Cytotoxic T Lymphocytes Populations Research Articles

Mutational Features and Tumor Microenvironment Alterations in High-Grade Appendiceal Cancers Treated With Iterative Hyperthermic Intraperitoneal Chemotherapy.

High-grade appendiceal adenocarcinomas (HGAA) with peritoneal metastases (PMs) are associated with poor survival. Hyperthermic intraperitoneal chemotherapy (HIPEC) is a novel treatment approach for unresectable HGAA-PM. However, its influence on immunogenomic profiles has not yet been fully explored. We obtained 79 samples of metastatic peritoneal tumor deposits from patients diagnosed with HGAA and performed whole-exome sequencing, RNA sequencing, and immunoprofiling before and after HIPEC. Tumor biopsies were subjected to immunogenomic profiling to detect mutational signatures and immune populations associated with oncologic outcomes. Fifteen patients with HGAA-PMs were included in the study. The median progression-free survival (PFS) was 6.7 months (2.7-25.3) and the median overall survival was 11.4 months (4.7-42). Mucin-associated genes (MUC16, MUC3A, and MUC5AC) and titin (TTN) had the highest mutation frequencies. Mutational signatures such as single-base substitution 29 and doublet-base substitution 11 were present in >50% of single-base and double-base mutations. Higher PD-L1 coexpression on CD8+ T cells demonstrated a higher PFS both intratumorally (P = .019) and at the margin (P = .025). HIPEC-associated mutational signatures were identified in HGAA-PMs. Elevated PD-L1+ cytotoxic T-cell populations after HIPEC had better PFS, offering valuable insights for prognostication in the context of HIPEC treatment.

Single-cell RNA sequencing defines disease-specific differences between chronic nodular prurigo and atopic dermatitis

Chronic nodular prurigo (CNPG) is an inflammatory skin disease that is maintained by a chronic itch-scratch cycle likely rooted in neuroimmunological dysregulation. This condition may be associated with atopy in some patients, and there are now promising therapeutic results from blocking type 2 cytokines such as IL-4, IL-13, and IL-31. This study aimed to improve the understanding of pathomechanisms underlying CNPG as well as molecular relationships between CNPG and atopic dermatitis (AD). We profiled skin lesions from patients with CNPG in comparison with AD and healthy control individuals using single-cell RNA sequencing combined with T-cell receptor sequencing. We found type 2 immune skewing in both CNPG and AD, as evidenced by CD4+ helper T cells expressing IL13. However, only AD harbored an additional, oligoclonally expanded CD8A+IL9R+IL13+ cytotoxic T-cell population, and immune activation pathways were highly upregulated in AD, but less so in CNPG. Conversely, CNPG showed signatures of extracellular matrix organization, collagen synthesis, and fibrosis, including a unique population of CXCL14-IL24+ secretory papillary fibroblasts. Besides known itch mediators such as IL31 and oncostatin M, we also detected increased levels of neuromedin Bin fibroblasts of CNPG lesions compared with AD and HC, with neuromedin B receptors detectable on some nerve endings. These data show that CNPG does not harbor the strong disease-specific immune activation pathways that are typically found in AD but is rather characterized by upregulated stromal remodeling mechanisms that might have a direct impact on itch fibers.

Immune-mediated liver injury following COVID-19 vaccination.

Liver injury secondary to vaccination is a rare adverse event that has recently come under attention thanks to the continuous pharmacovigilance following the widespread implementation of coronavirus disease 2019 (COVID-19) vaccination protocols. All three most widely distributed severe acute respiratory syndrome coronavirus 2 vaccine formulations, e.g., BNT162b2, mRNA-1273, and ChAdOx1-S, can induce liver injury that may involve immune-mediated pathways and result in autoimmune hepatitis-like presentation that may require therapeutic intervention in the form of corticosteroid administration. Various mechanisms have been proposed in an attempt to highlight immune checkpoint inhibition and thus establish causality with vaccination. The autoimmune features of such a reaction also prompt an in-depth investigation of the newly employed vaccine technologies. Novel vaccine delivery platforms, e.g., mRNA-containing lipid nanoparticles and adenoviral vectors, contribute to the inflammatory background that leads to an exaggerated immune response, while patterns of molecular mimicry between the spike (S) protein and prominent liver antigens may account for the autoimmune presentation. Immune mediators triggered by vaccination or vaccine ingredients per se, including autoreactive antibodies, cytokines, and cytotoxic T-cell populations, may inflict hepatocellular damage through well-established pathways. We aim to review available data associated with immune-mediated liver injury associated with COVID-19 vaccination and elucidate potential mechanisms underlying its pathogenesis.

