Facilitation of colonic T cell immune responses is associated with an exacerbation of dextran sodium sulfate–induced colitis in mice lacking microsomal prostaglandin E synthase-1

Background Microsomal prostaglandin E synthase-1 (mPGES-1) is a key enzyme that acts downstream of cyclooxygenase and plays a major role in inflammation by converting prostaglandin (PG) H2 to PGE2. The present study investigated the effect of genetic deletion of mPGES-1 on the development of immunologic responses to experimental colitis induced by dextran sodium sulfate (DSS), a well-established model of inflammatory bowel disease (IBD). Methods Colitis was induced in mice lacking mPGES-1 (mPGES-1−/− mice) and wild-type (WT) mice by administering DSS for 7 days. Colitis was assessed by body weight loss, diarrhea, fecal bleeding, and histological features. The colonic expression of mPGES-1 was determined by real-time PCR, western blotting, and immunohistochemistry. The impact of mPGES-1 deficiency on T cell immunity was determined by flow cytometry and T cell depletion in vivo. Results After administration of DSS, mPGES-1−/− mice exhibited more severe weight loss, diarrhea, and fecal bleeding than WT mice. Histological analysis further showed significant exacerbation of colonic inflammation in mPGES-1−/− mice. In WT mice, the colonic expression of mPGES-1 was highly induced on both mRNA and protein levels and colonic PGE2 increased significantly after DSS administration. Additionally, mPGES-1 protein was localized in the colonic mucosal epithelium and infiltrated inflammatory cells in underlying connective tissues and the lamina propria. The abnormalities consistent with colitis in mPGES-1−/− mice were associated with higher expression of colonic T-helper (Th)17 and Th1 cytokines, including interleukin 17A and interferon-γ. Furthermore, lack of mPGES-1 increased the numbers of Th17 and Th1 cells in the lamina propria mononuclear cells within the colon, even though the number of suppressive regulatory T cells also increased. CD4+ T cell depletion effectively reduced symptoms of colitis as well as colonic expression of Th17 and Th1 cytokines in mPGES-1−/− mice, suggesting the requirement of CD4+ T cells in the exacerbation of DSS-induced colitis under mPGES-1 deficiency. Conclusions These results demonstrate that mPGES-1 is the main enzyme responsible for colonic PGE2 production and deficiency of mPGES-1 facilitates the development of colitis by affecting the development of colonic T cell–mediated immunity. mPGES-1 might therefore impact both the intestinal inflammation and T cell–mediated immunity associated with IBD.

Haploidentical Allogeneic Stem Cell Transplantation in Sickle Cell Disease: A Systematic Review and Meta-Analysis

