scholarly journals Single-Cell Clonal Tracking in Allogeneic Hematopoietic Stem Cell Transplantation Reveals Time Dependent and Distinct Functional Patterns in Traceable Donor T Cell Clones

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 335-335
Author(s):  
Friedrich Wittenbecher ◽  
Luisa Keilholz ◽  
Benedikt Obermayer ◽  
Thomas Conrad ◽  
Marco Frentsch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Research Funding ◽  
Hematopoietic Stem ◽  
T Cell Clones ◽  
Time Points ◽  
Post Transplant ◽  
Cell Clones ◽  
Functional Patterns

Abstract Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only curative treatment option for various malignant hematological diseases. The therapeutic effect of alloHSCT is a long-lasting graft-versus-leukemia (GvL) effect of the transferred graft. T cells are important mediators of GvL and the longitudinal tracking of T-cell clones from donor to recipient is of particular interest in the setting of alloHSCT as this might provide further insight into mechanisms leading to survival and expansion of particular clones. In a broader sense, we used the unique setting of alloHSCT to study survival and expansion of mature T-cell clones after transfer into an immune cell depleted and allogeneic patient. We used single-cell RNA sequencing (scRNAseq) to integrate immune subset delineation, clone identification and transcriptome information of about 35500 single T cells in peripheral blood of 14 paired donor-recipient samples in four alloHSCT pairs. Donor samples were collected before and after treatment with Granulocyte-Colony Stimulating Factor (GCSF), and recipient samples were collected on days +90 and +180 post-transplant. Looking at common diversity scores of pooled donor versus pooled recipient time points we observe an expected decrease of TCR diversity after transplantation (median inv. Simpson's 379 in donor vs. 20 in recipient samples, p=0.011, Figure 1A). On single cell level, we observe a substantial decrease of unique T-cell clones after transplantation compared to donor samples, which in return means that certain TCR clones markedly expand, contributing to a skewing of the TCR repertoire in the post-transplant course. The majority of these cells represent CD8 effector memory T cells. Our main interest was a better understanding of traceable and persisting T-cell clones. In a first step, we looked at the overall clonal overlap between time points of the different donor-recipient pairs, using only combined TCR alpha and beta chain information to define specific T-cell clones. We find the highest overlaps of T-cell clones between time points within individuals (e.g., Morisita score 0.91 between preGCSF and postGCSF of donor 16 and Morisita score 0.65 between days +90 and +180 of recipient 16, Figure 1B). Additionally, we demonstrate an inter-individual overlap between donors and their respective recipients in all pairs on single cell level (Figure 1C). Next, we compared the differential gene expression of traceable and non-traceable T cell clones and found that the traceable T cell clones exhibit a distinct transcriptional program, characterized by upregulation of genes related to T cell proliferation and chemotaxis as well as antigen presentation, while housekeeping functions such as translation are downregulated. In order to examine the dynamic changes of the T-cell transcriptome, we looked at the differential gene expression at the consecutive time points of pooled traceable clones in all pairs. This shows an induction of an activation pattern during the donor-recipient transfer and post-transplant phase involving genes related to the cell cycle and graft-versus-host disease (Figure 1D). Phenotype analysis via antibody-derived tags accordingly revealed an upregulation of activation markers in the recipients. To our knowledge, this is the first time that longitudinal inter-individual (donor-to-recipient) overlap of single-cell TCR alpha/beta clones is demonstrated in the setting of alloHSCT revealing time point-dependent and distinct functional patterns in traceable donor T cell clones. Figure 1 Figure 1. Disclosures Penack: Neovii: Honoraria; MSD: Honoraria; Incyte: Research Funding; Priothera: Consultancy; Therakos: Honoraria; Gilead: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Takeda: Research Funding; Astellas: Honoraria; Jazz: Honoraria; Omeros: Consultancy; Shionogi: Consultancy. Bullinger: Amgen: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Hexal: Consultancy; Abbvie: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Astellas: Honoraria; Pfizer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bayer: Research Funding; Seattle Genetics: Honoraria; Novartis: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Menarini: Consultancy; Gilead: Consultancy; Celgene: Consultancy, Honoraria; Sanofi: Honoraria. Na: Bristol Myers Squibb: Research Funding; Shire/Takeda: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding.

