Immunological Control Research Articles

A neural network model for predicting the effectiveness of treatment in patients with neovascular glaucoma associated with diabetes mellitus.

The study hypothesizes that neural networks can be an effective tool for predicting treatment outcomes in patients with diabetic neovascular glaucoma (NVG), considering not only baseline intraocular pressure (IOP) values but also inflammation and intraocular microcirculation indicators. To investigate the diagnostic significance of inflammation and intraocular blood circulation indicators in a neural network model predicting the effectiveness of transscleral cyclophotocoagulation (TSC CPC) treatment in patients with NVG of diabetic origin. This retrospective cohort study included 127 patients (127 eyes; aged Me 65.0 years) with painful diabetic NVG and 20 healthy individuals (aged Me 61.5 years) as an immunological control. All patients underwent TSC CPC with a diode laser. Treatment success was defined as achieving an IOP level of ≤ 21 mmHg and maintaining or improving best-corrected visual acuity (BCVA) after 12 months of observation. Preoperative systemic immune-inflammation index (SII = platelets × [neutrophils/lymphocytes]) and systemic inflammation response index (SIRI = neutrophils × [monocytes/lymphocytes]) were calculated. We assessed the values of volumetric pulse blood filling, determined by the rheographic coefficient (RQ, 0/00), using the rheoophthalmography (ROG) method. Multiple regression analysis was used to conclude the significance of treatment efficacy based on initial clinical and laboratory indicators, followed by constructing a prediction model in the neural network. The development of the neural network model identified the most significant "input" parameters: SIRI (100%), RQ (85.7%), and SII (80.7%), which significantly influenced treatment success. The sensitivity of the neural network model was 100%, specificity was 30%, and the percentage of correctly predicted events during testing on the control group was 92.9%. Neural network-based prediction of transscleral cyclophotocoagulation effectiveness for patients with diabetic neovascular glaucoma allows for a sufficiently accurate forecast of treatment success with a probability of 92.9%. We believe the in-time correction of systemic inflammation and intraocular blood circulation can significantly reduce intraocular pressure, preserve visual acuity, and improve the quality of life in patients with diabetic NVG after TSC CPC. Further research is required to support these findings.

Interferon Signature's Members, a Novel Altered Correlation upon Interferon-β Treatment in Multiple Sclerosis Patients.

Multiple sclerosis (MS) is a chronic inflammatory disease that affects the central nervous system and is characterized by extensive brain damage and neurodegeneration. Immunological, genetic, and histological analyses of MS patients provide data in support of the concept that autoimmunity plays a crucial role in the condition's course. It has been proposed that MS may be treated with interferon (IFN)-β and other members of the type I family. Low levels of type I IFN in MS patients may affect immunological control, establish the threshold for an IFN therapeutic response, and be"primed" or "fixed" by IFN therapy. This study was conducted as a cross-sectional study. qRT-PCR was used to examine the expression of two critical IFN regulatory genes, IFI44 and MX1, in MS patients receiving IFN-β treatment. The findings demonstrated a considerable rise in the expression of both genes in MS patients treated with IFN-β compared to those newly diagnosed with the illness. In addition, IFI44 and MX1 might be positively associated with their expression after IFN-β therapy and be regarded as IFN-β responsiveness indicators. The IFI44/MX1 axis could act as one of the crucial regulators of the disease following IFN-β treatment.

HIV-1 diversity in viral reservoirs obtained from circulating T-cell subsets during early ART and beyond.

Even during extended periods of effective immunological control, a substantial dynamic of the viral genome can be observed in different cellular compartments in HIV-1 positive individuals, indicating the persistence of active viral reservoirs. To obtain further insights, we studied changes in the proviral as well as in the viral HIV-1 envelope (Env) sequence along with transcriptional, translational and viral outgrowth activity as indicators for viral dynamics and genomic intactness. Our study identified distinct reservoir patterns that either represented highly sequence-diverse HIV-1 populations or only a single / few persisting virus variants. The single dominating variants were more often found in individuals starting ART during early infection phases, indicating that early treatment might limit reservoir diversification. At the same time, more sequence-diverse HIV reservoirs correlated with a poorer immune status, indicated by lower CD4 count, a higher number of regimen changes and more co-morbidities. Furthermore, we noted that in T-cell populations in the peripheral blood, replication-competent HIV-1 is predominantly present in Lymph node homing TN (naïve) and TCM (central memory) T cells. Provirus genomes archived in TTM (transitional memory) and TEM (effector memory) T cells more frequently tended to carry inactivating mutations and, population-wise, possess changes in the genetic diversity. These discriminating properties of the viral reservoir in T-cell subsets may have important implications for new early therapy strategies, underscoring the critical role of early therapy in preserving robust immune surveillance and constraining the viral reservoir.

