Baseline Immune Profile Research Articles

Abstract 3718: ID8-Luc syngeneic ovarian cancer model for preclinical evaluation of immunomodulatory molecules

Abstract Ovarian cancer (OC) is a gynecological malignancy with a high mortality rate of over 14,000 deaths annually in the US. Although ~ 80% of OC patients initially go into remission after 1st line treatment (surgery and chemotherapy), more than 60% relapse within 16-18 months. Low neoantigen burden and the immunologically “cold” nature of OC makes it a challenging malignancy. Therefore, novel methods such as immunotherapy and/or combined therapies are essential to improve clinical outcome of patients. This work evaluated the ID8-Luc murine ovarian carcinoma model to determine sensitivity to immunomodulatory agents. C57BL/6 albino mice implanted intraperitoneally with ID8-Luc cells were evaluated for growth and sensitivity to checkpoint inhibitors anti-PD-1, anti-PD-L1, anti-CTLA-4 and the chemotherapy agent paclitaxel. Disease progression was monitored weekly by in vivo bioluminescence imaging (BLI). Baseline immune profile of the model was determined by collecting ascites from untreated mice and analyzed with panels for myeloid and lymphoid cell populations by flow cytometry. ID8-Luc cells successfully established tumors with 7-8 days doubling time, as determined by BLI, and a median overall survival time of ~ 40-50 days. Formation of ascites resulted in weight gain and extended abdomen at advanced disease state. Necropsy showed solid tumor nodules within the peritoneal cavity including involvement of pancreas, liver, spleen and abdominal wall. Baseline immune profiling of ascites indicated presence of B cells (23%), granulocytic myeloid derived suppressor cells (G-MDSCs; 15%), TAMs (25%) and a low percentage of T cells (3-4%). This was suggestive of a possibly “cold” tumor model. Paclitaxel is a front-line chemotherapy option in OC. We find that the ID8-Luc model has 60% tumor free survivors (TFS) following treatment. To determine how this model responds to immune checkpoint inhibition we tested response to anti-PD-1, anti-PD-L1 and anti-CTLA-4 antibodies. Treatment was initiated at either 7 (early) or 14 (late) days post tumor cell implant and we found that the overall response varied depending on the timing of treatment initiation. Early treatment with either anti-PD-1 or anti-PD-L1 resulted in complete regression of tumors (100% TFS) while the model was refractory to anti-CTLA-4 treatment. Late treatment initiation of anti-PD-1 or anti-PD-L1 resulted in an “all or nothing” response (40% complete response and 60% non-response). In addition, reducing the antibody dose from 10mg/kg to 5mg/kg did not make a material impact on the anti-tumor activity of these agents in the late treatment regimen. New models to further the preclinical testing of agents for OC is vital. This work characterizes the baseline immune profile of the murine ID8-Luc model along with response to checkpoint inhibitors to enable the rational design of combination strategies in the preclinical setting. Citation Format: Sumithra Urs, Sheri Barnes, Stacey Roys, Maryland R. Franklin. ID8-Luc syngeneic ovarian cancer model for preclinical evaluation of immunomodulatory molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3718.

Mass Cytometry Identifies Immunomic Shifts in the Bone Marrow Microenvironment of Multiple Myeloma and Light Chain Amyloidosis after Standard of Care First Line Therapies