Background Allogeneic hematopoietic stem cell transplantation (SCT) is the only established curative treatment option for patients with sickle cell disease (SCD). Transplantations with an HLA-identical matched sibling donor (MSD) have resulted in excellent disease-free survival of >90% and overall survival (OS) of >95%. However, lack of HLA-identical siblings is a limiting factor. The chance of finding a potential matched unrelated donor is low for patients with a non-Western ethnic background (< 20%). Haploidentical related donors are a promising pool of donors potentially extending SCT as a curative treatment to a larger group of SCD patients with no other meaningful treatment options. Myeloablative conditioning regimens (MAC) are not recommended for adult SCD patients, as cumulative SCD-related organ damage renders these patients susceptible to increased toxicity and higher risk of transplant-related mortality. Using reduced-intensity conditioning (RIC) or non-myeloablative conditioning (NMC) in both adult and pediatric patients has resulted in decreased transplant-related toxicity and mortality. However, while successful in hematologic malignancies, NMC has been associated with significant risk of graft failure in hemoglobinopathies. In the present study, we aimed to systematically review (1) the outcomes of haploidentical SCT (Haplo-SCT), (2) the effects of conditioning intensity and modes of T-cell depletion on Haplo-SCT outcomes and (3) comparative outcomes between matched sibling donor SCT (MSD-SCT) and Haplo-SCT in selected studies. Methods A comprehensive search was performed in MEDLINE/PubMed and Embase up to May 2021. Data was extracted by two reviewers independently and the Newcastle-Ottawa Quality Assessment Scale was used to assess the quality of the studies. Only studies reporting at least one of the outcomes: graft failure, OS, transplant-related mortality, and acute/chronic graft-versus-host disease (GvHD) were included. Fourteen studies met the inclusion criteria. To have an overview of the results of Haplo-SCT, we divided the included studies in four groups according to the conditioning intensity (MAC versus NMC/RIC) and the T-cell depletion method (in vivo (post-transplant cyclophosphamide (PTCy)) vs. in vitro). Results All included studies were observational cohort studies. A subgroup meta-analysis of the results of Haplo-SCT showed relatively low overall pooled proportions of graft failure (7%, 95% CI: 2 - 20), acute (4%, 95% CI: 2 - 12) and chronic (11%, 95% CI: 7 - 16) GvHD. Overall survival was high (91%, 95% CI: 85 - 94). Graft failure in MAC-in vitro, MAC-in vivo, NMC/RIC-in vitro and NMC/RIC-in vivo groups was 4% (95% CI: 1 - 14), 0% (95% CI: 0 - 100), 25% (95% CI: 10 - 51) and 11% (95% CI: 3 - 36) respectively (Figure 1). OS was 100% and 93% for MAC and NMC/RIC groups with PTCy (in vivo T-cell depletion) respectively. In patients with in vitro T-cell depletion, OS was 87% and 81% in MAC and NMC/RIC groups respectively (Figure 2). Based on a comparative meta-analysis of the three studies that included both haploidentical and MSD transplantation, graft failure was significantly higher in the haploidentical group than in the MSD group (odds ratio 5.3, 95% CI: 1.0 - 27.6). Overall survival, transplant-related mortality and acute/chronic GvHD were not significantly different between the groups. Conclusions This systematic review shows that modifications in the intensity of the conditioning regimen and improved T-cell depletion approaches in Haplo-SCT in SCD have led to reduced transplantation-related toxicity while keeping graft failure rates low. Both in vitro and in vivo (PTCy) T-cell depletions result in very low transplantation-related mortality, though in vitro T-cell depletion is associated with a higher incidence of viral reactivations and other infectious complications. Haploidentical stem cell transplantation is becoming a viable alternative curative option for SCD, extending the availability of allogeneic SCT as a treatment option to many more transplant eligible SCD patients. Novel immunosuppression (immunoablation) and improvement in supportive care have allowed the use of RIC or NMC regimens, resulting in low risk of transplantation-related complications and improvement in engraftment rates. Figure 1 Figure 1. Disclosures Biemond: Global Blood Therapeutics: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; CSL Behring: Honoraria; Sanquin: Research Funding; Novo Nordisk: Honoraria; Celgene: Honoraria. Nur: Celgene: Speakers Bureau; Roche: Speakers Bureau; Novartis: Research Funding, Speakers Bureau.

Graft-Vs-Host Disease (GvHD) Prophylaxis with Post-Transplant Cyclophosphamide (PTCy) and Thymoglobulin (ATG) Are More Important Determinants of Cytomegalovirus (CMV) Reactivation after Allogeneic Hematopoietic Cell Transplantation (HCT) Than Ex-Vivo T-Cell Depletion in the Era of Pre-Emptive Therapy

