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SUMMARYMycobacterium-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 Mw+ChAdOx1 group, only 2 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 TLR2-MYD88 pathway and concomitant attenuation of downstream inflammatory pathways. This might have resulted in robust protection during the pandemic with the delta variant.

Allogenic hematopoietic cell transplant (HCT) is the best curative approach patients with severe aplastic anemia (SAA). Outcome of HCT from haploidentical family donor (HFD) has improved, making it a feasible option for patients lacking HLA-identical donor. However, data on HFD-HCT for younger patients with SAA is sparse. In multicentre retrospective study, we report outcome of 79 patients undergoing HFD-HCT for SAA. All were heavily pre-transfused, median time to HCT12 months and 67% had failed previous therapies. Conditioning was based on Flu-Cy-ATG/TBI with or without thiotepa/melphalan (TT/Mel). Post-transplantation cyclophosphamide (PTCy) and CNI/Sirolimus were employed as GvHD prophylaxis with or without abatacept. Primary graft failure (PGF) was 16.43%, less in those conditioned with TT/Mel. Incidence of acute and chronic GVHD were 26.4% and 18.9%. 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/EFS were better in TT/Mel group and with abatacept as GVHD prophylaxis. On multivariate analysis, use of Abatacept was found to favourably 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, where optimisation of conditioning and GVHD prophylaxis might improve outcomes further.

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.

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., In a cohort of patients with severe COVID‐19 lung disease who continued to have severe illness even after clearance of virus, lack of recovery of NKG2C+ adaptive NK cells correlated with cytomegalovirus viral load and increased mortality.

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

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.

We prospectively studied the impact of preemptive granulocyte infusions (pGIs) in 69 patients colonized with carbapenem-resistant gram-negative bacteria (CRGNB) undergoing haploidentical hematopoietic cell transplantation (HCT) compared with a previous cohort of 33 patients who received only antimicrobials directed toward CRGNB at the onset of neutropenic fever (non-pGI group). All patients developed neutropenic fever at a median of day +8 (range, –4 to +12) after transplantation. Engraftment kinetics were similar for both groups. The median number of GIs was 2 (range, 1 to 7), and the median dose of granulocytes infused was 5 × 1010 granulocytes per infusion (range, 1 to 30). The overall incidence of CRGNB bloodstream infections (BSIs) was 21.2% in non-pGI group (7/33) and 17.5% (12/69) in the pGI group (P = .8). However, the CRGNB-related mortality among those with BSI was 100% (7/7) in the non-pGI group versus 16.6% (2/12) in the pGI group (P = .001). The day 100 (4.4% versus 24.4%, P = .002) and 2-year nonrelapse mortality (7.5% versus 35.6%, P = .0001) were significantly reduced in the pGI group. The overall survival at 2 years was 75.6% in the pGI group versus 21.2% in the non-pGI group (P = .0001). Colonization and subsequent BSI with CRGNB are associated with a high incidence of mortality in patients undergoing HCT. pGI reduced early mortality associated with CRGNB in colonized patients undergoing post-transplant cyclophosphamide–based haploidentical HCT.

Background – Diffuse Large B Cell Lymphoma (DLBCL) is the most common type of Non- Hodgkin Lymphoma (NHL) and is categorized into the Germinal Centre B-cell (GCB) and Activated B-cell (ABC/ Non-GCB) subtypes as per the Cell-of-origin (COO) model with the help of Gene Expression Profiling/ Immunohistochemistry. The non -GCB subtype has been found to correlate with the Double Expressor (DE) phenotype (i.e. co-expression of bcl-2 and c-myc by IHC) and this correlation was substantiated and proven to be statistically significant in the present study.Results- A retrospective study of 50 patients was carried out with the help of archival material filed in the Department of Anatomic Pathology at Dharamshila Narayana Superspeciality Hospital from 1st January 2019 to 30th June 2020. The study cohort was divided into two Groups – Group-A with nodal presentation and Group -B with extranodal presentation. By using the Hans and Choi IHC algorithms, the cases were categorized into the GCB and non – GCB subtypes in both the groups. The DE phenotype was determined in each case by the co-expression of c-myc (>40%) and bcl-2 (>50%) by IHC.Conclusion – Due to the heterogeneity inherent in DLBCL, prediction of the Double Expressor phenotype and the COO by IHC is a sensitive tool and helps in the prognostication and therapeutic triage of patients. Hence in all cases diagnosed as DLBCL, a detailed morphological and IHC work up is mandatory to determine prognosis and possibly tailor therapy according to cell of origin and double expressor phenotype.

