Generation and Large‐Scale Expansion of Human Inducible Regulatory T Cells That Suppress Graft‐Versus‐Host Disease

Adoptive transfer of thymus‐derived natural regulatory T cells (nTregs) effectively suppresses disease in murine models of autoimmunity and graft‐versus‐host disease (GVHD). TGFß induces Foxp3 expression and suppressive function in stimulated murine CD4+25‐ T cells, and these induced Treg (iTregs), like nTreg, suppress auto‐ and allo‐reactivity in vivo. However, while TGFß induces Foxp3 expression in stimulated human T cells, the expanded cells lack suppressor cell function. Here we show that Rapamycin (Rapa) enhances TGFß‐dependent Foxp3 expression and induces a potent suppressor function in naive (CD4+ 25–45RA+) T cells. Rapa/TGFß iTregs are anergic, express CD25 at levels higher than expanded nTregs and few cells secrete IL‐2, IFNγ or IL‐17 even after PMA and Ionomycin stimulation in vitro. Unlike other published methods of inducing Treg function, Rapa/TGFß induces suppressive function even in the presence of memory CD4+ T cells. A single apheresis unit of blood yields an average ～240 × 10⁹ (range ～70–560 × 10⁹) iTregs from CD4+25‐ T cells in ≤2 weeks of culture. Most importantly, Rapa/TGFß iTregs suppress disease in a xenogeneic model of GVHD. This study opens the door for iTreg cellular therapy for human diseases.     

Rotavirus infection following post‐transplantation cyclophosphamide based haploidentical hematopoietic cell transplantation in children is associated with hemophagocytic syndrome and high mortality

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.     

Microchimerism in labial salivary glands of hematopoietic stem cell transplanted patients

Oral Diseases (2011) 17 , 484–488 Objective: Microchimerism has been extensively investigated in autoimmune diseases, which display similarities with graft‐vs‐host disease. This study was conducted to investigate the presence of microchimerism in minor salivary glands of hematopoietic stem cell transplanted patients, one of the targets of graft‐vs‐host disease. Methods: Labial salivary glands biopsy specimens from 11 stem cell transplanted patients were analysed. The samples were grouped in control (five specimens from a female‐to‐female transplantation) and study group (five glands from male‐to‐female transplantation). One male transplanted patient was used as a positive control. Fluorescence in situ hybridization with Y‐chromosome probe and immunofluorescence with anticytokeratin AE1/AE3 and CD45 were used to identify Y‐chromosome positive glandular epithelial cells from allogeneic hematopoietic stem cell transplanted patients. Results: In the study group, all samples were positive to Y‐chromosome and cytokeratin AE1/AE3, in agreement with the pattern exhibited by male labial salivary gland. None of the samples from control group were positive to Y‐chromosome despite being positive to cytokeratin AE1/AE3. Positivity to CD45 was not relevant. Conclusion: Microchimerism in the labial salivary glands of sex‐mismatched stem cell transplanted patients is a real phenomenon. Further studies are necessary to elucidate the impact of this phenomenon on the clinical status of stem cell transplanted patients.     

Contribution of B7RP‐1/ICOS co‐stimulation to lethal acute GVHD

Fujimura J, Takeda K, Kaduka Y, Saito M, Akiba H, Yagita H, Yamashiro Y, Shimizu T, Okumura K. Contribution of B7RP‐1/ICOS co‐stimulation to lethal acute GVHD.
Pediatr Transplantation 2010: 14:540–548. © 2010 John Wiley & Sons A/S. Abstract: Co‐stimulatory molecules expressed on T cells critically regulate donor T‐cell activation and are implicated in acute GVHD after allogeneic BMT. We here investigated the role of interaction between B7‐related protein‐1 (B7RP‐1) and ICOS in murine acute GVHD model that received T cell‐depleted BM cells and splenocytes. Administration of blocking anti‐B7RP‐1 mAb significantly reduced the lethality and symptoms in acute GVHD. A significant hypo‐responsiveness of splenocytes to host alloantigen was observed in the recipient mice treated with anti‐B7RP‐1 mAb. Moreover, acute GVHD was significantly reduced in the recipients of T cells composed of ICOS‐deficient CD8 T cells and WT CD4 T cells compared with that in the recipients of T cells composed of WT CD8 T cells and ICOS‐deficient CD4 T cells. These results suggested that B7RP‐1/ICOS co‐stimulatory signal plays a role in the activation of alloantigen‐reactive donor T cells, particularly in CD8 T cells, in murine acute GVHD model, and that the blockade of B7RP‐1/ICOS interaction may be useful for selectively manipulating allo‐reactive T cells in the recipients with acute GVHD.     

