Unknown,Transcriptomics,Genomics,Proteomics

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Modification of T cell responses by stem cell mobilization requires direct signalling of the T cell by G-CSF and IL-10

ABSTRACT: The majority of allogeneic stem cell transplants are currently undertaken using G-CSF mobilized peripheral blood stem cells. G-CSF has diverse biological effects on a broad range of cells and IL-10 is a key regulator of many of these effects. Using mixed radiation chimeras in which the haematopoietic or non-haematopoietic compartments were wild-type (WT), IL-10–/–, G-CSFR–/– or combinations thereof we demonstrated that the attenuation of alloreactive T cell responses after with G-CSF mobilization required direct signalling of the T cell by both G-CSF and IL-10. IL-10 was generated principally by radio-resistant tissue, and was not required to be produced by T cells. G-CSF mobilization significantly modulated the transcription profile of CD4+CD25+ regulatory T cells, promoted their expansion in the donor and recipient and their depletion significantly increased graft-versus-host disease (GVHD). In contrast, stem cell mobilization with the CXCR4 antagonist AMD3100 did not alter the donor T cell’s ability to induce acute GVHD. These studies provide an explanation for the effects of G-CSF on T cell function and demonstrate that IL-10 is required to license regulatory function but T cell production of IL-10 is not itself required for the attenuation GVHD. Although administration of CXCR4 antagonists is an efficient means of stem cell mobilization, this fails to evoke the immunomodulatory effects seen during G-CSF mobilization. These data provide a compelling rationale for considering the immunological benefits of G-CSF in selecting mobilization protocols for allogeneic stem cell transplantation. Single colour, Illumina MouseRef-8 v2.0 Beadarrays.

ORGANISM(S): Mus musculus

SUBMITTER: Glen Boyle

PROVIDER: E-GEOD-54616 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Regulatory T (Treg) cells play an important role in the induction and maintenance of peripheral tolerance. Treg cells also suppress a variety of other immune responses, including anti-tumor and alloimmune responses. We have previously reported that tumor-activated Treg cells express granzyme B and that granzyme B is important for Treg cell-mediated suppression of anti-tumor immune responses (GSE13409). Here, we report that allogeneic mismatch also induces the expression of granzyme B. Granzyme B-deficient mice challenged with fully mismatched allogeneic P815 mastocytoma cells have markedly improved survival compared to WT and other granzyme- or perforin-deficient mice, suggesting an immunoregulatory role for granzyme B in this setting. Treg cells harvested from the tumor environment of P815-challenged mice express granzyme B. Treg cells also express granzyme B in vitro during mixed lymphocyte reactions and in vivo in a mouse model of graft-versus-host disease (GVHD). However, in contrast to findings from our previously published tumor model, granzyme B is not required for the suppression of effector T cell (Teff) proliferation in in vitro Treg suppression assays stimulated by either Concanavalin A or allogeneic antigen presenting cells. Additionally, in an ex vivo assay, sort-purified in vivo-activated CD4+Foxp3+ Treg cells from mice with active GVHD -- under conditions known to induce granzyme B expression in Treg cells -- suppressed Teff cell proliferation in a granzyme B-independent manner. Adoptive transfer of naive granzyme B-deficient CD4+CD25+ Treg cells into a mouse model of GVHD rescued hosts from lethatlity equivalently to naive wild-type Treg cells. Serum analysis of GVHD-associated cytokine production in these recipients also demonstrated that Treg cells suppressed production of IL-2, IL-4, IL-5, GM-CSF, and IFN-gamma in a granzyme B-independent manner. In order to determine whether the context in which Treg cells are activated alters the intrinsic properties of Treg cells, we used Foxp3 reporter mice to obtain gene expression profiles of CD4+Foxp3+ Treg cells purifed from naive resting spleens, spleens from mice with acute GVHD, and from ascites fluid of mice challenged intraperitoneally with allogeneic P815 tumor cells. Unsupervised analyses revealed distinct activation signatures of Treg cells among the 3 experimental groups. Taken together, these findings demonstrate that granzyme B is not required for Treg cell-mediated suppression of GVHD, which is in contrast to what we have previously reported for Treg cell function in the setting of tumor challenge. Cell intrinsic differences could partially account for these differential phenotypes. These data also suggest the therapeutic potential of targeting specific Treg cell suppressive functions in order to segregate GVHD and graft-versus-tumor effector functions. Experiment Overall Design: Six replicates of Naive CD4+Foxp3+ Treg cells were purified from resting spleens, five replicates of allogeneic tumor-activated Treg cells and three samples of GVHD-activated Treg cells. Experiment Overall Design: Naive reps 1-3 are controls for the GVHD-activated samples. Experiment Overall Design: Naive reps 4-6 are controls for the Allogeneic tumor-activated samples.

Project description:Granulocyte-colony stimulating factor (G-CSF) is used to boost granulocyte counts in immunocompromised patients, but its effects on the immune system may be counter productive. We tested the hypothesis that G-CSF mobilized peripheral blood stem cell (PBSC) products are immunologically down regulated based on gene microarray analysis. Ten peripheral blood samples from normal donors for allogeneic PBSC transplantation were obtained before and after administration of G-CSF and tested on Affymetrix Human U133 Plus 2.0 GeneChipÂ® microarrays. Significant changes in gene expression after G-CSF mobilization were reported by controlling the false discovery rate at 5%. Immune-related genes were isolated from the data set and categorized according to probe set annotations and a thorough, independent literature search. We found that G-CSF up-regulated inflammatory and neutrophil activation pathway gene expression; however, adaptive immune-related gene expression, such as antigen presentation, co-stimulation, T cell activation and cytolytic effector pathways, were generally down-regulated. Thus, despite significant increases in stem cells, lymphocytes and antigen presenting cells, G-CSF mobilized PBSC allografts exhibit a suppressive adaptive immune-related gene expression profile. Our data provides an explanation for the potentially immunosuppressive effects observed after G-CSF administration. Experiment Overall Design: This study was approved by the University of Florida Institutional Review Board. Five healthy donors (A-E) for allogeneic PBSC transplantation consented and were mobilized with rHu G-CSF at 10Âµ/kg/day for five days. Approximately 4mL of venous blood was collected before (Pre-G-CSF) and after (Post-G-CSF) mobilization. Whole blood leukocyte RNA was purified from each sample and used to generate cRNAs which were subsequently hybridized onto Affymetrix Human U133 Plus 2.0 GeneChipÂ® microarrays. Representative genes were successfully validated with quantitative RT-PCR.

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Modification of T cell responses by stem cell mobilization requires direct signalling of the T cell by G-CSF and IL-10

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