Sterner, Rosalie M.

Abstract Description Common variable immunodeficiency (CVID) is a common inborn error of immunity, but a monogenic cause is rare. Biallelic loss-of-function pathogenic variants in the gene encoding BAFF-Receptor (TNFRSF13C) is one such cause. A common missense variant in BAFF-R, p.Pro21Arg, was proposed as contributing to the pathogenesis of non-monogenic CVID by Peiper et al. 2014 with an allelic frequency of 6.7% in controls versus 10.2% in CVID patients (OR 1.57, p = 0.0026) and Block et al. 2023 with an 11.36% allele frequency in CVID when compared to the Non-Finnish European population in the gnomAD database (OR = 1.316, p = 0.0145) and to an internal cohort of controls (OR = 1.316, p = 0.0458). Exact population composition matching is critical, as the frequency of this variant is highly variable even in Europe. We found the allelic frequency of BAFF-R p.Arg21Pro to be 10% from 90 non-familial CVID patients from the University of Utah Health Sciences Center, similar to Pieper et al. (10.2%) and Block et al. (11.36%), but comparing our Utah CVID patients to the 1000 Genomes project Utah Residents (CEU) with Northern and Western European Ancestry, which matches our CVID cohort (9.6% allelic frequency); we obtained an OR of 1.0468 (95% CI: 0.5309 to 2.0638; p = 0.8950). For further studies on BAFF-R p.Pro21Arg, we recommend closely matching the reference populations, and not using a broad European or other control populations. Topic Categories Translational and Interventional Immunology (TI)

AbstractBackgroundFlow cytometry protocols for counting fresh CD34+ cell samples are not ideal for cryopreserved products due to cryoprotectant cytotoxicity. For cryopreserved samples, often large volumes of hypotonic solutions, which can cause cell death, are used to remove the cryoprotectant with a post‐thaw wash. We recently developed a novel multistep dilution method with subsequent flow cytometry analysis to allow for accurate and reproducible results. The previous method involved washing steps which invalidate the ability to enumerate cell recovery, and success had to be gauged solely on viability. The new method allows for assessment of total cell recovery and viable cell recovery.Study Design and MethodsApheresis products were cryopreserved in 10% DMSO at a target WBC concentration of 300 × 106/mL. Cryovials from these products were thawed at 37°C, and samples were diluted 1:2 by three additions of 1/3 sample volume using 1%‐Human Albumin in Dextran 40 (10% Low Molecular Weight Dextran in 0.9% NaCl) separated by 5 min between each addition. A 1:10 dilution was performed to obtain the correct cell concentration for flow cytometric analysis resulting in a 1:20 dilution. End WBC concentrations were ~15 × 106/mL with DMSO diluted to 0.5%.ResultsFifty‐two samples were tested with this new method. Total and viable cell recoveries were calculated based on pre‐cryopreservation data. Median total cell recoveries for CD34 and CD3 were >85%, while median viable cell recoveries were >75%.DiscussionCryopreserved samples can be reliably prepared for flow cytometric testing using a step‐wise dilution to preserve cell integrity and robust recoveries.

AbstractBackgroundStandard flow cytometry protocols for CD34+ cell enumeration designed for fresh samples are not appropriate for cryopreserved products. Special protocols have been developed to remove the cryoprotectant by quickly washing a freshly thawed sample. Exposing cells to a large volume of hypotonic solution and subsequent washing process was hypothesized to cause lab‐induced cell death. Moreover, standard gating strategies must be altered to avoid reporting falsely high viabilities.Study Design and MethodsWe developed a novel method whereby thawed samples were diluted step‐wise to 1:2 by 3 additions of 1/3 sample volume using 1% Human Albumin in Dextran 40 (10% Low Molecular Weight Dextran in 0.9% NaCl) separated by 5 min between each addition. An additional 1:10 dilution was required to obtain a desired cell concentration for flow cytometry testing resulting in a 1:20 dilution.ResultsTwenty samples were tested simultaneously in a method comparison; the new method demonstrated significant increases in mean cell viabilities for white blood cells, hematopoietic progenitor cells, and T cells as well as reduced standard deviations for each parameter.DiscussionSlow, step‐wise dilutions of freshly thawed samples of cryopreserved apheresis products to 1:20 yielded higher and more precise viability measurements compared to quickly washing samples to remove DMSO.