To evaluate ability of modulated innate immune response to provide resistance to development of symptomatic RT-PCR confirmed COVID-19, 96 inpatient front line health care workers (HCW) were cohorted in 1:2 ratio to receive TLR2 agonist (heat killed Mycobacterium w, Mw; n=32) as innate immune response modulator or observation (n=64). All were followed up for 100 days. The incidence of COVID-19 was 31 (32.3%) for the entire cohort, with only one developing COVID-19 in Mw group (3.1% vs 46.8%. protective efficacy - 93.33%, p=0.0001; 95% CI 53.3-99.1). Self-limiting local injection site reaction was the only side effect and was seen in 14 HCW. Findings from the study suggest the potential for providing resistance against novel pathogen like SARS-CoV-2 by modulating innate immune response.

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 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.

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 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.

The management of myeloma has evolved dramatically in the last two decades. High dose melphalan and autologous hematopoietic stem cell transplantation (HSCT) marked the beginning of this journey. This was followed by an explosion of novel agents which were approved for management of myeloma. Allogeneic HSCT which was deemed as the only curative option was largely abhorred due to high transplant-related mortality (TRM) until the advent of reduced intensity conditioning (RIC). An approach of tandem autologous and RIC-allogeneic transplantations has showed the best promise for cure for this condition, particularly for those with high-risk cytogenetics. Yet, allogeneic HSCT seems to have fallen out of favor due to the projected high TRM and late relapses, even though the alternatives do not offer a cure, but merely prolong survival. Offering an allogeneic HSCT as a final resort in unlikely to yield gratifying results. At the same time, allogeneic HSCT needs to evolve in a disease-specific manner to address the relevant concerns regarding TRM and relapse. With the introduction of effective GVHD prophylaxis in the form of post-transplantation cyclophosphamide, transplantation from a haploidentical family donor has become a reality. The challenge lies in segregating graft-vs-myeloma effect from a graft-versus-host effect. We discuss the pro-survival and anti-apoptotic pathways via CD28-CD86 interactions which confer survival advantages to myeloma cells and the possibility of disruption of this pathway in the context of haploidentical transplantation through the use of CTLA4Ig without incurring T cell alloreactivity.

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.

To understand the phenomenon of early alloreactivity (EA) in younger children undergoing post-transplantation cyclophosphamide (PTCy)-based haploidentical transplantation, we studied the graft composition and the immune reconstitution in 32 consecutive patients (aged 2 to 25 years) undergoing PTCy and T cell costimulation blockade based peripheral blood stem cell transplantation with emphasis on CD45RA+ subset of regulatory T cells (Tregs). All but 1 engrafted, and 14 patients experienced EA (acute graft-versus-host disease grades II to IV, n = 8; and post-transplantation hemophagocytic syndrome, n = 6) with a cumulative incidence of 43.7%; 42% developed mild chronic graft-versus-host disease. The overall survival was 70.2% with a nonrelapse mortality of 16.8% at a median of 19 months. Age 45 years, and poor recovery of Tregs correlated with EA. Not Tregs but higher CD45RA+ Tregs in the graft was associated with less EA (11.7% versus 32.5%, P = .0001). Higher donor age correlated with a lower CD45RA+ Tregs in the graft (P = .01). However, only higher CD45RA+ Treg percentage in the graft favorably impacted EA as well as nonrelapse mortality and overall survival. Our study demonstrates a critical role for CD45RA+ Tregs in determining EA and outcome after PTCy-based haploidentical peripheral blood stem cell transplantation, and the age-related physiologic decline in this population might be responsible for adverse impact of donor age.