Vascular endothelial growth factor inhibitors promote antitumor responses via tumor microenvironment immunosuppression in advanced colorectal cancer

Purpose This study aims to investigate changes in the tumor immune environment of patients who underwent therapy with a vascular endothelial growth factor (VEGF) inhibitor for advanced colorectal cancer. Methods Patients (n = 135) with T3 or T4 colorectal cancer were included in this retrospective study. They were classified as follows: patients who had not received preoperative treatment (UPFRONT group, n = 54), who had received FOLFOX as preoperative chemotherapy (FOLFOX group, n = 55), and who had undergone resection after combination FOLFOX and bevacizumab as unresectable colorectal cancer (B-MAB group, n = 26). The number of cytotoxic T lymphocytes (CTLs), FOXP3+ lymphocytes (including regulatory T cells (Tregs)), CD163+ monocytes (including M2-type tumor-associated macrophages (TAM-M2 type)), and programmed cell death 1 (PD-1)+ lymphocytes was evaluated immunohistochemically in the cancer cell area (CC) and stromal cell area (ST) of surgical specimens, and compared among the three groups. Results The CTL population did not differ among the three groups in both areas. In the B-MAB group, the numbers of PD-1+ cells in the ST, FOXP3+ lymphocytes in both areas, and CD163+monocytes in the ST was lower than that in the other two groups, and a correlation with the histological therapeutic effect was observed. Conclusions In advanced colorectal cancer, VEGF inhibitors may decrease the number of PD-1+ cells and inhibit the infiltration of FOXP3+ lymphocytes and CD163+monocytes into the tumor environment.

Combination therapies for the optimisation of Bispecific T-cell Engagers in cancer treatment.

Bispecific T-cell engagers (BiTEs) redirect endogenous T-cell populations to cells expressing tumour-associated antigens to induce tumour cell killing. This inherently relies upon a cytotoxic T-cell population that is able to be recruited. In many cancers, immune checkpoints and other immunosuppressive factors in the tumour microenvironment lead to a population of anergic T-cells which cannot be redirected to tumour killing and thus impede the efficacy of BiTE therapy. Furthermore, there is evidence that BiTE therapy itself can increase immune checkpoint expression, and this is thought to be a major escape mechanism for the BiTE therapy blinatumomab. To overcome these inadequate T-cell responses, BiTEs may be combined with checkpoint inhibitors, chemotherapy, costimulatory molecules or oncolytic viruses. Study of these combinations is needed to expand the use of BiTEs in solid malignancies. This review covers the rationale, preclinical evidence and any clinical trials for these combination therapies and a few other less-studied combinations.

SMAD4 and TGFβ are architects of inverse genetic programs during fate determination of antiviral CTLs.

Transforming growth factor β (TGFβ) is an important differentiation factor for cytotoxic T lymphocytes (CTLs) and alters the expression levels of several of homing receptors during infection. SMAD4 is part of the canonical signaling network used by members of the transforming growth factor family. For this study, genetically modified mice were used to determine how SMAD4 and TGFβ receptor II (TGFβRII) participate in transcriptional programming of pathogen-specific CTLs. We show that these molecules are essential components of opposing signaling mechanisms, and cooperatively regulate a collection of genes that determine whether specialized populations of pathogen-specific CTLs circulate around the body, or settle in peripheral tissues. TGFβ uses a canonical SMAD-dependent signaling pathway to downregulate Eomesodermin (EOMES), KLRG1, and CD62L, while CD103 is induced. Conversely, in vivo and in vitro data show that EOMES, KLRG1, CX3CR1, and CD62L are positively regulated via SMAD4, while CD103 and Hobit are downregulated. Intravascular staining also shows that signaling via SMAD4 promotes formation of long-lived terminally differentiated CTLs that localize in the vasculature. Our data show that inflammatory molecules play a key role in lineage determination of pathogen-specific CTLs, and use SMAD-dependent signaling to alter the expression levels of multiple homing receptors and transcription factors with known functions during memory formation.