In depth analysis of the HIV reservoir confirms effectiveness and safety of DTG/3TC in a phase 4 randomized controlled switch trial (RUMBA).

Reducing the number of active compounds for lifelong HIV treatment is of interest, especially to reduce potential long-term side effects. So far, available data assessing viral control, support the robustness and safety of 2DR (2-drug regimen) ART compared to 3DR. However, further in-depth investigations of the viral reservoirs are mandatory to guarantee long-term safety of these regimens regarding stable intact HIV-1 DNA copies, HIV-1 RNA transcripts and sustained immunological control. The Rumba study is the first prospective randomized controlled trial evaluating the impact of switch from 3DR to 2DR on the viral reservoir. Participants on any stable 2nd generation INSTI-based 3DR regimen with HIV-1 RNA<50 copies/ml plasma for at least 3 months were randomized to switch to dolutegravir/lamivudine (DTG/3TC, N=89) or to switch or stay on bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF, N=45). After 48 weeks, virological, immunological and metabolic parameters were evaluated. We did not observe a significant difference in change over time in the mean number of intact HIV-1 DNA copies/million CD4+ T cells with DTG/3TC compared to B/F/TAF. There was no evidence in this study that switching to DTG/3TC increased the active reservoir by HIV-1 transcription. No significant changes in pro-inflammatory cytokines or major immune cell subsets were observed. Changes in exhaustion and activation of specific cellular subsets were small and bidirectional. Metabolic outcomes are similar between the treatment regimens. This study confirms the safety of DTG/3TC compared to B/F/TAF through viral control after in-depth investigations of the intact HIV-1 reservoir, HIV-1 transcription and inflammatory markers.

The Effect of Mesenchymal Stem Cells on the Wound Infection.

Wound infection often requires a long period of care and an onerous treatment process. Also, the rich environment makes the wound an ideal niche for microbial growth. Stable structures, like biofilm, and drug-resistant strains cause a delay in the healing process, which has become one of the important challenges in wound treatment. Many studies have focused on alternative methods to deal the wound infections. One of the novel and highly potential ways is mesenchymal stromal cells (MSCs). MSCs are mesoderm-derived pluripotent adult stem cells with the capacity for self-renewal, multidirectional differentiation, and immunological control. Also, MSCs have anti-inflammatory and antiapoptotic effects. MScs, as pluripotent stromal cells, differentiate into many mature cells. Also, MSCs produce antimicrobial compounds, such as antimicrobial peptides (AMP), as well as secrete immune modulators, which are two basic features considered in wound healing. Despite the advantages, preserving the structure and activity of MSCs is considered one of the most important points in the treatment. MSCs' antimicrobial effects on microorganisms involved in wound infection have been confirmed in various studies. In this review, we aimed to discuss the antimicrobial and therapeutic applications of MSCs in the infected wound healing processes.

Identification and management of infections caused by Giardia sp., Trichuris sp. and Demodex sp. in captive Brazilian porcupines (Coendou prehensilis)

The Brazilian porcupine (Coendou prehensilis, Rodentia, Erethizontidae) is an arboreal South American nocturnal rodent. Switzerland is home to one of the largest captive colonies in Europe. In June 2022, most of the animals in this colony showed severe diarrhoea, and Giardia sp. cysts were detected. All the animals were treated with metronidazole (75 mg/animal/day orally) for five days, repeating after two weeks. The diarrhoea continued, sometimes containing blood, and further analyses revealed Giardia sp. cysts and Trichuris sp. eggs with a particular undulating eggshell in pooled samples. The soil layer of some enclosures was removed to thoroughly clean and disinfect the underlying concrete floor. The animals were treated with fenbendazole (50 mg/kg/day orally) for 5 days repeating after three days. Giardia sp. cysts were not further detected. However, Trichuris sp. eggs were detected in branch bark samples and in six animals 2–3 months after treatment. The treatment with fenbendazole was repeated and no further Trichuris sp. eggs were detected. A 18S rRNA fragment consensus sequence showed 98.58% identity with Trichuris fossor. The Trichuris sp. in C. prehensilis may represent a new species, specific for arboreal porcupines. Demodex mites were observed in faecal flotations and thereafter in skin scrapings from five animals (four of them being family-related). A 16S consensus sequence showed 86.4% identity with other Demodex species. The animals were initially treated with moxidectin (0.4 and 0.8 mg/kg orally) and afterwards with sarolaner (10 mg/animal) but the treatments were not completely effective since in control scrapings, two animals evidenced few non-motile mites. An individual susceptibility and poor immunological control of the infection is suggested. Treatment with fenbendazole was effective against Giardia sp. and Trichuris sp. infections; however, reinfections may occur if the enclosures and tree branches are not deep cleaned and disinfected or replaced.

