scholarly journals CTIM-29. CRUCIAL APPLICATION OF SINGLE CELL GENE EXPRESSION FOR MONITORING CHANGES IN GLIOBLASTOMA PATIENTS WITH SUSTAINED RADIOGRAPHIC RESPONSE TO CMV pp65-LAMP RNA-PULSED DENDRITIC CELL-BASED VACCINES

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii39-ii39
Author(s):  
Oleg Yegorov ◽  
Changlin Yang ◽  
Anjelika Dechkovskaia ◽  
Maryam Rahman ◽  
Ashley Ghiaseddin ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Dendritic Cell ◽  
Single Cell ◽  
Immune Cell ◽  
Nkt Cells ◽  
Durable Response ◽  
Radiographic Response ◽  
Dc Vaccines

Abstract BACKGROUND The application of single cell sequencing as a novel immune monitoring platform can be used to identify the molecular mechanisms of immune response to dendritic cell- based vaccines, trace the cell types and states involved, and uncover novel biomarkers for immunotherapy. We applied single-cell RNA Seq analysis of longitudinal peripheral blood mononuclear cells (PBMCs) in patients with newly-diagnosed GBM enrolled on the ATTAC II clinical trial (FDA IND BB-16530; Clinicaltrials.gov # NCT02465268) who experienced a sustained radiographic response to autologous CMV pp65-LAMP RNA-pulsed DC vaccines plus GM-CSF and tetanus-diphtheria booster administered during adjuvant cycles of dose-intensified temozolomide. METHODS We constructed 5’ gene expression libraries and T cell receptor enriched libraries for 10x Genomics single-cell 5’ and VDJ sequencing, generated from PBMCs collected prior to and during patient immunization using dendritic cells loaded with messenger ribonucleic acid encoding the human cytomegalovirus (CMV) matrix protein pp65 conjugated with the lysosomal associated membrane protein (LAMP) sequence. RESULTS Overall, we sequenced a total of 189,808 single-cell transcriptomes from 5 patients. We leveraged these transcriptome-wide features to distinguish 15 peripheral immune cell subtypes in tested PBMCs. Analysis revealed dynamic changes in immune cell subsets over the course of first three vaccines, including increases in cytotoxic CD8 T cells, CD4 T cells, and NK cell subsets. Increased markers of T cell activation were observed during vaccination. Surprisingly, we observed a very high-level frequency of natural killer T (NKT) cells in the patient with a complete durable response compared to other patients. After three DC vaccines, the level of NKT cells in PBMC of this patient increased up to 10%. CONCLUSIONS These results emphasize the importance of subset specific profiling to achieve higher resolution in monitoring immune responses compared with bulk expression profiling in patients receiving immunotherapeutic treatment.

scholarly journals ATIM-34. SINGLE-CELL RNA SEQUENCING REVEALS DYNAMIC IMMUNE RESPONSE CHANGES IN GLIOBLASTOMA PATIENT WITH DURABLE COMPLETE RESPONSE TO CMV PP65-LAMP RNA-PULSED DENDRITIC VACCINES

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi9-vi9
Author(s):  
Oleg Yegorov ◽  
Changlin Yang ◽  
Maryam Rahman ◽  
Ashley Ghiaseddin ◽  
Anjelika Dechkovskaia ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cytotoxic T Cells ◽  
Complete Response ◽  
Nkt Cells ◽  
Single Cell Sequencing ◽  
Single Cell Rna Sequencing ◽  
Dc Vaccines

