CTL Cells Research Articles

Designing a multi-epitope influenza vaccine: an immunoinformatics approach

Influenza continues to be one of the top public health problems since it creates annual epidemics and can start a worldwide pandemic. The virus’s rapid evolution allows the virus to evade the host defense, and then seasonal vaccines need to be reformulated nearly annually. However, it takes almost half a year for the influenza vaccine to become accessible. This delay is especially concerning in the event of a pandemic breakout. By producing the vaccine through reverse vaccinology and phage display vaccines, this time can be reduced. In this study, epitopes of B lymphocytes, cytotoxic T lymphocytes, and helper T lymphocytes of HA, NA, NP, and M2 proteins from two strains of Influenza A were anticipated. We found two proper epitopes (ASFIYNGRL and LHLILWITDRLFFKC) in Influenza virus proteins for CTL and HTL cells, respectively. Optimal epitopes and linkers in silico were cloned into the N-terminal end of M13 protein III (pIII) to create a multi-epitope-pIII construct, i.e., phage display vaccine. Also, prediction of tertiary structure, molecular docking, molecular dynamics simulation, and immune simulation were performed and showed that the designed multi-epitope vaccine can bind to the receptors and stimulate the immune system response.

IL-21- and CXCL9-engineered GPC3-specific CAR-T cells combined with PD-1 blockade enhance cytotoxic activities against hepatocellular carcinoma

The application of CAR-T cells in solid tumors poses several challenges, including poor T cell homing ability, limited infiltration of T cells and an immunosuppressive tumor environment. In this study, we developed a novel approach to address these obstacles by designing GPC3-specific CAR-T cell that co-express IL-21 and CXCL9 (21 × 9 GPC3 CAR-T cells) and blocking the PD-1 expression on it. The proliferation, cell phenotype, cytokine secretion and cell migration of indicated CAR-T cells were evaluated in vitro. The cytotoxic activities of genetically engineered CAR-T cells were accessed in vitro and in vivo. Compared to conventional GPC3 CAR-T cells, the 21 × 9 GPC3 CAR-T cells demonstrated superior proliferation, cytokine secretion and chemotaxis capabilities in vitro. Furthermore, when combined with PD-1 blockade, the 21 × 9 GPC3 CAR-T cells exhibited enhanced proliferation, cytokine secretion and enrichment of effector T cells such as CTL, NKT and TEM cells. In xenograft tumor models, the PD-1 blocked 21 × 9 GPC3 CAR-T cells effectively suppressed HCC xenograft growth and increased T cell infiltration. Overall, our study successfully generated GPC3 CAR-T cells expressing both IL-21 and CXCL9, demonstrated that combining PD-1 blockade can further enhance CAR-T cell function by promoting proliferation, cytokine secretion, chemotaxis and antitumor activity. These findings present a hopeful and potentially effective strategy for GPC3-positive HCC patients.

Machine Learning-Directed Conversion of Glioblastoma Cells to Dendritic Cell-Like Antigen-Presenting Cells as Cancer Immunotherapy.

Immunotherapy has limited efficacy in glioblastoma (GBM) due to the blood-brain barrier and the immunosuppressed or "cold" tumor microenvironment (TME) of GBM, which is dominated by immune-inhibitory cells and depleted of CTL and dendritic cells (DC). Here, we report the development and application of a machine learning precision method to identify cell fate determinants (CFD) that specifically reprogram GBM cells into induced antigen-presenting cells with DC-like functions (iDC-APC). In murine GBM models, iDC-APCs acquired DC-like morphology, regulatory gene expression profile, and functions comparable to natural DCs. Among these acquired functions were phagocytosis, direct presentation of endogenous antigens, and cross-presentation of exogenous antigens. The latter endowed the iDC-APCs with the ability to prime naïve CD8+ CTLs, a hallmark DC function critical for antitumor immunity. Intratumor iDC-APCs reduced tumor growth and improved survival only in immunocompetent animals, which coincided with extensive infiltration of CD4+ T cells and activated CD8+ CTLs in the TME. The reactivated TME synergized with an intratumor soluble PD1 decoy immunotherapy and a DC-based GBM vaccine, resulting in robust killing of highly resistant GBM cells by tumor-specific CD8+ CTLs and significantly extended survival. Lastly, we defined a unique CFD combination specifically for the human GBM to iDC-APC conversion of both glioma stem-like cells and non-stem-like cell GBM cells, confirming the clinical utility of a computationally directed, tumor-specific conversion immunotherapy for GBM and potentially other solid tumors.

Exploratory phase I study of HF1K16 for the treatment of patients with refractory/recurrent advanced glioma: Preliminary efficacy and mechanism as a monotherapy.

