Cells In PBMC Research Articles

Phenotypic changes of γδ T cells in Plasmodium falciparum placental malaria and pregnancy outcomes in women at delivery in Cameroon.

Depending on the microenvironment, γδ T cells may assume characteristics similar to those of Th1, Th2, Th17, regulatory T cells or antigen presenting cells. Despite the wide documentation of the effect of Th1/Th2 balance on pregnancy associated malaria and outcomes, there are no reports on the relationship between γδ T cell phenotype change and Placental Malaria (PM) with pregnancy outcomes. This study sought to investigate the involvement of γδ T cells and its subsets in placental Plasmodium falciparum malaria. In a case-control study conducted in Yaoundé, Cameroon from March 2022 to May 2023, peripheral, placental and cord blood samples were collected from 50 women at delivery (29 PM negative: PM- and 21 PM positive: PM+; as diagnosed by light microscopy). Hemoglobin levels were measured using hemoglobinometer. PBMCs, IVBMCs and CBMCs were isolated using histopaque-1077 and used to characterize total γδ T cell populations and subsets (Vδ1+, Vδ2+, Vδ1-Vδ2-) by flow cytometry. Placental Plasmodium falciparum infection was associated with significant increase in the frequency of total γδ T cells in IVBMC and of the Vδ1+ subset in PBMC and IVBMC, but decreased frequency of the Vδ2+ subset in PBMC and IVBMC. The expression of the activation marker: HLA-DR, and the exhaustion markers (PD1 and TIM3) within total γδ T cells and subsets were significantly up-regulated in PM+ compared to PM- group. The frequency of total γδ T cells in IVBMC, TIM-3 expression within total γδ T cells and subsets in IVBMC, as well as HLA-DR expression within total γδ T cells and Vδ2+ subset in IVBMC were negatively associated with maternal hemoglobin levels. Furthermore, the frequency of total γδ T cells in PBMC and PD1 expression within the Vδ2+ subset in CBMC were negatively associated with birth weight contrary to the frequency of Vδ1-Vδ2- subset in PBMC and HLA-DR expression within the Vδ2+ subset in IVBMC which positively associated with maternal hemoglobin level and birth weight, respectively. The data indicate up-regulation of activated and exhausted γδ T cells in Plasmodium falciparum placental malaria, with effects on pregnancy outcomes including maternal hemoglobin level and birth weight.

Increased CD123 + HLA-DR- Granulocytes in Allergic Rhinitis and Influence of Allergens on Expression of Cell Membrane Markers.

It is reported that CD123 + HLA-DR- cells in PBMC are basophils, and CD203c, CD63, and FcεRI molecules are activation markers of basophils. However, little is known of CD123 + HLA-DR-cells in blood granulocytes. To investigate the presence of CD123 + HLA-DR- cells in the blood granulocytes and peripheral PBMC of patients with allergic rhinitis (AR), as well as the impact of allergens on the cell membrane markers of basophils. Flow cytometry was used to detect the expression of the membrane molecules. While CD123 + HLA-DR- PBMCs are representative of basophils, their presence did not significantly change in patients with AR. In contrast, both the percentage and number of CD123 + HLA-DR- granulocytes, which make up only up to 50% of basophils, were significantly increased in patients with seasonal (sAR) and perennial AR (pAR). CD63+, CD203c+, and FcεRIα+ cells within CD123 + HLA-DR- granulocytes also showed enhanced activity in patients with AR. Allergen extracts from house dust mite allergen extract (HDME) and Artemisia sieversiana wild extract further increased the number of CD123 + HLA-DR- cells in granulocytes of sAR and pAR patients, as well as in PBMCs of pAR patients. The use of CD123 + HLA-DR- granulocytes and PBMC may not be sufficient for diagnosing AR. Allergens could potentially contribute to the development of AR by influencing the number of CD123 + HLA-DR- cells, as well as the expression of CD63, CD203c, and FcεRIαin these cells.

Durable cellular immune response against inactivated ZIKV and envelope proteins in ZIKV-infected women during pregnancy

IntroductionZika virus (ZIKV) infection has been associated to Guillain-Barré syndrome in adults and congenital malformations during pregnancy, leading to the manifestation of congenital Zika syndrome (CZS). The ZIKV envelope protein (EZIKV), prominently displayed on the virus surface, is a primary target for the humoral immune response. However, limited information exists regarding its capacity to induce cellular immunity, particularly in pregnant women with a history of ZIKV infection. The EZIKV protein comprises three domains: the central domain (EDI), a dimerization domain (EDII), and a domain responsible for binding to the cell surface receptor (EDIII). To examine the regions of EZIKV targeted by cellular immunity, we examined cellular immune responses in a cohort of mothers infected with ZIKV, whose infants exhibited microcephaly.MethodsTo assess the ZIKV-specific response, we used inactivated virus and different recombinant viral envelope proteins (EZIKV, EDI/IIZIKV and EDIIIZIKV). All women in the study contracted the infection during pregnancy, with 72% experiencing symptoms such as fever, rash, joint pain, and retro-orbital pain. Peripheral blood mononuclear cells (PMBC) were collected post- ZIKV diagnosis confirmation, with a median time of 18 months (IQR 13.5-19) after parturition. Using the ELISpot assay, we quantified specific interferon-gamma (IFNγ) producing cells by stimulating PBMC with either inactivated ZIKV particles or equimolar amounts of recombinant EZIKV, EDI/IIZIKV and EDIIIZIKV.Results and discussionOur findings demonstrate the induction of IFN-γ producing cells in PBMC from ZIKV-convalescent mothers, whose infants manifested microcephaly, upon stimulation with both inactivated ZIKV particles and recombinant proteins. The identification of immunodominant regions within ZIKV can contribute for the development of targeted treatments and vaccine candidates tailored for pregnant women.

