scholarly journals Dominant Expression of PVRIG and TIGIT Inhibitory Pathways in Bone Marrow of Multiple Myeloma Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4717-4717
Author(s):  
Masha Frenkel ◽  
Zoya Alteber ◽  
Ning Xu ◽  
Mingjie Li ◽  
Haiming Chen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Inhibitory Receptor ◽  
Preclinical Models ◽  
Cell Numbers

Abstract Introduction Blocking inhibitory immune checkpoints holds promise to treat multiple myeloma (MM) patients. However, currently available checkpoint inhibitors have not shown significant clinical benefits for MM patients, warranting the need for alternative checkpoint blockers. The immune checkpoint TIGIT was recently shown to be the most upregulated immune inhibitory receptor on CD8+ T cells in MM patients' bone marrow (BM), compared to other checkpoints (Guillerey C., Blood. 2018). Preclinical models demonstrated the dominant effects of TIGIT blockade or depletion, by significantly improving mice survival, reducing myeloma cell numbers and exhausted T cell hallmarks (Minnie S., Blood. 2018). As a result, several clinical trials using anti-TIGIT monoclonal antibodies have been recently initiated in MM patients. The DNAM-1 family, in addition to TIGIT, also includes the inhibitory receptor PVRIG, that competes with the co-activating receptor DNAM-1 for the binding to the shared ligand PVRL2, similarly to the TIGIT/PVR/DNAM-1 interaction. Accordingly, TIGIT and PVRIG co-blockade were shown to synergize in enhancing T cell activity and anti-tumor activity in preclinical models (Whelan S., Cancer Immunol. Res. 2019). PVRL2 together with PVR (ligand of TIGIT) were shown to be highly expressed on plasma cells and on CD14+ cells in BM of MM patients (Lozano E., Clin. Cancer Res. 2020). This study aimed at evaluating DNAM-1 axis receptors expression in MM patients' BM. Methods Fresh BM aspirates were collected from 21 MM patients with progressive disease (PD) or in complete response (CR) after obtaining IRB approval. BM mononuclear cells were isolated and single cell suspensions were obtained followed by staining with anti-human antibodies to evaluate DNAM-1 axis members and PD-1 expression. BM biopsies from 6 MM patients (each patient had 4 core on the Tissue Micro-Array T291 USBiomax) were stained for PVRL2 expression by immuno-histochemistry (IHC). Results Flow cytometry analysis of PD-1 and DNAM-1 axis receptors revealed a significant lower fraction of PD1+ cells among cell populations examined compared with other receptors. TIGIT expression was the highest on NK, CD8+ and NKT cells compared to CD4+ T cells, which is in line with previous published data (Lozano E. Clin. Cancer Res. 2020). In contrast, DNAM-1 was expressed on CD8+ T, NK and NKT cells with prominent high expression on CD4+ T cells (Fig 1A). The highest expression among the receptors was of PVRIG on all lymphoid populations, except CD4+ where DNAM-1 was more highly expressed. Importantly, 50% of CD8+ T cells co-expressed TIGIT and PVRIG, supporting a combinatorial therapeutic approach (Fig. 1B). Additionally, the expression of the PVRL2 ligand on MM plasma and endothelial cells was demonstrated by IHC. FACS analysis further supported PVRL2 expression on plasma cells in MM BM (Fig 2). A higher expression of PVRIG, TIGIT and PD-1 was present on DNAM-1 negative CD8+ T cells (Fig 3A, B), suggesting accumulation of exhausted cells in MM tumor microenvironment (TME) as previously described (Minnie S., Blood. 2018). PVRIG had significantly higher expression on DNAM+ cells, compared to PD-1 and TIGIT (Fig 3C), suggesting the potential of its blockade to enhance DNAM-1 activation and subsequent proliferation of earlier differentiated memory cells in MM TME. Finally, CR patients had a trend for higher DNAM-1 expression on CD8+ T cells compared to those with PD (Fig 3D). This is consistent with other reports in mice showing that the expression of DNAM-1 negatively correlates with BM myeloma cell numbers (Minnie S., Blood. 2018). Conclusions DNAM-1 axis receptors are dominantly expressed on lymphocytes in BM of MM patients, with PVRIG exhibiting the most prominent expression. The reduced expression of DNAM-1 in PD patients' TME, compared to CR patients, suggests a link between DNAM-1 axis and clinical outcomes. Recent data suggest TIGIT is an attractive target for blockade in MM. Our new findings highlight for the first time the dominant expression of PVRIG, as well as TIGIT, and suggest that combined blockade of TIGIT with PVRIG may potentially benefit MM patients, placing the DNAM-1 axis as a dominant pathway in MM therapy. Figure 1 Figure 1. Disclosures Frenkel: Compugen Ltd.: Current Employment, Other: in the event of frontal participation, I will be reimbursed for my travel expenses by Compugen Ltd.. Alteber: Compugen Ltd.: Current Employment. Cojocaru: Compugen Ltd.: Current Employment. Ophir: Compugen Ltd.: Current Employment.