e14030 Background: The absence of I.O success in advanced glioma treatment suggests that a deeper understanding of the brain tumor and the immune microenvironment is critical. It is well-documented that refractory/recurrent glioma has an immunosuppressive nature. One frequent observation is the high level of circulating myeloid-derived suppressor cells (MDSCs). We conducted a exploratory study to assess an immunomodulatory approach for relieving immune suppression employing HF1K16, a liposome all-trans retinoic acid (ATRA) suspension. Methods: This Phase I study (NCT05388487) comprise a conventional 3+3 dose escalation plus an advanced glioma-specific expansion arm. Eligible patients had prior confirmed advanced solid tumor and failed standard treatment. HF1K16 infusions were administered in 21-day cycles (q.o.d days 1-14). Prior to and during treatment, peripheral blood mononuclear cells were collected and analyzed with flow cytometry to monitor the changes in myeloid cell phenotype and T cell composition. Survival data were calculated as it is difficult to determine the timing of progression for immunotherapies owing to pseudoprogression. Results: As of Feb 2th, 2024, 14 recurrent/refractory glioma patients including 6 males and 8 females had been enrolled in this study. 2 of them withdrew before finished 1st cycle. 7 had received treatment for at least 3 cycles and 1 had achieved CR after 15 cycles. For the 8 primary diagnosed grade IV glioma patients, the mOS is 7.8m, and 6 (75%) are alive. The mOS for the primary dignosed grade II/III is 13.7m and 100% patients are alive (n=4). There is 1 recurrent GBM patent who had received 5 cycles of treatment. MRI showed an enlargement of tumor lesion while KPS score remained >90. A surgical resection was performed and we subsequently studied the posttreatment tumor specimens (window of opportunity). IHC and flow cytometry analysis revealed prominent perivascular and intratumoral inflammatory infiltrate, containing CD8+CTL cells and HLA-DR expressing cells. Conclusions: While the interpretation of our data is limited by the small sample size, the results provide an encouraging signal and warrants further assessment. Patient recruitment continues with anticipated completion in 2024. Clinical trial information: NCT05388487 . [Table: see text]

NFAT5 upregulates osmolyte transporters to protect IMCD3 cells against hyperosmolarity

The kidney inner medulla is one of the only places in the body exposed to extremely high osmolarity, reaching up to 1300 mOsm, while the iso-osmotic cortex and plasma are ≍300m mOsm. Renal epithelial cells need a mechanism to address the stress that comes with hyperosmolarity. One possible mechanism is regulation of osmolyte transporters to increase intracellular osmolyte concentration and reduce osmotic stress. NFAT5 is an important factor in regulating renal epithelial cell responses to high osmolarity. We hypothesize that NFAT5 is protective in inner medullary collecting duct cells (IMCD3) via upregulation of key transporters. To test this hypothesis, we used CRISPR to delete NFAT5 in IMCD3 cells. IMCD3 -NFAT5 null (NFAT5 null) and control (Ctl, IMCD3) cells were used and cell death assessed. Cells were plated at the same concentrations, in media (320mmol) with additional osmolarities of: urea (160mmol), mannitol (160mmol) and salt (40 and 80 mmol). Cell death was measured over 5 days using Janus Green B, which permeates living cell membranes. Cells were incubated with Janus Green B, lysed and then absorbance measured with a spectrophotometer to determine concentration. Data were analyzed as mean ± SEM; cell confluence, two-way ANOVA, n=3-5. Total mRNA was also isolated for qPCR analysis and data expressed as a fold change (DCT) over housekeeping (PPIA). At iso-osmolarity (»300mOsm), no differences were observed between NFAT5 null and Ctl cells. However, by day 3, NFAT5 null cells had reduced cell growth in all hyperosmolar conditions: urea 160 mmol (1.192 ± 0.225 vs. 1.953 ± 0.167 Wt ****p<0.0001, n=4), mannitol 160 mmol (0.852 ± 0.135 vs. 1.683 ± 0.283 Wt ****p<0.0001, n=3-4), NaCl 40 mmol (1.745 ± 0.210 vs. 2.020 ± 0.252 Wt ****p<0.0001, n=4-5), NaCl 80 mmol (0.385 ± 0.059 vs. 1.089 ± 0.191 Wt ****p<0.0001, n=4-5). Interestingly, NFAT5 null cells recovered growth patterns similar to Ctl cells by day 5. This suggests that although NFAT5 is essential in the cellular response hyperosmolarity, other mechanisms are present that allow for adaptation over time. To address a possible mechanism, we performed qPCR investigating common osmo-protective genes. We hypothesized that expression is lower in the NFAT5 null cells as compared to Wt. When cultured with 40 mmol of NaCl (n=2), we found that the NFAT5 null cells showed a 3.31 fold reduction for SMIT1, a 2924.98 fold reduction for TauT, and a 4.91 fold reduction for BGT1 as compared to Wt, each of which are osmolyte transporters. Additionally, we saw a 161.46 fold reduction for AQP1, a water channel and a 4.91 fold reduction for Akr1b3, which influences water homeostasis, as compared to Wt. Together, these results suggest that NFAT5 is a critical regulator of both osmolyte and transporters. Here, we show that loss of NFAT5 reduces IMCD cell viability in the face of hyperosmotic stress, possibly through a reduction in key osmolyte transporters. These data suggest that NFAT5 is key in the renal epithelial cell response to, and survival in, the hyperosmolar environment of the inner medulla. Undergraduate Research Opportunity Program grant NIH NIDDK DK115660-01A1 University of Utah DOIM, UCares. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract LB426: Evidence for a role of GPR35 in IDO1-mediated tumor immune escape by regulating Hippo-YAP pathway