We conducted a prospective study on T and natural killer (NK) cell subset composition of graft and transplant outcomes in T cell–replete haploidentical transplantation with a single dose of subcutaneous plerixafor (Px) added to granulocyte colony-stimulating factor (G-CSF)-based mobilization in allogeneic donors to collect 10 × 106/kg CD34+ hematopoietic stem cells (HSCs) at single apheresis. Twnety-six donors received G-CSF + Px and 25 G-CSF alone for mobilization. Despite significantly lower peripheral blood (PB) CD34+ HSCs on day 4 in the G-CSF + Px group (33 [range, 6-47] cells/µL versus 81 [range, 50-168] cells/µL in the G-CSF group; P = .0001), PB CD34+ HSC count (median 136 versus 139 cells/µL) on day 5 as well as that in the graft (2.7 versus 2.3 × 106/mL, P = .1) were comparable between the 2 groups. The total nucleated cell count was higher (3.4 versus 3.1 × 108/mL, P = .05), but CD4+ T cells (2.3 versus 2.7 × 107/mL, P = .09) were lower in the G-CSF group with mobilization of regulatory T cells being similar. NK cells were skewed toward the CD56+/16− subset in both groups, varying significantly from the steady-state NK subset ratio in PB. The time to engraftment, incidences of acute and chronic graft-versus-host disease, nonrelapse mortality, and overall survival were also similar. Addition of single-dose Px to G-CSF mobilization improves CD34 recovery and does not significantly alter the T and NK cell composition of the graft, including regulatory T cells, with no adverse impact on transplant outcomes.

We conducted a pilot study employing extended T cell costimulation blockade (COSBL) with Abatacept along with sirolimus and post-transplantation cyclophosphamide (PTCy) in 10 patients (median age 12) with severe aplastic anemia (SAA). Nine patients engrafted in the COSBL group, compared to all 10 patients (median 14 vs 13days) treated on PTCy protocols without abatacept (CONTROL group). The incidence of acute graft-versus-host disease (GVHD) was 10.5% in the COSBL group compared to 50% in the CONTROL group (p=0.04). Chronic GVHD (12.5% vs 56%, p=0.02) and CMV reactivation (30% vs 80%, p=0.03) were also reduced in the COSBL group. T and NK cell subset analysis revealed higher CD56brightCD16- NK cells in the CONTROL group (p=0.004), but similar CD56dimCD16+ NK cells in both groups at day+30. Tregs (CD4+CD25+CD127dim/- FoxP3+) were markedly higher in the COSBL group at day+30 (8.4% vs 1.1%) and the trend was maintained through day+90 (p

We conducted a pilot study on the feasibility of CD56-enriched donor cell infusion after post-transplantation cyclophosphamide (PTCy) for 10 patients with advanced myeloid malignancies undergoing haploidentical peripheral blood stem cell transplantation with cyclosporine alone as graft-versus-host disease (GVHD) prophylaxis and compared the outcome and immune reconstitution with a control group of 20 patients undergoing the same without CD56-enriched donor cell infusion. An early and rapid surge of mature NK cells as well as CD4 + T cells and regulatory T cells (Tregs) was noted compared with the control group. KIR of donor phenotype reconstituted as early as day 30 with expression of CD56 dim CD16 + NKG2A − KIR + phenotype. None experienced viral or fungal infections, and non-relapse mortality was 10% only. The incidence of grade 2–4 acute GVHD was 50% in the control group with none in the CD56 group ( P = 0.01). Only two had de novo chronic GVHD in each group. Relapse occurred in five patients in CD56 group with a median follow-up of 12 months, similar to the control group. Our preliminary data show that CD56 + donor cell infusion after PTCy and short-course cyclosporine is feasible with prompt engraftment, rapid reconstitution of CD4 + T cells, Tregs and NK cells and reduced incidence of acute GVHD.

