Memory Cytotoxic T Lymphocytes Research Articles

Differentiation of BCMA-specific induced pluripotent stem cells into rejuvenated CD8αβ+ T cells targeting multiple myeloma

AbstractA major hurdle in adoptive T-cell therapy is cell exhaustion and failure to maintain antitumor responses. Here, we introduce an induced pluripotent stem cell (iPSC) strategy for reprogramming and revitalizing precursor exhausted B-cell maturation antigen (BCMA)-specific T cells to effectively target multiple myeloma (MM). Heteroclitic BCMA72-80 (YLMFLLRKI)–specific CD8+ memory cytotoxic T lymphocytes (CTL) were epigenetically reprogrammed to a pluripotent state, developed into hematopoietic progenitor cells (CD34+ CD43+/CD14− CD235a−), differentiated into the T-cell lineage and evaluated for their polyfunctional activities against MM. The final T-cell products demonstrated (1) mature CD8αβ+ memory phenotype, (2) high expression of activation or costimulatory molecules (CD38, CD28, and 41BB), (3) no expression of immune checkpoint and senescence markers (CTLA4, PD1, LAG3, and TIM3; CD57), and (4) robust proliferation and polyfunctional immune responses to MM. The BCMA-specific iPSC–T cells possessed a single T-cell receptor clonotype with cognate BCMA peptide recognition and specificity for targeting MM. RNA sequencing analyses revealed distinct genome-wide shifts and a distinctive transcriptional profile in selected iPSC clones, which can develop CD8αβ+ memory T cells. This includes a repertoire of gene regulators promoting T-cell lineage development, memory CTL activation, and immune response regulation (LCK, IL7R, 4-1BB, TRAIL, GZMB, FOXF1, and ITGA1). This study highlights the potential application of iPSC technology to an adaptive T-cell therapy protocol and identifies specific transcriptional patterns that could serve as a biomarker for selection of suitable iPSC clones for the successful development of antigen-specific CD8αβ+ memory T cells to improve the outcome in patients with MM.

Enhanced antitumor activity of a novel, oral, helper epitope-containing WT1 protein vaccine in a model of murine leukemia

BackgroundA Wilms’ tumor 1 (WT1) oral vaccine, Bifidobacterium longum (B. longum) 420, in which the bacterium is used as a vector for WT1 protein, triggers immune responses through cellular immunity consisting of cytotoxic T lymphocytes (CTLs) and other immunocompetent cells (e.g., helper T cells). We developed a novel, oral, helper epitope-containing WT1 protein vaccine (B. longum 2656) to examine whether or not B. longum 420/2656 combination further accelerates the CD4+ T cell help-enhanced antitumor activity in a model of murine leukemia.MethodsC1498-murine WT1—a genetically-engineered, murine leukemia cell line to express murine WT1—was used as tumor cell. Female C57BL/6 J mice were allocated to the B. longum 420, 2656, and 420/2656 combination groups. The day of subcutaneous inoculation of tumor cells was considered as day 0, and successful engraftment was verified on day 7. The oral administration of the vaccine by gavage was initiated on day 8. Tumor volume, the frequency and phenotypes of WT1-specific CTLs in CD8+ T cells in peripheral blood (PB) and tumor-infiltrating lymphocytes (TILs), as well as the proportion of interferon-gamma (INF-γ)-producing CD3+CD4+ T cells pulsed with WT135–52 peptide in splenocytes and TILs were determined.ResultsTumor volume was significantly smaller (p < 0.01) in the B. longum 420/2656 combination group than in the B. longum 420 group on day 24. WT1-specific CTL frequency in CD8+ T cells in PB was significantly greater in the B. longum 420/2656 combination group than in the B. longum 420 group at weeks 4 (p < 0.05) and 6 (p < 0.01). The proportion of WT1-specific, effector memory CTLs in PB increased significantly in the B. longum 420/2656 combination group than in the B. longum 420 group at weeks 4 and 6 (p < 0.05 each). WT1-specific CTL frequency in intratumoral CD8+ T cells and the proportion of IFN-γ-producing CD3+CD4+ T cells in intratumoral CD4+ T cells increased significantly (p < 0.05 each) in the B. longum 420/2656 combination group than in the 420 group.ConclusionsB. longum 420/2656 combination further accelerated antitumor activity that relies on WT1-specific CTLs in the tumor compared with B. longum 420.

Mycobacterium bovis BCG Given at Birth Followed by Inactivated Respiratory Syncytial Virus Vaccine Prevents Vaccine-Enhanced Disease by Promoting Trained Macrophages and Resident Memory T Cells.