Tigit, CD226 and PD-L1/PD-1 Are Highly Expressed By Marrow-Infiltrating T Cells in Patients with Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2102-2102 ◽  
Author(s):  
Mahesh Yadav ◽  
Cherie Green ◽  
Connie Ma ◽  
Alberto Robert ◽  
Andrew Glibicky ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Plasma Cells ◽  
Color Flow

Abstract Introduction:TIGIT (T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif [ITIM] domain) is an inhibitory immunoreceptor expressed by T and natural killer (NK) cells that is an important regulator of anti-tumor and anti-viral immunity. TIGIT shares its high-affinity ligand PVR (CD155) with the activating receptor CD226 (DNAM-1). We have recently shown that TIGIT blockade, together with PD-L1/PD-1 blockade, provides robust efficacy in syngeneic tumor and chronic viral infection models. Importantly, CD226 blockade abrogates the benefit of TIGIT blockade, suggesting additional benefit of TIGIT blockade through elaboration of CD226-mediated anti-tumor immunity, analogous to CTLA-4/CD28 regulation of T-cell immunity. Whether TIGIT and CD226 are expressed in patients with multiple myeloma (MM) and how TIGIT expression relates to PD-L1/PD-1 expression is unknown. Here we evaluate expression of TIGIT, CD226, PD-1 and PD-L1 in patients with MM to inform novel immunotherapy combinations. Methods:We performed multi-color flow cytometry (n = 25 patients), and multiplex qRT-PCR (n = 7) on bone marrow specimens from patients with MM to assess expression of TIGIT, CD226, PD-1, and PD-L1 on tumor and immune cells. Cells were stained with fluorescently conjugated monoclonal antibodies to label T cells (CD3, CD4, CD8), NK cells (CD56, CD3), plasma cells (CD38, CD45, CD319, CD56), inhibitory/activating receptors (PD-1, TIGIT, PD-L1, CD226), and an amine-reactive viability dye (7-AAD). Stained and fixed cells were analyzed by flow cytometry using BD FACSCanto™ and BD LSRFortessa™. Results:TIGIT, CD226 and PD-L1/PD-1 were detectable by flow cytometry in all patients with MM who were tested, with some overlapping and distinct expression patterns. TIGIT was commonly expressed by marrow-infiltrating CD8+ T cells (median, 65% of cells), CD4+ T cells (median, 12%) and NK cells. In contrast, CD226 was more commonly expressed by marrow-infiltrating CD4+ T cells (median, 74%) compared with CD8+ T cells (median, 38%). PD-1 was expressed by marrow-infiltrating CD8+ T cells (median 38%) and CD4+ T cells (median, 16%). TIGIT was co-expressed with PD-1 on CD8+ T cells (67%-97% TIGIT+ among PD-1+), although many PD-1-negative CD8+ T cells also expressed TIGIT (39%-78% of PD-1-negative). PD-L1 was also expressed by CD8+ (median, 23%) and CD4+ (median, 8%) T cells in addition to MM plasma cells (median, 95%), albeit with significantly lower intensity on T cells compared with plasma cells. The expression of TIGIT and PD-L1 mRNA was highly correlated (R2 = 0.80). Analysis of PVR expression will also be presented. Conclusions: TIGIT, CD226, PD-1, and PD-L1 were commonly expressed in MM bone marrow, but with different patterns. Among CD8+ T cells, the frequency of TIGIT+ T cells was almost twice that of PD-1+ T cells, whereas the majority of CD4+ T cells expressed CD226. TIGIT blockade may complement anti-PD-L1/PD-1 immunotherapy by activating distinct T-cell/NK-cell subsets with synergistic clinical benefit. These results provide new insight into the immune microenvironment of MM and rationale for targeting both the PD-L1/PD-1 interaction and TIGIT in MM. Disclosures Yadav: Genentech, Inc.: Employment. Green:Genentech, Inc.: Employment. Ma:Genentech, Inc.: Employment. Robert:Genentech, Inc.: Employment. Glibicky:Makro Technologies Inc.: Employment; Genentech, Inc.: Consultancy. Nakamura:Genentech, Inc.: Employment. Sumiyoshi:Genentech, Inc.: Employment. Meng:Genentech, Inc.: Employment, Equity Ownership. Chu:Genentech Inc.: Employment. Wu:Genentech: Employment. Byon:Genentech, Inc.: Employment. Woodard:Genentech, Inc.: Employment. Adamkewicz:Genentech, Inc.: Employment. Grogan:Genentech, Inc.: Employment. Venstrom:Roche-Genentech: Employment.

