Cytotoxic T Lymphocytes Attack Research Articles

An exploration of the natural and acquired immunological mechanisms to high-risk human papillomavirus infection and unmasking immune escape in cervical cancer: A concise synopsis.

The most common STD that triggers cervical cancer is the human papillomavirus. More than 20 types of human papillomavirus (HPV) can induce uterine cervical cancer. Almost all women acquire genital HPV infection soon after their first intercourse, with most of them clearing the virus within 3 years. An immune response is necessary to clear. The first responders to HPV infection are the innate immune system elements composed of macrophages, keratinocytes, natural killer cells, and natural killer T-lymphocytic (NKT) cells. Cytotoxic T lymphocytes (CTLs) comprise the second line of defense and kill HPV16-infected cells expressing various peptides derived from their transforming early viral oncoproteins, mainly E2•E6. Even though HPV can manage to trick away our immune systems, first of all, it is important to emphasize that HPV replication does not kill the host cells. It does not replicate viral antigens or cause inflammation. The HPV16 E6 and E7 genes suppress host cell type 1 interferons (IFNs), which are detectable after infection. The patient may have immunological tolerance; hence, there are no costimulatory signals from inflammatory cytokines like IFNs during antigen recognition. Evidence shows that HlA class I generations have been inhibited by HPV16 E5, which could protect this tumor cell from CTL attack. HPV16 E7 is responsible for initiating immunotolerance and increasing regulatory T cells (Treg) to repress immunological regression. Evasion from immune system protection plays a critical role in the outcome of persistent HPV infection and the development of cervical cancer. Vaccination against HPV16 and 18 during adolescence is the most effective method for preventing cervical cancer in women, considering the immunological processes involved.

Abstract 3964: A cancer survival program against activated T cell attack

Abstract The immune-cancer interactions downstream of immune checkpoint inhibition present the next targetable interface of cancer immunity. Despite evidence for cytotoxic T lymphocyte (CTL) infiltration and cytolytic activity within immunotherapy-responsive tumors, partial responses and acquired resistance are common, indicating additional barriers for complete tumor elimination. We report a survival program engaged within residual cancer cells exposed to weeks of activated CTLs. Contrary to immune-evasion mechanisms, we observe a residual antigenic cancer cell subpopulation which activates CTLs, survives within IFNγ-high environments, and withstands CTL-induced apoptosis. This cancer survival program, termed immune-induced persistence, is reversibly-driven (non-genetic) and prevents the complete CTL-mediated elimination of cancer cell populations by supporting the survival of quiescent cancer ‘persister’ cells against activated CTL attack. Persister cells display sublethal caspase activity and caspase-dependent DNA damage, consistent with CTL-induced genomic instability, and depend on the inhibitors of apoptosis proteins for survival. Notably, immune-induced persistence presents a markedly distinct survival program than drug-induced persistence, including distinctive apoptotic profiles, stress response pathways, and vulnerabilities. Thus, cancer cell populations can transiently adopt non-redundant survival programs in response to various cytotoxic environments. Features of immune-induced persistence are observed within CTL-treated human cancer cell lines, immune-treated human tumor slice cultures, and intratumor transcriptional heterogeneity programs. Under continued CTL exposure, persistent cancer populations give rise to genetically distinct cancer clones, including stably resistant immune escape variants, modeling acquired resistance to cancer immunotherapies. We propose sublethal interactions between effector immune cells and cancer persister cells as a critical and targetable variable at the interface between activated immunity and cancer. Citation Format: Michael X. Wang, Brandon E. Mauch, August F. Williams, Tania Barazande-Pour, Filipe Araujo Hoffmann, Sophie H. Harris, Cooper P. Lathrop, Claire E. Turkal, Michelle H. Paw, David A. Gervasio, Tiffany Tran, Theresa Guo, Gregory A. Daniels, Soo J. Park, Matthew J. Hangauer. A cancer survival program against activated T cell attack [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3964.

TSC2 regulates tumor susceptibility to TRAIL-mediated T-cell killing by orchestrating mTOR signaling.

Resistance to cancer immunotherapy continues to impair common clinical benefit. Here, we use whole-genome CRISPR-Cas9 knockout data to uncover an important role for Tuberous Sclerosis Complex 2 (TSC2) in determining tumor susceptibility to cytotoxic T lymphocyte (CTL) killing in human melanoma cells. TSC2-depleted tumor cells had disrupted mTOR regulation following CTL attack, which was associated with enhanced cell death. Wild-type tumor cells adapted to CTL attack by shifting their mTOR signaling balance toward increased mTORC2 activity, circumventing apoptosis, and necroptosis. TSC2 ablation strongly augmented tumor cell sensitivity to CTL attack invitro and invivo, suggesting one of its functions is to critically protect tumor cells. Mechanistically, TSC2 inactivation caused elevation of TRAIL receptor expression, cooperating with mTORC1-S6 signaling to induce tumor cell death. Clinically, we found a negative correlation between TSC2 expression and TRAIL signaling in TCGA patient cohorts. Moreover, a lower TSC2 immune response signature was observed in melanomas from patients responding to immune checkpoint blockade. Our study uncovers a pivotal role for TSC2 in the cancer immune response by governing crosstalk between TSC2-mTOR and TRAIL signaling, aiding future therapeutic exploration of this pathway in immuno-oncology.

