Valganciclovir as Add-On Therapy Modifies the Frequency of NK and NKT Cell Subpopulations in Disseminated Kaposi Sarcoma Patients

Human herpesvirus-8 infection (HHV-8) is the causative agent of Kaposi sarcoma (KS) and is highly prevalent among people living with HIV (KS/HIV). It has been reported that valganciclovir (VGC) reduces HHV-8 replication in KS/HIV patients. However, currently it is unclear if VGC modifies the frequency and induces changes in markers of immune regulation of immune cells necessary to eliminate HHV8-infected cells, such as Natural Killer (NK) and NK T cells (NKT). This study evaluated the effect of VGC used as antiviral HHV8 therapy in KS patients on the frequency of NK and NKT subpopulations based on the CD27 and CD57 expression, and the immunosenescence markers, PD-1 and KLRG1. Twenty KS/HIV patients were followed-up at baseline (W0), 4 (W4), and 12 weeks (W12) of the study protocol. Among them, 10 patients received a conventional treatment scheme (CT), solely antiretroviral therapy (ART), and 10 patients received a modified treatment regime (MT), including VGC plus ART. In both groups, bleomycin/vincristine was administrated according to the treating physician’s decision. The soluble levels of IL-15, PD-L1, PD-L2, and E-cadherin were quantified across the follow-up. Our results showed that the higher IL-15 levels and lower NK frequencies cells in KS/HIV patients reach almost normal values with both treatments regimes at W12. CD27+ NK and NKT cell frequencies increased since W4 on KS/HIV patients with MT. Furthermore, PD-1 expression decreased while KLRG1 increased on NK and NKT subpopulations at W12, and it is accompanied by increased PD-L1 plasma level since W4. Our study highlights the disruption of NK and NKT subpopulations in patients with KS/HIV and explores VGC treatment’s contribution to immune reconstitution during the first weeks of treatment.

926 Immune profiling of KEAP1-deficient NSCLC: development of therapeutic strategies to overcome resistance to immunotherapy

BackgroundIn LUAD, KEAP1 is the third most common tumor suppressor and loss-of-function mutations in KEAP1 commonly co-occur with STK11/LKB1 and KRAS mutations. KEAP1 protein that regulates the degradation of the antioxidant transcription factor NRF2. The role of STK11/LKB1 mutations in immunotherapy resistance has been characterized, however the mechanistic understanding of KEAP1 deficiency in shaping LUAD phenotype and therapy response is still very limited. Recent clinical data has been reported suggesting that mutations in STK11/LKB1 and KEAP1 are strongly associated with immune checkpoint blockade resistance in LUAD, particularly those with KRAS mutations. Nevertheless, the biology of KEAP1-deficient tumors and the immune suppression mechanisms are to be characterized.MethodsWe have first validated response to anti-PD1 treatment in vivo using subcutaneous murine models, and performed a deep profiling and characterization of tumor microenvironment (TME) heterogeneity of KRAS-mutant (K) and LKB1 (KL), and/or KEAP1 deficient (KK and KLK) tumors using single-cell RNA sequencing (scRNA-seq) and multiplex staining. Data from pre-clinical models has been used to survey the immune genomic data available from the MD Anderson ICON study (a cohort of early stage lung cancer untreated 148 resected tumors) and TCGA lung cohorts to further validate our findings.ResultsWhile K tumors showed significant response to anti-PD1 treatment, KEAP1 loss completely impaired therapeutic response to this immunotherapy. KEAP1-deficient tumors were characterized by low immune infiltration while displayed an enrichment of cancer associated fibroblasts (CAFs) and endothelial cells. scRNA-seq data indicated a significant reduction of T cell infiltration, in particularly, CD8 and NK T cells, pronounced decreased of B cell population and a marked M2 macrophages polarization. Likewise, IHC and multiplex analysis of CD3 and F4/80 markers confirmed these previous findings. In TCGA lung cancer cohort, CD8B expression was dramatically decreased while MIF (macrophage migration inhibitory factor) was upregulated in KK compared to K LUADs tumors, and expression of KEAP1 inversely correlated with CD163, ARG2 and IL10, which are mainly secreted by macrophages. Concordantly, KEAP1-deficient pre-clinical tumors showed a significant upregulation of MIF expression and secretion, and CRISPR-Cas9 deletion of MIF dramatically impaired in vivo tumor growth in KK and KLK but not in K or KL models.ConclusionsThese findings indicate that loss of KEAP1, alone or in combination with STK11/LKB1 alterations, unfavorably reprograms TME. These changes appear to be mediated at least in part through MIF upregulation, providing a potential therapeutic strategy for overcoming KEAP1-dependent resistance to immunotherapy.