Mechanistic Pathways and Clinical Implications of GLP-1 Receptor Agonists in Type 1 Diabetes Management.

GLP-1 receptor agonists, which were initially intended to treat type 2 diabetes patients, have demonstrated promise as an adjuvant therapy for type 1 diabetes (T1D). These medications can manage T1D by improving β-cell function, reducing glucose fluctuation, and providing cardioprotective effects. Recent research suggests that boosting cell proliferation and lowering apoptosis can help maintain the bulk of β-cells. Furthermore, GLP-1 receptor agonists have potent anti-inflammatory characteristics, improving immunological control and lowering systemic inflammation, both of which are critical for reducing autoimmune damage in T1D. Beyond glucose control, these agonists have neuroprotective qualities and aid in weight management. Combining these medications with insulin could significantly change how T1D is managed. The clinical data and biological mechanisms discussed in this review support the potential use of GLP-1 receptor agonists in T1D.

Mass cytometric single cell immune profiles of peripheral blood from acute myeloid leukemia patients in complete remission with measurable residual disease.

Measurable residual disease (MRD) is detected in approximately a quarter of AML chemotherapy responders, serving as a predictor for relapse and shorter survival. Immunological control of residual disease is suggested to prevent relapse, but the mechanisms involved are not fully understood. We present a peripheral blood single cell immune profiling by mass cytometry using a 42-antibody panel with particular emphasis on markers of cellular immune response. Six healthy donors were compared with four AML patients with MRD (MRD+) in first complete remission (CR1MRD+). Three of four patients demonstrated a favorable genetic risk profile, while the fourth patient had an unfavorable risk profile (complex karyotype, TP53-mutation) and a high level of MRD. Unsupervised clustering using self-organizing maps and dimensional reduction analysis was performed for visualization and analysis of immune cell subsets. CD57+ natural killer (NK)-cell subsets were found to be less abundant in patients than in healthy donors. Both T and NK cells demonstrated elevated expression of activity and maturation markers (CD44, granzyme B, and phosho-STAT5 Y694) in patients. Although mass cytometry remains an expensive method with limited scalability, our data suggest the utility for employing a 42-plex profiling for cellular immune surveillance in whole blood, and possibly as a biomarker platform in future clinical trials. The findings encourage further investigations of single cell immune profiling in CR1MRD+ AML-patients.

Stimulation-induced cytokine polyfunctionality as a dynamic concept

Cytokine polyfunctionality is a well-established concept in immune cells, especially T cells, and their ability to concurrently produce multiple cytokines has been associated with better immunological disease control and subsequent effectiveness during infection and disease. To date, only little is known about the secretion dynamics of those cells, masked by the widespread deployment of mainly time-integrated endpoint measurement techniques that do not easily differentiate between concurrent and sequential secretion. Here, we employed a single-cell microfluidic platform capable of resolving the secretion dynamics of individual PBMCs. To study the dynamics of poly-cytokine secretion, as well as the dynamics of concurrent and sequential polyfunctionality, we analyzed the response at different time points after ex vivo activation. First, we observed the simultaneous secretion of cytokines over the measurement time for most stimulants in a subpopulation of cells only. Second, polyfunctionality generally decreased with prolonged stimulation times and revealed no correlation with the concentration of secreted cytokines in response to stimulation. However, we observed a general trend towards higher cytokine secretion in polyfunctional cells, with their secretion dynamics being distinctly different from mono-cytokine-secreting cells. This study provided insights into the distinct secretion behavior of heterogenous cell populations after stimulation with well-described agents and such a system could provide a better understanding of various immune dynamics in therapy and disease.

Stimulation-induced cytokine polyfunctionality as a dynamic concept.