Abstract 7239: Eradication of B cell lymphoma in a mouse model through in vivo transduction of T cells with an aCD19-CAR utilizing a CD3-targeted lentiviral vector

Abstract One of the challenges in CAR-T cell therapies is the need for ex vivo processing of autologous T cells. The generation of CAR-T cells directly within the patient has therefore become an attractive alternative. Here, we pseudotyped lentiviral vectors with a VSV-G protein engineered to de-target the LDL receptor and to bind to CD3 via a displayed CD3-specific single-chain antibody (G-CD3), allowing for targeted recognition of T cells. In ex vivo experiments, we demonstrated that engineered G-CD3-LV specifically recognizes and transduces T cells in PBMC and whole blood without apparent off-target transduction of other cells. Furthermore, unlike G-WT-LV, which necessitates T cell activation for transduction, G-CD3-LV does not require pre-activation or the presence of cytokines for successful T cell transduction. The presence of CD3 antibodies on the surface of G-CD3-LV alone was sufficient to activate T cells, as evidenced by CD25 expression and the induction of T cell proliferation. For in vivo evaluation of retargeted LV efficiency NSG mice implanted with NALM6 B-lymphoma were treated with G-CD3 LV encoding αCD19-CAR/GFP (G-CD3/LV-CAR). The tumor was established by tail vein injection of NALM6/F-Luc cells, and 3 or 10 days later animals were humanized via intraperitoneal (IP) injection of human PBMC. Four hours after PBMC transfer, G-CD3/LV-CAR was delivered by IP route. Blood was drawn every week to monitor the generation of CAR-T cells, cytokine levels, and vector copy numbers. Tumor burden was monitored by bioluminescence imaging. Treatment with G-CD3/LV-CAR at both time points resulted in complete tumor eradication. Subsequently, significant levels of CAR-T cells were observed in the blood through Flow analysis, peaking at two weeks after LV injection. Importantly, mice treated on day 10 after tumor establishment reached higher levels of CAR-T cells compared to those treated on day 3 (CAR-T levels were 38±6.5% vs. 10±2.38% of hCD45+ cells, respectively). These results suggest that CAR-T cells proliferated proportionally in response to the level of tumor antigen. With the eradication of the tumor, CAR-T levels in the blood diminished. We also demonstrated that the levels of CAR-T cells correlated well with vector copy numbers. Throughout the treatment, mice did not exhibit any signs of toxicity or body weight loss. Additionally, by measuring cytokines in the blood produced by human cells, we found only a moderate increase in hIFNγ (0.852±0.681 ng/ml) at the peak of CAR-T cell levels in the blood, while the levels of hIL-2 and hTNFα were below the detection limit. In summary, these data highlight the efficiency and potential applicability of fully retargeted, serum stable LV vectors for in vivo CAR-T generation, offering promising prospects to optimize CAR-T cells therapy applications. Citation Format: Karina Krotova, Gopal Naik Nenavath, Nandakumar Packiriswamy, Rianna Vandergaast, Yumeng McDaniel, Melissa Moy, Zachary Larson, Luke Schnebeck, Chia-Hsuan Chin, Kyle Gromer, Kah-Whye Peng, Stephen J. Russell. Eradication of B cell lymphoma in a mouse model through in vivo transduction of T cells with an aCD19-CAR utilizing a CD3-targeted lentiviral vector [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 7239.

Granulysin-mediated reduction of PDZRN3 induces Cx43 gap junctions activity exacerbating skin damage in trichloroethylene hypersensitivity syndrome

Trichloroethylene (TCE)-induced hypersensitivity syndrome (THS) has been a concern for many researchers in the field of environmental and occupational health. Currently, there is no specific treatment for THS, leaving patients to contend with severe infections arising from extensive skin lesions, consequently leading to serious adverse effects. However, the pathogenesis of severe skin damage in THS remains unclear. This study aims to investigate the specific danger signals and mechanisms underlying skin damage in THS through in vivo and in vitro experiments. We identified that cell supernatant containing 15 kDa granulysin (GNLY), released from activated CD3-CD56+NK cells or CD3+CD56+NKT cells in PBMC induced by TCE or its metabolite, promoted apoptosis in HaCaT cells. The apoptosis level decreased upon neutralization of GNLY in the supernatant by a GNLY-neutralizing antibody in HaCaT cells. Subcutaneous injection of recombinant 15 kDa GNLY exacerbated skin damage in the THS mouse model and better mimicked patients’ disease states. Recombinant 15 kDa GNLY could directly induce cellular communication disorders, inflammation, and apoptosis in HaCaT cells. In addition to its cytotoxic effects, GNLY released from TCE-activated NK cells and NKT cells or synthesized GNLY alone could induce aberrant expression of the E3 ubiquitin ligase PDZRN3, causing dysregulation of the ubiquitination of the cell itself. Consequently, this resulted in the persistent opening of gap junctions composed of connexin43, thereby intensifying cellular inflammation and apoptosis through the “bystander effect”. This study provides experimental evidence elucidating the mechanisms of THS skin damage and offers a novel theoretical foundation for the development of effective therapies targeting severe dermatitis induced by chemicals or drugs.

Immunological Landscapes in Lung Transplantation: Insights from T Cell Profiling in BAL and PBMC.

