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Simultaneous measurement of surface proteins and gene expression within single cells offers high resolution snapshots of complex cell populations. These methods rely on staining cells with oligo-conjugated antibodies analogous to staining for flow- and mass cytometry. Unlike flow- and mass cytometry, signal from oligo-conjugated antibodies is not hampered by spectral overlap or limited by the number of metal isotopes, making it a highly sensitive and scalable approach. Signal from oligo-conjugated antibodies is quantified by counting reads from high-throughput sequencing. Consequently, cost of sequencing is strictly dependent on the signal intensities and background from the pool of antibodies used in analysis. Thus, considering the "cost-of-signal" as well as optimizing "signal-to-noise", makes titration of oligo-conjugated antibody panels more complex and even more important than for flow- and mass cytometry. In this study, we investigated the titration response of a panel of oligo-conjugated antibodies towards four variables: Antibody concentration, staining volume, cell number at staining, and tissue of origin. We find that staining with high antibody concentrations recommended by published protocols and commercial vendors cause unnecessarily high background signal and that concentrations of many antibodies can be drastically reduced without loss of biological information. Reducing staining volume only affects antibodies targeting highly abundant epitopes used at low concentrations and can be counteracted by reducing cell numbers at staining. We find that background signal from empty droplets can account for a major fraction of the total sequencing reads and is primarily derived from antibodies used at high concentrations. Together, this study provides new insight into the titration response and background signal of oligo-conjugated antibodies and offers concrete guidelines on how such panels can be improved.

We conducted a review of the literature describing the most up-to-date diagnosis and treatment options of chronic bacterial prostatitis. Recurrence after oral antimicrobial therapy is common, due in part to the rising rates of antimicrobial resistance and inability to completely clear the offending bacteria from the prostate following prostatitis. Recent literature has described various treatment options for chronic bacterial prostatitis refractory to conventional antimicrobial agents, including the use of alternative agents such as fosfomycin, direct antimicrobial injections into the prostate, surgical removal of infected prostatic tissue, chronic oral antibiotic suppression, and an emerging novel therapy utilizing bacteriophages to target antibiotic resistant bacteria. Management of chronic bacterial prostatitis, especially recurrence after oral antimicrobial treatment, remains challenging. This review highlights an urgent need for further evidence assessing the efficacy and safety of treatment modalities for chronic bacterial prostatitis refractory to conventional oral antimicrobials.

INTRODUCTION AND OBJECTIVE:Immune checkpoint inhibitors (ICIs) are frequently used in the treatment of metastatic melanoma, clear cell renal cell carcinoma, and an increasing number of other cancer...

Recently identified SARS-CoV-2 variants Mu and C.1.2 have spike proteins with mutations that might confer resistance to natural and vaccine-elicited antibodies. Analysis of neutralizing antibody titers in the sera of vaccinated individuals without previous history of infection and from convalescent individuals showed partial resistance of the viruses. In contrast, sera from individuals with a previous history of SARS-CoV-2 infection who were subsequently vaccinated, neutralized variants with titers 4-11-fold higher, providing a rationale for vaccination of infection-experienced individuals. The heavily mutated C.1.2 spike was the most antibody neutralization-resistant spike to date; however, the avidity of C.1.2 spike protein for ACE2 was low. This finding suggests that the virus evolved to escape the humoral response but has a decrease in fitness suggesting that it may cause milder disease or be less transmissible. It may be difficult for the spike protein to evolve to escape neutralizing antibodies while maintaining high affinity for ACE2., Graphical Abstract, Tada et al. show that infection with SARS-CoV-2 followed by vaccination results in broadly neutralizing antibody. The C.1.2 variant has a highly mutated spike and is the most neutralization-resistant variant; however, its affinity for ACE2 is decreased. Thus, the virus cannot evolve to escape humoral response without becoming less fit.

