Pharmacological Immunosuppression Research Articles

Efficacy and limitations of combined A2.CAR Treg and anti-CD154 therapy in a mouse model of haplo-mismatched heart transplantation.

The ability to induce allograft specific tolerance would reduce the need for daily pharmacological immunosuppression, improve post-transplant quality of life and transplant outcomes. Adoptive cell therapy with regulatory T cells expressing donor-specific Chimeric Antigen Receptors (CAR-Tregs) is a promising strategy, but monotherapy has not resulted in prolonged survival of grafts with multiple MHC mismatches. We used a clinically-relevant, haplo-mismatched model of heart transplantation in immune-competent C57BL/6 recipients to test the ability of HLA-A2-specific (A2) CAR Tregs to synergize with CD154 blockade to enhance graft survival. We found that in combination with a single low dose of anti-CD154, A2.CAR Tregs significantly prolonged heart allograft survival. Through use of grafts expressing the 2W-OVA transgene, tetramer tracking of 2W- and OVA-specific cells revealed that in mice with accepted grafts, the effects of A2.CAR Tregs included inhibition of endogenous donor-specific CD4 + and CD8 + T cell expansion, and B cell and antibody responses. Moreover, within the 2W-specific CD4 + T cell population, there was a significant increase in the proportion of FoxP3 pos cells, suggestive of infectious tolerance. In mice where CD154 blockade and A2.CAR Tregs failed to prolong graft survival, there was preferential expansion of FoxP3 neg A2.CAR T cells within the rejecting allograft. Thus, this study provides the first evidence for a synergistic effect between CAR Tregs and CD40-pathway blockade and supports the further refinement of this strategy as a promising future direction towards the goal of transplantation tolerance.

Advancing Allogeneic Hematopoietic Stem Cell Transplantation Outcomes through Immunotherapy: A Comprehensive Review of Optimizing Non-CAR Donor T-Lymphocyte Infusion Strategies.

Optimized use of prophylactic or therapeutic donor lymphocyte infusions (DLI) is aimed at improving clinical outcomes in patients with malignant and non-malignant hematological diseases who have undergone allogeneic hematopoietic stem cell transplantation (allo-HSCT). Memory T-lymphocytes (CD45RA-/CD45RO+) play a crucial role in immune reconstitution post-HSCT. The infusion of memory T cells is proven to be safe and effective in improving outcomes due to the enhanced reconstitution of immunity and increased protection against viremia, without exacerbating graft-versus-host disease (GVHD) risks. Studies indicate their persistence and efficacy in combating viral pathogens, suggesting a viable therapeutic avenue for patients. Conversely, using virus-specific T cells for viremia control presents challenges, such as regulatory hurdles, cost, and production time compared to CD45RA-memory T lymphocytes. Additionally, the modulation of regulatory T cells (Tregs) for therapeutic use has become an important area of investigation in GVHD, playing a pivotal role in immune tolerance modulation, potentially mitigating GVHD and reducing pharmacological immunosuppression requirements. Finally, donor T cell-mediated graft-versus-leukemia immune responses hold promise in curbing relapse rates post-HSCT, providing a multifaceted approach to therapeutic intervention in high-risk disease scenarios. This comprehensive review underscores the multifaceted roles of T lymphocytes in HSCT outcomes and identifies avenues for further research and clinical application.

Spatiotemporal immune atlas of a clinical-grade gene-edited pig-to-human kidney xenotransplant

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. Here, we transplant a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and study the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells are uncommon in the porcine kidney cortex early after xenotransplantation and consist of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages express genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft is detectable. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression may be able to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.

Risk Factors Associated to Severe Episodes of Infection in Myelodysplastic Syndromes

