Memory CTL Research Articles

A diversity of novel type-2 innate lymphoid cell subpopulations revealed during tumour expansion

Type 2 innate lymphoid cells (ILC2s) perform vital functions in orchestrating humoral immune responses, facilitating tissue remodelling, and ensuring tissue homeostasis. Additionally, in a role that has garnered considerably less attention, ILC2s can also enhance Th1-related cytolytic T lymphocyte immune responses against tumours. Studies have thus far generally failed to address the mystery of how one ILC2 cell-type can participate in a multiplicity of functions. Here we utilized single cell RNA sequencing analysis to create the first comprehensive atlas of naïve and tumour-associated lung ILC2s and discover multiple unique subtypes of ILC2s equipped with developmental gene programs that become skewed during tumour expansion favouring inflammation, antigen processing, immunological memory and Th1-related anti-tumour CTL responses. The discovery of these new subtypes of ILC2s challenges current paradigms of ILC2 biology and provides an explanation for their diversity of function.

Active maintenance of CD8+ T cell naivety through regulation of global genome architecture

The differentiation of naive CD8+ T lymphocytes into cytotoxic effector and memory CTL results in large-scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organization underpin these transcriptional programs. We use Hi-C to map changes in the spatial organization of long-range genome contacts within naive, effector, and memory virus-specific CD8+ Tcells. We observe that the architecture of the naive CD8+ Tcell genome is distinct from effector and memory genome configurations, with extensive changes within discrete functional chromatin domains associated with effector/memory differentiation. Deletion of BACH2, or to a lesser extent, reducing SATB1 DNA binding, within naive CD8+ Tcells results in a chromatin architecture more reminiscent of effector/memory states. This suggests that key transcription factors within naive CD8+ Tcells act to restrain Tcell differentiation by actively enforcing a unique naive chromatin state.

Cutting Edge: Polycomb Repressive Complex 1 Subunit Cbx4 Positively Regulates Effector Responses in CD8 T Cells.

CTL differentiation is controlled by the crosstalk of various transcription factors and epigenetic modulators. Uncovering this process is fundamental to improving immunotherapy and designing novel therapeutic approaches. In this study, we show that polycomb repressive complex 1 subunit chromobox (Cbx)4 favors effector CTL differentiation in a murine model. Cbx4 deficiency in CTLs induced a transcriptional signature of memory cells and increased the memory CTL population during acute viral infection. It has previously been shown that besides binding to H3K27me3 through its chromodomain, Cbx4 functions as a small ubiquitin-like modifier (SUMO) E3 ligase in a SUMO-interacting motifs (SIM)-dependent way. Overexpression of Cbx4 mutants in distinct domains showed that this protein regulates CTL differentiation primarily in an SIM-dependent way and partially through its chromodomain. Our data suggest a novel role of a polycomb group protein Cbx4 controlling CTL differentiation and indicated SUMOylation as a key molecular mechanism connected to chromatin modification in this process.

FURIN regulates cytotoxic T lymphocyte effector function and memory cell transition in mice.

The proprotein convertase subtilisin/kexins (PCSKs) regulate biological actions by cleaving immature substrate proteins. The archetype PCSK, FURIN, promotes the pathogenicity of viruses by proteolytically processing viral proteins. FURIN has also important regulatory functions in both innate and adaptive immune responses but its role in the CD8+ CTLs remains enigmatic. We used a T cell specific FURIN deletion in vivo to demonstrate that FURIN promotes host response against the CTL-dependent lymphocytic choriomeningitis virus by virtue of restricting viral burden and augmenting IFNG production. We also characterized Furin KO CD8+ T cells ex vivo, including after their activation with FURIN regulating cytokines IL12 or TGFB1. Furin KO CD8+ T cells show an inherently activated phenotype characterized by the up-regulation of effector genes and increased frequencies of CD44+, TNF+ and IFNG+ cells. In the activated CTLs FURIN regulates the productions of IL2, TNF and GZMB and the genes associated with the TGFBR-signaling pathway. FURIN also controls the expression of Eomes, Foxo1 and Bcl6 and the levels of ITGAE and CD62L, which implies a role in the development of CTL memory. Collectively, our data suggest that the T cell expressed FURIN is important for host responses in viral infections, CTL homeostasis/activation and memory development. This article is protected by copyright. All rights reserved.

