Galectin-1 is associated with hematopoietic cell engraftment in murine MHC-mismatched allotransplantation.

Haploidentical hematopoietic cell transplantation (haplo-HCT) is associated with an increased risk of allograft rejection. Here, we employed a major histocompatibility complex (MHC)-mismatched allogeneic HCT (allo-HCT) murine model to better understand the role of Gal-1 in immune tolerance. Transplanted mice were classified into either rejected or engrafted based on donor chimerism levels. We noted significantly higher frequencies of CD4+ T cells, CD8+ T cells, natural killer cells, IFN-γ and TNF-α producing CD4+ T cells, and IFN-γ producing dendritic cells and macrophages in rejected mice. Conversely, we found significantly increased frequencies of regulatory T cells (Tregs), predominantly Helios+, IL-10-producing CD4+ T cells, type 1 regulatory (Tr1) cells, and the proportion of Tr1+Gal-1+ cells in engrafted mice. Further, Gal-1 specific blockade in Tregs reduced suppression of effector T cells in engrafted mice. Lastly, effector T cells from engrafted mice were more prone to undergo apoptosis. Collectively, we have shown that Gal-1 may favor HSC engraftment in an MHC-mismatched murine model. Our results demonstrate that Gal-1-expressing Tregs, especially at earlier time points post-transplant, are associated with inducing immune tolerance and stable mixed chimerism after HCT.

Pulmonary Function after Nonmyeloablative Hematopoietic Cell Transplant for Sickle Cell Disease.

Rationale: Sickle cell disease (SCD) is a monogenetic condition with recurring vasoocclusive events causing lifelong pulmonary morbidity and mortality. There is increasing access to curative therapies, such as hematopoietic cell transplant (HCT), for people living with SCD. However, more information on pulmonary function in adults with SCD after HCT is needed to best guide decisions for HCT and post-HCT care. Objectives: To test the hypothesis that forced expiratory volume in 1 second (FEV1) and other pulmonary function testing (PFT) parameters remain stable 3 years after HCT. Methods: People living with SCD undergoing nonmyeloablative HCT in a prospective cohort at the NIH Clinical Center from 2004 to 2019 were evaluated for enrollment. Global Lung Function Initiative reference equations and descriptive statistics were calculated before HCT and annually for 3 years. Six-minute-walk distance (6MWD) testing was performed. Generalized estimating equations were employed to evaluate interindividual changes in PFT parameters and 6MWD. Results: Of 97 patients with SCD undergoing HCT, 41 (42%) were female with median (25th, 75th percentile) age 31.8 (24.8, 38.0) years. Each year of measurement included the following numbers of subjects available for analysis with PFTs: baseline (n = 97), Year 1 (n = 91), Year 2 (n = 72), and Year 3 (n = 55); and the following numbers of subjects available for analysis with 6MWD: baseline (n = 79), Year 1 (n = 73), Year 2 (n = 57), and Year 3 (n = 41). Pre-HCT FEV1 was median (25th, 75th percentile) 68.3% (61.3%, 80.3%) and 69.2% (60.8%, 77.7%) 3 years after HCT, and pre-HCT diffusing capacity of the lung for carbon monoxide (DlCO) was 60.5% (53.0%, 66.3%) and 64.6% (55.1%, 73.4%) 3 years after HCT. Generalized estimating equations estimated that DlCO percent predicted increased significantly by 3.7% (95% confidence interval, 1.0%, 6.3%), and the 6MWD significantly increased by 25.9 (6.6, 45.2) meters 3 years after HCT, whereas there was no significant change in percent predicted FEV1 or FVC compared with before HCT. Conclusions: Overall, PFT results remained stable and there was an improvement in DlCO and 6MWD in this predominantly adult cohort undergoing nonmyeloablative HCT for SCD. Allogeneic HCT for SCD may cease the cycle of vasoocclusive pulmonary injury and prevent continued damage. Multicenter studies are needed to evaluate the long-term lung health effects of HCT for SCD in adults and children.

Low dose post-transplant cyclophosphamide and sirolimus induce mixed chimerism with CTLA4-Ig or lymphocyte depletion in an MHC-mismatched murine allotransplantation model.