The bone marrow microenvironment acts as a protective niche for malignant cells. Within the bone marrow microenvironment is the endosteal niche, which is the area where the ossified surface of the bone and marrow cavity interact. The endosteal niche provides the opportunity for hematological malignancies such as acute myeloid leukemia (AML) of the bone marrow to interact with osteoblasts from the bone. To study the interaction between AML cells and osteoblasts, our lab developed a co‐culture model of osteoblasts (MC3T3 osteoblast cell line) and AML cells (KG1a cell line or patient samples from bone marrow aspirates). AML cells expressing high levels of CXCR4 were cultured alone or with osteoblasts and then challenged with SDF‐1, the ligand to CXCR4 and a chemokine readily abundant in the bone marrow microenvironment that our lab has previously shown actually induces apoptosis in AML cells expressing high levels of CXCR4 and that osteoblasts are able to protect AML cells from SDF‐1 induced apoptosis. Our lab now shows that treating osteoblasts with cyclosporine (CSA) prevents their ability to effectively protect AML cells from SDF‐1 induced cell death as assayed by annexin‐V staining of AML cells by flow cytometry. CSA treatment of osteoblasts decreased protein levels of tissue non‐specific alkaline phosphatase (ALPL), a phosphatase necessary for osteoblast mineralization, compared to vehicle control. As such, osteoblasts were treated with control siRNA or ALPL siRNA. CXCR4 expressing AML cells were then co‐cultured with the transfected osteoblasts and challenged with SDF‐1. siRNA knockdown of ALPL in osteoblasts was sufficient to prevent protection of AML cells from the apoptotic stimuli. These studies indicate that osteoblast differentiation plays a role in mediating the protection of AML cells. Therefore, targeting specific differentiation factors in osteoblasts may help to make to bone marrow microenvironment a more hostile environment for AML cells and aid in better malignancy eradication. Support or Funding Information This study was supported by NIH R21 CA194217 (to K.E.H.). R.M.S. is supported by the Mayo Clinic Medical Scientist Training Program Robert L. Howell Physician‐Scientist Scholarship.

Abstract Antibiotics are critical complements to the immune response in combatting infection. Anaerobic bacteria are increasingly common causes of infection, and antibiotic resistance of anaerobes has been rising. Most laboratories do not perform antimicrobial susceptibility testing of anaerobes, and even fewer have compiled antibiogram data. Here we investigated antimicrobial susceptibility results of anaerobes from January 2012 to December 2021 obtained from inpatients at Mayo Clinic using reference agar dilution testing. Actinomyces (60–70 isolates annually), Bacteroides fragilis (30–80), Finegoldia magna (40–60), and Cutibacterium acnes (70–110) were included. Penicillin, clindamycin, metronidazole, ertapenem, piperacillin-tazobactam, and moxifloxacin were tested. Clinical and Laboratory Standards (CLSI) breakpoints were followed. B. fragilis showed ≥90% and Actinomyces 100% susceptibility to piperacillin-tazobactam and ertapenem. All F. magna and >95% B. fragilis were susceptible to metronidazole. The most striking fluctuations in percent susceptibility from year to year were observed for clindamycin, with Actinomyces ranging from 66–79%, B. fragilis 34–67%, and F. magna 47–85%. In the absence of local anaerobe antibiograms, providers may refer to such antibiogram data to guide empiric anti-anaerobic therapy at their institutions. In conclusion, anaerobe antibiotic susceptibility patterns must be monitored to ensure appropriate antibiotic empiric therapy and stewardship.