Recipient NOD2/CARD15 status affects cellular infiltrates in human intestinal graft‐versus‐host disease

Nucleotide‐binding oligomerization domain 2/caspase recruitment domain 15 (NOD2/CARD15) polymorphisms have been identified as risk factors of both Crohn's disease and graft‐versus‐host disease (GVHD) following allogeneic stem cell transplantation. However, the role of these receptors of innate immunity in the pathophysiology of gastrointestinal GVHD is still poorly defined. Immunohistological features of intestinal GVHD were analysed in gastrointestinal biopsies from 58 patients obtained at the time of first onset of intestinal symptoms. The observed changes were correlated with concomitant risk factors and the presence of polymorphisms within the pathogen recognition receptor gene NOD2/CARD15. Intestinal GVHD was associated with a stage‐dependent decrease in CD4 T cell infiltrates and an increase in CD8 T cells in the lamina propria; CD8 infiltrates correlated with extent of apoptosis and consecutive epithelial proliferation. The presence of NOD2/CARD15 variants in the recipient was associated with a significant loss of CD4 T cells: in a semiquantitative analysis, the median CD4 score for patients with wild‐type NOD2/CARD15 was 1·1 (range 3), but only 0·4 (range 2) for patients with variants (P = 0·002). This observation was independent from severity of GVHD in multivariate analyses and could not be explained by the loss of forkhead box P3⁺ T cells. Our results suggest a loss of protective CD4 T cells in intestinal GVHD which is enhanced further by the presence of NOD2/CARD15 variants. Our study might help to identify more selective therapeutic strategies in the future.     

Modified Donor Lymphocyte Infusion after HLA-Mismatched/Haploidentical T Cell-replete Hematopoietic Stem Cell Transplantation for Prophylaxis of Relapse of Leukemia in Patients with Advanced Leukemia

We evaluated the safety and efficacy of donor lymphocyte infusion (DLI) with granulocyte colony-stimulating factor priming and short-term immunosuppressive agents for prophylaxis of relapse in patients with advanced leukemia after human leukocyte antigen (HLA)-mismatched T cell-replete hematopoietic stem cell transplantation (HCT). Twenty-nine patients received prophylactic DLI at a median 75 (33–120) days after HCT. Acute graft-vs-host disease (GVHD) grades 3–4 occurred in six patients, and all cases were controlled. Eleven patients were alive and relapse-free with a probability of leukemia-free survival (LFS) of 37.3 ± 9.6% at 3 years. Chronic GVHD was associated with a lower relapse rate and higher probability of LFS. Prophylactic-modified DLI is feasible in patients with advanced leukemia to prevent relapse after HLA-mismatched HCT. Xiao-Jun Huang: involved in conception and design, revising the article critically, and final approval of the version to be published; Dai-Hong Liu: performed research, analysis, and interpretation of data and drafting of the article and gave final approval of the version to be published; the other authors: performed research and gave final approval of the version to be published; the authors reported no potential conflicts of interest.     

Sirolimus demonstrates activity in the primary therapy of acute graft-versus-host disease without systemic glucocorticoids

Background

Advances in acute graft-versus-host disease therapy are needed.

Design and Methods

We examined the efficacy of sirolimus as primary therapy for acute graft-versus-host disease in 32 patients.