Allogeneic hematopoietic cell transplantation (allo-HCT) offers a curative option for patients with certain non-malignant hematological diseases. High-dose post-transplant cyclophosphamide (PT-Cy) (200 mg/kg) and sirolimus (3 mg/kg), (HiC) synergistically induce stable mixed chimerism. Further, sirolimus and cytotoxic T lymphocyte-associated antigen-4 immunoglobulin (CTLA4-Ig), also known as Abatacept (Aba), promote immune tolerance and allograft survival. Here, in a major histocompatibility complex (MHC)-mismatched allo-HCT murine model, we combined Aba and/or T-cell depleting anti-Thy1.2 (Thy) with a lower dose of PT-Cy (50 mg/kg) and Sirolimus (3 mg/kg), (LoC). While mice in the LoC group showed graft rejection, the addition of Thy to LoC induced similar donor chimerism levels when compared to the HiC group. However, the addition of Aba to LoC led to graft acceptance only in younger mice. When Thy was added to the LoC+Aba setting, graft acceptance was restored in both age groups. Engrafted groups displayed significantly reduced frequencies of recipient-specific interferon-γ-producing T cells as well as an increased frequency in regulatory T cells (Tregs) except in the LoC+Aba group. Splenocytes from engrafted mice showed no proliferation upon restimulation with Balb/c stimulators. Collectively, in combination with Aba or Thy, LoC may be considered to reduce graft rejection in patients who undergo allo-HCT.

TP53 mutation screening for patients at risk of myeloid malignancy.

There is increasing recognition of the risk of developing therapy-related myeloid malignancy, including after cellular therapy. While retrospective studies have implicated pre-existing TP53 mutated hematopoietic clones as a common causative mechanism, no prospective screening to identify those patients at greatest risk is currently possible. We demonstrate that ultradeep DNA-sequencing prior to therapy may be used for discovery of TP53 mutations that are subsequently associated with malignancy.

Improvement in Cardiac Morphology Demonstrated by Cardiac Magnetic Resonance Imaging and Echocardiography after Haploidentical Hematopoietic Cell Transplantation in Adults with Sickle Cell Disease.

Cardiopulmonary complications account for approximately 40% of deaths in patients with sickle cell disease (SCD). Diffuse myocardial fibrosis, elevated tricuspid regurgitant jet velocity (TRV) and iron overload are all associated with early mortality. Although HLA-matched sibling hematopoietic cell transplantation (HCT) offers a potential cure, less than 20% of patients have a suitable donor. Haploidentical HCT allows for an increased donor pool and has recently demonstrated improved safety and efficacy. Our group has reported improved cardiac morphology via echocardiography at 1 year after HCT. Here we describe the first use of cardiac magnetic resonance imaging (CMR), the gold standard for measuring volume, mass, and ventricular function, to evaluate changes in cardiac morphology post-HCT in adults with SCD. We analyzed baseline and 1-year data from 12 adults with SCD who underwent nonmyeloablative haploidentical peripheral blood HCT at the National Institutes of Health. Patients underwent noncontrast CMR at 3 T, echocardiography, and laboratory studies. At 1 year after HCT, patients showed marked improvement in cardiac chamber morphology by CMR, including left ventricular (LV) mass (70.2 to 60.1 g/m2; P = .02) and volume (114.5 to 90.6 mL/m2; P = .001). Furthermore, mean TRV normalized by 1 year, suggesting that HCT may offer a survival benefit. Fewer patients had pathologically prolonged native myocardial T1 times, an indirect marker of myocardial fibrosis at 1 year; these data showed a trend toward significance. In this small sample, CMR was very sensitive in detecting cardiac mass and volume changes after HCT and provided complementary information to echocardiography. Notably, post-HCT improvement in cardiac parameters can be attributed only in part to the resolution of anemia; further studies are needed to determine the roles of myocardial fibrosis reversal, improved blood flow, and survival impact after HCT for SCD.

Thyroid and Adrenal Dysfunction in Hemoglobinopathies Before and After Allogeneic Hematopoietic Cell Transplant.