The Molecular Characteristics in CDR3 of TCR Vα and Vβ Genes Associated with cGVHD in Patients after Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3247-3247
Author(s):  
Xiuli Wu ◽  
Yangqiu Li ◽  
Kanger Zhu ◽  
Xin Du ◽  
Shaohua Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
T Cell Immunity ◽  
Molecular Characteristics ◽  
Hematopoietic Stem ◽  
T Cell Clones ◽  
New Name ◽  
Cell Immunity ◽  
Cell Clones

Abstract Successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) requires reconstitution of normal T-cell immunity. Chronic graft-versus-host disease (cGVHD) is one of the major complications following allo-HSCT. The poor reconstitution of T-cell immunity (including the reconstitution of recent thymic output function and T-cell receptor (TCR) repertoire) was associated with cGVHD. In the previous study, we found that cGVHD predicted low TCR rearrangement excision circles (TRECs) levels and slow naïve T-cell recovery. Because GVHD displayed as clonal proliferation of special T-cells clones, which was triggered by donor T cells to recognize the host’s allogene antigen, in the present study we analyzed the TCR Vα and Vβ repertoire and cloanlity in patients with cGVHD, in order to find the special T-cell clones associated with cGVHD and evaluate the molecular characteristics of the CDR3 of TCR Vα and Vβ repertoire of GVHD-associated T-cell clones. Peripheral blood mononuclear cells (PBMNCs) were obtained from 5 leukemia patients with cGVHD after allo-HSCT. The expression and cloanlity analysis of TCR Vα and Vβ repertoire were detected by RT-PCR and genescan technique. Six donors served as controls. Almost all of TCR Vα and Vβ repertoire with polyclonal pattern were identified in normal controls. However, the skew expression pattern of TCR Vα and Vβ repertoire could be detected in patients with cGVHD even more than 4 year after allo-HSCT. Among 29 Vα and 24 Vβ subfamilies, there were only 4∼12 Vα and 4∼11 Vβ subfamilies expressed in patients with cGVHD. Oligoclonal or monoclonal expanded T cells were identified in TCR Vα 2, 3, 6, 10, 12, 14, 15, 25, 26 and TCR Vβ 1, 3, 7∼9, 13, 17, 19, 20 subfamilies respectively. The CDR3 sequences were further analyzed and all the sequences were blasted by internet (http://www.ncbi.nlm.nih.gov) and confirmed that it belonged to specific TCR Vα or Vβ gene rearrangement. The lengths of CDR3 were ranged from 12 to 15 amino acids. The molecular characteristics of the CDR3 of TCR Vα and Vβ genes rearrangement were TCRVα 3 (new name: Vα 17*01)-N-Jα 48*01-Cα (motif: CATEVDFGNEKLIF), TCRVα 2 (new name: Vα 12–2*01)-N-Jα 20*01-Cα (motif: CAVNLNDYKLIF), TCRVβ 1 (new name: Vβ 9*01)-N-Dβ 2*01-N-Jβ 2–1*01-Cβ 2 (motif: CASSDPPETYNEQFF), TCRVβ 7 (new name: Vβ 4–3*01)-N-Dβ 1*01-Jβ 1–1*01-Cβ 1 (motif: CASSHESGNTEAFF). Some TCR subfamily genes shared similarity in CDR3 amino acid motif. The role of specific sequences of CDR3 of TCR Vα and Vβ repertoire and T-cell clones will be confirmed in vivo by animal models.

scholarly journals Single-cell immune profiling reveals the impact of antiretroviral therapy on HIV-1-induced immune dysfunction, T cell clonal expansion, and HIV-1 persistence in vivo

2021 ◽  
Author(s):  
Jack A. Collora ◽  
Delia Pinto-Santini ◽  
Siavash Pasalar ◽  
Neal Ravindra ◽  
Carmela Ganoza ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antiretroviral Therapy ◽  
Single Cell ◽  
Cytotoxic T Cell ◽  
T Cell Clones ◽  
Cell Clones ◽  
Immune Profiling ◽  
Hiv 1