Oligoclonal T-cell expansion in a patient with bone marrow failure after CD19 CAR-T therapy for Richter-transformed DLBCL

The advent of chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment landscape of refractory B-cell malignancies. Real-world evidence has highlighted the high incidence of hematological toxicity, including prolonged and profound cytopenia. Here, we present a case of bone marrow (BM) failure in a 57-year old man with DLBCL-type Richter transformation receiving tisagenlecleucel in a standard-of-care setting. His clinical course was notable for underlying BM infiltration, severe cytokine release syndrome, and multiple infectious complications in the setting of prolonged, profound neutropenia. Cytokine profiling revealed a signature consistent with acquired aplastic anemia - including downregulation of CD40-L and EGF. Flow cytometric analyses demonstrated migration of CAR T-cells to the BM, as well as peripheral blood expansion of both CAR and non-CAR bearing CD8+CD57+ T-cells, an immunophenotype linked to oligoclonality in aplastic anemia. From single-cell level transcriptomics, this cytotoxic T-cell population was characterized by T-cell receptor Vβ oligoclonal expansion and post-CAR clonal drift. Gene expression profiling revealed upregulation of exhaustion markers on (CAR) T-cells and decreased expression of genes involved in STAT signalling and inflammatory response. In conclusion, this case highlights the complex nature of CAR-T-related hematological toxicity and introduces oligoclonal (CAR) T-cell expansion as a potential contributing pathophysiologic mechanism.

Improving anticancer effect of aPD-L1 through lowering neutrophil infiltration by PLAG in tumor implanted with MB49 mouse urothelial carcinoma

BackgroundThe PD-L1 antibody is an immune checkpoint inhibitor (ICI) attracting attention. The third-generation anticancer drug has been proven to be very effective due to fewer side effects and higher tumor-specific reactions than conventional anticancer drugs. However, as tumors produce additional resistance in the host immune system, the effectiveness of ICI is gradually weakening. Therefore, it is very important to develop a combination therapy that increases the anticancer effect of ICI by removing anticancer resistance factors present around the tumor.Methods The syngeneic model was used (n = 6) to investigate the enhanced anti-tumor effect of PD-L1 antibody with the addition of PLAG. MB49 murine urothelial cancer cells were implanted into the C57BL/6 mice subcutaneously. PLAG at different dosages (50/100 mpk) was daily administered orally for another 4 weeks with or without 5 mpk PD-L1 antibody (10F.9G2). PD-L1 antibody was delivered via IP injection once a week.ResultsThe aPD-L1 monotherapy group inhibited tumor growth of 56% compared to the positive group, while the PLAG and aPD-L1 co-treatment inhibited by 89%. PLAG treatment effectively reduced neutrophils infiltrating localized in tumor and converted to a tumor microenvironment with anti-tumor effective T-cells. PLAG increased tumor infiltration of CD8 positive cytotoxic T-cell populations while effectively inhibiting the infiltration of neoplastic T-cells such as CD4/FoxP3. Eventually, neutrophil-induced tumor ICI resistance was resolved by restoring the neutrophil-to-lymphocyte ratio to the normal range. In addition, regulation of cytokine and chemokine factors that inhibit neutrophil infiltration and increase the killing activity of cytotoxic T cells was observed in the tumors of mice treated with PLAG + aPD-L1.ConclusionsPLAG effectively turned the tumor-promoting microenvironment into a tumor-suppressing microenvironment. As a molecule that increases the anti-tumor effectiveness of aPD-L1, PLAG has the potential to be an essential and effective ICI co-therapeutic agent.

Insight Into Novel Anti-tuberculosis Vaccines by Using Immunoinformatics Approaches.