INTRODUCTION. Mortality differs according to the Revised International Prognostic Scoring System (IPSS-R) risk categories in patients afflicted with myelodysplastic syndromes (MDS). Historically, high-risk (HR)-MDS (IPSS-R >3.5 points) is related to death caused by progression to acute myeloid leukemia (AML), while low-risk (LR)-MDS is associated with mortality due to severe infections (Nachtkamp K, et al. 2016), probably caused by neutrophile dysfunction and hindered oxidative burst. Studies of causes of death (COD) in MDS are scarce. A MD Anderson study in LR-MDS patients identified infections as the leading COD, followed by progression (Dayyani F, et al, 2010), while the Düsseldorf registry recognized infections as the main COD across all MDS categories (Neukirchen, J. et al, 2011). Considering that information in low- and medium-income countries is especially limited, this study aims to describe the characteristics and identify the risk factors for severe infectious events in patients afflicted by MDS in a third-level center in Mexico. METHODS. In this single-center retrospective study, we analyzed severe infection episodes, associated risk factors, and the COD in MDS patients.We included patients diagnosed with MDS from January 2000 to December 2021, according to the 2016 WHO criteria. We registered baseline characteristics and described the episodes of severe infection, defined as hospitalization events due to infectious diseases complicated with septic shock and/or respiratory failure requiring orotracheal intubation. We conducted uni- and multivariate analyses based on the following variables: neutropenia (≤0.5x10 9/L), monocytopenia (≤0.2x10 9/L), lymphopenia (≤1.0x10 9/L), ferritin (≥1,000ng/mL), autoimmune disease, MDS comorbidity index, COPD, granulocyte dysplasia, and treatment with azacytidine or pharmacological immunosuppression. RESULTS. We included 156 patients with MDS treated at our institution. Median age at diagnosis was 65 years (IQR; 55-75 years), 53% of which were women. Multilineage dysplasia was the most prevalent MDS type (59%), followed by excess blasts type 2 (11.5%). LR-MDS represented 68% (n=106), while HR-MDS, 32% (n=50). Hypocellularity in bone marrow biopsy was found in 24.4%. Eighty-seven patients developed 156 infectious episodes, 64.7% of which were severe. A median of 1 serious event (IQR: 0.75-1 event) occurred in each patient, with a median time to diagnosis of 13 months (IQR: 3.5-34.5 months). Infection sites and etiologies are described in Figure 1. Of note, we identified 14 fungal infections, 8 of them by Aspergillus, with a 37.5% death rate. Risk factors associated with increased severe infection risk were neutropenia during follow-up (OR 3.2; 95% CI 1.5-6.4; p=0.001), neutrophil hypogranularity at diagnosis (OR 2.1; CI 95% 1.1-4.1; p=0.029), hyperferritinemia (OR 2.46; CI 95% 1.2-4.9; p=0.011), and azacitidine treatment (OR 3.1; CI 95% 1.01-9.6; p=0.047). Fifty patients (32%) died. In both HR-MDS and LR-MDS groups, the main COD was infection (13 in HR group, 48.1%, vs 10 in LR group, 43.5%; p=0.782), followed by progression to AML (3 in HR group, 11.1%, vs 4 in LR group, 21.7%; p=0.444). Median overall survival was lower in patients with severe neutropenia (62 vs. 210 months; p=0.009) and hyperferritinemia (62 months vs. non-reached, p<0.001) (Figure 2). CONCLUSIONS In our MDS population severe infections represent the leading COD, regardless of risk category. More than half developed at least one infectious episode, 64.7% of which were severe. Fungal infections were highly lethal, suggesting antifungal prophylactic interventions could prove helpful. We found neutropenia during follow-up and hypogranularity at diagnosis to be significant risk factors, the latter perhaps reflecting neutrophil disfunction which should be further evaluated. Hyperferritinemia's association with increased infection and mortality urges to further understand its role in MDS pathophysiology, and could potentially prove a therapeutic target in infections disease prevention. Additionally, there is currently no consensus on infectious disease for use of azacitidine, which might add to a worsening risk of infection.

1779. Trends in Anaplasmosis Over the Past Decade: A Review of Clinical Features, Laboratory Data and Outcomes

Abstract Background Human granulocytic anaplasmosis (HGA) is an emerging tick-borne illness endemic to the northeastern and midwestern United States. Risk factors for the severity of infection, predictors for hospitalization, temporal trends, co-infections, and outcomes are poorly studied. Methods We conducted a retrospective review of HGA at Mayo Clinic, Rochester, between 2011-2021. Cases were identified by positive Anaplasma PCR at our reference laboratory. Variables included patients’ characteristics, symptoms, risk factors for tick-borne illness, laboratory parameters, co-infections, treatment, and clinical outcomes. Results We identified 471 cases with a positive Anaplasma PCR, and 146 cases were included in this interim analysis. Table 1 shows a summary of the baseline characteristics. An increasing annual trend in diagnosis of HGA was noted from 2014 to 2016 with lower rates observed during COVID-19 pandemic (2020 to 2022) (Figure 1). Of the 146 patients, 19 (13%) were immunocompromised secondary to hematologic malignancies, solid organ malignancies, and pharmacological immunosuppression. Frequently reported symptoms included fever (79.5%), malaise (68.5%), chills (51.4%), and myalgia (50%). Tick exposure was documented in 79.2 % of cases. Forty-seven (33%) patients were hospitalized; older patients were more likely to be hospitalized (Table 2). The most common complications among the hospitalized patients included respiratory failure (50%), encephalopathy (44.4%) and renal failure (37.5%). One patient required ICU admission. Twenty-five patients (17.5%) and one patient (0.7%) were co-infected with Lyme and Ehrlichiosis, respectively. Higher rates of leukopenia and thrombocytopenia were associated with mono-infection with Anaplasma (p < 0.001). Follow up data was available for 136 patients; all of them had favorable response to doxycycline.Table 1:Summary of baseline characteristics, labs and treatment.Table 2:Predictors of HospitalizationFigure 1:Annual Trends in Anaplasmosis from 2011-2021. Conclusion Our study demonstrated comprehensive clinical characteristics of patients with HGA. Interestingly, tick exposure history was common, yet most patients did not have other tick-borne co-infections. Furthermore, only a minority of patients developed severe diseases and required hospitalization. The recent decline in cases during the early part of the COVID-19 pandemic is worth further investigation. Disclosures All Authors: No reported disclosures

Vaccination Recommendations in Solid Organ Transplant Adult Candidates and Recipients.