Dysfunctions of innate and adaptive immune tumor microenvironment in Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM) is a rare subtype of non-Hodgkin lymphoma characterized by malignant lymphoplasmacytic cells in the bone marrow (BM). To dissect the pathophysiology of WM, we evaluated clonal cells by mapping of B cell lymphomagenesis with adaptive and innate immune tumor microenvironment (TME) in the BM of WM patients using mass cytometry (CyTOF). In-depth immunophenotypic profiling of WM cells exhibited profound expansion of clonal cells in both unswitched and switched memory B cells and also plasma cells with aberrant expression variations. WM B lymphomagenesis was associated with reduction of most B cell precursors assessed with the same clonally restricted light chain and phenotypic changes. The immune TME was infiltrated by mature monocytes, neutrophils and adaptive T cells, preferentially subsets of effector T helper, effector CTL and effector memory CTL cells that were associated with superior overall survival (OS), in contrast to progenitors of T cells and myeloid/monocytic lineage subsets that were suppressed in WM cohort. Moreover, decrease in immature B and NKT cells was related to worse OS in WM patients. Innate and adaptive immune subsets of WM TME were modulated by immune checkpoints, including PD-1/PD-L1&PD-L2, TIGIT/PVR, CD137/CD137-L, CTLA-4, BTLA and KIR expression. The response of ibrutinib treatment to the reduction of clonal memory B cell was associated with high levels of immature B cells and effector memory CTL cells. Our study demonstrates that CyTOF technology is a powerful approach for characterizing the pathophysiology of WM at various stages, predicting patient risk and monitoring the effectiveness of treatment strategies.

Development of B-cell maturation antigen (BCMA)-specific CD8+ cytotoxic T lymphocytes using induced pluripotent stem cell technology for multiple myeloma.

2542 Background: A strategy for reversal of T cell exhaustion is reprograming of antigen-specific CTL to early lineage memory T cells with selective anti-tumor activities. To accomplish this goal, we epigenetically reprogrammed BCMA-specific CD8+ CTL to a pluripotent state through key defined transcription factors, established “induced Pluripotent Stem Cells (iPSC)” exhibiting transcriptional and epigenetic features, re-differentiated them back into antigen-specific CTL and evaluated their properties and functional activities against multiple myeloma (MM). Methods: Functionally activeIFN-g producing HLA-A2 heteroclitic BCMA72-80 (YLMFLLRKI)-specific CD8+ CTL were applied for iPSC via transduction of four reprogramming factors (OCT3/4, SOX2, KLF4, c-MYC). Upon characterization of the BCMA-specific iPSC with high pluripotency potential, embryoid body was formed from the iPSC and further polarized into mesoderm layer development as evidenced by upregulation of transcriptional regulators (ABCA4, BMP10, CDH5, FOXF1, HAND1, PLVAP, SNAI2, TBX3). Next, BCMA-specific embryoid body-derived hematopoietic progenitor cells (HPC; CD34+ CD43+/CD14- CD235a-) were sorted and induced to undergo T cell differentiation in the presence of Fc-DLL4 signaling and rectonectin. Results: Our RNAseq analyses demonstrated unique transcriptional profiles of HPC from different iPSC clones committing to CD8+ T cells or other cell lineages (monocytes/granulocytes, B lymphocytes/NK cells). Principal component analyses demonstrated a high similarity and low variability of transcription profiles within the replicates of HPC committed to the same cell lineage. In addition, distinct genome-wide shifts and differential gene expression profiles were detected in HPC committed to each specific cell differentiation pathway. Specifically, the HPC commit to CD8+ T cells utilized a diverse repertoire of modulators promoting development of T cell maturation, specific immune response regulation, memory T cells, cytotoxicity and interferon induction, which were significantly higher than shown in HPC that differentiate to other cell lineages. In parallel, specific repression genes were identified in the HPC commit to CD8+ T cells, which develop TGF-β receptor, rearrangement of Ig heavy chain genes and inhibitory receptors. The T cells differentiated were mainly CD45RO+ memory CTL and fully rejuvenated without immune checkpoints expression and regulatory T cells and with high anti-MM activities. Conclusions: These findings identify genetic and epigenetic mechanisms and regulatory elements, which play key roles during lineage specific commitment of HPC developed in iPSC into CD8+ CTL and help to further design a next generation of regenerative medicine that provide the appropriate signals for T cell lineage commitment from progenitor cells.

The downregulation of IL-18R defines bona fide kidney-resident CD8+ Tcells.

