Preclinical Evaluation Of Engineered T Cells In Multiple Myeloma: Uncovering a Mechanism Of Immune Escape

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4205-4205
Author(s):  
Zandra K. Klippel ◽  
Jeffrey Chou ◽  
Andrea M. H. Towlerton ◽  
Paul F Robbins ◽  
Lilien Voong ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Immune Escape ◽  
Adoptive Therapy ◽  
Myeloma Cells ◽  
Nsg Mice ◽  
Flow Cytometric

Abstract Introduction Adoptive immunotherapy is an increasingly effective modality of cancer therapy. The ability to redirect the antigenic specificity of patient-derived T cells toward autologous tumor cells through introduction of T-cell receptors (TCRs) or chimeric antigen receptors (CARs) enables reproducible manufacturing of tumor-reactive T cell products even in patients who carry few, if any, tumor-reactive T cells in their peripheral blood repertoire. We present the results of our pre-clinical studies of adoptive therapy with T cells transduced with a retroviral vector that encodes an enhanced-affinity (a95:LY) variant of the HLA-A*02:01-restricted, NY-ESO-1157-165-specific 1G4 TCR to redirect CD8+ T cells from HLA-A*02:01+ multiple myeloma patients to HLA-A*02:01+, NY-ESO-1-expressing myeloma cells. Methods CD3-stimulated peripheral blood mononuclear cells from HLA-A*02:01+ multiple myeloma patients were retrovirally transduced with the NY-ESO-1157-165-specific 1G4 a95:LY TCR. CD8+ TCR-transduced cells were isolated by flow cytometric sorting with a NY-ESO-1157-165/HLA-A*02:01 tetramer. The cytolytic activity of CD8+tetramer+ cells was evaluated by 51Cr release assay using as target cells the multiple myeloma cell lines U266 (HLA-A*02:01+ NY-ESO-1+) and UM-9 (HLA-A*02:01- NY-ESO-1+), and T2 cells with or without exogenous NY-ESO-1157-165 peptide. The U266 cell line was stably transduced with luciferase-containing retrovirus and used to develop a xenograft model of diffuse myeloma in NOD/scid/IL-2Rg-null (NSG) mice in order to evaluate the anti-myeloma activity of adoptive therapy with CD8+ TCR-transduced T cells. Mice that received TCR-transduced CD8+cells and developed disease were sacrificed, and human CD138+ cells were harvested from marrow and other sites for evaluation by flow cytometry, HLA-A typing, NY-ESO-1 expression, and loss of heterozygosity (LOH) analysis of the Major Histocompatibility Complex (MHC) on chromosome 6 with short tandem repeat (STR) probes to determine the mechanism of immune escape. Results CD8+ TCR-transduced cells were specifically cytolytic against HLA-A*02:01+, NY-ESO-1+ tumor cells. Intravenous injection of luciferase-transduced U266/Luc in sub-lethally irradiated NSG mice led to the development of a multiple myeloma-like disease. Mice that received U266/Luc without T cells (control) developed progressive disease within 2 weeks, and met criteria for euthanasia by week 9. Mice that received U266/Luc with sham-transduced cells developed myeloma more slowly, yet all met criteria for euthanasia by week 18 after U266/Luc injection. Of the 6 mice that received U266/Luc and NY-ESO-1-specific TCR-transduced CD8+ T cells, 4 did not have any evidence of myeloma by bioluminescence at the end of study (week 18), and 2 had low burden disease at that point. Kaplan-Meier survival analysis demonstrated significant improvement of overall survival in the mice that received TCR-transduced T cells (Log-rank test p< 0.0001). Flow cytometric analysis of human CD138+ cells isolated from the 2 mice that developed myeloma despite adoptive therapy with NY-ESO-1-specific T cells demonstrated selective loss of surface HLA-A*02 expression, with preserved expression of other MHC class I molecules. Real-time PCR analysis also confirmed preserved expression of HLA-A, B2M, and NY-ESO-1. Low resolution HLA-A typing of genomic DNA from myeloma cells from these 2 mice revealed loss of HLA-A*02, but retention of HLA-A*03. LOH analysis using 7 STR markers mapping to the MHC on chromosome 6p21.3 and 2 markers on chromosome 15 (control) demonstrated LOH in the MHC involving the HLA-A locus in myeloma cells from both of the mice that developed disease despite TCR-transduced T cells. The extent of LOH in the myeloma cells from the 2 mice was distinct. Conclusions LOH in the MHC as a mechanism of immune scape has been described in allogeneic transplantation for AML, but has not been described in multiple myeloma. We identified LOH affecting the HLA-A allele targeted by adoptively transferred TCR-transduced T cells. Given that NY-ESO-1-specific TCR-transduced cells have recently entered clinical testing, this mechanism of immune escape should be evaluated in patients that fail therapy despite persistence of adoptively transferred T cells. Disclosures: No relevant conflicts of interest to declare.