Tuberculosis (TB), an infectious disease, has been a leading cause of morbidity and mortality for decades. The causative agent of TB is the Mycobacterium tuberculosis (Mtb) which can infects various parts of the body, mainly the lungs in pulmonary TB cases. Mycobacterium bovis Bacillus Calmette–Guerin (BCG) is the only approved vaccine for TB, but its efficiency to combat pulmonary TB is limited. Multidrug-resistant (MDR) TB and extensive drug-resistant (XDR) TB requires the evolution of more potent vaccines. Therefore, this research aims to generate a universal TB subunit vaccine using advanced immunoinformatics techniques. In generating a novel multiepitope subunit vaccine, we selected the conserved and experimentally confirmed antigens Rv0058, Rv0101, and Rv3343. After a rigorous evaluation, the top candidates from predicted Helper T-lymphocytes (HTL), Cytotoxic T-lymphocytes (CTL), and B-cell epitopes were considered potential vaccine candidates. Immunogenicity was enhanced by the addition of an adjuvant to the ultimate construct of the vaccine. B-cell epitopes predictions guaranteed the eventual induction of a humoral response. Thereafter, dynamics simulations and molecular docking validated the vaccine-receptor complex’s stability and high affinity for the immune receptor TLR-3. Also, immune simulations revealed the significantly elevated levels of immunoglobulins such as IgM, cytokines such as interleukin-2, helper T (Th) cells, and cytotoxic T-cell populations. These results agreed with the actual inflammatory response and showed rapid antigen clearance after manifold exposure. Finally, the E. coli K12 strain was confirmed via in-silico cloning for quality expression. Nevertheless, in vivo experiments should be performed to validate the safety of the proposed vaccine and its inherent ability to prevent TB infection.

Deep phenotyping of surface stimulatory and inhibitory co-receptors on cancer-resident T and NK cells reveals cell subsets within the tumor-reactive CTL population that are uniquely defined by NKG2A expression.

The field of Immuno-Oncology (IO) is evolving to utilise novel antibody backbones that can co-target multiple cell-surface stimulatory and inhibitory co-receptors (SICR). This approach necessitates a better understanding of SICR co-expression at the single-cell level on IO-relevant tumor-infiltrating leukocyte (TIL) cell types such as T and natural killer (NK) cells. Using high-dimensional flow cytometry we established a comprehensive SICR profile for tumor-resident T and NK cells across a range of human solid tumors where there is a clear need for improved immunotherapeutic intervention. Leveraging the power of our large flow panel, we performed deep-phenotyping of the critical CD8+CD39+ Cytotoxic T Lymphocyte (CTL) population that is enriched for tumor-reactive cytotoxic cells, revealing subsets that are differentiated by their SICR profile, including three that are uniquely defined by NKG2A expression. This study establishes a comprehensive SICR phenotype for human TIL T and NK cells, providing insights to guide the design and application of the next generation of IO molecules.

The mitochondrial DNA constitution shaping T-cell immunity in patients with rectal cancer at high risk of metastatic progression

PurposeA significant percentage of colorectal cancer patients proceeds to metastatic disease. We hypothesised that mitochondrial DNA (mtDNA) polymorphisms, generated by the high mtDNA mutation rate of energy-demanding clonal immune cell expansions and assessable in peripheral blood, reflect how efficiently systemic immunity impedes metastasis.Patients and methodsWe studied 44 rectal cancer patients from a population-based prospective biomarker study, given curative-intent neoadjuvant radiation and radical surgery for high-risk tumour stage and followed for metastatic failure. Blood specimens were sampled at the time of diagnosis and analysed for the full-length mtDNA sequence, composition of immune cell subpopulations and damaged serum mtDNA.ResultsWhole blood total mtDNA variant number above the median value for the study cohort, coexisting with an mtDNA non-H haplogroup, was representative for the mtDNA of circulating immune cells and associated with low risk of a metastatic event. Abundant mtDNA variants correlated with proliferating helper T cells and cytotoxic effector T cells in the circulation. Patients without metastatic progression had high relative levels of circulating tumour-targeting effector T cells and, of note, the naïve (LAG-3+) helper T-cell population, with the proportion of LAG-3+ cells inversely correlating with cell-free damaged mtDNA in serum known to cause antagonising inflammation.ConclusionNumerous mtDNA polymorphisms in peripheral blood reflected clonal expansion of circulating helper and cytotoxic T-cell populations in patients without metastatic failure. The statistical associations suggested that patient’s constitutional mtDNA manifests the helper T-cell capacity to mount immunity that controls metastatic susceptibility.Trial registrationClinicalTrials.gov NCT01816607; registration date: 22 March 2013.

