anti-CD20 Treatment Research Articles

Mechanism of differentiation and unique repertoire of IgM secreting B-1 plasma cells

Abstract Natural antibodies are broadly reactive, mostly IgM antibodies that provide a first line of defense against pathogens. Distinct populations of fetal/neonatal-derived IgM-secreting B-1 cells, including non-terminally differentiated B-1 and B-1 plasma cells (PC) are present in bone marrow (BM) and spleen. B-1PC are transcriptionally and phenotypically similar to foreign antigen-induced, long-lived conventional PC, prompting us to hypothesize that both receive similar differentiation signals. To determine the extent to which natural IgM-producing B-1PC depend on T cell help, we studied TCRβδ−/− mice lacking either all T cells, or depleted of CD4 T cells by anti-CD4 mAb treatment. Both showed significantly reduced numbers of BM but not spleen B-1PC and slightly increased populations of B-1 cells. Consistent with those data, reconstitution of RAG-deficient mice with B-1 and CD4+ T cells, but not with B-1 cells alone, restored the BM B-1PC compartment and significantly enhanced serum IgM levels. B-1PC did not bind to PtC-containing liposomes and depletion of B-1 PC had minimal effect on the number of anti-PtC IgM ASC. In contrast, most anti-influenza natural IgM ASC were B-1PC, consistent with reduced serum levels influenza-binding natural IgM in TCRβδ−/− mice. Together, the study reveals that in contrast to previous assumptions, some natural IgM production is dependent on CD4 T cell help, suggesting that multiple mechanisms regulate induction of natural IgM secretion.

Proinflammatory CD8+ T‐Cells Contribute to Age‐Related Glucose Intolerance

Previously, we have found that T Cells play a role in age-related glucose intolerance. However, the specific T cell subtype that causes this impairment is unknown. Therefore, the purpose of this study was to test the hypothesis that 1) CD8+ T cells that accumulate in metabolically active tissues of old mice exhibit a proinflammatory phenotype 2) depletion of CD8+ T-cells will improve glucose tolerance in old mice. This study was approved by the institutional animal care and use committee at the University of Texas at Arlington. First, we assessed CD8+ cell infiltration and phenotype in epididymal white adipose tissue (eWAT) and skeletal muscle from young (4-6mo; n=9) and old (22-24mo; n=12) sedentary male mice. CD8+, Interferon-γ (IFN-γ) producing T cells, and Tumor Necrosis Factor-α (TNF-α) producing T cells were assessed by flow cytometry. Data are expressed as mean ± standard error. Group differences were assessed with an independent samples or paired samples T cells depending on experimental design. In eWAT there were significantly more total CD8+ (6232.1±2616.0, 713.5±215.2 cells per gram tissue, p=0.05) and TNF-α producing CD8+ T cells (2296.6±1167.3 cell/g, 206.6±74.7 cells per gram tissue, p=0.04) in the old compared to the young mice. There were no differences between old and young in IFN-γ producing T cells in eWAT. In the gastrocnemius there were no differences in the number of total CD8+ or IFN-γ producing CD8+ cells. However, the proportion of IFN-γ producing T cells (37.2±6.1%, 24.5±4.7% p=0.05) was greater in the old gastrocnemius compared to the young. Numbers of TNF-α producing T cells (268.6±144.5, 33.5±13.2 cells per gram tissue, p=0.07) tended to be greater in the old gastrocnemius compared to the young. To determine whether CD8+ T cells mediate age-related glucose intolerance, an additional cohort of old sedentary male and female mice (22-24mo) were injected (100μg, i.p. every 5 days for 28 days) with either an anti-CD8 (n=9) or control antibody (n=8). Glucose tolerance was assessed by i.p. injection of glucose (2g/kg) and blood glucose was measured over 120min using a handheld glucometer before and after the antibody treatment period. Anti-CD8 treatment significantly blunted the number of CD8+ cells in the eWAT (3420.2±1473.8, 17693.2±4745.8 cells per gram, p=0.03) compared to controls. There was also a lower number of CD8+ cells the quadriceps of the anti-CD8 group (116.0±48.5cell/g, 695.3±82 cells per gram, p<0.001) group compared to controls. There were no alterations in fasting glucose from pretreatment to posttreatment in the anti-CD8 (139.5±9.8, 142.1±7.4mg/dl, p=.40) or control group (128.9±5.8, 139±8.3 mg/dl, p=0.16). There was a significant reduction in the glucose tolerance test area under the curve (AUC) from pretreatment to posttreatment in the anti-CD8 group (30647.5±2282.1, 26077.5±2102.6 arbitrary units, p=0.04), suggesting an improvement in glucose tolerance. There were no alterations in the control group (27055.3±2488.5, 27666.6±2987.9 arbitrary units, p=0.4). In conclusion, these data suggest that increased infiltration of proinflammatory CD8+ cells in metabolically active tissues with age play an important role in age-related impairments in glucose tolerance.

