Cytotoxic Lymphocyte Precursors Research Articles

Stability and Hopf Bifurcation Analysis of an HIV Infection Model in the Evolution of Drug Resistance

In the course of reverse transcription, drug-sensitive strain can mutate to a drug-resistant one. Cell-to-cell transmission is proved to be more efficient than virus-to-cell infection. The population of Cytotoxic T Lymphocytes (CTLs) can be divided into CTL precursors and CTL effectors. Naive CTL precursors are those that never have contacted antigen, circulating around the body. Once the host is infected, CTL precursors (CTLp) begin to proliferate and differentiate into CTL effectors (CTLe), which have antiviral activity. In this paper, we study an HIV model with drug-sensitive and drug-resistant mixed strains, CTLp and CTLe, both viral and cellular infections. The basic reproduction ratios for each strain are derived and the stability of each feasible equilibrium is analyzed by using Routh–Hurwitz criteria. The periodic solutions may occur near the equilibrium with CTL immune response via Hopf bifurcation. By using normal form theory, we find more information about the limit cycles, such as the amplitude, phase and stability.

Utilizing TCR-Seq to Detect Tabelecleucel, an Allogeneic Epstein-Barr Virus (EBV)-Specific T-Cell Immunotherapy, Post-Infusion

Background: Tabelecleucel is an off-the-shelf, allogeneic EBV-specific T-cell immunotherapy that targets EBV-antigen presenting cells in an HLA restricted manner. It is not genetically modified and therefore lacks a distinct universal molecular moiety to detect it post-infusion. To date, the most effective measure of EBV reactive T-cells in patients (pts) treated with tabelecleucel is the EBV-cytotoxic T lymphocyte precursor (CTLp) assay, which measures the number of EBV reactive CTL precursors in each sample but cannot distinguish between the pt's endogenous CTLs or the infused product CTLs. We have previously shown that EBV-CTLp peak correlates with pt response. Prior methods to identify circulating product CTLs have relied on differences in the genomic profiles of the pt and dosing product lot. However, the limit of detection for these assays to date has not been sensitive enough to reliably detect product CTLs post-infusion. One aspect of T-cell biology that could be utilized to track product specific CTL clones is the unique T cell receptors (TCR) which impart antigen specificity. TCRs are highly diverse and only a small minority of TCR clones are found to be shared across unrelated individuals. Therefore, we leveraged TCRβ sequencing of the CDR3 region to barcode unique TCR clones in tabelecleucel product lots and pt derived samples to detect product T cell clones post-infusion, and annotate likely EBV-targeting T cell clones based upon sequence homology to previously reported EBV-targeting TCR clones. Using this approach, we aimed to identify corollaries associated with clinical outcomes. Methods: Peripheral blood mononuclear cell samples from pts enrolled in the ALLELE (NCT03394365) study were obtained pre-dose on cycle (C) 1 day (D)1/D8/D15, C2D1/D8/D15 and at safety follow-up. EBV-CTLp was used to measure circulating EBV reactive T-cells, and both the pre-infusion baseline timepoint, and the timepoint of peak on-treatment EBV-CTL expansion were selected for downstream TCRβ sequencing. TCR repertoires for pt samples and the tabelecleucel dosing lot were assessed using TCRβ immunoSEQ assay. The captured TCR repertoires of pt samples were compared to the TCR repertoire of the dosing tabelecleucel lot to annotate product TCR clones and were compared against publicly available databases of previously identified EBV-specific TCRβ sequences to annotate putative EBV targeting TCR clones. Results: Analyses for an initial subset of 7 pts with EBV-positive post-transplant lymphoproliferative disease have been completed and are described here. Six unique tabelecleucel lots were used to treat these pts and were included in the analysis. On average 0.15 ± 0.04% of TCRs detected were shared between multiple lots, allowing the ability to leverage TCRβ sequences as a unique barcode to identify specific product lots. All 7 pts tested had TCRβ clones matching previously identified EBV-specific TCRs detected at baseline (median [min, max]: 3498 [1855, 12486]) and peak EBV-CTLp; clones detected at peak EBV-CTLp were either those previously detected at baseline (532 [129,1627]) or newly emerged (3622, [1801,5347]). TCRβ clones matching the dosing lot were identified in 6 out of 7 pts. Three pts had TCRβ clones matching the dosing lot present at baseline prior to tabelecleucel infusion (2 [1,5]). Post-infusion emergence of TCRs matching the dosing lot was detected in 5 pts (1 [1,3]). TCRβ sequencing on additional pts is currently underway. Association of post-infusion detection of dosing lot specific TCR clones with pt best overall response is ongoing and will be reported at the time of presentation. Conclusions: Utilizing TCRβ sequencing we can identify putative EBV-targeting TCR clones and dosing product TCR clones in pt TCR repertoires. Overall, an average of 12.5 ± 1.6% of captured TCR repertoires were annotated as EBV-targeting TCR clones. As publicly available reference datasets for EBV-specific TCRs are currently limited, this is likely an underrepresentation of the totality of EBV-specific TCRs present within the samples. Additionally, an average of 75.8% of the dosing product TCR clones detected were found exclusively after tabelecleucel infusion. Altogether, these results confirm that tracking TCRβ clones can be utilized as a novel assessment to detect post-infusion non-genetically modified allogenic cell therapies and future associations with clinical efficacy will be determined.

