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Key Points HLA-haploidentical allogeneic blood or bone-marrow transplantation (haploBMT) has historically been associated with poor outcomes, owing to high rates of graft failure and graft-versus-host disease (GVHD) Several transplantation platforms have been developed that successfully overcome these historical barriers to haploBMT; three main approaches have been used extensively to conduct haploBMT procedures in patients T-cell depletion with 'megadose' CD34 + cells results in exceptionally low rates of GVHD, but is associated with poor T-cell function and thus high nonrelapse mortality (NRM), predominantly owing to infection The GIAC protocol, which involves in vivo modulation of T-cell-replete allografts, produces essentially universal engraftment with limited relapse and favourable survival, albeit with high rates of GVHD, particularly chronic GVHD Use of high-dose, post-transplantation cyclophosphamide after T-cell-replete allografting results in low rates of GVHD and NRM and favourable immune reconstitution, with somewhat higher rates of relapse, particularly after reduced-intensity conditioning No standard-of-care currently exists, as no completed prospective randomized studies have, thus far, compared any of these haploBMT approaches with each other or with transplantation approaches using other donor types In the past, only patients with fully HLA-matched donors were able to benefit from blood or bone-marrow transplantation (BMT) for a variety of haematological malignancies. Owing to the development of a variety of immunomodulatory strategies, patients with no HLA-matched donor, who can therefore receive an HLA-haploidentical BMT, can expect the same or similar outcomes as those receiving HLA-matched BMT. In this Review, the authors describe the new approaches to immunomodulation that have made HLA-haploidentical BMT a realistic therapeutic approach. Allogeneic blood or bone-marrow transplantation (alloBMT) is a potentially curative treatment for a variety of haematological malignancies and nonmalignant diseases. Historically, human leukocyte antigen (HLA)-matched siblings have been the preferred source of donor cells owing to superior outcomes compared with alloBMT using other donors. Although only approximately one-third of patients have an HLA-matched sibling, nearly all patients have HLA-haploidentical related donors. Early studies using HLA-haploidentical alloBMT resulted in unacceptably high rates of graft rejection and graft-versus-host disease (GVHD), leading to high nonrelapse mortality and consequently poor survival. Several novel approaches to HLA-haploidentical alloBMT have yielded encouraging results with high rates of successful engraftment, effective GVHD control and favourable outcomes. In fact, outcomes of several retrospective comparative studies seem similar to those seen using other allograft sources, including those of HLA-matched-sibling alloBMT. In this Review, we provide an overview of the three most-developed approaches to HLA-haploidentical alloBMT: T-cell depletion with 'megadose' CD34 + cells; granulocyte colony-stimulating factor-primed allografts combined with intensive pharmacological immunosuppression, including antithymocyte globulin; and high-dose, post-transplantation cyclophosphamide. We review the preclinical and biological data supporting each approach, results from major clinical studies, and completed or ongoing clinical studies comparing these approaches with other alloBMT platforms.

Post-transplantation cyclophosphamide (PTCy) has been highly successful at preventing severe acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). The clinical application of this approach was based on extensive studies in major histocompatibility complex (MHC)-matched murine skin allografting models, in which cyclophosphamide was believed to act via three main mechanisms: (1) selective elimination of alloreactive T cells; (2) intrathymic clonal deletion of alloreactive T-cell precursors; and (3) induction of suppressor T cells. In these models, cyclophosphamide was only effective in very specific contexts, requiring particular cell dose, cell source, PTCy dose, and recipient age. Achievement of transient mixed chimerism also was required. Furthermore, these studies showed differences in the impact of cyclophosphamide on transplanted cells (tumor) versus tissue (skin grafts), including the ability of cyclophosphamide to prevent rejection of the former but not the latter after MHC-mismatched transplants. Yet, clinically PTCy has demonstrated efficacy in MHC-matched or partially-MHC-mismatched HCT across a wide array of patients and HCT platforms. Importantly, clinically significant acute GVHD occurs frequently after PTCy, inconsistent with alloreactive T-cell elimination, whereas PTCy is most active against severe acute GVHD and chronic GVHD. These differences between murine skin allografting and clinical HCT suggest that the above-mentioned mechanisms may not be responsible for GVHD prevention by PTCy. Indeed, recent work by our group in murine HCT has shown that PTCy does not eliminate alloreactive T cells nor is the thymus necessary for PTCy's efficacy. Instead, other mechanisms appear to be playing important roles, including: (1) reduction of alloreactive CD4 effector T-cell proliferation; (2) induced functional impairment of surviving alloreactive CD4 and CD8 effector T cells; and (3) preferential recovery of CD4 regulatory T cells. Herein, we review the history of cyclophosphamide's use in preventing murine skin allograft rejection and our evolving new understanding of the mechanisms underlying its efficacy in preventing GVHD after HCT. Efforts are ongoing to more fully refine and elaborate this proposed new working model. The completion of this effort will provide critical insight relevant for the rational design of novel approaches to improve outcomes for PTCy-treated patients and for the induction of tolerance in other clinical contexts.