Dynamic switch of immunity and antitumor effects of metformin in rat spontaneous esophageal carcinogenesis

Chronic inflammation contributes to tumor development by creating a local microenvironment that facilitates neoplastic transformation and potentiates the progression of cancer. Esophageal cancer (EC) is an inflammation-associated malignancy with a poor prognosis. The nature of the switch between chronic inflammation of the esophagus and EC-related immunological changes remains unclear. Here, we examined the dynamic alterations of immune cells at different stages of chronic esophagitis, Barrett’s esophagus (BE) and EC using an esophageal spontaneous carcinogenesis rat model. We also investigated the anticancer effects of metformin. To stimulate EC carcinogenesis, chronic gastroduodenal reflux esophagitis via esophagojejunostomy was induced in 120 rats in metformin-treated and non-treated (control) groups. After 40 weeks, BE and EC developed in 96.7% and 63.3% of the control group, and in 66.7% and 23.3% of the metformin-treated group, respectively. Flow cytometric analysis demonstrated that the balance of M1/M2-polarized or phospho-Stat3-positive macrophages, regulatory T, cytotoxic T, natural killer (NK), NK T cells, and Th17 T cells was dynamically changed at each stage of the disease and were resolved by metformin treatment. These findings clarify the immunity in esophageal carcinogenesis and suggest that metformin could suppress this disease by improving the immunosuppressive tumor microenvironment and immune evasion.

Cellular Basis for the Enhanced Efficacy of the Fms-Like Tyrosine Kinase 3 Ligand (FL) Adjuvanted VCG-Based Chlamydia abortus Vaccine

Efficacious vaccines are needed to control genital chlamydial diseases in humans and the veterinary industry. We previously reported a C. abortus (Cab) vaccine comprising recombinant Vibrio cholerae ghosts (rVCG) expressing the conserved and immunogenic N-terminal region of the Cab polymorphic membrane protein D (rVCG-Pmp18.1) protein that protected mice against intravaginal challenge. In this study, we investigated the immunomodulatory effect of the hematopoietic progenitor activator cytokine, Fms-like tyrosine kinase 3-ligand (FL) when co-administered with the rVCG-Pmp18.1 vaccine as a strategy to enhance the protective efficacy and the potential mechanism of immunomodulation. Groups of female C57BL/6J mice were immunized and boosted twice intranasally (IN) with rVCG-PmpD18.1 with and without FL or purified rPmp18.1 or rVCG-gD2 (antigen control) or PBS (medium) per mouse. The results revealed that co-administration of the vaccine with FL enhanced antigen-specific cellular and humoral immune responses and protected against live Cab genital infection. Comparative analysis of immune cell phenotypes infiltrating mucosal and systemic immune inductive tissue sites following immunization revealed that co-administration of rVCG-Pmp18.1 with FL significantly enhanced the number of macrophages, dendritic and NK cells, γδ and NK T cells in the spleen (systemic) and iliac lymph nodes (ILN) draining the genital tract (mucosal) tissues compared to rVCG-Pmp18.1 alone. Furthermore, FL enhanced monocyte infiltration in the ILN, while CD19+ B cells and CD4+ T cells were enhanced in the spleen. These results indicate that the immunomodulatory effect of FL is associated with its ability to mobilize innate immune cells and subsequent activation of robust antigen-specific immune effectors in mucosal and systemic lymphoid tissues.

