Memory CTL Research Articles

Neoantigen Expression in Steady-State Langerhans Cells Induces CTL Tolerance.

The skin hosts a variety of dendritic cells (DCs), which act as professional APC to control cutaneous immunity. Langerhans cells (LCs) are the only DC subset in the healthy epidermis. However, due to the complexity of the skin DC network, their relative contribution to either immune activation or immune tolerance is still not entirely understood. To specifically study the function of LCs in vivo, without altering the DC subset composition in the skin, we have generated transgenic mouse models for tamoxifen-inducible de novo expression of Ags in LCs but no other langerin+ DCs. Therefore, this system allows for LC-restricted Ag presentation to T cells. Presentation of nonsecreted OVA (GFPOVA) by steady-state LCs resulted in transient activation of endogenous CTL in transgenic mice. However, when these mice were challenged with OVA by gene gun immunization in the contraction phase of the primary CTL response they did not respond with a recall of CTL memory but, instead, with robust Ag-specific CTL tolerance. We found regulatory T cells (Tregs) enriched in the skin of tolerized mice, and depletion of Tregs or adoptive experiments revealed that Tregs were critically involved in CTL tolerance. By contrast, when OVA was presented by activated LCs, a recallable CTL memory response developed in transgenic mice. Thus, neoantigen presentation by epidermal LCs results in either robust CTL tolerance or CTL memory, and this decision-making depends on the activation state of the presenting LCs.

Recruitment of Immune Effector Cells against Glioblastoma-Multiforme by a MHC-Chlorotoxin Chimeric Protein

Glioblastoma Multiforme is the most common malignant primary brain tumor, having a mean overall survival <2 years. The lack of an efficient immune response against the tumor have been attributed to its immunosuppressive capabilities and an immunosuppressing local environment. Aim: We set out to design a chimeric molecule that recognizes and binds tissue inducible metalloproteinase known to be induced in GBM cells (MMP-2) on one end. Its other end, the effector domain, mobilizes and recruits cytotoxic T-cells to mount an effective anti-tumor reaction. Methods: The targeting moiety is the small 36-amino acids Chlorotoxin, derived from the venom of the Israeli Yellow scorpion. The effector end is a single chain HLA-A2 (Human leukocyte antigen subtype A2) covalently bound to phosphoprotein-65 derived from the cytomegalovirus, to which most of the human population has developed a specific immune response. Results: The molecular construct was cloned and expressed in E.coli. The protein product was isolated, purified, and then folded in vitro. Various activity assays employed demonstrated retained activity of each domain, including flow-cytometry, intracellular staining, fluorescence immunohistochemistry, radiolabeled toxicity assays etc. Initial in-vivo studies show great promise. Conclusions: We present a proof of concept study for a new immunotherapy approach to battle GBM. A molecular construct which contains a non-antibody compact and highly specific targeting domain, combined with the ability to recruit anti-CMV T-cell lymphocyte population. The recruitment of potent memory CTL’s to the tumor’s milieu may prove resistant to the previously described local immunosuppressive environment brought about by the tumor.

Major-histocompatibility complex harnessing and lymphocytic recruitment: a new tool for glioblastoma multiforme immunotherapy.

Glioblastoma multiforme (GBM) is the most common malignant primary brain neoplasm, notorious for being able to evade and suppress the immune-system. We present a new immunotherapeutic approach that can potentially overcome or circumvent many of the GBM laden obstacles for an efficient antitumor immune response. In this molecular construct, a soluble major-histocompatibility complex (MHC), presenting an immunogenic peptide is directed towards GBM cells. This soluble complex is known to be able to activate relevant cytotoxic T-lymphocyte (CTL's) populations, mounting an effective local immune response. The chimeric protein consists of a targeting domain, a function that can be fulfilled by an antibody or other small molecule that binds tumor associated antigens, and an effector domain. A single chain MHC directly linked to an immunogenic Cytomegalovirus derived peptide (phosphoprotein 65) can be used as such an effector domain. Targeting MHC complexes to the tumor enables to recruit different lymphocyte populations using MHC-molecules bearing a single, highly antigenic peptide derived from immunogenic T cell epitopes. Moreover, the recruited potent memory CTL's at the tumor's milieu may prove resistant to the previously described local immunosuppressive environment, and may enable the shift to TH1 cytokine profile resulting in specific massive tumor destruction.

IL-10 promotes homeostatic proliferation of human CD8(+) memory Tcells and, when produced by CD1c(+) DCs, shapes naive CD8(+) T-cell priming.