Foxp3 is regarded as the major transcription factor for T regulatory (T reg ) 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 (T conv ) 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 T reg marker in man. Helios, a member of the Ikaros gene family, has been shown to be expressed by 80-90% of human Foxp3 + T reg cells and can potentially serve as a marker of human T reg . Here, we confirm that Foxp3 expression is readily upregulated by T conv upon TCR stimulation in vitro , while Helios expression is not altered. More importantly, we show that Foxp3 expression is not elevated by stimulation of hT conv 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 T conv cell activation in vivo and that Foxp3 expression alone can be used to quantitate T reg cells in humans. Nevertheless, the combined use of Foxp3 and Helios expression provides a more reliable approach for the characterization of T reg in humans.

Prophylactic immunosuppressants for graft-versus-host disease (GVHD) in allogeneic hematopoietic cell transplantation (alloHCT), including post-transplant cyclophosphamide (PTCy) and mycophenolate mofetil (MMF), exhibit complex pharmacokinetic profiles. Interindividual variations in pharmacokinetic exposure to these immunosuppressants or their metabolites may interfere with treatment outcomes. A feasibility study (n = 11) was conducted to investigate the pharmacokinetic/pharmacodynamic relationship in patients undergoing HLA-haploidentical alloHCT with standard high-dose PTCy (50 mg·kg ·day on days +3/+4) combined with MMF and tacrolimus or sirolimus. Blood samples were collected to assess the variability in pharmacokinetic biomarkers, including exposures [areas under the curve (AUCs)] to cyclophosphamide (Cy), carboxycyclophosphamide (cepm), -dechloroethyl cyclophosphamide (dccy), 4-ketocyclophosphamide (ketocy), mycophenolic acid (MPA), and mycophenolic acid glucuronide (MPAG). Serial dynamic changes in immune cell populations, including regulatory T cells (Tregs), over the first 3 post-transplant weeks were monitored. A transient reduction in the proliferation (Ki-67 ) of activated (HLA-DR ) T cells coincided with Cy treatment. The ratio of Tregs to the CD4 T-cell population increased in a time-dependent manner within the first 21 days post-transplant. We observed moderate interindividual variability across all pharmacokinetic biomarkers. Serum creatinine and blood urea nitrogen levels positively correlated with exposure to Cy and MMF metabolites, including cepm, MPA, and MPAG. Using correlation analysis, we further confirmed the negative association between pharmacokinetic (PK) biomarkers and lymphocyte count, but not Treg percentage, suggesting that careful optimization of Cy and MMF dosing may have the potential to support immune recovery, although this requires further validation in larger studies. The relationship of pharmacokinetic biomarkers to immune and clinical outcomes warrants further investigation in larger studies but holds promise for personalizing dosing of GVHD prophylaxis to improve patient outcomes after alloHCT.