Spike-specific immune response induced by BNT162b2 mRNA vaccine in former COVID-19 patients and high responsive subjects

Background: The worldwide escalation of Coronavirus Disease 2019 (COVID-19) has urgently required the development of safe and effective vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of disease. The BNT162b2 (Pfizer–BioNTech) RNA-based vaccine confers 95% protection against COVID-19 by encoding a mutated isoform of SARS-CoV-2 full-length spike (S) protein. Objective: Here, we report the antigen-specific immune profile against SARS-CoV-2 S protein after vaccination with a single dose of BNT162b2 in order to define the immunological landscape required for an efficient response to the SARS-CoV-2 vaccine. Methods: We determined the levels of antibodies and antigen-specific B, T and NK-T cells against a recombinant GFP tagged SARS-CoV-2 S protein in subjects up to 20 days after injection of a single dose of BNT162b2 vaccine using a combined approach involving serological assays and flow cytometry analyses. Former COVID-19 patients have been also included in this study to evaluate the effect of vaccine after exposition to SARS-CoV-2. Results: The level of antigen-specific helper T-cells against SARS-CoV-2 S protein was reduced in subjects, low responsive or unresponsive to vaccination with respect to the highly responsive individuals, while the numbers of antigen-specific regulatory and cytotoxic T-cells were comparable. Of interest, in former COVID-19 patients, a single dose of BNT162b2 vaccine induced a significant increase of antibody production simultaneous with an antigen-specific B and NK-T cell response. Conclusion: Taken together, these results suggest that favorable immune profiles support the progression and an effective reaction to BNT162b2 vaccination.

Distinct hepatic myeloid and lymphoid cell repertoires are associated with susceptibility and resistance to Ascaris infection

The soil-transmitted helminth Ascaris lumbricoides infects ~800 million people worldwide. Some people are heavily infected, harbouring many worms, whereas others are only lightly infected. The mechanisms behind this difference are unknown. We used a mouse model of hepatic resistance to Ascaris, with C57BL/6J mice as a model for heavy infection and CBA/Ca mice as a model for light infection. The mice were infected with the porcine ascarid, Ascaris suum or the human ascarid, A. lumbricoides and immune cells in their livers and spleens were enumerated using flow cytometry. Compared to uninfected C57BL/6J mice, uninfected CBA/Ca mice had higher splenic CD4+ and γδ T cell counts and lower hepatic eosinophil, Kupffer cell and B cell counts. Infection with A. suum led to expansions of eosinophils, Kupffer cells, monocytes and dendritic cells in the livers of both mouse strains and depletions of hepatic natural killer (NK) cells in CBA/Ca mice only. Infection with A. lumbricoides led to expansions of hepatic eosinophils, monocytes and dendritic cells and depletions of CD8+, αβ, NK and NK T cells in CBA/Ca mice, but not in C57BL/6J mice where only monocytes expanded. Thus, susceptibility and resistance to Ascaris infection are governed, in part, by the hepatic immune system.

A Rise in Percentages of Circulating Lymphocyte Subsets Correlates With Acute Rejection in Liver Transplant Recipients