Prevention of infections is crucial in solid organ transplant (SOT) candidates and recipients. These patients are exposed to an increased infectious risk due to previous organ insufficiency and to pharmacologic immunosuppression. Besides infectious-related morbidity and mortality, this vulnerable group of patients is also exposed to the risk of acute decompensation and organ rejection or failure in the pre- and post-transplant period, respectively, since antimicrobial treatments are less effective than in the immunocompetent patients. Vaccination represents a major preventive measure against specific infectious risks in this population but as responses to vaccines are reduced, especially in the early post-transplant period or after treatment for rejection, an optimal vaccination status should be obtained prior to transplantation whenever possible. This review reports the currently available data on the indications and protocols of vaccination in SOT adult candidates and recipients.

Initial exploration of a novel fusion protein, IL-4/IL-34/IL-10, which promotes cardiac allograft survival mice through alloregulation.

Immune mediated graft loss still represents a major risk to transplant recipients. Creative approaches to immunosuppression that exploit the recipient's own alloregulatory mechanisms could reduce the need for pharmacologic immunosuppression and potentially induce immune tolerance. In the process of studying recipient derived myeloid derived suppressor cells (MDSCs), we identified key alloregulatory MDSC mechanisms, mediated by isolatable proteins IL-4, IL-34, and IL-10. We sought to purify these proteins and fuse them for subsequent infusion into transplant recipients as a means of inducing an alloregulatory response. In this introductory investigation, we leveraged molecular engineering technology to create a fusion protein (FP) of three cytokine coding sequences of IL-4, IL-34, and IL-10 and demonstrated their expressions by Western Blot analysis. Following purification, we tested whether FP IL-4/IL-34/IL-10 (FP1) can protect heart transplant allografts. Injection of FP1 significantly prolonged allogeneic cardiac graft survival in a dose-dependent fashion and the increase of graft survival time exceeded survival attributable to IL-34 alone. In vitro, MDSCs cells were expanded by FP1 treatment. However, FP1 did not directly inhibit T cell proliferation in vitro. In conclusion, newly developed FP1 improves the graft survival in cardiac transplantation mouse model. Significant additional work to optimize FP1 or include other novel proteins could supplement current treatment options for transplant patients.

Safety and Effectiveness of Intensive Treatments Administered Outside the Intensive Care Unit to Hematological Critically Ill Patients: An Intensive Care without Walls Trial.

Historically, the admission of hematological patients in the ICU shortly after the start of a critical illness is associated with better survival rates. Early intensive interventions administered by MET could play a role in the management of hematological critically ill patients, eventually reducing the ICU admission rate. In this retrospective and monocentric study, we evaluate the safety and effectiveness of intensive treatments administered by the MET in a medical ward frame. The administered interventions were mainly helmet CPAP and pharmacological cardiovascular support. Frequent reassessment by the MET at least every 8 to 12 h was guaranteed. We analyzed data from 133 hematological patients who required MET intervention. In-hospital mortality was 38%; mortality does not increase in patients not immediately transferred to the ICU. Only three patients died without a former admission to the ICU; in these cases, mortality was not related to the acute illness. Moreover, 37% of patients overcame the critical episode in the hematological ward. Higher SOFA and MEWS scores were associated with a worse survival rate, while neutropenia and pharmacological immunosuppression were not. The MET approach seems to be safe and effective. SOFA and MEWS were confirmed to be effective tools for prognostication.

Route of dexamethasone administration influences parasite burden in Strongyloides hyperinfection model.

Rodents infected with Strongyloides venezuelensis are experimental models applied to strongyloidiasis research. This study evaluated oral and subcutaneous dexamethasone (DEX) treatments to establish immunosuppression in an experimental model of Strongyloides hyperinfection. Rattus norvegicus Wistar were divided: G I (-): untreated and uninfected animals, G II (+): untreated and infected, G III (o -) orally treated and uninfected, G IV (o +) orally treated and infected, G V (sc -) subcutaneously treated and uninfected, G VI (sc +) subcutaneously treated and infected. For oral administration, DEX was diluted in sterile water (5µg/ml) and made available to the animals on intervals in experimental days - 5-0, 8-13 and 21-26. For subcutaneous administration, animals received daily injections of DEX disodium phosphate (2mg/kg). Infection was established by the subcutaneous inoculation of 3000 S. venezuelensis filarioid larvae. Groups were evaluated by egg per gram of feces and parasite females counts and IgG, IgG1 and IgG2a detection. GIV (o +) had egg peaks count on days 13 and 26 and maintained egg elimination until the last experimental day. Parasitic females recovery at day 30 was significantly higher in G IV (o +) when compared to G VI (sc +). Levels of IgG, IgG1 and IgG2a of all groups, except the positive control GII (+), were below the detection threshold. Pharmacological immunosuppression induced by oral administration of DEX produced high parasitic burden, and is a noninvasive method, useful to establish immunosuppression in strongyloidiasis hyperinfection model in rats.

