Increase In CD4 Research Articles

Preliminary translational immune and stromal correlates in a randomized phase II trial of pembrolizumab with or without defactinib for resectable pancreatic ductal adenocarcinoma (PDAC).

4024 Background: PDAC is an aggressive cancer and refractory to immunotherapy due to its immunosuppressive tumor microenvironment (TME). Focal adhesion kinase (FAK) is a master regulator of the TME and associated with TME immune suppression. Our current randomized phase II trial evaluates the use of pembrolizumab, a programmed cell death 1 (PD-1) immune checkpoint inhibitor, with or without defactinib, a FAK inhibitor (FAKi), as sequential neoadjuvant and adjuvant therapy in patients with high risk resectable PDAC. We hypothesize that the patients receiving pembrolizumab and defactinib treatment will exhibit a decrease in immunosuppressive fibroblasts and myeloid subtype populations, leading to increased CD8+ T-cell infiltration into the TME compared to patients receiving pembrolizumab alone. Methods: We performed quantitative analysis of multiplex immunohistochemistry (mIHC) using 40 biomarkers, evaluating immune and stromal cell types on 14 pre-treatment biopsies and post-treatment resections of PDAC patients enrolled in our platform neoadjuvant clinical trial (NCT03727880). All patients received 2 cycles of gemcitabine+nab-paclitaxel neoadjuvant chemotherapy and underwent a biopsy after completion of chemotherapy. Patients randomized to Arm A received 2 cycles of pembrolizumab 200 mg IV every 3 weeks and defactinib 400 mg PO BID, and those randomized to Arm B received pembrolizumab alone, followed by surgical resection. Image cytometry was used to quantify immune cell populations and colocalize biomarker expression in distinct cell types. Cell populations were compared using unpaired T-tests. Results: Lower FAP+ fibroblast density was significantly associated with higher CD8+ T-Cell infiltration in Arm A (p=0.002), but not in Arm B. Patients in Arm A demonstrated a 5.44-fold average increase in CD8+ T-cell percentage between pre-immunotherapy treatment and post-immunotherapy treatment specimens compared to a 2.01-fold increase in patients in Arm B (p=0.02, p=0.09, respectively). Only patients in Arm A showed a significant increase in M1 macrophage cell density (p=0.02) after treatment. Both arms increased in CXCR4+ cell percentage (p=0.011, p=0.06) following neoadjuvant immunotherapy; however, the increase in Arm A was less than in Arm B (0.72 and 0.84-fold, respectively). Conclusions: In this analysis, pembrolizumab combined with defactinib was associated with lower fibroblast infiltration, higher anti-tumor M1 macrophage expression and increased CD8+ T-cell infiltration into the TME, versus pembrolizumab alone. The increased expression of CXCR4 across both treatment arms may represent a resistance mechanism and supports CXCR4 as an additional TME target. These preliminary findings warrant continued research into FAK inhibition and immune checkpoint combinatorial strategies. Clinical trial information: NCT03727880 .

Cellular and Molecular Mechanisms of Liver Fibrosis in Patients with NAFLD.

The expression of immune- and cancer-related genes was measured in liver biopsies from 107 NAFLD patients. The strongest difference in overall gene expression was between liver fibrosis stages F3 and F4, with 162 cirrhosis-associated genes identified. Strong correlations with fibrosis progression from F1 to F4 were observed for 91 genes, including CCL21, CCL2, CXCL6, and CCL19. In addition, the expression of 21 genes was associated with fast progression to F3/F4 in an independent group of eight NAFLD patients. These included the four chemokines, SPP1, HAMP, CXCL2, and IL-8. A six-gene signature including SOX9, THY-1, and CD3D had the highest performance detecting the progressors among F1/F2 NAFLD patients. We also characterized immune cell changes using multiplex immunofluorescence platforms. Fibrotic areas were strongly enriched in CD3+ T cells compared to CD68+ macrophages. While the number of CD68+ macrophages increased with fibrosis severity, the increase in CD3+ T-cell density was more substantial and progressive from F1 to F4. The strongest correlation with fibrosis progression was observed for CD3+CD45R0+ memory T cells, while the most significant increase in density between F1/F2 and F3/F4 was for CD3+CD45RO+FOXP3+CD8- and CD3+CD45RO-FOXP3+CD8- regulatory T cells. A specific increase in the density of CD68+CD11b+ Kupffer cells with liver fibrosis progression was also observed.

Cerebrospinal fluid CD14++CD16+monocytes in HIV-1 subtype C compared with subtype B.

