Cytolytic T-cell Response Research Articles

Biomarkers of tumor microenvironment of malignant neoplasms of kidneys, urinary bladder, and prostate gland (literature review)

The development of malignant tissue transformation is accompanied by the accumulation of immune system cells or tumor microenvironment cells (MCO) in it. Three variants of immune cell accumulation were identified: the ‘immune desert’ phenotype, ‘hot’ tumors, with a cytolytic T-cell response. The review presents immunotherapeutic strategies of exposure in order to enhance the ability of McO to initiate immune mechanisms capable of blocking the development of tumor tissue. The analysis of the presented data on the importance of immuno-oncological biomarkers as laboratory indicators of the therapeutic effectiveness of drug therapy aimed at restoring key immune defense pathways in oncourological diseases was carried out. The results of the study of the effectiveness of immuno-oncological biomarkers for assessing the state of antitumor immunity in malignant neoplasms of the bladder, kidneys, prostate gland are summarized.

Abstract 268: DNA-launched HPV E7 nanoparticle vaccine induces potent anti-tumor cytolytic T-cell responses

Abstract Nanotechnology-based approaches have provided promising tools for development of novel cancer immunotherapeutic immunogens through the decoration of multiple antigens on nanoparticle scaffolds. However, challenges with inducing robust cytolytic T cell responses critical to anti-tumor immunity, as well as purification and scalability, have hampered their clinical translation. To address these challenges, here we present the design and characterization of a DNA-launched nanoparticle vaccine displaying the HPV E7 epitope for inducing anti-tumor immune responses. We designed a nanovaccine construct by scaffolding the HPV E7 epitope with bacterial enzyme lumazine synthase. We demonstrate that through electroporation-facilitated DNA vaccination encoding the antigen-scaffold construct, this nanovaccine self-assembles in vivo into nanoparticles displaying 60 copies of the antigen on its surface. Compared to vaccination with CpG-adjuvanted HPV E7 peptide, the DNA-launched nanovaccine induced significantly greater antigen-specific CD8+ T cell responses upon stimulation with the HPV E7 peptide as well as with the epitope natively expressed on the target tumor cells. In a flank model of TC-1 lung cancer, the DNA-launched nanovaccine achieved complete tumor protection in 70% of mice when administered prophylactically. With therapeutic treatment, the nanovaccine achieved complete tumor regression in 60% of mice. In addition, anti-tumor immunity was retained upon rechallenge over 100 days after initial tumor challenge, supporting the induction of immune memory. These promising results, along with its facile characterization and scalability, support that DNA-launched nanovaccines that self-assemble in vivo can induce significant anti-tumor cellular immunity and are ideally positioned for study in clinical translation. Citation Format: Kevin Liaw, Ziyang Xu, Nicholas Tursi, Neethu Chokkalingam, Daniel Kulp, David Weiner. DNA-launched HPV E7 nanoparticle vaccine induces potent anti-tumor cytolytic T-cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 268.

Serial Stimulation of Invariant Natural Killer T Cells with Covalently Stabilized Bispecific T-cell Engagers Generates Antitumor Immunity While Avoiding Anergy.

CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong antitumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T-cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogues of the glycolipid α-galactosylceramide. Here we characterize the in vivo activities of iNKT cell-specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8+ cytolytic T-cell responses. The antitumor effects of iNKT-cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell-specific BiTEs activated iNKT cells without causing iNKT cell anergy or exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens. SIGNIFICANCE: Covalently stabilized conjugates that engage the antigen receptors of iNKT cells and target a tumor antigen activate potent antitumor immunity without induction of anergy or depletion of the responding iNKT cells.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses.

