ALBERT

Description: Lifelong interactions between host and the ubiquitous and persistent cytomegalovirus (CMV) have been proposed to contribute to the age-related decline in immunity. Prior work from us and others found some support for that idea, yet evidence that this led to increased vulnerability to other infections was not obtained. Moreover, evidence has accumulated that CMV infection can be beneficial to immune defense in young/adult mice and humans, dominantly via enhanced innate immunity. Here, we describe an unexpected impact of murine CMV (MCMV) upon the T cell response of old mice to Listeria monocytogenes expressing the model antigen, OVA (Lm-OVA). Single-cell sequencing of the OVA-specific CD8 T cell receptor β (TCRβ) repertoire of old mice demonstrated that old MCMV-infected mice recruited many diverse clonotypes that afforded broad and often more efficient recognition of antigenic peptide variants. This stood in contrast to old control mice, which exhibited strong narrowing and homogenization of the elicited repertoire. High-throughput sequencing of the total naïve CD8 TCRβ repertoire showed that many of these diverse OVA-specific clonotypes were present in the naïve CD8 repertoire of mice in all groups (adult, old control, and old MCMV+) yet were only recruited into the Lm-OVA response in MCMV+ old mice. These results have profound implications for our understanding of T cell immunity over a life span and suggest that our coevolution with CMV may include surprising, potentially positive impacts on adaptive heterologous immunity in late life.

Description: The reconstitution of specific immune niches after bone marrow transplant is mediated by donor immune cells repopulating specific microenvironments. Here, we demonstrate that type 2 innate lymphoid cells (ILC2) in the GI tract are rapidly eliminated by irradiation and/or chemotherapy and are not repopulated in the first month post-transplant by donor bone marrow cells. In contrast, ILC2 cells in the lung are not sensitive to conditioning therapy and quickly recover quantitatively post-transplant. Donor ILC2 cell infusion with donor T cells and TCD bone marrow has been shown by us to markedly diminish lethality and clinical score associated with acute GvHD. However, the mechanism for that finding was unclear. Here, we demonstrate that donor ILC2 infusion enhances the persistence of GI tract, but not splenic or pulmonary MDSCs (Fig 1a). This persistence required the expression of IL-13 by the ILC2 cells (Fig 1b). Absence of IL-13 production by ILC2 cells greatly diminished the activity of those cells to prevent GvHD (Fig 1c). ILC2 cell infusion decreased the number of T cells generating IFN-γ and IL-17A in the GI tract. However, we found no difference in the number of IFN-γ or IL-17A generating T cells in the GI tract after the infusion of IL-13-/- ILC2 cells. To evaluate the function of ILC2 cells in MDSC biology we performed in vitro quantitation of MDSCs in co-culture with ILC2 cells. MDSCs cultured with WT ILC2s, survived significantly better than MDSCs cultured alone, MDSCs cultured with IL-13 and MDSC cultured in the presence of IL-7 and IL-33. In addition, MDSCs co-cultured with WT ILC2s survived significantly better than those cultured with IL-13-/- ILC2s. Surprisingly, enhancement of MDSC survival requires cell-to-cell contact with ILC2s, as seen when either WT or IL-13-/- ILC2s and MDSCs are cultured in plates across semi-permeable membranes. ILC2s have been shown to have a role in intestinal barrier repair via amphiregulin (Areg). When compared to untreated recipients, ILC2 treated recipients showed significantly enhance barrier function which can be observed as early as 12 days post-transplant and as late as day 20. Interestingly, recipients of Areg deficient ILC2s show improved survival compared to control treatment but diminished survival compared to recipients of WT ILC2 cells. Several cell types have been shown to suppress aGvHD, including MDSCs and regulatory T cells (Treg). To evaluate the function of ILC2 cells as treatment, we compared them to MDSC and Treg infusion. When used to treat aGvHD on day 7 post-transplant, IL-13 derived MDSCs have no effect on recipient survival. In contrast ILC2 infusion results in a significant prolongation of survival compared to MDSC infusion with approximately 35% of recipient mice surviving past day 50. When compared to Treg infusion (1:1 ratio with Tcons) both ILC2 and Treg infusions block aGvHD. However, unlike ILC2 infusion in which no mice develop P815 tumors post-transplant, 80% of mice given Tregs developed tumors. At a lower Treg dose (1:4) the GvL response was restored but without the beneficial effect on GvHD. In summary, ILC2s represent a novel cell population that are required for recovery of GI tract homeostasis following allo-SCT. ILC2s play a pivotal role in the development of donor MDSCs and intestinal barrier repair. Additional investigation should be focused on the recovery of specific immunosuppressive cell populations from donor bone marrow cells after allo-SCT. IL-13 dependent ILC2 induced expansion of donor MDSCs. (a) Frequencies CD11b+/GR-1+/Ly-6C+/Ly-6G+ MDSCs in the LP of the colon of allo-SCT recipients 12 days post-transplant with or without WT ILC2s. These represent 2 independent experiments, bar graphs are average ± SEM, student's t test using GraphPad Prism, * p

Description: Key Points Extended donor Treg survival is required for protection from GVHD; donor Treg longevity depends on Treg CCR8 expression. Donor CD11c+ APCs promote Treg longevity in vivo; host CD11c+ APCs do not appear to contribute to donor Treg reconstitution.