Lung transplant recipients frequently encounter immune-related complications, including chronic lung allograft dysfunction (CLAD). Monitoring immune cells within the lung microenvironment is pivotal for optimizing post-transplant outcomes. This study examined the proportion of T cell subsets in paired bronchoalveolar lavage (BAL) and peripheral PBMC comparing healthy (n = 4) and lung transplantation patients (n = 6, no CLAD and n = 14 CLAD) using 14-color flow cytometry. CD4+ T cell proportions were reduced in CD3 cells in both PBMC and BAL, and positive correlations were discerned between T cell populations in peripheral PBMC and BAL, suggesting the prospect of employing less invasive PBMC sampling as a means of monitoring lung T cells. Furthermore, regulatory T cells (Tregs) were enriched in BAL when compared to peripheral PBMC for transplant recipients. A parallel positive correlation emerged between Treg proportions in BAL and peripheral PBMC, underscoring potential avenues for monitoring lung Tregs. Finally, the most promising biomarker was the Teff (CD8+Granzyme B+)-Treg ratio, which was higher in both the PBMC and BAL of transplant recipients compared to healthy individuals, and increased in the patients with CLAD compared to no CLAD and healthy patients. Conclusions: Distinct T cell profiles in BAL and peripheral PBMC underscore the significance of localized immune monitoring in lung transplantation. The Teff (CD8+granzyme B+)-Treg ratio, particularly within the context of CLAD, emerges as a promising blood and BAL biomarker reflective of inflammation and transplant-related complications. These findings emphasize the imperative need for personalized immune monitoring strategies that tailored to address the unique immunological milieu in post-transplant lungs.

Toll-like Receptor 2 Mediated Immune Regulation in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

Toll-like receptors (TLRs) are crucial to the innate immune response. They regulate inflammatory reactions by initiating the production of pro-inflammatory cytokines and chemokines. TLRs also play a role in shaping the adaptive immune responses. While this protective response is important for eliminating infectious pathogens, persistent activation of TLRs may result in chronic immune activation, leading to detrimental effects. The role of TLR2 in regulating HIV-1 infection in vivo has yet to be well described. In this study, we used an SIV-infected rhesus macaque model to simulate HIV infection in humans. We evaluated the plasma of the macaques longitudinally and found a significant increase in the soluble TLR2 (sTLR2) level after SIV infection. We also observed an increase in membrane-bound TLR2 (mb-TLR2) in cytotoxic T cells, B cells, and NK cells in PBMC and NK cells in the gut after infection. Our results suggest that sTLR2 regulates the production of various cytokines and chemokines, including IL-18, IL-1RA, IL-15, IL-13, IL-9, TPO, FLT3L, and IL-17F, as well as chemokines, including IP-10, MCP-1, MCP-2, ENA-78, GRO-α, I-TAC, Fractalkine, SDF-1α, and MIP-3α. Interestingly, these cytokines and chemokines were also upregulated after the infection. The positive correlation between SIV copy number and sTLR2 in the plasma indicated the involvement of TLR2 in the regulation of viral replication. These cytokines and chemokines could directly or indirectly regulate viral replication through the TLR2 signaling pathways. When we stimulated PBMC with the TLR2 agonist in vitro, we observed a direct induction of various cytokines and chemokines. Some of these cytokines and chemokines, such as IL-1RA, IL-9, IL-15, GRO-α, and ENA-78, were positively correlated with sTLR2 in vivo, highlighting the direct involvement of TLR2 in the regulation of the production of these factors. Our findings suggest that TLR2 expression may be a target for developing new therapeutic strategies to combat HIV infection.

Humoral and cellular immunity in three different types of COVID-19 vaccines against SARS-CoV-2 variants in a real-world data analysis.

An effective vaccine response is currently a critical issue in the control of COVID-19. Little is known about humoral and cellular immunity comparing protein-based vaccine with other types of vaccines. The relevance of basal immunity to antibody production is also unknown. Seventy-eight individuals were enrolled in the study. The primary outcome were the level of spike-specific antibodies and neutralizing antibodies measured by ELISA. Secondary measures included memory T cells and basal immunity estimated by flow cytometry and ELISA. Correlations for all parameters were calculated using the nonparametric Spearman correlation method. We observed that two doses of mRNA-based Moderna mRNA-1273 (Moderna) vaccine produced the highest total spike-binding antibody and neutralizing ability against the wild-type (WT), Delta, and Omicron variants. The protein-based MVC-COV1901 (MVC) vaccine developed in Taiwan produced higher spike-binding antibodies against Delta and Omicron variants and neutralizing ability against the WT strain than the adenovirus-based AstraZeneca-Oxford AZD1222 (AZ) vaccine. Moderna and AZ vaccination produced more central memory T cells in PBMC than the MVC vaccine. However, the MVC vaccine had the lowest adverse effects compared to the Moderna and AZ vaccines. Surprisingly, the basal immunity represented by TNF-α, IFN-γ, and IL-2 prior to vaccination was negatively correlated with the production of spike-binding antibodies and neutralizing ability. This study compared memory T cells, total spike-binding antibody levels, and neutralizing capacity against WT, Delta, and Omicron variants between the MVC vaccine and the widely used Moderna and AZ vaccines, which provides valuable information for future vaccine development strategies.