Cytokine polyfunctionality is a well-established concept in immune cells, especially T cells, and their ability to concurrently produce multiple cytokines has been associated with better immunological disease control and subsequent effectiveness during infection and disease. To date, only little is known about the secretion dynamics of those cells, masked by the widespread deployment of mainly time-integrated endpoint measurement techniques that do not easily differentiate between concurrent and sequential secretion. Here, we employed a single-cell microfluidic platform capable of resolving the secretion dynamics of individual PBMCs. To study the dynamics of poly-cytokine secretion, as well as the dynamics of concurrent and sequential polyfunctionality, we analyzed the response at different time points after ex vivo activation. First, we observed the simultaneous secretion of cytokines over the measurement time for most stimulants in a subpopulation of cells only. Second, polyfunctionality generally decreased with prolonged stimulation times and revealed no correlation with the concentration of secreted cytokines in response to stimulation. However, we observed a general trend towards higher cytokine secretion in polyfunctional cells, with their secretion dynamics being distinctly different from mono-cytokine-secreting cells. This study provided insights into the distinct secretion behavior of heterogenous cell populations after stimulation with well-described agents and such a system could provide a better understanding of various immune dynamics in therapy and disease.

The immunology of systemic lupus erythematosus.

Understanding the pathogenesis and clinical manifestations of systemic lupus erythematosus (SLE) has been a great challenge. Reductionist approaches to understand the nature of the disease have identified many pathogenetic contributors that parallel clinical heterogeneity. This Review outlines the immunological control of SLE and looks to experimental tools and approaches that are improving our understanding of the complex contribution of interacting genetics, environment, sex and immunoregulatory factors and their interface with processes inherent to tissue parenchymal cells. Efforts to advance precision medicine in the care of patients with SLE along with treatment strategies to correct the immune system hold hope and are also examined.

Hepatitis B cure: Current situation and prospects.

Achievement of a 'clinical cure' in chronic hepatitis B (CHB) implies sustained virological suppression and immunological control over the infection, which is the ideal treatment goal according to domestic and international CHB management guidelines. Clinical practice has shown encouraging results for specific patient cohorts using tailored treatment regimens. These regimens incorporate either nucleos(t)ide analogs, immunomodulatory agents such as pegylated interferon α, or a strategic combination of both, sequentially or concurrently administered. Despite these advancements in the clinical handling of hepatitis B, achieving a clinical cure remains elusive for a considerable subset of patients due to the number of challenges that preclude the realization of optimal treatment outcomes. These include, but are not limited to, the emergence of antiviral resistance, incomplete immune recovery, and the persistence of covalently closed circular DNA. Moreover, the variance in response to interferon therapy and the lack of definitive biomarkers for treatment cessation also contribute to the complexity of achieving a clinical cure. This article briefly overviews the current research progress and existing issues in pursuing a clinical cure for hepatitis B.

Pelareorep driven blood TIL expansion in patients with pancreatic, breast and colon cancer.

e14625 Background: Tumor infiltrating lymphocytes (TILs) represent a major immunological tumor control mechanism that is associated with better prognosis in cancer. One emerging cancer treatment approach to leverage the therapeutic potential of TILs is the expansion and adoptive cell transfer of autologous TILs, commonly referred to as autologous adoptive therapy (ACT). ACT clinical studies have shown encouraging results. While the majority of these trials have targeted melanoma, impressive clinical benefit has also been observed in cervical cancer, with preliminary efficacy in colorectal cancer (CRC), cholangiocarcinoma, non-small cell lung cancer, and breast cancer. Pelareorep (pela) is a live, replication competent reovirus (T3D) that selectively replicates in cancer cells and represents a new class of immunotherapy currently being explored in multiple clinical settings. To examine the effect of pela therapy on TIL expansion we applied T cell receptor sequencing of matched tumor tissue and whole blood pre- and post-treatment in a subset of breast, pancreatic, and (CRC) subjects receiving chemotherapy and atezolizumab, an anti-PD-L1 therapy, and intravenous treatment with pela. Methods: Identification of TIL clones was performed by immunosequencing of the CDR3 regions of human T-cell receptor-β (TCRβ) chains (ImmunoSEQ Assay, Adaptive Biotechnologies). DNA was isolated from tissue and blood at baseline and from blood collected post-treatment. TCRβ CDR3 regions were amplified by a multiplex, bias-controlled PCR with primers targeting the V and J genes of T cells as well as primers targeting housekeeping genes to quantitate the total nucleated cells in each sample. PCR products were sequenced on an Illumina NextSeq. Results: Pela treatment was observed to increase the expansion of pre-existing and new TIL clones in the blood in from all tumor samples after one cycle of treatment. We also observed that pre-existing TIL clonal expansion in the blood seemed to correlate with reductions in tumor volume in pancreatic cancer and to a lesser extent in CRC patients who received multiple cycles of therapy. Interestingly, unlike atezolizumab, pela’s clinical activity was lost when combined with avelumab, a PD-LI inhibitor capable of binding Fc receptors and inducing ADCC. The addition of avelumab eliminated pre-existing TIL expansion in the blood highlighting the importance of TIL expansion following pela therapy. Conclusions: These findings, while preliminary, suggest that the clinical benefits observed following pela immunotherapy may be mechanistically analogous to ACT. Accordingly, pela therapy may offer a means to directly expand TILs without the need for tumor resection, ex vivo TIL expansion, T cell ablation and IL-2 therapy. Clinical trial information: Eudra-CT: 2020-003996-16 .