Abstract BACKGROUND The application of single cell sequencing as a novel immune monitoring platform can enhance the ability to interrogate at unprecedented depth the single-cell level dynamic phenotypic and functional attributes of immune cells in patients undergoing cancer immunotherapy treatment. We applied single-cell sequencing analysis of PBMCs in a patient with newly-diagnosed GBM enrolled on the ATTAC II clinical trial (FDA IND BB-16530) who experienced a sustained complete radiographic response to autologous CMV pp65-LAMP RNA-pulsed DC vaccines plus GM-CSF and tetanus-diphtheria booster administered during adjuvant cycles of dose-intensified TMZ. METHODS We constructed 5’ gene expression libraries and T cell receptor enriched libraries for 10x Genomics single-cell sequencing, generated from PBMCs collected prior to and during patient immunization using dendritic cells loaded with RNA encoding the CMV matrix protein pp65 conjugated with the lysosomal associated membrane protein (LAMP) sequence. RESULTS RNA-Seq revealed dynamic changes in immune cell subsets over course of first three vaccines, including increases in cytotoxic T cells and memory T cell subsets. Increased markers of T cell activation were observed during vaccination including enhanced T cell signaling pathways, proliferative signals, and cytokine production. We found that proportion of cytotoxic T cells increased from 3.42% to 11.74% in whole PBMCs after immunization. Surprisingly, we observed very high level of frequency natural killer T (NKT) cells comprising 4.75 % of this patient’s PBMCs at baseline. After three DC vaccines, the level of NKT cells in PBMC increased up to 10%. CONCLUSIONS Single cell RNA sequencing of a patient with sustained complete response to DC vaccination during cycles of dose-intensified TMZ reveals dynamic immune activation and expansion within the cytotoxic T cells and NKT compartments. These results emphasize the importance of subset specific profiling to achieve higher resolution in monitoring immune responses compared with bulk expression profiling in patients receiving immunotherapeutic treatment.

scholarly journals Single-cell Multiomics Reveals Clonal T-cell Expansions and Exhaustion in Blastic Plasmacytoid Dendritic Cell Neoplasm

2021 ◽  
Author(s):  
Erica A. K. DePasquale ◽  
Daniel Ssozi ◽  
Marina Ainciburu ◽  
Jonathan Good ◽  
Jenny Noel ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Dendritic Cell ◽  
Single Cell ◽  
Interferon Alpha ◽  
Immune Cell ◽  
Plasmacytoid Dendritic Cell ◽  
Cell Exhaustion ◽  
Tumor Environment

AbstractThe immune system represents a major barrier to cancer progression, driving the evolution of immunoregulatory interactions between malignant cells and T-cells in the tumor environment. Blastic plasmacytoid dendritic cell neoplasms (BPDCN), a rare acute leukemia with plasmacytoid dendritic cell (pDC) differentiation, provides a unique opportunity to study these interactions. pDCs are key producers of interferon alpha (IFNA) that play an important role in T-cell activation at the interface between the innate and adaptive immune system. To assess how uncontrolled proliferation of malignant BPDCN cells affects the tumor environment, we catalog immune cell heterogeneity in the bone marrow (BM) of five healthy controls and five BPDCN patients by analyzing 52,803 single-cell transcriptomes, including 18,779 T-cells. We test computational techniques for robust cell type classification and find that T-cells in BPDCN patients consistently upregulate interferon alpha (IFNA) response and downregulate tumor necrosis factor alpha (TNFA) pathways. Integrating transcriptional data with T-cell receptor sequencing via shared barcodes reveals significant T-cell exhaustion in BPDCN that is positively correlated with T-cell clonotype expansion. By highlighting new mechanisms of T-cell exhaustion and immune evasion in BPDCN, our results demonstrate the value of single-cell multiomics to understand immune cell interactions in the tumor environment.

IMMU-41. NEOADJUVANT PD-1 BLOCKADE INDUCES T CELL AND CDC1 ACTIVATION BUT FAILS TO OVERCOME THE IMMUNOSUPPRESSIVE TUMOR ASSOCIATED MACROPHAGES IN RECURRENT GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi101-vi101
Author(s):  
Alexander Lee ◽  
Lu Sun ◽  
Aaron Mochizuki ◽  
Jeremy Reynoso ◽  
Joey Orpilla ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Dendritic Cell ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Myeloid Cells ◽  
Interferon Γ ◽  
Cell Trafficking ◽  
Related Gene