Human induced pluripotent stem cells (hiPSC), enveloped in elastin-like recombinamers for cell therapy of type 1 diabetes mellitus (T1D): preliminary data.

Introduction: Therapeutic application and study of type 1 diabetes disease could benefit from the use of functional β islet-like cells derived from human induced pluripotent stem cells (hiPSCs). Considerable efforts have been made to develop increasingly effective hiPSC differentiation protocols, although critical issues related to cost, the percentage of differentiated cells that are obtained, and reproducibility remain open. In addition, transplantation of hiPSC would require immunoprotection within encapsulation devices, to make the construct invisible to the host's immune system and consequently avoid the recipient's general pharmacologic immunosuppression. Methods: For this work, a microencapsulation system based on the use of "human elastin-like recombinamers" (ELRs) was tested to envelop hiPSC. Special attention was devoted to in vitro and in vivo characterization of the hiPSCs upon coating with ERLs. Results and Discussion: We observed that ELRs coating did not interfere with viability and function and other biological properties of differentiated hiPSCs, while in vivo, ELRs seemed to afford immunoprotection to the cell grafts in preliminary in vivo study. The construct ability to correct hyperglycemia in vivo is in actual progress.

Anal high-risk human papillomavirus infection, squamous intraepithelial lesions and anal cancer in patients with inflammatory bowel disease: a systematic review and meta-analysis.

Ulcerative colitis (UC) and Crohn's disease (CD) can be associated with severe comorbidities, namely opportunistic infections and malignancies. We present the first systematic review and meta-analysis evaluating the burden of anal human papillomavirus disease in patients with UC and CD. PubMed, Web of Science and Scopus were searched until November 2022. Meta-analyses were performed using random effects models. The protocol was recorded at PROSPERO register with the number CRD42022356728. Six studies including 78 711 patients with UC with a total follow-up of 518 969 person-years described the anal cancer incidence rate. For anal cancer incidence rate in CD, six studies were selected including 56 845 patients with a total follow-up of 671 899 person-years. The incidence of anal cancer was 10.2 (95%CI 4.3-23.7) per 100 000 person-years in UC and 7.7 (3.5-17.1) per 100 000 person-years in CD. A subgroup analysis of anal cancer in perianal CD including 7 105 patients was calculated with incidence of 19.6 (12.2-31.6) per 100 000 person-years (three studies included). Few studies described prevalence of anal cytological abnormalities (four studies including 349 patients), and high-risk human papillomavirus (three studies including 210 patients), with high heterogeneity. Prevalence of cytological abnormalities or high-risk human papillomavirus was not associated with pharmacological immunosuppression in the studies included. The incidence of anal cancer is higher in UC than in CD, with the exception of perianal CD. There is limited and heterogenous data on anal high-risk human papillomavirus infection and squamous intraepithelial lesions prevalence in this population.

Pathogen Distribution, Drug Resistance Risk Factors, and Construction of Risk Prediction Model for Drug-Resistant Bacterial Infection in Hospitalized Patients at the Respiratory Department During the COVID-19 Pandemic.

To investigate the distribution and drug resistance of pathogens among hospitalized patients in the respiratory unit during the COVID-19 pandemic, analyze the risk factors of drug resistance, construct a risk prediction model. This study isolated 791 strains from 489 patients admitted to the Affiliated Hospital of Chengdu University, who were retrospectively enrolled between December 2019 and June 2021. The patients were divided into training and validation sets based on a random number table method (8:2). The baseline information, clinical characteristics, and culture results were collected using an electronic database and WHONET 5.6 software and compared between the two groups. A risk prediction model for drug-resistant bacteria was constructed using multi-factor logistic regression. K. pneumoniae (24.78%), P. aeruginosa (17.19%), A. baumannii (10.37%), and E. coli (10.37%) were the most abundant bacterial isolates. 174 isolates of drug-resistant bacteria were collected, ie, Carbapenem-resistant organism-strains, ESBL-producing strains, methicillin-resistant S. aureus, multi-drug resistance constituting 38.51%, 50.57%, 6.32%, 4.60%, respectively. The nosocomial infection prediction model of drug-resistant bacteria was developed based on the combined use of antimicrobials, pharmacological immunosuppression, PCT>0.5 ng/mL, CKD stage 4-5, indwelling catheter, and age > 60 years. The AUC under the ROC curve of the training and validation sets were 0.768 (95% CI: 0.624-0.817) and 0.753 (95% CI: 0.657-0.785), respectively. Our model revealed an acceptable prediction demonstrated by a non-significant Hosmer-Lemeshow test (training set, p=0.54; validation set, p=0.88). K. pneumoniae, P. aeruginosa, A. baumannii, and E. coli were the most abundant bacterial isolates. Antimicrobial resistance among the common isolates was high for most routinely used antimicrobials and carbapenems. COVID-19 did not increase the drug resistance pressure of the main strains. The risk prediction model of drug-resistant bacterial infection is expected to improve the prevention and control of antibacterial-resistant bacterial infection in hospital settings.