SummaryStepwise induction of CD69 and CD103 marks distinct differentiation stages of mucosal Trms. But the majority of non-mucosal Trm lacks CD103 expression. The expression of CD69 alone cannot faithfully define Trm cells in heavily vascularized non-mucosal tissues, such as the kidney. Here, we found that a subset of kidney Trms downregulated IL-18 receptor during differentiation. Via global transcriptional analysis and parabiosis experiments, we have discovered that the downregulation of interleukin-18 receptor (IL-18R) is associated with the establishment of tissue residency. Together with the expression of CD69, IL-18Rlo exclusively identify tissue-resident cells whereas IL-18Rhi population contains both tissue-resident and migratory ones. Local cytokines including transforming growth factor β (TGF-β) and interferon α (IFN-α)/β as well as TGF-β-dependent suppression of transcription factor Tcf-1 are essential for IL-18R downregulation during kidney Trm differentiation. Together, we identified a convenient surface marker to distinguish bona fide kidney-resident CD8+ T cells as well as underlying molecular mechanisms controlling this differentiation process.

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.

CD4+ T cells support polyfunctionality of cytotoxic CD8+ T cells with memory potential in immunological control of tumor.

Polyfunctionality/multifunctionality of effector T cells at the single cell level has been shown as an important parameter to predict the quality of T cell response and immunological control of infectious disease and malignancy. However, the fate of polyfunctional CD8+ CTLs and the factors that control the polyfunctionality of T cells remain largely unknown. Here we show that the acquisition of polyfunctionality on the initial stimulation is a sensitive immune correlate of CTL survival and memory formation. CD8+ T cells with high polyfunctionality, assessed with γ‐interferon and tumor necrosis factor‐α production and surface mobilization of the degranulation marker CD107a, showed enhanced Bcl‐2 expression, low apoptosis, and increased CD127highKLRG1low memory precursor phenotype. Consistent with these observations, CD8+ T cells were found to acquire high frequency of cells with polyfunctionality when stimulated in conditions known to enhance memory formation, such as the presence of CD4+ T cells, interleukin (IL)‐2, or IL‐21. Utilizing T‐cell receptor (TCR) transgenic mouse‐derived CD8+ T cells that express a TCR specific for a tumor‐derived neoantigen, we showed that polyfunctional tumor‐specific CTLs generated in the presence of CD4+ T cells showed long persistence in vivo and induced enhanced tumor regression when adoptively transferred into mice with progressing tumor. Acquisition of polyfunctionality thus impacts CTL survival and memory formation associated with immunological control of tumor.

Splenic Red Pulp Macrophages Cross-Prime Early Effector CTL That Provide Rapid Defense against Viral Infections.

Cross-presentation allows dendritic cells (DCs) to present peptides derived from endocytosed Ags on MHC class I molecules, which is important for activating CTL against viral infections and tumors. Type 1 classical DCs (cDC1), which depend on the transcription factor Batf3, are considered the main cross-presenting cells. In this study, we report that soluble Ags are efficiently cross-presented also by transcription factor SpiC-dependent red pulp macrophages (RPM) of the spleen. In contrast to cDC1, RPM used the mannose receptor for Ag uptake and employed the proteasome- and TAP-dependent cytosolic cross-presentation pathway, previously shown to be used in vitro by bone marrow-derived DCs. In an in vivo vaccination model, both cDC1 and RPM cross-primed CTL efficiently but with distinct kinetics. Within a few days, RPM induced very early effector CTL of a distinct phenotype (Ly6A/E+ Ly6C(+) KLRG1- CD127- CX3CR1- Grz-B+). In an adenoviral infection model, such CTL contained the early viral spread, whereas cDC1 induced short-lived effector CTL that eventually cleared the virus. RPM-induced early effector CTL also contributed to the endogenous antiviral response but not to CTL memory generation. In conclusion, RPM can contribute to antiviral immunity by generating a rapid CTL defense force that contains the virus until cDC1-induced CTL are available to eliminate it. This function can be harnessed for improving vaccination strategies aimed at inducing CTL.

Analysis of the CD8+ IL-10+ T cell response elicited by vaccination with the oncogenic tumor-self protein D52