Active Vaccination with DKK1 Induces Protective and Therapeutic Antitumor Immunity In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3040-3040
Author(s):  
Jianfei Qian ◽  
Sungyoul Hong ◽  
Hong Qin ◽  
Ji Wang ◽  
Jinsong Wei ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Tumor Cells ◽  
Dna Vaccine ◽  
Cd8 T Cells ◽  
Dna Vaccines ◽  
Therapeutic Effects ◽  
Tumor Challenge ◽  
Myeloma Cells ◽  
Active Vaccination

Abstract Abstract 3040 Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by the tumor cells of almost all patients with multiple myeloma (MM) and may be responsible for suppressed osteoblast formation. In our previous studies, we demonstrated that DKK-1 is a potent tumor-associated antigen in MM recognized by cytotoxic T lymphocytes (CTLs), which can effectively lyse autologous myeloma tumor cells in vitro (Qian et al., Blood 2007;110:1587-1594) and eradicate established patient-derived primary myeloma in SCID-hu mice upon adoptive transfer. To examine the potential of DKK1-based immunotherapy in MM, we investigated the efficacy of active vaccination with (murine) DKK1 DNA vaccine in a murine (MOPC-21) myeloma model. A plasmid DNA construct encoding defensin2-sFv was generated, and DKK1 full-length cDNA was cloned from DKK1-expressing mouse stromal cells by reverse transcription–polymerase chain reaction and genetically fused with defensin2 (DNA-vac). We first examined the ability of the vaccines to protect mice from developing myeloma. While 100% of mice vaccinated with vector control or PBS (10 mice for each group) developed tumors, 70% and 40% mice vaccinated with DNA-vac or DNA-vac with CpG (ODN 1826), respectively, developed tumors. These results clearly show that active vaccination with DKK1 DNA vaccines was able to protect mice from tumor challenge, and that combination with CpG was more effective than DNA vaccine alone. On day 90 after tumor challenge, all surviving tumor-free mice were rechallenged with the same myeloma cells and followed for tumor development. By day 180, All surviving mice that were vaccinated with DNA-vac or DNA-vac with CpG have no developed tumors, indicating that active vaccination with DNA vaccines induced strong tumor-specific memory immune responses to protect mice from tumor rechallenge. Next, we examined the therapeutic effects of DNA vaccines in our myeloma mouse model. DNA vaccine alone eradicated established myeloma in 1 out of 5 mice, while DNA vaccine plus CpG eradicated myeloma in 3 out of 5 mice bearing intermediate tumors (≥ 5 mm in diameter). These results indicate that DNA vaccine plus CpG was much more effective at eradicating established myeloma than DNA vaccine alone. Finally, the mechanisms of tumor protection induced by DNA vaccines were investigated. By depleting CD4+ or CD8+ T cells, we showed that CD8+ T cells are required for DNA-induced antimyeloma responses. In DNA-vaccinated mice, splenocytes contained increased numbers of DKK1-specific, IFN-g-secreting and proliferative T cells. The splenic CD8+ T cells exhibited enhanced cytotoxicity against myeloma cells. Furthermore, DKK1-specific CD8+ T cells were shown to be increased in DKK1-DNA vaccinated mice by using DKK1-peptide (P11, P15 and P210) tetramer staining. These results demonstrate the presence of myeloma-specific CTLs in vaccinated mice and show that DKK1 DNA vaccine can induce a potent CTL response capable of killing myeloma cells. Together, our study lays a basis for future clinical trials in MM by using DKK1 as a vaccine for all patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Anti-CD38-blocked ricin: an immunotoxin for the treatment of multiple myeloma [see comments]