T lymphocyte depletion ameliorates age-related metabolic impairments in mice.

Both glucose tolerance and adaptive immune function exhibit significant age-related alterations. The influence of the immune system on obesity-associated glucose intolerance is well characterized; however, whether the immune system contributes to age-related glucose intolerance is not as well understood. Here, we report that advancing age results in an increase in T cell infiltration in the epididymal white adipose tissue (eWAT), liver, and skeletal muscle. Subtype analyses show that both CD4+, CD8+ T cells are greater with advancing age in each of these tissues and that aging results in a blunted CD4 to CD8 ratio. Anti-CD3 F(ab')2 fragments depleted CD4+ and CD8+ cells in eWAT, CD4+ cells only in the liver, and did not deplete quadriceps T cells. In old mice, T cells producing both interferon-γ and tumor necrosis factor-α are accumulated in the eWAT and liver, and a greater proportion of skeletal muscle T cells produced interferon-γ. Aging resulted in increased proportion and numbers of T regulatory cells in eWAT, but not in the liver or muscle. Aging also resulted in greater numbers of eWAT and quadriceps CD206- macrophages and eWAT, liver and quadriceps B cells; neither cell type was altered by anti-CD3 treatment. Anti-CD3 treatment improved glucose tolerance in old mice and was accompanied by improved signaling related to liver and skeletal muscle insulin utilization and decreased gluconeogenesis-related gene expression in the liver. Our findings indicate a critical role of the adaptive immune system in the age-related metabolic dysfunction.

A Milestone in Multiple Sclerosis Therapy: Monoclonal Antibodies Against CD20\u2014Yet Progress Continues

Multiple sclerosis (MS), which is a chronic inflammatory disease of the central nervous system, still represents one of the most common causes of persisting disability with an early disease onset. Growing evidence suggests B cells to play a crucial role in its pathogenesis and progression. Over the last decades, monoclonal antibodies (mabs) against the surface protein CD20 have been intensively studied as a B cell targeting therapy in relapsing MS (RMS) as well as primary progressive MS (PPMS). Pivotal studies on anti-CD20 therapy in RMS showed remarkable clinical and radiological effects, especially on acute inflammation and relapse biology. These results paved the way for further research on the implication of B cells in the pathogenesis of MS. Besides controlling relapse development in RMS, ocrelizumab (OCR) also showed clinical benefits in patients with PPMS and became the first approved drug for this disease course. In this review, we provide an overview of the current anti-CD20 mabs used or tested for the treatment of MS—namely rituximab (RTX), OCR, ofatumumab (OFA), and ublituximab (UB). Besides their effectiveness, we also discuss possible limitations and safety concerns especially in regard to long-term treatment, both for this class of drugs overall as well as for each anti-CD20 mab individually. Additionally, we elucidate to what extent anti-CD20 therapy may alter the function of other immune cells, both directly or indirectly. Finally, we cover the current knowledge on repopulation of CD20+ cells after cessation of anti-CD20 treatment and discuss future aspirations towards alternative, further developed B cell silencing therapies.

Targeting resistance to radiation-immunotherapy in cold HNSCCs by modulating the Treg-dendritic cell axis

BackgroundNumerous trials combining radiation therapy (RT) and immunotherapy in head and neck squamous cell carcinoma (HNSCC) are failing. Using preclinical immune cold models of HNSCC resistant to RT-immune checkpoint inhibitors,...

The deficiency of FKBP-5 inhibited hepatocellular progression by increasing the infiltration of distinct immune cells and inhibiting obesity-associated gut microbial metabolite.