Development of a Simple and Sensitive Enzyme-linked Immunosorbent Assay for Sinomenine

Sinomenine (SIN) is a major component contained in extracts of the Chinese medicinal herb Sinomenium acutum. SIN has various pharmacological properties, including cytoprotection, immunosuppression and anti-inflammation effects. Furthermore, recent studies have reported that SIN has anti-tumor and antidepressant effects, which has created a strong need for SIN kinetic studies. This paper reports a simple and sensitive competitive enzyme-linked immunosorbent assay (ELISA) for the pharmacokinetic evaluation of SIN. Anti-SIN serum was obtained by immunizing mice with an antigen conjugated with bovine serum albumin and carboxylic modified SIN using the N-succinimidyl ester method. Enzyme labeling of SIN with horseradish peroxidase was similarly performed using carboxylic modified SIN. Under optimized conditions, this ELISA shows a linear detection range from 40 to 5000 pg/mL, and a limit of detection of 12.1 pg/mL for 50-µL samples. This assay was specific for SIN and showed very slight cross-reactivity with dextromethorphan (0.45%), dimemorfan (0.22%) and codeine (0.01%), but no cross-reactivity with 2-methoxycyclohex-2-enone (<0.001%). Using this ELISA, SIN levels were easily determined in the blood of mice after oral administration of Kampo medicine, Boiogito. The ELISA may be a valuable tool for studies of the biological and pharmacological properties of SIN.

Clinical adoptive regulatory T Cell therapy: State of the art, challenges, and prospective.

Rejection of solid organ transplant and graft versus host disease (GvHD) continue to be challenging in post transplantation management. The introduction of calcineurin inhibitors dramatically improved recipients' short-term prognosis. However, long-term clinical outlook remains poor, moreover, the lifelong dependency on these toxic drugs leads to chronic deterioration of graft function, in particular the renal function, infections and de-novo malignancies. These observations led investigators to identify alternative therapeutic options to promote long-term graft survival, which could be used concomitantly, but preferably, replace pharmacologic immunosuppression as standard of care. Adoptive T cell (ATC) therapy has evolved as one of the most promising approaches in regenerative medicine in the recent years. A range of cell types with disparate immunoregulatory and regenerative properties are actively being investigated as potential therapeutic agents for specific transplant rejection, autoimmunity or injury-related indications. A significant body of data from preclinical models pointed to efficacy of cellular therapies. Significantly, early clinical trial observations have confirmed safety and tolerability, and yielded promising data in support of efficacy of the cellular therapeutics. The first class of these therapeutic agents commonly referred to as advanced therapy medicinal products have been approved and are now available for clinical use. Specifically, clinical trials have supported the utility of CD4+CD25+FOXP3+ regulatory T cells (Tregs) to minimize unwanted or overshooting immune responses and reduce the level of pharmacological immunosuppression in transplant recipients. Tregs are recognized as the principal orchestrators of maintaining peripheral tolerance, thereby blocking excessive immune responses and prevent autoimmunity. Here, we summarize rationale for the adoptive Treg therapy, challenges in manufacturing and clinical experiences with this novel living drug and outline future perspectives of its use in transplantation.

Cotransplantation of marginal mass allogeneic islets with 3D culture-derived adult human skin cells improves glycemia in diabetic mice.

