Granzyme A Required for Regulatory T-Cell Mediated Prevention of Gastrointestinal Graft-versus-Host Disease

In our previous work we could identify defects in human regulatory T cells (Tregs) likely favoring the development of graft-versus-host disease (GvHD) following allogeneic stem cell transplantation (SCT). Treg transcriptome analyses comparing GvHD and immune tolerant patients uncovered regulated gene transcripts highly relevant for Treg cell function. Moreover, granzyme A (GZMA) also showed a significant lower expression at the protein level in Tregs of GvHD patients. GZMA induces cytolysis in a perforin-dependent, FAS- FASL independent manner and represents a cell-contact dependent mechanism for Tregs to control immune responses. We therefore analyzed the functional role of GZMA in a murine standard model for GvHD. For this purpose, adoptively transferred CD4+CD25+ Tregs from gzmA-/- mice were analyzed in comparison to their wild type counterparts for their capability to prevent murine GvHD. GzmA-/- Tregs home efficiently to secondary lymphoid organs and do not show phenotypic alterations with respect to activation and migration properties to inflammatory sites. Whereas gzmA-/- Tregs are highly suppressive in vitro, Tregs require GZMA to rescue hosts from murine GvHD, especially regarding gastrointestinal target organ damage. We herewith identify GZMA as critical effector molecule of human Treg function for gastrointestinal immune response in an experimental GvHD model

Granzyme A Is Required for Regulatory T-Cell Mediated Prevention of Gastrointestinal Graft-versus-Host Disease

<div><p>In our previous work we could identify defects in human regulatory T cells (Tregs) likely favoring the development of graft-versus-host disease (GvHD) following allogeneic stem cell transplantation (SCT). Treg transcriptome analyses comparing GvHD and immune tolerant patients uncovered regulated gene transcripts highly relevant for Treg cell function. Moreover, granzyme A (GZMA) also showed a significant lower expression at the protein level in Tregs of GvHD patients. GZMA induces cytolysis in a perforin-dependent, FAS-FASL independent manner and represents a cell-contact dependent mechanism for Tregs to control immune responses. We therefore analyzed the functional role of GZMA in a murine standard model for GvHD. For this purpose, adoptively transferred CD4⁺CD25⁺ Tregs from <i>gzmA</i>^-/- mice were analyzed in comparison to their wild type counterparts for their capability to prevent murine GvHD. <i>GzmA</i>^-/- Tregs home efficiently to secondary lymphoid organs and do not show phenotypic alterations with respect to activation and migration properties to inflammatory sites. Whereas <i>gzmA</i>^-/- Tregs are highly suppressive <i>in vitro</i>, Tregs require GZMA to rescue hosts from murine GvHD, especially regarding gastrointestinal target organ damage. We herewith identify GZMA as critical effector molecule of human Treg function for gastrointestinal immune response in an experimental GvHD model.</p></div

<i>GzmA</i>^<i>-/-</i> Tregs show no phenotypic alteration with respect to activation and migration.

<p>WT Tregs (white bars) and <i>gzmA</i>^<i>-/-</i> Tregs (grey bars) were isolated from spleen (A) and peripheral lymph nodes (B) and analyzed by FACS. Expression values were calculated as a percentage of the CD4⁺ T lymphocyte population and mean values are presented as bar graphs. Students t-test did not reveal any significant differences between WT and <i>gzmA</i>^<i>-/-</i> Tregs. Results are shown for n = 3 WT and <i>gzmA</i>^<i>-/-</i> mice each.</p

<i>GzmA</i>^<i>-/-</i> Tregs are required for prevention of gastrointestinal GvHD.