Phase I dose escalation safety and feasibility study of autologous WT1-sensitized T cells for the treatment of patients with recurrent ovarian cancer

BackgroundThis phase I dose escalation trial evaluated the feasibility of production, safety, maximum tolerated dose, and preliminary efficacy of autologous T cells sensitized with peptides encoding Wilms’ tumor protein 1...

Towards Safer Allogeneic HSCT in Autoimmunity: Proof of Concept in Diabetes-Prone Mice

R.S.K and B.N.L contributed equally to this study. The induction of tolerance towards pancreas autoantigens is a promising approach in the treatment of type 1 diabetes mellitus (T1DM). This goal can be partially attained by immuno-ablation followed by autologous HSCT which mitigates the immune reaction leading to diabetes, at least temporarily, if performed soon after diagnosis. However, most T1DM patients transplanted with autologous HSCT eventually relapse (E Snarski et al., Bone marrow Transplantation; 2016; 51, 398-402). Thus, developing methodologies for allogeneic HSCT, to provide a durable non-autoimmune TCR repertoire, could be a promising treatment approach provided that such protocols can safely achieve donor type chimerism. Accordingly, transplantation of T cell-depleted allogeneic HSCT (TD-HSCT) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option. However, overcoming rejection after reduced conditioning in T1DM represents a major challenge. We previously demonstrated in wild type mice that rejection of TD-HSCT can be prevented using donor-derived veto cells. Here, we show proof of concept of the safety and efficacy of veto cell mediated non-myeloablative mismatched allogeneic TD-HSCT, in the established NOD mouse T1DM model. Veto activity, first defined by Miller (Miller, R. G; Nature; 1980; 287; 544-54), is based on the ability of specific cell populations to attack host CTL-precursors (CTLp) that are directed against the antigens presented by the veto cells themselves. This response spares cells that are not targeted against the veto cells, including those recognizing pathogens. Among different veto cell populations described in the literature, central memory CD8 T cells exhibit the most robust veto activity upon transplantation, but are also endowed with marked GvH activity. We overcame GvH reactivity by expanding naïve or memory CD8 T cells against 3rd party MHC or viral antigens, under culture conditions favoring expression of the central memory phenotype. Such anti-3rd party central memory CD8 T cells (Tcm) are endowed with marked veto activity, while effectively depleted of GVH reactivity in fully mis-matched recipients (Reviewed in Reisner Y, Or-Geva N. Semin. Hematol. 2019; 56(3): 173-182). In this study, Tcm veto cells were generated from splenocytes obtained from Balb/c donors (H2d) cultured against irradiated third-party splenocytes (FVB; H2q), under cytokine deprivation. The selective expansion of CD8 mouse T cells against 3rd party stimulators under these conditions leads to selective 'death by neglect' of bystander anti-host T cell clones that could mediate GvHD; such cells are further diluted out by subsequent expansion of anti-3rd party T clones during further culture in the presence of IL15. In addition to causing selective loss of GvH reactive T cells, these culture conditions induce a central memory phenotype shown to be crucial for robust veto activity in vivo. To evaluate the safety and efficacy of such a transplant for diabetes therapy, 8 week old NOD mice (before diabetes onset) were treated with 4.5 Gy TBI conditioning at day -1, anti-3rd party veto Tcm and megadose nude bone marrow (NuBM) on day 0, and Rapamycin treatment from day -1 to day +4 (Scheme 1). Controls were untreated or received conditioning with no transplant. In four independent experiments (n= 35 NOD mice in the transplanted group), high chimerism from 83.5% to 99.6 % was found in all transplanted mice at 6 months post-transplant. Notably, with a follow-up of 200 days, 72.4% mice in the untreated group, and 96% in the conditioned group died of diabetes, while only 8.5% diabetes-associated mortality (Fig.1A) and no GvHD or other transplant-related mortality was observed in the transplanted mice (Fig.1B). Our results demonstrate a proof of concept for the safety and efficacy of non-myeloablative allogeneic TD-HSCT in type 1 diabetes. A clinical protocol testing the safety and efficacy of anti-3rd party veto cells in the context of low toxicity non-myeloablative TD-HSCT in hematological malignancies is commencing at MD Anderson Cancer Center. If successful, our results support further extension of this platform to autoimmune diabetes and other autoimmune diseases. Disclosures Lustig: Yeda Ltd.: Patents & Royalties. Reisner:Cell Source, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Proof of Concept for Repair of Sickle Cell Disease By Non-Myeloablative MHC Disparate T Cell Depleted HSCT Combined with Donor-Derived Veto Cells