Respiratory syncytial virus (RSV) infects more than 60% of infants in their first year of life. Since an experimental formalin-inactivated (FI) RSV vaccine tested in the 1960s caused enhanced respiratory disease (ERD), few attempts have been made to vaccinate infants. ERD is characterized by Th2-biased responses, lung inflammation, and poor protective immune memory. Innate immune memory displays an increased nonspecific effector function upon restimulation, a process called trained immunity, or a repressed effector function upon restimulation, a process called tolerance, which participates in host defense and inflammatory disease. Mycobacterium bovis bacillus Calmette-Guérin (BCG) given at birth can induce trained immunity as well as heterologous Th1 responses. We speculate that BCG given at birth followed by FI-RSV may alleviate ERD and enhance protection through promoting trained immunity and balanced Th immune memory. Neonatal mice were given BCG at birth and then vaccinated with FI-RSV+Al(OH)3. BCG/FI-RSV+Al(OH)3 induced trained macrophages, tissue-resident memory T cells (TRM), and specific cytotoxic T lymphocytes (CTL) in lungs and inhibited Th2 and Th17 cell immune memory, all of which contributed to inhibition of ERD and increased protection. Notably, FI-RSV+Al(OH)3 induced tolerant macrophages, while BCG/FI-RSV+Al(OH)3 prevented the innate tolerance through promoting trained macrophages. Moreover, inhibition of ERD was attributed to trained macrophages or TRM in lungs but not memory T cells in spleens. Therefore, BCG given at birth to regulate trained immunity and TRM may be a new strategy for developing safe and effective RSV killed vaccines for young infants. IMPORTANCE RSV is the leading cause of severe lower respiratory tract infection of infants. ERD, characterized by Th2-biased responses, inflammation, and poor immune memory, has been an obstacle to the development of safe and effective killed RSV vaccines. Innate immune memory participates in host defense and inflammatory disease. BCG given at birth can induce trained immunity as well as heterologous Th1 responses. Our results showed that BCG/FI-RSV+Al(OH)3 induced trained macrophages, TRM, specific CTL, and balanced Th cell immune memory, which contributed to inhibition of ERD and increased protection. Notably, FI-RSV+Al(OH)3 induced tolerant macrophages, while BCG/FI-RSV+Al(OH)3 prevented tolerance through promoting trained macrophages. Moreover, inhibition of ERD was attributed to trained macrophages or TRM in lungs but not memory T cells in spleens. BCG at birth as an adjuvant to regulate trained immunity and TRM may be a new strategy for developing safe and effective RSV killed vaccines for young infants.

Vaccination with a shared oncogenic tumor-self antigen elicits a population of CD8+ T cells with a regulatory phenotype

ABSTRACT Cancer immunotherapy is a powerful tool for inducing antigen-specific antitumor cytotoxic T lymphocytes (CTLs). Next-generation strategies may include vaccination against overexpressed oncogenic tumor-self antigens. Previously, we reported vaccination against the oncogenic tumor-self antigen D52 (D52) was effective in preventing tumor growth. We recently reported that D52-vaccinated IL-10-deficient mice generated a significant memory response against tumor recurrence compared to wild-type mice and that vaccine-induced CD8+ IL-10+ T cells may possess regulatory function. Herein, we extended these studies by testing the hypothesis that D52-vaccine-elicited CD8+ IL-10+ T cells represent a distinct T cell population with a regulatory phenotype. C57Black/6J mice deficient in IL-10 or IFN-γ were vaccinated with the murine orthologue of D52; vaccination of wild-type (wt) mice served as a control for comparison. T cells were isolated from all three groups of vaccinated mice, and RNA was extracted from purified CD8+ T cells for deep sequencing and expression analysis. Chemokine receptor 8 (CCR8) and inducible co-stimulator (ICOS) were overexpressed in CD8+ T cells that produced IL-10 but not IFN-γ. These surface markers are associated with IL-10 producing CD4+ T regulatory cells thus supporting the possibility that CD8+ IL-10+ T cells elicited by D52 vaccination represent a unique regulatory T cell subset. The current phenotypic analyses of D52 vaccine elicited CD8+ T cells strengthen our premise that CD8+ IL-10+ T cells elicited by D52 tumor-self protein vaccination likely contribute to the suppression of memory CTL responses and inhibition of durable tumor immunity.

Abstract 6347: Immunotherapeutic application of induced pluripotent stem cell technology: Rejuvenated BCMA-specific CD8+T cells for multiple myeloma

Abstract T cell regenerative medicine represents an immunotherapeutic approach using antigen-specific induced Pluripotent Stem Cells (iPSC) to rejuvenate CD8+ cytotoxic T lymphocytes (CTL). Here, we report an iPSC-derived therapeutic strategy targeting B-Cell Maturation Antigen (BCMA) to overcome exhaustion of antigen-specific CTL and revitalize them to fully functional antigen-specific memory T cells to target multiple myeloma (MM). First, we established the iPSC by reprogramming IFN-γ producing heteroclitic BCMA72-80 (YLMFLLRKI) peptide-specific CD8+ CTL via Sendai virus transduction of transcription factors, OCT3/4, SOX2, KLF4 and c-MYC. The BCMA-specific iPSC demonstrated high pluripotency potential and ability to differentiate into three key germ layers, as evidenced by expression of stem cell markers (SSEA-4, TRA1-60), germ differentiation markers (SOX-17 on Endoderm, Brachyury on Mesoderm, and Pax-6 on Ectoderm) and alkaline phosphatase. The polarization of iPSC was followed during embryoid body formation into mesoderm development, evidenced by activation of transcriptional regulators SNAI2, TBX3, PLVAP, HAND1 and CDX2. Furthermore, hematopoietic progenitor cells (HPC; CD34+ CD43+/CD14- CD235a-) were sorted and induced to undergo T cell development under feeder-free culture conditions in the presence of rectonectin. Upon differentiation, phenotypic characterization revealed fully mature T cells with high expression (&amp;gt; 95%) of CD3, CD45, TCRαβ and CD8αβ, which were predominantly CD45RO+ memory T cells with high activation (CD38) and costimulatory (CD28) molecule expression, while lacking immune checkpoints (CTLA4, PD1, LAG3, Tim3). This phenotype was aligned with their high proliferative (1,800-fold increase) capacity and effective anti-tumor cytotoxicity and Th1 cytokine (IFN-γ, IL-2, TNF-α) production against MM patients’ tumor cells in antigen-specific and HLA-A2-restricted manner. Their anti-MM activities were specifically directed against the parent heteroclitic BCMA72-80 peptide via a distinct sole T cell receptor clonotype. RNAseq analyses identified specific transcriptional pathways utilized by BCMA-specific HPC during their differentiation into CD8+ CTL, which include upregulation of transcriptional regulators determining CD4/CD8 T cell differentiation ratio, memory CTL formation, NF-kappa-B/JNK pathway activation, as well as downregulation of regulators controlling B and T cell interactions or CD4+ Th cells and inhibitory receptor development. In summary, these results highlight the processes and pathways mediating somatic T cell epigenetic reprogramming and differentiation into rejuvenated BCMA-specific CD8+ CTL with high proliferation and functional anti-MM activities, providing the framework for regenerative medicine as an adoptive immunotherapy to improve patient outcome in MM. Citation Format: Jooeun Bae, Shuichi Kitayama, Zach Herbert, Laurence Daheron, Nikhil Munshi, Shin Kaneko, Jerome Ritz, Kenneth Anderson. Immunotherapeutic application of induced pluripotent stem cell technology: Rejuvenated BCMA-specific CD8+T cells for multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6347.