Abstract In a small phase I clinical trial, neoadjuvant anti-PD-1 (neo-aPD1) improved survival in glioblastoma patients (GBM) and was associated with increased interferon-γ-related gene expression and an associated decrease in cell cycle-related gene expression in the tumor. Despite this, neo-aPD1 was not curative. To better understand how neo-aPD1 alters the GBM tumor microenvironment and to discover new therapeutic axes, we used CyTOF and single-cell RNAsequencing to analyze the tumor-infiltrating immune populations of 70 GBM patients, 20 of whom had received neo-aPD1. In patients treated with neo-aPD1 the proportion and number of tumor-infiltrating T cells increased and these T cells had increased expression of the cytokines CCL5 and XCL1, which promote dendritic cell migration. Among the infiltrating T cells was a population of progenitor exhausted CD8+ T cells expressing the genes TCF, SLAM6, CCR7, IL7R, and GZMB. Single-cell TCR analysis revealed that these CD8+ T cells arose from a population of early activated, cytotoxic T cells that had expanded in the peripheral blood and trafficked into the tumor. Within the tumor-infiltrating myeloid cells, we found that neo-aPD1 upregulated genes in the interferon-γ response pathways; namely genes related to T cell trafficking and immune suppression, such as CD274 (PD-L1), TREM2, GPNMB, and IL10, indicating a maladaptive response by some myeloid cells to the immune response triggered by neo-aPD1. Finally, we identified a new migratory and interferon-γ activated tumor-infiltrating conventional dendritic cell population that was increased with neo-aPD1 and that notably expressed XCR1, the receptor for XCL1. Additionally, our results provide future basis to study if modalities that enhance the T cell:DC axis or that inhibit the inhibitory myeloid populations may improve survival in patients with GBM when used in conjunction with neo-aPD1. In sum, we characterized the immune landscape in GBM and how it changes with neo-aPD1 at single cell resolution.

540 Transcriptionally defined immune landscape in human gliomas

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A576-A576
Author(s):  
Pravesh Gupta ◽  
Minghao Dang ◽  
Krishna Bojja ◽  
Huma Shehwana ◽  
Tuan Tran ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Immune Cell ◽  
Myeloid Cell ◽  
Wild Type ◽  
Cell Clusters ◽  
Md Anderson ◽  
Antigen Presenting

BackgroundBrain immunity is largely myeloid cell dominated rather than lymphoid cells in healthy and diseased state including malignancies of glial origins called as gliomas. Despite this skewed myeloid centric immune contexture, immune checkpoint and T cell based therapeutic modalities are generalizably pursued in gliomas ignoring the following facts i) T cells are sparse in tumor brain ii) glioma patients are lymphopenic iii) gliomas harbor abundant and highly complex myeloid cell repertoire. We recognized these paradoxes pertaining to fundamental understanding of constituent immune cells and their functional states in the tumor immune microenvironment (TIME) of gliomas, which remains elusive beyond a priori cell types and/or states.MethodsTo dissect the TIME in gliomas, we performed single-cell RNA-sequencing on ~123,000 tumor-derived sorted CD45+ leukocytes from fifteen genomically classified patients comprising IDH-mutant primary (IMP; n=4), IDH-mutant recurrent (IMR; n=4), IDH-wild type primary (IWP; n=3), or IDH-wild type recurrent (IWR; n=4) gliomas (hereafter referred as glioma subtypes) and two non-glioma brains (NGBs) as controls.ResultsUnsupervised clustering analyses delineated predominant 34-myeloid cell clusters (~75%) over 28-lymphoid cell clusters (~25%) reflecting enormous heterogeneity within and across glioma subtypes. The glioma immune diversity spanned functionally imprinted phagocytic, antigen-presenting, hypoxia, angiogenesis and, tumoricidal myeloid to classical cytotoxic lymphoid subpopulations. Specifically, IDH-mutant gliomas were predominantly enriched for brain-resident microglial subpopulations in contrast to enriched bone barrow-derived infiltrates in IDH-wild type especially in a recurrent setting. Microglia attrition in IWP and IWR gliomas were concomitant with invading monocyte-derived cells with semblance to dendritic cell and macrophage like transcriptomic features. Additionally, microglial functional diversification was noted with disease severity and mostly converged to inflammatory states in IWR gliomas. Beyond dendritic cells, multiple antigen-presenting cellular states expanded with glioma severity especially in IWP and IWR gliomas. Furthermore, we noted differential microglia and dendritic cell inherent antigen presentation axis viz, osteopontin, and classical HLAs in IDH subtypes and, glioma-wide non-PD1 checkpoints associations in T cells like Galectin9 and Tim-3. As a general utility, our immune cell deconvolution approach with single-cell-matched bulk RNA sequencing data faithfully resolved 58-cell states which provides glioma specific immune reference for digital cytometry application to genomics datasets.ConclusionsAltogether, we identified prognosticator immune cell-signatures from TCGA cohorts as one of many potential immune responsiveness applications of the curated signatures for basic and translational immune-genomics efforts. Thus, we not only provide an unprecedented insight of glioma TIME but also present an immune data resource that can be exploited for immunotherapy applications.Ethics ApprovalThe brain tumor/tissue samples were collected as per MD Anderson internal review board (IRB)-approved protocol numbers LAB03-0687 and, LAB04-0001. One non-tumor brain tissue sample was collected from patient undergoing neurosurgery for epilepsy as per Baylor College of Medicine IRB-approved protocol number H-13798. All experiments were compliant with the review board of MD Anderson Cancer Center, USA.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal

Single Cell Profiling Reveals Unique CXCL13 Positive T Cell Subsets in the Tumor Microenvironment of Lymphocyte Rich Classic Hodgkin Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-33
Author(s):  
Tomohiro Aoki ◽  
Lauren C. Chong ◽  
Katsuyoshi Takata ◽  
Katy Milne ◽  
Elizabeth Chavez ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Single Cell ◽  
Immune Cells ◽  
Research Funding ◽  
Immune Cell ◽  
Expression Patterns ◽  
The Other ◽  
Tfh Cells

Introduction: Classic Hodgkin lymphoma (CHL) features a unique crosstalk between malignant cells and different types of normal immune cells in the tumor-microenvironment (TME). On the basis of histomorphologic and immunophenotypic features of the malignant Hodgkin and Reed-Sternberg (HRS) cells and infiltrating immune cells, four histological subtypes of CHL are recognized: Nodular sclerosing (NS), Mixed cellularity, Lymphocyte-rich (LR) and Lymphocyte-depleted CHL. Recently, our group described the high abundance of various types of immunosuppressive CD4+ T cells including LAG3+ and/or CTLA4+ cells in the TME of CHL using single cell RNA sequencing (scRNAseq). However, the TME of LR-CHL has not been well characterized due to the rarity of the disease. In this study, we aimed at characterizing the immune cell profile of LR-CHL at single cell resolution. METHODS: We performed scRNAseq on cell suspensions collected from lymph nodes of 28 primary CHL patients, including 11 NS, 9 MC and 8 LR samples, with 5 reactive lymph nodes (RLN) serving as normal controls. We merged the expression data from all cells (CHL and RLN) and performed batch correction and normalization. We also performed single- and multi-color immunohistochemistry (IHC) on tissue microarray (TMA) slides from the same patients. In addition, an independent validation cohort of 31 pre-treatment LR-CHL samples assembled on a TMA, were also evaluated by IHC. Results: A total of 23 phenotypic cell clusters were identified using unsupervised clustering (PhenoGraph). We assigned each cluster to a cell type based on the expression of genes described in published transcriptome data of sorted immune cells and known canonical markers. While most immune cell phenotypes were present in all pathological subtypes, we observed a lower abundance of regulatory T cells (Tregs) in LR-CHL in comparison to the other CHL subtypes. Conversely, we found that B cells were enriched in LR-CHL when compared to the other subtypes and specifically, all four naïve B-cell clusters were quantitatively dominated by cells derived from the LR-CHL samples. T follicular helper (TFH) cells support antibody response and differentiation of B cells. Our data show the preferential enrichment of TFH in LR-CHL as compared to other CHL subtypes, but TFH cells were still less frequent compared to RLN. Of note, Chemokine C-X-C motif ligand 13 (CXCL13) was identified as the most up-regulated gene in LR compared to RLN. CXCL13, which is a ligand of C-X-C motif receptor 5 (CXCR5) is well known as a B-cell attractant via the CXCR5-CXCL13 axis. Analyzing co-expression patterns on the single cell level revealed that the majority of CXCL13+ T cells co-expressed PD-1 and ICOS, which is known as a universal TFH marker, but co-expression of CXCR5, another common TFH marker, was variable. Notably, classical TFH cells co-expressing CXCR5 and PD-1 were significantly enriched in RLN, whereas PD-1+ CXCL13+ CXCR5- CD4+ T cells were significantly enriched in LR-CHL. These co-expression patterns were validated using flow cytometry. Moreover, the expression of CXCR5 on naïve B cells in the TME was increased in LR-CHL compared to the other CHL subtypes We next sought to understand the spatial relationship between CXCL13+ T cells and malignant HRS cells. IHC of all cases revealed that CXCL13+ T cells were significantly enriched in the LR-CHL TME compared to other subtypes of CHL, and 46% of the LR-CHL cases showed CXCL13+ T cell rosettes closely surrounding HRS cells. Since PD-1+ T cell rosettes are known as a specific feature of LR-CHL, we confirmed co-expression of PD-1 in the rosetting cells by IHC in these cases. Conclusions: Our results reveal a unique TME composition in LR-CHL. LR-CHL seems to be distinctly characterized among the CHL subtypes by enrichment of CXCR5+ naïve B cells and CD4+ CXCL13+ PD-1+ T cells, indicating the importance of the CXCR5-CXCL13 axis in the pathogenesis of LR-CHL. Figure Disclosures Savage: BeiGene: Other: Steering Committee; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie: Honoraria; Roche (institutional): Research Funding; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie, Servier: Consultancy. Scott:Janssen: Consultancy, Research Funding; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding; NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy. Steidl:AbbVie: Consultancy; Roche: Consultancy; Curis Inc: Consultancy; Juno Therapeutics: Consultancy; Bayer: Consultancy; Seattle Genetics: Consultancy; Bristol-Myers Squibb: Research Funding.