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 "immunodominance" in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

The Mevalonate Pathway Is a Druggable Target for Vaccine Adjuvant Discovery

Motivated by the clinical observation that interruption of the mevalonate pathway stimulates immune responses, we hypothesized that this pathway may function as a druggable target for vaccine adjuvant discovery. We found that lipophilic statin drugs and rationally designed bisphosphonates that target three distinct enzymes in the mevalonate pathway have potent adjuvant activities in mice and cynomolgus monkeys. These inhibitors function independently of conventional "danger sensing." Instead, they inhibit the geranylgeranylation of small GTPases, including Rab5 in antigen-presenting cells, resulting in arrested endosomal maturation, prolonged antigen retention, enhanced antigen presentation, and Tcell activation. Additionally, inhibiting the mevalonate pathway enhances antigen-specific anti-tumor immunity, inducing both Th1 and cytolytic Tcell responses. As demonstrated in multiple mouse cancer models, the mevalonate pathway inhibitors are robust for cancer vaccinations and synergize with anti-PD-1 antibodies. Our research thus defines the mevalonate pathway as a druggable target for vaccine adjuvants and cancer immunotherapies.

Sipuleucel-T (sip-T) to induce cytolytic T lymphocyte (CTL) activity against target antigens in men with hormone-sensitive and castration-resistant prostate cancer (CRPC).

5046 Background: Sip-T is an FDA-approved autologous cellular immunotherapy for patients with asymptomatic or minimally symptomatic metastatic CRPC (mCRPC), manufactured from peripheral blood mononuclear cells (PBMCs) cultured with the immunogen PA2024 (prostatic acid phosphatase [PAP] conjugated to granulocyte macrophage colony-stimulating factor). Treatment with sip-T induces cellular and humoral immune responses to both PA2024 and PAP (Antonarakis, ASCO 2015; Petrylak, ECC 2015; Small Clin Cancer Res 2015). To further elucidate the mechanism of sip-T–induced immune responses, we evaluated proliferative and cytolytic characteristics of PA2024- and PAP-specific T cells. Methods: Antigen-specific proliferation of CD4 (T helper [TH]) and CD8 (CTL) T cells was quantified using flow cytometry. To assess CTL lytic activity, cell-surface CD107a expression, indicating lysosomal fusion with the cell membrane and expulsion of lytic enzymes, was measured after stimulation with PAP or PA2024. CTL lytic activity was assessed in 13 patients (from 3 sip-T trials [NCT01431391, NCT01981122, and NCT01487863] in hormone-sensitive PC and mCRPC patients) and 1 healthy donor (control). Results: In sip-T–treated patients, a significant increase in proliferation of both CTLs and TH cells was detected in response to PA2024 (p < 0.05) and PAP (p < 0.10) at week 6 compared with baseline. Increased PA2024-specific proliferation was maintained in TH cells through week 26 (p < 0.05). CTL CD107a expression increased in sip-T patient samples stimulated with PAP and PA2024 but not in the healthy donor control. Preliminary analyses suggest that patients with longer overall survival (OS) may have greater CTL CD107a activity. Conclusions: Sip-T treatment was associated with well-maintained antigen-specific cellular responses, including robust CTL responses, demonstrating sip-T induces antigen-specific tumor lysis and providing a biologic basis for OS benefit in mCRPC patients. Ongoing work will examine whether patients with longer OS display greater CTL activity. Clinical trial information: NCT01431391; NCT01981122; NCT01487863.

1541P - Presence of tumor-specific cytolytic T cells in human primary breast carcinoma: consequences for immunotherapy

Immunotherapy through stimulatory antibodies targeting the CTLA-4 or PD-1 pathways has a demonstrated clinical efficacy in a fraction of patients with various cancers. It is likely that the main immune effectors of these therapies are CD8+ cytolytic T lymphocytes (CTL) recognizing tumor-specific antigens. Highly antigenic tumors such as metastatic melanomas are often immunogenic, i.e. they stimulate spontaneous anti-tumor CTL responses. This immunogenicity, of which the presence of tumor-infiltrating T cells (TILs) is probably a surrogate marker, might be a predictive marker for clinical benefit to immunostimulatory antibodies. The immunogenicity of primary breast carcinomas for CD8+ T cells has not been studied, but the amounts of TILs have been positively correlated with patients' survival. Here we wished to obtain evidence for the presence of tumor-specific CD8+ T cells in TILs from primary breast carcinomas. From 5 tumors (2 ER+/HER2-, 2 ER+/HER2+, 1 ER-/HER2-) we isolated TILs and derived a random set of ±100 CD8+ T cell clones that we screened for recognition of candidate tumor-specific antigenic peptides selected through tumor exome sequencing and gene expression profiling. These peptides were encoded either by mutated genes or by cancer-germline genes. For 4 tumors, we screened 61-142 T cell clonotypes for recognition of 23-61 candidate mutated peptides, without any positive result. For the last tumor (ER+/HER2-), 6 out of 57 T cell clonotypes recognized 4 out of 109 candidate mutated peptides but not the corresponding wild-type peptides. This tumor contained more mutations than the other four, and displayed microsatellite instability. Some human primary breast carcinomas are immunogenic, as one tumor contained at least 10% of tumor-specific cells among the CD8+ TILs. Our observation corroborates the association between high mutation burden and CTL response to mutated tumor antigens. The presence of tumor-specific CD8+ suggests that the corresponding patient could benefit from the currently used immunostimulatory antibodies.