Adaptive antitumor immune response stimulated by bio-nanoparticle based vaccine and checkpoint blockade

BackgroundInteractions between tumor and microenvironment determine individual response to immunotherapy. Triple negative breast cancer (TNBC) and hepatocellular carcinoma (HCC) have exhibited suboptimal responses to immune checkpoint inhibitors (ICIs). Aspartate β-hydroxylase (ASPH), an oncofetal protein and tumor associated antigen (TAA), is a potential target for immunotherapy.MethodsSubcutaneous HCC and orthotopic TNBC murine models were established in immunocompetent BALB/c mice with injection of BNL-T3 and 4 T1 cells, respectively. Immunohistochemistry, immunofluorescence, H&E, flow cytometry, ELISA and in vitro cytotoxicity assays were performed.ResultsThe ASPH-MYC signaling cascade upregulates PD-L1 expression on breast and liver tumor cells. A bio-nanoparticle based λ phage vaccine targeting ASPH was administrated to mice harboring syngeneic HCC or TNBC tumors, either alone or in combination with PD-1 blockade. In control, autocrine chemokine ligand 13 (CXCL13)-C-X-C chemokine receptor type 5 (CXCR5) axis promoted tumor development and progression in HCC and TNBC. Interactions between PD-L1+ cancer cells and PD-1+ T cells resulted in T cell exhaustion and apoptosis, causing immune evasion of cancer cells. In contrast, combination therapy (Vaccine+PD-1 inhibitor) significantly suppressed primary hepatic or mammary tumor growth (with distant pulmonary metastases in TNBC). Adaptive immune responses were attributed to expansion of activated CD4+ T helper type 1 (Th1)/CD8+ cytotoxic T cells (CTLs) that displayed enhanced effector functions, and maturation of plasma cells that secreted high titers of ASPH-specific antibody. Combination therapy significantly reduced tumor infiltration of immunosuppressive CD4+/CD25+/FOXP3+ Tregs. When the PD-1/PD-L1 signal was inhibited, CXCL13 produced by ASPH+ cancer cells recruited CXCR5+/CD8+ T lymphocytes to tertiary lymphoid structures (TLSs), comprising effector and memory CTLs, T follicular helper cells, B cell germinal center, and follicular dendritic cells. TLSs facilitate activation and maturation of DCs and actively recruit immune subsets to tumor microenvironment. These CTLs secreted CXCL13 to recruit more CXCR5+ immune cells and to lyse CXCR5+ cancer cells. Upon combination treatment, formation of TLSs predicts sensitivity to ICI blockade. Combination therapy substantially prolonged overall survival of mice with HCC or TNBC.ConclusionsSynergistic antitumor efficacy attributable to a λ phage vaccine specifically targeting ASPH, an ideal TAA, combined with ICIs, inhibits tumor growth and progression of TNBC and HCC.

HTLV-1 Tax-specific memory cytotoxic T lymphocytes in long-term survivors of aggressive-type adult T-cell leukemia/lymphoma.

PurposeAdult T‐cell leukemia/lymphoma (ATLL) is a relatively refractory peripheral T‐cell lymphoma caused by human T‐cell lymphotropic virus type 1 (HTLV‐1). The objective of this study was to investigate the characteristics of long‐term survivors with ATLL.MethodsWe conducted an observational study of 75 aggressive‐type ATLL patients. Flow cytometry was conducted to analyze HTLV‐1 Tax‐specific cytotoxic T‐lymphocytes (CTLs) and T‐cell receptor Vβ gene repertoire.ResultsWe first evaluated six long‐term survivors among 37 patients who were newly diagnosed with ATLL and then treated with intensive chemotherapy without mogamulizumab, a monoclonal antibody for C‐C chemokine receptor four antigen. Reversal of the CD4‐to‐CD8 ratio (CD4/CD8) in peripheral mononuclear cells was observed in all six patients. Three of these six patients showed reversed CD4/CD8 immediately after herpes virus infection. Four of these six patients who could be examined demonstrated long‐term maintenance of HTLV‐1 Tax‐specific CTLs. We subsequently identified four long‐term survivors among 38 patients who were newly diagnosed with ATLL and then treated with intensive chemotherapy plus mogamulizumab. All four patients showed reversed CD4/CD8, and three of the four patients contracted herpes virus infection during immunochemotherapy. Six of the total 10 patients were subjected to CTL analyses. Tax‐specific CTLs were observed, and the CTLs that were almost entirely composed of memory CTLs in all patients were recorded. HTLV‐1 provirus was also detected in all six patients.ConclusionsThese data suggest that Tax‐specific memory CTLs probably, together with anticancer agents, eradicate ATLL cells and exhibit long‐term preventive effects from relapse ATLL. Thus, the strong activation of cellular immunity, such as herpes virus infection, seems to be necessary to induce such a potent number of Tax‐specific CTLs.

The endothelial diapedesis synapse regulates transcellular migration of human T lymphocytes in a CX3CL1- and SNAP23-dependent manner.