Inflammatory cytokines released by activated lymphocytes and innate cells in the context of cellular therapy can cause fever, vasodilatation and end organ damage, so-called cytokine release syndrome (CRS). CRS can occur after allogeneic blood or marrow transplantation, but especially after HLA-haploidentical (haplo) peripheral blood transplantation (PBT). We reviewed charts of all patients who underwent haplo PBT between October 1, 2013 and September 1, 2017 and graded CRS. A total of 146 consecutive related haplo PBTs were analyzed. CRS occurred in 130 (89%) of the patients, but most was of mild (Grades 0–2) severity. Severe CRS (Grades 3–5) occurred in 25 patients (17% of PB haploPBT). In this group with severe CRS, 13 had encephalopathy; 12 required hemodialysis, and 11 were intubated. Death from the immediate complications of CRS occurred in 6 patients (24% of the severe CRS group and 4% of the entire haplo PBT cohort). The cumulative probability of non-relapse mortality (NRM) for patients with severe CRS was 38% at 6 months; it was 8% in the 121 out of 146 patients without severe CRS. In conclusion, CRS occurs in nearly 90% of haploidentical peripheral blood stem cell transplants. Older recipients (OR 2.4, 95% CI 0.83–6.75, p=0.11) of haploidentical PBT and those with a history of XRT (OR=3.85, 95% CI 1.32–11.24, p=0.01) have a higher risk of developing severe CRS. Most recipients of haplo PBT develop CRS, but less than 20% experience severe complications. The development of severe CRS significantly increases NRM.

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.

We evaluated the incidences and consequences of rotavirus induced diarrhea in a cohort of 115 patients undergoing T-cell replete haploidentical transplantation. Four out of 115 patients developed rotavirus-induced diarrhea between 47 and 147 days. The incidence of rotavirus infection was 9.7% in children compared to none in adults (P = .01). This was 25.3% in those with GVHD compared to 1.2% in those without GVHD (P = .001). Rotavirus infection was followed by post-transplantation hemophagocytic syndrome (PTHPS) at a median of 4 days (range, 3-10 days) in all four patients. Three patients succumbed to the complications related to PTHPS. Only one patient, who is long-term survivor, was able to eliminate this virus after 2 weeks. Children undergoing T-replete haploidentical hematopoietic cell transplantation who develop GVHD are at a higher risk of community-acquired rotavirus infection which was strongly associated with PTHPS with poor outcome.

Haemophagocytosis is a dysregulated immune condition characterised by both inflammation and uncontrolled activation of macrophages and T-cells, which causes aberrant cytokine release, leading to cytokine storm [1] it can be primary or secondary, depending upon the etiology.

The outcome of hyperacute grade 3-4 steroid-refractory graft-versus-host-disease (SR-GVHD) remains dismal despite a plethora of agents being tried alone or in combination. Following T replete haploidentical transplantation with post-transplantation cyclophosphamide on 75 patients, 10 patients (13%) aged 2-20years, developed hyperacute SR-GVHD. We report on the outcome of two different regimens for treatment of SR-GVHD on the outcome of these patients. Five patients were treated in Regimen A consisting of anti-thymocyte globulin, Etanercept and Basiliximab. The next 5 patients were treated combining T cell costimulation blockade with Abatacept along with Etanercept and Basiliximab. The overall response at days 29 and 56 were 40% and 0% with Regimen A and100% and 40% with Regimen B. The major cause of treatment failure was progression of GVHD and opportunistic infections. Two of the patients achieving a complete remission on Regimen B are long term disease free survivors off immunosuppression. Our study demonstrates the dismal outcome of early onset SR-GVHD in children following T replete haploidentical transplantation. However, the combination of Abatacept with anticytokine agents seems to produce encouraging early response and might warrant further investigation.

Post-transplantation cyclophosphamide (PTCY) therapy has made haploidentical transplantation a global reality in adults, but the literature is largely silent on the feasibility of this approach in children. We conducted a prospective study of 20 patients (median age, 12 years; range, 2-20 years) with advanced acute leukemia to evaluate the feasibility of PTCY-based haploidentical peripheral blood stem cell (PBSC) transplantation in children. The conditioning regimen comprised fludarabine, i.v. busulfan, and melphalan (Flu-Bu-Mel). PTCY on days +3 and +4 was followed by mycophenolate mofetil for 14-21 days and cyclosporine for 60 days. Thirteen patients (65%) had refractory or relapsed myelogenous leukemia, and the remainder had high-risk lymphoblastic leukemia. Prompt engraftment was noted at a median of 14 days, with full donor chimerism by day +28. The cumulative incidence of acute and chronic graft-versus-host disease was 35% and 5%, respectively. Nonrelapse mortality at 1 year was 20%. The incidence of disease progression was 25.7%. The actuarial overall survival at 2 years was 64.3% (95% confidence interval, 53.4%-75.2%). Our data suggest that Flu-Bu-Mel–based conditioning followed by PTCY-based haploidentical PBSC transplantation with reduced duration of immunosuppression is feasible in pediatric patients with advanced leukemia.