Human primary immunodeficiencies are inherited diseases that can provide valuable insight into the immune system. Calcium (Ca2+) is a vital secondary messenger in T lymphocytes regulating a vast array of important events including maturation, homeostasis, activation, and apoptosis and can enter the cell through CRAC, TRP, and Cav channels. Here we describe three Cav1.4-deficient siblings presenting with X-linked incomplete congenital stationary night blindness as well as an immune phenotype characterized by several recurrent infections. Complete exome sequencing demonstrated that the patients share only a single pathogenic allele; a R625X (p.Arg625Ter) point mutation that leads to a premature stop codon in theCACNA1Fgene encoding the L-type Ca2+channel Cav1.4. The subjects uniformly exhibited an expansion of central and effector memory T lymphocytes, and evidence of T lymphocytes exhaustion with corresponding upregulation of inhibitory receptors. Moreover, the sustained elevated levels of activation markers on B lymphocytes suggest that they are in a chronic state of activation. Finally, the T lymphocytes from patients andCACNA1Fknockdown Jurkat T lymphocytes exhibited a reduced Ca2+flux, compared to controls. This is the first example where the mutation of any Cav channel causes a primary immunodeficiency in humans and establishes the physiological importance of Cav channels in the human immune system.

Background The Institute of Medicine Report and Department of Health and Human Services Healthy People 2020 Initiative have called for steps to address health disparities facing sexual minorities. Aim We sought to characterize the practice patterns of sexual health specialists as they relate to men who have sex with men (MSM). Methods Surveys were electronically mailed to 696 members of the Sexual Medicine Society of North America (SMSNA). Responses were compared using descriptive statistics and χ2 analysis with Yates correction where appropriate. Outcomes Outcomes were SMSNA members' demographics, their assessment of their patients' sexual orientation, and adaptation of care to address the specific needs of their MSM patients. Results 92 (13.2%) Members responded. While 93.3% of respondents reported treating MSM patients, only 51.7% routinely asked about sexual orientation. Of those that do not ask, 41.9% responded that sexual orientation is irrelevant to their patients' care and 25.6% responded that patients will disclose this information if the patient thinks it is important. Practitioners inquiring about sexual orientation were more likely to practice in urban settings; more likely to inquire about a greater number of sexual behaviors; more likely to tailor their care to lesbian, gay, bisexual, and transgender needs; and more likely to endorse the notion that homosexual/bisexual patients have unique sexual dysfunction concerns. Clinical Translation Limited and uneven inquiry about sexual orientation necessitates efforts aimed at tailoring care to the needs of sexual minority patients. Strengths & Limitations This survey addresses a gap in the literature by investigating predictors and practical consequences of practitioner inquiry about sexual orientation. Limitations include a low response rate, disparate study population, the potential influence of respondent social desirability biases, and spurious associations due to a multiplicity of statistical tests. Conclusion Only about half of surveyed SMSNA members ask their patients' sexual orientation; inquiry about sexual orientation was associated with practice setting and with provider practice patterns and attitudes.

Sayin et al. describe the spatial distribution and functional characteristics of T follicular regulatory (Tfr) cells in human lymph nodes. Contrary to existing paradigms, Tfr-mediated suppression appears to be most efficient at the T-B border and within the follicle, not in the germinal center., T follicular regulatory (Tfr) cells are a population of CD4+ T cells that express regulatory T cell markers and have been shown to suppress humoral immunity. However, the precise mechanisms and location of Tfr-mediated suppression in the lymph node (LN) microenvironment are unknown. Using highly multiplexed quantitative imaging and functional assays, we examined the spatial distribution, suppressive function, and preferred interacting partners of Tfr cells in human mesenteric LNs. We find that the majority of Tfr cells express low levels of PD-1 and reside at the border between the T cell zone and B cell follicle, with very few found in the germinal centers (GCs). Although PD-1+ Tfr cells expressed higher levels of CD38, CTLA-4, and GARP than PD-1Neg Tfr cells, both potently suppressed antibody production in vitro. These findings highlight the phenotypic diversity of human Tfr cells and suggest that Tfr-mediated suppression is most efficient at the T-B border and within the follicle, not in the GC., Graphical Abstract

The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56hi versus CD56dim defined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation stages. Such comprehensive cytotoxic profiling may identify distinct patterns of cytotoxic potential relevant for specific infections, autoimmunity or tumor settings.