Background: Cytomegalovirus (CMV) is a common cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT) despite major advances in diagnostic techniques and antiviral prophylactic strategies. The relative impacts of donor/recipient CMV serologic status, disease-specific and transplant-related prognostic factors on the risk of CMV reactivation and survival are undefined. Methods: We studied the outcome of 199 patients (median age, 46 years; range 17-71 years) receiving allogeneic HCT at National University Cancer Institute of Singapore (NCIS) between January 2016 and December 2020. Their hematologic diseases included AML (n=92), ALL (n=46), MDS (n=19), lymphomas (n=19), MPN (n=7) and others (n=16) such as refractory myelomas and aplastic anemias. The conditioning regimens used were either myeloablative (n=80) or reduced intensity conditioning (n=119) prior to an allograft from different donor sources. T-cell depletion (TCD) was used for GVHD prophylaxis in 124 patients; and this included post-transplant cyclophosphamide (PTCy, n=31), ex-vivo T-cell receptor alpha-beta / CD45RA depletion (TCRab/CD45RA) (ex-vivo TCD, n=31) for haploidentical HCT, or thymoglobulin (ATG, n=62) for matched unrelated donor (MUD) HCT. Results: With a median follow-up duration of 15.6 months (range, 0.2-63.6 months), 136 (68.3%) patients had CMV reactivation (median onset, 27.5 days) while 6 (3.0 %) patients developed clinically significant CMV disease, such as colitis, retinitis and encephalitis. The cumulative incidences of CMV reactivation within the first 100 days among the recipients of matched unrelated donor (MUD) (n=60), mismatched related donor or unrelated donor (MMRD/MMUD) (n=60), umbilical cord blood (UCB) (n=18) and matched related donor (MRD) (n=61) HCT were 71.6 %, 61.7 %, 50.0 % and 32.7 %, respectively (p<0.001). There were no statistically significant differences in overall survival (OS, p=0.830) and disease-free survival (DFS, p=0.983) at 5 years between CMV-seropositive (D+/R+ or D-/R+, n=181) and CMV-seronegative recipients (D-/R- or D+/R-, n=18). There were also no significant differences in the cumulative incidences of CMV reactivation within 100 days (p=0.879), CMV end-organ disease (p=0.522) and non-relapse mortality (NRM, p=0.202), respectively. HCT-CI score of ≥1 (p=0.005) and the use of reduced intensity conditioning regimen (p<0.001) were associated with a higher NRM at 2 years. There was also a trend towards higher NRM among patients with peak CMV DNA titers of above 1000 IU/ml, but this did not reach statistical significance (p=0.188). The secondary objective of this study was to determine the risk factors associated with CMV reactivation within the first 100 days post-transplant. There was no statistically significant impact of the donor or recipient CMV serostatus (p=0.790) on the risk of CMV reactivation. In multivariable analysis, the use of any T-cell depletion (p<0.001) was a significant predictor of CMV reactivation. In a subset analysis comparing the 3 different methods of TCD, the use of ATG (p=0.004) and PTCy (p=0.005) was found to be associated with an increased risk of CMV reactivation, but not in patients receiving ex-vivo TCD (p=0.184) (Figure 1). Notably, patients receiving ex-vivo TCD haploidentical HCT was not associated with a higher risk of CMV reactivation as compared to the recipients of MRD HCT without any TCD. Conclusions: Our study concluded that CMV serologic status did not affect the incidence of CMV reactivation, NRM, OS and DFS in patients undergoing allogeneic HCT. The use of PTCy and ATG for GVHD prophylaxis, remains the most important risk factor for CMV reactivation in the era of pre-emptive therapy and hence, the need for aggressive prevention strategies in this vulnerable group of patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Overall Survival Improvement Following ALLO-SCT in Patients Older THAN 60 YEARS: A Gruppo Italiano Trapianto DI Midollo Osseo (GITMO) Registry Study