IL-10 is an anti-inflammatory cytokine that inhibits maturation and cytokine production of dendritic cells (DCs). Although mature DCs have the unique capacity to prime CD8(+) CTL, IL-10 can promote CTL responses. To understand these paradoxic findings, we analyzed the role of IL-10 produced by human APC subsets in T-cell responses. IL-10 production was restricted to CD1c(+) DCs and CD14(+) monocytes. Interestingly, it was differentially regulated, since R848 induced IL-10 in DCs, but inhibited IL-10 in monocytes. Autocrine IL-10 had only a weak inhibitory effect on DC maturation, cytokine production, and CTL priming with high-affinity peptides. Nevertheless, it completely blocked cross-priming and priming with low-affinity peptides of a self/tumor-antigen. IL-10 also inhibited CD1c(+) DC-induced CD4(+) T-cell priming and enhanced Foxp3 induction, but was insufficient to induce T-cell IL-10 production. CD1c(+) DC-derived IL-10 had also no effect on DC-induced secondary expansions of memory CTL. However, IL-15-driven, TCR-independent proliferation of memory CTL was enhanced by IL-10. We conclude that DC-derived IL-10 selects high-affinity CTL upon priming. Moreover, IL-10 preserves established CTL memory by enhancing IL-15-dependent homeostatic proliferation. These combined effects on CTL priming and memory maintenance provide a plausible mechanism how IL-10 promotes CTL responses in humans.

PD-1 inhibition results in reduced dendritic cell (DC) activation of HIV-1-specific de novo CTL and enhancement of DC-induced CTL memory recall responses

Abstract Combination strategies consisting of DC vaccines and suppression of immunoregulatory pathways such as PD-1 blockade have been advocated to elicit optimal CTL responses in chronic diseases including cancer and HIV infection. We have shown that inactivated HIV-1 virus loaded, high IL-12 producing, mature, type-1 polarized DC (DC1) can activate CTL from naïve CD8+ T cell precursors that more effectively kill HIV-1-infected cells than CTL derived from memory CD8+ T cells. Here we tested DC1 transfected with an adenoviral (Ad) vector encoding anti-PD-1 antibody (DC.αPD1) enhancement of both primary as well as memory CTL responses to HIV-1. Ad-αPD1-transduced DC1 (DC.αPD1) secreted high levels of functional anti-PD1 Ab without affecting their phenotype or IL-12p70-producing capacity. We next compared HIV Gag peptide epitope-loaded DC.αPD1 to DC1 that were transfected with an empty vector for their ability to activate either purified, autologous naïve or memory HIV-1-specific CD8+ T cells from chronic HIV-1-infected participants of the Multicenter AIDS Cohort Study. When compared to the control DC1, antigen-loaded DC.αPD1 enhanced the overall magnitude of HIV-1-specific CTL responses induced, as determined by the expansion of HIV-1-peptide responsive CD107a and IFNγ expressing T cells. In contrast, the overall number HIV-1 antigen-reactive CTL derived from the naïve CD8+ T cell fraction sharply decreased when using the DC.αPD1-based approach. These results suggest previously unrecognized, opposing roles of the inhibitory receptor PD-1 in DC1-induced primary versus memory recall CD8+ T cell responses to HIV-1.

The Differentiation and Protective Function of Cytolytic CD4 T Cells in Influenza Infection.

CD4 T cells that recognize peptide antigen in the context of class II MHC can differentiate into various subsets that are characterized by their helper functions. However, increasing evidence indicates that CD4 cells with direct cytolytic activity (CD4 CTL) play a role in chronic as well as acute infections, such as influenza A virus (IAV) infection. In the last couple of decades, techniques to measure the frequency and activity of these cytolytic cells has demonstrated their abundance in infections, such as human immunodeficiency virus, mouse pox, murine gamma herpes virus, cytomegalovirus, Epstein–Barr virus, and influenza among others. We now appreciate a greater role for CD4 CTL as direct effectors in viral infections and antitumor immunity through their ability to acquire perforin-mediated cytolytic activity and contribution to lysis of virally infected targets or tumors. As early as the 1980s, CD4 T cell clones with cytolytic potential were identified after influenza virus infection, yet much of this early work was dependent on in vitro culture and little was known about the physiological relevance of CD4 CTL. Here, we discuss the direct role CD4 CTL play in protection against lethal IAV infection and the factors that drive the generation of perforin-mediated lytic activity in CD4 cells in vivo during IAV infection. While focusing on CD4 CTL generated during IAV infection, we pull comparisons from the literature in other antiviral and antitumor systems. Further, we highlight what is currently known about CD4 CTL secondary and memory responses, as well as vaccination strategies to induce these potent killer cells that provide an extra layer of cell-mediated immune protection against heterosubtypic IAV infection.