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.

Carcinoembryonic-Antigen-Related Cell Adhesion Molecule (CEACAM) Expression on Multiple Myelomas Inhibits Their Recognition by Autologous Memory CD8 T Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 738-738
Author(s):  
Mathias Witzens-Harig ◽  
Dirk Hose ◽  
Michael Hundemer ◽  
Simone Jünger ◽  
Anthony D. Ho ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Cell Responses ◽  
Healthy Donors

Abstract Introduction. Previous studies clearly demonstrated the spontaneous induction and accumulation of functionally competent myeloma antigen–specific memory CD8 T cell responses in the bone marrow of a large proportion of myeloma patients. However, other studies convincingly demonstrated that CD8 T cells from myeloma patients were incabable of lysing autologous myeloma cells. An explanation for this apparent discrepancy is still lacking. CEACAMs are induced on T cells during TCR-specific activation and mediate a rapid blocking of T cell effector functions upon homophilic binding with CEACAMs expressed on target cells. We here addressed the question if myeloma cells might escape recognition by autologous, myeloma-specific CD8 T cells through CEACAM expression. Methods. Presence of myeloma-specific CD8 T cells was analyzed by IFN-y Elispot assays using separated, bone marrow derived CD8 T cells and myeloma-associated antigen-pulsed autologous DCs or autolgous myeloma cells as antigen presenting cells. Expression of CEACAMs 1–21 was analyzed by differential gene expression profiling of sorted CD138+ myeloma cells from bone marrow of 140 myeloma patients and of respective plasma cells from 14 healthy donors. In addition, the expression of CEACAMs 1, 5, 6 and 8 was analyzed by flowcytometry on the myeloma cell line MM8226 and on CD138+ myeloma cells from altogether 7 myeloma patients. A role of CEACAM on T cell recognition of autologous myeloma cells was analyzed by coculture of CD8 T cells and sorted, autologous myeloma cells in the presence or absence of blocking antibodies against CEACAMs by IFN-γ Elispot assay. Results. We identified the presence of myeloma-specifcic CD8 T cells in app. 40% of tested myeloma patients (N=20). However, in none of the tested cases T cells were able to directly recognize autologous myeloma cells. Over expression of CEACAM mRNA was found for CEACAM1, 6 and 8 in myeloma cells of up to 20% of patients, but not in plasma cells of healthy donors. Flowcytometric analysis revealed the protein expression of CEACAMs 1 and 6 on 25–50 % of the myeloma cells of all 7 tested patients and on the myeloma cell line MM8226. Blocking of CEACAMs on sorted myeloma cells before their coculture with autologous CD8 T cells resulted in significant T cell responses to myeloma cells in all tested patients (N=6), while in none of these cases the T cells were able to respond to unblocked myeloma cells. Conclusions. We here demonstrate for the first time the expression of CEACAMs 1 and 6 on freshly isolated myeloma cells. Blocking of these CEACAMs resulted in a spontaneous CD8 T cell response against cocultured autologous myeloma cells which was undetectable in case of unblocked CEACAM expression despite the presence of myeloma-reactive memory T cells. We suggest that CEACAMs on myeloma cells inhibit the re-activation of tumour antigen specific CD8 T cells upon interaction with myeloma cells and may contribute to the well characterized immune resistance of multiple myeloma.