CD14++CD16+ monocytes are susceptible to HIV-1 infection, and cross the blood-brain barrier. HIV-1 subtype C (HIV-1C) shows reduced Tat protein chemoattractant activity compared to HIV-1B, which might influence monocyte trafficking into the CNS. We hypothesized that the proportion of monocytes in CSF in HIV-1C is lower than HIV-1B group. We sought to assess differences in monocyte proportions in cerebrospinal fluid (CSF) and peripheral blood (PB) between people with HIV (PWH) and without HIV (PWoH), and by HIV-1B and -C subtypes. Immunophenotyping was performed by flow cytometry, monocytes were analyzed within CD45 + and CD64 + gated regions and classified in classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14lowCD16+). Among PWH, the median [IQR] CD4 nadir was 219 [32-531] cell/mm3; plasma HIV RNA (log10) was 1.60 [1.60-3.21], and 68% were on antiretroviral therapy (ART). Participants with HIV-1C and -B were comparable in terms of age, duration of infection, CD4 nadir, plasma HIV RNA, and ART. The proportion of CSF CD14++CD16+ monocytes was higher in participants with HIV-1C than those with HIV-1B [2.00(0.00-2.80) vs. 0.00(0.00-0.60) respectively, p = 0.03 after BH correction p = 0.10]. Despite viral suppression, the proportion of total monocytes in PB increased in PWH, due to the increase in CD14++CD16+ and CD14lowCD16+ monocytes. The HIV-1C Tat substitution (C30S31) did not interfere with the migration of CD14++CD16+ monocytes to the CNS. This is the first study to evaluate these monocytes in the CSF and PB and compare their proportions according to HIV subtype.

The Tumor Microenvironment in Classic Hodgkin's Lymphoma in Responder and No-Responder Patients to First Line ABVD Therapy.

Although classical Hodgkin lymphoma (CHL) is typically curable, 15-25% of individuals eventually experience a relapse and pass away from their disease. In CHL, the cellular microenvironment is constituted by few percent of H/RS (Hodgkin/Reed-Sternberg) tumor cells surrounded from a heterogeneous infiltration of inflammatory cells. The interplay of H/RS cells with other immune cells in the microenvironment may provide novel strategies for targeted immunotherapies. In this paper we analyzed the microenvironment content in CHL patients with responsive disease (RESP) and patients with relapsed/refractory disease to treatment (REL). Our results indicate the increase of CD68+ and CD163+ macrophages, the increase of PDL-1+ cells and of CD34+ microvessels in REL patients respective to RESP patients. In contrast we also found the decrease of CD3+ and of CD8+ lymphocytes in REL patients respective to RESP patients. Finally, in REL patients our results show the positive correlation between CD68+ macrophages and PDL-1+ cells as well as a negative correlation between CD163+ and CD3+.

Hepatic proinflammatory myeloid phenotypes are a hallmark of Ebola virus Kikwit pathogenesis in rhesus monkeys.

The liver is an early systemic target of Ebola virus (EBOV), but characterization beyond routine histopathology and viral antigen distribution is limited. We hypothesized Ebola virus disease (EVD) systemic proinflammatory responses would be reflected in temporally altered liver myeloid phenotypes. We utilized multiplex fluorescent immunohistochemistry (mfIHC), multispectral whole slide imaging, and image analysis to quantify molecular phenotypes of myeloid cells in the liver of rhesus macaques (Macaca mulatta; n = 21) infected with EBOV Kikwit. Liver samples included uninfected controls (n = 3), 3 days postinoculation (DPI; n = 3), 4 DPI (n = 3), 5 DPI (n = 3), 6 DPI (n = 3), and terminal disease (6-8 DPI; n = 6). Alterations in hepatic macrophages occurred at ≥ 5 DPI characterized by a 1.4-fold increase in CD68+ immunoreactivity and a transition from primarily CD14-CD16+ to CD14+CD16- macrophages, with a 2.1-fold decrease in CD163 expression in terminal animals compared with uninfected controls. An increase in the neutrophil chemoattractant and alarmin S100A9 occurred within hepatic myeloid cells at 5 DPI, followed by rapid neutrophil influx at ≥ 6 DPI. An acute rise in the antiviral myxovirus resistance protein 1 (MxA) occurred at ≥ 4 DPI, with a predilection for enhanced expression in uninfected cells. Distinctive expression of major histocompatibility complex (MHC) class II was observed in hepatocytes during terminal disease. Results illustrate that EBOV causes macrophage phenotype alterations as well as neutrophil influx and prominent activation of interferon host responses in the liver. Results offer insight into potential therapeutic strategies to prevent and/or modulate the host proinflammatory response to normalize hepatic myeloid functionality.