INTRODUCTION: Traditional cytometry methods have been unable to capture the immense heterogeneity of the tumor immune microenvironment in various malignancies including multiple myeloma (MM). Cytometry by time of flight (CyTOF) can, to some extent, overcome this limitation. However, the computational challenges that come with analyzing these complex datasets in a reproducible manner remain. In this study, using a large cohort of patients, we compare the bone marrow immunomes from patients with MGUS, multiple myeloma (MM), smoldering MM (SMM) and light chain amyloidosis (AL) at diagnosis, after induction therapy with lenalidomide and dexamethasone and after autologous transplant (ASCT). METHODS: We studied a total of 118 cryopreserved samples as follows: 14 healthy donors, 43 AL (27 newly diagnosed-ND, of which 13 with <10% bone marrow plasma cells, 16 matched samples post ASCT), 12 with ND MGUS, 11 with SMM (of which 6 were ND), 14 with ND MM, 13 paired MM samples post induction therapy and 11 paired MM samples post ASCT. Our CyTOF surface staining panel included the following 33 markers: CD45, HLA-DR, CD19, CD3, CD4, CD8, CD14, CCR6, CD11a, Cd123, CCR5, CD7, ICOS, CD25, CD57, CD45RA, CD163, PD-1, PDL-1, CXCR3, CCR4, CCR7, CD28, CTLA4, CD11c, CD56, CD45RO, CD44, CD27, CD138, CD38, CD-127 and CD16. Data processing and analysis was performed using Cytobank. Live cells were identified based on Pt195 and Ir193 staining. Myeloma cells and CD45- cells were excluded and only CD45+ cells were used for subsequent analyses. Single-cell data were downsampled using VisNE, a permutation of t-Distributed Stochastic Neighbor Embedding (tSNE) and clustered using CITRUS (using 10,000 events per sample with a minimum cluster size of 1%). A Significance analysis of microarrays (SAM) analysis was performed to ascertain differences between groups. Significance was inferred for a false discovery rate <1% . All CITRUS analyses were repeated at least 3 times and only clusters found to differ consistently across runs were considered. RESULTS: The proportions of immune subsets identified to vary by CITRUS before and after induction therapy and ASCT for MM and AL are shown in the table. No differences were identified between MGUS, SMM and NDMM. The proportion of a subset (369850) of CD14+/C16- monocytes, a group of cells shown to correlate positively with survival and response to therapy in solid malignancies, increased after induction therapy in MM. Naïve B cells increased dramatically post ASCT in both AL (429918) and MM (369948), consistent with expected immune reconstitution patterns, although a CCR6+ B Cell subset (429940) shown to mediate effective antibody responses, decreased (in grey). A subset (369980) of functionally exhausted (PD1/CTLA4+) central memory (CM) CD4 T cells decreased after induction therapy in MM but recovered early post ASCT whereas a CM CD4+ subset (369986) lacking major activation markers (CD28, CD25) decreased gradually with therapy and post ASCT. A subset (369953) of naïve CD8 T cells shown to be actively recruited in tumor sites (CXCR3+) decreased after ASCT. CD57+ senescent effector memory (EM) CD8 T cells (369962) decreased with induction therapy but recovered post ASCT. EM CD8 T cells associated with long term immune memory (CCR5+, CD127+, cluster 369959), also decreased after ASCT. LIMITATIONS: Include a) No barcoding b) batch effects were difficult to avoid when processing large number of samples c) use of cryopreserved samples d) need for downsampling to cope with the computational burden. The latter could have been one of the reasons we did not identify any differences between MGUS, SMM and MM. At the time of the meeting we will present confirmatory analyses using conceptually different methods of clustering and of performing across group comparisons as well as analyses that do not include downsampling. CONCLUSIONS: Mass cytometry can provide a more granular view of the bone marrow immunome in plasma cell dyscrasias. Novel agent induction therapy can create an immunologically favorable anti-tumor microenvironment although, in some cases, these favorable immunomic shifts are temporarily reversed by ASCT. Confirmatory analyses, baseline immune profiles, comparisons with healthy donors and correlation with other clinically relevant patient characteristics and outcomes will be reported at the meeting. [Display omitted] DisclosuresDispenzieri:Celgene, Takeda, Prothena, Jannsen, Pfizer, Alnylam, GSK: Research Funding. Kumar:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Dimethyl fumarate as a first- vs second-line therapy in MS

ObjectiveTo elucidate the immunomodulatory effects of dimethyl fumarate (DMF) on B cells in patients with relapsing MS receiving DMF as a “1st-line” vs “2nd-line” therapy.MethodsB cells were isolated from 43 patients with MS at baseline and after 15-week DMF therapy. Phenotype and functional markers and cytokine profile were assessed by flow cytometry. Analysis included clinical and MRI parameters recorded during a 1-year follow-up.Results1st-line and 2nd-line patients presented several differences in their baseline immune profile, which corresponded with differences in their immunologic response to DMF treatment. DMF reduced the proportions of B cells and CD8 T cells whereas increased monocytes. DMF reduced memory B cells, including plasma cells in 2nd-line patients only, whereas strongly increased transitional B cells. Several IL10+ B-cell subsets and TGFβ+ B cells were increased. Proinflammatory LTα+ and TNFα+ B cells were reduced, while IL4+ B cells elevated, whereas IFNγ+ B cells showed opposite effects in 1st-line and 2nd-line patients. HLA and ICAM-1 expression was increased, but % CD86+ B cells reduced. The expression of B-cell activating factor receptor and the proportion of activated CD69 B cells were increased.ConclusionsDMF is associated with increased transitional and IL10+ and TGFβ+ regulatory B cells and a shift toward a more anti-inflammatory immune profile. Cell activation with reduced costimulatory capacity may induce immune hyporesponsiveness. Carryover effects of preceding therapies in 2nd-line patients and the stage of disease influence the immune profile of the patients and the immunomodulatory effects of DMF.