Interdependence of sequential cytotoxic T lymphocyte and natural killer cell cytotoxicity against melanoma cells.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells recognize and eliminate cancer cells. However, immune evasion, downregulation of immune function by the tumour microenvironment and resistance of cancer cells are major problems. Although CTL and NK cells are both important to eliminate cancer, most studies address them individually. We quantified sequential primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, NK cells or melan-A (MART-1)-specific CTL eliminated all SK-Mel-5 cells within 24h, indicating that SK-Mel-5 cells are not resistant initially. However, at lower effector-to-target ratios, which resemble numbers of the immune contexture in human cancer, a substantial number of SK-Mel-5 cells survived. Pre-exposure to CTL induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, and pre-exposure to NK cells induced resistance in surviving SK-Mel-5 cells to NK cells. Higher human leucocyte antigen class I expression or interleukin-6 levels were correlated with resistance to NK cells, whereas reduction in MART-1 antigen expression was correlated with reduced CTL cytotoxicity. The CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. In contrast to the other three combinations, CTL cytotoxicity against SK-Mel-5 cells was enhanced following NK cell pre-exposure. Our assay allows quantification of sequential CTL and NK cell cytotoxicity and might guide strategies for efficient CTL-NK cell anti-melanoma therapies. KEY POINTS: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. Both CTL and NK cells attack the same targets, but most studies address them individually. In a sequential cytotoxicity model, the interdependence of antigen-specific CTL and NK cell cytotoxicity against melanoma is quantified. High numbers of antigen-specific CTL and NK cells eliminate all melanoma cells. However, lower numbers induce resistance if secondary CTL or NK cell exposure follows initial CTL exposure or if secondary NK cell exposure follows initial NK cell exposure. On the contrary, if secondary CTL exposure follows initial NK cell exposure, cytotoxicity is enhanced. Alterations in human leucocyte antigen class I expression and interleukin-6 levels are correlated with resistance to NK cells, whereas a reduction in antigen expression is correlated with reduced CTL cytotoxicity; CTL cytotoxicity is rescued beyond control levels by exogenous antigen. This assay and the results on interdependencies will help us to understand and optimize immune therapies against cancer.

Greek Fire, Poison Arrows, and Scorpion Bombs: How Tumor Cells Defend Against the Siege Weapons of Cytotoxic T Lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) are the main cellular effectors of the adaptive immune response against cancer cells, which in turn have evolved sophisticated cellular defense mechanisms to withstand CTL attack. Herein we provide a critical review of the pertinent literature on early and late attack/defense events taking place at the CTL/target cell lytic synapse. We examine the earliest steps of CTL-mediated cytotoxicity (“the poison arrows”) elicited within seconds of CTL/target cell encounter, which face commensurately rapid synaptic repair mechanisms on the tumor cell side, providing the first formidable barrier to CTL attack. We examine how breach of this first defensive barrier unleashes the inextinguishable “Greek fire” in the form of granzymes whose broad cytotoxic potential is linked to activation of cell death executioners, injury of vital organelles, and destruction of intracellular homeostasis. Herein tumor cells deploy slower but no less sophisticated defensive mechanisms in the form of enhanced autophagy, increased reparative capacity, and dysregulation of cell death pathways. We discuss how the newly discovered supra-molecular attack particles (SMAPs, the “scorpion bombs”), seek to overcome the robust defensive mechanisms that confer tumor cell resistance. Finally, we discuss the implications of the aforementioned attack/defense mechanisms on the induction of regulated cell death (RCD), and how different contemporary RCD modalities (including apoptosis, pyroptosis, and ferroptosis) may have profound implications for immunotherapy. Thus, we propose that understanding and targeting multiple steps of the attack/defense process will be instrumental to enhance the efficacy of CTL anti-tumor activity and meet the outstanding challenges in clinical immunotherapy.

Ultrarapid lytic granule release from CTLs activates Ca2+-dependent synaptic resistance pathways in melanoma cells.

Human cytotoxic T lymphocytes (CTLs) exhibit ultrarapid lytic granule secretion, but whether melanoma cells mobilize defense mechanisms with commensurate rapidity remains unknown. We used single-cell time-lapse microscopy to offer high spatiotemporal resolution analyses of subcellular events in melanoma cells upon CTL attack. Target cell perforation initiated an intracellular Ca2+ wave that propagated outward from the synapse within milliseconds and triggered lysosomal mobilization to the synapse, facilitating membrane repair and conferring resistance to CTL induced cytotoxicity. Inhibition of Ca2+ flux and silencing of synaptotagmin VII limited synaptic lysosomal exposure and enhanced cytotoxicity. Multiplexed immunohistochemistry of patient melanoma nodules combined with automated image analysis showed that melanoma cells facing CD8+ CTLs in the tumor periphery or peritumoral area exhibited significant lysosomal enrichment. Our results identified synaptic Ca2+ entry as the definitive trigger for lysosomal deployment to the synapse upon CTL attack and highlighted an unpredicted defensive topology of lysosome distribution in melanoma nodules.

