Marilia Cascalho

Acute respiratory distress syndrome (ARDS) is a devastating complication of respiratory infections; however, the biological mechanisms that initiate its onset are poorly defined. Here we show that TNFRSF13B polymorphisms increase the risk of ARDS following SARS-CoV-2 infection up to 7.4-fold compared to the WT genotype. The increased risk was not due to immune-deficiency or impaired virus neutralization. On the contrary, TNFRSF13B mutant subjects mounted better antibody neutralization compared to subjects with WT TNFRSF13B. However, IgG from subjects expressing TNFRSF13B variants had less sialic acid, terminal galactose, and fucose than IgG from subjects with a WT genotype. Moreover, IgG from TNFRSF13B mutant subjects exhibited increased recruitment of complement factors. Thus, besides well-known actions governing plasma cell differentiation, TNFRSF13B impacts both affinity maturation and effector functions of IgG in ways that independently govern complement activation controlling inflammatory responses known to trigger ARDS.

Anticancer treatments may cause depletion of the nonrenewable ovarian reserve of follicles, the source of key hormones, leading to premature ovarian insufficiency (POI) and substantial endocrine, musculoskeletal, and neurological complications. Hormone replacement therapy, the only approved treatment for POI, poorly mimics ovarian physiology. Here, we examined whether human ovarian tissue implanted in an immune-isolating hydrogel-based capsule restored ovarian endocrine function in ovariectomized immunodeficient mice. Daily vaginal cytology, hormone measurements, and histological analysis demonstrated that the encapsulated xenografts integrated into the murine hypothalamic-pituitary-gonad axis and responded to circulating gonadotropins. Without exogenous stimulation, regular estrous cyclicity resumed 12 weeks postimplantation, and mice experienced three to eight estrous cycles in 20 weeks. Large antral follicles, ~3 millimeters in diameter, were present in the grafts, consistent with regular cyclicity and increasing levels of estradiol that reached 50 picograms per milliliter, demonstrating integration with the host physiology and restoration of ovarian endocrine function, similar to the nonencapsulated controls.

Heart transplantation is the gold standard treatment for patients with end-stage heart failure. However, there is a shortage of donor hearts available. The short tolerable cold ischemic time for delivering donor hearts to matching recipients is closely responsible for this shortage. Here we uncover the phenomenon of mineralocorticoid receptor (MR) phase separation, which exacerbates injury to the murine and human donor heart during cold storage and can be modulated with pharmacological inhibition to improve preservation quality. Interestingly, donor cardiomyocytes strongly expressed MR, which undergoes preservation-related phase separation. The phenomenon of macromolecular phase separation is not limited to the heart or MR during preservation. Cold preservation of the lung, liver and kidney also displays phase separation of other transcriptional regulators including histone deacetylase 1 (HDAC1), bromodomain-containing 4 (BRD4) and MR. Our results reveal an understudied area of preservation biology that may be further exploited to improve the preservation of multiple solid organs.

Immune-surveillance depends in part on the recognition of peptide variants by T cell antigen receptors. Given that both normal B cells and malignant B cells accumulate mutations we chose a murine model of multiple myeloma to test conditions to induce cell-mediated immunity targeting malignant plasma cell (PC) clones but sparing of normal PCs. Revealing a novel function for intracellular C3d, we discovered that C3d engaged T cell responses against malignant plasma cells in the bone marrow of mice that had developed multiple myeloma spontaneously. Our results show that C3d internalized by cells augments immune surveillance by several mechanisms. In one, C3d induces a master transcription regulator, E2f1, to increase the expression of long non-coding (lnc) RNAs, to generate peptides for MHC-I presentation and increase MHC-I expression. In another, C3d increases expression of RNAs encoding ribosomal proteins linked to processing of defective ribosomal products (DRiPs) that arise from non-canonical translation and known to promote immunosurveillance. Cancer cells are uniquely susceptible to increased expression and presentation of mutant peptides given the extent of protein misfolding and accumulation of somatic mutations. Accordingly, although C3d can be internalized by any cell, C3d preferentially targets malignant clones by evoking specific T cell mediated immunity (CMI) and sparing most non-transformed polyclonal B cells and plasma cells with lower mutation loads. Malignant plasma cell deletion was blocked by cyclosporin or by CD8 depletion confirming that endogenous T cells mediated malignant clone clearance. Besides the potential for therapeutic application our results highlight how intracellular C3d modifies cellular metabolism to augment immune surveillance. One Sentence Summary: We show that intracellular soluble fragment 3d of complement (C3d) induces regression of spontaneous multiple myeloma in mice reducing tumor burden by 10 fold, after 8 weeks. C3d enables cell-mediated immunity to target multiple myeloma clones sparing non-transformed polyclonal B cells and plasma cells with lower mutation loads. We show that C3d increases the expression of ribosomal subunits associated with the translation of defective ribosomal products (DRiPs). C3d also decreases expression of protein arginine methyl transferase (PRMT) 5 which in turn relieves E2f1 repression increasing the expression of Lnc RNAs and derived peptides that evoke anti-tumor cellular immunity. The approach increases MHC-I expression by tumor cells and generates a CMI response that overcomes tumor immune-evasion strategies. Significance: Tumors are immunogenic in part because of somatic mutations that originate novel peptides that once presented on MHC engage cell-mediated immunity (CMI). However, in spite of the higher mutation load most tumors evade immunity. We discovered that a component of the complement system (C3d) overcomes tumor immune evasion by augmenting expression of ribosomal proteins and lncRNAs linked to the presentation of novel peptides by tumor cells. C3d induced CMI targets cancer cells sparing non transformed cells uncovering a novel function for complement in immune surveillance.