ABSTRACT Development of cancer vaccines targeting tumor self-antigens is complex and challenging due to the difficulty of overcoming immune tolerance to self-proteins. Vaccination against tumor self-protein D52 (D52) has been successful, although complete protection appears impaired by immune regulation. Our previous studies suggest that vaccine elicited CD8 + T cells producing interleukin 10 (IL-10) may have a negative impact on tumor protection. Understanding the role CD8+ IL-10 + T cells play in the immune response following vaccination with D52 could result in a more potent vaccine. To address this, we vaccinated IL-10 deficient mice with the murine orthologue of D52; vaccination of wild type (wt) C57BL/6J served as a control for comparison. In separate experiments, D52 vaccinated wt mice were administered IL-10R-specific mAb to neutralize IL-10 function. Interestingly, we observed similar protection against primary tumor challenge in the experimental groups compared to the controls. However, individual IL-10 deficient mice that rejected the primary tumor challenge were re-challenged 140 days post-primary challenge to access vaccine durability and immunologic memory against tumor recurrence. Mice deficient in IL-10 demonstrated a memory response in which 100% of the mice were protected from secondary tumor challenge, while wt mice had diminished recall response (25%) against tumor recurrence. These results with analysis of vaccine-elicited CD8 + T cells for tumor-specific killing and regulatory cell marker expression, add further support to our premise that CD8+ IL-10 + T cells elicited by D52 tumor-self protein vaccine contribute to the suppression of a memory CTL responses and durable tumor immunity.

Transcutaneous immunization with CD40 ligation boosts cytotoxic T lymphocyte mediated antitumor immunity independent of CD4 helper cells in mice

Transcutaneous immunization (TCI) is a novel vaccination strategy that utilizes skin-associated lymphatic tissue to induce immune responses. Employing T-cell epitopes and the TLR7 agonist imiquimod onto intact skin mounts strong primary, but limited memory CTL responses. To overcome this limitation, we developed a novel imiquimod-containing vaccination platform (IMI-Sol) rendering superior primary CD8+ and CD4+ T-cell responses. However, it has been unclear whether IMI-Sol per se is restricted in terms of memory formation and tumor protection. In our present work, we demonstrate that the combined administration of IMI-Sol and CD40 ligation unleashes fullblown specific T-cell responses in the priming and memory phase, strongly enhancing antitumor protection in mice. Interestingly, these effects were entirely CD4+ T cell independent, bypassing the necessity of helper T cells. Moreover, blockade of CD70 in vivo abrogated the boosting effect of CD40 ligation, indicating that the adjuvant effect of CD40 in TCI is mediated via CD70 on professional APCs. Furthermore, this work highlights the so far underappreciated importance of the CD70/CD27 interaction as a promising adjuvant target in TCI. Summing up, we demonstrate that the novel formulation IMI-Sol represents a powerful vaccination platform when applied in combination with sufficient adjuvant thereby overcoming current limitations of TCI.

Abstract 4041: Coupling neoantigen specific T cell clonotypes and their molecular phenotypes at the single cell level in resectable anti-PD-1 treated NSCLC

Abstract We recently completed a phase II study evaluating the safety and efficacy of neoadjuvant anti-PD-1 in non-small cell lung cancer (NSCLC). To evaluate anti-tumor T cell responses, we used the recently described MANAFEST (Mutation Associated Neoantigen Functional Expansion of Specific T cells) assay, which links antigen specificity with unique CD8+ TCR Vβ CDR3 identities, in 7 patients from the trial. We then carried out single cell sequencing of tumor infiltrating T lymphocytes (TIL) to enumerate the genome wide digital gene expression of each single cell (VDJ+DGE analysis), and more particularly those with Vβ CDR3 regions identical to those identified by MANAFEST. Neoantigen-specific TCRs were detected in all patients with major pathological response (MPR) and in 3 of 4 patients without MPR. VDJ+5’DGE single cell sequencing demonstrated differential phenotypes of TIL obtained from surgical resection, and these T cells had varying degrees of peripheral perturbations during treatment. In PBMC from patient MD043-011, the MANAFEST assay detected a T cell response specific for a CARM1 R208W mutation, despite no evidence of pathological response in this patient. These neoantigen-specific T cells possessed a single TCR Vβ CDR3 at the aa level and represented 3.4% of TIL. Upon analysis of TIL with the VDJ+DGE platform, the same CDR3 was detected in 32 TIL, all of which possessed the identical TCR Vα at the aa level. However, analysis of CDR3 sequences at the nucleotide level revealed 3 distinct clones encoding this antigen-specific TCR. Differential gene expression profiling demonstrated a unique and discriminating expression profile for each clonotype, with one clone expressing CTL activation markers such as granzyme k, 4-1BB, and PD-1, the second clone expressing IL10 family member IL26 and genes that inhibit effector and memory CTL function, such as CISH, TOX, and SLAMF1, and the third clone expressing no known activation markers. These findings demonstrate the existence of different intratumoral T cell clones with distinct functions yet identical specificity for a tumor neoantigen. We propose that these differential expression programs are induced because these clones were differentially activated under different conditions in the TME, primed in distinct anatomical locations, or at different times during disease progression despite recognizing the same antigen. Analyses to evaluate the prevalence of this phenomenon in the larger cohort are ongoing. In conclusion, the coupling of MANAFEST with single cell VDJ+ DGE analysis enabled the first comprehensive phenotypic profiling of neoantigen-specific T cells using the TCR as a molecular barcode. Ultimately, this integrative approach may provide key insights in predicting and understanding not only clinical response to neoadjuvant PD-1 blockade in NSCLC, but also other early stage cancers agnostic of tissue of origin. Citation Format: Justina X. Caushi, Zhicheng Ji, Jiajia Zhang, Margueritta El Asmar, Valsamo Anagnostou, Tricia R. Cottrell, Hok Yee Chan, Haidan Guo, Taha Merghoub, Jedd D. Wolchok, Jarushka Naidoo, Kristen A. Marrone, Jamie E. Chaft, Matthew D. Hellmann, Edward Gabrielson, Janis M. Taube, Julie R. Brahmer, Victor E. Velculescu, Ni Zhao, Ludmila Danilova, Leslie Cope, Patrick M. Forde, Drew M. Pardoll, Hongkai Ji, Srinivasan Yegnasubramanian, Kellie N. Smith. Coupling neoantigen specific T cell clonotypes and their molecular phenotypes at the single cell level in resectable anti-PD-1 treated NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4041.