scholarly journals Ex Vivo Evidence for the Combination of Immune Checkpoint Inhibition with TGF-β Blockade to Enhance Anti-Tumor T Cell Responses in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3198-3198
Author(s):  
Yoon Seok Choi ◽  
Minsuk Kwon ◽  
Ik-Chan Song ◽  
Deog-Yeon Jo ◽  
Eui-Cheol Shin
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Immune Checkpoint Blockade ◽  
High Level

Abstract Immunosuppressive milieu of multiple myeloma is associated with various cellular and non-cellular factors that foster immune escape leading to tumor progression. While immune checkpoint inhibitors have achieved significant clinical success in many types of solid tumors, recent clinical trials of immune checkpoint blockade performed in patients with multiple myeloma failed to demonstrate significant anti-tumor efficacy. To enhance the clinical efficacy of immune checkpoint blockade in multiple myeloma, elaborate characterization of tumor antigen-specific T cells is an essential prerequisite. In the present study, we investigated the immunophenotypic and functional characteristics of tumor antigen-specific T cells in patients with multiple myeloma. In addition, using direct ex vivo experimental techniques, we tried to examine how to manipulate the immunosuppressive microenvironment to maximize anti-myeloma responses of the tumor-specific T cells. We first tried to define and characterize CD8+ T cell population capable of specifically recognizing and reacting to myeloma cells in bone marrow of myeloma patients using MHC multimer technique. In selected patients, we could successfully define CD8+ T cell population specifically recognizing the HLA-A*0201-restricted epitopes (either "SLLMWITQC" or "LLLGIGILV"), included in myeloma tumor antigens NY-ESO-1 and HM1.24. The vast majority of myeloma-antigen specific CD8+ T cells expressed high level of PD-1 and also co-expressed other types T cell inhibitory receptors. More strikingly, PD-1+ myeloma-specific CD8+ T cells had a distinct pattern of transcriptional factor expression, high level of Eomes and low level of T-bet (EomeshiT-betlo), indicating that they were profoundly exhausted functionally and that a simple blockade of PD-1/PD-L1 binding might not be enough to reinvigorate their anti-myeloma activity. Consistent with the immunophenotypes of myeloma-specific CD8+ T cells, malignant plasma cells in bone marrow of myeloma patients, defined as CD14-CD19-CD138+CS1+CD56hi, also expressed PD-L1 abundantly, compared to normal plasma cells, suggesting that PD-1/PD-L1 axis plays a major role in making myeloma-recognizing T cells unresponsive to TCR stimulation. Interestingly, in addition to tumor cells, various types of immune cells comprising myeloma microenvironment also highly express PD-L1. Indeed, in response to ex vivo TCR stimulation with anti-CD3, CD8+ T cells from myeloma bone marrow showed lower proliferation and reduced production of anti-tumor effector cytokines (INF-γ and TNF-α), compared to marrow-infiltrating CD8+ T cells of diffuse large B cell lymphoma and Hodgkin lymphoma patients with extensive bone marrow involvement. However, even in the presence of anti-PD-1, myeloma-specific responses of marrow-infiltrating CD8+ T cells was only modestly improved in terms of proliferation and cytokine production. Next, we investigated whether blocking TGF-β signaling in combination with PD-1/PD-L1 axis blockade could restore the function of marrow-infiltrating CD8+ T cells of myeloma patients, since TGF-β produced by clonal plasma cells and bone marrow stromal cells is critical in immunosuppressive microenvironment of multiple myeloma. To this end, we found that combination of TGF-β signaling blockade (either anti-TGF-β1 or Galunisertib, a small molecule inhibitor of TGF-β receptor I) with anti-PD-1 significantly increased the frequencies of IFN-γ- and/or TNF-α-producing CD8+ T cells in response to ex vivo TCR stimulation, compared to a single PD-1 or a single TGF-β blockade. Likewise, myeloma antigen-specific proliferation of CD8+ T cells was significantly enhanced with addition of TGF-β signaling blockade. Taken together, although PD-1/PD-L1 axis acts as a major component of immunosuppressive milieu in multiple myeloma, the efficacy of PD-1 blockades in multiple myeloma might be hampered by complicated microenvironment consisting of T cell-intrinsic and -extrinsic factors. Our results provide an ex vivo evidence of incorporating TGF- β signaling blockade to immune checkpoint inhibition to enhance anti-tumor T cell responses in multiple myeloma Disclosures No relevant conflicts of interest to declare.