Purpose: To determine the rate and clinical characteristics associated with abnormal thyroid and adrenal function in recipients of nonmyeloablative hematopoietic cell transplantation (HCT) for sickle cell disease (SCD) and beta-thalassemia. Methods: We retrospectively reviewed patients who enrolled in 4 nonmyeloablative HCT regimens with alemtuzumab and total body irradiation (TBI). Baseline and annual post-HCT data were compared, which included age, sex, sickle phenotype, thyroid panel (total T3, free T4, thyroid stimulating hormone, antithyroid antibodies), cortisol level, ACTH stimulation testing, ferritin, medications, and other relevant medical history. Results: Among 43 patients in haploidentical transplant and 84 patients in the matched related donor protocols with mostly SCD, the rate of any thyroid disorder pre-HCT was 3.1% (all subclinical hypothyroidism) and post-HCT was 29% (10 hypothyroidism, 4 Grave's disease, and 22 subclinical hypothyroidism). Ninety-two (72%) patients had ferritin >1000 ng/dL, of which 33 patients (35.8%) had thyroid dysfunction. Iron overload was noted in 6 of 10 patients with hypothyroidism and 12 of 22 patients with subclinical hypothyroidism.Sixty-one percent were on narcotics for pain control. With respect to adrenal insufficiency (AI) pre-HCT, 2 patients were maintained on corticosteroids for underlying rheumatologic disorder and 8 had AI diagnosed during pre-HCT ACTH stimulation testing (total 10, 7.9%). Post-HCT, an additional 4 (3%) developed AI from corticosteroid use for acute graft vs host disease, Evans syndrome, or hemolytic anemia. Conclusion: Although iron overload was common in SCD, thyroid dysfunction pre-HCT related to excess iron was less common. Exposure to alemtuzumab or TBI increased the rates of thyroid dysfunction post-HCT. In contrast, AI was more common pre-HCT, but no risk factor was identified. AI post-HCT was infrequent and associated with corticosteroid use for HCT-related complications.

Co-expression of Foxp3 and Helios facilitates the identification of human T regulatory cells in health and disease.

Foxp3 is regarded as the major transcription factor for T regulatory (Treg) cells and expression of Foxp3 is used to identify and quantitate Treg cells in mouse models. However, several studies have demonstrated that human CD4+ T conventional (Tconv) cells activated in vitro by T cell receptor (TCR) stimulation can express Foxp3. This observation has raised doubt as to the suitability of Foxp3 as a Treg marker in man. Helios, a member of the Ikaros gene family, has been shown to be expressed by 80-90% of human Foxp3+ Treg cells and can potentially serve as a marker of human Treg. Here, we confirm that Foxp3 expression is readily upregulated by Tconv upon TCR stimulation in vitro, while Helios expression is not altered. More importantly, we show that Foxp3 expression is not elevated by stimulation of hTconv in a humanized mouse model of graft versus host disease (GVHD) and in patients with a wide variety of acute and chronic inflammatory diseases including sickle cell disease, acute and chronic GVHD, systemic lupus erythematosus, as well as critical COVID-19. In all patients studied, an excellent correlation was observed between the percentage of CD4+ T cells expressing Foxp3 and the percentage expressing Helios. Taken together, these studies demonstrate that Foxp3 is not induced upon Tconv cell activation in vivo and that Foxp3 expression alone can be used to quantitate Treg cells in humans. Nevertheless, the combined use of Foxp3 and Helios expression provides a more reliable approach for the characterization of Treg in humans.

Secondary Neoplasms After Hematopoietic Cell Transplant for Sickle Cell Disease.