Suppression of inducible CD4 regulatory cells by MHC class I-restricted human tumor epitope specific TCR engineered multifunctional CD4 T cells

Regulatory T cells (Treg) can interfere with the generation and function of anti-tumor immune effectors. Accordingly, ways that could block Treg function would be useful in cancer immunotherapy. We have previously shown that incorporation of CD4+CD25-ve T cells in an in vitro cytolytic T lymphocyte (CTL) generation assay leads to generation of induced regulatory T cells (iTregs), and that these iTreg block the generation of productive CTL response (Chattopadhyay et al., 2006). We here show that human CD4 T cells engineered to express MHC class I-restricted human melanoma associated epitope, MART-127–35, specific T cell receptor (TCR), that can simultaneously exhibit helper as well as cytolytic effector functions (Chhabra et al., 2008, Ray et al., 2010), can interfere with the generation of inducible Treg, block iTreg-mediated suppression, and allow the activation and expansion of MART-127–35 specific CTL responses, in vitro. We also show that mitigation of Treg generation by TCR engineered CD4 T cells is not mediated by a soluble factor and may involve “licensing/conditioning” of the dendritic cells (DC). Our data offer novel insights on the biology of MHC class I restricted TCReng CD4 T cells and have translational implications.

Tumor-Infiltrating Plasma Cells Are Associated with Tertiary Lymphoid Structures, Cytolytic T-Cell Responses, and Superior Prognosis in Ovarian Cancer.

CD8(+) tumor-infiltrating lymphocytes (TIL) are key mediators of antitumor immunity and are strongly associated with survival in virtually all solid tumors. However, the prognostic effect of CD8(+) TIL is markedly higher in the presence of CD20(+) B cells, suggesting that cooperative interactions between these lymphocyte subsets lead to more potent antitumor immunity. We assessed the colocalization patterns, phenotypes, and gene expression profiles of tumor-associated T- and B-lineage cells in high-grade serous ovarian cancer (HGSC) by multicolor IHC, flow cytometry, and bioinformatic analysis of gene expression data from The Cancer Genome Atlas. T cells and B cells colocalized in four types of lymphoid aggregate, ranging from small, diffuse clusters to large, well-organized tertiary lymphoid structures (TLS) resembling activated lymph nodes. TLS were frequently surrounded by dense infiltrates of plasma cells (PC), which comprised up to 90% of tumor stroma. PCs expressed mature, oligoclonal IgG transcripts, indicative of antigen-specific responses. PCs were associated with the highest levels of CD8(+), CD4(+), and CD20(+) TIL, as well as numerous cytotoxicity-related gene products. CD8(+) TIL carried prognostic benefit only in the presence of PCs and these other TIL subsets. PCs were independent of mutation load, BRCA1/2 status, and differentiation antigens but positively associated with cancer-testis antigens. PCs are associated with the most robust, prognostically favorable CD8(+) TIL responses in HGSC. We propose that TLS facilitate coordinated antitumor responses involving the combined actions of cytolytic T cells and antibody-producing PCs. Clin Cancer Res; 22(12); 3005-15. ©2016 AACR.

Mammary-tumor-educated B cells acquire LAP/TGF-β and PD-L1 expression and suppress anti-tumor immune responses.