Gut microbiota has a number of essential roles in nutrition metabolism and immune homeostasis, and is closely related to hepatocellular progression. In recent years, studies have also shown that FK506 binding protein 5 (FKBP-5) plays a crucial role in immune regulation. However, it is not yet clear whether FKBP-5 promotes the development of hepatocellular carcinoma (HCC) by affecting immune function and gut microbiota. FKBP-5 expression was verified by immunochemistry and western blot and reverse transcription polymerase chain reaction (RT-qPCR) assays. After treatment in WT and FKBP-5-/- mice, the histological characteristic of mice liver tissue was assessed by H&E staining, and hepatic leukocytes and hepatic NKT cells were identified by flow cytometer. Meanwhile, primary bile acids (BAs), secondary BAs, serum total cholesterol, and the weight of abdomen adipose tissues were examined, and the gut microbiota was evaluated by 16S ribosomal ribonucleic acid (rRNA) sequencing. We discovered that FKBP-5 was highly expressed in HCC tissues. Meanwhile, FKBP-5 deletion inhibited tumor progression by increasing CD8+ T, CD4+ T, NKT and CD4+NKT cells in mice after diethylnitrosamine (DEN) injection. Besides, we proved that FKBP-5 deletion generated rapid and significant reductions in the intestinal BAs, the weight of abdomen adipose tissues and the serum total cholesterol. FKBP-5 deletion also led to a change in the composition of gut microbiota, suggesting that BAs are the main dietary factor regulating gut microbiota, which could be affected by FKBP-5 deletion. Further, we uncovered that anti-CD4 and anti-CD8 treatments facilitated hepatocellular progression by modulating gut microbiota composition in FKBP-5-/- mice. Therefore, we demonstrated that FKBP-5 deletion inhibited hepatocellular progression by modulating immune response and gut microbiome-mediated BAs metabolism.

Anti-CD20 therapy corrects a CD8 regulatory T cell deficit in multiple sclerosis

Objective: To determine the effect of long-term anti-CD20 B-cell-depleting treatment on regulatory T cell immune subsets that are subnormal in untreated MS patients. Methods: 30 clinically stable MS patients, before and over 38 months of ocrelizumab treatment, were compared to 13 healthy controls, 29 therapy-naïve MS, 9 interferon-β-treated MS, 3 rituximab-treated MS, and 3 rituximab-treated patients with other autoimmune inflammatory diseases. CD8, CD28, CD4, and FOXP3 expression in peripheral blood mononuclear cells was quantitated with flow cytometry. Results: CD8+ CD28− regulatory cells rose from one-third of healthy control levels before ocrelizumab treatment (2.68% vs 7.98%), normalized by 12 months (13.5%), and rose to 2.4-fold above healthy controls after 18 months of ocrelizumab therapy (19.0%). CD4+ FOXP3+ regulatory cells were lower in MS than in healthy controls (7.98%) and showed slight long-term decreases with ocrelizumab. CD8+ CD28− and CD4+ FOXP3+ regulatory T cell percentages in IFN-β-treated MS patients were between those of untreated MS and healthy controls. Interpretation: Long-term treatment with ocrelizumab markedly enriches CD8+ CD28− regulatory T cells and corrects the low levels seen in MS before treatment, while slightly decreasing CD4+ FOXP3+ regulatory T cells. Homeostatic enrichment of regulatory CD8 T cells provides a mechanism, in addition to B cell depletion, for the benefits of anti-CD20 treatment in MS.

Targeting B cells in multiple sclerosis

Treatments targeting B cells are increasingly used for patients with multiple sclerosis (MS). We review the mechanisms of action, clinical effectiveness and safety of treatment, with emphasis on recently published studies. Several monoclonal antibodies targeting the surface molecule CD20 on B cells are approved or being developed for treatment of MS. Overall, they seem comparable in terms of strongly suppressing radiological disease activity and relapse biology. Novel approaches include anti-CD19 antibody therapy and treatment with oral drugs targeting Bruton's tyrosine kinase (BTK). The main safety issue with persistent B cell depletion is an increased risk of infections - possibly including an increased risk of severe COVID-19. Vaccine responses are also blunted in patients treated with anti-CD20 antibodies. Lower doses or longer infusion intervals may be sufficient for control of disease activity. Whether this might also improve the safety of treatment and increase vaccination responses remains to be determined. Available data support the widespread use of therapies targeting B cells in MS. Whether novel approaches targeting CD19 or BTK will have advantages compared to anti-CD20 antibody therapy remains to be established. Furthermore, trials investigating alternative dosing regimens for anti-CD20 antibody treatment are warranted.

A Protective Role of Donor B Cells against Ischemia-Reperfusion Injury in a Minor-Mismatched Mouse Lung Transplant Model