Regulation of dendritic cell maturation in osimertinib-treated lung adenocarcinoma patients

Osimertinib (OSI), a third-generation tyrosine kinase inhibitor (TKI), efficiently benefits lung adenocarcinoma (LUAD) patients with epidermal growth factor receptor (EGFR) mutations. However, combined OSI and immune checkpoint inhibitor in EGFR-mutant patients increases the incidence of interstitial lung disease (ILD), although the mechanism is unknown. Here, we investigated the interaction between dendritic cells (DCs), a potential critical player in ILD, and OSI. Seventeen LUAD patients received TKI therapy, and only the OSI therapy group (N=10) showed a significant increase in CD40 and CD83 on immature DCs (iDCs), and an elevated trend for both markers on mature DCs (mDCs) during short- and long-term OSI therapy. Our results indicated that OSI therapy may potentially activate DC functions, which might increase the potential immune toxicity when combined with onco-immunotherapy.

Preliminary analysis of double-negative T, double-positive T, and natural killer T-like cells in B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the expansion of CD5+ malignant B lymphocytes. Recent discoveries have shown that double-negative T (DNT) cells, double-positive T (DPT) cells, and natural killer T (NKT)-cells may be involved in tumor surveillance. A detailed immunophenotypic analysis of the peripheral blood T-cell compartment of 50 patients with B-CLL (classified in three prognostic groups) and 38 healthy donors (as controls) matched for age was performed. The samples were analyzed by flow cytometry using a stain-lyse-no wash technique and a comprehensive six-color antibody panels. Our data confirmed a reduction in percentage values and an increase in absolute values of T lymphocytes in patients with B-CLL, as already reported. In particular, DNT, DPT, and NKT-like percentages were significantly lower than in the controls, except for NKT-like in the low-risk prognostic group. Moreover, a significant rise in the absolute counts of DNT cells in each prognostic group and in the low-risk prognostic group of NKT-like cells was found. A significant correlation of the absolute values of NKT-like cells in the intermediate-risk prognostic group versus B cells was observed. Furthermore, we analyzed whether the increase in T cells was related to the subpopulations of interest. Only DNT cells were positively correlated with the increase in CD3+ T lymphocytes, regardless of the stage of the disease, supporting the hypothesis that this T-cell subset plays a key role in the immune T response in B-CLL. These early results supported that DNT, DPT, and NKT-like subsets may be related to disease progression and should encourage further studies aimed at identifying the potential immune surveillance role of these minority T subpopulations.

Treatment with doxorubicin and PD-1/CTLA-4 blockade improves T cell activation and anti-tumor efficacy in MCA-205 murine fibrosarcoma

Abstract Immune checkpoint inhibitors (ICIs) against PD-1 and CTLA-4 produce responses in about 20% of adult soft tissue sarcomas (STSs), however, most patients do not respond, potentially from poor T cell activation. Standard chemotherapy for STSs includes the anthracycline doxorubicin (DOX) which has been shown in preclinical cancer models to induce immunogenic cell death (ICD) via tumor production of type I interferons (IFN). We hypothesized that induction of ICD and IFN release by doxorubicin would enhance T cell activation, infiltration, and boost anti-tumor efficacy when combined with dual anti-PD-1/CTLA-4 blockade in the MCA-205 murine fibrosarcoma model. MCA-205 tumors were generated in C57BL/6 mice and were treated with DOX, anti-PD1 (RMP1–14), anti-CTLA4 (9D9 mouse IgG2b) or a mouse surrogate of botensilimab, an anti-CTLA4 (9D9 mouse IgG2b.DLE) antibody with enhanced binding to FcγR, or combination DOX plus anti-PD-1/CTLA-4. We found reduction of tumor growth and improved survival (median survival not reached) with DOX/PD-1/CTLA-4 compared to DOX (median survival 29.5 days). This correlated with a 2-fold increased influx of TILs as measured by flow cytometry and immunohistochemistry, with a marked increase in CD8:CD4 T cell ratio with increased expression of activation markers (PD-1, 4–1BB and TIM3). TCR Vβ clonotyping revealed a shift in clonotype frequency with DOX/PD-1/CTLA-4 relative to other treatment groups. Pathway analysis from bulk RNA sequencing demonstrated upregulation of T cell activation in DOX/PD-1/CTLA-4 tumors compared to control tumors. Further work will correlate these findings with human STS patients receiving DOX plus CTLA-4/PD-1 blockade in an ongoing clinical trial (NCT04028063). This work was supported by grants from the University of Colorado Cancer Center (P30CA046934) and the University of Colorado School of Medicine.