B lymphocytes play a role in inhibiting the immune response against certain tumors, but the underlying mechanisms are poorly understood. EMT-6 mammary tumors grow well in wild-type (WT) mice but show reduced growth in B-cell-deficient μ(-/-) BALB/c mice (BCDM). WT mice demonstrate extensive B-cell infiltration into the tumor bed, reduced CD8(+) T cell and CD49(+) NK cell infiltration, and markedly reduced cytolytic T-cell response relative to BCDM. Expression of LAP/TGF-β1, CD80, CD86 and PD-L1 is significantly increased in tumor-infiltrating B cells (TIL-B) relative to splenic B cells. LAP/TGF-β1 expression on TIL-B progressively increased from 5.4±1.7% on day 8 to 43.1±6.1% by day 21 post tumor implantation. Co-culture of EMT-6 tumor cells with Naive-B cells ex vivo generated B cells (EMT6-B) with a similar immunophenotype to TIL-B. Purified TIL-B, or in-vitro-generated EMT6-B suppressed CD4(+), CD8(+) and CD4(+)CD25(-) T-cell proliferation, and Th1 cytokine secretion, and also suppressed purified NK-cell proliferation in response to IL-15, compared to naive splenic B cells. Acquired B regulatory function required direct tumor cell: B-cell contact, and was partially reversed by antibody to TGF-β or PD-L1, leading to tumor rejection in vivo B-cell acquisition of a suppressive phenotype following tumor infiltration may result in profound inhibition of T-cell anti-tumor responses.

Abstract P4-04-08: Presence of tumor-specific cytolytic T cells in human primary breast carcinoma: Consequences for immunotherapy

Abstract Background: Immunotherapy through stimulatory antibodies targeting the CTLA-4 or PD-1 pathways has a clear clinical efficacy in a fraction of patients with various cancers. It is likely that the main immune effectors of these therapies are CD8+ cytolytic T lymphocytes (CTL) recognizing tumor-specific antigens. The antigenicity of human tumors has been demonstrated with studies conducted mostly on melanomas. However the genetic mechanisms leading to antigenicity, notably point mutations in the tumor cells, apply to all cancer types. Thus primary breast carcinoma cells do certainly bear tumor-specific antigens, even though the extent of this antigenicity is unknown. Most melanomas, which are highly antigenic tumors, are also immunogenic, i.e. they stimulate spontaneous anti-tumor CTL responses. This immunogenicity, of which the presence of tumor-infiltrating T cells (TILs) is probably a surrogate marker, might be a predictive marker for clinical benefit to immunostimulatory antibodies. Whether primary breast carcinomas are immunogenic is not known, mainly due the absence of autologous tumor cell lines to analyze patients' T cells. However even in the absence of T-cell aimed immunotherapy the amounts of TILs have been positively correlated with patients' survival. Here we wished to obtain evidence for the presence of tumor-specific CD8+ T cells in TILs from primary breast carcinomas. Methods: From each tumor we isolated TILs and derived a random set of ±100 CD8+ clones maintained in culture by stimulation with anti-CD3 antibodies, thus irrespective of their antigenic specificity. We screened these clones for recognition of tumor-specific antigens present on the autologous tumor. In the absence of autologous tumor lines we restricted our analysis to mutated antigens selected on the basis of tumor exome sequencing and gene expression profiling. Indels and non-synonymous base substitutions were selected to synthesize candidate mutated peptides. Results: Thus far we have analyzed two hormone receptor-positive HER2-negative primary carcinomas. For one patient we screened 144 T cell clones for recognition of 40 candidate mutated peptides, without any positive result. For the other patient, 6 out of 98 T cell clones recognized 4 out of 119 candidate mutated peptides. Two peptides were recognized by two different T cell clones, i.e. with different T cell receptor sequences. These 4 'antigenic' mutations appear to be passenger, i.e. the four genes have a low published mutation frequency. Conclusions: We conclude that some human primary breast carcinomas are immunogenic, as one tumor contained at least 6% of tumor-specific T cells among the CD8+ TILs. It suggests that the corresponding patient could benefit from the currently used immunostimulatory antibodies. More work is required to understand the reasons for the negative results in the first patient. We are pursuing the work on 2 HER2-positive and 2 triple-negative tumors, in which TILs are better correlated with prognosis. Our results warrant more investigations on the activation or inhibition of tumor-specific T cells at early stages of human breast cancer development. Citation Format: Schröder DJ, Bricard O, Hames G, Remy N, Carrasco J, Canon J-L, Berlière M, Galant C, Coulie PG. Presence of tumor-specific cytolytic T cells in human primary breast carcinoma: Consequences for immunotherapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-08.