Post-transplantation cyclophosphamide (PTCy) reduces the incidence and severity of graft-versus-host disease (GVHD), thereby improving the safety and accessibility of allogeneic hematopoietic cell transplantation (HCT). We have shown that PTCy works by inducing functional impairment and suppression of alloreactive T cells. We also have identified that reduced proliferation of alloreactive CD4 + T cells at day +7 and preferential recovery of CD4 + CD25 + Foxp3 + regulatory T cells (T regs ) at day +21 are potential biomarkers associated with optimal PTCy dosing and timing in our B6C3F1→B6D2F1 MHC-haploidentical murine HCT model. To understand whether the effects of PTCy are unique and also to understand better the biology of GVHD prevention by PTCy, here we tested the relative impact of cyclophosphamide compared with five other optimally dosed chemotherapeutics (methotrexate, bendamustine, paclitaxel, vincristine, and cytarabine) that vary in mechanisms of action and drug resistance. Only cyclophosphamide, methotrexate, and cytarabine were effective in preventing fatal GVHD, but cyclophosphamide was superior in ameliorating both clinical and histopathological GVHD. Flow cytometric analyses of blood and spleens revealed that these three chemotherapeutics were distinct in constraining conventional T-cell numerical recovery and facilitating preferential T reg recovery at day +21. However, cyclophosphamide was unique in consistently reducing proliferation and expression of the activation marker CD25 by alloreactive CD4 + Foxp3 - conventional T cells at day +7. Furthermore, cyclophosphamide restrained the differentiation of alloreactive CD4 + Foxp3 - conventional T cells at both days +7 and +21, whereas methotrexate and cytarabine only restrained differentiation at day +7. No chemotherapeutic selectively eliminated alloreactive T cells. These data suggest that constrained alloreactive CD4 + Foxp3 - conventional T-cell numerical recovery and associated preferential CD4 + CD25 + Foxp3 + T reg reconstitution at day +21 may be potential biomarkers of effective GVHD prevention. Additionally, these results reveal that PTCy uniquely restrains alloreactive CD4 + Foxp3 - conventional T-cell proliferation and differentiation, which may explain the superior effects of PTCy in preventing GVHD. Further study is needed to determine whether these findings also hold true in clinical HCT.

[...]results in patients with unfavorable prognosis, such as those with chemo-resistant disease, are still unsatisfactory, and relapse is still a major cause of treatment failure.Rovatti et al.discuss the mechanisms of immune evasion in allogeneic HCT, such as genomic loss or downregulation of HLA molecules, up-regulation of T-cell inhibitory ligands, release of mediators of T-cell exhaustion, and inhibition of the release of pro-inflammatory cytokines. The present Research Topic reports how haploHCT has become an effective and widespread treatment so much that survival rates are similar to those after HLA-matched sibling or unrelated donor HCT. [...]haploHCT is a feasible platform for further improvements, including the combination with innovative immunotherapies such as bi-specific antibodies and CAR-T cells and CAR-NK cells. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Posttransplantation cyclophosphamide (PTCy) recently has had a marked impact on human allogeneic hematopoietic cell transplantation (HCT). Yet our understanding of how PTCy prevents graft-versus- host disease (GVHD) largely has been extrapolated from MHC-matched murine skin-allografting models that were highly contextual in their efficacy. Herein, we developed a T cell-replete, MHC-haploidentical, murine HCT model (B6C3F'WB6D2F1) to test the putative underlying mechanisms: alloreactive T cell elimination, alloreactive T cell intrathymic clonal deletion, and suppressor T cell induction. In this model and as confirmed in four others, PTCy did not eliminate alloreactive T cells identified using either specific V[beta]s or the 2C or 4C T cell receptors. Furthermore, the thymus was not necessary for PTCy's efficacy. Rather, PTCy induced alloreactive T cell functional impairment, which was supported by highly active suppressive mechanisms established within one day after PTCy that were sufficient to prevent new donor T cells from causing GVHD. These suppressive mechanisms included the rapid, preferential recovery of [CD4.sup.+][CD25.sup.+][Foxp3.sup.+] regulatory T cells, including those that were alloantigen specific, which served an increasingly critical function over time. Our results prompt a paradigm shift in our mechanistic understanding of PTCy. These results have direct clinical implications for understanding tolerance induction and for rationally developing novel strategies to improve patient outcomes.