Background: Little is known about the shift of circulating lymphocyte subsets following liver transplantation and thus, its relationship with acute rejection. Methods: Liver transplant recipients were enrolled to assess the effect of primary liver diseases, gender, age, and follow-up periods on the shift of circulating lymphocyte subsets. Moreover, patients with rejection were paired to assess the effect of the shift on rejection.Results: When compared with patients from the middle-term group (29-180 d) and the long-term group (>180 d), patients from the short-term group (< 29 d) had the lowest absolute counts of T cell subsets, NK cells and NK T cells, and the lowest percentages of T cell subsets, B cells, NK cells and NK T cells but the highest percentage of DC. However, other factors did not affect circulating lymphocyte subsets. Percentages of T cells, CD4+T cells, B cells and NK T cells were higher in patients with acute rejection while percentages of T cell subsets and NK cells decreased after anti-rejection treatment. The percentage of NK T cells was identified to be the only independent predictor for acute rejection. The predicted probability was calculated using binary logistic with the area under the curve of 0.89, which had a sensitivity of 70.6% and a specificity of 94.1% at a cut-off value of 0.69.Conclusions: Circulating lymphocyte subsets gained a global recovery over the post-transplant period, leading to a sharp rise in percentages of circulating lymphocyte subsets, which was in close relation to the occurrence of acute rejection.

Detailed Immunophenotypic Profiling of Peripheral Blood Immune Cells in Patients with Hematologic Malignancies after Sars-Cov-2 Infection

Introduction:The spread of SARS-CoV-2 virus continues to pose a major public health threat. Patients with cancer are thought to be at increased risk from SARS-COV-2 infection due to the immunodeficiency that results from the underlying neoplasm and treatment. The immune response to this infection has been the subject of great interest, with an extreme variation in clinical severity between infected individuals. Variation in the immune cell response (B, T, and NK lymphocytes, monocytes, and myeloid derived suppressor cells (MDSCs), among others) and their function have been hypothesized to be responsible for this range of presentation. Methods:Two patients with a history of hematologic malignancies were matched with three non-cancer patients with similar baseline clinical characteristics and severity of COVID related illness. The critical group (CG) was defined as those requiring mechanical ventilation (MV) due to COVID related respiratory failure and the non-critical group (NCG) were hospitalized but did not require MV. All samples studied were obtained from peripheral blood and processed within 4-hours of collection. Peripheral blood mononuclear cell (PBMC) were isolated using ficoll density gradient separation. Flowcytometric analysis using CytekTM Aurora was done on fresh PBMC samples. Thirty antibody-based flow markers were used to identify 54 distinct immune cell populations. IRB approval was obtained. Results: Critical Group (CG):The CG included case 1, a 47 year-old (y.o.) female (F) with a history (hx) of acute myeloid leukemia and had an matched related donor allogeneic hematopoietic stem cell transplant (alloHSCT) 10-years prior remaining in remission, with hematologic recovery, and off immunosuppressants treated with remdesivir and coritcosteroids for COIVD directed therapy; and case 2, a 55 y.o. F with a hx of HIV treated with corticosteroids for COIVD directed therapy (see Figure 1a). Non-Critical Group (NCG):The NCG included case 3, a 73 y.o male (M) with hx of relapsed/refractory Philadelphia chromosome negative Acute Lymphoblastic Leukemia with loss of CD19 and CD22 expression following treatment with blinatumumab and inotuzumab, and most recently treated with decitabine/venetoclax; case 4, a 66 y.o. M with hx of cardiomyopathy; and case 6, a 54 y.o. M with hx of obesity. None of the NCG cases were treated with COVID directed therapy. See Table 1 for further clinical information. Immunophenotypic expression:Flow cytometry gating strategy done as outlined in Fig 1a. Case 1 had a high proportion of B-cells, CD8+ T-cells, and cells with exhaustion markers (CD8+CD94+ T-cells, CD4+PD1+ T-cells, CD4+PD1+CD94+ T-cells, PD1-CD94+ NK T-cells, Lag3+Cd11b- non-TB leukocytes) and MDSC immunophenotypes compared with matched case 2. Case 3 also had a high proportion of exhaustion markers (Lag3+CD39 low B-cells, CD8+PD1+ T-cells, CD8+CD94+PD1+ T-cells, CD4+PD1+CD94+ T-cells, PD1+CD94+ NK T-cells, PD1-CD94+ NK T-cells, Lag3+CD11b+, Lag3+CD11b- non-TB leukocytes) and high expression of immunosuppressive Treg and all MDSC; although high expression of granulocytic MDSC. Case 2 had a significant number of exhaustion and immunosuppressive cells as well. Cases 4 and 5 had a higher predominance of all T-cell subtypes and also had variable expression of exhaustion and immunosuppressive immunophenotypes (See Fib 1b). Conclusion:In our study of one critical and one non-critical patient with a history of hematologic malignancy matched with three non-cancer patients we demonstrate the high predominance of exhaustion markers (Lag3,PD1,CD94) and immunosuppressive cell types (Treg, granulocytic and monocytic MDSC). These findings are consistent with the fact that both CG and NCG, as hospitalized patients, represent the most severely ill COVID patient cohort. Of notable interest to the cancer population, cases 1 and 3 had a significant number of exhaustion and immunosuppressive immunophenotypes, suggestive of baseline exhaustion following alloHSCT even years after engraftment in case 1 and attenuated functional immunity in a patient undergoing active treatment in case 3. Interestingly, case 3 had lower expression of all MDSC, a known treatment effect of decitabine. Paired cytokine measurement and its effect on immunophenotype is underway. Additionally, we plan to present an atlas of the peripheral immune cell response on fifteen additional non-cancer COVID patients. Disclosures No relevant conflicts of interest to declare.