CD28-Mediated Regulation of Multiple Myeloma Cell Proliferation and Survival.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 355-355
Author(s):  
Kelvin P. Lee ◽  
Nizar J. Bahlis ◽  
Anne M. King ◽  
Despina Kolonias ◽  
Louise M. Carlson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
T Cells ◽  
Cell Survival ◽  
Plasma Cells ◽  
Survival Function ◽  
Myeloma Cell ◽  
Serum Starvation ◽  
Bone Marrow Biopsies

Abstract Although interactions with bone marrow stromal cells are essential for multiple myeloma (MM) cell survival, the specific molecular and cellular elements involved are largely unknown due to the complexity of the bone marrow microenvironment. The CD28 receptor, which costimulates survival signals in T cells, is also expressed on normal plasma cells and myeloma cells. In MM, CD28 expression correlates significantly with disease progression, also suggesting a pro-survival function. In contrast to T cells however, activation and function of CD28 in myeloma and plasma cells is almost entirely undefined. We found that direct activation of myeloma cell CD28 by anti-CD28 mAb alone induced activation of NFkappaB, suppressed MM cell proliferation and protected against serum starvation and dexamethasone-induced cell death. We hypothesized that the specific CD80/CD86 expressing stromal cell partner of this interaction is a professional antigen presenting cells, in particular dendritic cells. Histological studies demonstrated DC were extensively interdigitated throughout the myeloma infiltrates in patient bone marrow biopsies. In vitro coculture with DC also elicited CD28-mediated effects on MM survival and proliferation, and could be blocked by CD28Ig. Our findings suggest a previously undescribed myeloma:DC cell-cell interaction involving CD28 that may play an important role in myeloma cell survival within the bone marrow stroma. These data also suggest that CD28 may represent a therapeutic target in the treatment of multiple myeloma.

Spontaneous CD4 and CD8 Memory T Cell Responses Against MUC1 and Carcinoembryonic Antigen in Bone Marrow of Multiple Myeloma Patients.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3533-3533
Author(s):  
Mathias Witzens-Harig ◽  
Dirk Hose ◽  
Michael Hundemer ◽  
Simone Juenger ◽  
Anthony D. Ho ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Carcinoembryonic Antigen ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cd4 T Cell ◽  
Cell Responses

