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The Primary Immune Deficiency Treatment Consortium (PIDTC) performed a retrospective analysis of 662 patients with severe combined immunodeficiency (SCID) who received a hematopoietic cell transplantation (HCT) as first-line treatment between 1982 and 2012 in 33 North American institutions. Overall survival was higher after HCT from matched-sibling donors (MSDs). Among recipients of non-MSD HCT, multivariate analysis showed that the SCID genotype strongly influenced survival and immune reconstitution. Overall survival was similar for patients with RAG, IL2RG, or JAK3 defects and was significantly better compared with patients with ADA or DCLRE1C mutations. Patients with RAG or DCLRE1C mutations had poorer immune reconstitution than other genotypes. Although survival did not correlate with the type of conditioning regimen, recipients of reduced-intensity or myeloablative conditioning had a lower incidence of treatment failure and better T- and B-cell reconstitution, but a higher risk for graft-versus-host disease, compared with those receiving no conditioning or immunosuppression only. Infection-free status and younger age at HCT were associated with improved survival. Typical SCID, leaky SCID, and Omenn syndrome had similar outcomes. Landmark analysis identified CD4(+) and CD4(+)CD45RA(+) cell counts at 6 and 12 months post-HCT as biomarkers predictive of overall survival and long-term T-cell reconstitution. Our data emphasize the need for patient-tailored treatment strategies depending upon the underlying SCID genotype. The prognostic significance of CD4(+) cell counts as early as 6 months after HCT emphasizes the importance of close follow-up of immune reconstitution to identify patients who may need additional intervention to prevent poor long-term outcome.

Allogeneic hematopoietic stem cell transplant (HSCT) typically results in donor T-cell engraftment and function in patients with severe combined immunodeficiency (SCID), but humoral immunity, particularly when using donors other than matched siblings, is variable. B-cell function after HSCT for SCID depends on the genetic cause, the use of pre-HSCT conditioning, and whether donor B-cell chimerism is achieved. Patients with defects in IL2RG or JAK3 undergoing HSCT without conditioning often have poor B-cell function post-HSCT, perhaps as a result of impairment of IL-21 signaling in host-derived B cells. To investigate the effect of pre-HSCT conditioning on B-cell function, and the relationship of in vitro B-cell function to clinical humoral immune status, we analyzed 48 patients with IL2RG/JAK3 SCID who were older than 2 years after HSCT with donors other than matched siblings. T follicular helper cells (TFH) developed in these patients with kinetics similar to healthy young children; thus, poor B-cell function could not be attributed to a failure of TFH development. In vitro differentiation of B cells into plasmablasts and immunoglobulin secretion in response to IL-21 strongly correlated with the use of conditioning, donor B-cell engraftment, freedom from immunoglobulin replacement, and response to tetanus vaccine. Patients receiving immunoglobulin replacement who had normal serum immunoglobulin M showed poor response to IL-21 in vitro, similar to those with low serum IgM. In vitro response of B cells to IL-21 may predict clinically relevant humoral immune function in patients with IL2RG/JAK3 SCID after HSCT.