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 3017-3025 ◽  
Author(s):  
VS Goldmacher ◽  
LA Bourret ◽  
BA Levine ◽  
RA Rasmussen ◽  
M Pourshadi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Tumor Cell Line ◽  
Covalent Attachment ◽  
Myeloma Cells ◽  
Colony Forming Units

Abstract We report the development of a potent anti-CD38 immunotoxin capable of killing human myeloma and lymphoma cell lines. The immunotoxin is composed of an anti-CD38 antibody HB7 conjugated to a chemically modified ricin molecule wherein the binding sites of the B chain have been blocked by covalent attachment of affinity ligands (blocked ricin). Conjugation of blocked ricin to the HB7 antibody has minimal effect on the apparent affinity of the antibody and no effect on the ribosome-inactivating activity of the ricin A-chain moiety. Four to six logs of CD38+ tumor cell line kill was achieved at concentrations of HB7-blocked ricin in the range of 0.1 to 3 nmol/L. Low level of toxicity for normal bone marrow (BM) granulocyte-macrophage colony- forming units (CFU-GM), burst-forming units-erythroid (BFU-E), colony- forming units-granulocyte/erythroid/monocyte/macrophage (CFU-GEMM) cells was observed. Greater than two logs of CD38+ multiple myeloma cells were depleted from a 10-fold excess of normal BM mononuclear cells (BMMCs) after an exposure to HB7-blocked ricin under conditions (0.3 nmol/L) that were not very toxic for the normal BM precursors. HB7- blocked ricin was tested for its ability to inhibit protein synthesis in fresh patients' multiple myeloma cells and in normal BMMCs isolated from two healthy volunteers; tumor cells from four of five patients were 100-fold to 500-fold more sensitive to the inhibitory effect of HB7-blocked ricin than the normal BM cells. HB7 antibody does not activate normal resting peripheral blood lymphocytes, and HB7-blocked ricin is not cytotoxic toward these cells at concentrations of up to 1 nmol/L. The potent killing of antigen-bearing tumor cells coupled with a lack of effects on peripheral blood T cells or on hematopoietic progenitor cells suggests that HB7-blocked ricin may have clinical utility for the in vivo or in vitro purging of human multiple myeloma cells.

scholarly journals Distinct changes of in BTLA, ICOS, PD-1, and TIGIT expression on peripheral blood and decidual CD8+ T cells in women with unexplained recurrent spontaneous abortion†

2020 ◽  
Vol 103 (5) ◽  
pp. 1012-1017
Author(s):  
Qianqian Liang ◽  
Lingxia Tong ◽  
Liping Xiang ◽  
Sujuan Shen ◽  
Chenhuan Pan ◽  
...  
Keyword(s):  
T Cells ◽  
Spontaneous Abortion ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Peripheral Blood ◽  
Immune Escape ◽  
Recurrent Spontaneous Abortion ◽  
Decidual Tissue ◽  
Unexplained Recurrent Spontaneous Abortion ◽  
The Difference