Abstract Introduction: The bone marrow (BM) is a site of induction of tumour antigen specific T cell responses in many malignancies. We have demonstrated in the BM of myeloma patients high frequencies of spontaneously generated CD8 memory T cells with specificity for the myeloma-associated antigen MUC1, which were not detectable in the peripheral blood (PB). Besides MUC1, carcinoembryonic antigen was recently identified as a tumour-associated antigen in a patient with multiple myeloma. Up to now, spontaneous CD4 T cell responses against myeloma-associated antigens have not been reported. We undertook this study to evaluate to what extent spontaneous CD4 T cell responses against myeloma antigens occur during myeloma progression and if MUC1 or carcinoembryonic antigen represent immunogenic targets of spontaneous CD4 and CD8 T cell responses. Methods: Altogether, 78 patients with multiple myeloma were included into the study. Presence of functionally competent antigen specific T cells was evaluated by ex vivo short term (40 h) IFN-γ Elispot analyses. CD4 T cell responses against MUC1 were assessed by stimulation of purified CD4 T cell fractions with antigen pulsed, autologous dendritic cells (DCs) pulsed with two synthetic 100 meric polypeptides (pp1-100ss and (137–157)5 tr) that can be processed and presented via multiple HLA-II alleles. CD4- or CD8 T cell reactivity against carcinoembryonic antigen was assessed on purified CD4- and CD8 T cell fractions by pulsing DCs with highly purified CEA derived from culture supernatants of an epithelial carcinoma cell line. CD8 responses against MUC1 were analyzed by stimulation of HLA-A2+ patients derived purified T cells with DCs loaded with HLA-A2 restricted MUC1-derived nonameric peptide LLLLTVLTV. As negative control antigen for MUC1 polypeptides and CEA human IgG was used for pulsing DCs at identical concentrations while HLA-A2-restricted peptide SLYNTVATL derived from HIV was used as control antigen for LLLLTVLTV. Test antigen specific reactivity was defined by significantly increased numbers of IFN-γ spots in triplicate test wells compared to control wells (p<0.05, students T test). Results: 8 out of 19 tested patients (42%) contained MUC1 specific CD8 T cells in their bone marrow, while MUC1 specific CD4 T cells were detected in the BM of 30% of the cases (3/10). Interestingly, in peripheral blood (PB) CD8 reactivity against MUC1 was detectable in only 1 out of 10 patients while CD4 reactivity in PB was not detectable at all (0/10). CEA was specifically recognized by BM CD8 T cells from 5 out of 30 patients (17%) and by BM CD4 T cells from 5 out of 18 patients (28%). CEA was not recognized by CD4 and CD8 T cells in the PB of the same patients (0/13). Conclusion: Spontaneous T helper responses against tumour-associated antigens occur in the BM at similar levels as antigen specific CD8 T cells responses while they are virtually undetectable in the PB. Compared to CEA, MUC1 induces CD8 T cell responses in a much higher proportion of myeloma patients. Nevertheless, our data suggest that CEA may trigger spontaneous T cell responses against multiple myeloma in a considerable number of patients. Thus, systematic functional analyses of this potential tumour antigen in multiple myeloma appears to be justified.

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.

scholarly journals The Combination of Anti-Tigit and Lenalidomide Promotes Synergistic Myeloma-Specific Immunity after ASCT

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 328-328
Author(s):  
Simone A Minnie ◽  
Nicole S Nemychenkov ◽  
Kathleen S Ensbey ◽  
Christine R Schmidt ◽  
Gregory Driessens ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Median Survival ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Molecular Transport ◽  
Immunomodulatory Drugs ◽  
Preclinical Models ◽  
Specific Immunity