AbstractDespite antiretroviral therapy (ART), HIV-1 persists in proliferating T cell clones that increase over time. To understand whether early ART affects HIV-1 persistence in vivo, we performed single-cell ECCITE-seq and profiled 89,279 CD4+ T cells in paired samples during viremia and after immediate versus delayed ART in six people in the randomized interventional Sabes study. We found that immediate ART partially reverted TNF responses while delayed ART did not. Antigen and TNF responses persisted despite immediate ART and shaped the transcriptional landscape of CD4+ T cells, HIV-1 RNA+ cells, and T cell clones harboring them (cloneHIV-1). Some HIV-1 RNA+ cells reside in the most clonally expanded cytotoxic T cell populations (GZMB and GZMK Th1 cells). CloneHIV-1+ were larger in clone size, persisted despite ART, and exhibited transcriptional signatures of antigen, cytotoxic effector, and cytokine responses. Using machine-learning algorithms, we identified markers for HIV-1 RNA+ cells and cloneHIV-1+ as potential therapeutic targets. Overall, by combining single-cell immune profiling and T cell expansion dynamics tracking, we identified drivers of HIV-1 persistence in vivo.

Molecular Follow-Up of Patients Treated with HSV-TK/ΔLNGFR Engineered Donor Lymphocytes after Allogeneic Stem Cell Transplantation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1741-1741
Author(s):  
Chiara Bonini ◽  
Zulma Magnani ◽  
Alessandra Recchia ◽  
Fabrizia Urbinati ◽  
Sara Muraro ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Pcr Analysis ◽  
Hematopoietic Stem ◽  
T Cell Clones ◽  
Cell Clones

Abstract Donor lymphocyte infusions (DLI) play a crucial role in promoting immune reconstitution and anti-tumor activity in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). The efficacy of DLI is, however, limited by the occurrence of graft-versus-host disease (GvHD). In three different clinical trials, we showed that the infusion of lymphocytes transduced by a retroviral vector expressing a suicide gene (HSV-TK) and a surface marker (ΔLNGFR) allows to control GvHD in 100% of cases, while preserving anti-tumor and antiviral activities. In >40 patients treated with TK-cells in the context of HLA identical and haplo-identical HSCT, we observed consistent expansion (up to 40% of circulating cells) and long-term persistence (>10 years) of transduced cells. No acute or chronic adverse or toxic effect due to the gene transfer procedure was observed in these patients, who were treated with a total of >1011 cells generated by >60 independent transductions. Analysis of vector integration sites was preformed by LM-PCR on lymphocytes obtained up to 10 years after treatment from 4 patients, and selected for ΔLNGFR expression. 60% of the sequences met our validity criteria and were unambiguously mapped onto the human genome by Ensembl BLAST analysis. Transduced T-cells were highly polyclonal, with vector integrations occurring preferentially within genes (52%), and particularly within the first intron (25%). Quantitative PCR analysis of selected integrations was performed to follow the dynamics of individual T-cell clones during time. Microchip analysis of the gene expression profile showed that <200 out of >22,000 genes (0.9%) were differentially expressed in ΔLNGFR+ vs. ΔLNGFR- T-cells from two different patients, suggesting that no significant perturbation was induced by retroviral transduction or HSV-TK/ΔLNGFR expression in human lymphocyte populations. Interestingly, these data also show the substantial biological identity of T-cell population generated by HSCT and those administered as DLI. Finally, the influence of proviral integration on the expression of targeted genes was studied in individual T-cell clones transduced in vitro or obtained ex vivo from treated patients by quantitative gene expression analysis.

Functional Characterization of Aspergillus Fumigatus Specific T-Cells Clones.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3223-3223
Author(s):  
Thomas Lehrnbecher ◽  
Ulrike Koehl ◽  
Emmanuel Roilides ◽  
Maria Simitsopoulou ◽  
Mitra Hanisch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Aspergillus Fumigatus ◽  
Functional Characterization ◽  
Cd4 Cells ◽  
Hematopoietic Stem ◽  
T Cell Clones ◽  
Th1 Response ◽  
Cell Clones ◽  
Ifn Γ