Although life extending medical treatments are available for sickle cell disease (SCD), allogenic hematopoietic stem cell transplantation (HSCT) is considered a treatment of choice (Ozdogu et al., Bone Marrow Transplant 2018; 53(7): 880-890). However, HSCT is associated with several limitations, including conditioning-related toxicity and graft-versus-host disease (GvHD), especially when using MHC disparate transplants. Thus, the development of safe transplantation protocols for MHC disparate HSCT in sickle cell disease is vital. While the risk of GvHD and conditioning toxicity can be effectively reduced by the use of T-cell-depleted HSCT (TD-HSCT) under reduced intensity conditioning (RIC), rejection of TD-HSCT remains a challenge. We previously demonstrated in wild type mice that this barrier can be overcome using donor-derived veto cells. Here, we demonstrate the safety and efficacy of this approach in a well-defined murine model for sickle cells disease. Veto activity is based on the ability of certain cells to attack host CTL-precursors (CTLp) which are directed against antigens expressed on the veto cells themselves, sparing cells that are not targeted against the veto cells including T cells needed for defense against pathogens. Central memory CD8 T cells exhibit the most robust veto activity upon transplantation; however, these cells are also endowed with marked GvH activity. We overcame this issue by expanding naïve or memory CD8 T cells against 3rd party MHC or viral antigens, respectively, under culture conditions favoring expression of central memory phenotype. Such anti-3rd party central memoryCD8 T cells (Tcm), which are endowed with marked veto activity, also exhibit reduced risk for GvHD in fully mis-matched recipients (Reviewed in Reisner Y, Or-Geva N. Semin Hematol. 2019; 56(3): 173-182.) To generate Tcm veto cells, splenocytes obtained from Balb/c donors (H2d) were cultured against irradiated third-party splenocytes (FVB; H2q) under cytokine deprivation. The selective expansion of CD8 mouse T cells against 3rd party stimulators leads to selective 'death by neglect' of bystander anti-host T cell clones potentialy mediating GvHD, and these are further diluted out by subsequent expansion of anti-3rd party T clones during continued culture in the presence of IL-15. Apart from selective loss of GvH reactive T cells, these culture conditions induce a central memory phenotype shown to be important for attaining robust veto activity in vivo (Ophir et al., Blood. 2013; 121(7): 1220-8). We first calibrated the optimal irradiation dose for sickle mice (Berkeley model, H2b), comparing 4.5 Gy versus 5 Gy TBI in a conditioning protocol also including short term rapmycin treatment (Scheme 1). Higher levels of engraftment and chimerism were found in the group receiving 5Gy TBI (Fig. 1). All mice of both treatment groups survived >140 days with no evidence of GvHD. To further evaluate this treatment approach, 8 week old sickle mice (N=7) were given bone marrow transplants using the protocol described in Scheme 1, including conditioning with 5 Gy TBI (day -1), rapamycin treatment (day -1 to day 4), and transplantation of NuBM (day 0) plus veto cells (day 7; Scheme 1). Notably, at 44 days post-transplant, 6 out of 7 mice receiving NuBM + TCM + Rapa (85.7%) showed donor chimerism in the peripheral blood, ranging between 77-94% (Fig.2A-B), while no chimerism was detected in mice receiving conditioning alone, or conditioning and transplantation with only NuBM or only veto cells . All mice in all groups survived (N=26) , and no GvHD was detected with a follow up of 77 days, even in the transplanted group which exhibited high donor-derived chimerism. Furthermore, reversal of sickle disease symptoms was observed, including reticulocyte levels (p=0.001;Fig.2C) and expression of wild type hemoglobin ( Fig.2D) in all engrafted mice. Our results offer a proof of concept for the treatment of sickle disease by MHC disparate non-myeloablative T cell depleted HSCT in conjunction with anti-3rd party central memory veto CD8 T cells. A clinical trial testing the safety and efficacy of anti-3rd party veto cells in the context of low toxicity non-myeloablative TD-HSCT in hematological malignancies is currently ongoing at MD Anderson Cancer Center. If successful, our present results support further evaluation of this platform for sickle cell disease. Disclosures Lustig: Yeda Ltd.: Patents & Royalties. Reisner:Cell Source, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Correlation of circulating EBV-targeted cytotoxic T lymphocyte precursors (EBV-CTLp) and clinical response following tabelecleucel (tab-cel) infusion in patients with EBV-driven disease.

2532 Background: EBV is implicated in a variety of diseases. Tab-cel is an investigational off-the-shelf, allogeneic T-cell immunotherapy utilizing endogenous T cell receptors targeting EBV antigens. We hypothesized the clinical activity of tab-cel is mediated by expansion and persistence of EBV-specific T cells. Therefore, we quantified circulating EBV- CTLp after tab-cel administration and examined the correlation between expansion and clinical response. Methods: Samples from 10 patients with EBV+ post-transplant lymphoproliferative disease (PTLD) and other EBV-associated diseases enrolled in an multicenter expanded access protocol (EAP) study ( NCT02822495 ) were analyzed. To evaluate CTLp frequencies, limited dilution analysis was performed on samples taken at baseline and day 34 post first tab-cel dose (end cycle 1). The day 34 persistence of circulating EBV-CTLp from best overall response to initial tab-cel product was tested using the two-tailed Mann-Whitney test. Changes in inflammatory cytokines were also measured. Results: Responders represented in this sampling (n=6; 2 PR and 4 CR) showed a median 5.8-fold increase in circulating CTLp between baseline and day 34 (range: 0.8 to 133-fold). Five of 6 responders showed an increase in EBV-CTLp at day 34 of ≥ 3.8-fold while 1 pt showed no change in CTLp (0.8-fold change). In contrast, the 4 non-responders (3 SD; 1 PD) showed a median 0.3-fold decrease in EBV-CTLp from baseline (range: 1.2 to 0.02-fold; ns). Cumulative analyses revealed a statistically significant correlation between the fold-change of circulating CTLp at day 34 and clinical response (p=0.038) which did not appear to correlate with the type of the EBV-associated disease. Inflammatory cytokines showed no meaningful change from baseline. The safety profile remains consistent with previously reported data. Conclusions: These data support the correlation of clinical activity of tab-cel with the expansion and persistence of EBV-specific T-cells at day 34 post-treatment, as well as the use of circulating CTLp as a biomarker for response in clinical studies. Clinical trial information: NCT02822495.