Abstract The two-way communication between the mother and the fetus is accomplished by immune cells. CD8+ T cells of normal pregnant (NP) women express progesterone receptor (PR). Binding of PR to progesterone (P) and the production of progesterone-induced blocking factor (PIBF) can aid immune escape, which is an important factor in the maternal immune response. We detected the proportion of CD8+ T cells and the expression of the surface costimulatory molecules BTLA, TIGIT, ICOS, and PD-1 in peripheral blood and decidual tissues of women with unexplained recurrent spontaneous abortion (URSA) and in NP women. All patients were at 8 -10 weeks of gestation. The results showed that there was no change in the proportions of CD8+ T cells in peripheral blood and decidual tissues of URSA patients compared to those of NP women. In peripheral blood, compared with the NP group, the URSA group showed decreased expression of BTLA + CD8+ T cells and the difference was statistically significant, but there was no difference between the groups in terms of TIGIT + CD8+, PD-1 + CD8+, and ICOS + CD8+ T cells. There was no change in the levels of TIGIT + CD8+, PD-1 + CD8+, ICOS + CD8+, and BTLA + CD8+ T cells in decidual tissue. These data confirm that the number of CD8+ T cells in peripheral blood and decidual tissue is not the main factor leading to the pathogenesis of URSA, and other immune cells may play an important role in URSA, but this hypothesis needs further exploration and research.

Tumor-specific recognition of human myeloma cells by idiotype-induced CD8+ T cells

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2828-2833 ◽  
Author(s):  
Yiwen Li ◽  
Maurizio Bendandi ◽  
Yuping Deng ◽  
Cynthia Dunbar ◽  
Nikhil Munshi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
Class I ◽  
Specific Lysis ◽  
Myeloma Cells ◽  
Id Protein ◽  
Human Myeloma Cells

Immunoglobulin secreted by myeloma cells contains a unique antigenic determinant (idiotype [Id]) that may serve as a tumor-specific antigen. Although Id-protein–specific T-cell responses have been reported in patients with myeloma, it is not known whether primary myeloma tumor cells can present naturally processed Id peptides on their surface as a target. We immunized 2 healthy human stem-cell donors with Id proteins from their recipients. T cells from the immunized donors released high levels of T-helper 1–type cytokines in response to stimulation with myeloma cells from their recipients. The T-cell–mediated cytokine response to tumor cells was blocked by a major histocompatibility complex (MHC) class I monoclonal antibody, whereas the response to soluble Id protein was dependent on MHC class II. To investigate whether Id-specific CD8+ T cells can recognize and kill autologous myeloma cells, we generated T cells from peripheral blood mononuclear cells from a third patient with myeloma by means of in vitro stimulation with autologous dendritic cells pulsed with Id protein. Tumor-specific lysis of myeloma cells was demonstrated by the lack of killing of autologous nonmalignant B cells or natural killer–sensitive K562 cells. Lysis of autologous myeloma targets was restricted by MHC class I molecules. These data represent the first report of class I–restricted T-cell recognition of fresh autologous myeloma targets and formally demonstrate that human myeloma cells can serve as targets of an Id-specific T-cell response.

Enrichment of functional CD8 memory T cells specific for MUC1 in bone marrow of patients with multiple myeloma

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 2132-2134 ◽  
Author(s):  
Carmen Choi ◽  
Mathias Witzens ◽  
Marianna Bucur ◽  
Markus Feuerer ◽  
Nora Sommerfeldt ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Hematologic Malignancies ◽  
Myeloma Cell ◽  
Interferon Γ ◽  
Multiple Myeloma Cell ◽  
Healthy Donors

AbstractMultiple myeloma (MM) is one of the most common hematologic malignancies. Despite extensive therapeutical approaches, cures remain rare exceptions. An important issue for future immunologic treatments is the characterization of appropriate tumor-associated antigens. Recently, a highly glycosylated mucin MUC1 was detected on a majority of multiple myeloma cell lines. We analyzed bone marrow and peripheral blood of 68 patients with HLA-A2–positive myeloma for the presence and functional activity of CD8 T cells specific for the MUC1-derived peptide LLLLTVLTV. Forty-four percent of the patients with MM contained elevated frequencies of MUC1-specific CD8 T cells in freshly isolated samples from peripheral blood (PB) or bone marrow (BM) compared with corresponding samples from healthy donors. BM-residing T cells possessed a higher functional capacity upon specific reactivation than PB-derived T cells with regard to interferon γ (IFN-γ) secretion, perforin production, and cytotoxicity.