Understanding how cytotoxic T lymphocytes (CTLs) efficiently leave the circulation to target cancer cells or contribute to inflammation is of high medical interest. Here, we demonstrate that human central memory CTLs cross the endothelium in a predominantly paracellular fashion, whereas effector and effector memory CTLs cross the endothelium preferably in a transcellular fashion. We find that effector CTLs show a round morphology upon adhesion and induce a synapse-like interaction with the endothelium where ICAM-1 is distributed at the periphery. Moreover, the interaction of ICAM-1:β2integrin and endothelial-derived CX3CL1:CX3CR1 enables transcellular migration. Mechanistically, we find that ICAM-1 clustering recruits the SNARE-family protein SNAP23, as well as syntaxin-3 and -4, for the local release of endothelial-derived chemokines like CXCL1/8/10. In line, silencing of endothelial SNAP23 drives CTLs across the endothelium in a paracellular fashion. In conclusion, our data suggest that CTLs trigger local chemokine release from the endothelium through ICAM-1-driven signals driving transcellular migration.

Evaluation of the peripheral blood T and B cell subsets and IRF-7 variants in adult patients with severe influenza virus infection.

Background and AimsInfluenza virus is one of the leading infections causing death among human being. Despite known risks, primary immune deficiency due to Interferon Regulatory Factor‐7 (IRF7) gene defect was reported as a possible cause of the risk factors for complicated influenza. We aimed to investigate the changes in peripheral T and B cell subsets in adult patients with severe seasonal influenza virus infection and the investigation of variants of IRF7 gene.MethodsIn this study, 32 patients, hospitalized due to influenza infection‐related acute respiratory failure were included.ResultsThe median age of the patients was 76 years (26‐96), and 13/32 (40.6%) were in the intensive care unit. Central memory Th, effector memory Th, TEMRA Th, cytotoxic T lymphocytes (CTL), central memory CTL of the patients were found to be increased, naive CTL were decreased. There was a significant increase in the percentage of effector memory Th, and a decrease in the percentage of naive CTL in patients ≥65 years‐old compared to patients <65 years old (P = .039, and P = .017, respectively). IRF7 gene analysis revealed two different nucleotide changes in three patients; c.535 A > G; p.Lys179Glu ( K179E ) and c584A > T; p.His195Leu ( H195L ), located in the fourth exon of the IRF7 gene.DiscussionThe increases in central and effector memory Th, central memory CTL and decrease of naive CTLs may be secondary to the virus infection. K179E (rs1061502) and H195L (rs139709725) variants were not reported to be related with susceptibility to an infection yet. It is conceivable to investigate for novel variants in other genes related to antiviral immunity.

Rejuvenated BCMA-Specific CD8 + Cytotoxic T Lymphocytes Derived from Antigen-Specific Induced Pluripotent Stem Cells : Immunotherapeutic Application in Multiple Myeloma

T cell regenerative medicine represents an emerging immunotherapeutic approach using antigen-specific Induced Pluripotent Stem Cells (iPSC) to rejuvenate CD8 + cytotoxic T lymphocytes (CTL). Here we report on an iPSC-derived therapeutic strategy targeting B-Cell Maturation Antigen (BCMA) against multiple myeloma (MM) via establishment of antigen-specific iPSC, followed by differentiation into highly functional BCMA-specific CD8 + CTL. The reprogrammed BCMA-specific iPSC displayed normal karyotypes and pluripotency potential as evidenced by expression of stem cell markers (SSEA-4, TRA1-60) and alkaline phosphatase, along with differentiation into three germ layers (Ectoderm, Mesoderm, Endoderm). During embryoid body formation, BCMA-specific iPSC further polarized into the mesoderm germ layer, evidenced by the activation of SNAI2, TBX3, PLVAP, HAND1 and CDX2 transcriptional regulators. Next, the BCMA-specific iPSC clones committed to CD8 + T cell differentiation were characterized by analyzing their hematopoietic progenitor cells (HPC; CD34 + CD43 +/CD14 - CD235a -) for specific transcriptional regulation. RNAseq analyses indicated a low variability and similar profiles of gene transcription within the iPSC clones committed to CD8 + CTL compared to increased transcriptional variability within iPSC clones committed to different cell types. The unique transcriptional profiles of the iPSC committed to CD8 + T cells included upregulation of transcriptional regulators controlling CD4/CD8 T cell differentiation ratio, memory CTL formation, NF-kappa-B/JNK pathway activation, and cytokine transporter/cytotoxic mediator development, as well as downregulation of regulators controlling B and T cell interactions, CD4 + Th cells, and inhibitory receptor development. Specifically, a major regulatory shift, indicated by upregulation of specific genes involved in immune function, was detected in HPC from the iPSC committed to CD8 + T cells. BCMA-specific T cells differentiated from the iPSC were characterized as displaying mature CTL phenotypes including high expression of CD3, CD8a, CD8b, TCRab, CD7 along with no CD4 expression (Fig. 1). In addition, the final BCMA iPSC-T cells were predominantly CD45RO + memory cells (central memory and effector memory cells) expressing high level of T cell activation (CD38, CD69) and costimulatory (CD28) molecules. Importantly, these BCMA iPSC-T cells lacked immune checkpoints (CTLA4, PD1, LAG3, Tim3) expression and regulatory T cells induction, distinct from other antigen-stimulated T cells. The rejuvenated BCMA iPSC-T cells demonstrated a high proliferative (1,000 folds increase) during the differentiation process as well as poly-functional anti-tumor activities and Th1 cytokine (IFN-g, IL-2, TNF-a) production triggered in response to MM patients' cells in HLA-A2-restricted manner (Fig. 2). Furthermore, the immune responses induced by these BCMA iPSC-T cells were specific to the parent heteroclitic BCMA 72-80 (YLMFLLRKI) peptide used to reprogram and establish the antigen-specific iPSC. Evaluation of 88 single cell Tetramer + CTL from the BCMA iPSC-T cells revealed a clonotype of unique T cell receptor (TCRa, TCRb) sequence. The BCMA-specific iPSC clones maintained their specific differentiation potential into the antigen-specific CD8 + memory T cells, following multiple subcloning in long-term cultures under feeder-free conditions or post-thaw after long-term (18 months) cryopreservation at -140 oC, which provides additional benefits to treat patients in a continuous manner. Taken together, rejuvenated CD8 + CTL differentiated from BCMA-specific iPSC were highly functional with significant (*p < 0.05) levels of anti-MM activities including proliferation, cytotoxic activity and Th-1 cytokine production. Therefore, the antigen-specific iPSC reprogramming and T cells rejuvenation process can provide an effective and long-term source of antigen-specific memory CTL lacking immune checkpoints and suppressors for clinical application in adoptive immunotherapy to improve patient outcome in MM. [Display omitted] DisclosuresMunshi: Amgen: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Legend: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Pfizer: Consultancy. Ritz: Amgen: Research Funding; Equillium: Research Funding; Kite/Gilead: Research Funding; Avrobio: Membership on an entity's Board of Directors or advisory committees; Akron: Consultancy; Biotech: Consultancy; Blackstone Life Sciences Advisor: Consultancy; Clade Therapeutics, Garuda Therapeutics: Consultancy; Immunitas Therapeutic: Consultancy; LifeVault Bio: Consultancy; Novartis: Consultancy; Rheos Medicines: Consultancy; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: 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; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