Abstract 5691: Evaluation of immunomodulatory agents in classically immunologically 'cold' cancers using syngeneic mouse models of breast and ovarian cancer

Abstract The clinical successes of immunotherapy in immunologically “hot” cancers such as kidney, bladder and lung cancers has lead researchers to pursue strategies to improve the initiation of immune responses in immunologically more “cold” cancers such as breast and ovarian cancer. We have characterized two syngeneic mouse models of breast cancer, 4T1 and E0771, and an ovarian cancer model, ID8, for their baseline immune profile and responsiveness to various immunotherapy approaches in an effort to enable rational combination therapy. The 4T1 model has useful traits for immuno-oncology research including a highly metastatic phenotype. However, our data illustrate that the tumors have a paucity of CD8 T cells and a highly immunosuppressed microenvironment with Tregs and ~80% G-MDSCs of CD11b+ cells leading to primary resistance to checkpoint blockade. Radiation can induce changes in an immunosuppressive microenvironment and radiotherapy remains an important clinical modality for the treatment of breast cancer. Treatment of 4T1 tumors with radiation resulted in a ~15% reduction of total MDSCs in the mice receiving 8Gy daily for three days. For the purpose of guiding future immunotherapy combinations, we established a focal beam radiation dose response and found single dose 5Gy had no activity while 10 or 20Gy resulted in increasing anti-tumor effects. The baseline immune cell profiling for E0771, a triple negative breast cancer, revealed notable differences to 4T1. While both models have a similar proportion of CD4 T cells and Tregs, 4T1 has ~55% G-MDSCs of CD11b+ cells while E0771 has ~0.4%. The content of M-MDSCs is nearly reciprocal with E0771 having ~49% M-MDSCs of CD11b+ cells and 4T1 having ~7%. The E0771 model also has a higher proportion of CD8 T cells than 4T1. Based on its immune profile, E0771could be a better candidate for responding to checkpoint inhibition. An efficacy study in the E0771 model demonstrated high sensitivity to inhibitors of PD-1, PD-L1, and CTLA-4, although the activity of anti-PD-1 on established tumors was more modest. Costimulatory agonistic antibodies to OX40, CD137 and GITR were also highly active. These data suggest that E0771 may represent a more immunologically “warm” breast cancer. We have developed the ID8-Luc-mCh-Puro syngeneic mouse cell line as an orthotopic (intraperitoneal) model of ovarian cancer wherein tumor burden is monitored by bioluminescence imaging. Immune profiling on ascites from ID8-Luc-mCh-Puro bearing mice shows heterogeneity in the profiles between mice; although, all mice have robust CD8+ T cell infiltration with favorable CD8+ T cell/Treg ratios. The activity of checkpoint inhibitors against orthotopic ID8-Luc-mCh-Puro is currently being evaluated. Together, these models of breast and ovarian cancer can be used to evaluate anti-tumor immune responses in immunologically more quiescent indications. Citation Format: Dylan Daniel, Sumithra Urs, Kevin Guley, Sarah Krueger, David Draper, Alden Wong, Hillary Evens, Claire Higginbottom, Dan Saims, Scott Wise, Maryland Rosenfeld Franklin. Evaluation of immunomodulatory agents in classically immunologically 'cold' cancers using syngeneic mouse models of breast and ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5691.

Baseline immune profile by CyTOF can predict response to an investigational adjuvanted vaccine in elderly adults

BackgroundMass cytometry, or CyTOF (Cytometry by Time-of-Flight), permits the simultaneous detection of over 40 phenotypic and functional immune markers in individual cells without the issues of spectral overlap seen in traditional flow cytometry.MethodsIn this study, we applied CyTOF to comprehensively characterize the circulating immune cell populations in elderly individuals both before and after administration of an investigational adjuvanted protein vaccine against respiratory syncytial virus (RSV) in a Phase 1a trial. Antigen-specific T cell responses to RSV by IFNγ ELISPOT had been observed in most but not all recipients in the highest dose cohort in this trial. Here, CyTOF was used to characterize the cellular response profile of ELISPOT responders and non-responders in this vaccine dose cohort.ResultsBoth CD4+ and CD8+ T cell antigen-specific IFNγ responses were observed. Principal components analysis revealed baseline differences between responders and non-responders, including differences in activated (HLA-DR+) CD4+ and CD8+ T cells, which were higher in non-responders versus responders. Using viSNE to analyze RSV-responsive CD4+ and CD8+ T cells, we also found increased expression of HLA-DR, CCR7, CD127 and CD69 in non-responders versus responders.ConclusionsHigh parameter CyTOF can help profile immune components associated with differential vaccine responsiveness.