PURPOSE: To report the incidence and risk factors for secondary neoplasm after transplantation for sickle cell disease. METHODS: Included are 1,096 transplants for sickle cell disease between 1991 and 2016. There were 22 secondary neoplasms. Types included leukemia/myelodysplastic syndrome (MDS; n = 15) and solid tumor (n = 7). Fine-Gray regression models examined for risk factors for leukemia/MDS and any secondary neoplasm. RESULTS: The 10-year incidence of leukemia/MDS was 1.7% (95% CI, 0.90 to 2.9) and of any secondary neoplasm was 2.4% (95% CI, 1.4 to 3.8). After adjusting for other risk factors, risks for leukemia/MDS (hazard ratio, 22.69; 95% CI, 4.34 to 118.66; P = .0002) or any secondary neoplasm (hazard ratio, 7.78; 95% CI, 2.20 to 27.53; P = .0015) were higher with low-intensity (nonmyeloablative) regimens compared with more intense regimens. All low-intensity regimens included total-body irradiation (TBI 300 or 400 cGy with alemtuzumab, TBI 300 or 400 cGy with cyclophosphamide, TBI 200, 300, or 400 cGy with cyclophosphamide and fludarabine, or TBI 200 cGy with fludarabine). None of the patients receiving myeloablative and only 23% of those receiving reduced-intensity regimens received TBI. CONCLUSION: Low-intensity regimens rely on tolerance induction and establishment of mixed-donor chimerism. Persistence of host cells exposed to low-dose radiation triggering myeloid malignancy is one plausible etiology. Pre-existing myeloid mutations and prior inflammation may also contribute but could not be studied using our data source. Choosing conditioning regimens likely to result in full-donor chimerism may in part mitigate the higher risk for leukemia/MDS.

Knowledge to date on secondary malignancy following hematopoietic cell transplantation for sickle cell disease.

Allogeneic hematopoietic cell transplantation, gene therapy, and gene editing offer a potential cure for sickle cell disease (SCD). Unfortunately, myelodysplastic syndrome and acute myeloid leukemia development have been higher than expected after graft rejection following nonmyeloablative conditioning and lentivirus-based gene therapy employing myeloablative busulfan for SCD. Somatic mutations discovered in 2 of 76 patients who rejected their grafts were identified at baseline at much lower levels. While a whole-genome sequencing analysis reported no difference between patients with SCD and controls, a study including whole-exome sequencing revealed a higher prevalence of clonal hematopoiesis in individuals with SCD compared with controls. Genetic risk factors for myeloid malignancy development after curative therapy for SCD are currently being explored. Once discovered, decisions could be made about whether gene therapy may be feasible vs allogeneic hematopoietic cell transplant, which results in full donor chimerism. In the meantime, care should be taken to perform a benefit/risk assessment to help patients identify the best curative approach for them. Long-term follow-up is necessary to monitor for myeloid malignancies and other adverse effects of curative therapies for SCD.

Allogeneic Transplant and Gene Therapy: Evolving Toward a Cure.

Curative therapies for sickle cell disease (SCD) include allogeneic human leukocyte antigen (HLA)- matched sibling and haploidentical hematopoietic cell transplant (HCT), gene therapy, and gene editing. However, comparative trial data that might facilitate selecting one curative therapy over another are unavailable. New strategies to decrease graft rejection and graft-versus-host disease (GVHD) risks are needed to expand haploidentical HCT. Myeloablative gene therapy and gene editing also has limitations. Herein, we review recent studies on curative therapies for SCD in the past 5 years.

Long-Term Health Effects of Curative Therapies on Heart, Lungs, and Kidneys for Individuals with Sickle Cell Disease Compared to Those with Hematologic Malignancies.

The goal of curing children and adults with sickle cell disease (SCD) is to maximize benefits and minimize intermediate and long-term adverse outcomes so that individuals can live an average life span with a high quality of life. While greater than 2000 individuals with SCD have been treated with curative therapy, systematic studies have not been performed to evaluate the long-term health effects of hematopoietic stem cell transplant (HSCT) in this population. Individuals with SCD suffer progressive heart, lung, and kidney disease prior to curative therapy. In adults, these sequalae are associated with earlier death. In comparison, individuals who undergo HSCT for cancer are heavily pretreated with chemotherapy, resulting in potential acute and chronic heart, lung, and kidney disease. The long-term health effects on the heart, lung, and kidney for children and adults undergoing HSCT for cancer have been extensively investigated. These studies provide the best available data to extrapolate the possible late health effects after curative therapy for SCD. Future research is needed to evaluate whether HSCT abates, stabilizes, or exacerbates heart, lung, kidney, and other diseases in children and adults with SCD receiving myeloablative and non-myeloablative conditioning regimens for curative therapy.