Abstract 5679: Tetra-specific antibody GNC-035: guidance and navigation control (GNC) molecule development for treatment of ROR1+ malignancies

Abstract Cancer-intrinsic immune escape mechanisms and immune cell suppression can progressively diminish the curative potential of currently available T cell-based therapies. Barriers to successful T cell checkpoint therapies may be addressed by redirection of T cells toward tumor antigens using T cell engagers that function independently of MHC presented T cell epitopes. Here we demonstrate that an octavalent, tetraspecific Guidance and Navigation Control (GNC) antibody, GNC-035, binds to ROR1, CD3, PD-L1, and 4-1BB and mediates redirected T cell cytolysis of human solid tumor and leukemia and lymphoma cell lines in a ROR1 specific manner. Experiments using GNC-035 to redirect T cell cytotoxicity toward ROR1+ cancer cell targets show the T cells in PBMC are highly functionalized by pre-exposure to GNC-035. This pre-exposure of PBMC to GNC-035 results in greater tumor cell killing efficacy compared to concurrent exposure of tumor cells in the presence of T cell effectors. This result suggests that the systemic delivery of GNC-035 can condition the T cell compartment to increase the therapeutic impact of T cells migrating to solid tumors, with or without preexisting infiltrating T cells. This beneficial conditioning of T cells by pre-exposure to GNC-035 is not observed with pre-exposure to CD3xROR1 bi-specific T cell engager controls. To evaluate the potential for GNC-035 to mediate cytokine release syndrome, the molecule is evaluated in soluble formats in the presence of PBMC and the ROR1+ A549 cancer cells, or HUVEC cells. Under these conditions, the cytolysis of A549 target cells is detectable after exposure to GNC-035 at 100 fM concentrations as well as the release of IFN-γ and certain other inflammatory cytokines at 24 or 48 hours post-treatment. However, consistent with Blinatumomab treatment, PBMC exposed to soluble GNC-035 for 24 or 48 hours on a monolayer of HUVEC cells, produced significantly greater amounts of IFN-γ and IL-6 at concentrations greater than 10 pM. These results indicate GNC-035 has a therapeutic window of activity that is ROR1 dependent, spanning cytolytic activity, and IFN-γ release without a production of IL-6 and which is wider than that indicated by Blinatumomab in PBMC. Collectively, the GNC-035 represents a class of multi-specific and multi-modal immune cell engagers with potential to mediate ROR1+ cancer regression, overcome TCR-based immune escape and reverse T cell immune suppression in tumor microenvironment. The clinical phase I-b study of GNC-035 is under way in breast cancer and hematologic cancers and the available data exhibit strong signals of efficacy with acceptable tolerability. Citation Format: Jahan Salar Khalili, Sa Xiao, Yi Zhu. Tetra-specific antibody GNC-035: guidance and navigation control (GNC) molecule development for treatment of ROR1+ malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5679.

Abstract 5681: Tetra-specific antibody GNC-038: guidance and navigation gontrol (GNC) molecule development for treatment of CD19+ malignancies

Abstract B cell malignancies treated with CD19-directed immunotherapies can relapse, in some cases due to clonal selection for reduced CD19 antigen expression or enhancement of immunosuppressive phenotypes. Here we demonstrate that a Guidance and Navigation Control (GNC) tetra-specific antibody, GNC-038, binds to CD19, CD3, PD-L1, and 4-1BB and mediates cytolysis of human leukemia and lymphoma cells by T cells. Redirected T cell cytolysis (RTCC) occurs in the presence of GNC-038 (Emfizatamab), resulting in the killing of CD19+ leukemia and lymphoma cell lines. The cytolytic functions induced by GNC-038 are similar to Blinatumomab in vitro, as indicated by T cell degranulation and production of IFN-gamma. Human T cells in PBMC exposed to GNC-038 in vitro proliferate in a dose-dependent fashion. Proliferation is further enhanced upon rechallenge with leukemic target cells. Proliferation of T cells from individuals with higher % PD-1+ and Effector polarized compartments is enhanced by GNC-038 compared to Blinatumomab. To evaluate the contribution of each binding domain of GNC-038 in mediating RTCC function, versions of GNC-038 were prepared, replacing each antigen binding domain with anti-FITC binding domains. Under assay conditions using PBMC for RTCC toward the CD19+ target cell line Nalm-6, the results demonstrate the contribution of each domain to the overall, anti-leukemic cytolytic activity. To evaluate the potential for GNC-038 to mediate cytokine release syndrome, the molecule is evaluated in soluble, and plate bound formats in the presence of PBMC and CD19+ leukemic targets cells. In comparison to Blinatumomab, the production of cytokines is comparable, with some notable differences. PBMC exposed to soluble GNC-038 for 48 hours produced more IFN-γ, IL-2 and TNF-α, while showing no significant difference in production of IL-6. Based on these results, the primary indicator of CRS, IL-6, does not suggest increased risk compared to Blinatumomab, while the type of T cell activity induced by GNC-038 in PBMC with leukemia cells is distinct. Collectively, the GNC-038 represents a class of multi-specific and multi-modal immune cell engagers with potential to mediate CD19+ cancer killing, while also increasing the T cell compartment’s therapeutic potential to respond to T cell redirection upon subsequent cycles of therapy. The clinical phase I-b study of GNC-038 is under way and the available data exhibit strong signals of efficacy with acceptable tolerability. Citation Format: Jahan Salar Khalili, Sa Xiao, Yi Zhu. Tetra-specific antibody GNC-038: guidance and navigation gontrol (GNC) molecule development for treatment of CD19+ malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5681.