Distinct Escape Mechanisms of HLA Class I Allele-Lacking Hematopoietic Stem Progenitor Cells (HSPCs) from GPI-Deficient HSPCs in Acquired Aplastic Anemia

[Background] In cases of immune-mediated bone marrow (BM) failure, such as acquired aplastic anemia (AA) and AA/PNH, aberrant hematopoietic stem progenitor cells (HSPCs) that acquire resistance to immune attack are thought to survive and support hematopoiesis in convalescent patients. Two representative progenies of such "escape" HSPC clones are HLA class I allele-lacking (HLA[-]) leukocytes and glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells. The mechanism underlying the immune selection of HLA(-) HSPCs is the failure of cytotoxic T lymphocyte (CTL) to recognize target antigens that are presented by particular HLA class I alleles of HSPCs. However, the mechanisms underlying the immune selection of GPI(-) HSPCs remain unclear. In addition, whether or not immune pressure that persists after immunosuppressive therapy (IST) contributes to the development and maintenance of clonal hematopoiesis by HLA(-) or GPI(-) HSPCs that are often seen in patients in long-term remission is also unknown. Phenotypical analyses of HSPCs that can be obtained from peripheral blood (PB) of AA patients who possess HLA(-) or GPI(-) leukocytes may provide a hint to elucidate these unsolved issues. [Objectives/Methods] We analyzed PB lineage-CD45dimCD34+CD38+ HSPCs of 15 AA patients who had 1%-99% HLA-A2(-) or HLA-A24(-) granulocytes (Gs) using flow cytometry (FCM). PB samples from 1 patient with severe AA were obtained before IST while the other 14 patients were in remission at the time of sampling; 10 were on cyclosporin (CsA) and eltrombopag (EPAG) (n=1), CsA and anabolic steroids (n=3), CyA (n=4), anabolic steroids (n=1) and EPAG alone (n=1); and 4 were free of therapy. We also determined the percentages of HLA(-) cells in different CD34+ subsets of BM, including HSCs (CD38-CD90+CD45RA-), MPPs, CMPs, GMPs, MEPs and CLPs for patients whose BM cells were available. Six AA/PNH patients whose GPI(-) Gs were 4-99% of the total Gs were subjected to the same PB HSPC analysis. For a separate group of seven AA patients who responded to CsA and had both HLA(-) and GPI(-) G populations, the percentages of each population were serially determined over one to 12 years. [Results] FCM identified 0.01%-0.4% (median 0.01%) CD45dimCD34+CD38+ HSPCs in the mononuclear cell population of the 15 AA patients, values that were significantly lower than those of seven healthy volunteers (0.19-0.78%, median 0.58%, P<0.05). In 4 of the 9 patients on treatment and in 4 patients who had been free of therapy, the percentages of HLA(-) cells in HSPCs were similar to those of Gs (1%/1%, 1%/1%, 14%/15% and 100%/98% in the 4 treated patients; 16%/11% in the untreated patient; 13%/13%, 95%/95%, 99%/99% and 99%/99% in the 4 patients in remission after therapy). BM subsets including HSCs of 2 patients whose Gs and PB HSPCs lacked HLA-A2 almost completely, were 99% negative for HLA-A2. In contrast, in the remaining 6 patients, the percentages of HLA(-) cells in HSPCs were markedly lower than those in Gs (0%/25%, 2%/7%, 4%/65%, 6%/77% [Case 1], 9%/75% and 10%/39% [Case 2]), suggesting the persistence of CTL attack against HPCs or more differentiated myeloid cells (Figure 1). In contrast to HLA(-) PB HSPCs, there was no discordance in the percentage of GPI(-) cells between PB Gs and HSPCs (4%/4%, 49%/50%, 94%/100%, 96%/96%, 96%/99% and 99%/92%) in all 6 AA/PNH patients including 3 responding to CsA. Lastly, serial changes in the percentage of PB HLA(-) and GPI(-) Gs during CsA therapy were compared in seven patients who possessed both aberrant cells. In 4 patients who were responding to CsA, the HLA(-) G percentage gradually decreased in 3 and remained stable in the remaining one, while the GPI(-) G percentage reciprocally increased in 3 and remained stable in one. In contrast, in 3 patients who obtained sustained remission after CsA therapy, the HLA(-) G percentage increased after cessation of CsA. The GPI(-) G percentages decreased in 1 and remained unchanged in 2 (Figure 2). [Conclusions] Immune selection that favors the survival of HLA(-) HSPCs or HPCs takes place even in AA patients who have been in remission for many years after successful IST and may contribute to clonal hematopoiesis by HLA(-) HSPCs. Given no signs of selection at the HSPC stage and the reciprocal increases in the percentage of GPI(-) Gs in AA/PNH patients responding to CsA therapy, the proliferation of GPI(-) HSPCs may not be affected by immune pressure in convalescent patients. Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Ono Pharmaceutical: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Novartis Pharma K.K: Honoraria; SynBio Pharmaceuticals: Consultancy; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Bristol-Myers Squibb: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Alaxion Pharmaceuticals: Honoraria; Kyowa Kirin: Honoraria.