Abstract Invasive aspergillosis (IA) remains a major cause of morbidity and mortality in patients with hematological malignancies, in particular in patients who have undergone allogeneic hematopoietic stem cell transplantation (SCT). There is a growing body of evidence that T-cells play an important role in the immunological response to Aspergillus fumigatus. Using the Aspergillus fumigatus antigen extract EC SAB and the IFN-γ secretion assay (Miltenyi Biotec, Germany), we generated Aspergillus fumigatus specific T-cell clones by limiting dilution (n=4). Flow cytometry revealed a cell population of CD3+/CD4+ cells (mean±SEM, 98.2±1.2%). Functional assessment by ICC revealed that an average of 8.7% of these cells (range, 6.6%–18.5%) specifically secreted IFN-γ on stimulation with EC SAB, which supports the TH1 response of the generated cells to Aspergillus fumigatus antigens. The antigenic components of EC SAB are one or more proteins, since the addition of proteinase completely suppressed the stimulating effect of this preparation. The percentage of IFN-γ producing CD3+/CD4+ cells was less than 1% upon activation with antigen extracts from Aspergillus flavus, Aspergillus niger, Alternaria alternata, Mucor racemosus, Penicillium notatum and Candida albicans, indicating that the generated T-cell clones are specific for Aspergillus fumigatus. A strong proliferation of the generated Aspergillus fumigatus specific T-cells was seen after re-stimulation with EC SAB, whereas alloreactivity was reduced compared to CD4+ T-cells of the original fraction. Hyphal damage of Aspergillus fumigatus was assessed by means of an XTT assay. Polymorphonuclear leukocytes (PMNs) showed a similar hyphal damage when tested alone (mean±SEM, 14.2±2.1%), in combination with antigen presenting cells (APCs) (15.1±1.4%), or in combination with Aspergillus fumigatus specific T-cells (15.0±2.0%). A comparable hyphal damage was seen when Aspergillus fumigatus specific T-cells were co-incubated with APCs (14.2±1.7%). In contrast, the combination of APCs and Aspergillus fumigatus specific T-cells with PMNs resulted in a significantly higher hyphal damage compared to all other settings (23.3±2.8%; P&lt;.0001). Interestingly, APCs alone or Aspergillus fumigatus specific T-cells alone showed a weak, but significant capacity to induce hyphal damage (7.4±1.1% and 11.3±1.8%, respectively). Before considering a clinical application, however, further studies need to focus on defining the optimal antigen(s) which reproducibly induce a TH1 response and elicit high antifungal activity, as well as to characterize the subpopulation of patients undergoing allogeneic SCT who ultimately benefit from either a prophylactic or a therapeutic adoptive transfer of Aspergillus fumigatus specific T-cells.

Analysis of T Cell Cloanlity of Ph+ Acute Lymphoblastic Leukemia with Chronic Gvhd in Continuous Remission after Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3941-3941
Author(s):  
Xiuli Wu ◽  
Qifa Liu ◽  
Yangqiu Li ◽  
Shaohua Chen ◽  
Xuan Du ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Blood Samples ◽  
T Cell Clones ◽  
Cell Clones ◽  
The Mean

Abstract Chronic graft-versus-host disease (cGVHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, recent study suggested that the presence of cGVHD might reduce the risk of relapse in Ph+ acute lymphoblastic leukemia (ALL) after allo-HSCT. GVHD is mediated by proliferation of special T-cell clones, we analyzed the T cell receptor (TCR) Vβ repertoire and cloanlity in Ph+ ALL patients undergone cGVHD after allo-HSCT in order to find the special T-cell clones associated with continuous remission. Allogeneic transplantations were performed in the first complete remission (CR) (6 ALLP190+ patients). The numbers of BCR-ABL copies in peripheral blood samples were assessed with the real-time quantitative RT-PCR (RQ-PCR) test at diagnosis, and every month after HSCT. The quantity of BCR-ABL transcript was normalized for normal ABL expression and the result was expressed as the ratio of BCR-ABL copies to ABL copies. With monitoring BCR-ABL copies of 6 BCR-ABLP190+ ALL patients after HSCT (the mean ratio of BCR-ABLP190/ABL at diagnose was 0.41 ± 0.39%), we found that 4 patients with extensive cutancous cGVHD had no evidence of leukemia recurrence, and the BCRABL transcripts of patients with cGVHD were remaining 0 copy, while 2 patients without cGVHD relapsed (the mean ratio of BCR-ABLP190/ABL was 7.56 ± 2.49%) and even dead within six months of HSCT. The expression and cloanlity analysis of TCR Vβ repertoire was detected in peripheral blood samples from patients with cGVHD by RT-PCR and genescan technique. TCR Vβ repertoires with polyclonal pattern were identified in normal controls and donor groups. However, the skew expression pattern of TCR Vβ repertoire could be detected in patients with cGVHD even more than 1 year after allo-HSCT. Among the 24 Vβ subfamilies, there were only 11~17 Vβ subfamilies expressed in cGVHD patients. Oligoclonal or monoclonal expanded T cells were identified in TCR Vβ 1, 2, 3, 7, 8, 12, 14, 16~20, 22 and 23 subfamilies in 4 ALL patients (BCR-ABLP190 remission) with cGVHD. As a contrast, oligoclonal or monoclonal expanded T cells were identified in TCR Vβ 2, 7, 8, 14, 17, 20, and 22 subfamilies in 2 chronic myeloid leukemia patients (BCR-ABLP210 remission) with cGVHD. In conclusion, the predominant usage and clonal expansion of TCR Vβ subfamily T cells could be found in Ph+ ALL patients with cGVHD and these groups of specific T-cell clones (Vβ 1, 3, 12, 16, 18, 19 and 23) were potentially associated with cGVHD in ALL and might be responsible for maintaining CR in Ph+ ALL patients after allo-HSCT. Research Funding.