CD4 + T cells play a critical role in the response to chronic viral infections during the acute phase and in the partial containment of infections once chronic infection is established. As infection persists, the virus-specific CD4 + T cell response begins to shift in phenotype. The predominant change described in both mouse and human studies of chronic viral infection is a decrease in detectable T helper type (Th)1 responses. Some Th1 loss is due to decreased proliferative potential and decreased cytokine production in the setting of chronic antigen exposure. However, recent data suggest that Th1 dysfunction is accompanied by a shift in the differentiation pathway of virus-specific CD4 + T cells, with enrichment for cells with a T follicular helper cell (Tfh) phenotype. A Tfh-like program during chronic infection has now been identified in virus-specific CD8 + T cells as well. In this review, we discuss what is known about CD4 + T cell differentiation in chronic viral infections, with a focus on the emergence of the Tfh program and the implications of this shift with respect to Tfh function and the host–pathogen interaction.

BackgroundDespite the success of immune checkpoint blockade (ICB), many patients still fail to achieve durable clinical benefit. Previous studies have shown that CD8 T cells are reinvigorated by ICB. However, not all patients with this immunological response experience an effective clinical response, suggesting additional parameters may be relevant.DNA damage and repair (DDR) has been extensively studied in the context of inducing cell death of highly-proliferating tumor cells. However, whether T cell-intrinsic DDR impacts T cell differentiation and function, and how the coordination of DDR affects immunological and clinical response to proliferation-inducing ICBs have been largely unexplored. We hypothesized that the T celI-intrinsic DDR responses to proliferative and genotoxic stress might contribute to the disparity between immunological and clinical response.MethodsTo understand the impact of cell-intrinsic DDR on T cell differentiation and responses to cancer therapies, we developed a novel high-dimensional cytometry platform. This DDR-Immune platform enables simultaneous analysis of T cell differentiation state and multiple DDR pathways at single cell resolution. We then investigated immune reinvigoration and its association with DDR, in a cohort of chemotherapy-resistant hypermutated or microsatellite instability-high (MSI-H) uterine cancer patients treated with nivolumab. Peripheral blood samples were examined every 2–4 weeks after initiating anti-PD-1 treatment (N = 21).ResultsThe DDR-Immune platform revealed consistent T cell subset specific patterns of DDR, as well as specific DDR pathways induced by different types of DNA damage, such as γ-irradiation (IR), UV irradiation (UV) or proliferative stress (i.e. anti-CD3/CD28 stimulation). For example, terminally differentiated effector cells had higher DNA damage accumulation and cell death. In contrast, stem cell memory (TSCM) and regulatory T cells (Treg) displayed high DDR with less cell death, suggesting better cell-intrinsic DDR against genotoxic stress for survival advantage. In hypermutated MSI-H uterine cancer patients, CD8 T cells underwent rapid pharmacodynamic proliferation 2–4 weeks after starting PD-1 blockade, which did not correlate with clinical response. Application of the DDR-Immune platform to this cohort revealed, however, that in clinical responders but not clinical non-responders, Ki67+ CD8 T cells responding to PD-1 blockade had rapid induction of DDR represented as a spike increase of phosphorylated-ATM, presumably adapting T cell ‘fitness’ in response to proliferative stress induced by PD-1 blockade.ConclusionsCollectively, the new platform reveals previously unrecognized roles for T cell-intrinsic DDR as a novel determinant of immune responsiveness and clinical outcome to ICB and have potential application to other cancer therapies including chemotherapy and radiotherapy.Ethics ApprovalThe study was approved by MSKCC Ethics Board, approval number 17–180 (NCT03241745).