Background & Methods Post-transplantation cyclophosphamide (PTCy)-based HCT has been associated with an increased frequency of hemorrhagic cystitis (HC), dominantly associated with BK viruria. We prospectively evaluated the incidence of BK viruria and HC in 115 patients [(median age 20 years, 2-65), malignant(87) and non-malignant(28)], undergoing PTCy-based haploidentical HCT with CTLA4Ig (n=71) or without (n=44). Conditioning was predominantly myeloablative (74%). As prophylaxis for HC, continuous infusion of Mesna was used from 30 minutes prior to PTCy at 50% of the daily dose of Cy every 8 hourly for 72 hours. Development of HC before day+15 was termed as ‘early HC’ and later was referred to as ‘HC’. Results The overall incidence of BK viruria was 65.7%. None with BK viruria Overall BK viruria was significantly lower in those receiving CTLA4Ig (54.1% vs 89.4% in those without CTLA4Ig, p=0.001, Figure 2A). The incidences of both high BK viruria (4.3% with vs 11.9% without CTLA4Ig, p=0.08) and HC (2.9% with vs 9.5% without CTLA4Ig p=0.09, Figure 2B) tended to be lower in those receiving CTLA4Ig-based protocols. 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). Conclusions In conclusion, our study demonstrates that extended infusion of Mesna, along with reduction in alloreactivity with the use of CTLA4Ig were probably responsible for a much lower incidence of BK viruria and resultant HC, than previously reported following PTCy-based haploidentical HCT.

CTLA4Ig attenuates T cell activation by co-stimulation blockade, but natural killer (NK) cells are not only resistant to CTLA4Ig, they also may demonstrate better antileukemia effect in the presence of CTLA4Ig. To explore this phenomenon we used sequential CTLA4Ig primed donor lymphocyte infusion (DLI) after post-transplant cyclophosphamide–based haploidentical transplantation. Thirty patients (CTLA4Ig-DLI group) with advanced leukemia received CTLA4Ig on day –1 and subsequently on days +7, +21, and +35, followed 12hours later by DLI of 1 to 10 × 106 CD3+ T cells/kg containing .1 to 3.27 × 106/kg CD56+ NK cells, with low dose cyclosporine for 60days. The incidences of acute graft-versus-host disease (GVHD), chronic GVHD and nonrelapse mortality (NRM) were 6.7%, 21%, and 4.5 %, respectively, with disease progression of 23.3% and overall survival of 79% at 18 months. Patients without disease progression had a significant early surge in CD56dimCD16+NK cells with lower NKG2A expression. CTLA4Ig primed DLI was associated with an upregulation of CD86 in mature NK cells that was not witnessed with CTLA4Ig administration alone. Thus, CTLA4Ig primed DLI resulted in early proliferation of mature NK cells with cytotoxic potential enabling early institution of adoptive immunotherapy to mitigate the risk of relapse in advanced leukemia with reduced GVHD and NRM.

Background: Gut colonisation with Carbapenem Resistant Enterobacteriaceae (CRE) is a risk factor for CRE bacteremia and patients with haematological malignancies (HM) are at the highest risk of mortality.Methods: We conducted a prospective surveillance study of gut colonisation with CRE and its impact on the outcome on 225 consecutive patients of HM over 28 months. Results: The median age of the cohort was 46 years, majority with Acute Leukemia. 48 (21%) patients were colonized with CRE on admission (CAD). Another 46 patients were colonized with CRE in the hospital (CIH). The risk factors for CAD and CIH were diagnosis of acute leukemia and duration of hospital stay respectively. CRE accounted for 77% of non-relapse mortality (NRM). CRE bacteremia occurred only in colonized patients with 100% mortality. NRM was 35.3% in CIH group compared to 10.5% in the CAD group (p=0.0001). NRM was highest in those with AML and CIH (54.9% p=0.0001). On multivariate analysis, CIH was the most important risk factor for NRM (HR-7.2).Conclusion: Our data demonstrates that a substantial proportion of patients with HM are colonized with CRE without prior hospitalization, but those with nosocomial colonization have the highest risk of mortality, particularly in those with Acute Myeloid Leukemia.