Long-Term Follow-Up of Abatacept, Post-Transplantation Cyclophosphamide, and Sirolimus-Based Haploidentical Transplantation in Younger Patients with Nonmalignant Diseases.

Haploidentical (haplo) hematopoietic cell transplantation (HCT) for nonmalignant disease (NMD) carries inherent challenges of both alloreactivity and graft failure. Building on promising results from pilot studies in which abatacept was combined with post-transplantation cyclophosphamide (PTCy) and sirolimus (AbaCyS) in younger NMD patients undergoing haplo-HCT, we present the long-term outcomes of this protocol. On the back of uniform disease-specific conditioning regimens containing antithymocyte globulin 4.5 mg/kg from day -9 to day -7, GVHD prophylaxis with AbaCyS consisted of abatacept administered on days 0, +5, +20, +35, and monthly until 180 days with PTCy and sirolimus. The patients were followed up with longitudinal assessment of immune reconstitution, growth, and reproductive development and quality of life (QoL) analyses. Among 40 patients (aplastic anemia, n = 24; hemoglobinopathies, n = 14; and primary immunodeficiencies, n = 2) with a median age of 10 years (range, 2 to 25 years), 95% achieved sustained engraftment. Post-transplantation hemophagocytic syndrome was detected in 3 patients, leading to graft failure in 2 cases. The incidence of acute graft-versus-host disease (GVHD) was 2.6%, and that of chronic GVHD (cGVHD) was 14.3%. Cytomegalovirus, adenovirus, and Epstein-Barr virus infections were observed in 45%, 5%, and 0% respectively. Rates of nonrelapse mortality, overall survival, event-free survival, and GVHD-free, event-free survival were 5%, 95%, 90%, and 82%, respectively, at a median follow-up of 4.6 years. Absence of cGVHD correlated with younger patient age and early sustained recovery of regulatory T cells and mature natural killer cells, which in turn was associated with improved QoL and lack of late infections. The AbaCyS protocol was associated with excellent long-term survival, with attenuation of both early and late alloreactivity in >80% of younger patients undergoing haplo-HCT for NMD. This study sheds light on predispositions to cGVHD and its impact on QoL, warranting further optimization of this approach.

Augmenting Vaccine Efficacy against Delta Variant with 'Mycobacterium-w'-Mediated Modulation of NK-ADCC and TLR-MYD88 Pathways.

Mycobacterium-w (Mw) was shown to boost adaptive natural killer (ANK) cells and protect against COVID-19 during the first wave of the pandemic. As a follow-up of the trial, 50 healthcare workers (HCW) who had received Mw in September 2020 and subsequently received at least one dose of ChAdOx1 nCoV-19 vaccine (Mw + ChAdOx1 group) were monitored for symptomatic COVID-19 during a major outbreak with the delta variant of SARS-CoV-2 (April-June 2021), along with 201 HCW receiving both doses of the vaccine without Mw (ChAdOx1 group). Despite 48% having received just a single dose of the vaccine in the Mw + ChAdOx1 group, only two had mild COVID-19, compared to 36 infections in the ChAdOx1 group (HR-0.46, p = 0.009). Transcriptomic studies revealed an enhanced adaptive NK cell-dependent ADCC in the Mw + ChAdOx1 group, along with downregulation of the TLR2-MYD88 pathway and concomitant attenuation of downstream inflammatory pathways. This might have resulted in robust protection during the pandemic with the delta variant.

Impact of Conditioning Regimen and Graft-versus-Host Disease Prophylaxis on The Outcome of Haploidentical Peripheral Blood Stem Cell Transplantation for High-Risk Severe Aplastic Anemia in Children and Young Adults: A Report from the Pediatric Severe Aplastic Anemia Consortium of India.