Ubiquitin-like Molecule ISG15 Acts as an Immune Adjuvant to Enhance Antigen-specific CD8 T-cell Tumor Immunity.

ISG15 is an ubiquitin-like protein induced by type I interferon associated with antiviral activity. ISG15 is also secreted and known to function as an immunomodulatory molecule. However, ISG15's role in influencing the adaptive CD8 T-cell responses has not been studied. Here, we demonstrate the efficacy of ISG15 as a vaccine adjuvant, inducing human papilloma virus (HPV) E7-specific IFNγ responses as well as the percentage of polyfunctional, cytolytic, and effector CD8 T-cell responses. Vaccination with ISG15 conferred remarkable control and/or regression of established HPV-associated tumor-bearing mice. T-cell depletion coupled with adoptive transfer experiments revealed that ISG15 protective efficacy was CD8 T-cell mediated. Importantly, we demonstrate that ISG15 vaccine-induced responses could be generated independent of ISGylation, suggesting that responses were mostly influenced by free ISG15. Our results provide more insight into the immunomodulatory properties of ISG15 and its potential to serve as an effective immune adjuvant in a therapeutic tumor or infectious disease setting.

A Two-Component DNA-Prime/Protein-Boost Vaccination Strategy for Eliciting Long-Term, Protective T Cell Immunity against Trypanosoma cruzi.

In this study, we evaluated the long-term efficacy of a two-component subunit vaccine against Trypanosoma cruzi infection. C57BL/6 mice were immunized with TcG2/TcG4 vaccine delivered by a DNA-prime/Protein-boost (D/P) approach and challenged with T. cruzi at 120 or 180 days post-vaccination (dpv). We examined whether vaccine-primed T cell immunity was capable of rapid expansion and intercepting the infecting T. cruzi. Our data showed that D/P vaccine elicited CD4+ (30-38%) and CD8+ (22-42%) T cells maintained an effector phenotype up to 180 dpv, and were capable of responding to antigenic stimulus or challenge infection by a rapid expansion (CD8>CD4) with type 1 cytokine (IFNγ+ and TFNα+) production and cytolytic T lymphocyte (CTL) activity. Subsequently, challenge infection at 120 or 180 dpv, resulted in 2-3-fold lower parasite burden in vaccinated mice than was noted in unvaccinated/infected mice. Co-delivery of IL-12- and GMCSF-encoding expression plasmids provided no significant benefits in enhancing the anti-parasite efficacy of the vaccine-induced T cell immunity. Booster immunization (bi) with recombinant TcG2/TcG4 proteins 3-months after primary vaccine enhanced the protective efficacy, evidenced by an enhanced expansion (1.2-2.8-fold increase) of parasite-specific, type 1 CD4+ and CD8+ T cells and a potent CTL response capable of providing significantly improved (3-4.5-fold) control of infecting T. cruzi. Further, CD8+T cells in vaccinated/bi mice were predominantly of central memory phenotype, and capable of responding to challenge infection 4-6-months post bi by a rapid expansion to a poly-functional effector phenotype, and providing a 1.5-2.3-fold reduction in tissue parasite replication. We conclude that the TcG2/TcG4 D/P vaccine provided long-term anti-T. cruzi T cell immunity, and bi would be an effective strategy to maintain or enhance the vaccine-induced protective immunity against T. cruzi infection and Chagas disease.

A lower viral set point but little immunological impact after early treatment during primary HIV infection.