Abstract IDO1 is an immune checkpoint regulator and mediator of tumor immune evasion (1). The cellular and molecular mechanisms of IDO1-mediated immune suppression are not fully understood. We describe here that GPR35, the putative receptor for kynurenic acid (KYNA), a metabolite of IDO1-mediated tryptophan catabolism, couples to Hippo-YAP pathway. GPR35 activation, either by overexpression or by treatment with tryptophan metabolites resulted in Hippo inactivation and YAP-directed transcription via TEAD family of transcription factors. Recently, YAP is found to be essential for tumor immune escape. Yap1 deficiency in T-lineage-specific knockout mice, or by dosing a YAP inhibitor, impairs Treg formation and function, leading to tumor growth inhibition (2). YAP also plays an inhibitory role in CD8 T cell function, especially in activated CTL usually found in the TME [3,4]. Through medicinal chemistry design and a structure-vs-activity relationship study effort, we discovered a GPR35-selective inhibitor, here termed TMER1i. TMER1i potently blocks GPR35 in Hippo suppression and YAP activation. We showed that GPR35 is expressed in the T-cell lineage of the immune cells including naïve CD4 T cells, Treg and CD8 T cells. Treatment of purified human naïve CD4 T cells with anti-CD3/anti-CD28 under an IDO1+ TME-like culture condition in vitro induced a robust T cell activation and differentiation toward Treg phenotype, as shown previously using mouse naïve CD4 T cells by others (5). Importantly, TMER1i efficiently blocked the Treg differentiation under these conditions. When dosed in human-PBMC reconstituted NCG mice bearing HT29 tumors, TMER1i dose-dependently increased the infiltration of human CD8 T cells within tumors in a statistically significant manner, and a trending lower Treg cell infiltration, accompanied by a tumor growth inhibition. qPCR analysis of the HT29 tumors grown in the PBMC-NCG mice indicated that IDO1 was highly expressed in the tumors. These results indicated that GPR35 selective inhibitor TMER1i enhanced an anti-tumor immune activity in an IDO1+ tumor microenvironment. Taken together, these studies provided evidence for a role of GPR35 in mediating IDO1 immune suppression by regulating Hippo-YAP pathway in Treg and CTL cells in the immune system. Thus, GPR35 inhibitors have a potential to be novel immuno-therapeutic agents effective in treating patients with IDO1-positive tumors. Reference1.Munn, DH, and Mellor, AL. (2007) Indoleamine 2,3-dioxygenase and tumor-induced tolerance. J. Clin. Invest. 117:1147-11542.Ni, X. et al. Yap is essential for Treg-mediated suppression of antitumor Immunity. Cancer Discovery 2018; 8:1026-10433.Lebid, A. et al. (2020) YAP attenuates CD8 T cell-mediated anti-tumor response. Frontiers in Immunology 11:1-84.Stampouloglou, E. et al. (2020) YAP suppresses T-cell function and infiltration in the tumor microenvironment. PLoS Biol 18(1): e30005915.Fallarino, F. et al. (2006) The combined effects of tryptophan starvation and tryptophan catabolites down-regulate T cell receptor æ-chain and induce a regulatory phenotype in naïve T cells. J Immunol 176:6752-6761 Citation Format: Hongmei He, Nam Hee Kim, Sangdon Han, Zhi Liang Chu. Evidence for a role of GPR35 in IDO1-mediated tumor immune escape by regulating Hippo-YAP pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB426.

Abstract 3741: Helper/killer hybrid epitope long peptide (H/K-HELP) cancer vaccine augments Th1-dependent CTL induction at tumor-draining lymph node to eradicate solid tumor: its application to develop an innovative combination cancer immunotherapy(iCCI)

Abstract We have initially demonstrated the pivotal role of IFN-γ-producing tumor-antigen-specific Th1 cells for the complete eradication of established tumor mass by CTL. The accumulated evidence has clearly demonstrated that induction of Th1-dependent full activation of CTL at tumor-draining lymph nodes (TDLN) are crucial for inducing complete rejection of solid tumor by CTL. To induce Th1 immunity efficiently at TDLN in tumor-bearing host, we developed an artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP), which conjugated killer and helper epitope peptides of cancer antigen by a glycine-linker. In mouse model, OVA-H/K-HELP was selectively presented in vivo by professional DC in vaccinated TDLN and sustained antigen presentation capability of DC to stimulate both CTL and Th1 cells. Thus, in contrast to short peptide, vaccination of OVA-expressing EG7-bearing mice with OVA-H/K-HELP caused a complete eradication of EG7 tumor mass. To apply these basic findings to clinical treatment of cancer patients, we then developed Survivin-H/K-HELP. Treatment of a patient with a triple-negative breast cancer patient with Survivin-H/K-HELP induced a complete response in a triple-negative breast cancer patient concomitantly with the induction of Th1-dependent cellular and humoral immune responses. In this clinical study, the patients showed Th1-dependent C’-fixing IgG antibody production and exhibited IFN-γ-producing Th1 cellular responses. Finally, we also applied H/K-HELP-pulsing DC-vaccine to develop an innovative combination cancer immunotherapy (iCCI). The iCCI consisting of radiation, low dose of immune check point inhibitors (ICIs:Nibolumab, Ipilimumab) and H/K-HELP-pulsed DC, all of which have been shown to initially modulate antitumor immunity at TDLN. Here, we described a successful outcome of a case of the stage IVA inoperable advanced hypopharyngeal squamous cell cancer patient with lymph node-metastasis. The iCCI treatment of the patient caused a complete disappearance of all cancers and the patient has been cancer free for over 15 months so far. Although the destruction mechanism of cancer is not determined, we image this iCCI might improve patient’s antitumor immune capability in immune microenvironment around tumor (IMAT) including TDLN and TME. Our developed iCCI will become a promising strategy to overcome inoperable advanced cancers in future. Citation Format: Takanori Iwasaki, Yuichiro Yazaki, Takashi Masuko, Takashi Nishimura. Helper/killer hybrid epitope long peptide (H/K-HELP) cancer vaccine augments Th1-dependent CTL induction at tumor-draining lymph node to eradicate solid tumor: its application to develop an innovative combination cancer immunotherapy(iCCI) [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 3741.