Primary cardiac angiosarcomas (PCA) are highly aggressive malignant heart tumors. Previous reports have shown a poor prognosis regardless of management, and no consensus or guidelines exist. It is necessary to clarify this information since patients with PCA have a short survival. Therefore, we aimed to systematically review clinical manifestations, management, and outcomes. We systematically searched in PubMed, Scopus, Web of Science, and EMBASE. We intended to include cross-sectional studies, case-control studies, cohort studies, and case series that reported clinical characteristics, management, and outcomes of patients with PCA. As a methodological approach, we used the Joanna Briggs Institute Critical Appraisal Checklist for Case Series and the Newcastle-Ottawa Scale for cohorts. We included six studies (five case series, one cohort). The mean/median age ranged from 39 to 48.9 years. Male sex was predominant. The most frequent manifestations were dyspnea (range: 50%-80%), pericardial effusion (29% & 56%), and chest pain (10%-39%). The mean tumor size ranged from 5.8 to 7.2 cm, with the majority of these localized in the right atrium (70-100%). The most common locations of metastasis were the lung (20%-55.6%), liver (10%-22.2%), and bone (10%-20%). Resection (22.9%-94%), and chemotherapy as neoadjuvant or adjuvant (30%-100%) were the most commonly used methods of treatment. Mortality ranged from 64.7% to 100%. PCA often presents late in its course and usually results in poor prognosis. We strongly recommend performing multi-institutional prospective cohorts to better study disease course and treatments to develop consensus, algorithms, and guidelines for this type of sarcoma.

Abstract The objective was to evaluate the impact of an interdisciplinary graduate level course on students’ knowledge and interests in immunotherapy and CAR-T cell therapy. It was a one week course conducted in November 2018. Students were surveyed before and after the course. The survey included questions regarding background, self-perceived knowledge/confidence in skills, and interests/predicted behaviors. Students were assigned to work in interdisciplinary teams to develop a research proposal. A total of 25 students taking the course for graduate level credit were surveyed. Of these, all 25 (100%) completed the surveys. Students came from variable backgrounds and were at different stages of graduate training. After completion of the course, there was a statistically significant increase in self-perceived knowledge of immunotherapy (mean±standard deviation score of 3.6±0.9 post-course vs 2.6±1.0 pre-course, on a 5 point Likert scale, P<0.001), knowledge of the benchtop research to clinic translational process (3.7±0.8 post-course vs 3.0±0.9 pre-course, P<0.001), confidence in critical reading skills (4.3±0.6 post-course vs 4.0±0.5 pre-course, P=0.008), confidence in immunotherapy focused grant writing skills (3.6±0.9 post-course vs 2.8±1.1 pre-course, P<0.001), interest in working in interdisciplinary teams (4.8±0.4 post-course vs 4.6±0.6 pre-course, P=0.02), and interest in attending immunotherapy educational events (3.5±1.3 post-course vs 3.2±1.3 pre-course, P≤0.05). In conclusion, this course helps trainees to build knowledge and interest in the fields of immunotherapy and CAR-T cell therapy and can potentially serve as a platform for future educational courses in interdisciplinary, translational research.

Purpose of review Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary tool in the treatment of cancer. CAR-T cells exhibit their effector functions through the recognition of their specific antigens on tumor cells and recruitment of other immune cells. However, this therapy is limited by the development of severe toxicities and modest antitumor activity in solid tumors. The host and tumor microenvironment interactions with CAR-T cells play an important role in orchestrating CAR-T-cell functions. Specifically, myeloid lineage cells and their cytokines critically influence the behavior of CAR-T cells. Here, we review the specific effects of myeloid cell interactions with CAR-T cells, their impact on CAR-T-cell response and toxicities, and potential efforts to modulate myeloid cell effects to enhance CAR-T-cell therapy efficacy and reduce toxicities. Recent findings Independent studies and correlative science from clinical trials indicate that inhibitory myeloid cells and cytokines contribute to the development of CAR-T-cell-associated toxicities and impairment of their effector functions. Summary These findings illuminate a novel way to reduce CAR-T-cell-associated toxicities and enhance their efficacy through the modulation of myeloid lineage cells and inhibitory cytokines.