PF766 T CELL EXHAUSTION PROFILE (CD4+/CD28/PER+/GRA+ CD8+/CD28/PER+/GRA+) IN STEM CELL TRANSPLANTATION, CMV RE ACTIVATIONS AND RESPIRATORY VIRUS INFECTION.CELLULAR DYNAMICS IN THE FIRST 100 DAYS POST SCT

Background:Recipients of allogeneic stem cells grafts have clonally expanded CD8+/CD28‐ T lymphocytes during the early period after SCT, this cellular dynamic is associated with the acquisition of a cytoxic phenotype. This scenario predisposes to continuous inflammation. On the other hand, respiratory viruses (RV) are recognized as the predominant leading to pneumonia after SCT, and CMV re activations were associated with morbi‐mortality in SCT patients.RVI and CMV re‐activations might contribute to the emergence of exhausted T cells phenotype.Aims:The present study aimed to evaluate the dynamics of T cell exhaustion profile during the +100d after SCT(auto, allo/patients with RVI and CMV re activations)Methods:Informed consent was obtained from all patients. PBs were obtained previous SCT and at day+100 post SCT from 37 patients (11 auto, 26 allo). To evaluate the expression of CD4+/CD28Per+/Gran+, CD8+/CD28Per+/Gran+. Flow cytometry analysis was performed: 100 μL of PB was labeled, with a panel of 8 AbMos: PERFORIN FITC, GRANZIME PE, CD4PerCP, CD28 APC, CD8 APCH7, CD16/56 V450 and CD45 V500. The molecular detection of RV was tested with theCLART® Pneumovir assay (Clinical Array Technology, Genomica, Spain). CMV detection was performed by quantitative CMV PCR (COBAS®Amplicor® CMV MonitorTM test) (qPCR CT). Statistical analysis was performed with IBM SPSS v24Results:Thirty seven patients were evaluated from November 2016 to December 2017 at the University Hospital of La Princesa, Madrid Spain. Patient characteristics:Table 1.The median percentages of baseline CD8+/CD28‐ cell line was 8,81%(range, 0,6–49,5) and the median percentages of CD8+/CD28‐ cellline at +100d were 29,2%(range, 1,2–60,8).Likewise, significant differences were found(p = 0.001).The group of patients who had an RVI and the group without RVI: in the CD8+/CD28+100d cell line, the median percentages of the RVI patients were 58.43%(range, 42.7–68.4)and the median percentages in the uninfected were 29.26%(range, 13.2–38.9)(p = 0.002). CMV re activations,the median percentages at +100d of CD8+/CD28‐ cell line with CMV‐ was 5,7%(range, 0,6–20,6)and the median percentage of CD8+/CD28‐with CMV+ at +100d was 36,7%(range, 13,5–68,7)p = 0,001.CMV reactivations and RVI, the median percentages at +100d of CD8+/CD28‐cell line with CMV/RVI was 6,82%(range, 0,6–20,6)and the medianpercentage of CD8+/CD28‐ with CMV+/RVI+ at +100d was 34,6%(range, 1,4–49,8)p = 0,012.Summary/Conclusion:We show a dynamic change of the T cell exhausted phenotype CD8+/CD28‐throughout the SCT, an also ID interactions (RVI, CMV re activations).There is a statistically significant difference when analyzing patients with RVI+ vs RVI‐ (baseline vs +100d), also might exist a synergic effect RVI+/CMV+.The CD8+/CD28‐Tcell population has been shown to contain virus specific memory CTL that respond to CMV and to other antigens.Thus, clonally expanded CD8+CD28‐ Tcells following STC may be derived from the T cells recognizing antigens that persistently exist in the host (CMV) or to the exposure to certain virus antigens in the first 100d after STC (synergic effect), this may contribute to the appearance of populations of exhausted T cells CD8+/CD28‐ favoring an inflammatory environment and triggering certain immunological complications (immunedysregulation).We need to follow up these patients and correlate with other clinical epidemiological variables in SCT(aGVHD,cGVHD,pulmonary functional tests,ID complications)image