P-083: Rejuvenated BCMA-specific CD8+ Cytotoxic T lymphocytes derived from induced pluripotent stem cells for treatment of Multiple Myeloma

<h3>Background</h3> This study reports on the reprogramming of B-Cell Maturation Antigen (BCMA)-specific CD8+ cytotoxic T lymphocytes (CTL) to induced pluripotent stem cells (iPSC) and their differentiation into rejuvenated antigen-specific CD8+ CTL as a potential therapeutic option to effectively treat cancer patients. Along with characterization of BCMA-specific iPSC by key stem cell markers, pluripotency potential and normal karyotypes, we further detected their polarization into mesoderm development associated with activation of transcriptional regulators SNAI2, TBX3, PLVAP, HAND1 and CDX2 during embryoid body formation. BCMA-specific iPSC clones utilized distinctive commitment pathways during T cells re-differentiation. RNAseq analyses of the "iPSC committed to rejuvenated memory CD8+ T cells" showed unique transcriptional profiles as evidenced by upregulation of transcriptional regulators determining CD4/CD8 T cell differentiation ratio, memory CTL formation, NF-kappa-B / JNK pathway activation, and cytokine transporter/cytotoxic mediator development. In parallel, regulators controlling B and T cell interactions or CD4+ Th cells and inhibitory receptor development were downregulated. The rejuvenated CD8+ BCMA-specific CTL re-differentiated from the iPSC demonstrated (1) mature T cell phenotype and highly enriched central and effector memory T cells without induction of checkpoint molecules; (2) high proliferation and poly-functional anti-myeloma activities in an antigen-specific and HLA-A2-restricted manner; (3) specific immune recognition of cognate HLA-A2 heteroclitic BCMA72-80 (YLMFLLRKI) peptide; and (4) distinct sole clonotype for T cell receptor. Furthermore, the specific iPSC clones maintained their differentiation potential into CD8+ T cells upon sub-cloning or long-term culture under feeder-free culture conditions. <h3>Conclusion</h3> In conclusion, these results establish a framework for iPSC-based regenerative medicine to provide rejuvenated and highly functional memory CD8+ BCMA-specific CTL as an adoptive immunotherapy to improve patient outcome in multiple myeloma.

Abstract 523: Modulation of immune suppressive cells by toll-like 7 agonist DSP-0509 which leads to potentiate anti-tumor activity of radiotherapy

Abstract Toll-like receptors (TLRs) are a family of pattern-recognition receptors that recognize pathogen-associated molecular patterns. TLR7 stimulation in pDCs (plasmacytoid dendritic cell) induces type I interferon, which results in innate immune activation. We previously showed that intravenously injectable DSP-0509 activated anti-tumor immunity in combination with immune checkpoint inhibitors. In this study, we present that DSP-0509 can enhance anti-tumor activity of radiation therapy (RT). RT is generally known to induce not only anti-tumor effector T cells through immunogenic cell death, but also cause immune suppression. We assessed the modulatory activity of DSP-0509 on immune suppressive cells. In CD8+ T cell suppression assay in which CD8+ T cells were cocultured with mMDSC (monocytic myeloid-derived suppressor cell, CD11b+Gr-1low), DSP-0509 restored the proliferation of CD8+ T cell suppressed by mMDSC. In Treg differentiation assay, in which Treg (CD4+CD25+Foxp3+) was differentiated from naïve CD4+ T cell in the presence of TGF-β, DSP-0509 suppressed the Treg differentiation. Treg function was assessed in coculture system, in which proliferation of CTV-labeled (CellTrace™ Violet) CD4+ T cell was suppressed by Treg. DSP-0509 restored the proliferation of CD4+ T cell. As a next step, to confirm a combination effect with RT, DSR-0509 (5 mg/kg, weekly) was administered with RT (5 Gy x 5 consecutive day). The combination administration extended the survival of CT26-bearing mice. The ratio of effector memory T cell (CD8+CD62L-CD127+) in CD8+ T cell was increased after administration of combination of DSP-0509 with RT. ELISpot assay using splenocytes collected from CT26-bearing mice after the administration of combination of DSP-0509 with RT was conducted. As a result, the number of IFNγ secreting cytotoxic T lymphocytes (CTLs) that responded to gp70 peptide was increased in combination of DSP-0509 with RT. When CT26 cells were re-challenged to the mice that completely rejected tumor after combination of DSP-0509 with RT, no tumor growth was observed in all mice. Anti-CD8 antibody treatment prior to re-challenge of CT26 cells cancelled this memory effect. qPCR analysis using mRNA isolated from treated tumor was conducted. RT monotherapy increased the expression of Cd8b1, but addition of DSP-0509 further enhanced the expression of Gzmb and Il12b in tumor. We showed two novel effects of DSP-0509 in this study, 1) modulation of immune suppressive cells and 2) extension of the survival of tumor-bearing mice in combination with RT, which would be explained by the additive CTL activation and memory T cell induction. Taken together, these results supported the hypothesis that combination of DSP-0509 with RT may enhance the anti-tumor immune activity by modulating CTL activity as well as immune suppressive cells. Citation Format: Yosuke Ota, Yasuhiro Nagai, Masashi Goto, Jun Oishi, Setsuko Yamamoto. Modulation of immune suppressive cells by toll-like 7 agonist DSP-0509 which leads to potentiate anti-tumor activity of radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 523.

