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Posttransplant cyclophosphamide (PTCy) is associated with a low incidence of chronic graft -versus-host disease (cGVHD) following hematopoietic stem cell (HSC) transplantation. Previous studies have shown the important roles of B cell immunity in cGVHD development. Here, we investigated the long-term reconstitution of B lymphopoiesis after PTCy using murine models. We first demonstrated that the immune homeostatic abnormality leading to cGVHD is characterized by an initial increase in effector T cells in the bone marrow and subsequent B and Treg cytopenia. PTCy, but not cyclosporine A or rapamycin, inhibits the initial alloreactive T cell response, which restores intra-bone marrow B lymphogenesis with a concomitant vigorous increase in Tregs. This leads to profound changes in posttransplant B cell homeostasis, including decreased B cell activating factors, increased transitional and regulatory B cells, and decreased germinal center B cells. To identify the cells responsible for PTCy-induced B cell tolerance, we selectively depleted Treg populations that were graft or HSC derived using DEREG mice. Deletion of either Treg population without PTCy resulted in critical B cytopenia. PTCy rescued B lymphopoiesis from graft-derived Treg deletion. In contrast, the negative effect of HSC-derived Treg deletion could not be overcome by PTCy, indicating that HSC-derived Tregs are essential for maintaining favorable B lymphopoiesis following PTCy. These findings define the mechanisms by which PTCy restores homeostasis of the B cell lineage and reestablishes immune tolerance.

Background: Granulocyte transfusion therapy is a rational therapeutic option for patients with prolonged, severe neutropenia. Although high molecular weight hydroxyethyl starch (hHES) facilitates the separation of red blood cells during granulocyte collection, renal dysfunction has been noted as a potential side effect. HES130/0.4 (Voluven®) is a medium molecular weight HES (mHES) with superior safety profiles compared to hHES. Although HES130/0.4 is reportedly effective in the collection of granulocytes, we lack studies comparing the efficiency of granulocyte collection using HES130/0.4 and hHES. Study Design and Methods: We retrospectively collected the data from 60 consecutive apheresis procedures performed on 40 healthy donors at the Okayama University Hospital between July 2013 and December 2021. All procedures were performed using the Spectra Optia system. Based on the HES130/0.4 concentration in the separation chamber, granulocyte collection methods using HES130/0.4 were classified into m0.46, m0.44, m0.37, and m0.8 groups. We used HES130/0.4 and hHES groups to compare the various sample collection methods. Results: The median granulocyte collection efficiency (CE) was approximately 24.0% and 28.1% in the m0.8 and hHES groups, respectively, which were significantly higher than those in the m0.46, m0.44, and m0.37 groups. One month following granulocyte collection with HES130/0.4, no significant changes were observed in serum creatinine levels compared to those before the donation. Conclusion: Therefore, we propose a granulocyte collection approach employing HES130/0.4, which is comparable to the use of hHES in terms of the granulocyte CE. A high concentration of HES130/0.4 in the separation chamber was considered crucial for granulocyte collection.

CD4(+)Foxp3(+) regulatory T cells (Tregs) play a central role in the maintenance of immune tolerance after allogeneic hematopoietic stem cell transplantation (HSCT). Tregs promptly respond to low concentrations of IL-2 through the constitutive expression of high-affinity IL-2 receptors. It has been reported that low-dose IL-2 therapy increased circulating Tregs and improved clinical symptoms of chronic GVHD. Clinical studies of IL-2 therapy so far have mainly targeted patients in the chronic phase of transplantation when acute immune responses has subsided. However, the biological and clinical effects of exogenous IL-2 in an acute immune environment have not been well investigated. In the current study, we investigated the impact of exogenous IL-2 therapy on the post-transplant homeostasis of T cell subsets which influence the balance between GVHD and GVL in the acute phase, by setting the various immune environments early after HSCT in murine model. We initially found that 5,000 IU of IL-2 was enough to induce the active proliferation of Treg without influencing other conventional T cells (Tcons) when administered to normal mice. However, activated Tcons showed the response to the same dose of IL-2 in recipients after allogeneic HSCT. In a mild inflammatory environment within a threshold, exogenous IL-2 could effectively modulate Treg homeostasis with just limited influence to activated T cells, which resulted in an efficient GVHD suppression. In contrast, in a severely inflammatory environment, exogenous IL-2 enhanced activated T cells rather than Tregs, which resulted in the exacerbation of GVHD. Of interest, in an immune-tolerant state after transplant, exogenous IL-2 triggered effector T-cells to exert an anti-tumor effect with maintaining GVHD suppression. These data suggested that the responses of Tregs and effector T cells to exogenous IL-2 differ depending on the immune environment in the host, and the mutual balance of the response to IL-2 between T-cell s

Background and Aim: Chronic graft-versus-host disease (cGVHD) is a major cause of nonrelapse morbidity and mortality following hematopoietic stem cell transplantation (HSCT). alpha-Galactosylceramide (alpha-GC) is a synthetic glycolipid that is recognized by the invariant T-cell receptor of invariant natural killer T (iNKT) cells in a CD1d-restricted manner. Stimulation of iNKT cells by alpha-GC leads to the production of not only immune-stimulatory cytokines but also immune-regulatory cytokines followed by regulatory T-cell (Treg) expansion in vivo. Methods: We investigated the effect of iNKT stimulation by liposomal alpha-GC just after transplant on the subsequent immune reconstitution and the development of sclerodermatous cGVHD. Results: Our study showed that multiple administrations of liposomal alpha-GC modulated both host- and donor-derived iNKT cell homeostasis and induced an early expansion of donor Tregs. We also demonstrated that the immune modulation of the acute phase was followed by the decreased levels of CXCL13 in plasma and follicular helper T cells in lymph nodes, which inhibited germinal center formation, resulting in the efficient prevention of sclerodermatous cGVHD. Conclusions: These data demonstrated an important coordination of T- and B-cell immunity in the pathogenesis of cGVHD and may provide a novel clinical strategy for the induction of immune tolerance after allogeneic HSCT.

Posttransplantation cyclophosphamide (PTCy) has become a popular option for haploidentical hematopoietic stem cell transplantation (HSCT). However, personalized methods to adjust immune intensity after PTCy for each patient's condition have not been well studied. Here, we investigated the effects of reducing the dose of PTCy followed by alpha -galactosylceramide (alpha -GC), a ligand of iNKT cells, on the reciprocal balance between graft-versus-host disease (GVHD) and the graft-versus-leukemia (GVL) effect. In a murine haploidentical HSCT model, insufficient GVHD prevention after reduced-dose PTCy was efficiently compensated for by multiple administrations of alpha -GC. The ligand treatment maintained the enhanced GVL effect after reduced-dose PTCy. Phenotypic analyses revealed that donor-derived B cells presented the ligand and induced preferential skewing to the NKT2 phenotype rather than the NKT1 phenotype, which was followed by the early recovery of all T cell subsets, especially CD4(+)Foxp3(+) regulatory T cells. These studies indicate that alpha -GC administration soon after reduced-dose PTCy restores GVHD-preventing activity and maintains the GVL effect, which is enhanced by reducing the dose of PTCy. Our results provide important information for the development of a novel strategy to optimize PTCy-based transplantation, particularly in patients with a potential relapse risk.