Abstract 4695: Murine tumor growth suppression through CD8+CTLs via activated DEC-205+dendritic cells by sequential administration of α-galactosylceramide in vivo

Abstract Cancer immunotherapy is mediated by the effective priming and activation of tumor-specific class I major histocompatibility complex molecule-restricted CD8+ cytotoxic T lymphocytes. DEC-205+ dendritic cells can cross-present the epitopes of captured tumor antigens associated with class I MHC molecules alongside co-stimulatory molecules to prime and activate tumor-specific CD8+ CTLs. Immunosuppressive tolerogenic DCs with reduced co-stimulatory molecules may be a cause of insufficient CTL induction. Hepa1-6-1 cells were established from the mouse hepatoma cell line Hepa1-6; these cells grow continuously after subcutaneous implantation into syngeneic C57BL/6 mice and do not prime CD8+ CTLs. Here, we show that the growing of tumors was suppressed by activated CD8+ CTLs with tumor-specific cytotoxicity through the administration of the glycolipid α-galactosylceramide, which is a compound known to stimulate invariant natural killer T cells and selectively activate DEC-205+ DCs. Moreover, we demonstrated that sequential repetitive intraperitoneal inoculation of α-GalCer every 48 hour appeared to convert tolerogenic DEC-205+ DCs into immunogenic DCs with a higher expression of co-stimulatory molecules and a greater cross-presentation capacity, which primed CTL precursors and induced tumor-specific CD8+ CTLs within the tumor environment without activating iNKT cells. These findings provide a new basis for cancer immunotherapy to convert tolerogenic DEC-205+ DCs within tumors into immunogenic DCs via the sequential administration of an immuno-potent lipid/glycolipid, and activated immunogenic DCs expressing sufficient suitable co-stimulatory molecules will prime and activate tumor-specific CD8+ CTLs within the tumor to control tumor growing. Citation Format: Hideki Kogo, Hidemi Takahashi, Eiji Uchida. Murine tumor growth suppression through CD8+CTLs via activated DEC-205+dendritic cells by sequential administration of α-galactosylceramide in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4695.

Hematopoiesis by iPSC-derived hematopoietic stem cells of aplastic anemia that escape cytotoxic T-cell attack

AbstractHematopoietic stem cells (HSCs) that lack HLA-class I alleles as a result of copy-number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or HLA allelic mutations often constitute hematopoiesis in patients with acquired aplastic anemia (AA), but the precise mechanisms underlying clonal hematopoiesis induced by these HLA-lacking (HLA−) HSCs remain unknown. To address this issue, we generated induced pluripotent stem cells (iPSCs) from an AA patient who possessed HLA-B4002–lacking (B4002−) leukocytes. Three different iPSC clones (wild-type [WT], 6pLOH+, and B*40:02-mutant) were established from the patient's monocytes. Three-week cultures of the iPSCs in the presence of various growth factors produced hematopoietic cells that make up 50% to 70% of the CD34+ cells of each phenotype. When 106 iPSC-derived CD34+ (iCD34+) cells with the 3 different genotypes were injected into the femoral bone of C57BL/6.Rag2 mice, 2.1% to 7.3% human multilineage CD45+ cells of each HLA phenotype were detected in the bone marrow, spleen, and peripheral blood of the mice at 9 to 12 weeks after the injection, with no significant difference in the human:mouse chimerism ratio among the 3 groups. Stimulation of the patient's CD8+ T cells with the WT iCD34+ cells generated a cytotoxic T lymphocyte (CTL) line capable of killing WT iCD34+ cells but not B4002− iCD34+ cells. These data suggest that B4002− iCD34+ cells show a repopulating ability similar to that of WT iCD34+ cells when autologous T cells are absent and CTL precursors capable of selectively killing WT HSCs are present in the patient's peripheral blood.

Mannosylated T/Tn with Freund's adjuvant induces cellular immunity.