505 Activation of NK T cells promote the inflammatory tumor microenvironment and control the growth of solid tumor

BackgroundType I NK T cells, also known as iNKT cells, can recognize self or microbial lipids presented through CD1d molecules on antigen-presenting cells. Activation of NKT cells induces inflammatory cytokines and help in mounting anti-tumor immunity. How does stimulation of iNKT cells in vivo alter the tumor microenvironment is not clearly understood.MethodsC57BL/6 mice were given a subcutaneous injection of B16F10 melanoma cell line (1 X 106 cells). Mice were given intraperitoneal injection of alpha-galactosylceramide (a-GalCer, a ligand for iNKT cells; 2 microgram/injection) on day +1, +5, +10, +15 and +20. NK cells, Gr1+ cells and F4/80+ macrophages in mice were depleted using cell-specific antibodies. The growth of tumors was monitored, and immune cells were characterized using flow cytometry and immunofluorescence staining. Student’s t-test and one-way ANOVA were used for statistical analysis.ResultsOur results showed that intratumoral NK T cells had significantly low expression of CD25, CD69, CD122, and IFN-gamma receptor molecules and produced lower inflammatory cytokines (IFN-gamma, TNF-alpha, and GM-CSF) as compared to splenic NK T cells. The soluble factor produced by B16F10 cells reduces the expression of these cytokines and cytokine receptors in vitro on the NK T cells purified from the spleen. Treatment of tumor-bearing mice with a-GalCer significantly increased the IFN-gamma-producing NK T cells, CD8+ T cells, and effector Th1 cells in secondary lymphoid organs, and tumors, also significantly reduced the tumor growth. Furthermore, a-GalCer treatment significantly increased the iNOS+CD206- M1-macrophages and reduced the iNOS-CD206+ M2-macrophages in the spleen and tumor. The depletion of F4/80+ macrophages prevented the a-GalCer-induced reduction of tumor growth.ConclusionsOur results showed that tumor produced soluble factors alter the phenotype of NK T cells. Activation of NKT cells with a-GalCer promotes the M1-macrophages, and effector CD8+ T cells, Th1 cells in the secondary lymphoid organs and tumor microenvironment. This finding suggests that activation of NKT cells may provide an effective anti-tumor response.AcknowledgementsThis work was supported by the Science and Engineering Research Board grant (EMR/2016/007108) and SwarnJayanti Fellowship (DST/SJF/LSA-01/2017–18) from the Department Science and Technology (DST), Government of India.Ethics ApprovalAll the procedures performed in the experiments involving mice were in accordance with the ethical standards of (NCCS) Institutional Ethics Committee of Animals Usage (Approval ID: EAF/B-166/2011 and EAF/B-256/2016).