Background & Aim: Mesenchymal stromal cells (MSCs) are an investigational cell therapy for inflammatory diseases. Although they have robust anti-inflammatory properties, their success has been variable in clinical trials due to an unclear understanding of their mechanism. Once injected, a majority of MSCs traffic to the lung, where they are rapidly cleared, signifying an opportunity to target lung inflammatory [Figure presented] conditions such as acute respiratory distress syndrome (ARDS). ARDS is a catastrophic condition of the lungs, involving pulmonary inflammation that develops with severe SARS-CoV2 and other respiratory infections. MSCs are expected to prevent alveolar damage by suppressing the immune response and there is evidence that MSCs protect in phase I/IIa trials for ARDS associated with COVID-19. While these results are promising, understanding the mechanism is critical to determine dosing, maximize efficacy, and ultimately lead to an approved product. Methods, Results & Conclusion: We and others have demonstrated that within their short time in the lung, MSCs interact with monocytes and macrophages. Through direct cell contact, MSCs transfer cytoplasmic components, notably cytoplasmic processing-bodies (p-bodies) to monocytes and macrophages. P-bodies are membrane-less organelles that contain RNA binding proteins, microRNAs, and mRNAs enriched for genes that regulate the transcriptional landscape of cells. MSC interactions result in long-term transcriptional reprogramming of monocytes and macrophages to suppress a helper T cell response and upregulate tissue repair pathways. To investigate the mechanisms of MSCs in vivo, we utilized 2 mouse models of lung inflammation: 1. intranasal lipopolysaccharide (LPS) to study general acute inflammation and 2. an engineered vesicular stomatitis virus (VSV) with a SARS-CoV2 Spike protein. Using these models, we demonstrated that during inflammation cytoplasm of MSCs transferred to lung macrophages to decrease activation and the expression of MHC-II. Further, MSCs prevented a decrease in resident alveolar macrophages, suppressed proinflammatory macrophages, and blocked an influx in infiltrating monocytes (Fig 1). Depleting p-bodies from MSCs abolished the beneficial effects, despite transfer cytoplasmic component to macrophages at similar levels of control MSCs. Overall, our data suggest a novel form of cell communication that could explain how MSCs could lead to long-term beneficial effects on lung inflammation despite being rapidly cleared. [Figure presented]

Background & Aim We have developed an umbilical cord-blood derived, macrophage-like cell therapy, DUOC-01, to treat demyelinating conditions of the central nervous system. Previously, we showed that DUOC-01 accelerated remyelination, decreased gliosis, and reduced cellular infiltration in the brain of immune-incompetent mice exposed to the demyelinating agent, cuprizone. To further explore the mechanism of action and investigate whether DUOC-01 will be effective in other experimental models of demyelination, we tested DUOC-01 in the experimental autoimmune encephalomyelitis (EAE) model, an animal model used to study multiple sclerosis (MS). Methods, Results & Conclusion EAE was induced in C57BL/6 mice by immunizing with myelin oligodendrocyte glycoprotein peptide (MOG35-55) in complete Freund's adjuvant. We have previously established in a phase I trial of intrathecally administered DUOC-01 cells in patients with leukodystrophies, that DUOC-01 is formulated in hydrocortisone (HC) for infusion in the clinic. Therefore, to mimic the practice in the clinic, DUOC-01 cells were incubated in Ringer's Lactate with 3mg/mL HC at a dilution of 1 × 106 cells/ml for 2 hours at room temperature prior to injection. DUOC-01 cells were then administered at a dose of 3 × 105cells/mouse into the cerebrospinal fluid by a single intra-cisterna magna injection at the point when mice started showing early disease symptoms of EAE. Clinical score was recorded for next several days. Compared to mice injected with Ringer's (n=9), mice injected with DUOC-01 (n=9) had decreased severity of the disease, as indicated by lower clinical scores based on severity of ascending paralysis. The spinal cords of mice in the cohorts were studied histopathologically and those with mice injected with DUOC-01 had reduced inflammation and lower cellular infiltration compared to spinal cords of mice injected with Ringer's. The overall number of CD45+ immune cells were less in spinal cords of mice treated with DUOC-01 cells compared to the Ringer's injected mice. Most strikingly, among the various cell types, the number of neutrophils present in the spinal cords from the DUOC-01 treated group was less than the Ringer's treated samples. Presently we are exploring the mechanism(s) through which DUOC-01 cells promote remyelination and decrease immune cell infiltration. In brief, our data suggest that DUOC-01 cell therapy product could be beneficial in treating MS and other diverse neurological conditions with demyelination.