Islet transplantation represents a therapeutic option for type 1 diabetes (T1D). Long-term viability of transplanted islets requires improvement. Mesenchymal stromal cells (MSCs) have been proposed as adjuvants for islet transplantation facilitating grafting and functionality. Stem cell aggregation provides physiological interactions between cells and enhances the in situ concentration of modulators of inflammation and immunity. We established a hanging-drop culture of adult human skin fibroblast-like cells as spheroids, and skin spheroid-derived cells (SphCs) were characterized. We assessed the potential of SphCs in improving islet functionality by cotransplantation with a marginal mass of allogeneic islets in an experimental diabetic mouse model and characterized the secretome of SphCs by mass spectrometry-based proteomics. SphCs were characterized as multipotent progenitors and their coculture with anti-CD3 stimulated mouse splenocytes decreased CD4+ T cell proliferation with skewed cytokine secretion through an increase in the Th2/Th1 ratio profile. SphCs-conditioned media attenuated apoptosis of islets induced by cytokine challenge in vitro and importantly, intratesticular SphCs administration did not show tumorigenicity in immune-deficient mice. Moreover, SphCs improved glycemic control when cotransplanted with a marginal mass of allogeneic islets in a diabetic mouse model without pharmacological immunosuppression. SphCs' protein secretome differed from its paired skin fibroblast-like counterpart in containing 70% of up- and downregulated proteins and biological processes that overall positively influenced islets such as cytoprotection, cellular stress, metabolism, and survival. In summary, SphCs improved the performance of transplanted allogeneic islets in an experimental T1D model, without pharmacological immunosuppression. Future research is warranted to identify SphCs-secreted factors responsible for islets' endurance.

Molecular and anatomical roadmap of stroke pathology in immunodeficient mice.

Stroke remains a leading cause of disability and death worldwide. It has become apparent that inflammation and immune mediators have a pre-dominant role in initial tissue damage and long-term recovery. Still, different immunosuppressed mouse models are necessary in stroke research e.g., to evaluate therapies using human cell grafts. Despite mounting evidence delineating the importance of inflammation in the stroke pathology, it is poorly described to what extent immune deficiency influences overall stroke outcome. Here, we assessed the stroke pathology of popular genetic immunodeficient mouse models, i.e., NOD scid gamma (NSG) and recombination activating gene 2 (Rag2-/-) mice as well as pharmacologically immunosuppressed mice and compared them to immune competent, wildtype (WT) C57BL/6J mice three weeks after injury. We performed histology, gene expression, blood serum and behavioural analysis to identify the impact of immunosuppression on stroke progression. We detected changes in microglia activation/macrophage infiltration, scar-forming and vascular repair in immune-suppressed mice three weeks after injury. Transcriptomic analysis of stroked tissue revealed the strongest deviation from WT was observed in NSG mice affecting immunological and angiogenic pathways. Pharmacological immunosuppression resulted in the least variation in gene expression compared with the WT. These anatomical and genetic changes did not affect functional recovery in a time course of three weeks. To determine whether timing of immunosuppression is critical, we compared mice with acute and delayed pharmacological immunosuppression after stroke. Mice with delayed immunosuppression (7d) showed increased inflammatory and scarring responses compared to animals acutely treated with tacrolimus, thus more closely resembling WT pathology. Transplantation of human cells in the brains of immunosuppressed mice led to prolonged cell survival in all immunosuppressed mouse models, which was most consistent in NSG and Rag2-/- mice. We detected distinct anatomical and molecular changes in the stroke pathology between individual immunosuppressed mouse models that should be considered when selecting an appropriate mouse model for stroke research.

A Phase 1/1b Multicenter Ascending Dose Study to Evaluate the Safety of HA-1 Minor Histocompatibility Antigen-Reactive TCR-Modified T Cells (BSB-1001) in Patients Undergoing HLA-Matched Allogenic Hematopoietic Stem Cell Transplant (alloSCT) for MRD+ AML or ALL or Poor/Very Poor Risk MDS