HIV rapid tests immunological internal control can be misleading

Objective: To assess whether internal controls (IC) of HIV rapid tests (HRT) and a rapid confirmatory assay (Geenius) were designed to solely prevent procedural missteps or wether they can also alert for insufficient sample quantity.Material and methods: Thirteen tests were evaluated using distilled water, phosphate buffered saline, and HIV-1 positive serum samples : Autotest VIH® (Autotest) (AAZ-LMB), INSTITM HIV-1/HIV-2 Antibody Test (INSTI) (Biolytical), MULTISURE® HIV Rapid Test (MULTISURE) (MP Diagnostics Asia Pacific Pte. CE Ltd), Biosynex® HIVTOP (Biosynex), Exacto HIVTOP® (Biosynex), Exacto® TEST HIV PRO (Biosynex), GenieTM Fast HIV 1/2 (Bio-Rad laboratories), GeeniusTM HIV 1/2 Confirmatory Assay (Geenius) (Bio-Rad laboratories), VIKIA® HIV 1/2 (bioMérieux), FIRST RESPONSE® Test VIH 1-2.O CARTE (Premier Medical Corporation), Hexagon HIV (Human), DetermineTM HIV EARLY DETECT (Determine) (Abbott), and ONE STEP Anti-HIV 1&2 (InTec PRODUCTS, Xiamen, Fujian P. R. China).Results: Among the 13 tests, 4 were invalid when tested without human serum sample: Autotest, Geenius, INSTI and MULTISURE. The Autotest and INSTI accurately labelled diluted samples, with a Test line positive at least one dilution below the IC. Geenius test line and IC were positive up to the same dilution with automated reading. However, MULTISURE IC was positive up to 1000-fold dilution higher than its test line, making it unsuitable to alert for low sample quantity.

T Cell Homeostasis Disturbances in a Cohort of Long-Term Elite Controllers of HIV Infection.

Elite controllers (ECs) are people living with HIV (PLWH) able to control HIV replication without antiretroviral therapy and have been proposed as a model of a functional HIV cure. Much evidence suggests that this spontaneous control of HIV has a cost in terms of T cell homeostasis alterations. We performed a deep phenotypic study to obtain insight into T cell homeostasis disturbances in ECs maintaining long-term virologic and immunologic control of HIV (long-term elite controllers; LTECs). Forty-seven PLWH were included: 22 LTECs, 15 non-controllers under successful antiretroviral therapy (onART), and 10 non-controllers not receiving ART (offART). Twenty uninfected participants (UCs) were included as a reference. T cell homeostasis was analyzed by spectral flow cytometry and data were analyzed using dimensionality reduction and clustering using R software v3.3.2. Dimensionality reduction and clustering yielded 57 and 54 different CD4 and CD8 T cell clusters, respectively. The offART group showed the highest perturbation of T cell homeostasis, with 18 CD4 clusters and 15 CD8 clusters significantly different from those of UCs. Most of these alterations were reverted in the onART group. Interestingly, LTECs presented several disturbances of T cell homeostasis with 15 CD4 clusters and 13 CD8 clusters different from UC. Moreover, there was a specific profile of T cell homeostasis alterations associated with LTECs, characterized by increases in clusters of naïve T cells, increases in clusters of non-senescent effector CD8 cells, and increases in clusters of central memory CD4 cells. These results demonstrate that, compared to ART-mediated control of HIV, the spontaneous control of HIV is associated with several disturbances in CD4 and CD8 T cell homeostasis. These alterations could be related to the existence of a potent and efficient virus-specific T cell response, and to the ability to halt disease progression by maintaining an adequate pool of CD4 T cells.