Recovery of Paired T Cell Receptors from Single-cell Seq-Well Libraries

2019 ◽  
Author(s):  
Ang A. Tu ◽  
Todd M. Gierahn ◽  
Brinda Monian ◽  
Duncan M. Morgan ◽  
Naveen K. Mehta ◽  
...  
Keyword(s):  
T Cells ◽  
Food Allergy ◽  
T Cell ◽  
Single Cell ◽  
T Cell Receptors ◽  
Immune Cell ◽  
Cost Effective ◽  
Antigen Specificity ◽  
Activated T Cells ◽  
Cell Receptors

Abstract High-throughput 3’ single-cell RNA-Sequencing (scRNA-Seq) allows for cost-effective, detailed characterization of thousands of individual immune cells from healthy and diseased tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including the variable sequences of T cell receptors (TCRs) that confer antigen specificity in T cells. Here, we present an enrichment strategy that enables simultaneous analysis of TCR variable sequences and corresponding full transcriptomes from 3’ barcoded scRNA-Seq samples. This approach is compatible with common 3’ scRNA-Seq methods, and adaptable to processed samples post hoc. We applied the technique to resolve clonotype-to-phenotype relationships among antigen-activated T cells from immunized mice and from patients with food allergy. We observed diverse but preferential cellular phenotypes manifest among subsets of expanded clonotypes, including functional Th2 states associated with food allergy. These results demonstrate the utility of our method when studying complex diseases in which clonotype-driven immune responses are critical to understanding the underlying biology.

scholarly journals Global immune characterization of HBV/HCV-related hepatocellular carcinoma identifies macrophage and T-cell subsets associated with disease progression

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Guohe Song ◽  
Yang Shi ◽  
Meiying Zhang ◽  
Shyamal Goswami ◽  
Saifullah Afridi ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Immune Cells ◽  
Immune Cell ◽  
T Cell Subsets ◽  
M2 Macrophage ◽  
Advanced Hcc ◽  
Immune Cell Subsets