<h3>Purpose</h3> We showed that recipient B cells are necessary for the development of chronic allograft fibrosis after ischemia-reperfusion injury (IRI) in a C57BL/10 (B10, H-2^b)-to-C57BL/6 (B6, H-2^b) minor alloantigen-mismatched mouse orthotopic lung transplant model. Whether donor-derived B cells play a specific role in this phenomenon is unknown. We hypothesized that donor B cells play a minimal role in the recipient. <h3>Methods</h3> We performed B10-to-B6 lung transplants with either prolonged (6h at 4°C followed by 45 min at 37°C and 15min anastomosis time) or minimal (1h at 4°C followed by 15min anastomosis time) antecedent storage using congenic donors and recipients. Donors were given anti-CD20 antibody or isotype control on day -2 (Fig 1A). Necropsies were performed at days +3 and +28. Allografts were histologically assessed in a blinded manner. Flow cytometry was used to evaluate immune cell populations at days 0 (pre-transplant donor samples), +3, and +28 (post-transplant samples). <h3>Results</h3> In the context of prolonged storage, anti-CD20 treatment was associated with early postoperative deaths not seen in the other groups (Fig 1B). At day 28, prolonged storage allografts exhibited fibrosis, with anti-CD20 treatment enhancing vascular fibrosis in surviving mice, but without impact on immune cell populations (Fig 1C). To better understand how anti-CD20 donor-treatment is impacting the allograft early post-transplant, we assessed the grafts at day 3: Both prolonged storage groups had severe IRI although, somewhat surprisingly, acute lung injury score was decreased by anti-CD20. Donor-derived B cells were reduced, and donor-derived neutrophils and monocytes were increased in the anti-CD20 group (Fig 1D). <h3>Conclusion</h3> Unexpectedly, our results suggest that donor-derived B cells have a protective role against IRI possibly via augmentation of neutrophils and monocytes in the donor graft. Further investigation is now underway.

Trafficking and Repopulation of Donor B Cells in a Minor-Mismatched Mouse Lung Transplant Model

<h3>Purpose</h3> Previous work has shown that B cells are necessary for the development of chronic allograft fibrosis after ischemia-reperfusion injury (IRI) in a C57BL/10 (B10, H-2^b)-to-C57BL/6 (B6, H-2^b) minor alloantigen-mismatched mouse orthotopic lung transplant model. It remains unknown whether donor-derived B cells play a specific role in this phenomenon and how long they survive in the recipient. We hypothesized that donor B cells are short-lived and thus play a minimal role in the recipient. <h3>Methods</h3> We conducted B10-to-B6 lung transplants (without IRI) using congenic donors and recipients. Donors were treated 2 days before transplantation with an anti-CD20 antibody or its isotype control. We used flow cytometry to track B cell populations in the donor and recipient before and up to 28 days after transplantation. <h3>Results</h3> Anti-CD20 antibody effectively eliminated >85% of B cells from donor lungs prior to transplantation (Fig 1A). We observed low frequencies of donor (CD45.2⁺) B cells in the recipient (CD45.1⁺) peripheral blood after transplantation. Despite profound B cell depletion in donor lungs, donor-derived leukocytes were unexpectedly still present in the allograft (median 3.6%, range 2.3-10%), thoracic lymph nodes (median 0.5%, range 0.2-1.6%) and spleen (median 1.2%, range 0.7-1.8%) at day 28 irrespective of anti-CD20 treatment. B and plasma cells constituted >80% of donor-derived leukocytes observed in the recipient spleen at day 28 (Fig 1B). As expected, given the lack of IRI, allograft histology showed minimal rejection and fibrosis with no effect of anti-CD20 antibody observed (Fig 1C). <h3>Conclusion</h3> Donor-derived B cells repopulated in the recipient allograft and secondary lymphoid organs by day 28, to a similar extent with and without anti-CD20 donor treatment. These findings suggest donor-derived B cells may play important functional roles after transplantation. An examination of the role of donor- and recipient-derived B cells in IRI-augmented lung allograft fibrosis is now underway.

EASL position paper on the use of COVID-19 vaccines in patients with chronic liver diseases, hepatobiliary cancer and liver transplant recipients

According to a recent World Health Organization estimate, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, which originated in China in 2019, has spread globally, infecting nearly 100 million people worldwide by January 2021. Patients with chronic liver diseases (CLD), particularly cirrhosis, hepatobiliary malignancies, candidates for liver transplantation, and immunosuppressed individuals after liver transplantation appear to be at increased risk of infections in general, which in turn translates into increased mortality. This is also the case for SARS-CoV-2 infection, where patients with cirrhosis, in particular, are at high risk of a severe COVID-19 course. Therefore, vaccination against various pathogens including SARS-CoV-2, administered as early as possible in patients with CLD, is an important protective measure. However, due to impaired immune responses in these patients, the immediate and long-term protective response through immunisation may be incomplete. The current SARS-CoV-2 pandemic has led to the exceptionally fast development of several vaccine candidates. A small number of these SARS-CoV-2 vaccine candidates have already undergone phase III, placebo-controlled, clinical trials in healthy individuals with proof of short-term safety, immunogenicity and efficacy. However, although regulatory agencies in the US and Europe have already approved some of these vaccines for clinical use, information on immunogenicity, duration of protection and long-term safety in patients with CLD, cirrhosis, hepatobiliary cancer and liver transplant recipients has yet to be generated. This review summarises the data on vaccine safety, immunogenicity, and efficacy in this patient population in general and discusses the implications of this knowledge on the introduction of the new SARS-CoV-2 vaccines.