MAGE-A3 Specific T-Cell Receptor Adoptive Cell Transfer of Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1843-1843 ◽  
Author(s):  
Jeesun Park ◽  
Shi Zhong ◽  
Michelle Krogsgaard ◽  
Amitabha Mazumder
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Fold Increase ◽  
Cell Receptor ◽  
Tumor Cell Lines ◽  
Peptide Concentration

Abstract Abstract 1843 Background: Multiple myeloma (MM) is a cancer of plasma cells and the second most common blood cancer. Current treatment strategies such as high dose chemotherapy, autologous stem cell rescue, and allogeneic transplantation have improved response rates and increased survival. However, these treatments often include high procedure-related morbidity and mortality and can only be applied to a small minority of myeloma patients. Therefore, safe broadly applicable immunologic strategies for myeloma, such as Adoptive Cell Therapy (ACT) are urgently needed. Methods: In this study we focused on aHLA-A*0201-restricted cancer testis antigen MAGE-A3:112–120, which is widely expressed in many forms of cancers such as metastatic melanoma, non-small cell lung cancer and MM, but not expressed in most normal tissues. To develop a system of effective strategies for T-cell therapy of multiple myeloma, we employed T-cell engineering technology using a MAGE-A3specific T-cell receptor (TCR)obtained from Dr. Steven Rosenberg at the National Cancer Institute. MAGE-A3 specific TCR was sub-cloned into a lentiviral vector and tranduced into purified CD8+ T-cells from human peripheral blood mononucleocytes (hPBMCs). To test the effector functionality of the MAGE-A3 specific TCR, the MAGE-A3 TCR-transduced CD8+ T-cells were subjected to cytokine release and chromium release assays after being co-cultured with MAGE-A3 peptide-loaded T2 cells, and U266 (MAGE-A3+/HLA-A*0201+), MM1.r (MAGE-A3+/HLA-A*0201-), KAS6 (MAGE-A3-/HLA-A*0201+), and KMS11(MAGE-A3-/HLA-A*0201-) MM tumor cell lines. Results: We observedcytokine production of INF-g and IL-2 in the MAGE-A3 TCR-transduced CD8+ T-cells generally in a dose-dependent manner to the MAGE-A3 peptide-loaded T2 cells. For example, the difference of INF-g secretion bythe MAGE-A3 TCR-transduced CD8+ T-cells wasa 10-fold increase from 0.001 uM to 0.02 uM of the loaded MAGE-A3 peptide. IL-2 secretion was also increasedby 7-fold from 0.001 uM to 0.1 uM of the MAGE-A3 peptide concentration. At 10uM of the peptide concentration, there was a 29-fold increase of the IL-2 production as compared to the 0.001 uM peptide concentration. Between 10uM and 100 uMof the peptide concentration, there was a decrease in IL-2 secretion by 2-fold, which is commonly observed at high peptide concentrations presumably due to cytotoxicity. Specific lysis of tumor cells by the MAGE-A3 TCR-transduced CD8+ T-cellswas observed in all four MM tumor cell lines, and we detected higher percentage of cell lysisin U266 (38%) and MM1.r (51%) cell lines as compared to the KAS6 (11%) and KMS11(21%) cell lines. Conclusions: Our findings suggest that the MAGE-A3 TCR-engineered CD 8+ T-cells are able to specifically recognize MAGE-A3 antigen, produce IL-2 and IFN-g, and destroy MM tumor cells loaded with the MAGE-A3 antigen. This potentially could further translate into effective MAGE-A3 specific targeted tumor rejection in vivo. We also plan to transduce the MAGE-A3 TCR into hematopoietic stem cells to and test the effector function of those cells against MM tumor cells and eventually against MM patient samples. Disclosures: No relevant conflicts of interest to declare.