AbstractDiverse immune cells in the tumor microenvironment form a complex ecosystem, but our knowledge of their heterogeneity and dynamics within hepatocellular carcinoma (HCC) still remains limited. To assess the plasticity and phenotypes of immune cells within HBV/HCV-related HCC microenvironment at single-cell level, we performed single-cell RNA sequencing on 41,698 immune cells from seven pairs of HBV/HCV-related HCC tumors and non-tumor liver tissues. We combined bio-informatic analyses, flow cytometry, and multiplex immunohistochemistry to assess the heterogeneity of different immune cell subsets in functional characteristics, transcriptional regulation, phenotypic switching, and interactions. We identified 29 immune cell subsets of myeloid cells, NK cells, and lymphocytes with unique transcriptomic profiles in HCC. A highly complex immunological network was shaped by diverse immune cell subsets that can transit among different states and mutually interact. Notably, we identified a subset of M2 macrophage with high expression of CCL18 and transcription factor CREM that was enriched in advanced HCC patients, and potentially participated in tumor progression. We also detected a new subset of activated CD8+ T cells highly expressing XCL1 that correlated with better patient survival rates. Meanwhile, distinct transcriptomic signatures, cytotoxic phenotypes, and evolution trajectory of effector CD8+ T cells from early-stage to advanced HCC were also identified. Our study provides insight into the immune microenvironment in HBV/HCV-related HCC and highlights novel macrophage and T-cell subsets that could be further exploited in future immunotherapy.

Subclinical GVHD Impairs Responses to Dendritic Cell Vaccines Following Allogeneic Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 571-571
Author(s):  
Sarah Herby ◽  
Matthew Milliron ◽  
Crystal Mackall ◽  
Terry Fry
Keyword(s):  
T Cells ◽  
T Cell ◽  
Dendritic Cell ◽  
Physical Symptoms ◽  
Third Party ◽  
Donor Strain ◽  
Vaccine Responses ◽  
Dc Vaccine ◽  
Dc Vaccines ◽  
A Minor

Abstract Background: Although allogeneic graft versus tumor responses represent the most potent form of immunotherapy, recurrent malignancy remains a major cause of post-transplant mortality. Thus, strategies to enhance GVT are needed to improve outcomes. Dendritic cell (DC) vaccines can potently enhance anti-tumor immunity in the autologous setting. The impact of alloreactivity on DC vaccine responses is not known, but the potency of the GVT effect suggests that mild alloreactivity might enhance vaccine responses to third party antigens. Methods: To determine the effect of a minor histocompatibility mismatch on DC vaccine responses, lethally irradiated female C3H.SWxC57BL/6 and C3H.SW recipients were transplanted with T cell depleted (TCD) C3H.SW bone marrow (BM). On days 14 and 28, recipients received donor-type lymph node cells containing mature T cells (DLI, 1 x 10^6, 5 x 10^6, or 10 x 10^6 IV). To immunize against the male antigenic complex (HY), BM-derived, anti-CD40 activated donor strain male DC were administered IP at the same time as the DLI. At Day 35, HY specific immune responses were measured using interferon γ ELISPOT against the dominant (UTY) and subdominant (SMCY) class I epitopes and dominant class II epitope (DBY). In some experiments, rhIL-7 and rhIL-15 were administered (5 mcg/day IP) from day 14 to 35. Results: In syngeneic recipients, robust HY responses were observed at all DLI doses, consistent with rapid T cell reconstitution via the thymus. Allogeneic recipients demonstrated mild weight loss and decreased splenic cellularity as the DLI dose was increased, consistent with the development of mild GVHD. However, even recipients receiving the highest DLI dose recovered completely and had none of the major physical symptoms of GVHD. Increasing DLI dose from 1 x 10^6 to 10 x 10^6 paradoxically led to a marked decrease in the median total number of T cells/spleen responding to UTY, SMCY, and DBY (9196 at 1 x 10^6 vs 897 at 10 x 10^6, p = .01; 15662 vs 2854, p= .02; 10257 vs 4227, p = .02). To test whether the presence of alloreactive T cells were necessary for this effect, C3H.SW minor antigen reactive T cells were deleted from B6-derived DLI. TCD B6 BM was transplanted into C3H.SWxC57BL/6 mice and at 5 weeks LN cells were collected and transferred as DLI. Strikingly, transfer of 10 x 10^6 tolerized DLI did not result in a loss of HY vaccine responses when compared to transfer of non-tolerized DLI (UTY, 3888 vs 21075, p= .05; SMCY, 7135 vs 21126, p = .018; DBY, 7117 vs 29201, p= .006). Although IL-7 and IL-15 can potently enhance DC vaccine responses in the syngeneic setting, these cytokines did not improve vaccine responses in allogeneic recipients. Conclusions: Using a minor mismatched allogeneic transplant model, we have shown that mild GVHD can diminish the ability to respond to third party antigens expressed on activated DC vaccines. This suggests that the potency of GVT does not result from the alloreactive milieu, but rather from the nature of the antigens targeted. The mechanisms involved in the suppression of DC vaccine responses in allogeneic recipients are currently under study. These findings have important implications for the use of tumor antigen expressing vaccines as a modality to improve the graft versus tumor effect.