Abstract 2507: Bivalent adenylate cyclase (CYA)-based therapeutic vaccines: eradication of tumor cells expressing different antigens over time

Abstract Introduction: ProCervix is a clinical stage HPV16 E7/HPV18 E7 bivalent adenylate cyclase (CyaA)-based therapeutic vaccine targeting HPV16/18 infection in women prior to cervical high grade lesion development. We report here on the ability of ProCervix to induce E7-specific CD8+ T lymphocytes which can be both functional effector and memory cytotoxic CD8+ T lymphocytes with (CTL) and we bring the proof of concept that co-delivery of HPV16 E7 protein with non-viral Ag including OVA or MAGE-A3 result in an effective therapeutic effect while providing broad prophylaxis. Methods: Three vaccines bearing oncogenes or model Ag were prepared, ProCervix (CyaA-HPV16 E7 and CyaA-HPV18 E7), CyaA-HPV16 E7/CyaA-MAGEA3 and CyaA-HPV16 E7/CyaA-OVA. ProCervix was adjuvanted with imiquimod 5% cream applied on the skin dermis at the injection site, or admixed with Poly-ICLC (Oncovir, USA). The two other vaccines were adjuvanted with Poly-ICLC. All vaccines were injected intradermally in C57BL/6 mice. Induction of E7-specific CD8+ T cells was evaluated by IFN-γ ELISpot and E7-specific memory CTL by in vivo killing. Therapeutic efficacy was performed in TC-1 tumor bearing mice (expressing HPV16 E7). The evaluation of memory to HPV18 was subsequently performed in TC-1 tumor-free mice rechallenged with a newly engineered LL2 cell line expressing HPV18 E7. Dual vaccination effects were further evaluated with 2 vaccine combinations in TC-1-tumor bearing mice, CyaA-HPV16 E7/CyaA-OVA (EG7 rechallenge) or CyaA-HPV16 E7/CyaA-MAGEA3 (B16-MAGEA3 rechallenge). Results: ProCervix adjuvanted by Imiquimod 5% cream or Poly-ICLC produced similar therapeutic efficacy. ProCervix-vaccinated mice developed a long-lasting HPV16 E7-specific CD8+ T cell response leading to HPV16 E7-expressing tumor eradication. Functional HPV18 E7-specific memory CTL were also identified in these mice leading to the Ag-specific protection against a challenge with HPV18 E7-expressing tumor cells. Similar results were observed with eradication of TC-1 tumors followed by Ag-specific tumor no takes of either EG7 or B16-MAGEA3, with CyaA-HPV16E7/CyaA-OVA and CyaA-HPV16 E7/CyaA-MAGEA3 vaccinations, respectively. Conclusion: CyaA technology is amenable to multivalent/multi-Ag therapeutic vaccination approaches as shown by the sequential successful T-cell based therapy, and the protective memory achieved in an Ag-specific fashion. We could anticipate that ProCervix-treated patients could clear their HPV16 infection while being protected against a later infection with HPV18. This dual mechanism opens the possibility to protect/treat patients with several different Ag for a given cancer with appropriately designed CyaA-based therapeutic vaccine. Citation Format: Michaël Esquerré, Marie Momot, Anne Goubier, Yolande Misseri, Marie-Christine Bissery. Bivalent adenylate cyclase (CYA)-based therapeutic vaccines: eradication of tumor cells expressing different antigens over time. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2507. doi:10.1158/1538-7445.AM2015-2507

The Role of Invariant Natural Killer T Cells in Dendritic Cell Licensing, Cross-Priming, and Memory CD8(+) T Cell Generation.

New vaccination strategies focus on achieving CD8+ T cell (CTL) immunity rather than on induction of protective antibody responses. While the requirement of CD4+ T (Th) cell help in dendritic cell (DC) activation and licensing, and in CTL memory induction has been described in several disease models, CTL responses may occur in a Th cell help-independent manner. Invariant natural killer T cells (iNKT cells) can substitute for Th cell help and license DC as well. iNKT cells produce a broad spectrum of Th1 and Th2 cytokines, thereby inducing a similar set of costimulatory molecules and cytokines in DC. This form of licensing differs from Th cell help by inducing other chemokines, while Th cell-licensed DCs produce CCR5 ligands, iNKT cell-licensed DCs produce CCL17, which attracts CCR4+ CD8+ T cells for subsequent activation. It has recently been shown that iNKT cells do not only enhance immune responses against bacterial pathogens or parasites but also play a role in viral infections. The inclusion of iNKT cell ligands in influenza virus vaccines enhanced memory CTL generation and protective immunity in a mouse model. This review will focus on the role of iNKT cells in the cross-talk with cross-priming DC and memory CD8+ T cell formation.