Enhanced CD138 peptide-specific cytotoxic T lymphocyte activities against breast, colon and pancreatic cancers in combination with pembrolizumab (anti-PD1).

e14302 Background: We investigated the ability of CD138 antigen-specific CD8+ cytotoxic T lymphocytes (CD138-CTL), generated using a novel HLA-A2-specific CD138260-268 (GLVGLIFAV) peptide, in combination with various immune checkpoint inhibitors, to target breast, colon, and pancreatic cancer. Methods: This study was designed to evaluate anti-tumor activities of CD138-CTL, alone or in combination with Pembrolizumab (anti-PD1), against solid tumors. Results: Upregulation of CD138 protein expression was detected on breast, colon, and pancreatic cancer cells. CD138-CTL induced ex vivo by repeated stimulations with HLA-A2-specific CD138260-268 (GLVGLIFAV) peptide were enriched for CD45RO+ memory CTL and demonstrated antigen-specific and HLA-A2-restricted anti-tumor activities. Treatment of CD138-CTL with anti-PD1, anti-TIM3, or anti-LAG3 or tumor cells with anti-PDL1, anti-Gal9 or anti-LAG3 resulted in an increased proliferation of CD138-specific CD8+ CTL, which express costimulatory and activation markers (CD28, CD40L, 41BB, CD69, CD38). However, expression of immune checkpoints (PD1, LAG3, CTLA4) was also induced on these CD138-CTL upon stimulation. Importantly, anti-tumor activities of CD138 peptide-CTL was significantly enhanced in combination with Pembrolizumab, especially within the antigen-specific memory CD8+ T cell subsets (central memory > effector memory), directed against HLA-A2+ breast cancer, colon cancer and pancreatic cancer. Conclusions: These data provide the framework for an immunotherapeutic strategy encompassing a CD138260-268 (GLVGLIFAV) peptide-based cancer vaccine in combination with Pembrolizumab to enhance antigen-specific central memory cells with more potent and long-lasting CD8+ CTL immune responses in patients with solid tumors. Based on these result, clinical trials evaluating CD138260-268 (GLVGLIFAV) in multi-peptide cancer vaccine are on-going in patients with triple negative breast cancer in combination with anti-PD1 (Pembrolizumab) in metastatic setting (NCT 03362060) or anti-PD-L1 (Durvalumab) in stage II/III, adjuvant setting (NCT 02826434).

Selective targeting of multiple myeloma by B cell maturation antigen (BCMA)-specific central memory CD8+ cytotoxic T lymphocytes: immunotherapeutic application in vaccination and adoptive immunotherapy.

To expand the breadth and extent of current multiple myeloma (MM)-specific immunotherapy, we have identified various antigens on CD138+ tumor cells from newly diagnosed MM patients (n = 616) and confirmed B-cell maturation antigen (BCMA) as a key myeloma-associated antigen. The aim of this study is to target the BCMA, which promotes MM cell growth and survival, by generating BCMA-specific memory CD8+ CTL that mediate effective and long-lasting immunity against MM. Here we report the identification of novel engineered peptides specific to BCMA, BCMA72-80 (YLMFLLRKI), and BCMA54-62 (YILWTCLGL), which display improved affinity/stability to HLA-A2 compared to their native peptides and induce highly functional BCMA-specific CTL with increased activation (CD38, CD69) and co-stimulatory (CD40L, OX40, GITR) molecule expression. Importantly, the heteroclitic BCMA72-80 specific CTL demonstrated poly-functional Th1-specific immune activities [IFN-γ/IL-2/TNF-α production, proliferation, cytotoxicity] against MM, which were correlated with expansion of Tetramer+ and memory CD8+ CTL. Additionally, heteroclitic BCMA72-80 specific CTL treated with anti-OX40 (immune agonist) or anti-LAG-3 (checkpoint inhibitor) display increased immune function, mainly by central memory CTL. These results provide the framework for clinical application of heteroclitic BCMA72-80 peptide, alone and in combination with anti-LAG3 and/or anti-OX40 therapy, in vaccination and/or adoptive immunotherapeutic strategies to generate long-lasting anti-tumor immunity in patients with MM or other BCMA expressing tumors.