Rituximab and obinutuzumab differentially hijack the B cell receptor and NOTCH1 signaling pathways

SummaryThe anti-CD20 monoclonal antibodies rituximab and obinutuzumab differ in their mechanisms of action, with obinutuzumab evoking greater direct B cell death. To characterize the signaling processes responsible for improved B cell killing by obinutuzumab, we undertook a phosphoproteomics approach and demonstrate that rituximab and obinutuzumab differentially activate pathways downstream of the B cell receptor. Although both antibodies induce strong ERK and MYC activation sufficient to promote cell-cycle arrest and B cell death, obinutuzumab exceeds rituximab in supporting apoptosis induction by means of aberrant SYK phosphorylation. In contrast, rituximab elicits stronger anti-apoptotic signals by activating AKT, by impairing pro-apoptotic BAD, and by releasing membrane-bound NOTCH1 to up-regulate pro-survival target genes. As a consequence, rituximab appears to reinforce BCL2-mediated apoptosis resistance. The unexpected complexity and differences by which rituximab and obinutuzumab interfere with signaling pathways essential for lymphoma pathogenesis and treatment provide important impetus to optimize and personalize the application of different anti-CD20 treatments.

COVID-19 in multiple sclerosis patients: susceptibility, severity risk factors and serological response.

Information regarding multiple sclerosis (MS) patients with the 2019 novel coronavirus disease (COVID-19) is scarce. The study objective was to describe the incidence and characteristics of MS patients with COVID-19, to identify susceptibility and severity risk factors and to assess the proportion of positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serologies according to disease-modifying treatments. This was a retrospective study of an MS cohort analysing data collected between February and May 2020. Cases were identified through an email survey and clinical visits. The relationship of demographic and MS characteristics with COVID-19 and of the disease-modifying treatments with SARS-CoV-2 serostatus were examined. Data from 48 suspected cases out of 758 valid respondents and from 45 COVID-19 cases identified through clinical visits were collected. Incidence was 6.3%. Nineteen (20.3%) patients were hospitalized and two (2.2%) died. Multivariable models determined that age (odds ratio [OR] per 10years 0.53, 95% confidence interval [CI] 0.34-0.85), contact with a confirmed case (OR 197.02, 95% CI 56.36-688.79), residence in Barcelona (OR 2.23, 95% CI 1.03-4.80), MS duration (OR per 5years 1.41, 95% CI 1.09-1.83) and time on anti-CD20 treatment (OR per 2years 3.48, 95% CI 1.44-8.45) were independent factors for presenting COVID-19 and age (OR per 10years 2.71, 95% CI 1.13-6.53) for a severe COVID-19. Out of the 79 (84.9%) with serological test, 45.6% generated antibodies, but only 17.6% of those on anti-CD20 therapies. Lymphopaenia or immunoglobulin levels did not relate to COVID-19. Multiple sclerosis patients present similar incidence, risk factors and outcomes for COVID-19 as the general population. Patients treated with an anti-CD20 therapy for a longer period of time might be at a higher risk of COVID-19 and less than 20% generate an antibody response. Only age was related to severity.

Survival and Incidence of COVID-19 After SARS-CoV-2 Vaccination in a Series of 2391 Cancer Patients

Rationale: Patients with cancer are at high risk for severe or lethal CoVID-19. The impact of SARS-COV-2 vaccination on the risk of developing COVID-19 was investigated in an exhaustive series of patients from a comprehensive cancer center.Methods: This is a study of the exhaustive population of 2391 cancer patients who were prescribed SARS-COV-2 vaccination until 09/21. Patient characteristics, documented SARS-COV-2 infection with RT-PCR, and survival were collected. The primary endpoint was the rate of COVID-19 after vaccination. Secondary endpoints included risk factors to develop COVID-19 after vaccination, with a comparison with the cohort of vaccinated health care workers (HCW), and risk factors for death.Findings: From Jan to Sept 2021, among 2391 patients with cancer under active treatment prescribed a SARS-COV-2 vaccine, 659 (28%), 1498 (63%) and 139 (6%) received 1, 2 and 3 doses respectively. 95 patients received a single dose of vaccine after a previous COVID-19. 2285 health care workers (HCW) received one (N=17, 0.7%), 2-3 (N=2026, 88.7%) vaccine doses and one dose after COVID-19 (N=242, 10.6%). With a median follow-up of 142 days and 199 for patients and HCW respectively, 39 (1.6%) patients and 35 (1.5%) HCW developed COVID-19 after vaccination. 6 of 39 cancer patients and no HCW died of COVID-19 within 50 days after diagnosis. Independent risk factors for COVID-19 in vaccinated patients were age, single dose of vaccine and anti-CD20 treatment in the last 3 months. Independent risk factors for any cause of death included metastatic disease, gender, cancer type, but also documented COVID-19 before vaccination.Interpretation: Patients receiving two or more doses of COVID-19 vaccine have reduced risk of COVID-19. The risk of death of vaccinated cancer patients presenting COVID-19 remains high. COVID-19 before vaccination is associated with an increased overall risk of death.Funding Information: LYRICAN (INCA-DGOS-INSERM 12563), NetSARC (INCA & DGOS), InterSARC (INCA), LabEx DEvweCAN (ANR-10-LABX 0061), PIA Institut Convergence Francois Rabelais PLAsCAN (PLASCAN, 17-CONV-0002), Fondation ARC contre le Cancer, La Ligue contre le Cancer (Canopée), EURACAN (EC 739521) contributed to fund this study.Declaration of Interests: The authors have declared no conflicts of interestEthics Approval Statement: The study was approved by the Institutional review board of the Centre Leon Berard on March 2021.