The Primo-SHM trial, a multicenter randomized trial comparing no treatment with 24 or 60 weeks of combination antiretroviral therapy (cART) during primary human immunodeficiency virus (HIV) infection (PHI), recently demonstrated that temporary early cART lowered the viral set point and deferred the need for re-initiation of cART during chronic HIV infection. This study examined whether the beneficial effect of early treatment was caused by preservation of immunological responses. Twenty-seven treated and 20 untreated PHI individuals participating in the Primo-SHM trial were compared at viral set point, that is, 36 weeks after baseline or after treatment interruption, respectively, for a diverse set of immunological parameters. The results show no differences between treated and untreated individuals at the level of effector T-cell formation or replication capacity of the T-cells; regulation of various T, B, natural killer, or dendritic cells; polyfunctionality of the CD8 T-cells; preservation of CD4 T-cells in the gut associated lymphoid tissue; or immune activation. There were subtle differences in the quality of the cytolytic CD4 T-cell response: 11% (median) of CD4 T-cells of the early treated individuals produced the cytolytic molecule perforin compared to 5% in untreated individuals (p=0.046), and treatment caused a modest increase in CD4 T-cells expressing both perforin and granzyme B (median 9% vs. 4% of CD4 T-cells; p=0.045). Early treatment had a modest positive effect on the quality of the CD4 T-cell response. It remains unclear, however, whether these subtle immunological differences were the cause or a result of the lower viral set point in patients who received early treatment.

Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations.

Despite antiretroviral therapy (ART), HIV-1 persists in a stable latent reservoir1, 2, primarily in resting memory CD4+ T cells3, 4. This reservoir presents a major barrier to the cure of HIV-1 infection. To purge the reservoir, pharmacological reactivation of latent HIV-1 has been proposed5 and tested both in vitro and in vivo6–8. A key remaining question is whether virus-specific immune mechanisms including cytolytic T lymphocytes (CTL) can clear infected cells in ART-treated patients after latency is reversed. Here we show that there is a striking all or none pattern for CTL escape mutations in HIV-1 Gag epitopes. Unless ART is started early, the vast majority (>98%) of latent viruses carry CTL escape mutations that render infected cells insensitive to CTLs directed at common epitopes. To solve this problem, we identified CTLs that could recognize epitopes from latent HIV-1 that were unmutated in every chronically infected patient tested. Upon stimulation, these CTLs eliminated target cells infected with autologous virus derived from the latent reservoir, both in vitro and in patient-derived humanized mice. The predominance of CTL-resistant viruses in the latent reservoir poses a major challenge to viral eradication. Our results demonstrate that chronically infected patients retain a broad spectrum viral-specific CTL response and that appropriate boosting of this response may be required for the elimination of the latent reservoir.

ERAP1 functions override the intrinsic selection of specific antigens as immunodominant peptides, thereby altering the potency of antigen-specific cytolytic and effector memory T-cell responses.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a critical component of the adaptive immune system that has been shown to increase or decrease the presentation of specific peptides on MHC class I molecules. Here, we have demonstrated that ERAP1 functions are not only important during the presentation of antigen-derived peptides, but these functions can also completely change which antigen-derived peptides ultimately become selected as immunodominant T-cell epitopes. Our results suggest that ERAP1 may do this by destroying epitopes that would otherwise become immunodominant in the absence of adequate ERAP1 functionality. We further establish that ERAP1-mediated influences on T-cell functions are both qualitative and quantitative, by demonstrating that loss of ERAP1 function redirects CTL killing toward a different set of antigen-derived epitopes and increases the percent of antigen-specific memory T cells elicited by antigen exposure. As a result, our studies suggest that normal ERAP1 activity can act to suppress the numbers of T effector memory cells that respond to a given antigen. This unique finding may shed light on why certain ERAP1 single nucleotide polymorphisms are associated with several autoimmune diseases, for example, by significantly altering the robustness and quality of CD8+ T-cell memory responses to antigen-derived peptides.