Bystander Proliferation of Piga-Mutated Hematopoietic Progenitor Cells in Acquired Aplastic Anemia Patients Possessing HLA Class I Allele-Lacking Leukocytes

[Background] Hematopoietic stem progenitor cells (HSPCs) with PIGA mutations are thought to acquire a survival advantage over normal HSPCs under immune attack against HSPCs and produce glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells in patients with acquired aplastic anemia (AA). Various underlying mechanisms of the survival advantage of PIGA-mutated HSPCs have been proposed; however, it remains still unclear how PIGA-mutated HSPCs are immunologically selected in AA. Approximately 15% of AA patients with increased GPI(-) cells possess another aberrant leukocyte subset that lacks the expression of the HLA-class I allele due to a copy number-neutral loss of heterozygosity of the HLA haplotype, which occurs in the short arm of chromosome 6 (6pLOH) as a result of uniparental disomy, or HLA allelic mutations. The presence of HLA-class I allele-lacking leukocytes (HLA-LLs) is considered to be the most compelling evidence to support the involvement of cytotoxic T lymphocytes (CTLs) in the development of bone marrow failure. Charactering GPI(-) leukocytes and platelets in AA patients with HLA-LLs may provide an insight into the mechanism underlying the immune selection of PIGA-mutated HSPCs.[Patients and Methods] We investigated the presence of GPI(-) leukocytes, erythrocytes, and platelets in 63 patients with AA using high-sensitivity flow cytometry (FCM). For the platelet analysis, platelet rich plasma (PRP) was obtained by centrifuging anticoagulated blood at 1000 rpm for 7 minutes with the brake turned off. Thirty microliters of PRP was incubated with monoclonal antibodies specific to CD55-PE, CD59-PE, CD41a-APC and HLA-A2 or A24-FITC for 20 minutes at room temperature in the dark. To prevent doublets, samples were diluted 1 to 100 in PBS and filtered with mesh immediately before the FCM analysis. Thirty of the 63 patients were heterozygous for the HLA-A allele with A24 and A2, and thus the presence of both HLA-LLs and HLA-A allele-lacking platelets could be evaluated by FCM. The lack of the HLA-A allele due to 6pLOH or allelic mutations in all HLA-LL(+) patients was confirmed by a droplet digital PCR or deep sequencing.[Results] Increased GPI(-) granulocytes, which accounted for 0.01-99.8% of the total granulocytes, were detected in 37 (58.7%) patients while HLA-A24 or A2-lacking granulocytes accounted for 0.39-98.3% of the total granulocytes in 20 (66.7%) of the 30 patients. Eight patients possessed both GPI(-) cells and HLA-LLs. In all 8 of these patients, the two aberrant cell populations were mutually exclusive. The analyses of different cell lineages revealed HLA-A allele-lacking cells in all lineages of cells, including granulocytes (Gs), monocytes (Ms), T cells (Ts), B cells (Bs), NK cells (NKs), and platelets (Ps) in 7 of the 8 patients; the remaining one patient had the GMTP pattern. In contrast, the lineage diversity of GPI(-) in the 8 patients was more restricted; GMTBNKP was only detected in 2 patients; the combinations in the other 6 patients were GT (n=1), GMBNKP (n=2), GMTNKP (n= 1) and GMTBP (n= 2). In Case 1, GPI(-) cells were not detected in T cells while HLA-A24(-) cells were detected in all lineages of cells including T cells (Figure 1). The limited lineage diversity of GPI(-) cells was also evident in 6 patients who did not possess HLA-LLs (GMP, GMBP, GMBNKP, GMTNKP, GMTBP) with GPI(-) granulocytes>10% while the GMTBNKP pattern was common in 10 HLA-LL(+) patients who did not possess GPI(-) cells, regardless of their percentage of HLA-A allele-lacking granulocytes. Longitudinal follow-up of 5 patients over a period of 8-27 years showed a decline in the percentage of GPI(-) granulocytes (39.2 to 0.00%, 11.4 to 0.04%, 3.50 to 0.30%, 1.77 to 0.00% and 0.79 to 0.11%) and a reciprocal increase in the percentage of HLA-A allele-lacking granulocytes (80.0 to 95.2%, 92.0 to 99.1%, 24.0 to 24.4%) in 3 patients who had been placed under observation; in two patients (Cases 2 and 3) whose GPI(-) granulocyte percentages had been >10%, the PNH clones were completely replaced by HLA-LL clones during 6 and 8 years, respectively (Figure 2).[Conclusions] The limited diversity of the blood cell lineage and spontaneous decline of GPI(-) cells that coexisted with HLA-LLs suggest that GPI(-) cells are derived from the PIGA-mutated hematopoietic progenitor cells that were allowed to proliferate as a bystander in the environment where the CTL attack against HSPCs is taking place. [Display omitted] DisclosuresNakao:Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria.