Inducing cancer-specific cellular immune responses has become an attractive strategy in cancer treatment. In this study, we investigated the role of several adjuvants in eliciting T/Tn-specific cellular immunity and protection against T/Tn expressing tumor challenge. T/Tn (9:1) antigen was purified from blood type “O” erythrocytes donated from healthy Korean volunteers. Immunization was performed using: T/Tn only, T/Tn mixed with Freund’s adjuvant (T/Tn + FA), keyhole limpet hemocyanin (KLH)-conjugated T/Tn mixed with FA (KLH-T/Tn + FA), and oxidized mannan-conjugated T/Tn mixed with FA (ox-M-T/Tn + FA). Mice immunized with ox-M-T/Tn + FA generated T/Tn-specific CD3, helper T (Th) cells, major histocompatibility complex (MHC) II, and MHC I; T/Tn presentation was significantly high and tolerogenic CD11b+ was the lowest among the tumor models. To verify Th type, we stained intracellular cytokines (interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, and IL-10) using CD3 co-staining. Th1 (IFN-γ and GM-CSF) cytokines were highly expressed and showed high FasL/Fas ratios, cytotoxic T lymphocyte (CTL) activity, and cytotoxic T lymphocyte precursor (CTLp) activity in mice immunized with ox-M-T/Tn + FA. Lymphocyte infiltration was highest in mice immunized with ox-M-T/Tn + FA. Additionally, we monitored FasL, MHC I, CD301, and T/Tn expression levels using immunohistochemistry (IHC) on macrophage and tumor sites. The expression of all markers was highest in the ox-M-T/Tn + FA group. Furthermore, tumor retardation and survival rate were highest in the ox-M-T/Tn + FA group. These results demonstrate that a vaccine formulation of T/Tn conjugated with ox-M and mixed with FA-induced cellular immunity and sustained a humoral immune response without over-activating the immune system, thus effectively inhibiting tumor growth.

Abstract 4571: Advantage of dendritic cells for the therapeutic cancer vaccine

Abstract Background: To develop effective cancer vaccine, we examined CTLs induced by peptide vaccination (CTLpep) and DC vaccination (CTLDC) in terms of their antitumor effects. Methods: For preparation of peptide vaccine, 50 µg hgp100 peptide (KVPRNQDWL) and 20 µg CpG were emulsified with 50 µl CFA or IFA. Bone marrow-derived DCs were matured with LPS and pulsed with hgp100 peptide at 1 µg/ml and used as DC vaccine. To induce CTLpep and CTLDC, C57BL/6 mice were first injected with 1 x 107 naive spleen cells from Pmel-1 TCR transgenic mice to increase hgp100-specific CTL precursors. Then mice were vaccinated twice with 2 weeks interval. The induction of CTLs were evaluated 2 weeks after the second vaccination. To evaluate their antitumor activity, 1 x 106 B16F10 were inoculated into vaccinated mice (prophylactic model). For therapeutic model, 1 x 106 B16F10 were inoculated into mice on day 0. Five days later 1 x 107 Pmel-1 splenocytes were transferred. Then, mice were vaccinated on days 5 and 12, followed by the measurement of tumor growth. Results: DC vaccine induced 5-times more CTLs than peptide vaccine. The numbers of CTLpep and CTLDC obtained in the spleen were 2.4±0.2x105 and 1.2±0.4x106, respectively. CTLpep expressed PD-1 and Tim-3 at higher level than CTLDC. Most of CTLpep showed effector memory phenotype (CD44hiCD62L-), while CTLDC contained a considerable fraction of central memory cells (CD44hiCD62L+). Although both CTLpep and CTLDC produce IFN-γ and TNF-α at similar level, more CD107a were detected on CTLDC than CTLpep upon re-stimulation. CTLDC displayed better proliferation potential than CTLpep. These phenotypic differences were confirmed by the comprehensive gene expression analysis. Transcriptome analysis revealed that considerable portion of CTLpep and CTLDC were comparable and different from that of naïve CTLs. However, several genes were expressed differentially. CTLpep expressed higher amount of inhibitory receptors, such as Pdcd1, Lag3, Ctla4 and Tigit than CTLDC. CTLDC expressed Fbp1, which inhibits glycolysis, and Cpt1a, which is the rate-limiting enzyme of mitochondrial fatty acid oxidation, suggesting that their metabolic statuses were shifted from glycolysis to fatty acid oxidation. DC vaccine completely inhibited the tumor growth in prophylactic vaccine setting, while peptide vaccine delayed tumor growth compared to control mice. In therapeutic protocol, peptide vaccine showed no antitumor effect, while DC vaccine significantly suppressed tumor growth. The numbers of intratumoral CTLpep and CTLDC on day 19 were 5.2±2.8 x 104/g and 5.0±1.4 x 105/g, respectively. Intratumoral CTLDC expressed lower levels of PD-1, Tim-3 and LAG-3 and more Ki67 than CTLpep. Furthermore, Intratumoral CTLDC produce more IFN-γ and TNF-α than CTLpep. Conclusion: DC vaccine induced intratumoral CTLs with multi-functionality and high proliferation potential. Therefore, DC vaccine is our choice for the therapeutic cancer vaccine. Citation Format: Koji Nagaoka, Akihiro Hosoi, Tamaki Iino, Hirokazu Matsushita, Kazuhiro Kakimi. Advantage of dendritic cells for the therapeutic cancer vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4571. doi:10.1158/1538-7445.AM2017-4571

Suppression of murine tumour growth through CD8+ cytotoxic T lymphocytes via activated DEC-205+ dendritic cells by sequential administration of α-galactosylceramide in vivo.