Allogenic hematopoietic cell transplantation (HCT) is the best curative approach for patients with severe aplastic anemia (SAA). The outcomes of HCT from haploidentical family donors (HFDs) have improved, making it a feasible option for patients lacking an HLA-identical donor. However, data on HFD-HCT for younger patients with SAA is sparse. In this multicenter retrospective study, we evaluated the outcomes of 79 patients undergoing HFD-HCT for SAA. All the patients were heavily pretransfused, the median time to HCT was >12 months, and 67% had failed previous therapies. Conditioning was based on fludarabine (Flu)-cyclophosphamide (Cy)-antithymocyte globulin (ATG)/total body irradiation (TBI) with or without thiotepa/melphalan (TT/Mel). Post-transplantation Cy (PTCy) and calcineurin inhibitors (CNIs)/sirolimus were used as graft-versus-host disease (GVHD) prophylaxis with or without abatacept. The rate of primary graft failure (PGF) was 16.43% overall, lower in patients conditioned with TT/Mel. The incidences of acute and chronic GVHD were 26.4% and 18.9%, respectively. At a median follow-up of 48 months, the overall survival (OS) and event-free survival (EFS) were 61.6% and 58.1%, respectively. Both OS and EFS were better in the TT/Mel recipients and with abatacept as GVHD prophylaxis. On multivariate analysis, the use of abatacept was found to favorably impact the outcome variables, including GVHD and EFS. Our study suggests that PTCy-based HFD-HCT is a reasonable option for young patients with high-risk SAA, in whom optimization of conditioning and GVHD prophylaxis might further improve outcomes.

Impact of an Immune Modulator Mycobacterium-w on Adaptive Natural Killer Cells and Protection Against COVID-19.

The kinetics of NKG2C+ adaptive natural killer (ANK) cells and NKG2A+inhibitory NK (iNK) cells with respect to the incidence of SARS-CoV-2 infection were studied for 6 months in a cohort of healthcare workers following the administration of the heat-killed Mycobacterium w (Mw group) in comparison to a control group. In both groups, corona virus disease 2019 (COVID-19) correlated with lower NKG2C+ANK cells at baseline. There was a significant upregulation of NKG2C expression and IFN-γ release in the Mw group (p=0.0009), particularly in those with a lower baseline NKG2C expression, along with the downregulation of iNK cells (p<0.0001). This translated to a significant reduction in the incidence and severity of COVID-19 in the Mw group (incidence risk ratio-0.15, p=0.0004). RNA-seq analysis at 6 months showed an upregulation of the ANK pathway genes and an enhanced ANK-mediated antibody-dependent cellular cytotoxicity (ADCC) signature. Thus, Mw was observed to have a salutary impact on the ANK cell profile and a long-term upregulation of ANK-ADCC pathways, which could have provided protection against COVID-19 in a non-immune high-risk population.

Nutritional Composition and In Vitro Starch Digestibility of Crackers Supplemented with Faba Bean Whole Flour, Starch Concentrate, Protein Concentrate and Protein Isolate.

The nutritional quality of common wheat-based foods can be improved by adding flours from whole pulses or their carbohydrate and protein constituents. Faba bean (Vicia faba L.) is a pulse with high protein concentration. In this study, prepared faba bean (FB) flours were added to wheat based baked crackers. Wheat cracker recipes were modified by substituting forty percent wheat flour with flours from whole faba bean, starch enriched flour (starch 60%), protein concentrate (protein 60%) or protein isolate (protein 90%). Baked crackers were ground into meal and analyzed for their macronutrient composition, starch characteristics and in vitro starch hydrolysis. Faba bean supplemented crackers had lower (p ≤ 0.001) total starch concentrations, but proportionally higher protein (16.8-43%), dietary fiber (6.7-12.1%), fat (4.8-7.1%) and resistant starch (3.2-6%) (p ≤ 0.001) than wheat crackers (protein: 16.2%, dietary fiber: 6.3%, fat: 4.2, resistant starch: 1.2%). The increased amylose, amylopectin B1- chain and fat concentration from faba bean flour and starch flour supplementation in cracker recipe contributed to increased resistant starch. Flours from whole faba bean, starch or protein fractions improved the nutritional properties and functional value of the wheat-based crackers. The analytical analysis describing protein, starch composition and structure and in vitro enzymatic hydrolysis advance understanding of factors that account for the in vivo benefits of faba bean flours added to crackers in human physiological functions as also previously shown for pasta. The findings can be used to guide development of improve nutritional quality of similar wheat-based food products.

Impact of adaptive natural killer cells, KLRC2 genotype and cytomegalovirus reactivation on late mortality in patients with severe COVID-19 lung disease.