Abstract 5668: The rational design and evaluation of a peptide inhibitor of the PD-1/PD-L1 interaction

Abstract Tumor cells exploit immune checkpoints by expressing checkpoint ligands such as PD-L1, effectively masking them from immune destruction. Programmed cell death- 1 (PD-1), plays a major role in tumor immune escape. The interaction of PD-1/PD-L1 inhibits cytotoxic T lymphocyte (CTL) proliferation, induces apoptosis of tumor-specific T cells, and promotes the resistance of tumor cells to CTL attack. Monoclonal antibodies to these proteins have been developed and approved for use in the clinic. Theoretically, peptide mimics, which have the benefit of lower half-life and significantly smaller size, are able to penetrate into solid tumors and tissues better than antibodies, and can function as inhibitors for the PD1/PD-L1 interaction. Here we report for the first time the rational design and validation of a peptide inhibitor to the PD-1/PD-L1 immune checkpoint as a viable alternative to current inhibitory antibodies. We demonstrated, by biolayer interferometry and in silico docking simulations, that a PD-L1 peptide mimetic (PL120131) can interfere with the PD-1/PD-L1 interaction by binding to PD-1. We established the binding constant for the interaction and PL120131 reduces association of PD-L1 in a dose dependent manner. We show that PL120131 is capable of inhibiting PD-1 mediated apoptotic signaling pathway and rescuing Jurkat cells and primary lymphocytes from apoptosis. Additionally, we show that PL120131 treatment allows for CTL anti-tumor activity. Furthermore, PL120131 can maintain co-culture survivability and activity of T Cells in a 3D co-culture model. Together, the characterization of this PD-1/PD-L1 inhibiting peptide provides insight regarding the ability to inhibit PD-L1 binding while maintaining CTL viability and activity. Understanding the mechanism(s) of action of this peptide has yielded information on the pathways that are drivers of immune evasion in a tumor microenvironment, which may improve and synergize with other forms of immunotherapy, thus increasing efficacy of cancer treatment. Outcomes from this study have provided a path forward to further development of small molecule inhibitors to the PD-1/PD-L1 interaction, and outlines a strategy for the design and development of small molecules to additional cell-surface checkpoint proteins. Citation Format: Rebecca J. Boohaker, Vijaya Sambandam, Mark Suto, Bo Xu. The rational design and evaluation of a peptide inhibitor of the PD-1/PD-L1 interaction [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 5668.

Sustained clonal hematopoiesis by HLA-lacking hematopoietic stem cells without driver mutations in aplastic anemia

AbstractClonal hematopoiesis by hematopoietic stem progenitor cells (HSPCs) that lack an HLA class I allele (HLA− HSPCs) is common in patients with acquired aplastic anemia (AA); however, it remains unknown whether the cytotoxic T lymphocyte (CTL) attack that allows for survival of HLA− HSPCs is directed at nonmutated HSPCs or HSPCs with somatic mutations or how escaped HLA− HSPC clones support sustained hematopoiesis. We investigated the presence of somatic mutations in HLA− granulocytes obtained from 15 AA patients in long-term remission (median, 13 years; range, 2-30 years). Targeted sequencing of HLA− granulocytes revealed somatic mutations (DNMT3A, n = 2; TET2, ZRSR2, and CBL, n = 1) in 3 elderly patients between 79 and 92 years of age, but not in 12 other patients aged 27 to 74 years (median, 51.5 years). The chronological and clonogenic analyses of the 3 cases revealed that ZRSR2 mutation in 1 case, which occurred in an HLA− HSPC with a DNMT3A mutation, was the only mutation associated with expansion of the HSPC clone. Whole-exome sequencing of the sorted HLA− granulocytes confirmed the absence of any driver mutations in 5 patients who had a particularly large loss of heterozygosity in chromosome 6p (6pLOH) clone size. Flow–fluorescence in situ hybridization analyses of sorted HLA+ and HLA− granulocytes showed no telomere attrition in HLA− granulocytes. The findings suggest that HLA− HSPC clones that escape CTL attack are essentially free from somatic mutations related to myeloid malignancies and are able to support long-term clonal hematopoiesis without developing driver mutations in AA patients unless HLA loss occurs in HSPCs with somatic mutations.

An Approach for a Synthetic CTL Vaccine Design against Zika Flavivirus Using Class I and Class II Epitopes Identified by Computer Modeling.