SummaryCancer immunity is mediated through the effective priming and activation of tumour‐specific class I MHC molecule‐restricted CD8+ cytotoxic T lymphocytes (CTLs). DEC‐205+ dendritic cells (DCs) can cross‐present the epitope(s) of captured tumour antigens associated with class I MHC molecules alongside co‐stimulatory molecules to prime and activate tumour‐specific CD8+ CTLs. Immunosuppressive tolerogenic DCs with reduced co‐stimulatory molecules may be a cause of impaired CTL induction. Hepa1‐6‐1 cells were established from the mouse hepatoma cell line Hepa1‐6; these cells grow continuously after subcutaneous implantation into syngeneic C57BL/6 (B6) mice and do not prime CD8+ CTLs. In this study, we show that the growth of ongoing tumours was suppressed by activated CD8+ CTLs with tumour‐specific cytotoxicity through the administration of the glycolipid α‐galactosylceramide (α‐GalCer), which is a compound known to stimulate invariant natural killer T (iNKT) cells and selectively activate DEC‐205+ DCs. Moreover, we demonstrated that sequential repetitive intraperitoneal inoculation with α‐GalCer every 48 hr appeared to convert tolerogenic DEC‐205+ DCs into immunogenic DCs with a higher expression of co‐stimulatory molecules and a stronger cross‐presentation capacity, which primed CTL precursors and induced tumour‐specific CD8+ CTLs within the tumour environment without activating iNKT cells. These findings provide a new basis for cancer immunotherapy to convert tolerogenic DEC‐205+ DCs within tumours into immunogenic DCs through the sequential administration of an immuno‐potent lipid/glycolipid, and then activated immunogenic DCs with sufficient expression of co‐stimulatory molecules prime and activate tumour‐specific CD8+ CTLs within the tumour to control tumour growth.

A novel approach to measuring cell-mediated lympholysis using quantitative flow and imaging cytometry

In this study, we established a novel isotope-free approach for the detection of cell-mediated lympholysis (CML) in MHC defined peripheral blood mononuclear cells (PBMCs) using multiparameter flow and imaging cytometry. CML is an established in vitro assay to detect the presence of cytotoxic effector T-lymphocytes precursors (CTLp). Current methods employed in the identification of CTLp in the context of transplantation are based upon the quantification of chromium (51Cr) released from target cells. In order to adapt the assay to flow cytometry, primary porcine PBMC targets were labeled with eFluor670 and incubated with major histocompatibility complex (MHC) mismatched effector cytotoxic lymphocytes (CTLs). With this method, we were able to detect target-specific lysis that was comparable to that observed with the 51Cr-based assay. In addition, the use of quantitative cell imaging demonstrates the presence of accessory cells involved in the cytotoxic pathway. This innovative technique improves upon the standard 51Cr release assay by eliminating the need for radioisotopes and provides enhanced characterization of the interactions between effector and target cells. This technique has wide applicability to numerous experimental and clinical models involved with effector–cell interactions.

Mesothelin-specific cell-based vaccine generates antigen-specific immunity and potent antitumor effects by combining with IL-12 immunomodulator.

Ovarian cancer is a gynecologic malignancy with a high mortality rate. In the present study, we developed a novel cell-based vaccine, Meso-VAX, to generate mesothelin antigen-specific immune responses and immunotherapy against ovarian cancer. Mesothelin, a secreted protein anchored at the cell membrane, has recently been identified as a potential new tumor antigen for ovarian cancer. In this study, mice vaccinated with Meso-VAX and adeno-associated virus (AAV)-IL-12 exhibited dramatic increases in the number of mesothelin-specific CD4(+) helper and CD8(+) cytotoxic T-cell precursors, higher titers of anti-mesothelin Abs and in vitro tumor killing activity, and all of these mice were tumor-free after 60 days of tumor challenge. In addition, a significant reduction in peritoneal tumors and longer survival were noted in the mice vaccinated with Meso-VAX combined with AAV-IL-12. CD4(+) helper and CD8(+) cytotoxic T lymphocytes were essential for the antitumor effect generated by Meso-VAX combined with AAV-IL-12. The post-vaccination sera of the mice vaccinated with Meso-VAX and AAV-IL-12 also showed mesothelin-specific complement-dependent cell-mediated cytotoxicity. Our results suggest that a Meso-VAX cell-based vaccine combined with AAV-IL-12 can generate antigen-specific immunological responses and antitumor effects on ovarian cancer.

Stability Analysis of an Improved HBV Model with CTL Immune Response.

To better understand the dynamics of the hepatitis B virus (HBV) infection, we introduce an improved HBV model with standard incidence function, cytotoxic T lymphocytes (CTL) immune response, and take into account the effect of the export of precursor CTL cells from the thymus and the role of cytolytic and noncytolytic mechanisms. The local stability of the disease-free equilibrium and the chronic infection equilibrium is obtained via characteristic equations. Furthermore, the global stability of both equilibria is established by using two techniques, the direct Lyapunov method for the disease-free equilibrium and the geometrical approach for the chronic infection equilibrium.

Association between CTL Precursor Frequency to HLA-C Mismatches and HLA-C Antigen Cell Surface Expression.