OBJECTIVE: SARS-CoV-2 infection results in severe lung disease in up to 50% of hospitalised patients. The aetiopathogenesis in a subset of such patients, who continue to have progressive pulmonary disease following virus clearance, remains unexplored. METHODS: We investigated the role of NKG2C+/NKG2A- adaptive natural killer (ANK) cells, KLRC2 genotype and cytomegalovirus (CMV) reactivation in 22 such patients. RESULTS: The median duration of virus positivity was 23 days, and the median duration of hospitalisation was 48 days. The overall survival at 60 days in this group was 50%. Older age and comorbidities impacted survival negatively. CMV viraemia was documented in 11 patients, with a survival of 25% vs 80% in those without viraemia with viral load correlating with mortality. Both NK and T cells were markedly depressed in all patients at day 15. However, only persistently low ANK cells at 30 days along with an inversely high NKG2C-/NKG2A+ inhibitory NK cells significantly correlated with high CMV viraemia and mortality, irrespective of KLRC2 genotype. However, day 30 ANK cells were significantly lower in the KLRC2 deletion group. The release of IFN-γ and perforin was severely compromised in all patients at day +15, with significant improvement in the survivors at day +30, but not in those with adverse outcome. CONCLUSION: Patients with progressive lung disease even after negative SARS-CoV-2 status, with persistently reduced and functionally compromised ANK cells, are more likely to have CMV reactivation and an adverse outcome, independent of KLRC2 genotype.

Early and Sustained Expansion of Adaptive Natural Killer Cells Following Haploidentical Transplantation and CTLA4Ig-Primed Donor Lymphocyte Infusions Dissociate Graft-versus-Leukemia and Graft-versus-Host Effects.

BACKGROUND: Adaptive or memory natural killer (NK) cells with epigenetic imprints similar to memory T cells have been shown to develop in response to cytomegalovirus (CMV) infection with upregulation of activating receptor NKG2C. These cells have been shown to possess strong anti-tumour efficacy both in-vitro as well as in-vivo. OBJECTIVES: To determine if reconstitution of adaptive NK cells (CD56dimNKG2C+NKG2A-) in patients with advanced leukemia undergoing haploidentical HCT had any impact on disease progression (DP). STUDY DESIGN: The study cohort comprised of 60 patients with advanced acute leukemia, aged 2-65 years, receiving myeloablative PTCy based haploidentical transplantation from CMV seropositive donors, followed by CTLA4Ig-primed donor lymphocyte infusions (DLI). They were evaluated for the kinetics of reconstitution of adaptive NK cells, both phenotypic and functional, at days +30,+60, +90 and at regular intervals, to 3 years of follow-up, in relation to DP. Reconstitution of adaptive NK cells was compared with a retrospective cohort of patients in the same protocol receiving DLI without CTLA4Ig. RESULTS: Non-relapse mortality, acute and chronic GVHD were 5.1%, 10.3% and 14.5%. DP was 17.5% at a median follow-up of 28 months. Adaptive NK cells were significantly higher in patients without DP at days+30, +60 and +90 (p = 0.0001), irrespective of CMV reactivation and remained elevated until 36 months post-HCT. These cells maintained their functional competence as measured by robust interferon-gamma production with higher expressions of KIR, NKG2D and CD57, without any increase in PD1 expression. Grafts from donors with higher adaptive NK cells were associated with a lower risk of DP (p = 0.0001). In multivariate analysis, adaptive NK cell recovery at day +90 had the most favorable impact on DP (HR-0.7). Tregs reconstituted briskly along with the adaptive NK cells and were sustained as well, without compromising the GVL effect. Comparison with a retrospective cohort receiving the same protocol with DLI without CTLA4Ig, showed a superior reconstitution of adaptive NK cells in those receiving CTLA4Ig-DLI (p < 0.0001). CONCLUSION: Our study suggests that myeloablative transplantation from CMV seropositive haploidentical donors augmented with CTLA4Ig-primed DLI might favor early and sustained expansion of functionally competent adaptive NK cells irrespective of CMV reactivation, with a favorable outcome.