The threat posed by severe congenital abnormalities related to Zika virus (ZKV) infection during pregnancy has turned development of a ZKV vaccine into an emergency. Recent work suggests that the cytotoxic T lymphocyte (CTL) response to infection is an important defense mechanism in response to ZKV. Here, we develop the rationale and strategy for a new approach to developing cytotoxic T lymphocyte (CTL) vaccines for ZKV flavivirus infection. The proposed approach is based on recent studies using a protein structure computer model for HIV epitope selection designed to select epitopes for CTL attack optimized for viruses that exhibit antigenic drift. Because naturally processed and presented human ZKV T cell epitopes have not yet been described, we identified predicted class I peptide sequences on ZKV matching previously identified DNV (Dengue) class I epitopes and by using a Major Histocompatibility Complex (MHC) binding prediction tool. A subset of those met the criteria for optimal CD8+ attack based on physical chemistry parameters determined by analysis of the ZKV protein structure encoded in open source Protein Data File (PDB) format files. We also identified candidate ZKV epitopes predicted to bind promiscuously to multiple HLA class II molecules that could provide help to the CTL responses. This work suggests that a CTL vaccine for ZKV may be possible even if ZKV exhibits significant antigenic drift. We have previously described a microsphere-based CTL vaccine platform capable of eliciting an immune response for class I epitopes in mice and are currently working toward in vivo testing of class I and class II epitope delivery directed against ZKV epitopes using the same microsphere-based vaccine.

Clinical significance and origin of leukocytes that lack HLA-A allele expression in patients with acquired aplastic anemia.

To gain insight into the origin and clinical significance of leukocytes that lack human leukocyte antigen A (HLA-A) allele expression caused by a copy-number-neutral loss of heterozygosity in the short arm of chromosome 6 in patients with acquired aplastic anemia (AA),weused a high-sensitivity flow cytometry assay to investigate the presence of HLA-A allele-lacking leukocytes(HLA-LLs) in 144 AA patients. HLA-LLs, accounting for0.2-99.8% of each leukocyte population, were detected in 18 of 71 (25.4%) newly diagnosed patientsand in 25 of 73 (34.2%) previously treated patients. The lineage combination patterns of the HLA-LLs in the 43 HLA-LL(+)patients were granulocytes (Gs), monocytes (Ms), Bcells (Bs), and Tcells (Ts; GMBT) in 13 cases, GMB in 16 cases, GM in 11 cases, and B alone in three cases. The response rate to antithymocyte globulin plus cyclosporine therapy (100%) and the 2-year, failure-free survivalrate (100%) in 8 newly diagnosed HLA-LL(+) patients were significantly higher than in 23 HLA-LL(-) patients (52.2% for both). These data suggest that HLA-LLs are a useful marker of the presence of immune pathophysiology in AA and that T-cell attacks against hematopoietic progenitor cells, rather than against hematopoietic stem cells, can trigger bone marrow failure in AA patients.

Melanoma cell lysosome secretory burst neutralizes the CTL-mediated cytotoxicity at the lytic synapse.

Human melanoma cells express various tumour antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo. However, natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that, on conjugation with CTL, human melanoma cells undergo an active late endosome/lysosome trafficking, which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking, pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance, we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients.

Evidence That T Cells Specific for Non-Hematopoietic Cells Trigger the Development of Acquired Aplastic Anemia

A T-cell attack against hematopoietic stem cells (HSCs) is believed to be the principal mechanism underlying the development of acquired aplastic anemia (AA). The presence of leukocytes that lack HLA class I alleles as a result of the copy number-neutral loss of heterozygosity of the HLA haplotype due to uniparental disomy in the short arm of chromosome 6 (6pLOH) is compelling evidence for the involvement of cytotoxic T lymphocytes (CTLs) in the HSC destruction; this is based on the high response rate to immunosuppressive therapy (IST) in 6pLOH(+) patients (Katagiri, et al. Blood, 2011). However, target cells of the putative CTLs have not yet been characterized because of the small number of patients analyzed via flow cytometry (FCM) in our previous study. FCM can be substituted for SNP arrays by detecting HLA-A allele-lacking leukocytes (HLA-LLs) caused by 6pLOH. To gain insight into the CTL target responsible for the development of AA, we examined a total of 223 patients (213 idiopathic and 10 hepatitis-associated AA; 93 severe and 130 non-severe AA) for the presence of HLA-LLs and determined the lineage combinations of the aberrant leukocyte population. Of the 223 patients, 145 (65.0%) were heterozygous for the HLA-A allele and could be assessed for the presence of HLA-LLs by FCM. Eighteen (25.4%; 10 with severe AA and 8 with non-severe AA) of the 71 pre-treatment patients, and 26 (35.1%; 13 with severe AA and 13 with non-severe AA) of the 74 post-treatment patients were found to be positive for HLA-LLs. The lineage combinations of HLA-LLs in the 44 HLA-LL(+) patients were granulocytes (Gs), monocytes (Ms), B cells (Bs), and T cells (Ts, GMBT) in 13, GMB in 16 and GM in 11 patients. Surprisingly, HLA-LLs were found in Bs alone in three patients, and in one patient, the lineage combination pattern was TB (Figure). The presence of 6pLOH was confirmed via deep sequencing of isolated Bs from one of the three Bs alone patients. These lineage combination patterns were not observed to change for 1-40 months in 22 of 23 patients whose blood samples were available for follow-up analyses. In one patient with the GMB pattern, HLA-LLs decreased from 11.7% before treatment to 0% after 12 months of ATG therapy. The response rate to IST in GMBT patients (3/4, 75%) and in patients with GMB or GM (9/10, 90%) were similarly higher than in patients without HLA-LLs (22/39, 56%) or in patients who were homozygous for the HLA-A allele (23/36, 64%). Two of the three Bs alone patients showed complete responses at the time of sampling three years after ATG therapy and 20 years after CsA therapy, and another patient had a secondary myelodysplastic syndrome two years after response to ATG. The TB alone patient developed AA 20 years earlier, but had not been treated until recently because there was no need for blood transfusions, and is now improving in response to eltrombapag. This study revealed that the targets of putative CTLs in more than half of AA patients are hematopoietic progenitor cells with limited differentiation but long-lasting capacity, and in some patients, they are lymphoid progenitor cells that do not contribute to hematopoiesis. This suggests that CTL attack against non-HSCs including lymphoid precursors could trigger BM failure. Consistent with our previous report, the bystander effects caused by the immune response to non-HSCs such as myelosuppressive cytokines may play a major role in the development of AA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