Tumor cells in multiple myeloma patients inhibit myeloma-reactive T cells through carcinoembryonic antigen-related cell adhesion molecule-6

Blood ◽  
2013 ◽  
Vol 121 (22) ◽  
pp. 4493-4503 ◽  
Author(s):  
Mathias Witzens-Harig ◽  
Dirk Hose ◽  
Simone Jünger ◽  
Christina Pfirschke ◽  
Nisit Khandelwal ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Cell Adhesion ◽  
Carcinoembryonic Antigen ◽  
Tumor Cells ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Myeloma Cells ◽  
Related Cell ◽  
Key Points

Key Points Multiple myeloma cells inhibit myeloma-specific T cells through expression of carcinoembryonic antigen-related cell adhesion molecule-6.

scholarly journals PD-1 Blockade Reinvigorates Bone Marrow CD8+ T Cells from Patients with Multiple Myeloma in the Presence of TGF-β Inhibitors

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3241-3241
Author(s):  
Minsuk Kwon ◽  
Eui-Cheol Shin ◽  
Yoon Seok Choi
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Cytokine Production ◽  
Plasma Cells ◽  
Myeloma Cells ◽  
Tgf Β1

Programmed cell death (PD)-1/PD-Ligand 1(PD-L1) blockade that reinvigorates exhausted T cells has been approved for the treatment of various solid tumors and hematological malignancies. However, in a clinical trial of multiple myeloma (MM) patients, anti-PD-1 monotherapy did not result in a clinical response. Furthermore, clinical trials of combining PD-1 blockade with immunomodulatory drugs or anti-CD38 monoclonal antibody failed to demonstrate clinical benefits in MM patients. To overcome the limitation of anti-PD-1 therapy in MM, the phenotype and differentiation of CD8+ T cells need to be characterized in the bone marrow (BM) of MM patients, particularly by analyzing myeloma antigen-specific CD8+ T cells. In addition, the role of immunosuppressive factors abundant in the MM microenvironment should be considered, including TGF-β. First, we confirmed the upregulation of PD-1 and PD-L1 expression in CD8+ T cells and myeloma cells, respectively, from the BM of MM patients. PD-1-expressing CD8+ T cells from the BM of MM patients co-expressed other checkpoint inhibitory receptors including Tim-3, LAG-3, and TIGIT. We also investigated the expression of T-cell transcription factors, such as T-bet, and EOMES, which are related to T-cell differentiation. In BM from MM patients, PD-1+CD8+ T cells had a higher percentage of EomeshiT-betlo cells than PD-1-CD8+ T cells. These data demonstrate that PD-1-expressing CD8+ T cells from the BM of MM patients exhibit a terminally differentiated phenotype with co-expression of multiple immune checkpoint inhibitory receptors. These results were also observed in BM CD8+ T cells specific to myeloma antigens NY-ESO-1 and HM1.24. Next, we investigated proliferation and cytokine production of BM CD8+ T cells from MM patients. BM CD8+ T cells from MM patients exhibited reduced proliferation and cytokine production upon T cell receptor (TCR) stimulation, compared to BM CD8+ T cells from other control group such as of undetermined significance. However, both anti-PD-1 alone and combined blockade of PD-1 with other immune checkpoint receptors, such as Tim-3, Lag-3, or TIGIT, did not increase the proliferation of BM CD8+ T cells from MM patients. Likewise, anti-PD-1 treatment failed to induce reinvigoration of BM CD8+ T cells stimulated with HLA-A*0201-restricted myeloma antigen peptides, including NY-ESO-1157-165 and HM1.2422-30 peptides. These data demonstrate that blocking PD-1 is not sufficient to restore the function of BM CD8+ T cells from MM patients. It has been known that TGF-β, which is actively secreted by malignant plasma cells and BM stromal cells, can inhibit T-cell responses. We confirmed that the major source of TGF- β1 is plasma cells including myeloma cells among BMMCs from MM patients, and the number of TGF- β1-producing plasma cells, including myeloma cells, is increased in the BM of MM patients. We investigated whether blocking TGF-β signaling enhances reinvigoration of BM CD8+ T cells from MM patients. The combined blockade of PD-1 and TGF- β significantly increased the proliferation of BM CD8+ T cells from MM patients in the presence of TCR stimulation. The production of IFN-γ and TNF by BM CD8+ T cells was also rescued by combined blockade of PD-1 and TGF-β. Moreover, combination of anti-PD-1 antibody and TGF-β inhibitors increased proliferative responses of BM CD8+ T cells from HLA-A2+ MM patients stimulated with a mixture of HLA-A*0201-restricted myeloma antigen peptides (NY-ESO-1157-165 and HM1.2422-30 peptides). Thus, PD-1 blockade reinvigorates BM CD8+ T cells from MM patients in the presence of TGF-β inhibitors. Taken together, BM CD8+ T cells and myeloma antigen-specific CD8+ T cells express increased levels of PD-1 and have a terminally exhausted phenotype in MM patients. Under TGF-β inhibition, anti-PD-1 reinvigorates BM CD8+ T cells from MM patients, but PD-1 blockade alone does not restore the function of BM CD8+ T cells. Blocking both TGF-β and PD-1 can be a promising therapeutic strategy for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clonally Expanded Bone Marrow T Cells Show Effector Differentiation and Rarely Recognize Disease-Associated Antigens in Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-7
Author(s):  
Carlotta Welters ◽  
Meng-Tung Hsu ◽  
Christian Alexander Stein ◽  
Livius Penter ◽  
María Fernanda Lammoglia Cobo ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Cell Expansion ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
T Cell Expansion