Faba bean meal, starch or protein fortification of durum wheat pasta differentially influence noodle composition, starch structure and in vitro digestibility.

Faba bean (Vicia faba L.) flour, starch concentrate (60% starch), protein concentrate (~60% protein) and protein isolate (~85% protein) were added to replace one-quarter of durum wheat semolina to enrich the nutritional quality and physiological functions of durum wheat (Triticum turgidum L.) pasta. The raw pasta samples prepared with protein concentrate or isolate had higher (p ≤ 0.001) protein and lower (p ≤ 0.001) total starch concentrations, along with increased total dietary fiber and slowly digestible starch (p ≤ 0.001) than durum wheat semolina control or those with added whole faba-bean flour or isolated starch. The faba bean fortified pasta had altered starch with increased proportion of medium B-type glucan chains and long C-type glucan chains, reduced starch digestibility and were associated with glycaemia related effects in the human diet. The faba bean fortified pasta had increased protein and dietary fiber that influenced food intake and satiety. The results suggest differential contributions of food ingredients in human health outcomes.

Safety and efficacy of Sofosbuvir and Velpatasvir in children with active hepatitis C virus infection undergoing haploidentical transplantation.

Direct Acting Antivirals (DAA) have changed the landscape of hepatitis C virus (HCV) infection with high cure rates across genotypes. However, the use of these agents in the setting of allogeneic hematopoietic cell transplantation (HCT) has been limited. In this context, we report the outcome of five children (5-12 years) with relapsed and refractory leukemia and active HCV infection (genotype 1b), who underwent urgent haploidentical HCT and were treated with Sofosbuvir and Velpatasvir (Sof-Vel) from initiation of treatment to 24 weeks post-HCT. All achieved complete virologic response (VR) at a median of 2 weeks, with normalization of liver enzymes. There were no adverse events related to the use of Sof-Vel, with no major fluctuations in cyclosporine levels. Two of the patients developed chronic GVHD and one relapsed. Sof-Vel was continued in one of them along with sirolimus without affecting drug levels. With a median follow-up of 15 months, four patients are disease free with sustained VR. Our study shows that combination of Sof-Vel might be effective in inducing rapid complete and sustained VR during HCT without any major untoward drug interaction.

CTLA4Ig-primed donor lymphocyte infusions following haploidentical transplantation improve outcome with a distinct pattern of early immune reconstitution as compared to conventional donor lymphocyte infusions in advanced hematological malignancies.

CTLA4Ig has a unique property to spare or even potentiate natural killer (NK) cell-mediated cytotoxicity, whilst inhibiting T cell activation. We explored the efficacy of prophylactic DLI following CTLA4Ig (CTLA4Ig-DLI group, n = 75), compared to conventional DLI (DLI group, n = 50), in patients with advanced hematological malignancies receiving PTCy-based haploidentical transplantation. Acute and chronic GVHD in the CTLA4Ig-DLI group were 9.6% and 15.3% compared to 18.8% [p = 0.09] and 36.5% [p = 0.01] in the DLI group. Both non-relapse mortality (4% vs 14.4%) and disease progression (DP) (15.7% vs 31.1%) were lower in CTLA4Ig-DLI group (p = 0.04). GVHD and progression-free survival was significantly improved in the CTLA4Ig-DLI group (p = 0.001). The recovery of CD56dimNK cells, NKG2A-KIR + NK subsets and Tregs was significantly better in the CTLA4Ig-DLI group at all time points and memory T cells at day +90. Immune recovery in relation to DP showed distinct patterns, with T cell subsets in the DLI group and NKG2A-KIR+NK cells in CTLA4Ig-DLI group having favorable impact. CTLA4Ig-DLI was thus associated with an improved outcome, possibly on account of the distinct pattern of immune recovery shown with this novel approach.

Brassinosteroid receptor mutation influences starch granule size distribution in barley grains.

