A randomized phase II study to evaluate tacrolimus in combination with sirolimus or methotrexate after allogeneic hematopoietic cell transplantation

Background

There is evidence suggesting that sirolimus, in combination with tacrolimus, is active in the prevention of graft-versus-host disease. Sirolimus-based immune suppression may suppress alloreactive T cells, while sparing the survival and function of regulatory T cells.

Design and Methods

We conducted a randomized trial to compare the impact of sirolimus/tacrolimus against that of methotrexate/tacrolimus on the prevention of graft-versus-host disease and regulatory T-cell reconstitution.

Results

Seventy-four patients were randomized 1:1 to sirolimus/tacrolimus or methotrexate/tacrolimus, stratified for type of donor (sibling or unrelated) and the patients'' age. The rate of grade II-IV acute graft-versus-host disease at 100 days was 43% (95% CI: 27-59%) in the sirolimus/tacrolimus group and 89% (95% CI: 72-96%) in the methotrexate/tacrolimus group (P<0.001). The rate of moderate/severe chronic graft-versus-host disease was 24% (95% CI: 7-47%) in the sirolimus/tacrolimus group and 64% (95% CI: 41-79%) in the methotrexate/tacrolimus group (P=0.008). Overall survival and patient-reported quality of life did not differ between the two groups. On days 30 and 90 post-transplant, sirolimus-treated patients had a significantly greater proportion of regulatory T cells among the CD4⁺ cells in the peripheral blood, and isolated regulatory T cells were functional.

Conclusions

These data demonstrate that sirolimus/tacrolimus prevents grade II-IV acute graft-versus-host disease and moderate-severe chronic graft-versus-host disease more effectively than does methotrexate/tacrolimus, and supports regulatory T-cell reconstitution following allogeneic hematopoietic cell transplantation. Trial registration: ({"type":"clinical-trial","attrs":{"text":"NCT00803010","term_id":"NCT00803010"}}NCT00803010)Key words: tacrolimus, sirolimus, methotrexate, combination therapy, GVHD prophylaxis     

Cloning and characterization of high mobility group box protein 1 (HMGB1) of Wuchereria bancrofti and Brugia malayi

A human homologue of high mobility group box 1 (HMGB1) protein was cloned and characterized from the human filarial parasites Wuchereria bancrofti and Brugia malayi. Sequence analysis showed that W. bancrofti HMGB1 (WbHMGB1) and B. malayi HMGB1 (BmHMGB1) proteins share 99 % sequence identity. Filarial HMGB1 showed typical architectural sequence characteristics of HMGB family of proteins and consisted of only a single HMG box domain that had significant sequence similarity to the pro-inflammatory B box domain of human HMGB1. When incubated with mouse peritoneal macrophages and human promyelocytic leukemia cells, rBmHMGB1 induced secretion of significant levels of pro-inflammatory cytokines such as TNF-α, GM-CSF, and IL-6. Functional analysis also showed that the filarial HMGB1 proteins can bind to supercoiled DNA similar to other HMG family of proteins. BmHMGB1 protein is expressed in the adult and microfilarial stages of the parasite and is found in the excretory secretions of the live parasites. These findings suggest that filarial HMGB1 may have a significant role in lymphatic pathology associated with lymphatic filariasis.     

A novel small heat shock protein 12.6 (HSP12.6) from Brugia malayi functions as a human IL-10 receptor binding protein

Phage display cDNA expression library of the third stage larvae (L3) of Brugia malayi was screened for identifying target(s) that bound to the human interleukin-10 receptor (huIL10R). This iterative screening identified an insert that showed significant homology to Caenorhabditis elegans HSP12.6. The gene was designated B. malayi HSP12.6 (BmHSP12.6) and has orthologues in several gastrointestinal nematode genome (Ancylostoma caninum, Ascaris lumbricoides and Ascaris suum) but the gene or gene product has not been studied further in these parasites. Structural analyses of BmHSP12.6 showed that it has a highly conserved alpha-crystallin central domain that is characteristic of other small heat shock proteins (HSPs). BmHSP12.6 has a short N-terminal domain and an unusually small C-terminal domain flanking the crystallin domain suggesting that this protein belongs to a novel class of small HSPs. BmHSP12.6 appears to be differentially transcribed with highest expression in the vertebrate stages of the parasite (L4, adult and mf) compared to its mosquito vector stage (L3). More importantly recombinant BmHSP12.6 bound to huIL10R in a dose dependent fashion and inhibited the binding of human IL-10 (huIL10) to huIL10R in vitro. rBmHSP12.6 also enhanced the growth and proliferation of MC/9 mast cells in vitro similar to huIL10. This study thus describes a novel small HSP from B. malayi that has the capacity to bind to huIL10R, block binding of huIL10 to huIL10R and function similar to huIL10.     

Ex vivo expansion of human Tregs specific for alloantigens presented directly or indirectly

Adoptive transfer of regulatory T cells (Tregs) prevents GVHD in experimental animals. Because antigen activation drives Treg function, we measured the frequency, growth requirements, and function of alloantigen-specific (allospecific) Tregs from human blood. When alloantigen was presented directly, the precursor frequency of allo-specific Tregs in normal individuals was 1.02% (95% confidence interval [95% CI]: 0.65-1.59) and non-Tregs 1.56% (95% CI: 0.94-2.55). When alloantigen was presented indirectly, the frequency of specific Tregs was approximately 100-fold less. Purified Tregs were expanded with APCs, rapamycin, IL-2, and IL-15. In 12 days, allo-specific Tregs expanded 793-fold (95% CI: 480-1107), with duplication approximately every 24 hours. Purified allo-specific Tregs suppressed responses to specific alloantigen selectively and were approximately 100-fold more potent than polyspecific Tregs and nonexpanded Tregs. Allo-specific Tregs maintained high expression of Foxp3, Bcl-2, lymphoid homing receptor CD62L, and chemokine receptor CCR7, predicting sustained function and migration to lymphoid tissues in vivo. Allo-specific Tregs produced TGF-β and IL-10 and expressed more cytoplasmic CTLA-4 compared with non-Tregs. These data provide a platform for the selective expansion of Tregs against major and possibly minor histocompatibility antigens and predict the feasibility of adoptive immunotherapy trials using Tregs with indirect allo-recognition for preventing GVHD while sparing GVL effects.