Lenalidomide Polarizes Th1-specific Anti-tumor Immune Response and Expands XBP1 Antigen-Specific Central Memory CD3+CD8+ T cells against Various Solid Tumors.

IntroductionEffective combination immunotherapeutic strategies may be required to enhance effector cells’ anti-tumor activities and improve clinical outcomes.MethodsXBP1 antigen-specific cytotoxic T lymphocytes (XBP1-CTL) generated using immunogenic heteroclitic XBP1 US184-192 (YISPWILAV) and XBP1 SP367-375 (YLFPQLISV) peptides or various solid tumor cells over-expressing XBP1 target antigen were evaluated, either alone or in combination with lenalidomide, for phenotype and immune functional activity.ResultsLenalidomide treatment of XBP1-CTL increased the proportion of CD45RO+ memory CD3+CD8+ T cells, but not the total CD3+CD8+ T cells. Lenalidomide upregulated critical T cell activation markers and costimulatory molecules (CD28, CD38, CD40L, CD69, ICOS), especially within the central memory CTL subset of XBP1-CTL, while decreasing TCRαβ and T cell checkpoint blockade (CTLA-4, PD-1). Lenalidomide increased the anti-tumor activities of XBP1-CTL memory subsets, which were associated with expression of Th1 transcriptional regulators (T-bet, Eomes) and Akt activation, thereby resulting in enhanced IFN-γ production, granzyme B upregulation and specific CD28/CD38-positive and CTLA-4/PD-1-negative cell proliferation.ConclusionsThese studies suggest the potential benefit of lenalidomide treatment to boost anti-tumor activities of XBP1-specific CTL against a variety of solid tumors and enhance response to an XBP1-directing cancer vaccine regime.

TLR9 and Dendritic Cells Are Required for CD8+ T Cell Responses to the AAV Capsid

CD8+ T cell responses to the adeno-associated virus (AAV) capsid have posed a significant barrier to transduction in clinical trials of AAV-mediated gene therapy for hemophilia B, as reactivation of a memory CTL response to the capsid is capable of eliminating transduced hepatocytes in the absence of immunosuppression. Recently, it has been suggested that innate immune responses induced by the toll-like receptor (TLR) pathway can influence the development of adaptive immune responses to AAV-mediated gene transfer. In particular, reports have implicated TLR2 (AAV capsid), TLR9 (AAV genome), and MyD88 (downstream signaling adaptor of both these TLRs). Herein, we have used a modified AAV2 with an insertion of the immunodominant MHC class I epitope of ovalbumin into the capsid (AAV2-SIINFEKL) to study the mechanism of CD8+ T cell responses to the AAV capsid. Using an H2-Kb-SIINFEKL tetramer reagent, we determined that anti-capsid CD8+ T cell responses depended on the TLR9-MyD88 pathway. While the frequency of circulating capsid-specific CD8+ T cells peaked around 7-10 days post-injection and subsided after about 21 days in wild type (WT) mice, tetramer-positive cells were not detected in TLR9-/- or MyD88-/- mice. The kinetics and magnitude of the response was unaltered in TLR2-/- mice. Mice deficient in STING, a downstream adaptor of multiple cytoplasmic DNA sensing pathways, also developed comparable capsid-specific CD8+ T cell frequencies to WT mice, suggesting that this is not a general effect of pattern recognition of DNA. Interestingly, the frequency of capsid-specific CD8+ T cells was not reduced in AP3-/- mice, which are deficient in type I IFN signaling downstream of TLR9. Adoptively transferred OVA-specific OT-1 T cells proliferated in WT but not TLR9-/- mice that received AAV2-SIINFEKL, confirming the importance of TLR9. The effect was antigen-specific, as OT-1 cells in WT mice that received AAV2 lacking SIINFEKL showed minimal proliferation comparable to TLR9-/- mice. In addition to pattern-recognition receptors, we also assessed the role of antigen-presenting cells in the CD8+ T cell response to capsid. The formation of capsid-specific CD8+ T cells was unaltered in mice that received gadolinium chloride to inactivate macrophages, or in B cell-deficient μMT mice. Depletion of B cells in WT mice prior to vector administration also failed to affect the anti-capsid CD8+ T cell response. However, transient depletion of dendritic cells (DCs) in CD11c-DTR mice resulted in a delayed development of capsid-specific CD8+ T cells. Seven days post-injection, DC-depleted mice had a significantly reduced frequency of tetramer-positive CD8+ T cells which recovered to normal by 10 days, likely due to the repopulation of DCs before the input capsid was completely cleared. Overall, our results show that TLR9 signaling, most likely in DCs, is required for the formation of de novo anti-capsid CD8+ T cell responses. DisclosuresHerzog:Genzyme: AAV-FIX technology Patents & Royalties.