Experience in Multiple Sclerosis Patients with COVID-19 and Disease-Modifying Therapies: A Review of 873 Published Cases

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic is a challenge for all participants in the healthcare system. At the beginning of the pandemic, many physicians asked themselves what risk their patients, especially those with chronic diseases, were exposed to. We present an overview of all patients with multiple sclerosis (MS) and SARS-CoV-2 infection published in the literature so far. In total, there are publications on 873 SARS-CoV-2 positive MS patients and information on the outcome can be given for 700 patients. With regard to the different disease modifying therapies (DMTs), by far the most cases were described under anti-CD20 treatment (n = 317). The mortality rate of all MS patients was 4% and a further 3% required invasive or non-invasive ventilation. When looking at the severe and fatal cases, it is particularly noticeable that patients without DMTs, with previous cardiovascular diseases, or with a severe degree of disability are at risk. Immunosuppressive therapy itself does not appear to be a substantial risk factor. Rather, it is reasonable to assume that the therapies could be protective, either directly, by mitigating the cytokine storm, or indirectly, by reducing the disease activity of MS.

CD28 Agonism Improves Survival in Immunologically Experienced Septic Mice via IL-10 Released by Foxp3+ Regulatory T Cells.

Immune dysregulation during sepsis is mediated by an imbalance of T cell costimulatory and coinhibitory signaling. CD28 is downregulated during sepsis and is significantly altered on memory versus naive T cells. Thus, to study the role of CD28 during sepsis in a more physiologically relevant context, we developed a "memory mouse" model in which animals are subjected to pathogen infections to generate immunologic memory, followed by sepsis induction via cecal ligation and puncture. Using this system, we show that agonistic anti-CD28 treatment resulted in worsened survival in naive septic animals but conferred a significant survival advantage in immunologically experienced septic animals. Mechanistically, this differential response was driven by the ability of CD28 agonism to elicit IL-10 production from regulatory T cells uniquely in memory but not naive mice. Moreover, elevated IL-10 released by activated regulatory T cells in memory mice inhibited sepsis-induced T cell apoptosis via the antiapoptotic protein Bcl-xL. Together, these data demonstrate that immunologic experience is an important parameter that affects sepsis pathophysiology and can fundamentally change the outcome of modulating the CD28 pathway during sepsis. This study suggests that testing therapeutic strategies in immunologically experienced hosts may be one way to increase the physiologic relevance of rodent models in sepsis research.

Long‐term porcine islet graft survival in diabetic non‐human primates treated with clinically available immunosuppressants

Although pancreatic islet transplantation is becoming an effective therapeutic option for patients with type 1 diabetes (T1D) who suffer from a substantially impaired awareness of hypoglycemia, its application is limited due to the lack of donors. Thus, pig-to-human islet xenotransplantation has been regarded as a promising alternative due to the unlimited number of "donor organs." Long-term xenogeneic islet graft survival in pig-to-non-human primate (NHP) models has mainly been achieved by administering the anti-CD154 mAb-based immunosuppressant regimen. Since the anti-CD154 mAb treatment has been associated with unexpected fatal thromboembolic complications in clinical trials, the establishment of a new immunosuppressant regimen that is able to be directly applied in clinical trials is an urgent need. We assessed an immunosuppressant regimen composed of clinically available agents at porcine islet transplantation in consecutive diabetic NHPs. Porcine islet graft survival in consecutive diabetic NHPs (n=7; >222, >200, 181, 89, 62, 55, and 34 days) without severe adverse events. We believe that our study could contribute greatly to the initiation of islet xenotransplantation clinical trials.