IMPS-13IMMUNOLOGIC PROFILING OF PEDIATRIC CNS MALIGNANCIES FOR THE RATIONAL SELECTION OF IMUNOTHERAPEUTIC TRIALS

BACKGROUND: Although clinical trials have resulted in significant improvements in childhood brain cancer treatments, further progress using strategically targeted, potentially less toxic modalities are needed. Immunotherapy is one such emerging treatment approach. The degree of tumor-infiltrating cytotoxic T-lymphocytes (CTLs) directly correlates with prognosis for several cancer types. MHC Class I is down-regulated in 40-90% of adult cancers, rendering them resistant to CTL attack, correlating with poorer prognosis. MHC I expression in medulloblastomas is associated with poor prognostic factors, a pattern opposite of most other cancer-MHC I associations described. Natural Killer (NK)-cell based therapies, dependent upon low MHC I expression, are therefore proposed as an immunotherapeutic approach to medulloblastomas. Very little literature exists classifying MHC I expression in other pediatric brain cancers, despite several ongoing trials using MHC I-dependent strategies. This lack of data raises uncertainty of which immunotherapies are optimal for these diseases. Toward this end, we classified key immunologic characteristics of pediatric brain tumors. METHODS: We interrogated pre-clinical and clinical mRNA microarray databases to identify tumor types which exhibit MHC I and the associated b-2-microglobulin molecule expression. We up-regulated MHC I expression in vitro in pediatric CNS tumor cell lines and identified intra-tumoral innate and adaptive immunologic cellular infiltrates within pediatric CNS tumor samples through immunohistochemistry. RESULTS: We found that atypical teratoid/rhabdoid tumors (AT/RT) and ependymomas exhibit decreased, whereas medulloblastomas and glioblastomas express higher MHC I mRNA levels than most other pediatric cancer types. We discovered MHC I can be up-regulated in vitro in medulloblastomas and AT/RT's, indicating a reversible epigenetic mechanism for down-regulation. There were decreased NK and regulatory T-cell, while increased CTL infiltrates in recurred versus diagnostic pediatric glioblastoma tumor samples. CONCLUSIONS: The immunologic profiling of pediatric brain cancers will guide rational selection of immune-based therapies, serving as an important biomarker for patient inclusion in immunotherapeutic trials.

A biased competition theory of cytotoxic T lymphocyte interaction with tumor nodules.

The dynamics of the interaction between Cytotoxic T Lymphocytes (CTL) and tumor cells has been addressed in depth, in particular using numerical simulations. However, stochastic mathematical models that take into account the competitive interaction between CTL and tumors undergoing immunoediting, a process of tumor cell escape from immunesurveillance, are presently missing. Here, we introduce a stochastic dynamical particle interaction model based on experimentally measured parameters that allows to describe CTL function during immunoediting. The model describes the competitive interaction between CTL and melanoma cell nodules and allows temporal and two-dimensional spatial progression. The model is designed to provide probabilistic estimates of tumor eradication through numerical simulations in which tunable parameters influencing CTL efficacy against a tumor nodule undergoing immunoediting are tested. Our model shows that the rate of CTL/tumor nodule productive collisions during the initial time of interaction determines the success of CTL in tumor eradication. It allows efficient cytotoxic function before the tumor cells acquire a substantial resistance to CTL attack, due to mutations stochastically occurring during cell division. Interestingly, a bias in CTL motility inducing a progressive attraction towards a few scout CTL, which have detected the nodule enhances early productive collisions and tumor eradication. Taken together, our results are compatible with a biased competition theory of CTL function in which CTL efficacy against a tumor nodule undergoing immunoediting is strongly dependent on guidance of CTL trajectories by scout siblings. They highlight unprecedented aspects of immune cell behavior that might inspire new CTL-based therapeutic strategies against tumors.