ADAM9 drives immunosuppressive environment for lung tumor progression

Abstract Cancer cells develop the immune suppressive tumor environment, but the underlying molecular mechanism for it is still elusive. Here, we demonstrated that ADAM9 contributed to lung tumor progression and promoted IFN-related gene expression in lung cancer cells that participated in immune regulation in the tumor environment. ADAM9 knockout (KO) lung cancer cells reduced lung cancer metastasis with a decrease in neutrophil infiltration and an increase in CD8+ T cell infiltration. By performing the genome-wide approach to explore ADAM9-regulated genes in lung cancer cells, we revealed that ADAM9 regulated cytokine production and lymphocyte activation. It enhanced the master transcription factor of IFN signaling, STAT1 activation in lung cancer cells. Moreover, ADAM9 promoted IL6/IL6 receptor-STAT3 axis signaling that lead to suppression of IL12p40 secretion and MHC class II expression in dendritic cells and macrophages. Notably, we found that type I IFN related-proteins and steroid biosynthesis-related proteins were related to ADAM9-mediated STAT signaling activation by LC-MS/MS analysis. We demonstrated that ADAM9 created an immune suppressive environment by regulating IFN signaling for tumor progression. Supported by grants from NHRI (NHRI-EX112-11219BI)

Targeting lL-38 triggers γδ T cell-dependent anti-tumor immunity

Abstract The IL-1 family receptor antagonist IL-38 suppresses auto-inflammatory reactions in murine disease models. Given the connection between auto-immunity and anti-tumor immunity, we wondered if targeting IL-38 would affect the latter. The transgenic PyMT mammary carcinoma model was employed to target IL-38 genetically or with a neutralizing antibody. IL-38-deficient (KO) PyMT mice had a markedly delayed tumor growth compared to WT mice, accompanied by increased NK cells, CD8+, NKT, and γδ T cell infiltrates. Accordingly, in human mammary carcinoma, IL-38 expression negatively correlated with infiltrating T cell subsets. This pattern was largely recapitulated with a IL-38-neutralizing antibody, where antibody-treated PyMT WT mice showed a strong decrease in tumor growth compared to IgG control treated animals, accompanied by an increase in CD8+ and γδ T cells. In a therapeutic model of chemoresistance, IL-38 antibody in combination with doxorubicin prevented tumor relapse, which was accompanied by increased in NK cells, CD8+, CD4+, and γδ T cells. To analyze the involvement of γδ T cells, we used γδ-TCR blocking antibody compared to IgG control in IL-38 KO mice. The delay in growth of IL-38 KO PyMT tumors compared to WT PyMT tumors was abolished upon γδ-TCR blockade. This was accompanied by a strong decrease in CD8+ cells upon effective γδ-TCR blockade, suggesting a cross talking between CD8+ and γδ T cells. Whole transcriptome sequencing analysis of PyMT tumors upon IL-38 neutralization and γδ-TCR blockage suggested NOTCH signaling to mediate the link between CD8+ and γδ T cells. Taken together, these results provide evidence for a therapeutic potential of anti-IL-38 antibodies to activate anti-tumor immunity in mammary carcinoma. Supported by a grant from Deutsche Krebshilfe (70114051)

Abstract A20: Inflammation remodels the immune microenvironment in acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a hematopoietic malignancy with poor prognosis and limited treatment options. Here we provide a comprehensive single cell RNA-Sequencing census of the bone marrow immune microenvironment in adult and pediatric AML patients. We characterize unique inflammation signatures in a subset of AML patients, associated with inferior outcomes. We identify atypical B cells, a dysfunctional B cell subtype enriched in high-inflammation AML patients, as well as an increase in CD8+ GZMK+ and regulatory T cells, accompanied by a reduction in T cell clonal expansion. We derive an inflammation-associated gene score (iScore) that associates with poor survival outcomes in AML patients. Addition of the iScore refines current risk stratifications for AML patients and may enable identification of patients in need of more aggressive treatment. This work provides a first framework for classifying AML patients based on their immune microenvironment and a rationale for consideration of the inflammatory state in clinical settings. Citation Format: Audrey Lasry, Bettina Nadorp, Maarten Fornerod, Deedra Nicolet, Huiyun Wu, Christopher J Walker, Zhengxi Sun, Matthew T Witkowski, Anastasia N Tikhonova, Maria Guillamot, Geraldine Cayanan, Anna Yeaton, Tanja A Gruber, Ann-Kathrin Eisfeld, Iannis Aifantis. Inflammation remodels the immune microenvironment in acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A20.