scholarly journals Divergent clonal differentiation trajectories of T cell exhaustion

2021 ◽  
Author(s):  
Bence Daniel ◽  
Kathryn E Yost ◽  
Katalin Sandor ◽  
Yu Xia ◽  
Yanyan Qi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Phenotypic Diversity ◽  
Killer Cell ◽  
T Cell Response ◽  
T Cell Exhaustion ◽  
T Cell Clones ◽  
Cell Exhaustion ◽  
Cell Clones

T cells activated by chronic antigen exposure in the setting of viral infections or cancer can adopt an exhausted T cell (Tex) state, characterized by reduced effector function and proliferative capacity, and the upregulation of inhibitory receptors. However, whether all antigen-specific T cell clones follow the same molecular and cellular Tex differentiation trajectory remains unclear. Here, we generate a single-cell multi-omic atlas of T cell exhaustion that redefines the phenotypic diversity and molecular regulation of Tex phenotypes. Longitudinal analysis during chronic viral infection identifies an early effector phenotype that is epigenetically primed for Tex differentiation and two late-stage Tex cell states with either a terminal exhaustion or a killer cell lectin-like receptor (KLR)-expressing cytotoxic gene signature. We define clonal trajectories of antigen-specific T cells using paired single-cell RNA and T cell receptor sequencing and reveal distinct differentiation trajectories resulting in terminal Tex-biased, KLR Tex-biased, or divergent clones that differentiate into both phenotypes. Comparison of Tex phenotypes among shared T cell clones that traffic to multiple organs reveals that clonal differentiation trajectories are maintained across tissues. Finally, we show that differences in clonal differentiation trajectory are driven by TCR signal strength, whereby high-affinity T cell clones preferentially adopt a terminal Tex fate, while low-affinity clones adopt an effector-like KLR Tex fate that is detectable long-term but depleted in high antigen settings. These findings reveal clonal heterogeneity in the T cell response to chronic antigen and genomic programs that underlie Tex fates and persistence.

Molecular Identification of Individual T Cell Clones Specific for Graft- Versus-Leukemia Reaction

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1249-1249
Author(s):  
Veronika Foltankova ◽  
Eva Matejkova ◽  
Milan Bartos ◽  
Milos Dendis ◽  
Dana Novotna ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mononuclear Cells ◽  
Cell Clone ◽  
Cell Receptor ◽  
Unique Sequence ◽  
Hematopoietic Stem ◽  
T Cell Clones ◽  
Cell Clones ◽  
T Cell Clone