Mucormycosis is a devastating disease with high morbidity and mortality in susceptible patients. Accurate diagnosis is required for timely clinical management since antifungal susceptibility differs between species. Irregular hyphal elements are usually taken as the hallmark of mucormycosis, but here, we show that some species may also produce yeast-like cells, potentially being mistaken for Candida or Paracoccidioides . We demonstrate that the dimorphic transition is common in Mucor species and can be driven by many factors. The multi-nucleate yeast-like cells provide an effective parameter to distinguish mucoralean infections from similar yeast-like species in clinical samples.

Dose optimization of sirolimus may further improve outcomes in allogeneic hematopoietic cell transplant (HCT) patients receiving post‐transplantation cyclophosphamide (PTCy) to prevent graft‐versus‐host disease (GVHD). Sirolimus exposure–response association studies in HCT patients (i.e., the association of trough concentration with clinical outcomes) have been conflicting. Sirolimus has important effects on T‐cells, including conventional (Tcons) and regulatory T‐cells (Tregs), both of which have been implicated in the mechanisms by which PTCy prevents GVHD, but there is an absence of validated biomarkers of sirolimus effects on these cell subsets. Considering the paucity of existing biomarkers and the complexities of the immune system, we conducted a literature review to inform a quantitative systems pharmacology (QSP) model of GVHD. The published literature presented multiple challenges. The sirolimus pharmacokinetic models insufficiently describe sirolimus distribution to relevant physiological compartments. Despite multiple publications describing sirolimus effects on Tcons and Tregs in preclinical and human ex vivo models, consistent parameters relating sirolimus concentrations to circulating Tcons and Tregs could not be found. Each aspect presents a challenge in building a QSP model of sirolimus and its temporal effects on T‐cell subsets and GVHD prevention. To optimize GVHD prevention regimens, phase I studies and systematic studies of immunosuppressant concentration–effect association are needed for QSP modeling.

Hepatic graft‐versus‐host disease (HGVHD) contributes significantly to morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Clinical findings and liver biomarkers are neither sensitive nor specific. The relationship between clinical and histologic diagnoses of HGVHD was assessed premortem and at autopsy. Medical records from patients who underwent HSCT at the National Institutes of Health (NIH) Clinical Center between 2000 and 2012 and expired with autopsy were reviewed, and laboratory tests within 45 days of death were divided into 15‐day periods. Clinical diagnosis of HGVHD was based on Keystone Criteria or NIH Consensus Criteria, histologic diagnosis based on bile duct injury without significant inflammation, and exclusion of other potential etiologies. We included 37 patients, 17 of whom had a cholestatic pattern of liver injury and two had a mixed pattern. Fifteen were clinically diagnosed with HGVHD, two showed HGVHD on autopsy, and 13 had histologic evidence of other processes but no HGVHD. Biopsy or clinical diagnosis of GVHD of other organs during life did not correlate with HGVHD on autopsy. The diagnostic accuracy of the current criteria was poor (κ = −0.20). A logistic regression model accounting for dynamic changes included peak bilirubin 15 days before death, and an increase from period −30 (days 30 to 16 before death) to period −15 (15 days before death) showed an area under the receiver operating characteristic curve of 0.77. Infection was the immediate cause of death in 68% of patients. In conclusion, liver biomarkers at baseline and GVHD elsewhere are poor predictors of HGVHD on autopsy, and current clinical diagnostic criteria have unsatisfactory performance. Peak bilirubin and cholestatic injury predicted HGVHD on autopsy. A predictive model was developed accounting for changes over time. Further validation is needed. Hepatic graft‐versus‐host disease (HGVHD) contributes significantly to morbidity and mortality post hematopoietic stem cell transplantation (HSCT). Liver biomarkers at baseline and GVHD elsewhere are poor predictors of HGVHD on autopsy, and current clinical diagnostic criteria have unsatisfactory performance. A predictive model was developed accounting for changes over time, and further validation is needed.