Real-World IgG Testing Frequency and Characteristics Associated with Hypogammaglobulinemia after Anti-CD20 Exposure in Patients with Chronic Lymphocytic Leukemia (CLL)

Introduction: Hypogammaglobulinemia is a known potential adverse event in patients with CLL receiving anti-CD20 monoclonal antibody (mAb) therapy. However, real-world immunoglobulin G (IgG) testing rates have not been extensively studied. We used a real-world database to investigate IgG testing rates in patients with CLL before and after anti-CD20 exposure. We further examined factors that increased the likelihood of new onset hypogammaglobulinemia following anti-CD20 therapy. Methods: The Flatiron Health EHR-derived de-identified database was used to select patients with abstraction-confirmed CLL diagnosed before 6/1/2020 with: (1) Exposure to an anti-CD20 mAb, (2) At least one IgG lab within 180 days prior to first anti-CD20 line start, and (3) No evidence of hypogammaglobulinemia (IgG &amp;lt; 7g/L) within 6 months prior to first anti-CD20 line start. A "hypogammaglobulinemia detection window" was defined for each patient as the time between first anti-CD20 administration through to 365 days thereafter. Patients were grouped into 3 categories: (A) "Unknown status": those with no IgG testing during the window, (B) "Confirmed hypogam": those with hypogammaglobulinemia detected on any test during the window, and (C) "No detected hypogam": those tested for IgG during the window with 0 tests meeting the hypogammaglobulinemia threshold. Demographic and clinical baseline characteristics were compared across the groups. A logistic regression was performed to compare factors associated with higher likelihood of detecting hypogammaglobulinemia among patients with an IgG test in the detection window. Factors included were: patient age at start of detection window, exposure to chemotherapy before detection window (Y/N), concurrent chemotherapy to anti-CD20 during the detection window (Y/N), and whether anti-CD20 was given in the first line (1L) setting (Y/N). We also performed a separate scenario analysis with outcome of severe hypogammaglobulinemia (IgG &amp;lt; 5 g/L, compared to &amp;lt; 7 g/L in primary analysis). Results: We found 5,838 anti-CD20 mAb-exposed patients with CLL, of which 1,927 (33%) had at least 1 IgG lab within 180 days of anti-CD20 start. Of those, 922 (48%) had hypogammaglobulinemia within 6 months prior to starting anti-CD20 therapy, leaving 1,005 patients in the cohort. 526 patients (52%) fell into the 'unknown status' category. 182 (18%) patients fell into the 'confirmed hypogam' category, and 297 (30%) fell into the 'no hypogam detected' category. Patients across these 3 categories had similar baseline characteristics (Table 1). Among the patients with an IgG test in the detection window (N=479), patients with 'confirmed hypogam' had more IgG tests during the window than the patients with 'no detected hypogam' (mean 3.9 vs. 2.8 tests, p&amp;lt;0.001). Results of the logistic regression for IgG &amp;lt; 7 g/L did not yield any factors significantly associated with higher likelihood of hypogammaglobulinemia (Table 2). However, the regression at IgG &amp;lt; 5 g/L (incl. changing cohort inclusion criteria 3, N=703) found that concurrent exposure to chemotherapy was associated with higher likelihood of severe documented hypogammaglobulinemia (odds ratio 1.89; 95% CI 1.23-2.96; p = 0.004). Conclusions: In this real-world cohort of patients with CLL with at least one IgG test within 6 months prior to anti-CD20 mAb initiation, hypogammaglobulinemia was common. A majority of the cohort was not tested for IgG post anti-CD20 mAb initiation, which likely reflects clinical practice of infrequently obtaining IgG levels in patients with normal pre-treatment IgG levels. As clinically expected, patients with hypogammaglobulinemia were tested for IgG more frequently than those who were not. This hypothesis-generating study suggests that administering chemotherapy concurrently to anti-CD20 mAbs increases the likelihood of documented severe hypogammaglobulinemia (IgG &amp;lt; 5g/L) in patients with CLL. Further research should confirm this. Our analyses did not account for patients who may have had transient hypogammaglobulinemia during anti-CD20 treatment, and did not stratify by intravenous immunoglobulin (IVIG) usage in this population. Further research should examine the effect of anti-CD20-associated hypogammaglobulinemia on outcomes, and determine if IgG surveillance should be standardized as part of clinical guidelines. Disclosures Hooley: Flatiron Health Inc: Current Employment, Research Funding; Roche Group: Current equity holder in publicly-traded company. Maignan:Flatiron Health Inc: Current Employment, Research Funding; Roche Group: Current equity holder in publicly-traded company. Jacob:Flatiron Health Inc: Current Employment, Research Funding; Roche Group: Current equity holder in publicly-traded company. Medina:Flatiron Health Inc: Current Employment, Research Funding; Roche Group: Current equity holder in publicly-traded company. Benderoff:Roche Group: Current equity holder in publicly-traded company; Flatiron Health Inc: Current Employment, Research Funding. Chen:Flatiron Health Inc: Current Employment, Research Funding; Roche Group: Current equity holder in publicly-traded company. Huntington:AbbVie: Consultancy; Astrazeneca: Honoraria; Bayer: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; DTRM: Research Funding; Genentech: Consultancy; Novartis: Consultancy; Pharmacyclics: Honoraria; TG Therapeutics: Research Funding.