The upper age-limit for patients with hematological malignancies eligible for allo-SCT progressed to 70-75 years. As a consequence, an increase of older patients submitted to allo-SCT has been observed worldwide and in Italy as well. This registry-based retrospective study on behalf of GITMO (GITMO AlloEld) describes the transplant activity among elderly patients in Italy, between 2000 and 2017. Thiry GITMO Centers participated to the Study. 2061 allo-SCTS in patients older than 60 years were exported from PROMISE database and 1996 first transplants were analysed. The median age of the patients at transplant was 63,5 years (59,5-77,8). The most commonly transplanted diseases were acute leukemias and myelodisplastic syndromes (67,5%). 28% and 27% of the patients showed a HCT-CI of 1-2 or grater than 3, respectively. The KPS was 100% in 27,2% and 90% in 42,9% of the cases. 32% of the patients received a myeloablative conditioning regimen and 55% of the patients received an in vivo T-cell depletion (either post transplant cyclophosphamide or ATG). With a median follow up of 10,4 years, the OS at 5 years significantly improved during time, moving from 28% between 2000-2005 to 37% between 2012-2017 (p=0,012). This was related to a significant reduction in RI (45% vs 30%, p<0,0001), whereas NRM remained stable over time (33% vs 35%, p=0,5). The following significant differences were observed across the years of the present study: 1) a longer 5 years OS in patients younger than 65 years (34%) vs those older than 70 years (19%) between 2000 and 2011 only (p=0,003) (Figure 1A and 1B); 2) a longer 5 years OS (p<0.001) and a reduced NRM (p=0.02) for HCT-CI 0 vs 1-2 vs >3 between 2000-2011 only (Figure 2A and 2B); 3) a different 5 years OS according to donor type between 2000 and 2011 only (19% for haplo vs 31% for sibling vs 33% for mismatch UD and 38% for MUD; p<0,0001) (Figure 3A and 3B); 4) a reduction in the incidence of extensive cGVHD at 1 year (15,6% between 2000 and 2005 vs 10,3% between 2012 and 2017, p=0,004) (Figure 4). Comparing 2000-2005 vs 2012-2017, the major significant differences of the patients regard: the baseline disease (more AL/MDS: 40% vs 77%; p<0,001); the disease status at SCT (more CR: 29% vs 54%, p<0,0001), the intensity of conditioning (MAC in 14% vs 42%, p<0,001), the use of alkylator-base conditioning regimen (49% vs 91%; p<0,001) and the use of in vivo T-cell depletion (17% vs 43%, p<0,0001). Moreover, the percentage of patients with HCT-CI > 3 moved from 10% to 30% (p<0,001), in parallel with an increase in patients with KPS 100 (10% vs 30%; p<0,001) By multivariate analysis MUD donor or UCB, no response at the time of SCT and male recipient significantly impaired OS, whereas HCT-CI <1, KPS 90-100 and transplant between 2011-2017 significantly improved OS. These retrospective data showed that the transplant procedure for elderly patients became safer and more effective over time, for a reduction of the RI related to a better selection of patients (more acute leukemias in CR) and a better selection of conditionings (more MAC and more alkylators). Nowdays, the HCT-CI score is probably not sufficient for estimate elderly patients probability of OS and NRM. Figure 1 Figure 1. Disclosures Luppi: Abbvie: Honoraria; Novartis: Honoraria; Sanofi: Honoraria; MSD: Honoraria; Gilead Science: Honoraria, Other: Travel grant; Daiichi-Sankyo: Honoraria; Jazz Pharma: Honoraria. Ciceri: IRCCS Ospedale San Raffaele: Current Employment.

Cryptococcus neoformans Δsgl1 Vaccination Requires Either CD4+ or CD8+ T Cells for Complete Host Protection

Cryptococcus neoformans is a fungal pathogen causing life-threatening meningoencephalitis in susceptible individuals. Fungal vaccine development has been hampered by the fact that cryptococcosis occurs during immunodeficiency. We previously reported that a C. neoformans mutant (Δsgl1) accumulating sterylglucosides (SGs) is avirulent and provides complete protection to WT challenge, even under CD4+ T cell depletion, an immunodeficient condition commonly associated with cryptococcosis. We found high levels of SGs in the lungs post-immunization with Δsgl1 that decreased upon fungal clearance. Th1 cytokines increased whereas Th2 cytokines concurrently decreased, coinciding with a large recruitment of leukocytes to the lungs. Depletion of B or CD8+ T cells did not affect either Δsgl1 clearance or protection from WT challenge. Although CD4+ T cell depletion affected clearance, mice were still protected indicating that clearance of the mutant was not necessary for host protection. Protection was lost only when both CD4+ and CD8+ T cells were depleted, highlighting a previously unexplored role of fungal-derived SGs as an immunoadjuvant for host protection against cryptococcosis.