Abstract 2731: Cell-type deconvolution of African American breast tumors reveals spatial heterogeneity of the immune microenvironment

Abstract Background: As immunotherapy emerges as a possible treatment for triple negative breast cancer (TNBC), it is crucial to understand heterogeneity and the complexities of the tumor microenvironment. GeoMx, a new platform developed by NanoString, allows for gene expression analysis within localized segments of the tumor, and is therefore ideal for determining the spatial distribution of immune cell-types. To test the feasibility of applying spatial transcriptomic to breast tumors from diverse populations, we selected ten paraffin embedded blocks from a cohort of self-reported African American patients with TNBC in the Chicago Multi-Ethnic Breast Cancer Study (ChiMEC). Methods: Tissue sections were immunostained with four antibodies (PanCK, CD3E, CD20 and DAPI) and regions of interest (ROIs) were manually selected based on specific morphology, resulting in 10 to 39 ROIs per slide (with a total of 234 ROIs across the ten slides). Using the GeoMx Cancer Transcriptome Atlas assay, expression levels of 1825 genes were measured within each ROI. For this cell-type deconvolution analysis, we used the Spatial Deconvolution algorithm, developed specifically for GeoMx data. We first normalized read counts across all ROIs of a slide and removed the background expression levels. In order to account for cancer cell types in the deconvolution process, we selected ROIs with high tumor content to predict the gene expression profiles of these tumor cells. We then performed deconvolution using both cancer and immune expression profiles, and clustered ROIs into immune signature groups. Results: As expected, we saw a higher abundance of immune cells in the stroma compared with the tumor, which resulted in lower immune cell-type diversity within the tumor segments. Tumor segments were CD8+ T-cell and neutrophil enriched: 20.3% of immune cells in tumor segments were neutrophils, and 23.7% were CD8+ T-cells, whereas in the stroma, only 3.6% and 13.8% were neutrophils and CD8+ T-cells respectively. The proportion of naïve versus memory cytotoxic T-cells differed between the tumor and the stroma, with 84.1% of CD8+ T-cells in the stroma identified as memory versus only 23.9% for tumor segments. We were also able to compare two slides from the same patient and found similar immune patterns across the two sections of the tumor. Clustering of immune cell-type distribution revealed three major immune signatures within both sections of this tumor: macrophage-rich, B-cell rich, and CD8+ T-cell rich ROIs. Conclusion: Spatial transcriptomics allows for granular analysis of the tumor microenvironment. This level of detail is crucial to understanding the cancer-immune relationship, as our analysis revealed high heterogeneity of the immune landscape within tumors. Future work will compare the tumor microenvironment within and across racial groups. Citation Format: Jean-Baptiste Reynier, Jovian Yu, Anna Biernacka, Galina Khramtsova, Toshio Yoshimatsu, Qun Niu, Anna Woodard, Yonglan Zheng, Jeffrey Mueller, Mengjie Chen, Olufunmilayo I. Olopade. Cell-type deconvolution of African American breast tumors reveals spatial heterogeneity of the immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2731.

Pembrolizumab (anti-PD-1) Treatment Increases Anti-Tumor Activities of XBP1/CD138/CS1-Specific Cytotoxic T Lymphocytes Against Multiple Myeloma