Abstract Graft-verus-leukemia (GVL) effect in hematopoietic stem cell transplantation (HSCT) is usually complicated by the alloreactivity of donor T cells which leads to acute graft-versus-host (GVH) disease. GVL and GVH reactions are proved to be mediated by different T cell clones. The objective of this study was to identify and characterize T cells clones with specific antileukemia activity without mediating GVHD. We have performed primary mixed leukocyte reaction (MLR) using patient non-leukemic irradiated peripherial blood mononuclear cells (PBMC) as stimulators and donor PBMC as responders. To prepare GVL specific T cells, activated alloreactive T cells were first selectively depleted with an anti-CD25 immunotoxin (Michalek, et al. PNAS2003, 100: 1180–4). Allodepleted T cells were then stimulated in secondary MLR using irradiated leukemia cells from the same patient. Activated leukemia-reactive cells were purified by immunomagnetic selection or by FACS based on INF-γ or CD25 expression, respectively. Clonotypic assay was used for identification of individual leukemia-specific T cell clones (Michalek, et al. Lancet2003, 361: 1183–5; Michalek, et al. J Immunol2007, 178: 6789– 5). This highly sensitive assay is based on detailed analysis of T cell receptor β VDJ unique sequence (TCRB-VDJ). mRNA was extracted from sortred activated cells and cDNA synthetized by anchored reverse transcription. Target TCRB-VDJ gene sequence was amplified by anchor PCR and used to transform bacteria. Bacterial colonies were picked for plasmid isolation and subsequent direct automated sequencing of the TCRBVDJ sequences. We assume that the frequency of particular TCRB-VDJ sequences among bacterial clones after transformation are proportional to the frequency of those sequences in the original population of T cells activated by GVH or GVL reaction. We investigated the presence of individual antileukemic T cell clones in patients with acute myeloid leukemia (AML) and chronic lymphatic leukemia (CLL), and defined them by the TCRB-VDJ unique sequence. The sequences that occured in more than 10% bacterial colonies are likely to represent the most immunodominant clones. Populations of antileukemic T cell clones were oligoclonal, i.e. we observed limited number of individual immunodominat clones which plays important role in GVL reaction. In first CLL patient who had undergone HSCT, six antileukemic T cell clones were identified, four of them are considered to be immunodominant. In second CLL patient after HSCT, only one highly immunodominat autileukemic T cell clone was observed. This specific clone was further monitored by quantitative real-time PCR in patients peripherial blood.

scholarly journals Safety and Efficacy of Multiantigen-Targeted T Cells for Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1014-1014 ◽  
Author(s):  
Premal Lulla ◽  
Ifigeneia Tzannou ◽  
George Carrum ◽  
Carlos A. Ramos ◽  
Rammurti Kamble ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Advisory Committees ◽  
T Cell Clones ◽  
Cell Clones ◽  
Equity Ownership ◽  
T Cell Lines