Premature loss of ovarian function (POI) is associated with numerous negative side effects, including vasomotor symptoms, sleep and mood disturbances, disrupted urinary function, and increased risks for osteoporosis and heart disease. Hormone replacement therapy (HRT), the standard of care for POI, delivers only a subset of ovarian hormones and fails to mimic the monthly cyclicity and daily pulsatility characteristic of healthy ovarian tissue in reproductive-aged individuals whose ovarian tissue contains thousands of ovarian follicles. Ovarian tissue allografts have the potential to serve as an alternative, cell-based HRT, capable of producing the full panel of ovarian hormones at physiologically relevant doses and intervals. However, the risks associated with systemic immune suppression (IS) required to prevent allograft rejection outweigh the potential benefits of comprehensive and dynamic hormone therapy. This work investigates whether the age of ovarian tissue donor animals affects the function of, and immune response to, subcutaneous ovarian grafts. We performed syngeneic and semi-allogeneic ovarian transplants using tissue from mice aged 6–8 (D7) or 20–22 (D21) days and evaluated ovarian endocrine function and immune response in a mouse model of POI. Our results revealed that tissue derived from D7 donors, containing an ample and homogeneous primordial follicle reserve, was more effective in fully restoring hypothalamic–pituitary–ovarian feedback. In contrast, tissue derived from D21 donors elicited anti-donor antibodies with higher avidity compared to tissue from younger donors, suggesting that greater immunogenicity may be a trade-off of using mature donors. This work contributes to our understanding of the criteria donor tissue must meet to effectively function as a cell-based HRT and explores the importance of donor age as a factor in ovarian allograft rejection.

Transplantation of allogeneic donor ovarian tissue holds great potential for female cancer survivors who often experience premature ovarian insufficiency. To avoid complications associated with immune suppression and to protect transplanted ovarian allografts from immune-mediated injury, we have developed an immunoisolating hydrogel-based capsule that supports the function of ovarian allografts without triggering an immune response. Encapsulated ovarian allografts implanted in naïve ovariectomized BALB/c mice responded to the circulating gonadotropins and maintained function for 4 months, as evident by regular estrous cycles and the presence of antral follicles in the retrieved grafts. In contrast to non-encapsulated controls, repeated implantations of encapsulated mouse ovarian allografts did not sensitize naïve BALB/c mice, which was confirmed with undetectable levels of alloantibodies. Further, encapsulated allografts implanted in hosts previously sensitized by the implantation of non-encapsulated allografts restored estrous cycles similarly to our results in naïve recipients. Next, we tested the translational potential and efficiency of the immune-isolating capsule in a rhesus monkey model by implanting encapsulated ovarian auto- and allografts in young ovariectomized animals. The encapsulated ovarian grafts survived and restored basal levels of urinary estrone conjugate and pregnanediol 3-glucuronide during the 4- and 5-month observation periods. We demonstrate, for the first time, that encapsulated ovarian allografts functioned for months in young rhesus monkeys and sensitized mice, while the immunoisolating capsule prevented sensitization and protected the allograft from rejection.