The Hitchhiker Guide to CD4+ T-Cell Depletion in Lentiviral Infection. A Critical Review of the Dynamics of the CD4+ T Cells in SIV and HIV Infection

CD4+ T-cell depletion is pathognomonic for AIDS in both HIV and simian immunodeficiency virus (SIV) infections. It occurs early, is massive at mucosal sites, and is not entirely reverted by antiretroviral therapy (ART), particularly if initiated when T-cell functions are compromised. HIV/SIV infect and kill activated CCR5-expressing memory and effector CD4+ T-cells from the intestinal lamina propria. Acute CD4+ T-cell depletion is substantial in progressive, nonprogressive and controlled infections. Clinical outcome is predicted by the mucosal CD4+ T-cell recovery during chronic infection, with no recovery occurring in rapid progressors, and partial, transient recovery, the degree of which depends on the virus control, in normal and long-term progressors. The nonprogressive infection of African nonhuman primate SIV hosts is characterized by partial mucosal CD4+ T-cell restoration, despite high viral replication. Complete, albeit very slow, recovery of mucosal CD4+ T-cells occurs in controllers. Early ART does not prevent acute mucosal CD4+ T-cell depletion, yet it greatly improves their restoration, sometimes to preinfection levels. Comparative studies of the different models of SIV infection support a critical role of immune activation/inflammation (IA/INFL), in addition to viral replication, in CD4+ T-cell depletion, with immune restoration occurring only when these parameters are kept at bay. CD4+ T-cell depletion is persistent, and the recovery is very slow, even when both the virus and IA/INFL are completely controlled. Nevertheless, partial mucosal CD4+ T-cell recovery is sufficient for a healthy life in natural hosts. Cell death and loss of CD4+ T-cell subsets critical for gut health contribute to mucosal inflammation and enteropathy, which weaken the mucosal barrier, leading to microbial translocation, a major driver of IA/INFL. In turn, IA/INFL trigger CD4+ T-cells to become either viral targets or apoptotic, fueling their loss. CD4+ T-cell depletion also drives opportunistic infections, cancers, and comorbidities. It is thus critical to preserve CD4+ T cells (through early ART) during HIV/SIV infection. Even in early-treated subjects, residual IA/INFL can persist, preventing/delaying CD4+ T-cell restoration. New therapeutic strategies limiting mucosal pathology, microbial translocation and IA/INFL, to improve CD4+ T-cell recovery and the overall HIV prognosis are needed, and SIV models are extensively used to this goal.

Novel GVHD resistant humanized-PBMC mouse model for preclinical HIV research

Humanized mouse models are based on the engraftment of human cells in immunodeficient mouse strains, most notably the NSG strain. Most used models have a major limitation in common, the development of graft-versus-host disease (GVHD). GVHD not only introduces variabilities into the research data but also leads to animal welfare concerns. A new mouse strain, B6.129S-Rag2tm1Fwa CD47tm1Fpl Il2rgtm1Wjl/J which lacks Rag1, IL2rg, and CD47 (triple knockout or TKO), is resistant to GVHD development. We transplanted TKO mice with human peripheral blood mononuclear cells (PBMCs) to establish a new humanized PBMC (hu-PBMC) mouse model. A cohort of these mice was infected with HIV-1 and monitored for plasma HIV viremia and CD4+ T cell depletion. The onset and progression of GVHD were monitored by clinical signs. This study demonstrates that TKO mice transplanted with human PBMCs support engraftment of human immune cells in primary and secondary lymphoid tissues, rectum, and brain. Moreover, the TKO hu-PBMC model supports HIV-1 infection via intraperitoneal, rectal, or vaginal routes, as confirmed by robust plasma HIV viremia and CD4+ T cell depletion. Lastly, TKO mice showed a delayed onset of GVHD clinical signs (~21 days) and exhibited significant decreases in plasma levels of TNFβ. Based on these results, the TKO hu-PBMC mouse model not only supports humanization and HIV-1 infection but is also resistant to GVHD development, making this model a valuable tool in HIV research.