Overcoming the Immunosuppressive Tumor Microenvironment in Multiple Myeloma.

Simple SummaryThis article provides a comprehensive review of new and emerging treatment strategies against multiple myeloma that employ precision medicines and/or drugs capable of improving the ability of the immune system to prevent or slow down the progression of multiple myeloma. These rationally designed new treatment methods have the potential to change the therapeutic landscape in multiple myeloma and improve the long-term survival outcome.SeverFigurel cellular elements of the bone marrow (BM) microenvironment in multiple myeloma (MM) patients contribute to the immune evasion, proliferation, and drug resistance of MM cells, including myeloid-derived suppressor cells (MDSCs), tumor-associated M2-like, “alternatively activated” macrophages, CD38+ regulatory B-cells (Bregs), and regulatory T-cells (Tregs). These immunosuppressive elements in bidirectional and multi-directional crosstalk with each other inhibit both memory and cytotoxic effector T-cell populations as well as natural killer (NK) cells. Immunomodulatory imide drugs (IMiDs), protease inhibitors (PI), monoclonal antibodies (MoAb), adoptive T-cell/NK cell therapy, and inhibitors of anti-apoptotic signaling pathways have emerged as promising therapeutic platforms that can be employed in various combinations as part of a rationally designed immunomodulatory strategy against an immunosuppressive tumor microenvironment (TME) in MM. These platforms provide the foundation for a new therapeutic paradigm for achieving improved survival of high-risk newly diagnosed as well as relapsed/refractory MM patients. Here we review the scientific rationale and clinical proof of concept for each of these platforms.

Dysregulation of murine immune functions on inhalational exposure to ammonia, dimethyl disulfide, 3-methylindole, or propionic acid.

Animal husbandry workers are exposed to various malodorous compounds in the workplace. Although these compounds cause severe nuisance, no systemic investigation of their effects on the immune system has been conducted. To address this issue, we evaluated the effects of inhalational exposure to ammonia, dimethyl disulfide, 3-methylindole (3-MI), and propionic acid (PA), representing four major groups of malodorous compounds, on humoral and cellular immunity in mice. Mice were exposed to the substances (low dose: 10 µL and high dose: 200 µL) for 10 min/day for 4 weeks in a modified standard mouse cage. Neutrophil% and splenic cytotoxic T cell% were significantly lower in the high-dose ammonia group than in the vehicle control. Exposure to ammonia and 3-MI increased immature thymic T lymphocyte% relative to control and concomitantly decreased both mature helper and cytotoxic T-cell populations in the thymus. In the ammonia exposure group, levels of serum immunoglobulin E and immunoglobulin A were elevated, and the IgG2a:IgG1 ratio in the serum was reduced in a dose-dependent manner. Splenic natural killer cell activity was significantly less in the PA exposure group than in the control. Overall, our findings suggest that inhalational exposure to these malodorous substances disturbs immune homeostasis in vivo.

HISTOLOGICAL AND IMMUNOHISTOCHEMICAL FEATURES OF BONE TISSUE AND RED BONE MARROW IN ACUTE HEMATOGENOUS 132 OSTEOMYELITIS IN CHILDREN

Acute hematogenous osteomyelitis (AHO) is a severe disease of childhood and leads to disability in 70-87 % of cases. Despite the achievements of modern science, research methods are not always able to diagnose CSO in children in time. The number of scientific works devoted to the early diagnosis of CSO in children based on the use of morphological and functional characteristics of bone tissue is limited. The material for the study was a biopsy of bone tissue and red bone marrow obtained after osteoperforation in children with CSO. After preliminary processing of the biopsy, histological and immuno-histochemical studies were performed, followed by an assessment of the prognostic value of the data obtained. The study revealed histological changes in bone tissue characteristic of the intramedullary and extramedullary phases of CSO, IHC indicators of an increase in the population of cytotoxic T-lymphocytes in the red bone marrow, indicating the development of a severe inflammatory process.