Abstract Multiple myeloma is a largely incurable bone marrow (BM) resident plasma cell malignancy that is increasing in incidence. Autologous stem cell transplantation (ASCT) is the current standard consolidation therapy and a subset of patients achieve durable progression free survival that is suggestive of long-term immune control. Utilizing novel preclinical models, we have provided definitive evidence that this is largely mediated by T cell-dependent myeloma-specific immunity. In both patients and preclinical models, myeloma progression is associated with T cell dysfunction and expression of multiple inhibitory receptors suggesting a loss of immunosurveillance. In mice, we have demonstrated potent anti-myeloma efficacy of TIGIT blockade in both ASCT and non-transplant settings. Here we utilized identical TIGIT Abs that do or do not Fc bind to demonstrate that immunological efficacy after ASCT was absolutely dependent on ADCC (median survival was unreached (&gt;110 days) in Fc-binding vs 73 days in Fc-dead and 71 days in control Ig (cIg)-treated mice). Since TIGIT inhibition does not protect against myeloma relapse in all mice, it is apparent that combinational approaches are required to target non-responders. Therefore, we hypothesized that TIGIT blockade could be combined with immunomodulatory drugs (IMiDs) to provide synergistic anti-myeloma activity after ASCT. To that end, we utilized CRBN transgenic mice to investigate the efficacy of TIGIT blockade in combination with lenalidomide, the standard of care IMiD used in maintenance therapy after clinical ASCT. Briefly, B6 Vk*MYC myeloma-bearing (MM-bearing) mice were lethally irradiated and transplanted with B6 bone marrow (BM) and a suboptimal dose of T cells followed by anti-TIGIT or control Ig (100 mg twice weekly) for 5 weeks with lenalidomide (50 mg/kg daily gavage) or control diluent from D+14 for 3 weeks (Figure 1A). The combination of anti-TIGIT and lenalidomide provided synergistic anti-myeloma efficacy evidenced by prolonged median survival (109 days in combination vs &lt; 60 days in monotherapy/control-treated mice, p&lt;0.01; Figure B). Myeloma M bands were also suppressed in the combination treated mice relative to monotherapy or cIg-treated mice (p&lt;0.01; Figure 1). Analysis of BM CD8 T cells 6 weeks after ASCT demonstrated that combination therapy significantly decreased terminal exhaustion (TOX + TIM3 + CD101 + PD-1 + DNAM-1 ─) with an average of only 20% of CD8 T cells with an exhausted phenotype in the combination group compared to greater than 50% exhausted CD8 T cells in monotherapy or cIg-treated mice (p&lt;0.05; Figure 1C-D). The combination also increased the frequency of central memory and tissue-resident memory subsets (CD49b +CD69 +; p&lt;0.05; Figure 1C-D), and increased IFNγ production from activated (PD-1 +; p&lt;0.05; Figure 1E) cells compared to monotherapy or control Ig-treated mice. Importantly, these phenotypic changes were specific to the BM tumor microenvironment as we observed no effect of combination or monotherapy treatment on CD8 or CD4 T cells in peripheral blood. In sum, these data provide a strong rationale for combining TIGIT inhibition with immunomodulatory drugs to prevent the progression of myeloma. Figure 1 Figure 1. Disclosures Driessens: iTeos Therapeutics: Current Employment, Current equity holder in publicly-traded company. Holmberg: Up-To-Date: Patents & Royalties; Bristol Myers Squibb: Research Funding; Janssen: Research Funding; Merck: Research Funding; Millennium-Takeda: Research Funding; Sanofi: Research Funding; Seattle Genetics: Research Funding. Hill: NeoLeukin Therapeutics: Consultancy; Compass Therapeutics: Research Funding; NapaJen Pharma: Consultancy; Generon Corporation: Consultancy; Roche: Research Funding; iTeos Therapeutics: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding; Applied Molecular Transport: Research Funding.

scholarly journals REGN5458, a Bispecific BCMAxCD3 T Cell Engaging Antibody, Demonstrates Robust In Vitro and In Vivo Anti-Tumor Efficacy in Multiple Myeloma Models, Comparable to That of BCMA CAR T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1944-1944 ◽  
Author(s):  
David J Dilillo ◽  
Kara Olson ◽  
Katja Mohrs ◽  
T. Craig Meagher ◽  
Kevin Bray ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T