Defining the Drivers of Idelalisib-Related Early Autoimmune Toxicity in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) patients with disease resistant to BTK and BCL-2 inhibitors can potentially be treated with PI3K inhibitors to overcome resistance and improve outcomes. PI3K inhibitors such as idelalisib and duvelisib have shown promising efficacy in CLL patients, but severe toxicities remain a significant hurdle to their implementation in the clinic (PMID: 24615777). By investigating the molecular mechanisms that lead to severe early autoimmune toxicity with idelalisib, we hope to predict or prevent toxicity, thus expanding the use of this efficacious PI3K inhibitor class of drugs. To this end, the Brown lab showed that idelalisib-induced hepatitis is autoimmune. CyTOF analysis in CLL patients with early severe toxicity in a front-line idelalisib clinical trial cohort showed a significant decrease in CD4+ T regulatory cells (Treg) with a relative shift in favor of T effectors in peripheral blood. In addition, the Brown lab showed that the Th17 pathway is more active at baseline, with a further increase in patients with toxicity (PMID: 27247136, 36732326). Since the CYTOF data is restricted to fewer mostly T-cell markers in our studies, an unbiased single cell RNA sequencing (scRNA-seq) approach is warranted to explore the full effects of idelalisib on immune cells and CLL cells in patients with and without autoimmune toxicity. The question remains what molecular mechanisms drive the idelalisib-induced decrease in the Treg population, its effect on natural killer (NK) and CD8 T-cell function, and the activation of the Th17 pathway in CLL patients with early toxicity. We therefore carried out the first systematic effort to understand the mechanisms of idelalisib toxicity in molecular detail using combined scRNA and scTCR-seq. In this study, we profile T-cell enriched PBMCs of matched pre-treatment and on-idelalisib treatment samples from CLL patients with early severe toxicity and without any toxicity (n=4 CLL patients from each group, a total of 16 samples) using 10X single cell transcriptomic and TCR profiling. Raw sequencing data files were processed using the Cell Ranger suite (version 7.1.0) from 10x Genomics. Sample identification based on SNP information was done using the Popscle suite. Seurat package (version 4.9.9.9) was used for the single cell analysis. After filtering cells with fewer than 600 features, less than 1000 UMIs, mitochondrial content higher than 15%, 51,487 cells were left for the downstream analysis. To explore transcriptional heterogeneity and perform initial cell clustering, principal component analysis and nonlinear dimensional reduction using UMAP were applied using 20 dimensions. Leiden algorithm was used for cluster identification. Cell types were identified using Azimuth with the PBMC reference dataset and confirmed with expression plots. The initial observations of the cell proportions show that baseline samples from patients with early idelalisib toxicity have fewer Tregs, NK, and CD4+ cytotoxic T (CTL) cells compared to patients without toxicity. In addition, we observed a decline in Tregs, gamma-delta (γδ) T, and CD4+ CTL cells and a concurrent increase in proliferative CD4+ and CD8+ T-cells and proliferative NK on idelalisib treatment in the early toxicity group. On the contrary, CD4+ CTL and CD4+ T effector memory (TEM) cells are enriched with idelalisib therapy in patients without toxicities. At the same time, more CD4+ T-Cell Memory (TCM), γδ T, CD8+ naive T, CD8+ TCM, and Mucosal-associated invariant T (MAIT) cells are present in pretreatment samples from patients with toxicity compared to patients without toxicity. Furthermore, proliferative T-cells are depleted with idelalisib therapy in patients without toxicity whereas they are enriched in patients with early toxicity. To further characterize idelalisib-induced depletion of Tregs, we reclustered them to identify distinct subgroups and found particular clusters of Treg were depleted in patients with early toxicity. In the non-toxicity group, more diversity of Tregs was identified with depletion of some clusters but also an increase in a cluster. At present, we are integrating the scRNA & scTCR-seq data to characterize the cellular diversity and heterogeneous phenotypic states of the T-cell populations and updated results unraveling molecular mechanisms that drive early severe toxicity in CLL patients with idelalisib treatment will be presented at the meeting.