In these studies, we investigated the role of Pembrolizumab (anti-PD-1)to enhance the function of myeloma-specific CD8+ cytotoxic T lymphocytes (CTL) and maintain their antigens-specific CTL activities against multiple myeloma cells. Bone marrow or peripheral blood mononuclear cells (BMMC, PBMC) from healthy donors or MGUS, smoldering multiple myeloma (SMM), newly diagnosed multiple myeloma (MM), relapsed MM, relapsed/refractory MM patients (N=5, respectively) were evaluated for expression of immune checkpoints that regulate antigen-specific CTL development and function. Active MM patients had higher levels of CD138+ tumor cells as compared to MGUS and SMM patients. PD-L1 and PD-L2 expression levels on myeloma cells were also higher in active MM patients than healthy donors. CD4 Treg (CD3+CD4+CD25+FoxP3+), but not CD8 Treg (CD3+CD8+CD25+FoxP3+), frequencies were highest in both BMMC and PBMC from MM patients. PD-L1, but not PD-L2 nor PD-1, was expressed on the CD4 Treg in BMMC and PBMC from MM patients. G-type MDSC (CD11b+CD33+HLA-DRlowCD15+), but not M-type MDSC (CD11b+CD33+HLA-DRlowCD14+), was increased in MM as compared to MGUS, SMM or healthy donors. The MDSC from active MM patients also had high levels of PD-L1 and PD-L2 expression. BMMC and PBMC from newly diagnosed MM patients (N=3) treated in vitro with Pembrolizumab enhanced the CD3+ T cell proliferation as compared to the untreated group. Interestingly, Pembrolizumab treatment induced proliferation of T cells expressing immune agonists (OX40, GITR) beneficial for cell function and immune checkpoint (LAG3) involved in down-regulating T cell activities. XBP1/CD138/CS1 antigens-specific CTL (XBP1/CD138/CS1-CTL) generated in vitro with a cocktail of immunogenic peptides [heteroclitic XBP1 US184-192 (YISPWILAV), heteroclitic XBP1 SP367-375 (YLFPQLISV), native CD138260-268 (GLVGLIFAV), and native CS1239-247 (SLFVLGLFL)] displayed increased proliferation of CD8+ CTL expressing the CD28 co-stimulatory and CD38 activation markers upon treatment with Pembrolizumab. Importantly, the XBP1/CD138/CS1-specific central memory and effector memory CTL subsets increased the cell proliferation, following treatment with the PD-1-specific antibody. In addition, XBP1/CD138/CS1-CTL treated with Pembrolizumab demonstrated increased cytotoxic activity and Th1-specific cytokines production against U266 myeloma cells. Thus, these studies suggest the potential benefit of Pembrolizumab (a-PD-1) therapy in MM patients to enhance XBP1/CD138/CS1 antigens-specific memory CTL proliferation and boost their anti-tumor activities through blockage of the PD-1 specific immune checkpoint specifically expressed on antigens-specific CD8+ CTL. These data provide the framework for an immunotherapeutic strategy encompassing a combination of multipeptide-based cancer vaccine with PD1-specific antibody to enhance the antigens-specific T cells efficacy and promote stronger and long-lasting CD8+ CTL immune responses in myeloma patients. DisclosuresBae:OncoPep: Other: Scientific Founder. Hideshima:C4 Therapeutics: Equity Ownership; Acetylon: Consultancy. Anderson:C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees.

Targeted Delivery of BZLF1 to DEC205 Drives EBV-Protective Immunity in a Spontaneous Model of EBV-Driven Lymphoproliferative Disease.

Epstein-Barr virus (EBV) is a human herpes virus that infects over 90% of the world’s population and is linked to development of cancer. In immune-competent individuals, EBV infection is mitigated by a highly efficient virus-specific memory T-cell response. Risk of EBV-driven cancers increases with immune suppression (IS). EBV-seronegative recipients of solid organ transplants are at high risk of developing post-transplant lymphoproliferative disease (PTLD) due to iatrogenic IS. While reducing the level of IS may improve EBV-specific immunity and regression of PTLD, patients are at high risk for allograft rejection and need for immune-chemotherapy. Strategies to prevent PTLD in this vulnerable patient population represents an unmet need. We have previously shown that BZLF1-specific cytotoxic T-cell (CTL) expansion following reduced IS correlated with immune-mediated PTLD regression and improved patient survival. We have developed a vaccine to bolster EBV-specific immunity to the BZLF1 protein and show that co-culture of dendritic cells (DCs) loaded with a αDEC205-BZLF1 fusion protein with peripheral blood mononuclear cells (PMBCs) leads to expansion and increased cytotoxic activity of central-effector memory CTLs against EBV-transformed B-cells. Human–murine chimeric Hu-PBL-SCID mice were vaccinated with DCs loaded with αDEC205-BZLF1 or control to assess prevention of fatal human EBV lymphoproliferative disease. Despite a profoundly immunosuppressive environment, vaccination with αDEC205-BZLF1 stimulated clonal expansion of antigen-specific T-cells that produced abundant IFNγ and significantly prolonged survival. These results support preclinical and clinical development of vaccine approaches using BZLF1 as an immunogen to harness adaptive cellular responses and prevent PTLD in vulnerable patient populations.

Dynamics of an HIV model with cytotoxic T-lymphocyte memory

We consider a four-dimensional HIV model that includes healthy cells, infected cells, primary cytotoxic T-lymphocyte response (CTLp), and secondary cytotoxic T-lymphocyte response (CTLe). The CTL memory generation depends on CD4+ T-cell help, and infection of CD4+ T cells results in impaired T-cell help. We show that the system has up to five equilibria. By the Routh–Hurwitz theorem and central manifold theorem we obtain some sufficient conditions for the local stability, globally stability of the equilibria, and the bifurcations. We still discover the bistability case where in the system there may coexist two stable equilibria or a stable equilibrium together with a stable limit cycle. Several numerical analyses are carried out to illustrate the validity of our theoretical results.

Abstract 399: Tertiary lymphoid structures features associate with outcome in non-small cell lung carcinoma