Association between cetuximab response and differential immunologic gene expression signatures in colorectal cancer metastases.

2016 ◽  
Vol 34 (4_suppl) ◽  
pp. 558-558 ◽  
Author(s):  
Michael Sangmin Lee ◽  
Benjamin Garrett Vincent ◽  
Autumn Jackson McRee ◽  
Hanna Kelly Sanoff
Keyword(s):  
Gene Expression ◽  
Colorectal Cancer ◽  
T Cells ◽  
T Cell ◽  
Immune Cell ◽  
Expression Profiles ◽  
Gene Set Enrichment Analysis ◽  
Activated T Cells ◽  
Gene Sets

558 Background: Different immune cell infiltrates into colorectal cancer (CRC) tumors are associated with different prognoses. Tumor-associated macrophages contribute to immune evasion and accelerated tumor progression. Conversely, tumor infiltrating lymphocytes at the invasive margin of CRC liver metastases are associated with improved outcomes with chemotherapy. Cetuximab is an IgG1 monoclonal antibody against epidermal growth factor receptor (EGFR) and stimulates antibody-dependent cellular cytotoxicity (ADCC) in vitro. However, it is unclear in humans if response to cetuximab is modulated by the immune response. We hypothesized that different immune patterns detected in gene expression profiles of CRC metastases are associated with different responses to cetuximab. Methods: We retrieved gene expression data from biopsies of metastases from 80 refractory CRC patients treated with cetuximab monotherapy (GEO GSE5851). Samples were dichotomized by cetuximab response as having either disease control (DC) or progressive disease (PD). We performed gene set enrichment analysis (GSEA) with GenePattern 3.9.4 using gene sets of immunologic signatures obtained from the Molecular Signatures Database v5.0. Results: Among the 68 patients with response annotated, 25 had DC and 43 had PD. In the PD cohort, 59/1910 immunologic gene sets had false discovery rate (FDR) < 0.1. Notably, multiple gene sets upregulated in monocyte signatures were associated with PD. Also, gene sets consistent with PD1-ligated T cells compared to control activated T cells (FDR = 0.052) or IL4-treated CD4 T cells compared to controls (FDR = 0.087) were associated with PD. Conclusions: Cetuximab-resistant patients tended to have baseline increased expression of gene signatures reflective of monocytic infiltrates, consistent with also having increased expression of the IL4-treated T-cell signature. Cetuximab resistance was also associated with increased expression of the PD1-ligated T cell signature. These preliminary findings support further evaluation of the effect of differential immune infiltrates in prognosis of metastatic CRC treated with cetuximab.

scholarly journals Single-Cell Profiling Reveals Contribution of Tumor Extrinsic and Intrinsic Factors to BCMA-Targeted CAR-T Cell Efficacy in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 326-326
Author(s):  
David T. Melnekoff ◽  
Yogita Ghodke-Puranik ◽  
Oliver Van Oekelen ◽  
Adolfo Aleman ◽  
Bhaskar Upadhyaya ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Poor Responders ◽  
Car T Cell ◽  
Car T