Previous studies showed the relevance of the cytotoxic T-cell precursor (CTLp) frequency assay for prediction of the outcome of HLA mismatched hematopoietic cell transplantation (HCT). Recently, it has been shown that HLA-C cell surface expression is correlated with virus specific cytotoxic T-cell responses and viremia control in HIV patients. The aim of the current study was to investigate the association between HLA-C antigen expression and the CTLp frequency to the mismatched HLA-C antigen. In total 115 recipient–donor pairs, for whom a successful CTLp assay was performed, were evaluated for this pilot study. All donor–recipient pairs were matched at 9/10 alleles with a single mismatch at the HLA-C locus. Antigen expression level of the mismatched HLA-C allele for each recipient and donor was based on the mean fluorescence intensity (MFI) values as described by Apps et al. (1). The cell surface expression of recipient’s mismatched HLA-C antigen was significantly lower among CTLp negative (n = 59) compared to CTLp positive (n = 56) pairs (154 and 193 MFI units, respectively, p = 0.0031). This difference was more pronounced in donor–recipient pairs that were mismatched for amino-acid residue-116 located in the groove of the HLA-C antigen, suggesting that the importance of peptide binding in the allo-recognition. Furthermore, in the particular case of low expression of the recipient mismatched HLA-C antigen (MFI < 115), CTLp reactivity depended on HLA-C expression level in the donor, the median MFI of donor’s mismatched HLA-C antigen was 114 in CTLp negative cases (n = 26), while in CTLp positive cases (n = 15) the median MFI of donor’s HLA-C antigen was 193 (p = 0.0093). We conclude that the expression level of the donor and recipient mismatched HLA-C antigens affect CTLp outcome. HLA-C antigen expression levels in combination with the CTLp assay may prove useful for the prediction of the clinical outcome of HLA-C mismatched HCT.

Transgenic 4-1BBL-engineered vaccine stimulates potent Gag-specific therapeutic and long-term immunity via increased priming of CD44(+)CD62L(high) IL-7R(+) CTLs with up- and downregulation of anti- and pro-apoptosis genes.

Human immunodeficiency virus type-1 (HIV-1)-specific dendritic cell (DC) vaccines have been used in clinical trials. However, they have been found to only induce some degree of immune responses in these studies. We previously demonstrated that the HIV-1 Gag-specific Gag-Texo vaccine stimulated Gag-specific effector CD8(+) cytotoxic T lymphocyte (CTL) responses, leading to completely protective, but very limited, therapeutic immunity. In this study, we constructed a recombinant adenoviral vector, adenovirus (AdV)4-1BBL, which expressed mouse 4-1BB ligand (4-1BBL), and generated transgenic 4-1BBL-engineered OVA-Texo/4-1BBL and Gag-Texo/4-1BBL vaccines by transfecting ovalbumin (OVA)-Texo and Gag-Texo cells with AdV4-1BBL, respectively. We demonstrate that the OVA-specific OVA-Texo/4-1BBL vaccine stimulates more efficient OVA-specific CTL responses (3.26%) compared to OVA-Texo-activated responses (1.98%) in wild-type C57BL/6 mice and the control OVA-Texo/Null vaccine without transgenic 4-1BBL expression, leading to enhanced therapeutic immunity against 6-day established OVA-expressing B16 melanoma BL6-10OVA cells. OVA-Texo/4-1BBL-stimulated CTLs, which have a CD44(+)CD62L(high) IL-7R(+) phenotype, are likely memory CTL precursors, demonstrating prolonged survival and enhanced differentiation into memory CTLs with functional recall responses and long-term immunity against BL6-10OVA melanoma. In addition, we demonstrate that OVA-Texo/4-1BBL-stimulated CTLs up- and downregulate the expression of anti-apoptosis (Bcl2l10, Naip1, Nol3, Pak7 and Tnfrsf11b) and pro-apoptosis (Casp12, Trp63 and Trp73) genes, respectively, by RT(2) Profiler PCR array analysis. Importantly, the Gag-specific Gag-Texo/4-1BBL vaccine also stimulates more efficient Gag-specific therapeutic and long-term immunity against HLA-A2/Gag-expressing B16 melanoma BL6-10Gag/A2 cells than the control Gag-Texo/Null vaccine in transgenic HLA-A2 mice. Taken together, our novel Gag-Texo/4-1BBL vaccine, which is capable of stimulating potent Gag-specific therapeutic and long-term immunity, may represent a new immunotherapeutic vaccine for controlling HIV-1 infection.

In Vivo RNA Interference Screens Identify Regulators of Antiviral CD4+ and CD8+ T Cell Differentiation

Classical genetic approaches to examine the requirements of genes for T cell differentiation during infection are time consuming. Here we developed a pooled approach to screen 30-100+ genes individually in separate antigen-specific T cells during infection using short hairpin RNAs in a microRNA context (shRNAmir). Independent screens using T cell receptor (TCR)-transgenic CD4(+) and CD8(+) T cells responding to lymphocytic choriomeningitis virus (LCMV) identified multiple genes that regulated development of follicular helper (Tfh) and T helper 1 (Th1) cells, and short-lived effector and memory precursor cytotoxic T lymphocytes (CTLs). Both screens revealed roles for the positive transcription elongation factor (P-TEFb) component Cyclin T1 (Ccnt1). Inhibiting expression of Cyclin T1, or its catalytic partner Cdk9, impaired development of Th1 cells and protective short-lived effector CTL and enhanced Tfh cell and memory precursor CTL formation in vivo. This pooled shRNA screening approach should have utility in numerous immunological studies.