Oligodendrocytes Enforce Immune Tolerance of the Uninfected Brain by Purging the Peripheral Repertoire of Autoreactive CD8+ T Cells

Myelin-specific CD8(+) Tcells are thought to contribute to the pathogenesis of multiple sclerosis. Here we modeled this contribution in mice with CD8(+) Tcells recognizing ovalbumin (OVA) expressed in oligodendrocytes (ODCs). Surprisingly, even very high numbers of activated OVA-reactive CD8(+) Tcells failed to induce disease and were cleared from the immune system after antigen encounter inthe centralnervous system (CNS). Peripheral infection with OVA-expressing Listeria (Lm-OVA) enabled CD8(+) Tcells to enter the CNS, leading to the deletion of OVA-specific clones after OVA recognition on ODCs. In contrast, intracerebral infection allowed OVA-reactive CD8(+) Tcells to cause demyelinating disease. Thus, in response to infection, CD8(+) Tcells also patrol the CNS. If the CNS itself is infected, they destroy ODCs upon cognate antigen recognition in pursuit of pathogen eradication. In the sterile brain, however, antigen recognition on ODCs results in their deletion, thereby maintaining tolerance.

The Coalescence of Intrahost HIV Lineages Under Symmetric CTL Attack

Cytotoxic T lymphocytes (CTLs) are immune system cells that are thought to play an important role in controlling HIV infection. We develop a stochastic ODE model of HIV-CTL interaction that extends current deterministic ODE models. Based on this stochastic model, we consider the effect of CTL attack on intrahost HIV lineages assuming that CTLs attack several epitopes with equal strength. In this setting, we introduce a limiting version of our stochastic ODE under which we show that the coalescence of HIV lineages can be described through Poisson-Dirichlet distributions. Through numerical experiments, we show that our results under the limiting stochastic ODE accurately reflect HIV lineages under CTL attack when the HIV population size is on the low end of its hypothesized range. Current techniques of HIV lineage construction depend on the Kingman coalescent. Our results give an explicit connection between CTL attack and HIV lineages.

Modulation of Human Mesenchymal Stem Cell Immunogenicity through Forced Expression of Human Cytomegalovirus US Proteins

BackgroundMesenchymal stem cells (MSC) are promising candidates for cell therapy, as they migrate to areas of injury, differentiate into a broad range of specialized cells, and have immunomodulatory properties. However, MSC are not invisible to the recipient's immune system, and upon in vivo administration, allogeneic MSC are able to trigger immune responses, resulting in rejection of the transplanted cells, precluding their full therapeutic potential. Human cytomegalovirus (HCMV) has developed several strategies to evade cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell recognition. Our goal is to exploit HCMV immunological evasion strategies to reduce MSC immunogenicity.Methodology/Principal FindingsWe genetically engineered human MSC to express HCMV proteins known to downregulate HLA-I expression, and investigated whether modified MSC were protected from CTL and NK attack. Flow cytometric analysis showed that amongst the US proteins tested, US6 and US11 efficiently reduced MSC HLA-I expression, and mixed lymphocyte reaction demonstrated a corresponding decrease in human and sheep mononuclear cell proliferation. NK killing assays showed that the decrease in HLA-I expression did not result in increased NK cytotoxicity, and that at certain NK∶MSC ratios, US11 conferred protection from NK cytotoxic effects. Transplantation of MSC-US6 or MSC-US11 into pre-immune fetal sheep resulted in increased liver engraftment when compared to control MSC, as demonstrated by qPCR and immunofluorescence analyses.Conclusions and SignificanceThese data demonstrate that engineering MSC to express US6 and US11 can be used as a means of decreasing recognition of MSC by the immune system, allowing higher levels of engraftment in an allogeneic transplantation setting. Since one of the major factors responsible for the failure of allogeneic-donor MSC to engraft is the mismatch of HLA-I molecules between the donor and the recipient, MSC-US6 and MSC-US11 could constitute an off-the-shelf product to overcome donor-recipient HLA-I mismatch.

Immune responses against human papillomavirus (HPV) infection and evasion of host defense in cervical cancer

Human papillomavirus (HPV) is the most important etiological factor for cervical cancer. A recent study demonstrated that more than 20 HPV types were thought to be oncogenic for uterine cervical cancer. Notably, more than one-half of women show cervical HPV infections soon after their sexual debut, and about 90% of such infections are cleared within 3years. Immunity against HPV might be important for elimination of the virus. The innate immune responses involving macrophages, natural killer cells, and natural killer T cells may play a role in the first line of defense against HPV infection. In the second line of defense, adaptive immunity via cytotoxic T lymphocytes (CTLs) targeting HPV16 E2 and E6 proteins appears to eliminate cells infected with HPV16. However, HPV can evade host immune responses. First, HPV does not kill host cells during viral replication and therefore neither presents viral antigen nor induces inflammation. HPV16 E6 and E7 proteins downregulate the expression of type-1 interferons (IFNs) in host cells. The lack of co-stimulatory signals by inflammatory cytokines including IFNs during antigen recognition may induce immune tolerance rather than the appropriate responses. Moreover, HPV16 E5 protein downregulates the expression of HLA-class 1, and it facilitates evasion of CTL attack. These mechanisms of immune evasion may eventually support the establishment of persistent HPV infection, leading to the induction of cervical cancer. Considering such immunological events, prophylactic HPV16 and 18 vaccine appears to be the best way to prevent cervical cancer in women who are immunized in adolescence.