γδ T cells are significantly suppressed in glioma patients but retain their cytotoxic potential

Abstract Complex interactions between tumor and immune cells influence γδ T cells in the course of glioma. The study aimed to characterise the γδ T cells in glioma patients and assess their potential for γδ T-based immunotherapy. Peripheral blood from 52 glioma patients and 30 healthy controls was collected. γδ T cells and their subsets were assessed by flow cytometry and compared between stages i.e. LGG and HGG. Cytokine expression and cytotoxicity against U251MG was tested on Vδ2 in vitro cultures. The co-culture of healthy donors PBMC with glioma patient plasma or 4 hours stimulations with U251MG were performed. Both total γδ T and Vδ1 cells were significantly expanded in glioma patients. While stimulatory molecules (CD226 and NKG2D) were downregulated, co-inhibitory molecules (PD-1, CTLA-4, TIGIT, TIM-3, LAG-3) were overexpressed. Suppression of γδ T cells seems to be promoted by circulating cytokines as PD-1, TIGIT, TIM-3, LAG-3 were upregulated in cultures with patient plasma. Healthy volunteer γδ T cells are usually significantly activated by U251MG as was demonstrated by an increase in CD69 and CD107a expression after short-term co-culture. Importantly, glioma patient-derived Vδ2 cells retained their cytotoxic potential against glioblastoma cells, even though they overexpressed anti-inflammatory cytokines (IL-10, TGF-β, IL-17). The results highlight the profoundly immunosuppressive peripheral blood environment in gliomas. Most importantly, this may significantly affect the general fitness of γδ T cells, lowering their potential for immunotherapy. On the other hand, those changes seem to be at least partially reversible as patient-derived γδ T cells retain their cytotoxic potential after in-vitro activation and culture. Supported by grant from Medical University of Lublin MG 25/2022.

Abstract A41: Single Cell Multiomic Analysis Reveals Association of TP53-mut Loss of Heterozygosity with Primitive Phenotype in Acute Myeloid Leukemia

Abstract TP53 mutations in acute myeloid leukemia (AML) are associated with copy number abnormalities (CNA), structural variants and high risk of relapse (Döhner et al., 2017; Giacomelli et al., 2018; Bernard et al. 2020). In spite of relatively high remission rates obtained by targeted therapies, TP53 mutant (TP53-mut) clones persist, invariably resulting in relapse (Short et al., 2021; Takahashi et al., 2016). Delineating the clonal architecture and the immunophenotypes of TP53-mut clones during AML therapy may provide a better understanding of the role of TP53-mutations in AML biology. Recent progress in sequencing technologies allows the integration of genotyping and phenotyping at the single cell level. Here, we took advantage of MissionBio Tapestri’s newest platform: single cell DNA + protein for simultaneous genotyping and phenotyping with 45 surface oligo-conjugated antibodies in 10 paired samples from 5 patients with TP53-mut AML before and after therapy. Samples with at least 70% viability were stained with the TotalSeq™-D Human Heme Oncology Cocktail, V1.0. Following surface marker staining, single-cell suspension, encapsulation, and barcoding were performed according to manufacturer’s instruction. For scDNA library preparation, we utilized a validated custom panel (Morita et al. 2020) consisting of 279 amplicons covering recurrent mutations in 37 genes in AML. We sequenced a total of 44,550 cells from 10 samples. We confirmed mutations reported by MD Anderson molecular diagnostic laboratory in the genes covered by the scDNA custom panel. The clonal architecture analysis distinguished between TP53-mut clones with or without loss of heterozygosity (LOH) of the normal TP53 allele. Our data show a primitive immunophenotype in TP53-mut with LOH (LOH+) clones in comparison to TP53-mut LOH- clones. We see 2.4 LOG2FC increase in CD34 and 1.8 LOG2FC increase in CD117 (p<0.001) in TP53-mut LOH+ clones in comparison to TP53-mut LOH- clones. Clonal evolution analysis shows that TP53-mut LOH+ clones are significantly more resistant to therapy than TP53-mut LOH-, consistent with previous publications. On the other hand, TP53-mut LOH- clones showed significantly higher levels of CD2, CD16, CD5, CD3, and CD8 among other lineage markers (LOG2FC= 1.1, 1.2, 1.4, 1.1, and 1.1 respectively; p.value <0.0001) compared to TP53-mut LOH+. This data indicates that TP53-mut LOH- cells can express lymphoid phenotypic markers. Single cell cytokine analysis (IsoPlexis) reveals profound lack of secreted cytokines in T-cells from TP53-mut AML. Further data from bulk-RNA sequencing, ddPCR, and CyTOF will be presented that validates a lymphoid phenotype of TP53-mut LOH-. In summary, we utilize a scDNA+protein multiomic approach to dissect clonal architecture and provide a link between genotype-phenotype in TP53-mut AML. We show that while TP53-mut LOH+ clones are exclusively primitive, TP53-mut LOH- clones retain the capacity to exist outside primitive immunophenotype and might lack differentiation block. Citation Format: Edward Ayoub, Vakul Mohanty, Yuki Nishida, Tallie Patsilevas, Mahesh Basyal, Russell Pourebrahim, Muharrem Muftuoglu, Ken Chen, Ghayas C. Issa, Michael Andreeff. Single Cell Multiomic Analysis Reveals Association of TP53-mut Loss of Heterozygosity with Primitive Phenotype in Acute Myeloid Leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A41.