Heteroclitic XBP1 peptides evoke tumor-specific memory cytotoxic T lymphocytes against breast cancer, colon cancer, and pancreatic cancer cells

XBP1 is a critical transcriptional activator of the unfolded protein response (UPR), which increases tumor cell survival under prolonged endoplasmic reticulum (ER) stress and hypoxic conditions.This study was designed to evaluate the immunogenicity of heteroclitic XBP1 unspliced (US)184–192 (YISPWILAV) and heteroclictic XBP1 spliced (SP)367–375 (YLFPQLISV) HLA-A2 peptides, and to characterize the specific activities of XBP1 peptides-specific cytotoxic T lymphocytes (XBP1-CTL) against breast cancer, colon cancer, and pancreatic cancer cells.The XBP1-CTL had upregulated expression of critical T cell markers and displayed HLA-A2-restricted and antigen-specific activities against breast cancer, colon cancer and pancreatic cancer cells. XBP1-CTL were enriched withCD45RO+ memory CTL, which showed high expression of critical T cell markers (CD28, ICOS, CD69, CD40L), cell proliferation and antitumor activities as compared to CD45RO− non-memory CTL. The effector memory (EM: CD45RO+CCR7−) subset had the highest level of cell proliferation while the central memory (CM: CD45RO+CCR7+) subset demonstrated enhanced functional activities (CD107a degranulation, IFNγ/IL-2 production) upon recognition of the respective tumor cells. Furthermore, both the EM and CM XBP1-CTL subsets expressed high levels of Th1 transcription regulators Tbet and Eomes. The highest frequencies of IFNγ or granzyme B producing cells were detected within CM XBP1-CTL subset that were either Tbet+ or Eomes+ in responding to the tumor cells.These results demonstrate the immunotherapeutic potential of a cocktail of immunogenic HLA-A2 specific heteroclitic XBP1 US184–192 and heteroclictic XBP1 SP367–375 peptides to induce CD3+CD8+ CTL enriched for CM and EM cells with specific antitumor activities against a variety of solid tumors.

Abstract 638: Lenalidomide treatment enhances the anti-tumor activities of XBP1 specific cytotoxic T lymphocytes by increasing the frequency and tumor-specific response of central memory CD3+CD8+ T cells

Abstract Introduction: Lenalidomide is an agent with immunomodulatory properties with demonstrated direct effects on the immune system and tumor microenvironment. The unfolded protein response (UPR) is a primary cellular adaptive mechanism that allows tumor cell survival under endoplasmic reticulum stress conditions by increasing its protein folding capacity. Thus, targeting the UPR is a rational approach for selective cancer cell killing. Here, we provide the basis for a novel immunotherapeutic approach by combining the antigen specific CTL against XBP1, an important transcriptional activator of the UPR, in combination with Lenalidomide, to boost the immune targeting against a variety of solid tumor cancer cells that over-express XBP1. Methods: XBP1 peptides-specific CTL (XBP1-CTL) were generated ex vivo from HLA-A2+ normal donors’ CD3+ T cells by repeated stimulation using a cocktail of heteroclitic XBP1 unspliced (US)184-192 (YISPWILAV) and XBP1 spliced (SP)367-375 (YLFPQLISV) peptides. XBP1-CTL were then incubated with or without Lenalidomide (5 µm), and evaluated for their immune responses and anti-tumor activities against breast, colon or pancreatic cancer cells. In addition, we characterized the XBP1-CTL, following lenalidamide treatment, for their enhanced expression of key T cell markers and transcriptional regulators, which are critical for immune function of effector and memory T cells. Results: An increased number of CD45RO+ memory CD3+CD8+ T cells, but not CD45RO- non-memory CD3+CD8+ T cells nor total CD3+CD8+ T cells, was observed within XBP1-CTL by Lenalidomide treatment. The combination of XBP1-CTL with Lenalidomide consistently enhanced the frequency and anti-tumor activity of the central memory CTL subset (CM: CD45RO+CCR7+/CD3+CD8+ T cells) as compared to the effector memory CTL subset (EM: CD45RO+CCR7-/CD3+CD8). In addition, Lenalidomide increased the expression of critical T cell surface markers including CD28, CD38, CD40L, CD69 and 41BB, but not ICOS nor TCRαβ. Lenalidomide treatment also enhanced anti-tumor activities of XBP1-CTL by increasing the frequencies of T-bet+/IFN-γ+, Eomes+/IFN-γ+, and Akt+ CD3+CD8+ T cells within both CM and EM subsets in response to HLA-A2+ breast (MB231), colon (LS180) or pancreatic (Panc1) cancer cell lines. Finally, the memory CD3+CD8+ T cells of XBP1-CTL displayed increased cytotoxicity (Granzyme B+) and IFN-γ production against the respective solid tumor cells when the XBP1-CTL were treated with Lenalidomide. Conclusions: This study provides the framework for using heteroclitic XBP1 US184-192 and XBP1 SP367-375 peptides as a vaccine to generate XBP1-CTL targeting a variety of solid tumors. Finally, it provides support for combination of the XBP1-based peptides vaccine with immunomodularoty drug Lenalidomide to improve patient outcome. Citation Format: Jooeun Bae, Rao Prabhala, Ruben Carrasco, Paul Richardson, Glen Dranoff, Kenneth C. Anderson, Nikhil C. Munshi. Lenalidomide treatment enhances the anti-tumor activities of XBP1 specific cytotoxic T lymphocytes by increasing the frequency and tumor-specific response of central memory CD3+CD8+ T cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 638. doi:10.1158/1538-7445.AM2014-638