Key Points: Ixazomib treatment resulted in decreases in B-cell subsets and bone marrow lymphocytes. Ixazomib treatment resulted in modest decreases in certain anti-HLA antibody specificities. Ixazomib treatment was tolerated, with modest adverse events. Background: Ixazomib is a second-generation oral proteasome inhibitor approved for treatment of refractory multiple myeloma. We conducted an open-label phase II trial, IXAzomib for DESensitization (IXADES), testing the safety of ixazomib treatment as an approach to decreasing the level and diversity of specificities of anti-HLA antibodies in subjects awaiting kidney transplantation. The trial (NCT03213158) enrolled highly sensitized kidney transplant candidates, defined as subjects with calculated panel reactive antibodies (cPRA) >80%, awaiting kidney transplantation >24 months. The subjects were treated with 12 monthly cycles of ixazomib 3 mg+dexamethasone 20 mg. Efficacy was defined as a decrease of cPRA >20% or kidney transplantation. The safety end point was tolerability. Methods: In ten enrolled subjects, no grade IV, five grade III, 11 grade II, and 43 grade I adverse events were noted. The adverse events included infection, transient paresthesia, nausea, vomiting, and diarrhea. The IXADES regimen was not associated with significant change in levels or diversity of anti-HLA antibodies (cPRA). Results: Although the IXADES regimen did not exhibit a clear impact on levels and diversity of anti-HLA antibodies in this small cohort, the prolonged half-life of IgG could necessitate a longer duration of treatment for accurate evaluation of efficacy. Conclusions: In conclusion, treatment with ixazomib/dexamethasone engendered mild-to-moderate toxicity. The impact on anti-HLA was modest and paradoxical in the case of anti-HLA-DR. Clinical trials combining ixazomib with other immunosuppressive agents may be more effective in addressing antibody-mediated processes in kidney transplantation.

Monoclonal antibodies that target SARS-CoV-2 with high affinity are valuable for a wide range of biomedical applications involving novel coronavirus disease (COVID-19) diagnosis, treatment, and prophylactic intervention. Strategies for the rapid and reliable isolation of these antibodies, especially potent neutralizing antibodies, are critical toward improved COVID-19 response and informed future response to emergent infectious diseases. In this study, single B cell screening was used to interrogate antibody repertoires of immunized mice and isolate antigen-specific IgG1⁺ memory B cells. Using these methods, high-affinity, potent neutralizing antibodies were identified that target the receptor-binding domain of SARS-CoV-2. Further engineering of the identified molecules to increase valency resulted in enhanced neutralizing activity. Mechanistic investigation revealed that these antibodies compete with ACE2 for binding to the receptor-binding domain of SARS-CoV-2. These antibodies may warrant further development for urgent COVID-19 applications. Overall, these results highlight the potential of single B cell screening for the rapid and reliable identification of high-affinity, potent neutralizing antibodies for infectious disease applications.

Host genes define the severity of inflammation and immunity but specific loci doing so are unknown. Here we show that TNF receptor superfamily member 13B ( TNFRSF13B ) variants, which enhance defense against certain pathogens, also control immune-mediated injury of transplants, by regulating innate B cells’ functions. Analysis of TNFRSF13B in human kidney transplant recipients revealed that 33% of those with antibody-mediated rejection (AMR) but fewer than 6% of those with stable graft function had TNFRSF13B missense mutations. To explore mechanisms underlying aggressive immune responses, we investigated alloimmunity and rejection in mice. Cardiac allografts in Tnfrsf13b- mutant mice underwent early and severe AMR. The dominance and precocity of AMR in Tnfrsf13b -deficient mice were not caused by increased alloantibodies. Rather, Tnfrsf13b mutations decreased “natural” IgM and compromised complement regulation, leading to complement deposition in allografted hearts and autogenous kidneys. Thus, WT TNFRSF13B and Tnfrsf13b support innate B cell functions that limit complement-associated inflammation; in contrast, common variants of these genes intensify inflammatory responses that help clear microbial infections but allow inadvertent tissue injury to ensue. The wide variation in inflammatory reactions associated with TNFRSF13B diversity suggests polymorphisms could underlie variation in host defense and explosive inflammatory responses that sometimes enhance morbidity associated with immune responses.