Abstract PO1-02-01: T Cell Receptor Sequencing to Monitor Pelareorep-Induced Expansion of Tumor Infiltrating Lymphocytes

Abstract Background: Tumor infiltrating lymphocytes (TILs) represent a major immunological tumor control mechanism that is associated with better prognosis in breast cancer. We have previously reported the effect of pelareorep (pela), an intravenously delivered, non-engineered oncolytic reovirus, on a composite measurement of TILs and tumor cellularity (CelTIL) from the AWARE-1 window-of-opportunity study in patients with early breast cancer (eBC). These results showed treatment increased in CelTIL scores especially in the atezolizumab group. To confirm and extend these findings we applied T cell receptor sequencing of matched tumor tissue and whole blood pre and post-treatment from the AWARE-1 study to further explore the effects of pela therapy on TILs. Methods: Newly diagnosed HR+/HER2- eBC patients were enrolled into two cohorts: Cohort 1: pela + letrozole (n=10); and Cohort 2: pela + letrozole + atezolizumab (n=10). Pela was administered on days 1, 2 and 8, 9, and atezolizumab was given on day 3. Tumor biopsies (FFPE samples) collected pre-treatment (~D-23) and on day ~21 when tumors were surgically removed. T cell fraction analysis and T cell receptor sequencing were performed by Adaptive Biotechnology (Seattle, Washington) Immunoseq protocol. Results: As was shown for the CelTIL scores, an increase in the tumor T cell fraction, a direct measurement of TILs, was observed at 1-month post-treatment in both cohorts with a mean percent increase of 21% for Cohort 1 and 67% for Cohort 2. Through TCR-sequencing of tumor tissue, we identified TIL clones and tracked their differential abundance in tumor tissue and blood post-treatment. The results of this analysis at Day 21 showed a mean post-treatment expansion of 10 tumor specific clones in the blood for Cohort 1, and a mean post-treatment increase of 40 tumor specific clones for Cohort 2. Conclusions: These results confirm the previously reported CelTIL results from AWARE-1 on pela-induced increases in TILs and demonstrate the expansion of these clones in both tumor and blood as a consequence of pela therapy for HR+/HER2- breast cancer. Citation Format: Richard Trauger, Houra Loghmani, Matt Coffey, Fernando Salvador, Joaquín Gavilá, Luis Manso, Tomás Pascual, Aleix Prat, Thomas Heineman. T Cell Receptor Sequencing to Monitor Pelareorep-Induced Expansion of Tumor Infiltrating Lymphocytes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-02-01.

MODERN ASPECTS OF CERVICAL CANCER TREATMENT IN WOMEN OF REPRODUCTIVE AGE

The problem of cervical cancer prevention (cervical cancer) is interdisciplinary since it can be solved only through the joint efforts of gynecologists, oncologists, virologists, and immunologists. The role of human papillomaviruses in the development of oncological processes is considered. The main emphasis is placed on the pathogenetic mechanisms of cervical cancer, where the etiotropic role of viruses is beyond doubt. the role of cellular protein p53 and hormonal factors (estrogens) in the carcinogenesis of HPV-infected cervical epithelial cells, immunological control of HPV-induced cervical cancer.

A hepatitis B virus (HBV) sequence variation graph improves alignment and sample-specific consensus sequence construction.

Nearly 300 million individuals live with chronic hepatitis B virus (HBV) infection (CHB), for which no curative therapy is available. As viral diversity is associated with pathogenesis and immunological control of infection, improved methods to characterize this diversity could aid drug development efforts. Conventionally, viral sequencing data are mapped/aligned to a reference genome, and only the aligned sequences are retained for analysis. Thus, reference selection is critical, yet selecting the most representative reference a priori remains difficult. We investigate an alternative pangenome approach which can combine multiple reference sequences into a graph which can be used during alignment. Using simulated short-read sequencing data generated from publicly available HBV genomes and real sequencing data from an individual living with CHB, we demonstrate alignment to a phylogenetically representative 'genome graph' can improve alignment, avoid issues of reference ambiguity, and facilitate the construction of sample-specific consensus sequences more genetically similar to the individual's infection. Graph-based methods can, therefore, improve efforts to characterize the genetics of viral pathogens, including HBV, and have broader implications in host-pathogen research.