Despite the remarkable activity of CD19 directed chimeric antigen receptor T cell (CART19) therapy in the treatment of B cell malignancies, the therapy is limited by the development of severe life-threatening complications such as neurotoxicity (NT) and cytokine release syndrome (CRS). Additionally, durable efficacy following CART19 therapy is not optimal. Emerging literature suggests that inhibitory myeloid cells and their cytokines play an important role in inducing CAR-T cell toxicities and also contribute to the inhibition of their effector functions. Specifically, GM-CSF was identified as a critical cytokine in the development of NT and CRS after CART19 therapy. Neutralization of GM-CSF in preclinical models has been shown to prevent CRS and enhance CART anti-tumor activity through modulation of myeloid cell behavior, resulting in reduction of tumor associated macrophages. In addition to the predominant effect of GM-CSF on myeloid cells, there appears to be a direct effect on CART19 cells. In this study, we aimed to evaluate the direct effect of GM-CSF on CART cells. Our initial finding of enhanced anti-tumor activity of CART19 cells after GM-CSF inhibition suggested a direct effect of GM-CSF on CART cells (Sterner et al. 2019, Blood). In these experiments, a guide RNA (gRNA) targeting exon 3 of GM-CSF in a CRISPR-Cas9 lentiviral vector was used to knock out GM-CSF during CART cell manufacturing. This resulted in a disruption efficiency of approximately 70% of the GM-CSF gene. Using a high tumor burden xenograft model for relapsed acute lymphoblastic leukemia established through the engraftment of the CD19+ luciferase+ NALM6 cell line (1x106 cells intravenously) in immunocompromised NOD-SCID-γ-/- mice, treatment with low doses of GM-CSFk/o CART19 resulted in improved anti-tumor activity and overall survival compared to GM-CSFwt CART19. The lack of myeloid cells in this model pointed to an intrinsic effect of GM-CSF on CAR-T cells. To ensure that this was not related to an off-target effect of the gRNA, whole exome sequencing (WES) of the modified cells was performed. There was no difference in the single nucleotide variants or indel counts between GM-CSFk/o CART19 and GM-CSFwt CART19 (Figure 1A). WES was significant for only two alterations in the exon 3 targeted by the gRNA (Figure 1B). The high efficiency and accuracy of targeting exon 3 of GM-CSF indicated that the improvement in CART function is unlikely related to an off-target effect of the gRNA and suggested the possibility of a direct interaction between GM-CSF and CART cells as a potential mechanism behind the improved anti-tumor activity. To investigate this interaction, we first assessed the expression of GM-CSF receptors on CART cells. While resting CART cells do not express any GM-CSF receptors, our analysis robustly indicates that activated CART cells significantly upregulate both α and β subunits of the GM-CSF receptor. This finding was significant both when CART cells are activated through their T cell receptor with CD3/CD28 beads (Figure 1C) or through the CAR with irradiated NALM6 cells (Figure 1D). Additionally, activated GM-CSFk/o CART19 cells also upregulated GM-CSF receptors, indicating this upregulation is induced by T cell stimulation. These results suggest a direct interaction between GM-CSF and upregulated GM-CSFR on activated CART cells. Having demonstrated that 1) GM-CSF depletion enhances CART19 efficacy in xenograft models in the absence of monocytes and 2) T cell activation increases GM-CSF receptor expression, we sought to uncover the downstream changes resulting from this effect. Transcriptome interrogation of GM-CSFk/o CART19 revealed a distinct signature including a significant inhibition of the Fas death pathway, a known critical pathway in inducing CART cell apoptosis. This suggests a potential mechanism for enhanced CART19 activity following GM-CSF depletion (Figure 1E). In summary, our results strongly indicate that CART cells increase expression of GM-CSF receptor subunits when activated, resulting in modulation of CART cell functions. Furthermore, GM-CSFk/o CART19 revealed a distinct transcriptome signature compared to GM-CSFwt CART19. These results illuminate a novel mechanism for a direct modulatory effect of GM-CSF on activated CART cells. Disclosures Cox: Humanigen: Patents & Royalties. Sterner:Humanigen: Patents & Royalties. Sakemura:Humanigen: Patents & Royalties. Ahmed:Humanigen: Employment. Chappell:Humanigen: Employment. Durrant:Humanigen: Employment. Parikh:Acerta Pharma: Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Genentech: Honoraria; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Kite/Gilead: Research Funding; Morphosys: Research Funding.