Results

Acute graft-versus-host disease involved the skin in 53% of cases, gastrointestinal tract in 66%, liver in 16%. The syndrome was overall grade 1 in 12% cases, grade 2 in 75%, and grade 3 in 13%. Sirolimus was targeted to achieve serum trough levels of 5–14 ng/mL. Sixteen (50%) patients achieved sustained, complete resolution of acute graft-versus-host disease with sirolimus alone. In contrast, 19 of 32 (59%) matched historical controls treated with standard 1 mg/kg steroids achieved complete response (P=0.47). With median follow-up time for surviving patients of 16 (range 6–26) months, one year overall survival was 56% (95% CI 38–74%). The cumulative incidence of relapse at one year was 37% (95% CI 23–60%), and mortality in remission was 20% (95% CI 10–42%). The cumulative incidence of chronic graft-versus-host disease was 55% (95% CI 39–79%). Thrombotic microangiopathy occurred in 3 cases (grade 1 n=1; grade 2 n=2), and responded to dose reduction of calcineurin inhibitor.

Conclusions

In this retrospective series, sirolimus demonstrates activity comparable to that of high-dose glucocorticoids in the primary therapy of acute graft-versus-host disease. Confirmation of this activity requires prospective clinical trials.     

Acute GVHD: New approaches to clinical trial monitoring

Acute GVHD occurs in nearly 50% of patients receiving hematopoietic cell transplantation (HCT), and is the major driver of mortality. However, progress in the development of new acute GVHD therapeutics has been slow, in part due to heterogeneity in acute GVHD data collection and interpretation among centers. Herein, we first describe the methods used by the Mount Sinai Acute GVHD International Consortium (MAGIC) to standardize acute GVHD data collection and curation. We then review the utility of serum biomarkers, specifically the MAGIC Algorithm Probability (MAP) that combines two GI biomarkers (ST2 and REG3α) that has been shown to be more accurate than changes in clinical symptom severity after GVHD treatment. We then present preliminary data on the feasibility of a surrogate clinical trial endpoint that combines clinical response and MAP two weeks after treatment. This novel endpoint is an earlier and potentially better predictor of non-relapse mortality than the current gold standard of clinical response four weeks after treatment.     

Administration of Anti-CD20 mAb Is Highly Effective in Preventing but Ineffective in Treating Chronic Graft-Versus-Host Disease While Preserving Strong Graft-Versus-Leukemia Effects

Chronic graft-versus-host disease (cGVHD) is an autoimmune-like syndrome, and donor B cells play important roles in augmenting its pathogenesis. B cell–depleting anti-CD20 mAb has been administered before or after cGVHD onset for preventing or treating cGVHD in the clinic. Although administration before onset appeared to be more effective, the effect is variable and sometimes minimal. Here, we used 2 mouse cGVHD models to evaluate the preventive and therapeutic effect of anti-CD20 mAb. With the model of DBA/2 donor to MHC-matched BALB/c recipient, 1 intravenous injection of anti-CD20 mAb (40 mg/kg) the following day or on day 7 after hematopoietic cell transplantation when serum autoantibodies were undetectable effectively prevented induction of cGVHD and preserved a strong graft-versus-leukemia (GVL) effect. The separation of GVL effect from GVHD was associated with a significant reduction of donor CD4⁺ T cell proliferation and expansion and protection of host thymic medullary epithelial cells. Anti-CD20 mAb administration also prevented expansion of donor T cells and induction of cGVHD in another mouse model of C57BL/6 donor to MHC-mismatched BALB/c recipients. In contrast, administration of anti-CD20 mAb after GVHD onset was not able to effectively deplete donor B cells or ameliorate cGVHD in either model. These results indicate that administration of anti-CD20 mAb before signs of cGVHD can prevent induction of autoimmune-like cGVHD while preserving a GVL effect; there is little effect if administered after cGVHD onset. This provides new insights into clinical prevention and therapy of cGVHD with B cell–depleting reagents.