Abstract 2894: Novel heteroclitic XBP1 peptides induce antigen-specific memory CD3+CD8+ T cells expressing critical T cell markers and transcription regulators

Abstract Background: XBP1 is a critical transcriptional activator of the unfolded protein response that enables tumors to survive prolonged endoplasmic reticulum stress due to nutrient deprivation, hypoxia or accumulation of unfolded/misfolded proteins. This provided us a rational to evaluate XBP1 as an antigen to develop a potential vaccine based immunotherapy against different types of solid tumors. Methods: XBP1 gene expression was investigated in a variety of primary tumor cells by searching the canEvlove and Oncomine databases and examined its differential expression level from normal cells. A cocktail of heteroclitic XBP1 unspliced (US)184-192 (YISPWILAV) and heteroclitic XBP1 spliced (SP)367-375 (YLFPQLISV) peptides were used to evoke XBP1 specific cytotoxic T Lymphocytes (XBP1-CTL) ex vivo to target breast, colon, and pancreatic cancer cells. XBP1-CTL were also evaluated for critical T cell markers, transcription regulators, and generation of specific memory CTL subsets. Results: Significantly higher XBP1 gene expression was detected in primary tumor cells from breast and colon cancer patients compared to the corresponding normal cells from healthy donors. Using a cocktail of immunogenic heteroclitic XBP1 US and SP peptides with improved HLA-A2 binding/stability as compared to their native counterparts, we induced XBP1-specific CTL from normal HLA-A2+ individuals' CD3+ T cells. The XBP1-CTL displayed an increased frequency of CD3+CD8+ T cells with enhanced expression (% positive cells, mean fluorescence intensity) of surface T cell markers including CD38, CD40L, CD69, 41BB (CD137), ICOS (CD278) and TCRαβ. In addition, the XBP1-CTL were enriched for central memory (CM: CD45RO+CCR7+) and effector memory (EM: CD45RO+CCR7-) CD3+CD8+ T cells as compared to baseline (unstimulated) CD3+CD8+ T cells. Functionally, the memory (CD45RO+) XBP1-CTL subsets showed increased levels of cellular proliferation, IFN-γ production, IL-2 production, and CD107a degranulation in response to HLA-A2+ breast (MB231), colon cancer (LS180) or pancreatic cancer (Panc1) cell lines. Finally, memory (CD45RO+) subset of XBP1-CTL expressed higher levels of Tbet and Eomes transcription regulators as compared to non-memory (CD45RO-) XBP1-CTL, with the highest frequencies of IFN-γ producing Tbet+, Eomes+ or Granzyme B+ cells in the CM subset. Conclusions: These results provide the rational for an immunotherapeutic vaccine comprised of a cocktail of heteroclitic XBP1 US184-192 and XBP1 SP367-375 HLA-A2 peptides to induce XBP1-CTL expressing critical T cell markers and transcription regulators as well as distinct memory CTL subsets with specific anti-tumor activities against breast, colon and pancreatic cancers. Citation Format: Jooeun Bae, Rao Prabhala, Ruben Carrasco, Ann-Hwee Lee, Alec Kimmelman, Kenneth C. Anderson, Nikhil Munshi. Novel heteroclitic XBP1 peptides induce antigen-specific memory CD3+CD8+ T cells expressing critical T cell markers and transcription regulators. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2894. doi:10.1158/1538-7445.AM2014-2894