Description: Minor histocompatibility antigens (mHA) mediate immune responses that can cause both graft vs. host disease (GvHD) and the graft vs. leukemia (GvL) effect observed in allogeneic stem cell transplantation (SCT). These responses result from donor T-cells being exposed to non-self peptides that are presented by HLA molecules on recipient cells. In contrast, cancer testis antigens (CTA) are peptide antigens derived from proteins, normally expressed only in male germ cells, but also aberrantly expressed in some tumors. These antigens can induce T-cell responses in an autologous setting without using T-cells from an allogeneic donor. Because, mHA that are expressed by hematopoietic tissue or leukemia could be therapeutic targets, which would enhance GVL without increasing the risk of GVHD we performed a genomics based mHA screen to identify high-value mHA. However, one of our predicted antigens, UNC-GRK4-1, was only expressed in testis and human AML, and has properties more consistent with a CTA. We used the Illumina NS-12 microarray platform to genotype 99 HLA-A0201 expressing myeloid leukemia patients and their HLA-matched donors at 13,917 non-synonymous coding single nucleotide polymorphisms (cSNPs). For both alleles in each of the 13,917 cSNPs we identified genetically predicted donor-recipient mHA responses, which were defined as the donor being homozygous for 1 allele and the recipient being either heterozygous or homozygous for the alternate allele. Using Fisher's exact test for each allele, we tested for the association between genetically predicted mHA responses and the clinical outcomes of remission vs. relapse over all 99 patients. Using the BioGPS database, we determined the tissue expression for the 261 alleles associated with remission to a p-value of

Description: Key Points NOTCH2 activation confers a marked increase in BCR responsiveness by cGVHD patient B cells that associates with increased BLNK. ATRA increases the IRF4-to-IRF8 ratio and blocks aberrant NOTCH2-BCR activation without affecting cGVHD patient B-cell viability/function.