Abstract Improving therapies for multiple myeloma (MM) remains a high medical need because of the significant morbidity and mortality of the disease. Targeted immunotherapies represent a promising opportunity to fill this clinical need. B cell maturation antigen (BCMA) is an attractive cell-surface target for MM due to its consistent expression on MM patient malignant plasma cells and expression limited in normal tissue primarily to plasma cells. Redirection of a patient's T cells to recognize tumors by CD3-binding bispecific molecules or through the generation of chimeric antigen receptor (CAR) T cells, has shown preliminary evidence of clinical activity. Bispecific antibodies concurrently engage a tumor antigen on cancer cells and the CD3 signaling machinery on T cells, bringing the tumor cell and T cell into proximity and facilitating T cell activation and tumor cell killing. By contrast, CAR T cell therapy involves re-infusion of the patient's own T cells after ex vivo engineering to express CARs targeting tumor antigens and triggering T cell signaling. Here we describe the generation of REGN5458, a human bispecific antibody that binds to BCMA and CD3. In vitro, REGN5458 efficiently activates T cells and induces polyclonal T cell killing of myeloma cell lines with a range of BCMA cell-surface densities, and also induces cytotoxicity of primary human plasma cells. Similar to gamma-sectretase inhibitors, incubation of myeloma cell lines with REGN5458 increased surface levels of BCMA. In xenogenic studies, after BCMAhigh NCI-H929 and BCMAlow MOLP-8 MM cells were co-implanted with PBMC and grown subcutaneously in immunodeficient NOD/SCID/L2Rgamma-deficient (NSG) mice, REGN5458 doses as low as 0.4 mg/kg significantly suppressed the growth of both tumors. Using aggressive, systemic xenogenic tumor models, in which NSG mice were engrafted with PBMC and intravenously injected with BCMAhigh OPM-2 cells or BCMAlow MOLP-8 cells expressing luciferase, REGN5458 reduced tumor burden and suppressed tumor growth at doses as low as 0.4 mg/kg. In immunocompetent mice genetically engineered to express human CD3, REGN5458 inhibited the growth of syngeneic murine tumors expressing human BCMA at doses as low as 0.04 mg/kg. Finally, as REGN5458 binds to cynomolgus CD3 and BCMA and mediates cytotoxicity of primary cynomolgus plasma cells, the pharmacology of REGN5458 was evaluated in cynomolgus monkeys. REGN5458 administration was well-tolerated, resulting in a mild inflammatory response characterized by transiently increased CRP and serum cytokines. Importantly, REGN5458 treatment led to the depletion of BCMA+ plasma cells in the bone marrow, demonstrating cytotoxic activity in non-human primates. The anti-tumor efficacy of REGN5458 was compared to BCMA-specific CAR T cells using 2nd generation CAR lentiviral constructs containing a single-chain variable fragment binding domain from REGN5458's BCMA binding arm and 4-1BB and CD3z signaling domains. Human PBMC-derived T cells were transduced to express this CAR and expanded. Both REGN5458 and the BCMA CAR T cells demonstrated similar targeted cytotoxicity of myeloma cell lines and primary patient blasts in vitro, and were capable of clearing established systemic OPM-2-luciferase myeloma tumors in NSG mice, but with different kinetics: treatment with REGN5458 resulted in rapid clearance of tumors within 4 days, whereas treatment with BCMA CAR T cells allowed tumors to continue to grow for 10-14 days following injection before rapidly inducing tumor clearance. Thus, REGN5458 exerts its therapeutic effect rapidly after injection, using effector T cells that are already in place. In contrast, BCMA CAR T cells require time to traffic to the tumor site and expand, before exerting anti-tumor effects. Collectively, these data demonstrate the potent pre-clinical anti-tumor activity of REGN5458 that is comparable to that of CAR T cells, and provide a strong rationale for clinical testing of REGN5458 in patients with MM. Disclosures Dilillo: Regeneron Pharmaceuticals: Employment. Olson:Regeneron Pharmaceuticals: Employment. Mohrs:Regeneron Pharmaceuticals: Employment. Meagher:Regeneron Pharmaceuticals: Employment. Bray:Regeneron Pharmaceuticals: Employment. Sineshchekova:Regeneron Pharmaceuticals: Employment. Startz:Regeneron Pharmaceuticals: Employment. Retter:Regeneron Pharmaceuticals: Employment. Godin:Regeneron Pharmaceuticals: Employment. Delfino:Regeneron Pharmaceuticals: Employment. Lin:Regeneron Pharmaceuticals: Employment. Smith:Regeneron Pharmaceuticals: Employment. Thurston:Regeneron Pharmaceuticals: Employment. Kirshner:Regeneron Pharmaceuticals: Employment.

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