Clonal Hematopoiesis and the Risk of Hematologic Malignancies after Curative Therapies for Sickle Cell Disease.

Sickle cell disease (SCD) is associated with severe morbidity and early mortality. Two large population studies found an increased risk for leukemia in individuals with SCD. Notably, while the relative risk of leukemia development is high, the absolute risk is low in individuals with SCD who do not receive cell-based therapies. However, the risk of leukemia in SCD is high after graft rejection and with gene therapy. Clonal hematopoiesis (CH) is a well-recognized premalignant condition in the general population and in patients after high-dose myelotoxic therapies. Recent studies suggest that CH may be more common in SCD than in the general population, outside the cell-based therapy setting. Here, we review risk factors for CH and progression to leukemia in SCD. We surmise why patients with SCD are at an increased risk for CH and why leukemia incidence is unexpectedly high after graft rejection and gene therapy for SCD. Currently, we are unable to reliably assess genetic risk factors for leukemia development after curative therapies for SCD. Given our current knowledge, we recommend counseling patients about leukemia risk and discussing the importance of an individualized benefit/risk assessment that incorporates leukemia risk in patients undergoing curative therapies for SCD.

Thrombospondin-1, Platelet Factor 4, and Galectin-1 Are Associated with Engraftment in Patients with Sickle Cell Disease who Underwent Haploidentical Hematopoietic Stem Cell Transplantation.

Sickle cell disease (SCD) is an inherited red blood cell disorder that leads to significant morbidity and early mortality. The most widely available curative approach remains allogeneic hematopoietic stem cell transplantation (HSCT). HLA-haploidentical (haplo) HSCT expands the donor pool considerably and is a practical alternative for these patients, but traditionally with an increased risk of allograft rejection. Biomarkers in patient plasma could potentially help predict HSCT outcome and allow treatment at an early stage to reverse or prevent graft rejection. Reliable, noninvasive methods to predict engraftment or rejection early after HSCT are needed. We sought to detect variations in the plasma proteomes of patients who engrafted compared with those who rejected their grafts. We used a mass spectrometry-based proteomics approach to identify candidate biomarkers associated with engraftment and rejection by comparing plasma samples obtained from 9 engrafted patients and 10 patients who experienced graft rejection. A total of 1378 proteins were identified, 45 of which were differentially expressed in the engrafted group compared with the rejected group. Based on bioinformatics analysis results, information from the literature, and immunoassay availability, 7 proteins-thrombospondin-1 (Tsp-1), platelet factor 4 (Pf-4), talin-1, moesin, cell division control protein 42 homolog (CDC42), galectin-1 (Gal-1), and CD9-were selected for further analysis. We compared these protein concentrations among 35 plasma samples (engrafted, n = 9; rejected, n = 10; healthy volunteers, n = 8; nontransplanted SCD, n = 8). ELISA analysis confirmed the significant up-regulation of Tsp-1, Pf-4, and Gal-1 in plasma samples from engrafted patients compared with rejected patients, healthy African American volunteers, and the nontransplanted SCD group (P < .01). By receiver operating characteristic analysis, these 3 proteins distinguished engrafted patients from the other groups (area under the curve, >0.8; P < .05). We then evaluated the concentration of these 3 proteins in samples collected pre-HSCT and at days +30, +60, +100, and +180 post-HSCT. The results demonstrate that Tsp-1 and Pf-4 stratified engrafted patients as early as day 60 post-HSCT (P < .01), and that Gal-1 was significantly higher in engrafted patients as early as day 30 post-HSCT (P < .01). We also divided the rejected group into those who experienced primary (n = 5) and secondary graft rejection (n = 5) and found that engrafted patients had significantly higher Tsp-1 levels compared with patients who developed primary graft rejection at days +60 and +100 (P < .05), as well as higher Pf-4 levels compared with patients who developed primary graft rejection at post-transplantation (PT) day 100. Furthermore, Tsp-1 levels were significantly higher at PT days 60 and 100 and Pf-4 levels were higher at PT day 100 in engrafted patients compared with those who experienced secondary graft rejection. Increased concentrations of plasma Gal-1, Tsp-1, and Pf-4 could reflect increased T regulatory cells, IL-10, and TGF-ß, which are essential players in the initiation of immunologic tolerance. These biomarkers may provide opportunities for preemptive intervention to minimize the incidence of graft rejection.