A novel CD4+ CTL subtype characterized by chemotaxis and inflammation is involved in the pathogenesis of Graves\u2019 orbitopathy

Graves’ orbitopathy (GO), the most severe manifestation of Graves’ hyperthyroidism (GH), is an autoimmune-mediated inflammatory disorder, and treatments often exhibit a low efficacy. CD4+ T cells have been reported to play vital roles in GO progression. To explore the pathogenic CD4+ T cell types that drive GO progression, we applied single-cell RNA sequencing (scRNA-Seq), T cell receptor sequencing (TCR-Seq), flow cytometry, immunofluorescence and mixed lymphocyte reaction (MLR) assays to evaluate CD4+ T cells from GO and GH patients. scRNA-Seq revealed the novel GO-specific cell type CD4+ cytotoxic T lymphocytes (CTLs), which are characterized by chemotactic and inflammatory features. The clonal expansion of this CD4+ CTL population, as demonstrated by TCR-Seq, along with their strong cytotoxic response to autoantigens, localization in orbital sites, and potential relationship with disease relapse provide strong evidence for the pathogenic roles of GZMB and IFN-γ-secreting CD4+ CTLs in GO. Therefore, cytotoxic pathways may become potential therapeutic targets for GO.

Cytotoxic T cells swarm by homotypic chemokine signalling.

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes.

Correlative studies investigating effects of PI3K inhibition on peripheral leukocytes in metastatic breast cancer: potential implications for immunotherapy.

Patients with localized breast cancer have a 5-year survival rate > 99% compared to patients with metastatic breast cancer (MBC) that have a 5-year survival rate of ~ 27%. Unregulated PI3K/AKT signaling is a common characteristic of MBC, making it a desirable therapeutic target for tumors with activating mutations in this pathway. Interestingly, inhibition of the PI3K/AKT pathway can affect signaling in immune cells, which could potentially alter the immune phenotype of patients undergoing therapy with these drugs. The purpose of this study is to evaluate how PI3K inhibition affects the immune cells of MBC patients during treatment. We investigated the effects of PI3K inhibition on the immune cell populations in peripheral blood of MBC patients enrolled in 4 different clinical trials utilizing PI3K inhibitors. Peripheral blood was drawn at different points in patient treatment cycles to record immune cell fluctuations in response to therapy. MBC patients who responded to treatment with a positive fold-change in cytotoxic T cell population, had an average duration of treatment response of 31.4months. In contrast, MBC patients who responded to treatment with a negative fold-change in cytotoxic T-cell population, had an average duration of therapeutic response of 5months. These data suggest that patients with a more robust, initial anti-tumor T cell response may have a longer therapeutic response compared to patients who do not have a robust, initial anti-tumor T cell response. These results highlight the potential for PI3K inhibition to sensitize tumors to immune checkpoint inhibitors, thus providing additional therapeutic options for patients with MBC.

Identification of two distinct populations of WT1-specific cytotoxic T lymphocytes in co-vaccination of WT1 killer and helper peptides.

Simultaneous induction of tumor antigen-specific cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs) is required for an optimal anti-tumor immune response. WT1332, a 16-mer WT1-derived helper peptide, induce HTLs in an HLA class II-restricted manner and enhance the induction of WT1-specific CTLs in vitro. However, in vivo immune reaction to WT1332 vaccination in tumor-bearing patients remained unclear. Here, a striking difference in WT1-specific T cell responses was shown between WT1 CTL + WT1 helper peptide and WT1 CTL peptide vaccines in patients with recurrent glioma. WT1-specific CTLs were more strongly induced in the patients who were immunized with WT1 CTL + WT1 helper peptide vaccine, compared to those who were immunized with WT1 CTL vaccine alone. Importantly, a clear correlation was demonstrated between WT1-specific CTL and WT1332-specific HTL responses. Interestingly, two novel distinct populations of WT1-tetramerlow WT1-TCRlow CD5low and WT1-tetramerhigh WT1-TCRhigh CD5high CTLs were dominantly detected in WT1 CTL + WT1 helper peptide vaccine. Although natural WT1 peptide-reactive CTLs in the latter population were evidently less than those in the former population, the latter population showed natural WT1 peptide-specific proliferation capacity comparable to the former population, suggesting that the latter population highly expressing CD5, a marker of resistance to activation-induced cell death, should strongly expand and persist for a long time in patients. These results demonstrated the advantage of WT1 helper peptide vaccine for the enhancement of WT1-specific CTL induction by WT1 CTL peptide vaccine.