Treatment of MS patients with BG-12 results in a decline in circulating memory T and B lymphocytes (HUM1P.317)

Abstract BG-12 (Tecfidera®) is a recently approved oral treatment for multiple sclerosis that has shown remarkable efficacy. The mechanisms by which BG-12 acts on the human immune system are poorly defined. In the current study, we analyzed the effects of BG-12 treatment on subsets of circulating human T and B lymphocytes using multicolor flow cytometry. Patients treated for four months with BG-12 had decreased total numbers of lymphocytes in their blood that did not reach significance, and no changes in their overall percentages of CD4+ T cells, CD8+ T cells, or CD19+ B cells compared to pretreatment levels. Significant differences were observed when subsets of T and B cells were evaluated. BG-12 treatment resulted in a decrease in percentages of circulating CD4+CD45RO+CD45RAneg memory TH cells and CD8+CD45RO+CD45RAneg memory CTL. Treatment with BG-12 also resulted in significant decreases in circulating CD27+CD19+ memory B cells, and a relative increase in the levels of CD24highCD38highCD19+ regulatory B cells. A CD27+CD24lowCD19+ B cell subset was present in the circulation of some MS patients prior to treatment, but returned to the very low levels found in healthy controls following four months of BG-12 treatment. The data suggest that BG-12 has in vivo down regulatory effects on some lymphocyte subsets, particularly memory T and B cells. Ongoing experiments are focused on measuring functional differences in T and B cell cytokine production in BG-12 treated patients.

Rescue of vaccine T cell memory following sublethal gamma irradiation (VAC3P.952)

Abstract Subsequent to sublethal γ radiation exposure, survivors reacquire immune competence, but lose vaccine immunity. Our goal is to rescue CD8+ T cell-mediated memory responses following γ radiation exposure. In a mouse model of Listeria monocytogenes (LM) infection, we found that rapid revaccination (rescue) of immunized mice following irradiation preserves CD8+ T cell mediated immunity. Rescued mice are able to survive a wild type LM challenge infection when the rescue vaccine strain, LM-deltaActA, is given 0-3 days following radiation exposure. A signature of rescue was revealed in gene profiling studies in which a group of cytokines and chemokines were uniquely up regulated in the spleens of LM-rescued animals as compared to mice irradiated without rescue. These included factors important in resistance to LM, such as proinflammatory factors CCL2, TNFα, and IL-6 and bacterocidal factors CXCL9, 10, and 11 as well as anti-apoptotic factors including IL-15 and IER3, which may have a role in rescue of memory CTL. RAW264.7, a macrophage cell line, produced a similar cytokine profile when irradiated and infected with LM, suggesting a probable myeloid source for the majority of these factors. Monocyte activation by the combination of irradiation and LM infection may thus create conditions of rescue by producing cytokines and chemokines that enhance clearance of LM and rescue CD8+ CTL mediating vaccine immunity.

Selective Induction of CTL Helper Rather Than Killer Activity by Natural Epitope Variants Promotes Dendritic Cell–Mediated HIV-1 Dissemination

The ability of HIV-1 to rapidly accumulate mutations provides the virus with an effective means of escaping CD8(+) CTL responses. In this study, we describe how subtle alterations in CTL epitopes expressed by naturally occurring HIV-1 variants can result in an incomplete escape from CTL recognition, providing the virus with a selective advantage. Rather than paralyzing the CTL response, these epitope modifications selectively induce the CTL to produce proinflammatory cytokines in the absence of target killing. Importantly, instead of dampening the immune response through CTL elimination of variant Ag-expressing immature dendritic cells (DC), a positive CTL-to-DC immune feedback loop dominates whereby the immature DC differentiate into mature proinflammatory DC. Moreover, these CTL-programmed DC exhibit a superior capacity to mediate HIV-1 trans-infection of T cells. This discordant induction of CTL helper activity in the absence of killing most likely contributes to the chronic immune activation associated with HIV-1 infection, and can be used by HIV-1 to promote viral dissemination and persistence. Our findings highlight the need to address the detrimental potential of eliciting dysfunctional cross-reactive memory CTL responses when designing and implementing anti-HIV-1 immunotherapies.