Description: Background: Despite recent advances in the therapeutic armamentarium for AML, outcomes remain dismal for patients (pts) with relapsed/refractory (R/R) AML. Response rates with high dose cytarabine (HiDAC) salvage chemotherapy are approximately 20%. Multiple immune aberrations in AML lead to immune suppression, exhaustion, and senescence. Programmed Death-1 (PD-1), a co-inhibitory receptor (IR) on immune cells, suppresses immune activation and is exploited by leukemic cells to evade immune surveillance. PD-1 and other IRs are up-regulated during disease progression. We hypothesized that pembrolizumab, a monoclonal antibody targeting PD-1, after HiDAC would stimulate a T-cell mediated anti-leukemic immune response. Methods: Eligibility for this study included R/R AML 18-70 years, ECOG PS 0-1 and adequate organ function. Treatment consisted of HiDAC (60 years: 1.5 gm/m2 IV Q12hours days 1-5) followed by pembrolizumab 200 mg IV on day 14. The primary objective of this study was to estimate the overall complete remission (CR + CRi) rate. Secondary objectives included assessment of safety, durability of CR, overall survival (OS) and biomarker correlates of response. Overall responders were eligible to receive maintenance phase pembrolizumab 200 mg IV Q3weeks for up to 2 years until progression. Allogeneic stem cell transplant (alloSCT) was permissible before or after maintenance phase. Results: Thirty-seven pts were enrolled and evaluable (Table 1). Sixteen (43%) pts had refractory disease and 16 (43%) pts had relapsed AML with CR1 duration 3: n=1), AST elevation (32%; Grade 〉3: n=1), fatigue (27%), alkaline phosphatase elevation (24%), and maculopapular rash (19%; Grade 〉3: n=2). Grade 〉3 immune-related adverse events (iRAE) were rare (maculopapular rash: n=2, AST/ALT increase: n=2, right upper quadrant pain with lymphocytic infiltrate in liver: n=1) and self-limiting. Five (14%) pts required steroid administration for grade 2 hyperbilirubinemia (n=1), grade 3 ALT elevation (n=1), grade 3 AST elevation with liver biopsy revealing no evidence of iRAE (n=1), grade 3 bilirubin subsequently deemed to be a delayed hemolytic transfusion reaction (n=1), and grade 3 systolic dysfunction without evidence of myocarditis by endomyocardial biopsy or cardiac MRI (n=1). Sixty-day mortality was 3% (1/37) due to progressive AML. Median time to full neutrophil (〉1x109/L) and platelet (〉100x109/L) recovery was 32 and 31 days, respectively. The overall response (ORR: CR+CRi+PR+MLFS) and composite CR (CR+CRi) rates were 46% [29%,63%] and 38% [22%,55%], respectively, meeting the primary endpoint of the study. Notably, 13/28 (46%) pts receiving HiDAC + pembrolizumab as their first salvage regimen achieved CR/CRi. Two pts refractory to HiDAC (administered within past 6 months) achieved CR including one pt who was refractory to HiDAC salvage 1 month prior to enrollment and ultimately achieved CR without evidence of minimal residual disease. Nine (24%) pts received an alloSCT. There were no instances of Grade 〉3 acute GVHD or veno-occlusive disease post-alloSCT. Nine (24%) pts received maintenance phase pembrolizumab (median # of cycles = 3; range: 1-12) for CR (n=8) or PR (n=1). Seven out of 9 pts relapsed/progressed after maintenance phase. Median follow-up among survivors, and median OS, event-free survival and disease-free survival was 7.8 months, 8.9 months [6.0,13.1], 6.9 months [4.2,11.5], and 5.7 months [1.9,7.3], respectively. Conclusions: Pembrolizumab can be safely administered after HiDAC salvage in R/R AML. Severe iRAE's were uncommon despite administration after cytotoxic chemotherapy. The addition of pembrolizumab to HiDAC led to an encouraging overall CR rate meeting the primary endpoint of the study. Immunogenomic biomarker analyses consisting of B cell receptor amplicon sequencing, RNA-seq of blasts and CD8+ T cells, CD8+ T cell receptor repertoire, whole exome sequencing and flow cytometry analyses are ongoing to determine predictors of response. These results warrant further investigation of IR blockade and other immunomodulatory therapeutic strategies after intensive cytotoxic chemotherapy in AML. Disclosures Zeidner: Takeda: Research Funding; Merck: Research Funding; AsystBio Laboratories: Consultancy; Pfizer: Honoraria; Tolero: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Agios: Honoraria; AbbVie: Honoraria. Vincent:Pharmacyclics: Research Funding; Merck: Research Funding. Foster:Bellicum Pharmaceuticals: Research Funding; Macrogenics: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Consultancy. Coombs:Dedham Group: Consultancy; Covance: Consultancy; Cowen & Co.: Consultancy; Octopharma: Honoraria; H3 Biomedicine: Honoraria; Loxo: Honoraria; Pharmacyclics: Honoraria; Medscape: Honoraria. Webster:Pfizer: Consultancy; Amgen: Consultancy; Genentech: Research Funding. DeZern:Astex Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy. Smith:Jazz: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Agios: Consultancy. Levis:Amgen: Consultancy, Honoraria; Astellas: Consultancy, Research Funding; FUJIFILM: Consultancy, Research Funding; Menarini: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Daiichi Sankyo Inc: Consultancy, Honoraria; Agios: Consultancy, Honoraria. Luznik:Merck: Research Funding, Speakers Bureau; Genentech: Research Funding; AbbVie: Consultancy; WindMiL Therapeutics: Patents & Royalties: Patent holder. Serody:Merck: Research Funding; GlaxoSmithKline: Research Funding. Gojo:Amphivena: Research Funding; Amgen Inc: Consultancy, Honoraria, Research Funding; Juno: Research Funding; Merck: Research Funding; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. OffLabel Disclosure: Pembrolizumab is investigational for AML.

Description: Background: CD8+ T-cells in AML pts co-express multiple inhibitory receptors (IRs), including PD1, and IR expression increases with disease progression (Knaus et al, JCI Insight 2018). AZA upregulates pathways related to immunity and immune evasion in tumor cells, including PD-L1, (Wrangle et al, Oncotarget 2013) providing rationale for exploring AZA/Pembro combination in AML. Aims: To assess safety and response to AZA/Pembro after minimum 2 cycles of therapy in relapsed/refractory (R/R) (Cohort 1) and newly diagnosed (dx) older AML (Cohort 2). Methods: Cohort 1: Pts must have failed prior AML therapy. The first 6 pts (run in phase) received AZA 75 mg/m2 Days (D) 1-7 with Pembro 200 mg beginning on D8 and every (q)3 weeks (wks) thereafter. AZA cycles were repeated q4wks. No pts experienced dose limiting toxicity after minimum 3 cycles observation. After safety was established with the dosing schedule, patients with prior allogeneic stem cell transplant (alloSCT) were included and Cohort 2 started enrollment. Cohort 2: Pts ≥65 years (yrs) with newly dx AML and not candidates, or unwilling to receive, intensive chemotherapy. Other eligibility criteria (Cohort 1 and 2): ECOG PS 0-2 (changed to PS 0-1), adequate organ function, and no autoimmune processes requiring systemic immunosuppression. Results: Efficacy: Cohort 1 : 37 R/R pts have been enrolled. Baseline characteristics are summarized in Table 1A. 29 (78%) pts completed at least 2 cycles and are evaluable for response: 4 achieved complete remission (CR)/CR with incomplete hematologic recovery (CRi) (2/2) (14%) (Table 1B), 1 partial remission (PR) (4%), 4 hematologic improvement (HI) (14%), and 7 stable disease (SD) for at least 6 cycles (24%). The median # of cycles to response was 4 (range, 2-6). The 4- and 8-week mortality were 8% [all with rapidly progressive disease (PD): 2 received AZA for 3 and 5 days only] and 13%, respectively. With a median follow-up of 14.9 months (mos), the median overall survival (OS) for the whole cohort, responders + SD, and CR/CRi/PR is 10.8 mos (40% 1-yr), 13.9 mos (51% 1-yr), and 17.2 mos (75% 1-yr). The median event-free survival (EFS) is 6 mos, for all responders + SD 8.7 mos versus 2.6 mos for others (P