Brassinosteroids (BR) are plant-based steroids which influence several morphogenetic and developmental processes. A barley (Hordeum vulgare L.) genotype Kinai Kyoshinkai-2 (KK-2) carrying the uzu mutation exhibited altered starch granule size distribution. Hybridizing KK-2 with a barley genotype CDC Kendall with bi-modal starch granules produced progeny lines (116, 144 and 168) with almost uni-modal starch granules. Bioassays correlated uzu mutation with defective BR perception. DNA sequence analysis of the BR receptor-1 (BRI-1) gene detected a single-nucleotide A > G substitution at the position 2612 in the kinase domain which resulted in the change of His (CAC) to Arg (CGC) at residue 857 in subdomain IV of the kinase domain of the respective polypeptide. The study focused on the development of barley grain, accumulation of starch and composition influenced by defective BR perception due to the mutation detected in KK-2 and three other barley-breeding lines (116, 144 and 168). Aberrant BRI-1 delayed grain development, amylose synthesis and starch accumulation in the endosperm. The barley breeding lines 116, 144 and 168 carrying the aberrant BRI-1, exhibited altered granule size distribution with significant shift in the diameter maxima, but insignificant differences in amylose concentration. The BRI-1 mutation also altered amylopectin fine structure in both B- and C- type small starch granules, resulting in an increased fraction of short A-type glucan chains (<10 DP) and decreased fraction of B2 chains (25-36 DP) in genotypes carrying the BRI-1 mutation. The results show the influence of BR on barley grain development, starch accumulation, granule size distribution and amylopectin structure.

CTLA4Ig in an Extended Schedule along with Sirolimus Improves Outcome with a Distinct Pattern of Immune Reconstitution Following Post-Transplantation Cyclophosphamide-Based Haploidentical Transplantation for Hemoglobinopathies.

The major hindrances to the success of a haploidentical hematopoietic cell transplantation for hemoglobinopathies are graft failure, early post-transplant hemophagocytic syndrome (PTHPS), and graft-versus-host disease (GVHD). Following the successful incorporation of CTLA4Ig (abatacept) in post-transplantation cyclophosphamide-based haploidentical transplantation, we piloted this approach in 10 patients (aged 3 to 19 years), with thalassemia major (TM, n=5) and sickle cell disease (n = 5). Pretransplant immunosuppressive therapy (pTIST) was administered for 10 weeks. Conditioning was myeloablative. CTLA4Ig was administered every 2 weeks during pTIST and on days -1, +5, +20, and +35 and every 4 weeks thereafter for 6 months, along with sirolimus. A short course of low-dose dexamethasone was given from day +6 for 14 days. Nine patients engrafted at a median of 15 days, with 1 patient with TM dying of sepsis on day +19. None of the patients developed acute or chronic GVHD. All 9 patients are alive and disease free at a median follow-up of 28 months. Only 4 patients had cytomegalovirus reactivation. The pattern of immune reconstitution showed a prompt and sustained recovery of T cell subsets with memory phenotype, along with early and sustained increase of Tregs and NKG2C+ natural killer (NK) cells. This novel approach, targeting CD80 and CD86 on monocytes/macrophages, promoted engraftment and limited early-onset PTHPS and graft failure. The lack of GVHD and serious infections with this approach reflects an early recovery of Tregs, memory T cells, and persistence of NKG2C+ NK cells.

Prophylactic oseltamivir during major seasonal influenza H1N1 outbreak might reduce both H1N1 and associated pulmonary aspergillosis in children undergoing haploidentical transplantation.

Following a major seasonal outbreak of H1N1 influenza in 2018 September, prophylactic oseltamivir for six months was initiated in children undergoing haploidentical HCT with regular monitoring for influenza and other respiratory virus infections. Influenza was not detected in 22 children undergoing prophylaxis, compared to 8 H1N1 infections in 21 adults without prophylaxis (P = .01). Four children on prophylaxis were detected to have other respiratory viruses, compared to 8 in those without prophylaxis. Invasive pulmonary aspergillosis (IPA) was observed only in association with H1N1 (4/8 with H1N1 vs 0/35 without H1N1, P = .001) and was thus lower in the prophylaxis group (P = .04). The overall incidence of episodes of respiratory illness and hospital stay were also lower in those on prophylaxis (P = .001). There were no untoward side effects associated with prophylactic oseltamivir. Prophylactic oseltamivir was safe and effective in prevention of H1N1 infection and subsequent IPA in children at-risk, early after haploidentical HCT.