Abstract Background: BCMA CAR-T cell therapy has shown great promise in relapsed/refractory multiple myeloma (RRMM) patients, even though there is unpredictable variability in the duration and depth of response. The mechanisms behind these divergent outcomes and relapse are not well understood and heterogeneity of MM patients at the level of both tumor genomics and tumor microenvironment (TME) likely contributes to this important knowledge gap. To explore this question, we performed a longitudinal high resolution single cell genomic and proteomic analysis of bone marrow (BM) and peripheral blood (PB) samples in MM patients treated with BCMA CAR-T. Methods: Longitudinal comprehensive immune phenotyping of 3.5 million peripheral blood mononuclear cells (PBMC, CD45+CD66b-) from 11 BCMA CAR-T (idecabtagene vicleucel, ide-cel) patients was achieved via mass cytometry (CyTOF) with a panel of 39 markers. In addition, a total of 45,161 bone marrow mononuclear cells (BMMC) were analyzed from 6 patients before initiation of ide-cel therapy and at relapse by unbiased mRNA profiling via single-cell RNA-seq (scRNA-seq) using the GemCode system (10x Genomics). Downstream analysis was performed using the CATALYST and Seurat R packages, respectively. Immune cell populations are reported as % of PBMC and CD138- BMMC respectively, unless noted otherwise. Reported p values correspond to non-parametric tests or paired t test where applicable. Results: We compared baseline immune cell populations in the PB and the TME (BM) with regards to depth of CAR-T response. In PB, good responders (≥VGPR) had a higher proportion of CD8+ T cells (37% in good vs 11% in poor responders (&lt;VGPR), p=0.08) and a lower proportion of CD14+ monocytes (30% vs 61%, p=0.28) and NK cells (2% vs 6%, p=0.08). In the TME, a similar trend was confirmed for CD8+ T cells and CD14+ monocytes. (Fig. 1A) Longitudinal analysis of PBMCs revealed phenotypic changes coinciding with CAR-T expansion; CD14+ monocytes declined from week 0 to week 4 after CAR-T infusion (40% vs 13%, p=0.04), while (non-CAR) CD8+ T cells expanded from week 0 to week 4 (32% vs 43%, p=0.15). The non-CAR CD8+ T cell expansion is characterized by differentiation towards a CD8+ effector-memory phenotype (EM, CCR7-CD45RA-) (73% vs 92% of CD8+ T cells, p=0.005). (Fig. 1B) BM samples at CAR-T relapse showed reversal of this shift: CD14+ monocyte levels remain constant or are slightly elevated, while non-CAR CD8+ T cells decrease at relapse. scRNA-seq of BMMC revealed significant gene expression changes between screening and relapse tumor samples, suggesting tumor-intrinsic factors of CAR-T response. For example, when comparing the pre and post tumor samples of a patient with durable response (PFS 652 days), we observed a significant upregulation of gene expression of pro-inflammatory chemokines (CCL3, CCL4), anti-apoptotic genes (MCL-1, FOSB, JUND), and NF-kB signaling genes (NFKBIA) in post tumor. Gene Set Enrichment Analysis (GSEA) of differentially expressed genes showed significant enrichment for TNFA signaling via NF-kB Hallmark Pathway (p.adj = 0.04). We observed similar statistically significant findings between other screening and relapse samples within our cohort, as well as upon comparison of baseline samples of poor vs good responders. (Fig. 1C, D) Thus, our data suggest that anti-apoptotic gene expression could be one of the tumor intrinsic mechanisms of CAR-T therapy resistance. Notably, we did not observe loss of BCMA expression in any tumor samples. Conclusion: Single cell immune profiling and transcriptomic sequencing highlights changes in the PB, TME and within the tumor, which in concert may influence CAR-T efficacy. Our integrated data analysis indicates general immune activation after CAR-T cell infusion that returns to baseline levels at relapse. Specifically, the expansion of non-CAR cytotoxic CD8+ EM T cells provides a rationale for co-administration of IMiDs to enhance CAR-T efficacy. Significant up-regulation of anti-apoptotic genes at baseline in poor responders, and at relapse in good responders, suggest a novel tumor-mediated escape mechanism. Targeting the MCL-1/BCL-2 axis may augment CAR-T efficacy by sensitizing tumor cells and enhancing the effect of CAR-T killing. We will confirm these findings in a longitudinal cohort of BMMC/PBMC CITE-seq patients (n=23) and will present results at the conference. Figure 1 Figure 1. Disclosures Sebra: Sema4: Current Employment. Parekh: Foundation Medicine Inc: Consultancy; Amgen: Research Funding; PFIZER: Research Funding; CELGENE: Research Funding; Karyopharm Inv: Research Funding.

Sign in / Sign up

Export Citation Format

Share Document