Insights into Large Granular Lymphocytic Leukemia-Associated Hemolytic Anemia: Clinical Associations, Therapeutic Responses, and Optimal Management Strategies from a Large Institutional Study

Introduction Large granular lymphocytic leukemia (LGLL) is characterized by a clonal proliferation of cytotoxic CD8+ T or CD3− natural killer (NK) cells. The disease typically presents with neutropenia in about 39-62% of patients, and anemia in up to 50% 1. While most cases of anemia in LGLL resemble pure red cell aplasia and present as reticulocytopenic and malproductive, we have also observed rare instances of hemolytic anemia (HA) with a high reticulocyte count. This unique and atypical presentation has prompted us to investigate the clinical features of LGLL with HA, aiming to gain insights into the pathogenesis of both conditions. In light of this, we conducted a comprehensive and systematic study of hemolysis within the context of LGLL. Methods We retrospectively examined clinical and molecular features of a large series of LGLL cases (n=262) diagnosed and managed at the Cleveland Clinic Foundation from 1998 to 2023 to identify those experiencing hemolysis. LGLL diagnosis was based on the presence of i) elevated LGL count (>0.5 × 10^9/L), ii) clonal TCR rearrangement, iii) VB expansion, iv) flow cytometric detection of aberrant CTL or NK cell proliferation and v) STAT3/5 mutation and vi) LGLL infiltration of the marrow. A diagnosis was established with >4/6 criteria fulfilled. HA diagnosis was based on evidence at any time during clinical course of laboratory signs of hemolysis. Results Overall, the median age at diagnosis was 63 years (IQR: 55-72) with a M:F ratio of 0.5. Majority of patients had been diagnosed with T-LGLL (n=225), whereas 22 had NK-LGLL. In 32/262 cases (12%), clinical and laboratory features were asserted to establish the diagnosis of HA. Of these, 22 (68%) had T-LGLL and 8 (25%) had NK-LGLL (Table 1). When comparing HA patients to the rest of the cohort, we observed a higher proportion of NK-LGLL cases with HA (25% vs 8%, p < 0.001). HA presented as the initial sign of the disease in majority of patients (88%), and 40% of them required transfusion support based on the degree of anemia and hemodynamics. The direct anti-globin test was positive in 30% of cases. Warm autoimmune HA (AIHA) was observed in 23% of patients, cold agglutinin disease in 7%, and one patient had both. Among 7 patients with IgG AIHA, 5 were also positive for anti-C3b, C3d antibodies. Two-thirds of the patients had multilineage cytopenia. Coexisting B-cell dyscrasia was identified in 13 cases, more frequently in HA than in those without (40% vs 26%, p=0.048), and autoimmune disorders in 8 cases (25%). HA in LGLL was also more likely to be associated with presence of splenomegaly (59% vs 21%, p<0.00001) and polyclonal hypergammaglobulinemia (22% vs 8%, p=0.02). Notably, STAT3 mutations were present in a higher proportion of HA patients (38% vs 22%, p=0.02), whereas median LGLL clone size (%) was similar between groups (68% vs 53%, p=0.5). Overall, 77% of HA patients received treatment, and 16% had still evidence of hemolysis at a median follow-up of 82 months. The majority (47%) were treated with steroids with a positive, albeit transient response (7 out of 15 treated). Other first-line treatments and their corresponding median duration of response (months) included cyclosporine (n=7, 72 months), cyclophosphamide (n=5, 72 months), IVIG (n=1, 16 months), and methotrexate (n=11, 21 months). Subsequent lines of therapy included campath (n=9), cyclosporine (CSA) (n=4), cyclophosphamide (n=9), intravenous immunoglobulin (IVIG) (n=2), MTX (n=4), rituximab (n=1), sirolimus (n=1), tacrolimus (n=2), and tofacitinib (n=2). Splenectomy was performed as definitive treatment for refractory cytopenia in 9 patients, with a durable response in 5 patients at last follow-up ( Fig.1). Conclusions Based on this large series, our study illustrates that LGLL represents a clonal lymphoproliferative disorder that is closely linked to HA. Notably, NK-LGLL cases exhibited a stronger association with HA. Our observations indicate that treatment with steroids and other therapeutic agents resulted in positive responses in a fraction of patients, while splenectomy demonstrated the most enduring response. These significant findings contribute substantially to advancing our comprehension of LGLL-associated HA and its optimal management strategies. AIHA in patients with LGLL points towards a global immune dysfunction in this disease beyond clonal cytotoxic T/NK cell expansion.

Bioinformatics analysis of Omp19 and Omp25 proteins for designing multi-epitope vaccines against Brucella.

Brucellosis is a zoonotic disease caused by Brucella. There is no effective vaccine against human brucellosis. Omp19 and Omp25 are the outer membrane proteins of Brucella. They are widely expressed and highly conserved in Brucella and have high immunogenicity. Herein, we aim to identify multi-epitope vaccine candidates based on Omp19 and Omp25. We analyzed the physicochemical properties and protein structure of Omp19 and Omp25, and predicted the corresponding B cell and T cell epitopes using bioinformatics analysis. Omp19 and Omp25 were composed of 177 amino acids and 213 amino acids, respectively. They were both stable hydrophilic proteins. The instability indices were 44.8 and 23, respectively. The hydrophilicity was -0.1 and -0.317, respectively. In the secondary structure of Omp19 and Omp25 proteins, the α-helix accounted for 12.43% and 23.94%, the β-sheet was 18.64% and 23.47%, the β-turn was 6.78% and 4.23%, and the random coil was 62.15% and 48.36%. Finally, 5 B cell epitopes, 3 Th-cell epitopes and 5 CTL cell epitopes of Omp19 protein, and 4 B cell epitopes, 3 Th-cell epitopes, and 5 CTL cell epitopes of Omp25 protein were selected as vaccine candidates. In conclusion, we obtained potential B cell and T cell epitopes of the Brucella outer membrane Omp19 and Omp25 proteins. This lays the foundation for the further design of multi-epitope vaccine of Brucella.

