoma.

Immunotherapy has emerged as an exciting strategy for cancer treatment. Therapeutic blockade of immune checkpoint regulators favors the ability of T cell responses to increase anti-tumor immunity. The cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death-1 (PD-1) are two T cell-inhibitory receptors with independent mechanisms of action. Immune checkpoint inhibitors targeting either CTLA-4, PD-1 or its ligand PD-L1 are currently yielding promising results in terms of efficacy in several clinical studies with melanoma patients and are being developed and tested as immunotherapy agents for multiple cancer types. To date, no reliable predictors of activity and efficacy of immunotherapy have yet been identified or validated. Even so, determining which patients derive clinical benefit from immune checkpoint agents remains an important clinical question and efforts to identify predictive markers of response are ongoing. This article reviews the current potential predictive factors for CTLA-4 and PD-1/PD-L1 immune checkpoints inhibitors in melanoma.

Tumor Necrosis Factor-like Cytokine TL1A and Its Receptors DR3 and DcR3: Important New Factors in Mucosal Homeostasis and Inflammation.

Tumor necrosis factor (TNF)-like cytokine 1A (TL1A) is a member of the TNF superfamily of proteins (TNFSF15), which signals through association with death domain receptor 3 (DR3). Decoy receptor 3 (DcR3) competes with DR3 for TL1A binding and inhibits functional signaling. These proteins are significantly upregulated in inflamed intestinal tissues, and their pathogenetic importance for inflammatory bowel disease (IBD) is suggested by accumulating evidence. TL1A/DR3 induce costimulatory signals to activated lymphocytes, including the gut-specific populations of CD4+CD161+ and CD4+CCR9+ cells, affecting all major effector pathways and inducing the mucosal upregulation of Th1, Th2, and Th17 factors. They may also participate in mucosal homeostasis and defense against pathogens through their effects on the development and function of the recently described innate lymphoid cells. T-regulatory lymphocytes highly express DR3, and they respond to TL1A stimulation also. Mechanistic studies by transgenic expression of TL1A, deletion of TL1A or DR3, and therapeutic blockade by anti-TL1A antibodies all support the critical involvement of the corresponding pathways in the pathogenesis of chronic mucosal inflammation. Wide genome association studies have identified IBD-specific polymorphisms in TNFSF15 gene, which have functional implications and serve as poor prognostic factors. Recently, TL1A blockade in mice was presented as a unique pharmacological treatment for the reversal of established intestinal fibrosis. Finally, TL1A/DR3 signaling seems to critically participate in extraintestinal inflammatory conditions that are frequently associated with IBD as part of the gut-joint-skin-eye axis. These converging lines of evidence make TL1A/DR3 a suitable model for personalized approaches to IBD therapy.

The checkpoint inhibitor TIGIT limits antitumor and antiviral CD8+ T cell responses

Success in eliciting tumor rejection by therapeutic blockade of T cell checkpoint inhibitors, such as PD-1/PD-L1 and CTLA-4, has spurred re-evaluation of additional receptors that regulate antitumor responses. Here we summarize our recent studies characterizing the role of co-inhibitory receptor TIGIT in antitumor and other chronic CD8+ T cell responses.