Abstract 5671: MB201, a CD8+ T cell-selective anti-PD-L1 x IL-2 variant fusion protein, exhibits a potent anti-tumor efficacy without peripheral toxicity

Abstract Interleukin-2 (IL-2), a cytokine with pleiotropic immune effects, is the first approved cancer immunotherapy. However, the clinical application of IL-2 is limited by systemic activation of unexpected immune cells, leading to severe toxicities including vascular leakage syndrome (VLS). These adverse effects were mainly caused by the activation of Treg and endothelial cells bearing IL-2 receptor (IL-2R) αβγ. Recently, studies have mainly focused on the optimization of IL-2Rs binding affinity to selectively invigorate CD8+ T cells in tumors, but the toxicity risk has yet to be solved. To overcome these limitations, we developed MB201, a novel anti-PD-L1 x IL-2v fusion protein, which selectively activates CD8+ T cells by attenuating binding affinity to IL-2Rβγ with no binding to IL-2Rα. MB201 showed a strong binding affinity to PD-L1 (KD value is 0.5 nM) and a weak binding affinity to IL-2Rβγ and IL-2Rαβγ (KD value is 23.8 nM and 30.6 nM, respectively), indicating the preferential targeting PD-L1+ tumor cells. MB201 exhibited a preferential activation of CD8+ T cells than Treg cells, which showed remarkably reduced activation compared to rhIL-2 (more than 100-fold based on EC50). Consistently with the result of T cell activation, MB201 only increased the expansion of CD8+ T cells rather than Treg cells in CD3-stimulated PBMC. Indeed, MB201 induced negligible expansion of immune cells and lower pro-inflammatory cytokine release compared to the rhIL-2 in CD3-unstimulated PBMC, indicating the reduced peripheral toxicity risk. When administered to MC38 syngeneic mice, MB201 showed superior anti-tumor efficacy without both body weight reduction and lung weight gain compared to the avelumab monotherapy or combination therapy with rhIL-2. These preclinical observations support that MB201 has characteristics of a superb safety profile and excellent anti-tumor efficacy, and it may offer an advantageous therapeutic strategy for the treatment of cancer. Citation Format: Bom Park, Hyojoo Bang, Youngjun Jung, Sunjung Cho, Mi Seong Kim, Seok Chan Kang, Sungyoub Jung, Youngjin Park. MB201, a CD8+ T cell-selective anti-PD-L1 x IL-2 variant fusion protein, exhibits a potent anti-tumor efficacy without peripheral toxicity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5671.

Abstract P1-12-08: Biomarker analysis of hepatotoxicity in a Phase II study of nivolumab, abemaciclib and endocrine therapy in patients with HR-positive, HER2-negative breast cancer: WJOG11418BTR NEWFLAME_TR

Abstract Background Previously, we reported the clinical outcomes of the combination of anti-PD-1 Ab, cyclin-dependent kinase 4/6 inhibitors, and endocrine therapy (ET) in patients with ER positive/HER2 negative advanced breast cancer in SABCS2020; biomarker analysis has been performed to provide insight into the hepatotoxicy frequently observed in the study. Methods Subjects received 240 mg nivolumab IV on days 1 and 15, 150 mg abemaciclib PO twice daily, and either 500 mg fulvestrant (FUL) on days 1, 15, 29, and every 4 weeks thereafter (FUL cohort) or 2.5 mg letrozole (LET) once daily (LET cohort). The primary endpoint was objective response rate and secondary endpoints included toxicity evaluated in the CTCAE along with an exploratory endpoint as related to the biomarker analysis. Archival tumor tissues were collected before study entry and blood and stool samples were collected at baseline and on cycle3 day1. Tumor tissues were subjected to IHC analysis and RNA sequencing followed by subtyping using NGS. High throughput cytokine analysis using ELISA-based assay were performed with serum samples and cell sorting analysis of PBMC was performed with FACS. Results From June 2019 to December 2019, 17 subjects were enrolled (FUL cohort [n = 12], LET cohort [n = 5]). The study was prematurely closed due to safety concerns such as hepatotoxicity and interstitial lung disease. AEs ≥ Grade 3 were observed in 91.7% and 100% of patients in the FUL and LET cohorts, respectively. The most frequent AEs ≥ Grade 3 were elevated liver function tests (LFT; FUL cohort: 50.0%, LET cohort: 60.0%). Serum cytokine analysis from the subjects with severe hepatotoxicity indicated cytokine storm with elevations of sCD30/TNFRSF8, IL-11, -34, Pentraxin-3, sTNF-R1, -R2, TSLP, which was supported by the findings of reduction of effector regulatory T cells in PBMC. IHC study in liver biopsy from three subjects with the toxicity revealed infiltration of CD8+ T cells and FOXP3+ T reg into the liver, suggesting the immune related liver injury upon the treatment with nivolumab and abemaciclib. HLA typing was performed in the 17 patients but no association between HLA type and ILD or hepatotoxicity were observed. Conclusions The frequent and severe immune related hepatotoxicity induced by the combination of anti-DD-1 and CDK 4/6 inhibitors might have been an immune-boosting therapy as suggested in the preclinical studies. This study was supported by the Ono Pharmaceutical Co., LTD. The registration number of the study is UMIN000036970. Citation Format: Junji Tsurutani, Jun Masuda, Norikazu Masuda, Yuko Tanabe, Tsutomu Iwasa, Masato Takahashi, Manabu Futamura, Koji Matsumoto, Kenjiro Aogi, Hiroji Iwata, Mari Hosonaga, Toru Mukohara, Kiyoshi Yoshimura, Chiyo K. Imamura, Sakiko Miura, Toshiko Yamochi, Kenichi Yoshimura, Toshimi Takano, Hidetaka Kawabata. Biomarker analysis of hepatotoxicity in a Phase II study of nivolumab, abemaciclib and endocrine therapy in patients with HR-positive, HER2-negative breast cancer: WJOG11418BTR NEWFLAME_TR [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-12-08.