Impact of extended infusional mesna prophylaxis on the incidence of BK viruria and hemorrhagic cystitis following post-transplantation cyclophosphamide and CTLA4Ig-based haploidentical transplantation.

Hemorrhagic cystitis (HC) has been reported with increased frequency following post-transplantation cyclophosphamide (PTCy)-based haploidentical hematopoietic cell transplantation (HCT) along with a strong association with BK viruria. We prospectively evaluated the incidence of BK viruria and HC in 115 patients (median age 20 years, 2-65) undergoing PTCy-based haploidentical HCT with (n = 71) or without (n = 44) CTLA4Ig. HC prophylaxis consisted of a continuous infusion of mesna 30 min prior and 48 h post-PTCy. The overall incidence of BK viruria was 65.7%. None with BK viruria < 104 copies/ml developed clinical symptoms (n = 65). The incidence of BK viruria ≥ 104 copies/ml was 7.1% (n = 8) and 75% developed HC. The incidence of HC was 5.4% at a median of 30 days. Both BK viruria ≥ 104 copies/ml and HC were strongly associated with acute GVHD (p < 0.001). A higher NRM was observed in those with BK viruria ≥ 104 copies/ml, related to GVHD and its complications (41.7% vs 12.6%, p = 0.04). The incidences of acute GVHD, vis-à-vis, overall BK viruria, BK viruria ≥ 104 copies/ml, and HC, tended to be lower in patients receiving CTLA4Ig. Thus, extended infusional mesna, coupled with significant reduction in alloreactivity along with possible preservation of antiviral immunity associated with the use of CTLA4Ig, was probably responsible for a much lower incidence of BK viruria and resultant HC than reported previously following PTCy-based haploidentical HCT.

Alterations in NKG2A and NKG2C Subsets of Natural Killer Cells Following Epstein-Barr Virus Reactivation in CTLA4Ig-based Haploidentical Transplantation Is Associated With Increased Chronic Graft-Versus-Host Disease.

BACKGROUND: The impact of newer approaches to haploidentical transplantation on Epstein-Barr virus (EBV) is largely unknown. METHODS: We prospectively evaluated the incidence of EBV reactivation and its impact on transplantation outcomes in 71 patients undergoing haploidentical transplantation with posttransplantation cyclophosphamide in combination with CTLA4Ig-based T-costimulation blockade. RESULTS: Eight patients developed EBV reactivation at a median of 96 days with no incidence of lymphoproliferative disorder. There was no impact of EBV reactivation on acute graft-versus-host disease (GVHD), nonrelapse mortality, progression-free, or overall survival. Despite an overall incidence of 19%, there was a significant increase in chronic GVHD following EBV reactivation (62.5% versus 8%; P = 0.01). NKG2A subset of CD56 natural killer cells increased substantially and persisted following EBV reactivation and chronic GVHD, with a reciprocal decrease in NKG2C subset, whereas the reverse was witnessed in those without chronic GVHD (P < 0.01). Increase in NKG2C subset and a decrease in the NKG2A subset were witnessed within 3 months of subsidence of chronic GVHD. CONCLUSIONS: Thus, CTLA4Ig-based haploidentical transplantation was associated with a low incidence of EBV reactivation without EBV-lymphoproliferative disorder. However, EBV reactivation was associated with a sustained alteration in NKG2A and NKG2C subsets of CD56 natural killer cells which might have a pathogenic role in chronic GVHD.

Natural killer cell-based immunotherapy with CTLA4Ig-primed donor lymphocytes following haploidentical transplantation.

NK cell-based immunotherapy is one of the more exciting propositions in the field of cellular therapy for hematological malignancies. Current protocols are largely based on expanded and activated NK cells which are used both with and without allogeneic transplantation. Based on our recent findings, we discuss the concept of CTLA4Ig-primed donor lymphocyte infusions following haploidentical transplantation as an effective tool to garner NK cell-mediated antitumor effect with abrogation of T cell-mediated alloreactivity. This approach might widen the possibility of immunotherapy following haploidentical transplantation without increase in graft-versus-host disease. Further studies would be needed to establish the veracity of this concept with better understanding of the antitumor effect via this pathway. Future studies would decide if CTLA4Ig might be used to augment NK-cell activation in vitro as well.