Wnt signaling inhibits CTL memory programming

Induction of functional CTLs is one of the major goals for vaccine development and cancer therapy. Inflammatory cytokines are critical for memory CTL generation. Wnt signaling is important for CTL priming and memory formation, but its role in cytokine-driven memory CTL programming is unclear. We found that wnt signaling inhibited IL-12-driven CTL activation and memory programming. This impaired memory CTL programming was attributed to up-regulation of eomes and down-regulation of T-bet. Wnt signaling suppressed the mTOR pathway during CTL activation, which was different to its effects on other cell types. Interestingly, the impaired memory CTL programming by wnt was partially rescued by mTOR inhibitor rapamycin. In conclusion, we found that crosstalk between wnt and the IL-12 signaling inhibits T-bet and mTOR pathways and impairs memory programming which can be recovered in part by rapamycin. In addition, direct inhibition of wnt signaling during CTL activation does not affect CTL memory programming. Therefore, wnt signaling may serve as a new tool for CTL manipulation in autoimmune diseases and immune therapy for certain cancers.

IL-12 is required for mTOR regulation of memory CTLs during virus infection (P6232)

Abstract Inflammatory cytokines are critical in the induction of memory CTLs, and inhibition of mTOR by rapamycin can enhance memory CTL generation. However, it is unknown if important inflammatory cytokines such as IL-12 are involved in the regulation of memory CTLs by mTOR during infection. We show here that inhibition of mTOR by rapamycin represses CTLs expansion and enhances memory CTL generation during vaccinia virus infection in mice. In addition, rapamycin promotes central memory phenotype in CTLs. However, the absence of IL-12 signal in CTLs diminishes rapamycin regulation on both CTL expansion and subsequent memory CTL formation, despite that rapamycin substantially increases memory CTLs when IL-12 signal is missing. Moreover, rapamycin-regulated memory CTLs in the absence of IL-12 are impaired in secondary expansion upon pathogen challenge, and subsequently form abolished secondary memory. In conclusion, IL-12 contributes to the strength of rapamycin regulation on primary memory CTLs, and lack of IL-12 signal leads to impaired secondary expansion of memory CTLs. Thus, the IL-12 signal is required for mTOR regulation on functional memory CTLs.

Histone deacetylase 11 is an epigenetic regulator of cytotoxic T-lymphocyte effector function and memory formation (P1404)

Abstract The roles of individual histone deacetylases in the regulation of T-cell development and function remain largely unknown. Here we provide evidence for HDAC11 as an epigenetic regulator of T-cell inflammatory response as well as CTL central memory formation. To investigate the role of HDAC11 in T-cells, an HDAC11 knockout (HDAC11KO) mouse model was utilized. HDAC11KO mice display no gross phenotypic abnormalities and no alterations in T-cell development or CD4+/CD8+ population distributions. However, HDAC11KO CTLs are hyper-proliferative and secrete significantly higher levels of IL-2, TNF, and IFN-γ upon activation, and HDAC11KO mice accumulate higher percentages of central memory CTLs. In an allogeneic bone marrow transfer model, HDAC11KO T-cells mediate more potent and robust graft vs host disease associated with increased inflammatory cytokines. Mechanistically, we provide evidence that in CTLs HDAC11 interacts with the Eomes gene promoter, a known regulator of IFN-γ production and memory formation. HDAC11KO CTLs, at both basal state and post stimulation, display higher levels of acetylation at the Eomes promoter, indicative of a permissive transcriptional state. Correspondingly, eomes mRNA levels in HDAC11KO cells are elevated. Finally, chromatin immunoprecipitation of HDAC11 reveals interaction between HDAC11 and the Eomes promoter in CTLs. These results support HDAC11 as an epigenetic regulator of CTL function and memory formation through epigenetic regulation of Eomes.

IL‐27 enhances the survival of tumor antigen‐specific CD8+ T cells and programs them into IL‐10‐producing, memory precursor‐like effector cells

IL-27 is a member of the IL-12 family of cytokines that is comprised of an IL-12 p40-related protein subunit, EBV-induced gene 3, and a p35-related subunit, p28. IL-27 functions through IL-27R and has been shown to have potent antitumor activity via activation of a variety of cellular components, including antitumor CD8(+) T-cell responses. However, the exact mechanisms of how IL-27 enhances antitumor CD8(+) T-cell responses remain unclear. Here we show that IL-27 significantly enhances the survival of activated tumor antigen-specific CD8(+) T cells in vitro and in vivo, and programs tumor antigen-specific CD8(+) T cells into memory precursor-like effector cells, characterized by upregulation of Bcl-6, SOCS3, Sca-1, and IL-10. While STAT3 activation and the CTL survival-enhancing effects can be independent of CTL IL-10 production, we show here that IL-27-induced CTL IL-10 production contributes to memory precursor cell phenotype induction, CTL memory, and tumor rejection. Thus, IL-27 enhances antitumor CTL responses via programming tumor antigen-specific CD8(+) T cells into a unique memory precursor type of effector cells characterized by a greater survival advantage. Our results have important implications for designing immunotherapy against human cancer.