Multiple myeloma is a malignancy of monoclonal plasma cells accumulating in the bone marrow. The critical influence of tumor-infiltrating T cells on disease control and therapeutic responses has been shown in a variety of malignancies, however, the role of multiple myeloma bone marrow-infiltrating T cells is incompletely understood. Although it has been shown that multiple myeloma neo-antigen-specific T cells can be expanded in vitro, little is known about functions and specificities of clonally expanded multiple myeloma-infiltrating bone marrow T cells. Here we asked at the single cell level whether clonally expanded T cells i) were detectable in multiple myeloma bone marrow and peripheral blood, ii) showed characteristic immune phenotypes, and iii) recognized antigens selectively presented on multiple myeloma cells. A total of 6,744 single bone marrow T cells from 13 treatment-naïve patients were index-sorted and sequenced using our methodologies for determination of paired T cell receptor (TCR) αβ sequences along with immune phenotype, transcription factor and cytokine expression. Clonal T cell expansion occurred predominantly within the CD8+ compartment. Phenotypes of clonally expanded T cells were distinctive of cytolytic effector differentiation and significantly different from non-expanded CD8+ T cells. Less than 25% of expanded CD8+ T cell clones expressed the immune checkpoint molecules programmed death-1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), or T cell immunoglobulin and mucin-domain containing-3 (TIM-3), while B and T lymphocyte attenuator (BTLA) was expressed on more than half of the expanded clones. Clonal T cell expansion did not correlate with neo-antigen load as determined by whole exome and RNA sequencing of purified multiple myeloma cells. Furthermore, peripheral blood TCRβ repertoire sequencing from five selected patients with substantial bone marrow T cell expansion identified 90% of expanded bone marrow T cell clones overlapping with peripheral blood. To determine whether clonally expanded bone marrow T cells recognized antigens selectively presented on multiple myeloma cells, 71 dominant TCRs from five selected patients with substantial clonal T cell expansion were re-expressed in 58α-β- T-hybridoma reporter T cells and co-incubated with CD38-enriched multiple myeloma cells from the same patients. Only one of these TCRs recognized antigens selectively presented on multiple myeloma cells and this TCR was not neo-antigen-specific. Hypothesizing that the target antigen was a non-mutated self-antigen, we could show that this TCR also recognized the plasma cell leukemia cell line U-266 in an HLA-A*02:01-restricted manner. In summary, clonally expanded T cells in multiple myeloma bone marrow of newly diagnosed patients show cytolytic effector differentiation. In the majority of patients, clonally expanded bone marrow T cells do not recognize antigens presented on multiple myeloma cells and are not neo-antigen-specific. Our findings are relevant for the design of future therapeutics and clinical trials. The identified TCR, which recognizes a multiple myeloma antigen shared with U-266 in an HLA-A*02:01-restricted manner, could be a promising candidate for T cell therapy. Disclosures Bullinger: Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Hexal: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees.

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