Early Myeloid Derived Suppressor Cells (eMDSCs) Are Associated With High Donor Myeloid Chimerism Following Haploidentical HSCT for Sickle Cell Disease.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a widely available curative option for patients with sickle cell disease (SCD). Our original non-myeloablative haplo-HSCT trial employing post-transplant (PT) cyclophosphamide had a low incidence of GVHD but had high rejection rates. Here, we aimed to evaluate immune reconstitution following haplo-HSCT and identify cytokines and cells associated with graft rejection/engraftment. 50 cytokines and 10 immune cell subsets were screened using multiplex-ELISA and flow cytometry, respectively, at baseline and PT-Days 30, 60, 100, and 180. We observed the most significant differences in cytokine levels between the engrafted and rejected groups at PT-Day 60, corresponding with clinical findings of secondary graft rejection. Of the 44 cytokines evaluated, plasma concentrations of 19 cytokines were different between the two groups at PT-Day 60. Factor analysis suggested two independent factors. The first factor (IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, and TGFb1 contributed significantly) was strongly associated with engraftment with OR = 2.7 (95%CI of 1.4 to 5.4), whereas the second factor (GROa and IL-18 contributed significantly) was not significantly associated with engraftment. Sufficient donor myeloid chimerism (DMC) is critical for the success of HSCT; here, we evaluated immune cells among high (H) DMC (DMC≥20%) and low (L) DMC (DMC<20%) groups along with engrafted and rejected groups. We found that early myeloid-derived suppressor cell (eMDSC) frequencies were elevated in engrafted patients and patients with HDMC at PT-Day 30 (P< 0.04 & P< 0.003, respectively). 9 of 20 patients were evaluated for the source of eMDSCs. The HDMC group had high mixed chimeric eMDSCs as compared to the LDMC group (P< 0.00001). We found a positive correlation between the frequencies of eMDSCs and Tregs at PT-Day 100 (r=0.72, P <0.0007); eMDSCs at BSL and Tregs at PT-Day 100 (r=0.63, P <0.004). Of 10 immune regulatory cells and 50 cytokines, we observed mixed chimeric eMDSCs and IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, TGFb1 as potential hits which could serve as prognostic markers in predicting allograft outcome towards engraftment following haploidentical HSCT employing post-transplant cyclophosphamide. The current findings need to be replicated and further explored in a larger cohort.

American Society of Hematology 2021 guidelines for sickle cell disease: stem cell transplantation.

BACKGROUND: Sickle cell disease (SCD) is a life-limiting inherited hemoglobinopathy that results in significant complications and affects quality of life. Hematopoietic stem cell transplantation (HSCT) is currently the only curative intervention for SCD; however, guidelines are needed to inform how to apply HSCT in clinical practice. OBJECTIVE: These evidence-based guidelines of the American Society of Hematology (ASH) are intended to support patients, clinicians, and health professionals in their decisions about HSCT for SCD. METHODS: The multidisciplinary guideline panel formed by ASH included 2 patient representatives and was balanced to minimize potential bias from conflicts of interest. The Mayo Evidence-Based Practice Research Program supported the guideline development process, including performing systematic evidence reviews (through 2019). The panel prioritized clinical questions and outcomes according to their importance for clinicians and patients. The panel used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach, including GRADE Evidence-to-Decision frameworks, to assess evidence and make recommendations, which were subject to public comment. RESULTS: The panel agreed on 8 recommendations to help patients and providers assess how individuals with SCD should consider the timing and type of HSCT. CONCLUSIONS: The evidence review yielded no randomized controlled clinical trials for HSCT in SCD; therefore, all recommendations are based on very low certainty in the evidence. Key recommendations include considering HSCT for those with neurologic injury or recurrent acute chest syndrome at an early age and to improve nonmyeloablative regimens. Future research should include the development of a robust SCD registry to serve as a comparator for HSCT studies.