Background: AlloSCT can cure patients with acute leukemia and high-risk myelodysplastic syndromes (MDS). However, post-transplant relapse remains the greatest single cause of post-transplant death, occurring in up to 80% of high-risk patients. In HLA-matched alloSCT, alloreactive donor T cells target minor histocompatibility antigens (miHAs), which are peptide products of coding polymorphisms that distinguish recipients from donors. These alloreactive T cells can target leukemic cells, mediating the graft-vs-leukemia (GVL) effect; unfortunately, they also can cause graft-vs-host disease (GVHD). Pre-clinical mouse models (Blood, 2011) demonstrate that CD8+ T cells that target miHAs with expression limited to hematopoietic cells can mediate GVL without causing GVHD. To translate this concept for how to safely augment GVL to the clinic, BlueSphere Bio (BSB) used its high throughput T cell receptor (TCR) discovery and characterization platforms to clone a panel of TCRs reactive against the HLA-A*02-restricted miHA, HA-1, which is expressed mostly in hematopoietic cells. These receptors were cloned from a multiparous A*02:01 HA-1- woman who was immunized to HA-1 through pregnancy. A high affinity TCR specific for HA-1 and not reactive against allogeneic HLA molecules was chosen to create a cellular therapeutic designed to mediate GVL and enhance donor engraftment with a lower risk for GVHD, relative to polyclonal donor T cells. The anti-HA-1 T cell product (BSB-1001) is composed of donor CD8+ T cells transduced with a lentivirus vector encoding the anti-HA-1 TCR and the RQR8 tag, which expresses an epitope from CD20, thereby enabling the in vivo killing of BSB-1001 cells with rituximab. CRISPR-cas9 editing of the CD8+ T cells deletes the endogenous TCRα and β chains with >95% efficiency. This increases expression of the TCR and reduces risk of GVHD caused by native TCRs, as well as possible off-target reactivities generated by pairing of endogenous and introduced TCR chains. BSB-1001 cells specifically kill HLA-A*02:01, HA-1+ targets, with no activity against cells that are HLA-A*02:01+ but HA-1-, or cells that are HA-1+ but are HLA-A*02:01-. BSB-1001 is also not reactive against a panel of B lymphoblastic cell lines (LCL) expressing allogeneic HLA molecules. Importantly, efficient target killing is seen at effector:target ratios as low as 0.1, and maintained after several rounds of ex-vivo expansion following target cell challenge, a likely predictor of potency and persistence in vivo. Study Design: The first in human Phase 1/1b study is a multicenter, open-label, single dose study with a 3+3 ascending design with the option to expand accrual at a promising dose. Subjects must have ALL, AML or high-risk MDS with evidence of disease after the last therapy, be HLA-A*02:01 HA-1+ and have a 10/10 HLA-matched HA-1- related or unrelated donor. GCSF-mobilized donors will undergo leukapheresis. From this product, a small fraction of cells will be removed for BSB-1001 manufacturing; the remainder will undergo CD34-selection and cryopreservation until SCT. After BSB-1001 manufacturing is complete, patients will undergo ablative conditioning (choice of three regimens), followed by infusion of the CD34-selected cells and then BSB-1001. Patients will not receive anti-T cell antibodies and will receive no prophylactic pharmacologic immunosuppression. Subjects must otherwise be suitable for ablative conditioning using standard criteria. The primary objective is to establish a recommended dose of BSB-1001 based on its dose-limiting toxicities and the magnitude of post-infusion BSB-1001 expansion. The primary endpoint is determining the incidence of treatment-emergent adverse events, including dose-limiting toxicities. Additional endpoints include rates of engraftment, incidences of cytokine release syndrome (CRS) and acute and chronic GVHD, quantitation of malignant hematopoiesis and lineage-specific donor chimerism. CRS and GVHD, should they occur, will be treated per institutional standards. Rituximab can be administered to kill BSB-1001 cells in case of refractory CRS or GVHD. Enrollment is anticipated to begin in 1Q2023. BSB is also developing panels of TCRs targeting additional relatively hematopoietically-restricted miHAs with prioritization based on the frequencies of the restricting HLAs and the immunogenic/nonimmunogenic miHA alleles.

Naïve T Cell-Depleted Hematopoietic Graft Combined with Solid Organ Transplantation As Strategy to Induce Tolerance By Transient Mixed Chimerism