<p>Lethally irradiated BALB/c mice (n = 9) received T cell depleted (TCD) bone marrow cells (BM) together with CD4⁺CD25^- T effector cells (squares, n = 3) and CD4⁺CD25⁺ WT Treg cells (circle, n = 3) or CD4⁺CD25⁺<i>gzmA</i>^<i>-/-</i> Treg cells (triangle, n = 3) from WT mice. (A) Body weight of the recipients was monitored daily. (B) Histopathological investigations of liver, small and large intestine of mice sacrified after 3 weeks. (C) Histopathological GvHD scoring 3 weeks after transplantation were graded for severity of inflammation and degree of organ destruction resulting in an organ-specific cumulative pathology score. For statistical analysis student`s t-test was performed and p values <0.05 (*) were considered significant, while p<0.01 (**) and p<0.001 (**) were considered highly significant.</p

<i>GzmA</i>^<i>-/-</i> Tregs home efficiently to secondary lymphoid organs.

<p>CD4⁺ (A) and CD4⁺CD25⁺ Tregs (B) were isolated from spleen and peripheral lymph node from wildtype (WT; n = 3) and <i>gzmA</i>^<i>-/-</i> mice (n = 3) and analyzed by FACS. Expression values were calculated as a percentage of the T lymphocyte population and mean values are presented as bar graphs. Students t-test did not reveal any significant differences between WT and <i>gzmA</i>^<i>-/-</i> Tregs.</p

Human Regulatory T Cells of G-CSF Mobilized Allogeneic Stem Cell Donors Qualify for Clinical Application

<div><p>Recent clinical studies demonstrate the high potency of regulatory T cells (Tregs) to control graft-versus-host disease in hematopoietic stem cell transplantation (SCT). However, the adoptive transfer of Tregs is limited by their low frequency in unstimulated donors and considerable concerns that G-CSF induced SC mobilization might have negative effects on the stability and function of Tregs. The isolation of Tregs from the G-CSF mobilized SC grafts would extend this novel strategy for tolerance induction to the unrelated setting and simplify global clinical application. We characterized CD4⁺CD25^highCD127⁻ Tregs from SC donors before and after G-CSF mobilization for their phenotype, function, and stability. After G-CSF application the Treg cell yield increased significantly. Donor Tregs retained their cytokine profile, phenotypic characteristics and <em>in vitro</em> expansion capacity after SC mobilization. Most importantly, <em>in vivo</em> G-CSF stimulated Tregs remained highly suppressive on the proliferation of effector T cells, also after <em>in vitro</em> expansion, and displayed a stable phenotype in epigenetic studies. The surface expression of CXCR3 is transiently reduced. However, donor-derived Tregs maintain their migratory properties after G-CSF stimulation. Therefore, the adoptive transfer of Tregs from G-CSF mobilized SC donors seems to be a feasible and safe strategy for clinical application in allogeneic SCT.</p> </div

Phenotypic analysis of <i>in vitro</i> expanded donor Tregs before and after SC mobilization.

<p>PBMCs were isolated before (n = 25) and after (n = 21) G-CSF mobilization. FACS isolated CD4⁺CD25^highCD127⁻Foxp3⁺ donor Tregs were expanded <i>ex vivo</i> for 14 days. FACS analysis was performed before (white bars) and after (grey bars) SC mobilization as well as after <i>in vitro</i> expansion of donor Tregs isolated before (white with black stripes) and after (grey with white stripes) SC mobilization. Expression values were calculated as percentage of CD4⁺CD25^highCD127⁻Foxp3⁺ Tregs and their mean values are presented as bar graphs with SD as error bars.</p

Donor Tregs maintain their migratory potency after SC mobilization.

<p>Freshly isolated PBMCs were cultured overnight in cell culture media. After 12 hours of <i>ex vivo</i> culture, PBMCs were reanalyzed by FACS for CXCR3 surface protein expression (A) and F-actin accumulation after challenging with the corresponding chemokine ligand IP10 (B). CXCR3 expression is shown as percentage of CD4⁺CD25^highCD127⁻Foxp3⁺ regulatory T cells analyzed before (black triangles) and after (white triangles) G-CSF <i>in vivo</i> mobilization as well as after overnight culture (grey triangles). Black lines indicate mean values (A).The phalloidin binding to F-actin following 30 seconds stimulation with 300 ng IP10 is shown as fold change of Phalloidin detection and presented as box whisker plots.</p