Background & Aim MSCs represent approximately 25% of all cell-based clinical trials with over 1,000 registered on clinicaltrials.gov. Although the safety of MSCs is well-documented, the success in trials over the past 25 years is variable. This is likely due to the differences in the source tissue to generate MSCs, manufacturing procedures, the heterogeneity of the MSC populations, and the disease being targeted. Regardless of these differences, it is absolutely critical to understand the mechanism of action in vivo to optimize the potential of MSCs as a FDA licensed cell therapy product. Recent reports highlight that MSCs are rapidly cleared in vivo yet modulate myeloid cell populations to limit inflammation well after they are no longer detectable. Human umbilical cord tissue is readily available and a rich source of rapidly proliferating MSCs. In this study, we hypothesized that human cord tissue-derived MSCs (hCT-MSCs) can reprogram inflammatory macrophages and limit disease progression in inflammatory diseases. Methods, Results & Conclusion Multiple lines of hCT-MSCs were manufactured from donated cord tissue in the Robertson GMP laboratory within the Marcus Center for Cellular Cures (MC3). Using cell culture assays, we determined that hCT-MSC indirectly suppressed T cells by directly interacting with macrophages. Macrophages did not phagocytosis live MSCs, rather they engulfed cytoplasmic components, which down-regulated genes responsible for presenting antigens and activating T cells. Since vast amounts of information can be stored within cytoplasmic granules call processing bodies (P-bodies), we tested their role in facilitating hCT-MSCs to reprogram macrophages. hCT-MSCs contained substantially numbers of P-bodies in the cytoplasm and when we genetically depleted P-bodies in MSCs, they failed to reprogram macrophages to suppress T cells (Fig. 1). Overall, our data demonstrate that hCT-MSCs reprogram macrophages to suppress a T cell response through P-bodies. This not only explains a potential in vivo mechanism how MSCs can confer long-term benefit after they are cleared but is a potential novel and manipulatable target to promote increased efficacy of MSCs in multiple inflammatory conditions.

Severe combined immunodeficiency (SCID) is a fatal syndrome caused by mutations in at least 13 different genes. It is characterized by the absence of T-cells. Immune reconstitution can be achieved through non-ablative related donor bone marrow transplantation. However, the first transplant may not provide sufficient immunity. In these cases, booster transplants may be helpful. A prospective/retrospective study was conducted of 49 SCID patients (28.7 percent of 171 SCIDs transplanted over 30 years) who had received booster transplants to define the long term outcome, factors contributing to a need for a booster and factors that predicted success. Of the 49 patients, 31 (63 percent) are alive for up to 28 years. Age at initial transplantation was found to have a significant effect on outcome (mean of 194 days old for patients currently alive, versus a mean of 273 days old for those now deceased, p=0.0401). Persistent viral infection was present in most deceased booster patients. In several patients, the use of two parents as sequential donors resulted in striking T and B cell immune reconstitution. A majority of the patients alive today have normal or adequate T-cell function and are healthy. Non-ablative booster bone marrow transplantation can be life-saving for SCID.

The persistence of transplacentally transferred maternal T cells is common in infants with severe combined immunodeficiency (SCID), occurring in more than half of patients with SCID undergoing transplantation at our institution. These T cells respond poorly to mitogens in vitro but can cause cutaneous graft-versus-host disease; however, other effects of these cells are unknown. We describe 2 infants with SCID who had unusual problems associated with transplacentally transferred maternal T cells. Patient 1 was a 5-month-old girl with Janus kinase 3-deficient SCID who had 4% circulating CD3(+) T cells but no lymphocyte proliferative response to mitogens. Although the number of T cells increased after 2 nonchemoablated, T cell-depleted, haploidentical, paternal bone marrow transplantations, T-cell function failed to develop, and she became pancytopenic. Restriction fragment length polymorphism studies of flow cytometry-sorted blood T cells revealed all to be of maternal origin. A subsequent nonchemoablated, T cell-depleted maternal transplantation resulted in normal T-cell function and marrow recovery. Patient 2 was a 9-month-old girl with IL-7Ralpha-deficient SCID who presented with autoimmune pancytopenia. She had 8% blood T cells (all CD45RO(+)) but no response to mitogens. High-resolution HLA sequence-specific priming typing detected both maternal haplotypes, indicating the presence of maternal cells. Her pancytopenia resolved after treatment with rituximab and was thought to be due to host B-cell activation by transplacentally acquired maternal T cells. Persistent transplacentally acquired maternal T cells in infants with SCID can mediate immunologic functions despite failing to respond to mitogens in vitro. We present evidence that these cells can cause allograft rejection and immune cytopenias.