Clinical and pathogenetic significance of immunity factors in congenital generalized infection caused by the herpes simplex virus

Intrauterine infections are serious diseases that largely determine the level of infant mortality. Newborns who have had intrauterine infections often have long-term consequences, leading to disability. One of the intrauterine infections is a congenital infection caused by the herpes simplex virus (neonatal herpes). Neonatal herpes occurs less frequently (1:2000 live births) than cytomegalovirus fection, but the clinical symptoms in this disease are characterized by multiorgan damage.Purpose. To determine the role of immune factors in the development of congenital generalized HSV infection.Material and methods. Twenty-two newborns with a severe form of congenital generalized infection caused by the herpes simplex virus infection were examined (group I). The control group consisted of 26 healthy newborns born to women with uncomplicated pregnancy and childbirth. Determination of the population and subpopulation composition of peripheral blood lymphocytes and monocytes, the level of expression of activation markers, T-regulatory cells (Treg) was carried out by laser fl w cytometry using reagents from Immunotex (France), Caltag (USA), HyCultbiotechnology (Netherlands): FITC (fl escein isothiocyanate) — labeled CD3+, CD4+, CD8+, CD 16+, CD19+, CD282+ and PE (phycoerythrin)-labeled CD95+, CD25+, CD14+. Determination of the number of lymphocytes that have entered apoptosis using a diagnostic kit including Annexin-V, labeled with FITC and propidium iodide (PI), (Caltag, USA). The concentration of IFN-γ, IFN-α, IL-12 in the blood serum of newborns was determined by ELISA using BenderMedsistems test systems.Results. The development of a congenital generalized infection caused by the herpes simplex virus is associated with a lack of IFN-α, IFN-γ, IL-12 production, a decrease in the number of monocytes expressing TLR-2, a decrease in the relative number of CD8+, CD16+ lymphocytes, CD25+ activation markers, on the surface of NK cells, in combination with an increase in CD16 + CD95 +, AnnexinV + PI +, the number of Tregs.Conclusion. The results of the work indicate suppression of the early stages of the innate immune response, impaired effector function of immunocompetent cells, apoptosis processes

A Systematic Review and Meta-Analysis on the Impact of Statin Treatment in HIV Patients on Antiretroviral Therapy

The rate of new human immunodeficiency virus (HIV) infections globally is alarming. Although antiretroviral therapy (ART) improves the quality of life among this group of patients, ARTs are associated with risk of cardiovascular diseases (CVD). Moreover, virally suppressed patients still experience immune activation associated with HIV migration from reservoir sites. Statins are widely recommended as therapeutic agents to control ART-related CVD; however, their impacts on the cluster of differentiation (CD)4 count and viral load are inconsistent. To assess the effect of statins on markers of HIV infections, immune activation and cholesterol, we thoroughly reviewed evidence from randomised controlled trials. We found 20 relevant trials from three databases with 1802 people living with HIV (PLHIV) on statin-placebo treatment. Our evidence showed no significant effect on CD4 T-cell count standardised mean difference (SMD): (-0.59, 95% confidence intervals (CI): (-1.38, 0.19), p = 0.14) following statin intervention in PLHIV on ART. We also found no significant difference in baseline CD4 T-cell count (SD: (-0.01, 95%CI: (-0.25, 0.23), p = 0.95). Our findings revealed no significant association between statins and risk of viral rebound in PLHIV with undetectable viral load risk ratio (RR): (1.01, 95% CI: (0.98, 1.04), p = 0.65). Additionally, we found a significant increase in CD8+CD38+HLA-DR+ T-cells (SMD (1.10, 95% CI: (0.93, 1.28), p < 0.00001) and CD4+CD38+HLA-DR+ T-cells (SMD (0.92, 95% CI: (0.32, 1.52), p = 0.003). Finally, compared to placebo, statins significantly reduced total cholesterol (SMD: (-2.87, 95% CI: (-4.08, -1.65), p < 0.0001)). Our results suggest that the statin lipid-lowering effect in PLHIV on ART may elevate immune activation without influencing the viral load and CD4 count. However, due to the limited evidence synthesised in this meta-analysis, we recommend that future powered trials with sufficient sample sizes evaluate statins' effect on CD4 count and viral load, especially in virally suppressed patients.