BACKGROUND The major challenge of solid organ transplantation (SOT) is to prevent graft rejection. The chronic use of pharmacological immunosuppression has been associated with increased rates of infection, cardiovascular, metabolic, endocrine disorders, and cancer. The induction of chimerism by donor hematopoietic stem cell transplantation (HSCT) together with SOT is an active tolerance induction approach. Here, we present a new model for inducing transient mixed chimerism in 2 patients undergoing SOT employing a non-myeloablative conditioning (NMA) regimen and a partially T cell-depleted (TCD) graft consisting of CD34+ hematopoietic progenitor cells and CD45RA− memory lymphocytes. METHODS SOT and HSCT were performed at University Hospital La Paz, Madrid. Bone marrow harvesting was performed on the iliac crest and lumbar and dorsal vertebrae of the deceased donor for patient#1 and mobilized leukoapheresis from sibling donor for patient#2. First we obtained the enriched by CD34+ selection using the CliniMACS system (Miltenyi Biotec) and afterwards the CD45RA− cells were purified. Lineage-specific chimerism analysis was analyzed using quantitative real-time polymerase chain reaction. Immune cell recovery was monitored by multiparametric flow cytometry. We obtained the permission of the Organización Nacional de Trasplantes and the informed consent was obtained from the patient and donor and/or caregivers. RESULTSPatient 1 A 15-year-old boy was diagnosed with intestinal epithelial dysplasia in 2008. After two previous intestinal and multivisceral acute cellular rejections forced a third MVT. With the imptemption to induce donor tolerance, a T cell-depleted bone marrow graft from the same deceased donor was employed. Bone marrow harvesting was performed obtaining a total volume of 141 ml with 4x109 total nucleated cells. Peripheral blood was obtained after unclasping the thoracic aorta. The stem cell fraction from bone marrow was enriched by CD34+ selection, obtaining 8.75x105/kg CD34+cells/kg. Memory lymphocytes were purified and aliquots containing 1x106/kg CD3+CD45RO+ and 2.1x104/kg CD3+CD45RA+ T cells. Both products were cryopreserved. The conditioning regimen before HSCT included fludarabine and cyclophosphamide. On day 88 after MVT, we infused all the CD34+ cells and 1x106/Kg CD3+CD45RO+ T cells. The CD3+CD45RO+ T cells were also infused periodically on a monthly basis for 1 year. Donor chimerism in CD3+ T cells ranged from 0.05 to 5.1 during the first year after HSCT. We observed a peak of the percentage of regulatory T cells (Tregs). No degree of graft-versus-host disease (GvHD) was observed, and the patient became independent of total parenteral and enteral nutrition. Serial follow-up intestinal biopsies did not show any sign of immune rejection. The patient continued to receive sirolimus to maintain drug concentrations at 6 ng/ml as the only immunosuppression treatment. No signs of rejection were detected after 27 months of follow-up. Patient 2 A 49-year-old woman was diagnosed with chronic kidney failure secondary to rapidly progressive glomerulonephritis. The patient had received 3 previous kidney transplants from deceased donors. A kidney transplant from her sibling was performed in March 2021. Hematopoietic progenitor cells were obtained from mobilized peripheral blood leukapheresis. A NMA conditioning regimen employing total lymphoid irradiation (8 Gy) with antithymocyte globulin (7.5 mg/kg). Two weeks after the kidney transplantation, we infused a graft consisting of 1.27x107/kg CD34+, 4.43x106/kg CD3+CD45RO+, and 5x104/kg CD3+CD45RA+ cells. Controls for hematopoietic chimerism demonstrated the presence of transient mixed hematopoietic chimerism during the first 2 months after SOT of between 0.1% and 40% CD3+ cells. The patient continued on monotherapy with tacrolimus. Immune reconstitution analysis showed a Treg peak. The patient did not have acute rejection, GvHD, viral reactivations or episodes of bacterial infection during the 17 months of follow-up. CONCLUSIONS We describe for the first time the use of non-myeloablative conditioning and a CD45RA− T cell-depleted graft to induce transitory mixed chimerism using both hematopoietic progenitors and a solid organ coming from the same donor, for tolerance induction purposes. The patients continue to progress favorably 17 months or more after SOT, receiving immunosuppressive monotherapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Islet allografts expressing a PD-L1 and IDO fusion protein evade immune rejection and reverse preexisting diabetes in immunocompetent mice without systemic immunosuppression

Allogeneic islet transplantation is a promising experimental therapy for poorly controlled diabetes. Despite pharmacological immunosuppression, long-term islet engraftment remains elusive. Here, we designed a synthetic fusion transgene coupling PD-L1 and indoleamine dioxygenase [hereafter PIDO] whose constitutive expression prevents immune destruction of genetically engineered islet allograft transplanted in immunocompetent mice. PIDO expressing murine islets maintain robust dynamic insulin secretion in vitro and when transplanted in allogeneic hyperglycemic murine recipients reverse pre-existing streptozotocin-induced and autoimmune diabetes in the absence of pharmacological immunosuppression for more than 50 and 8 weeks, respectively, and is dependent on host CD4 competence. Additionally, PIDO expression in allografts preserves endocrine functional viability of islets and promotes a localized tolerogenic milieu characterized by the suppression of host CD8 T cell and phagocyte recruitment and accumulation of FOXP3+ Tregs. Furthermore, in the canine model of xenogeneic islet transplantation, muscle implanted PIDO-expressing porcine islets displayed physiological glucose-responsive insulin secretion competency in euglycemic recipient for up to 20 weeks. In conclusion, the PIDO transgenic technology enables host CD4+ T cell-modulated immune evasiveness and long-term functional viability of islet allo- and xenografts in immune-competent recipients without the need for pharmacological immune suppression and would allow for improved outcomes for tissue transplantation.

Comprehensive investigation on the metabolism of emodin both in vivo and in vitro

Emodin is a natural anthraquinone, which displays numerous pharmacological activities, including anti-tumor, anti-inflammation and immunosuppression. However, there was no comprehensive study on its absorption, metabolism, distribution, and excretion. In order to further evaluation on the possibility of drug development of emodin, both in vivo and in vitro experiments were fulfilled in this study. The results showed that the absolute bioavailability of emodin is approximately 3.2%. Furthermore, about 56% of emodin was unabsorbed and mainly excreted into feces as prototype. The absorb constituent could be rapidly metabolized as hydroxylated and glucuronidated metabolites. Both prototype and metabolites of emodin absorbed into the body circulation were predominantly distributed in kidney. Hydroxyed metabolites were predominantly excreted via urine and feces and glucuronidated metabolites were predominantly excreted via urine and bile. CYP1A2, CYP2E1, UGT1A1, UGT1A9, and UGT2B7 played a key role in the metabolism of emodin in liver microsomes of rats. To the best of our knowledge, this is the first comprehensive study on the absorption, metabolism, distribution, and excretion of emodin, and our results could help to understand both pharmaceutical and toxicological effects of emodin greatly.