Live attenuated influenza A virus vaccine expressing an IgA-inducing protein protects pigs against replication and transmission

IntroductionThe rapid evolution of influenza A viruses (FLUAV) complicates disease control for animal and public health. Although vaccination is an effective way to control influenza, available vaccines for use in swine result in limited protection against the antigenically distinct FLUAV that currently co-circulate in pigs. Vaccines administered parenterally usually stimulate IgG antibodies but not strong mucosal IgA or cell-mediated responses, which are typically more cross-reactive.MethodsWe developed a live attenuated influenza virus (LAIV) vaccine containing IgA-inducing protein (IGIP) as a molecular marker and immunomodulator. This Flu-IGIP vaccine was tested in a bivalent formulation (H1N1 and H3N2) against challenge with antigenically drifted viruses in pigs. Pigs were vaccinated intranasally with either a bivalent Flu-IGIP or a bivalent Flu-att (control without IGIP) and boosted two weeks later. Three weeks post boost, pigs were challenged with antigenically drifted H1N1 or H3N2 virus.ResultsVaccinated pigs had increased numbers of influenza-specific IgA-secreting cells in PBMC two weeks post boost and higher numbers of total and influenza-specific IgA-secreting cells in bronchoalveolar lavage fluid (BALF) 5 days post inoculation (dpi) compared to naïve pigs. Pigs vaccinated with both Flu-IGIP and Flu-att shed significantly less virus after H1N1 or H3N2 challenge compared to non-vaccinated pigs. Vaccination with Flu-att reduced respiratory transmission, while Flu-IGIP fully blocked transmission regardless of challenge virus. Both Flu-IGIP and Flu-att vaccines reduced virus replication in the lungs and lung lesions after inoculation with either virus. IgG and IgA levels in BALF and nasal wash of vaccinated pigs were boosted after inoculation as soon as 5 dpi and remained high at 14 dpi.ConclusionOur results indicate that Flu-IGIP leads to protection from clinical signs, replication and shedding after antigenically drifted influenza virus infection.

MAIT cells predict long-term prognosis in liver failure patients.

Background:Liver failure (LF) is a life-threatening clinical syndrome characterized by intense systemic inflammation and organ failure(s), leading to a high mortality rate. The pathogenesis of LF is multifactorial, immune response, and gut bacterial translocation are thought to be major contributing factors. Mucosal-associated invariant T (MAIT) cells play a critical role in immune response and gut bacterial translocation. We aimed to investigate changes of the MAIT cell ratio in patients with LF and to explore the predictive value for long-term prognosis in patients with LF.Material and Method:We recruited 75 patients with LF from Nantong Third People’s Hospital, isolated peripheral blood mononuclear cells, and detected the proportion of circulating MAIT cells by flow cytometry. Statistical analyses were performed using the GraphPad Prism software.Results:Our data showed that the proportion of MAIT cells alterations was independent of the cause of viral infection in patients with LF. Kaplan-Meier survival analysis showed that LF patients with low level of MAIT cells had poor long-term prognosis. The area under the receiver operating characteristic curve of the MAIT cell proportion was larger than that of the Model for End-Stage Liver Disease (MELD) score. More importantly, the combination of MAIT cell proportion and MELD score had a better effect in predicting long-term prognosis of LF patients than any single index (AUC = 0.91, 95% CI:0.84–0.97), and multivariate logistic regression analysis indicated that the circulating MAIT cell proportion was an independent risk factor for LF.Conclusion:The proportion of MAIT cells in PBMC is an outstanding predictor for the long-term prognosis in patients with LF.

Abstract 4232: TLR2 agonist as a novel therapeutic approach to treat pancreatic adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with an overall 5-year survival rate of <10% due to late stage diagnosis and poor clinical outcome with existing therapies. PDAC displays a high degree of tumor heterogeneity and immunosuppressive tumor microenvironment with lower tumor infiltrating lymphocytes (TIL). These factors contribute to resistance against chemotherapies and immunotherapies. Immunotherapies have shown activities across different tumor types including chemotherapy or targeted therapy resistant cancers however these options have little efficacy in the PDAC tumor. Here, in this study we tested the efficacy of gemcitabine in combination with Cadi-05 (TLR2 agonist) against established murine PDAC tumor models (Pan02 in C57/BL6 and 688M in B6129SF1/J). Animals were randomized to receive gemcitabine, Cadi-05, their combination or no treatment when they had tumor size ≈ 50 mm3, 65mm3 and 100 mm3. The weekly treatment of gemcitabine, Cadi-05 and their combination inhibited the tumor progression across all tumor sizes. At the end of study, PBMC and tumors were collected and analyzed for different immune parameters. Results revealed that Cadi-05 treatment alone was associated with increase in CD4+ and CD8+ T cells in PBMC as well as amongst TIL with significant decrease in their expression of immunosuppressive markers like FOXP3 and PD-1. Its combination with gemcitabine synergized the effect of each other. There was further increase in absolute TIL as well as TIL expressing CD4+ and CD8+ markers with decrease in FOXP3 and PD-1 expressing CD4+ and CD8+ TIL. Inhibition of tumor progression was associated with improved effector function of PBMC. Synergy between Cadi-05 and gemcitabine was also evident in effector function. In presence of established tumor, TLR2 agonist Cadi-05 improves effector function, increases CD4+ and CD8+ expressing TIL, decreases immune suppressive TIL and retards tumor growth. Our study suggests that combination of gemcitabine and Cadi-05 inhibits the tumor growth irrespective of its size and it is coupled with increased anti-tumor response and decreased immunosuppressive function Citation Format: Arpit Dheeraj, Chandreshwar Shukla, Dhanir Tailor, Nayan K. Jain, Kirsten Stefan, Chintan D. Patel, Rajiv Modi, Bakulesh M. Khamar, Sanjay V. Malhotra. TLR2 agonist as a novel therapeutic approach to treat pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4232.