Immune Responses and Pathogenesis following Experimental SARS-CoV-2 Infection in Domestic Cats.

Several reports demonstrated the susceptibility of domestic cats to SARS-CoV-2 infection. Here, we describe a thorough investigation of the immune responses in cats after experimental SARS-CoV-2 inoculation, along with the characterization of infection kinetics and pathological lesions. Specific pathogen-free domestic cats (n = 12) were intranasally inoculated with SARS-CoV-2 and subsequently sacrificed on DPI (days post-inoculation) 2, 4, 7 and 14. None of the infected cats developed clinical signs. Only mild histopathologic lung changes associated with virus antigen expression were observed mainly on DPI 4 and 7. Viral RNA was present until DPI 7, predominantly in nasal and throat swabs. The infectious virus could be isolated from the nose, trachea and lungs until DPI 7. In the swab samples, no biologically relevant SARS-CoV-2 mutations were observed over time. From DPI 7 onwards, all cats developed a humoral immune response. The cellular immune responses were limited to DPI 7. Cats showed an increase in CD8+ cells, and the subsequent RNA sequence analysis of CD4+ and CD8+ subsets revealed a prominent upregulation of antiviral and inflammatory genes on DPI 2. In conclusion, infected domestic cats developed a strong antiviral response and cleared the virus within the first week after infection without overt clinical signs and relevant virus mutations.

OA11 Juvenile Sjögren’s syndrome is characterised by dysregulated of B and T memory cell frequencies: a pilot immunophenotyping analysis of this rare disease phenotype

Abstract Background/Aims Sjögren’s syndrome (SS) is an autoimmune rheumatic disease characterised by dryness resulting from chronic lymphocytic infiltration of the exocrine glands and extra glandular manifestations such as arthritis, anemia, and fatigue. The disease is more frequent in women aged 30-50. However, in rare cases, the disease starts in childhood, known as juvenile SS (jSS). Children have different clinical manifestations compared to adults, with dryness being less common, making the diagnosis very challenging. Our aim is to investigate in depth the immune cell profile of patients with jSS and their cytokine profile for better understanding of disease pathogenesis. Methods Peripheral blood was collected from a cohort of patients with jSS (n = 10) and age and sex-matched healthy controls (HC) (n = 10). None of the patients had received B-cell depletion therapy. Peripheral blood immune-phenotyping of 29 immune-cell subsets, including B and T cells, was performed using flow cytometry as we have reported previously for patients with adult-onset SS. Data were analyzed using multiple t-tests and compared with the adult SS immune phenotype. A panel of 15 serum cytokines associated with SS pathology were measured by Luminex, multiple t-tests were performed to detect statistically significant differences between jSS and HCs. Results Patients with jSS had an average age of 18 years (range 16-21) with an average age of disease onset at 14 years (range 12-18). 60% of patients had anti-Ro autoantibodies and 50% had anti-La autoantibodies. Patients with jSS had an altered immune profile compared to age-matched healthy controls (HCs) (mean age = 18 years, range 15-25). In the B cell compartment, jSS patients had higher frequencies of total CD19+ B cells (p = 0.0044), naïve B cells (CD19+IgD+CD27-) (p = 0.0183) and bm2 (CD19+IgD+CD38+) (p = 0.0490) whereas memory B cell subsets such as early bm5 (CD19+IgD-CD38+) and late bm5 (CD19+IgD-CD38-) were significantly reduced (p = 0.0249 and p = 0.0117, respectively). Interestingly, in the CD4+ T cell compartment, central memory T cells (CD4+CD27+CD45RA-) were significantly reduced (p = &amp;lt;0.0001) but effector memory (CD4+CD27-CD45-) and effector memory-re-expressing-CD45RA (EMRA, CD4+CD27-CD45RA+) T-cell subsets were significantly elevated (p = 0.0171 and p = 0.0002 respectively). There was also a significant increase in CD8+CD25-CD127+ responders T cells (p = 0.0392) in jSS patients versus HCs. Serum cytokines levels of APRIL and CCL8 were significantly elevated in jSS. Conclusion This is the first pilot study investigating the immune profile of patients with jSS. In line with our previous studies in adults we detected elevated naïve and reduced frequencies of memory B cells, suggesting an immunological rationale for the use of similar therapies across age. However, we also found a specific dysregulation of the responder CD8+ T cell subpopulation in jSS versus HCs, which requires further investigation. Our cytokine data could indicate an ongoing dysregulation of the homeostasis of mature B-cells and activation of different immune cells. Disclosure L. Martin-Gutierrez: None. H. Peckham: None. A. Radziszewska: None. J. Peng: None. O. Nettey: None. E. Jury: None. C. Ciurtin: None.