Abstract A21: Engineering antigen-specific natural killer cells against the melanoma-associated antigen tyrosinase via TCR gene transfer

Abstract Introduction of chimeric antigen receptors (CARs) to natural killer (NK) cells has so far been the only practical method for specific targeting of NK cells against surface antigens. In contrast, T-cell receptor (TCR) gene therapy can supply large populations of cytotoxic T-lymphocytes (CTL) genetically modified to express a TCR that can also target intracellular antigens. However, the mispairing of endogenous and genetically transferred TCR subunits constitutes a bottleneck in the development of safe therapies as it often leads to formation of TCRs with unknown specificity. In order to overcome this obstacle and enable intracellular antigen targeting, we propose the use of NK cells for TCR gene therapy. In this study, we approach the obstacles associated with TCR gene therapy from a unique perspective that results in MHC-I-restricted epitope-specific targeting of tumors cells through expression of a functional TCR complex on NK cells. Our results show that the ectopic expression of CD3δ, CD3γ, and CD3ϵ chains along with TCR α/β gene delivery to NK cells enables the functional expression of a TCR specific to the HLA-A2-restiricted tyrosinase-derived melanoma epitope, Tyr368-379. NK cells expressing a functional TCR exhibit the capacity to degranulate in an antigen-specific manner in response to engagement of the cognate peptide/MHC ligand on target cells. In addition, upon engagement of their TCR, NK cells are fully capable of producing proinflammatory cytokines IFNγ and TNF-α, a signature mark of NK cell activation and immune cell recruitment. Finally, NK-TCR cells exhibit MHC-I-restricted antigen detection and antigen-specific lysis of tumor cells both in vitro and in vivo. Antigen-specific targeting of NK cells via TCR gene delivery stands out as a unique discovery providing a novel tool in the field of adoptive immunotherapy that can also overcome the major hurdle of “mispairing” in TCR gene therapy. Clinical trials using NK cells, including genetically modified NK cells expressing activating receptors or CARs, have clearly demonstrated a significant benefit in patients with various malignancies. The overall safety profile and promising clinical benefits of NK cells combined with the exclusive antigen specificity of the TCR, all together provide a novel approach in the design of efficient antigen-specific adoptive immunotherapy. Citation Format: Ayhan Parlar, Ece C. Sayitoglu, Cevriye Pamukcu, Anna-Maria Georgoudaki, Didem Ozkazanc, Mertkaya Aras, Benjamin Josey, Michael Chrobok, Suzanne Branecki, Pegah Zahedimaram, Lolai Ikromzoda, Evren Alici, Batu Erman, Tolga Sutlu, Adil D. Duru. Engineering antigen-specific natural killer cells against the melanoma-associated antigen tyrosinase via TCR gene transfer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A21.

Correlation of Tumor-associated Leukocytes with Prognosis of Colorectal Carcinoma based on Pathologic Stage

Objectives To perform a pilot study investigating the presence of correlation between the different mean tumor-associated leukocyte counts and the prognosis of colorectal cancer based on pathologic stage. Methodology A cross-sectional study design, involving colorectal carcinoma cases in the Philippine General Hospital from 2015-2016. Proportional allocation stratified random sampling was done, with pathologic stage (AJCC 7th Edition) as the stratifying variable, collecting a total of 59 samples. Tissue sections from the samples were evaluated for the different tumor-associated lymphocyte counts. Correlation coefficients were computed to determine their correlation with pathologic stage as surrogate marker for prognosis. Results Of the myriad populations counted within and around the tumor mass, total lymphocyte, cytotoxic T-cell (CD8+ T-cell), neutrophil, macrophage, and plasma cell populations have significant correlation with pathologic stage as surrogate marker for prognosis of colorectal carcinoma. Conclusion The immune system appears to have a significant role in the natural history of colorectal carcinoma. The tumor-infiltrating lymphocytic population and especially the CD8+ T-cell subset, neutrophils, and macrophages are correlated with better prognosis. The same observation can be seen with the peritumoral CD8+ T-cells, neutrophils, macrophages, and plasma cells.