PD12-04 SURVEY OF B AND PLASMA CELLS IN BLADDER CANCER ACROSS TUMOR MICROENVIRONMENTS

You have accessJournal of UrologyCME1 May 2022PD12-04 SURVEY OF B AND PLASMA CELLS IN BLADDER CANCER ACROSS TUMOR MICROENVIRONMENTS Yuanshuo Wang, Jorge Daza, Emilie Grasset, Giuliana Magri, Daniel Ranti, Kristin Beaumont, Adam Farkas, Michelle Tran, Li Wang, Rachel Brody, Reza Mehrazin, Peter Wiklund, Robert Sebra, Jun Zhu, Matthew Galsky, Nina Bhardwaj, John Sfakianos, and Amir Horowitz Yuanshuo WangYuanshuo Wang More articles by this author , Jorge DazaJorge Daza More articles by this author , Emilie GrassetEmilie Grasset More articles by this author , Giuliana MagriGiuliana Magri More articles by this author , Daniel RantiDaniel Ranti More articles by this author , Kristin BeaumontKristin Beaumont More articles by this author , Adam FarkasAdam Farkas More articles by this author , Michelle TranMichelle Tran More articles by this author , Li WangLi Wang More articles by this author , Rachel BrodyRachel Brody More articles by this author , Reza MehrazinReza Mehrazin More articles by this author , Peter WiklundPeter Wiklund More articles by this author , Robert SebraRobert Sebra More articles by this author , Jun ZhuJun Zhu More articles by this author , Matthew GalskyMatthew Galsky More articles by this author , Nina BhardwajNina Bhardwaj More articles by this author , John SfakianosJohn Sfakianos More articles by this author , and Amir HorowitzAmir Horowitz More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002538.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Tumor infiltrating B cells in bladder cancer (BCa) have been associated with cancer invasion, but little is understood regarding their roles in different tumor microenvironments. In this study, we analyzed data from a large single cell (SC) RNA sequencing cohort of tumor (n=18) and PBMC (n=16) specimens from 26 BCa patients and PBMC samples from healthy donors (n=3) to profile B cells and plasma cells (PCs) in relation to BCa sample types. METHODS: SC data was imported into R (v4.03) using Seurat (v4.0.1). Genes expressed in fewer than 3 cells, cells with <200 or >2500 unique genes, and cells containing >15% mitochondrial genes were discarded. Data normalization, variable gene identification, integration, and scaling were performed. Known B cell and PC markers (MS4A1, IGHD, IGHM, IGHG1/2/3/4, IGHA1/2, MZB1, XBP1) were used to identify B cell and PC clusters after dimensionality reduction and graph-based clustering. RESULTS: We identified 15,008 naïve and memory B cells and PCs by their marker gene expression (Fig. 1A). Profiling these populations across sample types, we found a proportional decrease in memory B cells in BCa tumor and PBMC compared to healthy PBMCs (Fig. 1B). The high percentage of naïve B cells in patient PBMC suggest a possible stall in maturation to memory B cells, while the higher proportion of PCs in tumor suggest a possible competing maturation pathway between PCs and memory B cells from germinal center (GC) B cells. In addition, we observed differences in differentially expressed genes (DEGs) across sample types. Both memory B cells and PCs from BCa PBMC had significantly upregulated expression of ribosomal genes compared to healthy PBMC, and memory B cells and PCs also showed significantly higher expression of IGHG1/2/3/4 and IGHA1 in tumor relative to BCa PBMC (Fig. 1C-D). CONCLUSIONS: In our study of a large BCa SC cohort, we observed significant proportional differences in B cell and PCs across BCa tumors, PBMCs, and healthy PBMCs that could possibly be attributed to a stall in maturation from naïve to memory B cell in the BCa blood or competing maturation pathways between PCs and memory B cells from GC B cells in BCa tumor. We also observed significant increases in genes supporting upregulation of immunoglobulin production both in tumor and in BCa blood relative to BCa PBMCs and healthy PBMCs, respectively. Source of Funding: Institutional funds © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e196 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yuanshuo Wang More articles by this author Jorge Daza More articles by this author Emilie Grasset More articles by this author Giuliana Magri More articles by this author Daniel Ranti More articles by this author Kristin Beaumont More articles by this author Adam Farkas More articles by this author Michelle Tran More articles by this author Li Wang More articles by this author Rachel Brody More articles by this author Reza Mehrazin More articles by this author Peter Wiklund More articles by this author Robert Sebra More articles by this author Jun Zhu More articles by this author Matthew Galsky More articles by this author Nina Bhardwaj More articles by this author John Sfakianos More articles by this author Amir Horowitz More articles by this author Expand All Advertisement PDF DownloadLoading ...