Description: Abstract 2502 Leukemia stem cells (LSCs) in acute myeloid leukemia (AML) are resistant to conventional chemotherapy, able to sustain leukemia through treatment, and may regenerate disease after treatment completion. Understanding the biological differences between LSCs and non-LSC leukemic blasts will be important for developing improved and targeted AML therapies. We report here the results of an RNA-Seq analysis of differential gene and splice variant expression in LSC-enriched vs. non-enriched leukemia fractions in 8 AML patients. Using fluorescence activated cell sorting (FACS) we divided 8 adult AML patient blast samples into LSC-enriched (“LSC” = Lin-, CD34+, CD38-) and “non-LSC” fractions. We extracted total RNA from each sample, purified mRNA, and performed massively parallel RNA sequencing using the Illumina HiSeq platform. Hierarchical clustering based on RPKM gene expression values showed greater similarity between LSC and non-LSC fractions within each patient's leukemia than similarity of LSC and non-LSC fractions among patients. Three hundred seventy-one genes were differentially expressed between the LSC and non-LSC fractions, with the most prominent public differences being increased expression of myeloid differentiation markers (e.g. LYZ, MPO, HBA/B) in the non-LSC samples. The LSC fractions displayed increased expression of CD34, FLT3, N-ras, K-ras, AML1, cyclin D1, CD61, and M-SCF. Pathway analysis revealed statistically significant differences in gene expression in the KEGG-defined Acute Myeloid Leukemia, Cell Cycle, and Hematopoietic Cell Lineage pathways between the LSC and non-LSC fractions. Using the MapSplice software package we identified multiple novel splice variants present in ≥ 6 of the 8 AML patient samples. These splices were found in genes known to be associated with leukemogenesis including MYC, MTOR, and FLT3. In addition to the identification of novel splices in AML associated genes, we used the FDM software package to measure differential splice utilization between the LSC vs. non-LSC fractions. Using FDM, we measured statistically significant splice utilization between LSCs and non-LSCs in 87 genes including ZNF34, NUMA1, NBEAL2, BAT2D1, FGR, MAP3K6, RFX5, NFATC2IP, and LRMP. In addition to these globally differentially spliced genes, several splice variants were highly differentially expressed between LSC and non-LSC fractions in individual patients (e.g. MAGED4, CYBB, IL17RA). Somewhat unexpectedly, no genes were both significantly differentially expressed and differentially spliced in the LSC vs. non-LSC fractions. To our knowledge, the data reported here represent the first RNA-Seq analysis of LSC-enriched vs. non-enriched AML fractions. Our results confirm prior gene-expression microarray studies that showed differential gene expression in LSC-enriched vs. non-enriched fractions. We extend this work by reporting the discovery of novel splice variants in AML associated genes and observe, for the first time differential splice utilization between LSC and non-LSC fractions. These results should prove valuable for both understanding LSC biology and guiding development of LSC-specific therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.

Description: The PD-1/PD-L1 pathway plays an important role in regulation of alloimmune responses and in induction and maintenance of peripheral tolerance. Because GVHD is driven by donor T cells and PD-L1 expression can be markedly elevated on T cells during activation, we investigated the functional significance of PD-L1 expressed by donor T cells in regulating murine models of acute GVHD. PD-L1 expression was up-regulated on donor CD4 and CD8 T cells during GVHD. We considered the possibility that PD-L1 expression on activated donor T cells might inhibit GVHD by down regulating donor anti-host T cell responses, consistent with PD-L1 co-inhibitory activity when expressed on host parenchymal cells during GVHD. Surprisingly, T cell mediated GVHD lethality was markedly reduced in recipients of PD-L1-/- compared to WT donor T cells in both B6 to BALB/c model of GVHD(P