Intelligent solution predictive control strategy for nonlinear hepatitis B epidemic model with delay

This study exploits the artificial neural networks (ANNs) with backpropagation of the Levenberg-Marquardt method (LMM), i.e. ANNs-LMM, to explore the dynamics of a nonlinear hepatitis B epidemic model with the effect of delay (HBEMD). A system of three nonlinear ordinary differential equations having delay describes the susceptible liver cells, infected liver cells, and density of CTLs in the epidemic model. The referenced dataset is generated using the strength of the implicit Runge–Kutta numerical method in the variation of the incidence rate of disease, CTL immunological response rate of removing the disease-infected cells, virus specific CTL cells reproduction rate due to interaction with virally infected cells, time delay, white noise densities, and morality rate of individuals, infected cells death rate and virus specific CTL cells removing rate. The generated dataset is arbitrarily utilized for training, testing, and validation operations for each recurrent update in the Levenberg-Marquardt backpropagation to determine the numerical solutions for the dynamics of HBEMD. The effectiveness and reliability of the intelligent solution predictive control strategy of ANNs-LMM are endorsed with negligible absolute errors, mean square errors and near optimal regression measures.

SARS-CoV-2 Vaccination Effectiveness in Rituximab-Treated Patients Affected by Pemphigus Vulgaris

SARS-CoV-2 Vaccination Effectiveness in Rituximab-Treated Patients Affected by Pemphigus Vulgaris

Dysfunctions of innate and adaptive immune tumor microenvironment in Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM) is a rare subtype of non-Hodgkin lymphoma characterized by malignant lymphoplasmacytic cells in the bone marrow (BM). To dissect the pathophysiology of WM, we evaluated clonal cells by mapping of B cell lymphomagenesis with adaptive and innate immune tumor microenvironment (TME) in the BM of WM patients using mass cytometry (CyTOF). In-depth immunophenotypic profiling of WM cells exhibited profound expansion of clonal cells in both unswitched and switched memory B cells and also plasma cells with aberrant expression variations. WM B lymphomagenesis was associated with reduction of most B cell precursors assessed with the same clonally restricted light chain and phenotypic changes. The immune TME was infiltrated by mature monocytes, neutrophils and adaptive T cells, preferentially subsets of effector T helper, effector CTL and effector memory CTL cells that were associated with superior overall survival (OS), in contrast to progenitors of T cells and myeloid/monocytic lineage subsets that were suppressed in WM cohort. Moreover, decrease in immature B and NKT cells was related to worse OS in WM patients. Innate and adaptive immune subsets of WM TME were modulated by immune checkpoints, including PD-1/PD-L1&PD-L2, TIGIT/PVR, CD137/CD137-L, CTLA-4, BTLA and KIR expression. The response of ibrutinib treatment to the reduction of clonal memory B cell was associated with high levels of immature B cells and effector memory CTL cells. Our study demonstrates that CyTOF technology is a powerful approach for characterizing the pathophysiology of WM at various stages, predicting patient risk and monitoring the effectiveness of treatment strategies.

NK Cells Suppress Hyperfunction of CTLs Via ATM-Mediated NKG2D/NKG2DLs Activation in Severe Aplastic Anemia

Object: The activating receptor NKG2D on the surface of NK cells in peripheral blood of patients with severe aplastic anemia (SAA) was significantly increased, and NK cells may be activated during the pathogenesis of SAA. However, the immunomodulatory effect of NK cells on CTL cells remains unclear. ATM protein is a serine/threonine protein kinase mainly involved in cell cycle regulation, DNA damage recognition and repair. Activation of ATM may be the key to regulating the expression of NKG2DLs in T cells stimulated by superantigens. By detecting the changes of NKG2DLs in CTL cells of SAA patients under the regulation of ATM, we explored the immunoregulatory effect of NK cells on autologous CTL cells through the signaling axis NKG2D/NKG2DLs. Methods: We used flow cytometry, RT-qPCR and Western Blot to detect the expression of NKG2DLs (MICA, ULBP1, ULBP3) in CTL cells of untreated SAA patients, remission SAA patients and healthy controls under the intervention of CD3/CD28 antibody and ATM agonist (chloroquine phosphate). NK and CTL cells from SAA patients were co-cultured in vitro, and the function of CTL under the regulation of ATM was detected. Results: Under the intervention of CD3/CD28 antibody and chloroquine phosphate, the level of MICA in CTL cells detected by flow cytometry in untreated SAA patients was higher than that in convalescent SAA patients and healthy controls (Fig.A). RT-qPCR detection of mRNA levels of NKG2DLs (MICA, ULBP1 and ULBP3) in CTL cells of untreated SAA patients was also higher than that of remission SAA patients and healthy controls (Fig.B). When ATM agonist (chloroquine phosphate), ATM inhibitor (KU55933), and chloroquine phosphate + KU55933 were used to intervene T cells, Western Blot detected the correlation between ATM phosphorylation and NKG2DLs (MICA, ULBP1) expression(Fig.D). We co-cultured CTL cells from SAA patients with different interventions in vitro with autologous NK cells. The expression of IFN-γ in CTL cells in the ATM agonist group was significantly lower than that in the ATM inhibition group, and the apoptosis rate was higher than that in the ATM inhibition group and the control group (Fig.C). There was no significant difference in the expression of perforin and granzyme B. In addition, NK cell apoptosis did not differ among the groups.The expression levels of free MICA and ULBP3 in the co-culture supernatant were detected by ELISA, and the ATM agonist group was higher than the inhibition group(p<0.05). Conclusion: Activation of ATM increases the expression of NKG2DLs in CTL cells of SAA patients, which promotes the inhibitory effect of NK cells on CTL cells. Our study further confirms that ATM can regulate the relationship between NK cells and CTL cells in SAA disease, and inhibit the hyperactive state of CTL cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