Long-Term Host Immune Modulation Following Tisagenlecleucel Administration in Patients with Diffuse Large B-Cell Lymphoma and B-Lineage Acute Lymphoblastic Leukemia.

Background: Chimeric antigen receptor (CAR)-T cells represent a potentially curative strategy for patients with relapsed or refractory (R/R) B-cell malignancies. To elucidate a possible host immune activation following CAR-T-cell infusion, we investigated the effects of tisagenlecleucel administration on the patients' immune populations in 25 patients with R/R diffuse large B-cell lymphoma (DLBCL) and B-lineage acute lymphoblastic leukemia (B-ALL). Methods: The modulation of CAR-T cells over time, the numeric changes, as well as the cytokine production capability of different lymphocyte populations and circulating cytokine levels, were analyzed. Results: Our results confirmed the ability of tisagenlecleucel to control the disease, with an overall response observed in 84.6% of DLBCL and in 91.7% of B-ALL patients at 1-month post-infusion, and showed that most patients who subsequently relapsed could undergo further treatment. Interestingly, we could document a significant increase in CD3+, CD4+, CD8+, and NK cells over time, as well as a decrease in Treg cells, and an increased IFNγ and TNFα production by T lymphocytes. Conclusions: Taken together, our results indicate that in patients with DLBCL and B-ALL, the administration of tisagenlecleucel is capable of inducing a marked and prolonged in vivo modulation/reshaping of the host immune system, both in children and adults.

Fibroblast activation protein targeted radiotherapy induces an immunogenic tumor microenvironment and enhances the efficacy of PD-1 immune checkpoint inhibition

PurposeFAP is a membrane-bound protease under investigation as a pan-cancer target, given its high levels in tumors but limited expression in normal tissues. FAP-2286 is a radiopharmaceutical in clinical development for solid tumors that consists of two functional elements: a FAP-targeting peptide and a chelator used to attach radioisotopes. Preclinically, we evaluated the immune modulation and anti-tumor efficacy of FAP-2287, a murine surrogate for FAP-2286, conjugated to the radionuclide lutetium-177 (177Lu) as a monotherapy and in combination with a PD-1 targeting antibody.MethodsC57BL/6 mice bearing MCA205 mouse FAP-expressing tumors (MCA205-mFAP) were treated with 177Lu-FAP-2287, anti-PD-1, or both. Tumor uptake of 177Lu- FAP-2287 was assessed by SPECT/CT scanning, while therapeutic efficacy was measured by tumor volume and survival. Immune profiling of tumor infiltrates was evaluated through flow cytometry, RNA expression, and immunohistochemistry analyses.Results177Lu-FAP-2287 rapidly accumulated in MCA205-mFAP tumors leading to significant tumor growth inhibition (TGI) and longer survival time. Significant TGI was also observed from anti-PD-1 and the combination. In flow cytometry analysis of tumors, 177Lu-FAP-2287 increased CD8+ T cell infiltration which was maintained in the combination with anti-PD-1. The increase in CD8+ T cells was accompanied by an induction of STING-mediated type I interferon response and higher levels of co-stimulatory molecules such as CD86.ConclusionIn a preclinical model, FAP-targeted radiotherapy enhanced anti-PD-1-mediated TGI by modulating the TME and increasing the recruitment of tumor-infiltrating CD8+ T cells. These findings provide a rationale for clinical studies of combined 177Lu-FAP-2286 radiotherapy and immune checkpoint inhibition in FAP-positive tumors.

586 Increase in CD8+ effector memory cells re-expressing CD45RA (TEMRA) reflects disease activity and enables early prediction of therapeutic outcome in rapidly progressive alopecia areata

586 Increase in CD8+ effector memory cells re-expressing CD45RA (TEMRA) reflects disease activity and enables early prediction of therapeutic outcome in rapidly progressive alopecia areata