Abstract Despite an array of approved agents for the treatment of multiple myeloma (MM), most patients eventually relapse after conventional treatments. The adoptive transfer of tumor-targeted T cells has demonstrated efficacy in the treatment of patients with chemo-refractory hematological malignancies including MM. While the majority of T cell-based immunotherapeutic studies in the clinic explore genetically modified T cells that target a single tumor-expressed antigen, we have developed a strategy to generate non-engineered T cell lines that simultaneously target multiple MM-expressed antigens, thereby reducing the risk of tumor immune evasion. We manufacture multiTAA-specific T cells targeting the tumor-associated antigens PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin by culturing patient-derived PBMCs with autologous DCs loaded with pepmixes (15mer peptides overlapping by 11 aminos acids) spanning all 5 target antigens in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail. In our current clinical trial (NCT02291848), we have successfully generated multi-antigen-targeted lines from 18/ of 19 patients thus far, with one in production. The T cell lines comprise of CD3+ T cells (mean 95.6±2.2%) with a mixture of CD4+ (28.9±7.2%) and CD8+ (56.6±7.2%) T cells, which express central and effector memory markers (CD45RO+/CD62L+/CCR7+ -- 1.21±0.2%; CD45RO+/CD62L+/CCR7- -- 15.16±2.5%; CD45RO+/CD62L-/CCR7- -- 56.9±6.3%). All the expanded lines were specific for two to five target antigens with the majority of lines (13 of 18) specific for ≥3, (PRAME: Mean 45, range: 0 to 205 spot forming units (SFU)/2x105 input cells ; SSX2 mean: 57, 0 to 583, NYESO1: mean: 51, 0 to 125 , MAGE-A4 Mean: 67, 0 to 377 and Survivin mean: 53, 0 to 51), and did not react against non-malignant autologous recipient cells (2±3% specific lysis; E:T 20:1). We assessed the clonal diversity of the clinical product using TCR vβ deep sequencing analysis. We found both polyclonality and that the majority (mean 79%; range: 59 to 95%) represented rare T cell clones that were unique to the ex vivo expanded cell line and below levels of detection in the patients peripheral blood prior to infusion, thereby enabling in vivo tracking studies.. To date we have infused 18 patients with at least 2 infusions, 2 weeks apart of doses ranging from 0.5 to 2x107/m2. These patients had received a median of 4 lines of prior therapy including high dose chemotherapy with autologous stem cell rescue. Ten patients were refractory to their latest therapy and had active MM, while 8 were in remission at the time of infusion. At the 6 week evaluation period, of the 10 patients receiving multiTAA-specific T cells to treat active disease, 1 had a complete remission (CR) by the international myeloma working group (IMWG) response criteria, 1 had a partial remission (PR) and 8 others had stable disease (SD). Seven of these 10 patients were infused more than 1 year ago. Although 2 of the 7 subsequently had disease progression, the remaining 5 continue to respond, with sustained CR (1), PR (2) or SD (2). Of the 8 patients in CR at the time of T cell infusion, all remained in CR at the week 6 disease assessment and of the 6 evaluable patients who are >1 year post T cells, only one patient has relapsed, at 7 months after T cell infusion. These clinical responses correlated with the emergence and persistence (>6 months) of "line-exclusive" tumor-reactive T cells in patient peripheral blood, as assessed by longitudinal tracking of infused T cell clones using TCR deep sequencing. These infused product-derived T cells were detected in both peripheral blood (mean 0.43% ±SD of 0.3 of the total repertoire) and the marrow (mean 0.61%±0.24% of total repertoire). The expansion of product-derived T cell clones was higher among patients with active MM than in patients treated in remission (active: 0.60±0.39%, remission: 0.2±0.08%, p=0.048). Notably, no patient, including the complete responder, had infusion-related systemic- or neuro-toxicity. Thus, autologous multiTAA-targeted T cells directed to PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin can be safely administered to patients with MM, in whom they can subsequently be detected long-term in peripheral blood and marrow, and where they produce sustained tumor responses including CR. It will be of interest to discover whether larger or more frequent doses of these T cells can produce further benefit with maintained safety. Disclosures Brenner: Marker: Equity Ownership. Heslop:Marker: Equity Ownership; Viracyte: Equity Ownership; Cell Medica: Research Funding; Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding; Cytosen: Membership on an entity's Board of Directors or advisory committees. Vera:Marker: Equity Ownership. Leen:Marker: Equity Ownership.

scholarly journals Clonal replacement of tumor-specific T cells following PD-1 blockade

2019 ◽  
Author(s):  
Kathryn E. Yost ◽  
Ansuman T. Satpathy ◽  
Daniel K. Wells ◽  
Yanyan Qi ◽  
Chunlin Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Carcinoma ◽  
Single Cell ◽  
Cell Response ◽  
T Cell Response ◽  
Checkpoint Blockade ◽  
T Cell Clones ◽  
Cell Clones ◽  
Clonal Replacement

AbstractImmunotherapies that block inhibitory checkpoint receptors on T cells have transformed the clinical care of cancer patients. However, which tumor-specific T cells are mobilized following checkpoint blockade remains unclear. Here, we performed paired single-cell RNA- and T cell receptor (TCR)-sequencing on 79,046 cells from site-matched tumors from patients with basal cell carcinoma (BCC) or squamous cell carcinoma (SCC) pre- and post-anti-PD-1 therapy. Tracking TCR clones and transcriptional phenotypes revealed a coupling of tumor-recognition, clonal expansion, and T cell dysfunction: the T cell response to treatment was accompanied by clonal expansions of CD8+CD39+T cells, which co-expressed markers of chronic T cell activation and exhaustion. However, this expansion did not derive from pre-existing tumor infiltrating T cell clones; rather, it comprised novel clonotypes, which were not previously observed in the same tumor. Clonal replacement of T cells was preferentially observed in exhausted CD8+T cells, compared to other distinct T cell phenotypes, and was evident in BCC and SCC patients. These results, enabled by single-cell multi-omic profiling of clinical samples, demonstrate that pre-existing tumor-specific T cells may be limited in their capacity for re-invigoration, and that the T cell response to checkpoint blockade relies on the expansion of a distinct repertoire of T cell clones that may have just recently entered the tumor.

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