NcRNAs-mediated high expression of TICRR promotes tumor cell proliferation and migration and is correlated with poor prognosis and tumor immune infiltration of hepatocellular carcinoma

TICRR is a regulatory factor of DNA replication with ToPBP1 interaction. At present, the underlying function and mechanisms of TICRR remain unclear in LIHC. Our objective was to assess the function and prognosis of TICRR in LIHC. We conducted a differential expression analysis, GO/KEGG, and GSEA enrichment analysis of TICRR in LIHC. We also carried out the gene frequency and SCNA of TICRR. We found that TICRR could serve as an independent prognostic marker in LIHC by univariate and multivariate analysis. In addition, we observed that TICRR was related to immune infiltration, and TICRR had positive correlation with PD1/PD-L1 and CTLA-4 in LIHC. The hsa-miR-126-3p/IPO9-AS1 may be the candidate ncRNAs to regulate the expression of TICRR. The high rate of SCNV of TICRR might have critical effect on the function of CTL cells in LIHC. We further demonstrate through a series of experiments that TICRR facilitated the proliferation and metastasis of liver cancer cells in vitro. Altogether, TICRR might be a potential biomarker and therapeutic target in LIHC.

(-)-Epicatechin and its colonic metabolite hippuric acid protect against dexamethasone-induced atrophy in skeletal muscle cells

Cocoa flavanols have been shown to improve muscle function and may offer a novel approach to protect against muscle atrophy. Hippuric acid (HA) is a colonic metabolite of (-)-epicatechin (EPI), the primary bioactive compound of cocoa, and may be responsible for the associations between cocoa supplementation and muscle metabolic alterations. Accordingly, we investigated the effects of EPI and HA upon skeletal muscle morphology and metabolism within an in vitro model of muscle atrophy. Under atrophy-like conditions (24h 100μM dexamethasone (DEX)), C2C12 myotube diameter was significantly greater following co-incubation with either 25μM HA (11.19±0.39μm) or 25μM EPI (11.01±0.21μm) compared to the vehicle control (VC; 7.61±0.16μm, both P < .001). In basal and leucine-stimulated states, there was a significant reduction in myotube protein synthesis (MPS) rates following DEX treatment in VC (P=.024). Interestingly, co-incubation with EPI or HA abrogated the DEX-induced reductions in MPS rates, whereas no significant differences versus control treated myotubes (CTL) were noted. Furthermore, co-incubation with EPI or HA partially attenuated the increase in proteolysis seen in DEX-treated cells, preserving LC3 α/β II:I and caspase-3 protein expression in atrophy-like conditions. The protein content of PGC1α, ACC, and TFAM (regulators of mitochondrial function) were significantly lower in DEX-treated versus. CTL cells (all P < .050). However, co-incubation with EPI or HA was unable to prevent these DEX-induced alterations. For the first time we demonstrate that EPI and HA exert anti-atrophic effects on C2C12 myotubes, providing novel insight into the association between flavanol supplementation and favourable effects on muscle health.

Identification of SLAMF1 as an immune-related key gene associated with rheumatoid arthritis and verified in mice collagen-induced arthritis model.

BackgroundRheumatoid arthritis (RA) is the most common inflammatory arthropathy. Immune dysregulation was implicated in the pathogenesis of RA. Thus, the aim of the research was to determine the immune related biomarkers in RA.MethodsWe downloaded the gene expression data of RA in GSE89408 and GSE45291 from Gene Expression Omnibus public database (GEO). Differentially expressed genes (DEGs) were identified between RA and control groups. Infiltrating immune cells related genes were obtained by ssGSEA and weighted gene co-expression network analysis (WGCNA). We performed functional enrichment analysis of differentially expressed immunity-related genes (DEIRGs) by “clusterProfiler” R package, key genes screening by protein-protein interaction (PPI) network of DEIRGs. And mice collagen-induced arthritis (CIA) model was employed to verify these key genes.ResultsA total of 1,885 up-regulated and 1,899 down-regulated DEGs were identified in RA samples. The ssGSEA analysis showed that the infiltration of 25 cells was significantly different. 603 immune related genes were obtained by WGCNA, and 270 DEIRGs were obtained by taking the intersection of DEGs and immune related genes. Enrichment analyses indicated that DEIRGs were associated with immunity related biological processes. 4 candidate biomarkers (CCR7, KLRK1, TIGIT and SLAMF1) were identified from the PPI network of DEIRGs and literature research.In mice CIA model, the immunohistochemical stain showed SLAMF1 has a significantly high expression in diseased joints. And flow cytometry analysis shows the expression of SLAMF1 on CIA mice-derived CTL cells, Th, NK cells, NKT cells, classical dendritic cell (cDCs) and monocytes/macrophages was also significantly higher than corresponding immune cells from HC mice.ConclusionOur study identified SMLAF1 as a key biomarker in the development and progression of RA, which might provide new insight for exploring the pathogenesis of RA.