Anti-CD20-Mediated B Cell Depletion Is Associated with Reduced Osteoclastogenic Signals and Bone Mass Preservation: Clinical Observation in Patients with Follicular Lymphoma Supplemented By Animal Studies in a Murine Model

Background and aims: Immunotherapy with anti-CD20-specific antibodies (e.g. rituximab), has become the standard of care for B cell lymphoproliferative disorders and many autoimmune diseases. Despite previously demonstrated role for B cells in bone metabolism, the effect of anti-CD20-mediated B cell depletion on bone mass in human patients has not been thoroughly studied. For example, in rheumatological patients the effect of rituximab on bone mass yielded conflicting results, while in lymphoma patients it has not yet been described. Here, we describe the effect of treatment with anti-CD20-specific antibodies on bone mass in a cohort of patients with follicular lymphoma and propose a plausible mechanism using murine model. Methods: To assess the effect of rituximab on bone mass in lymphoma patients, we retrospectively studied the bone mass in patients with follicular lymphoma (FL) during the maintenance phase of chemoimunotherapy, i.e. when rituximab is administered as monotherapy. FL patients on no maintenance (historical controls) or patients with marginal zone lymphoma were include as a control group. Cross-sectional X-ray imaging (CT/PET-CT), performed at the completion of the induction phase and 6-12 months thereafter, were used to serially assess bone density. Wild-type female C57BL/6J mice and mouse anti-mouse CD20 antibody (Genentech) were used for the animal experimental system. Murine bone structure was assessed by the microCT method. Immunophenotyping of the bone marrow (BM), spleen and peripheral blood cells was performed. ELISA and "real-time" quantitative PCR were used to measure the levels of key mediators of bone remodeling, e.g. RANKL, OPG and TNFα. Standard osteoclastogenic assay was used to assesses the osteoclastogenic potential of BM cells. Results: Rituximab treatment prevented the decline in bone mass observed in patients who did not receive active maintenance therapy, both in the lumbar spine (-2.6% vs -8%) and femoral head (-0.5% vs -5.1%) (n=12 patients in each group, p&amp;lt;0.05 for the comparisons in the control group, calculated by Wilcoxon matched-pairs signed rank test). Anti-CD20-mediated B cell depletion in mice led to a significant increase in bone mass as reflected by: 7.7% increase in bone mineral density (whole femur) and ~5% increase in cortical as well as trabecular tissue mineral density (n=17-21 mice in each group, p&amp;lt;0.05). Mechanistically, treating mice with anti-CD20 antibodies significantly decreased the osteoclastogenic signals, including RANKL and TNFα, along with a substantial downregulation of RANK (the RANKL receptor). This correlated with nearly a 50% reduction in osteoclastogenic potential of BM cells derived from B-cell-depleted animals (p&amp;lt;0.05). The decline in the RANKL output was observed both at the bone level (≈25% relative reduction in the mRNA levels), as measured in the whole bone and BM cells, as well as in the serum (18% relative reduction) of the anti-CD20-treated mice as compared to diluent-treated controls (p&amp;lt;0.05 for all comparisons). No significant changes in the OPG levels were noted. Conclusions: While many lymphoma patients may suffer from bone loss due to advanced age and glucocorticoid administration, our data suggest additional favorable effect of anti-CD20 treatment in bone preservation. Importantly, our murine studies indicate that B cell depletion has a direct effect on bone remodeling, primarily by reducing the osteoclastogenic signals, thus potentially diminishing bone resorption. This novel unrecognized effect should be taken into consideration when maintenance treatment is considered. MM and DN contributed equally to this work. Figure Disclosures No relevant conflicts of interest to declare.

COVID-19 Impact on Lymphoma Patients' Clinical Outcomes - an Observational Cohort Study

COVID-19 Impact on Lymphoma Patients' Clinical Outcomes - an Observational Cohort Study