Among cationic polymers, chitosan (CS) is a promising non-viral vector due to its biocompatibility, biodegradability and safety profile. However, its application for gene delivery is hindered by its poor solubility at physiological pH. In this study, novel water-soluble chitosan derivatives containing triethylphosphonium chloride ((N-(1-carboxybutyl-4-triethylphosphonium chloride) chitosan = TEPB-CS)) or triethylammonium chloride ((N-(1-carboxybutyl-4-triethylammonium chloride) chitosan = TEAB-CS)) with 4-carbon spacers separating the cationic units from the chitosan backbone were synthesized by carbodiimide mediated coupling reactions. Two different degrees of substitution of quaternary phosphonium (43% and 21%) and ammonium (40.6% and 26%) were synthesized. The polymers with higher degrees of substitutions displayed higher solubility in aqueous media and were further grafted with mPEG (TEAB-CS-g-mPEG and TEPB-CS-g-mPEG) and used for DNA complexation, cytotoxicity, apoptosis, and gene transfection studies. Chemical characterization of the above polymers was performed by FTIR, 1H NMR, and 31P NMR, and the degree of substitution of the grafted phosphonium and ammonium and mPEG was calculated by 1H NMR. The polymers efficiently bound pDNA at weight ratio of polymer:pDNA of 2.8:1. From dynamic light scattering experiments, the mean diameters of the calf thymus DNA complexes with the polymers, in water and phosphate buffer (pH 7.4), were in the range of 140 – 330 nm. Zeta potential of these complexes in water became positive as the weight ratios of the polymers/DNA exceeded 2:1. Most of these polymers mildly affect the viability of HeLa cells with a final concentration of 10 µg/mL or higher. Cell viability was only modestly compromised in the presence of 100 µg/mL of TEPB-CS-g-mPEG, and this derivative was also found to display the highest transfection efficiencies among all four polymers. Apoptosis measurements by flow cytometry indicated that both TEAB-CS-g-mPEG and TEPB-CS-g-mPEG induce no perceptible and mild apoptosis with their concentration at 100 µg/mL, respectively. Together, the present study provides evidence that these PEGylated TEPB-CS and TEAB-CS are safe and effective vectors for efficient nucleic acid delivery.

237 Background: For patients of reproductive age with cancer, counseling regarding reproductive risks associated with systemic therapy and facilitating access to fertility services are essential to quality care. We conducted a quality improvement study to improve rates of fertility preservation counseling at the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center (SKCCC) in Baltimore, MD. Methods: We formed a multidisciplinary team as part of the ASCO Quality Training Program. We aimed to increase the rate of fertility preservation counseling for patients of reproductive age (18-40 years) with newly diagnosed cancer who were initiating systemic therapy from a baseline (June 2019- January 2020) of 36.7% (25/ 68) to 70.0% by February, 2021. Data sources included the electronic medical record and direct verification with patients by phone. We surveyed patients, oncologists, and experts in reproductive endocrinology and urology to identify barriers to optimal care. After considering a prioritization matrix, we implemented Plan-Do-Study-Act (PDSA) cycles. Results: We identified the following improvement opportunities: (1) oncologist under confidence about counseling, (2) oncologist lack of knowledge about local fertility preservation options/ processes, and (3) lack of a standardized referral mechanism to reproductive endocrinology/ urology. The first PDSA cycle was disrupted due to COVID-19; from February 2020-August 2020, we introduced the initiative at oncology disease site meetings (e.g., leukemia). In September 2020, we implemented a second PDSA cycle. Our interventions included (1) presenting the baseline data and fellow-led initiative at Oncology Grand Rounds (attended by 150 staff members), (2) creating and distributing paper and electronic informative pamphlets to oncologists and patients, and (3) implementing an electronic medical record order set. This order set included instructions for providers and patients, necessary laboratory studies, and a referral to reproductive endocrinology or orders for cryopreservation of sperm. It also added the following to a patient’s after visit summary: contact information for a dedicated fertility coordinator, estimated costs of services, and financial assistance programs. Post-implementation (September 2020- February 2021), the percentage of patients who reported receiving fertility preservation counseling increased from 36.7% to 80.9% (38/ 47). A sustained shift in the process was apparent on a control chart. Conclusions: Despite disruptions caused by the COVID-19 pandemic, we demonstrate how a trainee-led, patient-centered initiative improved fertility care services for people with cancer. Ongoing work focuses on ensuring sustainability of change, assessing the quality of counseling, and evaluating the impact on utilization of fertility care services.