IL-11 induces monocyte inflammasome activation and migration to the CNS in IL-11R+ cells in relapsing remitting multiple sclerosis (RRMS)

Abstract Objective To examine the effect of IL-11 on the transcriptome changes in the blood and cerebrospinal fluid (CSF)-derived cells from RRMS patients. Methods IL-11+ expressing cells in PBMC or CSF were characterized by flow cytometry (n=14 and 9 respectively). Single cell RNA sequencing (scRNAseq) was performed for IL-11R+ sorted peripheral blood-derived cells following IL-11 stimulation (1 hour) (n=3 RRMS patients) or CSF and blood samples (n=2 RRMS patients). The animal model of RRMS (RREAE) was used to test the therapeutic effect of anti-IL11 mAb. Results RRMS patients have an increased frequency of IL-11+ monocytes, IL-11+ and IL-11R+ CD4+ lymphocytes and IL-11R+ neutrophils in comparison to matched HCs. IL-11 expressing monocytes, CD4+ lymphocytes and neutrophils accumulate in the CSF of RRMS patients in comparison to paired blood samples. scRNAseq for IL-11R+ sorted cells following IL-11 stimulation, revealed the highest number of differentially expressed genes (DEGs) in classical monocytes with upregulation of NFKB1, NLRP3 and IL1B genes involved in the inflammasome activation. All CD4+ cell subsets had an increased expression of S100A8/9 genes involved in the inflammasome activation. ScRNAseq for IL-11R+-sorted cells from the CSF revealed that classical and intermediate monocytes upregulate multiple complement, inflammasome-induced (IL-18), and migratory genes. aIL-11 mAb in RREAE decreased clinical scores, CNS inflammatory infiltrates, demyelination, IL-11+ CD4+ cells and monocytes, and numbers of NFkBp65+, NLRP3+ and IL-1b+ monocytes within the CNS. Conclusion IL-11/IL-11R signaling in monocytes represents a therapeutic target in early RRMS. The study was supported by NIH 1R01AI131238-01A1 and PA Cure SAP4100083100 grant.

Identifying Function Determining Residues in Neuroimmune Semaphorin 4A.

Semaphorin 4A (Sema4A) exerts a stabilizing effect on human Treg cells in PBMC and CD4+ T cell cultures by engaging Plexin B1. Sema4A deficient mice display enhanced allergic airway inflammation accompanied by fewer Treg cells, while Sema4D deficient mice displayed reduced inflammation and increased Treg cell numbers even though both Sema4 subfamily members engage Plexin B1. The main objectives of this study were: 1. To compare the in vitro effects of Sema4A and Sema4D proteins on human Treg cells; and 2. To identify function-determining residues in Sema4A critical for binding to Plexin B1 based on Sema4D homology modeling. We report here that Sema4A and Sema4D display opposite effects on human Treg cells in in vitro PBMC cultures; Sema4D inhibited the CD4+CD25+Foxp3+ cell numbers and CD25/Foxp3 expression. Sema4A and Sema4D competitively bind to Plexin B1 in vitro and hence may be doing so in vivo as well. Bayesian Partitioning with Pattern Selection (BPPS) partitioned 4505 Sema domains from diverse organisms into subgroups based on distinguishing sequence patterns that are likely responsible for functional differences. BPPS groups Sema3 and Sema4 into one family and further separates Sema4A and Sema4D into distinct subfamilies. Residues distinctive of the Sema3,4 family and of Sema4A (and by homology of Sema4D) tend to cluster around the Plexin B1 binding site. This suggests that the residues both common to and distinctive of Sema4A and Sema4D may mediate binding to Plexin B1, with subfamily residues mediating functional specificity. We mutated the Sema4A-specific residues M198 and F223 to alanine; notably, F223 in Sema4A corresponds to alanine in Sema4D. Mutant proteins were assayed for Plexin B1-binding and Treg stimulation activities. The F223A mutant was unable to stimulate Treg stability in in vitro PBMC cultures despite binding Plexin B1 with an affinity similar to the WT protein. This research is a first step in generating potent mutant Sema4A molecules with stimulatory function for Treg cells with a view to designing immunotherapeutics for asthma.

Antibodies that react with bovine T lymphocytes expressing the T cell receptor β chain subgroup BV20 inhibit antigen recognition

In recent years, molecular studies have provided detailed information on the bovine T cell receptor (TCR) variable gene repertoire, both in resting T cells and during T cell responses. However, studies of the biological function of the receptor have been hampered by a lack of reagents that recognise the protein. Herein, we describe the characterisation of two antibodies (IL-A47 and IL-A98) that recognise T cells expressing the TCR VB20 subfamily of BV genes. These antibodies each recognise a small subset of αβ T cells in PBMC, including subsets of both CD4 and CD8 T cells. One of the antibodies (IL-A98) recognises a smaller subset of cells within the IL-A47+ population. When tested on a panel of T cell clones expressing different αβ TCR subfamilies of β chain genes, IL-A47 was found to react only with clones expressing the BV20 subfamily, which in cattle has undergone expansion due to gene duplication; IL-A98 reacted with a subset of the BV20 subfamily members. IL-A47 was shown to profoundly inhibit recognition of target cells by cytotoxic T cell clones, an effect that was mediated via the effector T cell rather than the target cells.

CD4+ and CD8+CD60+ T cells in PBMC of IgE+ allergic adults express IL-4 or IFN alpha but not IFN gamma, and some express receptors for complement split products (CSP) C3a and C5a

We previously reported that two distinct T cell subsets (CD4+ and CD8+CD60+) and IL-4, IFN alpha, and IFN gamma, but not IL-13, were required for human ragweed specific memory IgE responses. Nevertheless, the exact subsets producing these cytokines and their numbers are unknown, as are the numbers of C3a+ or C5a+ receptor T cells expressing IL-4.