Cytotoxic Immune Response Can be Obtained Earlier in Chronic Phase Chronic Myeloid Leukemia Patients Treated with Dasatinib Than with Other Tyrosine Kinase Inhibitors

Introduction: Generation of BCR-ABL fusion gene by reciprocal translocation of chromosomes 9 and 22 immortalizes hematopoietic stem cells by mechanisms such as activation of the JAK-STAT pathway, translational activation of BCL-XL, and inhibition of DNA repair, thereby leading to chronic myeloid leukemia (CML). Amazing improvement in the prognosis of CML has been achieved since the introduction of tyrosine kinase inhibitors (TKIs). Imatinib, a 1st-generation TKI, and dasatinib and nilotinib, 2nd-generation TKIs, are generally used for chronic phase (CP) CML as induction therapy. However, to date, no consensus about the cessation of TKIs in CP-CML patients has been obtained. We recently reported the case of a CP-CML patient with long-term complete molecular response (MR) after cessation of dasatinib, who has been maintaining memory CTLs with T cell receptor (TCR) clonality (Jo et al. Oncology Letters 15: 2935-2938, 2018). Here, we show that up-regulation of memory CTLs occurs early in dasatinib-treated patients compared with those treated with other TKIs.Methods: We examined the TCR V beta gene repertoire to analyze TCR clonality of CD8-positive T cells in TKI-treated CP-CML patients using flow cytometry.Results: Table 1 summarizes the data comparing patients treated with TKIs including dasatinib (Dasa group) and those treated with TKIs without dasatinib (non-Dasa group). Seven patients were treated with dasatinib only; 7, with imatinib only; 8, with multiple TKIs, including dasatinib; and 1, with multiple TKIs without dasatinib. The median age at first TKI administration was 57 years in the Dasa group and 69 years in the non-Dasa group. No significant statistical difference was observed in age at first TKI administration. The time of TCR clonality assay was significantly earlier in the Dasa group than in the non-Dasa group (P = 0.0013). There was no significant difference in the MR at the time of TCR clonality assay between the 2 groups. Figure 1 shows representative data of the TCR clonality assay of the patients in the non-Dasa group. We defined a TCR V beta gene percentage of 10% and above as being positive for monoclonality in this study. The time of analysis was at 116th month (Mo) after the 1st imatinib administration, and NK cell percentage was 30.2% at that time. TCR monoclonality was observed in neither effector CTLs (upper panel) nor memory CTLs (lower panel), although the patient had gained MR5. Figure 2 shows representative time-course data of the patients in the Dasa group. MR levels were MR4.5 (13th Mo), MR5 (16th Mo), and MR5 (19th Mo). Interestingly, memory CTL clonality with the TCR V beta 20 gene had already been observed in the 13th Mo, and it had been continuously observed in the 16th and 19th Mo. NK cell percentages were less than 24% throughout the observation period. Table 2 summarizes the CTL clonality assay results and NK cell percentages. There was no significant change in the NK cell percentages between the 2 groups. Although no statistical significance was observed in both effector and memory CTL clonality between the 2 groups, it is notable that approximately 73% and 87% positivity of effector and memory CTL clonality was observed in the Dasa group. Approximately 38% and 50% positivity of effector and memory CTL clonality was observed in the non-Dasa group, although the TKI exposure time for this group was significantly longer. Notably, the positive percentages of effector and memory CTL clonality in the non-Dasa group are quite similar to the overall probabilities of maintenance of deep MR reported in various imatinib-stop studies such as the STIM study (Mahon et al. Lancet Oncol 11: 1029-1035, 2010) and the TWISTER study (Ross et al. Blood 122: 515-522, 2013). These results suggest that acquisition of CTL clonality may correlate with treatment-free remission (TFR) in CP-CML patients treated with TKIs.Conclusions: Effector and memory CTL clonality was attained more rapidly and frequently in dasatinib-treated CP-CML patients than in patients treated with TKIs without dasatinib. There was no significant difference in the NK cell percentages. The positive percentages of CTL clonality resembled the percentages of TFR in various TKI-stop studies. These results suggest that the acquisition of CTL clonality may provide long-term remission and TFR to CP-CML patients and that cessation of TKIs should be considered in patients with clonal expansion of memory CTLs, irrespective of NK cells. [Display omitted] DisclosuresJo:Bristol-Myers Squibb: Honoraria.