BS-526-04 IMPAIRED REDOX RESPONSE TO INCREASED WORKLOAD IN ATRIAL MITOCHONDRIA FROM PATIENTS WITH ATRIAL FIBRILLATION

Calcium handling abnormalities contribute to the pathophysiology of atrial fibrillation (AF). In ventricular myocytes, Ca2+ has been shown to play an important role in mitochondrial response to increased workload by activating key dehydrogenases in the Krebs cycle, necessary for NAD(P)H regeneration and, ultimately, neutralisation. We hypothesise that workload response of mitochondria is impaired in atrial cardiomyocytes from patients with AF. Membrane currents (patch clamp), cytosolic Ca2+ (Fluo-3) and NAD(P)H/FAD autofluorescence were recorded in right atrial myocytes from sinus rhythm (CTL) or AF patients. Immunofluorescence labeling together with STED microscopy and EM microscopy were employed to characterize interaction between mitochondria and sarcoplasmic reticulum (SR). Diastolic [Ca2+]i of voltage-clamped myocytes was comparable, whereas amplitudes of L-type Ca2+ current and triggered of Ca2+ transients (CaTs) were decreased by 49% and 43%, respectively. Basal level of NAD(P)H at 0.5 Hz stimulation frequency was comparable in CTL and AF (73.08±2.62%, n=14/7 vs. 81.05±5.39%, n=4/3 respectively), suggesting a similar redox index. In all CTL cells, upon increasing stimulation frequency to 3 Hz and subsequent β-adrenergic stimulation, the NAD(P)H level initially decreased but recovered to a level comparable to basal state. In contrast, 35% of AF myocytes lost this capacity to recover. Electron and STED microscopy images (Figure) show a disturbance in mitochondrial organisation and their interaction with the SR. Impaired cytosolic Ca2+ handling and disturbed interaction between mitochondrial and Ca2+ release sites of the SR may contribute to the impaired redox response of mitochondria to increased workload which we observed in AF patients. This likely results in impaired ATP production and ROS neutralisation, which may finally contribute to atrial arrhythmogenesis in AF patients.

Circ_0004140 promotes LUAD tumor progression and immune resistance through circ_0004140/miR-1184/CCL22 axis

Lung adenocarcinoma (LUAD) is a highly prevalent cancer with high mortality. Immune resistance and tumor metastasis are the pivotal factors for the promotion of LUAD. CircRNAs have been revealed a crucial pre-clinical diagnostic and therapeutic potentials in LUAD. Herein, we identify a novel circRNA (circ_0004140), derived from the oncogene YAP1, which is up-regulated in LUAD. The high expression of circ_0004140 is correlated with poor prognosis and CTL cells dysfunction in LUAD patients. Knockdown of circ_0004140 regulated LUAD cells proliferation, migration, and apoptosis. Mechanistically, circ_0004140 served as a sponge of miR-1184 targeting C-C motif chemokine ligand 22(CCL22). Overexpression of CCL22 reversed the inhibitory effect induced by si-circ_0004140 on cells proliferation and migration. Moreover, we also revealed that elevated circ_ooo4140 was related to cytotoxic lymphocyte exhaustion, and a combination therapy of C-021 (CCL22/CCR4 axis inhibitor) and anti-PD-1 attenuated LUAD promotion and immune resistance. In conclusion, circ_0004140 may drive resistance to anti-PD-1 immunotherapy, providing a novel potential therapeutic target for LUAD treatment.

Interleukin-2 in the Complex Treatment of Surgical Patients

Immunotherapy for surgical diseases remains one of the urgent scientific and clinical issues. Immunodeficiency is manifested in patients with purulent wounds, pancreatic necrosis, osteomyelitis, oncological diseases, echinococcosis, gastroduodenal ulcers and other pathology. Drugs inducing interleukin-2 synthesis have shown high efficacy for the correction of immunodeficiency status. Clinical effects of rIL-2 are due to its ability to activate clonal proliferation of T- and B-lymphocytes, to enhance the effector potential of cytotoxic T-lymphocytes (CTL cells) and natural killer cells (NK cells), functional activity of mononuclear phagocytes and antigen-presenting cells, synthesis of specific immunoglobulins of most isotypes by plasma cells and to reduce apoptosis of mononuclears. This cytokine is a key link determining the development of cellular and humoral immunity. Interleukin-2 provides antitumor, antiviral, antibacterial and antifungal protection, being able to stimulate the processes of tissue regeneration and repair. The high clinical activity of the drug was revealed in the treatment of endotoxicosis, immunological insufficiency, inflammatory diseases in patients with surgical, oncological, traumatological, combustiological, resuscitation profiles, involving abdominal organs, pelvis and retroperitoneal space. It is rational to use recombinant interleukin-2 in case of previous inefficient conventional treatment and the process chronization. The drug has a number of positive aspects: rapid effect; reduced terms of treatment, frequency of infectious complications and sepsis; reduced mortality rate. The negative side of its administration is its high cost. Thus, immunosuppressive conditions are manifested in multiple surgical diseases. High clinical efficacy of recombinant interleukin 2, absence of complications, adverse reactions and undesirable effects, and favourable tolerability of the drug allow recommending it for wider use in surgical practice.