Abstract Introduction: Tertiary lymphoid structures (TLS) are ectopic lymphoid structures organized in a nodular pattern secondary to chronic inflammation. The presence of TLS has been associated with efficacious response to immune checkpoint blockade in a wide array of tumor types, including Non-Small Cell Lung Carcinoma (NSCLC). At early stages of NSCLC, TLS are observed in up to 70% of primary tumors where they are also associated with effective anti-tumor immune-responses and response to anti-PD-1 therapy. Despite complete surgical resection, however, up to 50% of patients with early stage NSCLC will eventually relapse. A systematic histomorphologic analysis of TLS and their association with relapse has not been well characterized. Design: Serial sections from Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimens from 33 patients with stage I NSCLC were obtained (23 adenocarcinomas and 10 squamous cell carcinomas). Slides were stained with hematoxylin and eosin (H&amp;E), immunohistochemistry for Vimentin, and 2 multiplex immunofluorescence (mIF) panels (Panel 1: cytokeratin (CK), CD3, CD8, CD68, PD-1, PD-L1 and DAPI Panel 2: CK, CD3 CD8, CD45RO, FOXP3, Granzyme B and DAPI). TLS were classified based on the H&amp;E and Vimentin analysis into lymphoid aggregates (LA), immature TLS (iTLS), and mature TLS (mTLS). Morphometric analysis (number, area, and distance to nearest malignant cell) of intratumoral (IT) and peritumoral (PT) TLS was performed. mIF slides were scanned and IT representative areas were selected for cell densities and percentage quantification of immune-phenotypes. Morphometric analysis of TLSs was correlated with clinical outcomes, and tumor infiltrating immune cells. Results: Patients who recurred had fewer IT mTLS (1.8 vs 6; p=0.02) and smaller area of mTLS (64541.7μm2 vs 149870.5 μm2; p=0.004) compared with patients who did not recurr.The cell density of IT antigen-experienced cytotoxic T-lymphocytes (CTLs), regulatory T-cells, memory CTLs, and memory CTLs expressing FOXP3 were inversely correlated with the mTLS area (r=-0.6, p≤0.02). Antigen-experienced CTLs (r=-0.61; p≤0.05) and non-CTLs (r=-0.63; p≤0.05) were inversely correlated with the number of IT mTLS. Conclusion: Detailed morphometric analysis of mTLS offers relevant prognostic information for recurrence at stage I of NSCLC. mTLSs are associated with reduced IT infiltration by PD-1+ TILs. Taken together, germinal center development in mTLSs might convey a protective immune response due to immuno-dominant neoantigen presentation. Supported in part by CPRIT RP160668 grant and UT Lung SPORE. Citation Format: Rossana Natalia Lazcano Segura, Andre Catao, Luisa M. Solis, Mei Jiang, Auriole Tamegnon, Junya Fujimoto, Jaime Rodriguez-Canales, Chi-Wan B. Chow, Carmen Behrens, Neda Kalhor, Annika Weissferdt, John Heymach, Stephen Swisher, Boris Sepesi, Jack Lee, Cesar Moran, Andrew Futreal, Jianjun Zhang, Edwin R. Parra, Ignacio I. Wistuba, Michael T. Tetzlaff, Alejandro Francisco-Cruz. Tertiary lymphoid structures features associate with outcome in non-small cell lung carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 399.

Single-cell RNA sequencing reveals compromised immune microenvironment in precursor stages of multiple myeloma.

Precursor states of Multiple Myeloma (MM) and its native tumor microenvironment need in-depth molecular characterization to better stratify and treat patients at risk. Using single-cell RNA sequencing of bone marrow cells from precursor stages, MGUS and smoldering myeloma (SMM), to full-blown MM alongside healthy donors, we demonstrate early immune changes during patient progression. We find NK cell abundance is frequently increased in early stages, and associated with altered chemokine receptor expression. As early as SMM, we show loss of GrK+ memory cytotoxic T-cells, and show their critical role in MM immunosurveillance in mouse models. Finally, we report MHC class II dysregulation in CD14+ monocytes, which results in T cell suppression in vitro. These results provide a comprehensive map of immune changes at play over the evolution of pre-malignant MM, which will help develop strategies for immune-based patient stratification.

Physiological expression and function of the MDR1 transporter in cytotoxic T lymphocytes.

Multidrug resistance-1 (MDR1) acts as a chemotherapeutic drug efflux pump in tumor cells, although its physiological functions remain enigmatic. Using a recently developed MDR1-knockin reporter allele (Abcb1aAME), we found that constitutive MDR1 expression among hematopoietic cells was observed in cytolytic lymphocytes-including CD8+ cytotoxic T lymphocytes (CTLs) and natural killer cells-and regulated by Runt-related (Runx) transcription factors. Whereas MDR1 was dispensable for naive CD8+ T cell development, it was required for both the normal accumulation of effector CTLs following acute viral infection and the protective function of memory CTLs following challenge with an intracellular bacterium. MDR1 acted early after naive CD8+ T cell activation to suppress oxidative stress, enforce survival, and safeguard mitochondrial function in nascent CTLs. These data highlight an important endogenous function of MDR1 in cell-mediated immune responses and suggest that ongoing efforts to intentionally inhibit MDR1 in cancer patients could be counterproductive.

Transcutaneous immunization with a highly active form of XCL1 as a vaccine adjuvant using a hydrophilic gel patch elicits long-term CD8+ T cell responses

Memory CD8+ cytotoxic T-lymphocytes (CTLs) play a key role in protective immunity against infection and cancer. However, the induction of memory CTLs with currently available vaccines remains difficult. The chemokine receptor XCR1 is predominantly expressed on CD103+ cross-presenting dendritic cells (DCs). Recently, we have demonstrated that a high activity form of murine lymphotactin/XCL1 (mXCL1-V21C/A59C), a ligand of XCR1, can induce antigen-specific memory CTLs by increasing the accumulation of CD103+ DCs in the vaccination site and the regional lymph nodes. Here, we combined a hydrophilic gel patch as a transcutaneous delivery device and mXCL1-V21C/A59C as an adjuvant to further enhance memory CTL responses. The transcutaneous delivery of ovalbumin (OVA) and mXCL1-V21C/A59C by the hydrophilic gel patch increased CD103+ DCs in the vaccination site and the regional lymph nodes for a prolonged period of time compared with the intradermal injection of OVA and mXCL1-V21C/A59C. Furthermore, the hydrophilic gel patch containing OVA and mXCL1-V21C/A59C strongly induced OVA-specific memory CTLs and efficiently inhibited the growth of OVA-expressing tumors more than the intradermal injection of OVA and mXCL1-V21C/A59C. Collectively, this type of hydrophilic gel patch and a high activity form of XCL1 may provide a useful tool for the induction of memory CTL responses.