A Highly Active Form of XCL1/Lymphotactin Functions as an Effective Adjuvant to Recruit Cross-Presenting Dendritic Cells for Induction of Effector and Memory CD8+ T Cells.

The chemokine receptor XCR1 is known to be selectively expressed by cross-presenting dendritic cells (DCs), while its ligand XCL1/lymphotactin is mainly produced by activated CD8+ T cells and natural killer cells. Recent studies have shown that XCL1-antigen fusion proteins efficiently induce CD8+ T cell responses by preferentially delivering antigens to XCR1+ DCs. However, XCL1 per se was found to be a poor adjuvant for induction of CD8+ T cell responses. XCL1 is unique because of its lack of one of the two disulfide bonds commonly conserved in all other chemokines and thus has an unstable structure with a relatively weak chemokine activity. In the present study, we generated a variant form of murine XCL1 termed mXCL1-V21C/A59C that contained a second disulfide bond to stabilize its chemokine structure. We confirmed that mXCL1-V21C/A59C had much more potent chemotactic and calcium mobilization activities than the wild type XCL1 (mXCL1-WT). Intradermal injection of mXCL1-V21C/A59C, but not that of mXCL1-WT, significantly increased the accumulation of XCR1+CD103+ DCs in the injection site, and most of the accumulated XCR1+CD103+ DCs were found to take up co-injected ovalbumin (OVA). Furthermore, recruited XCR1+CD103+ DCs efficiently migrated to the draining lymph nodes and stayed for a prolonged period of time. Consequently, mXCL1-V21C/A59C strongly induced OVA-specific CD8+ T cells. The combination of OVA and mXCL1-V21C/A59C well protected mice from E.G7-OVA tumor growth in both prophylactic and therapeutic protocols. Finally, memory CTL responses were efficiently induced in mice immunized with OVA and mXCL1-V21C/A59C. Although intradermal injection of OVA and polyinosinic-polycytidylic acid (poly(I:C)) as an adjuvant also induced CD8+ T cell responses to OVA, poly (I:C) poorly recruited XCR1+CD103+ DCs in the injection site and failed to induce significant memory CTL responses to OVA. Collectively, our findings demonstrate that a highly active form of XCL1 is a promising vaccine adjuvant for cross-presenting DCs to induce antigen-specific effector and memory CD8+ T cells.

An Antigen-Free, Plasmacytoid Dendritic Cell-Targeting Immunotherapy To Bolster Memory CD8+ T Cells in Nonhuman Primates.

The priming, boosting, and restoration of memory cytotoxic CD8+ T lymphocytes by vaccination or immunotherapy in vivo is an area of active research. Particularly, nucleic acid-based compounds have attracted attention due to their ability to elicit strong Ag-specific CTL responses as a vaccine adjuvant. Nucleic acid-based compounds have been shown to act as anticancer monotherapeutic agents even without coadministration of cancer Ag(s); however, so far they have lacked efficacy in clinical trials. We recently developed a second-generation TLR9 agonist, a humanized CpG DNA (K3) complexed with schizophyllan (SPG), K3-SPG, a nonagonistic Dectin-1 ligand. K3-SPG was previously shown to act as a potent monoimmunotherapeutic agent against established tumors in mice in vivo. In this study we extend the monoimmunotherapeutic potential of K3-SPG to a nonhuman primate model. K3-SPG activated monkey plasmacytoid dendritic cells to produce both IFN-α and IL-12/23 p40 in vitro and in vivo. A single injection s.c. or i.v. with K3-SPG significantly increased the frequencies of activated memory CD8+ T cells in circulation, including Ag-specific memory CTLs, in cynomolgus macaques. This increase did not occur in macaques injected with free CpG K3 or polyinosinic-polycytidylic acid. Injection of 2 mg K3-SPG induced mild systemic inflammation, however, levels of proinflammatory serum cytokines and circulating neutrophil influx were lower than those induced by the same dose of polyinosinic-polycytidylic acid. Therefore, even in the absence of specific Ags, we show that K3-SPG has potent Ag-specific memory CTL response-boosting capabilities, highlighting its potential as a monoimmunotherapeutic agent for chronic infectious diseases and cancer.

PP5.9 - Novel dual role of dendritic cells in priming de novo CTL responses while inhibiting memory CTL responses to HIV-1 through the PD-L1 pathway

PP5.9 - Novel dual role of dendritic cells in priming de novo CTL responses while inhibiting memory CTL responses to HIV-1 through the PD-L1 pathway