The Influenza Virus–Specific CTL Immunodominance Hierarchy in Mice Is Determined by the Relative Frequency of High-Avidity T Cells

Virus-specific CTL responses typically fall into reproducible hierarchies with particular epitopes eliciting either immunodominant or subdominant responses after viral challenge. The recently acquired capacity to directly enumerate naive CTL precursors (CTLps) in both mice and humans has implicated CTLp frequency as a key predictor of immune response magnitude after Ag challenge. However, recent studies have indicated that naive CTLp frequencies do not necessarily predict the size of the Ag-driven response, indicating an important role for differential CTLp recruitment and/or expansion. This study characterizes the early emergence of various influenza epitope-specific CTL responses at multiple sites in C57BL/6 mice, and probes the role of Ag dose and TCR avidity in dictating immune response hierarchies. Despite large naive CTLp numbers, subdominance was found to arise largely as a consequence of the abrupt and premature cessation of CTL proliferation, at least for one epitope specificity. Investigation into the possible drivers of the poor proliferation observed for subdominant specificities showed that the immunodominance hierarchy endured irrespective of epitope abundance, and correlated with the prevalence of high-avidity T cells in both the naive and immune compartments. Our study strongly indicates that the quality, and not simply the quantity, of antiviral CTLs dictate response magnitude.

Initial IL7R signaling regulate the induction of effector CD8+ T cells and subsequent anti-tumor immune response in lymphopenic host (CCR3P.208)

Abstract Induction of lymphopenia and adoptive transfer of T cells are followed by lymphopenia-induced proliferation (LIP), and generated a anti-tumor immune response. However, the distinct role of redundant IL7 in lymphopenic host and subsequent LIP of T cells for anti-tumor immune response has been unclear, since multiple mechanisms are involved in the anti-tumor immune response under lymphopenic conditions. To clarify the correlation between LIP and the anti-tumor effect, LIP was inhibited by IL7R blockade with anti-IL7Ra mAb at various stages, and the anti-tumor effect was assessed. When LIP was occurred, the growth of transplanted LLC expressing model antigen gp33, was markedly regressed. Using IL7R blockade with mAb at various stages, we found that IL7R signaling at the start of LIP is crucial for the anti-tumor immune response. Although some reports focused on the benefit of IL7 for the improvement of effector T cell function, later IL7R blockade did not arrest the expansion of CTLs, and anti-tumor effect was occurred. IFN-g production also depend on IL7R signaling during induction phase, but not later phase, and, the expression of co-inhibitory molecules, PD-1, on CTLs was unchanged in IL7R blockade hosts. In conclusion, our results demonstrated that initial IL7R signaling and subsequent LIP regulated the expansion of IL7Ra+ effector precursors mainly, but that the effector function of CTL precursors and migration into tumor did not require IL7R signaling.

IL-7 enhancement of antigen-driven activation/expansion of HIV-1-specific cytotoxic T lymphocyte precursors (CTLp).

CD8+ cytotoxic T lymphocytes are an important component in the immunologic control of human viral diseases. IL-7, a stromal cell-derived cytokine, has been demonstrated to enhance both anti-tumour and anti-viral CTL as well as lymphokine-activated killer (LAK) activity. We studied the ability of IL-7 to support the activation and the growth of in vitro antigen-specific CTL precursors (CTLp) present in peripheral blood mononuclear cells (PBMC) from HIV-infected patients. Results from these studies demonstrate that inclusion of IL-7 in a vaccinia/HIV-1 vector-based stimulation strategy greatly augmented overall CTL reactivities, whereas addition of IL-7 to unstimulated cultures failed to induce any significant anti-viral cytolytic activity. In four of six patients, HIV-specific lytic activities were significantly higher in cultures stimulated with antigen plus IL-7 compared with in vitro stimulation (IVS) with antigen alone. Cytotoxic activity was principally mediated by CD8+ effector cells, and CD3+/CD8+ cell expansion was increased by 2.7-fold in the presence of IL-7. In PBMC from seronegative donors, IL-7 enhanced anti-vaccinia CTL activities with less effect on cell proliferation. Furthermore, anti-gag CTL frequencies determined by limiting dilution analysis were increased by 2- and 10-fold in two asymptomatic patients following IVS plus IL-7 compared with antigen stimulation alone. Cytofluorimetric analysis revealed that IL-7 preferentially expanded CD8 memory cells (CD45RO+) and CD8+ lymphocytes expressing activation molecules. IL-7 was also able to support the growth of CD4+ lymphocytes, while having no effect on natural killer (NK)/K lymphocytes. Taken together, these data suggest that IL-7 acts cooperatively with the antigen supporting in vitro maturation of CTLp into functional cytotoxic effectors. Thus IL-7 may be an important biologic entity to consider as part of future immune-based therapies in which ex vivo expansion of antigen-driven CTL is an important determinant.