A novel multi-epitope peptide vaccine against cancer: An in silico approach

Cancer immunotherapy has an outstanding position in cancer prevention and treatment. In this kind of therapy, the immune system is activated to eliminate cancerous cells. Multi-epitope peptide cancer vaccines are manifesting as the next generation of cancer immunotherapy. In the present study, we have implemented various strategies to design an efficient multi-epitope vaccine. CD8+ cytolytic T lymphocytes (CTLs) epitopes, which have a pivotal role in cellular immune responses, helper epitopes and adjuvant, are three crucial components of peptide vaccine. CTL epitopes were determined from two high immunogenic protein Wilms tumor-1 (WT1) and human papillomavirus (HPV) E7 by various servers, which apply different algorithms. CTL epitopes were linked together by AAY and HEYGAEALERAG motifs to enhance epitope presentation. Pan HLA DR-binding epitope (PADRE) peptide sequence and helper epitopes, which have defined from Tetanus toxin fragment C (TTFrC) by various servers, were used to induce CD4+ helper T lymphocytes (HTLs) responses. Additionally, helper epitopes were conjugated together via GPGPG motifs that stimulate HTL immunity. Heparin-Binding Hemagglutinin (HBHA), a novel TLR4 agonist was employed as an adjuvant to polarize CD4+ T cells toward T-helper 1 to induce strong CTL responses. Moreover, the EAAAK linker was introduced to N and C terminals of HBHA for efficient separation. 3D model of protein was generated and predicted B cell epitopes were determined from the surface of built structure. Our protein contains several linear and conformational B cell epitopes, which suggests the antibody triggering property of this novel vaccine. Hence, our final protein can be used for prophylactic or therapeutic usages, because it can potentially stimulate both cellular and humoral immune responses.

Autophagy enhances antitumor immune responses induced by irradiated hepatocellular carcinoma cells engineered to express hepatitis B virus X protein

HepatitisB virusX protein (HBx) plays a critical role in malignancy transformation of hepatitisB virus (HBV)-related hepatocellular carcinoma (HCC). HBx sequence has been mapped with multi-epitopes which can elicit robust specific cytolytic T lymphocyte (CTL) responses. In our previous study, we developed an adenoviral vaccine against HBx oncoproteins to prevent growth of HBV-associated HCC. However, due to the weak immunogenicity of tumor antigen and pre-existing virus-neutralizing antibodies to the vaccine carrier preventing the vector from transducing target cells, the development of novel methods to enhance antigen presentation is urgently required. In the present study, we developed an adenoviral‑mediated genetic engineering of hepatoma cell vaccine to express HBx and to evaluate if the novel vaccine could elicit specific immune responses. Our data showed that the irradiated tumor cells engineered to express HBx could significantly induce antitumor immune responses invivo. The novel vaccine could induce a specific CTL response to recognize and lyse HBx-positive hepatoma cells invitro. Both CD8+ T and CD4+ Tlymphocytes are involved in the antitumor immune response induced by the novel vaccine. Furthermore, numerous autophagosomes and autolysosomes were found in the irradiated tumor cells engineered to express HBx. The results demonstrated that the irradiated HBx-modified tumor cell vaccine was a potent and promising therapeutic agent against HBx-positive HCC via induction of autophagy-enhanced CD8+ T and CD4+ Tlymphocyte-mediated antitumor immune responses. The present findings have implications for the development of clinical immunotherapy against HBV-associated HCC.

HIV-specific cytolytic CD4 T-cell responses effectively control HIV infection in macrophages

Methods Using a novel, fluorescence-based single-round viral suppression assay, we assessed the ability of CD4 T-cells from HIV infected subjects to lyse infected macrophages. Elimination of infected macrophages and CD4 cytolytic phenotype were determined by flow cytometry. In addition, HIV-specific CD4 T-cell clones were generated and their cytolytic ability examined by Cr51-release and viral inhibition assays.

A CD74-dependent MHC class I endolysosomal cross-presentation pathway

Immune responses are initiated and primed by dendritic cells (DCs) that cross-present exogenous antigen. The CD74 (invariant chain) chaperone protein is thought to exclusively promote DC priming in the context of MHC class II. However, we demonstrate herein a CD74-dependent MHC class I cross-presentation pathway in DCs that plays a major role in the generation of MHC class I restricted, cytolytic T lymphocyte (CTL) responses against viral protein- and cell-associated antigens. CD74 associates with MHC class I molecules in the endoplasmic reticulum of DCs and mediates trafficking of MHC class I to endolysosomal compartments for loading with exogenous peptides. We conclude that CD74 plays a hitherto, undiscovered physiological function in endolysosomal DC cross-presentation for priming MHC class I-mediated CTL responses.