Abstract 5278: Immunological control by PARP inhibitors for successful immunotherapy in metastatic ovarian carcinoma

Abstract Epithelial ovarian carcinoma (EOC) is among the top five causes of cancer-related death in women. Most women with EOC achieve indeed complete remission after primary or interval cytoreductive surgery combined with chemotherapy based on a platinum-taxane doublet. Homologous recombination (HR) defects imposed by germline or somatic BRCA1 DNA repair-associated (BRCA1) or BRCA2 mutations are not only key determinants of platinum sensitivity in EOC patients but also provide a strong rationale for maintenance therapy based on poly(ADP-ribose) polymerase (PARP) inhibitors, which is generally associated with improved progression-free survival (PFS). Besides direct cytotoxic and cytostatic properties, PARPi have been shown to mediate multipronged immunostimulatory effects, largely reflecting their ability to inhibit DNA repair in malignant cells and indicating the possibility to synergy with immune-check point inhibitors (ICIs). Here, using multiparametric flow cytometry, multiplex immunostaining, single cells transcriptomics and functional studies in experimental syngeneic BR5Brca1-/- mouse model and EOC tumor samples, we discuss PARPi immunomodulatory activity, with a specific focus on molecular and cellular pathways that can be harnessed to develop superior combinatorial regimens for clinical management. Thus, PARPi might enhance the mutational load of EOCs as consequence of unrepaired DNA damage and DAMPs exposure, favoring T cell infiltration, but also appear to drive robust type I IFN secretion downstream of cyclic GMP-AMP synthase and stimulator of IFN response cGAMP interactor 1 activation. In addition, the combination of PARPi with ICIs positively regulate the balance between adaptive anti-tumor immunity and innate myeloid components and significantly improve the cytotoxic T cell profile as shown in both experimental mice model and HGSOC tumor samples. We surmise that rationally designed combinations of PARPi and immunotherapeutic agents might be critical to unlock immunosuppression in the EOC microenvironment in support of clinical efficacy. Citation Format: Peter Holicek, Sarka Vosahlikova, Irena Moserova, Sandra Orsulic, Romana Mikyskova, Michal Hensler, Lenka Kasikova, Tereza Lanickova, Ondrej Novotny, Milan Reinis, Jana Drozenova, Ales Ryska, Jan Laco, Lukas Rob, Michael Halaska, David Cibula, Radek Spisek, Jitka Fucikova. Immunological control by PARP inhibitors for successful immunotherapy in metastatic ovarian carcinoma [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 5278.

Antibody-mediated depletion of select leukocyte subsets in blood and tissue of nonhuman primates.

Understanding the immunological control of pathogens requires a detailed evaluation of the mechanistic contributions of individual cell types within the immune system. While knockout mouse models that lack certain cell types have been used to help define the role of those cells, the biological and physiological characteristics of mice do not necessarily recapitulate that of a human. To overcome some of these differences, studies often look towards nonhuman primates (NHPs) due to their close phylogenetic relationship to humans. To evaluate the immunological role of select cell types, the NHP model provides distinct advantages since NHP more closely mirror the disease manifestations and immunological characteristics of humans. However, many of the experimental manipulations routinely used in mice (e.g., gene knock-out) cannot be used with the NHP model. As an alternative, the in vivo infusion of monoclonal antibodies that target surface proteins on specific cells to either functionally inhibit or deplete cells can be a useful tool. Such depleting antibodies have been used in NHP studies to address immunological mechanisms of action. In these studies, the extent of depletion has generally been reported for blood, but not thoroughly assessed in tissues. Here, we evaluated four depleting regimens that primarily target T cells in NHP: anti-CD4, anti-CD8α, anti-CD8β, and immunotoxin-conjugated anti-CD3. We evaluated these treatments in healthy unvaccinated and IV BCG-vaccinated NHP to measure the extent that vaccine-elicited T cells - which may be activated, increased in number, or resident in specific tissues - are depleted compared to resting populations in unvaccinated NHPs. We report quantitative measurements of in vivo depletion at multiple tissue sites providing insight into the range of cell types depleted by a given mAb. While we found substantial depletion of target cell types in blood and tissue of many animals, residual cells remained, often residing within tissue. Notably, we find that animal-to-animal variation is substantial and consequently studies that use these reagents should be powered accordingly.