Immune profiles of elderly breast cancer patients are altered by chemotherapy and relate to clinical frailty

BackgroundEffective therapeutic management of elderly patients with cancer, on an individual basis, remains a clinical challenge. Here, we identify novel biomarkers to assess elderly patients (≥70 years of age) with breast cancer undergoing treatment with or without chemotherapy.MethodsWe performed comprehensive geriatric assessment and measured markers sensitive to alteration in ageing, including leukocyte telomere length, CMV serostatus, levels of circulating growth factors and cytokines, and immune profiling of T cell and myeloid populations in blood before and at 3 months and 12 months after initiation of therapy, using flow cytometry.ResultsWe observed changes in immune profiles over time that were specific to patients receiving chemotherapy; these patients had elevated CD4+ T effector memory re-expressing CD45RA (TEMRA) cells and relatively lower CD8+ central memory cells at 3 months, with normalized levels after 12 months. Patients’ baseline immune profiles correlated with markers such as telomere length, cytomegalovirus (CMV) serostatus and levels of circulating cytokines. We also identified correlations between baseline immune profile and geriatric assessment, i.e. more frail patients had higher levels of granulocytic cells but lower levels of cells with suppressor phenotypes including myeloid-derived suppressor cells and regulatory T cells, although none of the examined immune populations correlated with chronological age. Importantly, immune profiles prior to therapy predicted unexpected hospitalizations in patients receiving chemotherapy.ConclusionThese findings suggest that immune profiling may represent a novel complementary approach to more accurately assess the global health status of the elderly patient with breast cancer and select the most appropriate individual treatment option.Trial registrationClinicalTrials.gov, NCT00849758. Registered on 20 February 2009.

Successful Rescue of Late-onset Antibody-mediated Rejection 12 Years After Living-donor Intestinal Transplantation: A Case Report

BackgroundAntibody-mediated rejection (ABMR) has recently surfaced as a potential form of graft dysfunction after intestinal transplantation. MethodsWe present a case of an intestinal transplant recipient who developed late-onset ABMR 12 years after living-donor transplantation. An 18-year-old male recipient with a history of extensive intestinal resection secondary to acute bowel volvulus exhibited an excellent baseline immune profile for transplantation, including ABO-identical and HLA-haploidentical to the donor; a negative cross-match with a panel reactive antibody of 3.0%. ResultsPost-transplantation immunosuppression consisted of tacrolimus, mycophenolate mofetil (MMF), and prednisone within the first year, followed by tacrolimus and MMF in the second year, and maintenance with tacrolimus monotherapy thereafter. The recipient experienced a single episode of indetermined acute cellular rejection 3 months after transplantation. Since then, he did not require any parenteral nutrition and had completely reintegrated with society. Twelve years later, the patient developed persistent diarrhea associated with transplant biopsy diffuse C4d deposition and circulating donor-specific antibodies. After the use of rituximab and intravenous immunoglobulin, the recipient stabilized 17 years after transplantation with complete recovery of intestinal mucosal damage. ConclusionLate-onset ABMR can emerge after transplantation and must be considered a possible cause of graft dysfunction in long-term intestinal transplantation survivors.

Effect of Absent Immune Cell Expression of Vitamin D Receptor on Cardiac Allograft Survival in Mice.

Vitamin D (VD) has immunomodulatory properties, but whether immune cell expression of the VD receptor (VDR) impacts costimulatory blockade induced cardiac allograft survival is not known. To localize effects of VDR deficiency to hematopoietic cells and to avoid the metabolic consequences of systemic VDR deficiency, we produced bone marrow (BM)-chimeric mice by transplanting lethally irradiated C57BL/6 mice with congenic VDR or wild type BM. After reconstitution, we characterized baseline immune profiles and transplanted chimeras with heterotopic cardiac allografts with or without costimulatory blockade using anti-CD154 (MR1) or CTLA4Ig, the latter approved for use in human kidney transplant recipients. Immune reconstitution occurred equivalently in chimeras with wild type and VDR BM. Untreated animals rejected class II disparate and fully allogeneic cardiac transplants with similar kinetics. Compared to untreated controls, treatment with either MR1 or CTLA4Ig induced significant and equivalent prolongation of graft survival in both groups of chimeric recipients. We observed no differences in induced antidonor cellular or humoral alloimmunity between groups. Our findings support the conclusion that absent immune cell VDR expression (a) does not impact the strength, phenotype, or kinetics of heart transplant rejection in mice and (b) does not impact the graft-prolonging effects of costimulatory blockade including that induced by clinically used CTLA4Ig.