Impact of animal strain on gene expression in a rat model of acute cardiac rejection

<p>Abstract</p> <p>Background</p> <p>The expression levels of many genes show wide natural variation among strains or populations. This study investigated the potential for animal strain-related genotypic differences to confound gene expression profiles in acute cellular rejection (ACR). Using a rat heart transplant model and 2 different rat strains (Dark Agouti, and Brown Norway), microarrays were performed on native hearts, transplanted hearts, and peripheral blood mononuclear cells (PBMC).</p> <p>Results</p> <p>In heart tissue, strain alone affected the expression of only 33 probesets while rejection affected the expression of 1368 probesets (FDR 10% and FC ≥ 3). Only 13 genes were affected by both strain and rejection, which was < 1% (13/1368) of all probesets differentially expressed in ACR. However, for PBMC, strain alone affected 265 probesets (FDR 10% and FC ≥ 3) and the addition of ACR had little further effect. Pathway analysis of these differentially expressed strain effect genes connected them with immune response, cell motility and cell death, functional themes that overlap with those related to ACR. After accounting for animal strain, additional analysis identified 30 PBMC candidate genes potentially associated with ACR.</p> <p>Conclusion</p> <p>In ACR, genetic background has a large impact on the transcriptome of immune cells, but not heart tissue. Gene expression studies of ACR should avoid study designs that require cross strain comparisons between leukocytes.</p

Host-Based Th2 Cell Therapy for Prolongation of Cardiac Allograft Viability

Donor T cell transfusion, which is a long-standing approach to prevent allograft rejection, operates indirectly by alteration of host T cell immunity. We therefore hypothesized that adoptive transfer of immune regulatory host Th2 cells would represent a novel intervention to enhance cardiac allograft survival. Using a well-described rat cardiac transplant model, we first developed a method for ex vivo manufacture of rat host-type Th2 cells in rapamycin, with subsequent injection of such Th2.R cells prior to class I and class II disparate cardiac allografting. Second, we determined whether Th2.R cell transfer polarized host immunity towards a Th2 phenotype. And third, we evaluated whether Th2.R cell therapy prolonged allograft viability when used alone or in combination with a short-course of cyclosporine (CSA) therapy. We found that host-type Th2.R cell therapy prior to cardiac allografting: (1) reduced the frequency of activated T cells in secondary lymphoid organs; (2) shifted post-transplant cytokines towards a Th2 phenotype; and (3) prolonged allograft viability when used in combination with short-course CSA therapy. These results provide further support for the rationale to use “direct” host T cell therapy for prolongation of allograft viability as an alternative to “indirect” therapy mediated by donor T cell infusion

Th2.R cell therapy plus cyclosporine prolongs cardiac allograft survival.

<p>Allograft recipients were monitored for cardiac viability through the 28-day post-transplant period of observation. Rejection and engraftment control cohorts received a 10-day short-course of cyclosporine A [“CSA(10]” or a daily 28-day course of “CSA [CSA(28)”]. Other cohorts received either no cyclosporine A or short-course CSA(10) in combination with Th2.R cell therapy at day −7 [“Th2.R(D-7)” or “CSA(10)+Th2.R(D-7)”], day 0 [“Th2.R(D0)” or “CSA(10)+Th2.R(D0)”], or day −7 plus day 0 [“Th2.R(D-7+D0)” or “CSA(10)+Th2.R(D-7+DO)”]. (a) Hematoxylin and eosin staining was performed at day 28 post-transplant. Left panels show a representative example of severe allograft rejection in recipients of short-course CSA(10) alone (characterized by diffuse inflammation and necrosis); right panels show a representative example of relatively preserved myocardial cell structure and reduced mononuclear cell infiltration in recipients of short-course CSA(10) therapy plus Th2.R cell therapy. (b) Cumulative histology score between various cohorts is shown. I = isograft, A = Allograft; IHC score = Immunohistochemistry score.</p

Th2.R cell therapy plus cyclosporine reduces host T cell activation, induces a host Th2 phenotype, and reduces intra-cardiac allospecific T cells.

<p>Rat allogeneic cardiac transplants were performed and assigned to one of eight treatment cohorts, including; 28-day daily cyclosporine A therapy [“CSA(28)”; engraftment control]; Th2.R adoptive cell therapy alone (“Th2.R”) at day 0 (D0), day-7 (D-7) or both (D-7+D0); 10-day, short-course CSA therapy alone [“CSA(10);” experimental control]; or a combination of short-course CSA plus Th2.R cell therapy [“CSA(10)+Th2.R”] at day 0 (D0), day-7 (D-7) or both (D-7+D0). (a) At day 28 post-transplant, transplant recipients were euthanized and splenocytes were harvested. The frequency of activated CD4⁺ and CD8⁺ T cells in each cohort at each organ site was then determined by flow cytometry (percent of CD4⁺ or CD8⁺ T cells that co-expressed CD25 in the absence of Foxp3 expression; results are mean ± SEM of 5 or 7 evaluated per cohort; *, indicates p<0.05). Splenocytes were harvested and subjected to either co-stimulation (b & c) or syngeneic and allogeneic APC stimulation (d & e; allospecific cytokine secretion is shown); resultant 24 h supernatants were then tested for cytokine content by Multiplex assay. (f) The frequency of activated CD4⁺ and CD8⁺ T cells in harvested cardiac tissue was determined by flow cytometry (percent of CD4⁺ or CD8⁺ T cells that co-expressed CD25 in the absence of Foxp3 expression; results are mean ± SEM of 7 evaluated per cohort; *, indicates p<0.05). (g) Intracardiac T cells were subjected to stimulation with allogeneic dendritic cells; resultant 24 h supernatants were then tested for cytokine content by Multiplex assay. (h) Survival of cardiac allografts between various cohorts is shown.</p

Evaluation of Th2.R cell timing and rapamycin co-administration.

<p>Host-type BN rats received an intravenous infusion of isogeneic Th2.R cells either at day −7 or day −14 prior to host euthanasia at “day 0”, with subsequent immune evaluation; Th2 cell therapy was administered either alone or in combination with in vivo rapamycin therapy. (a) Harvested spleen, inguinal lymph node, and mesenteric lymph node cells were harvested and subjected to co-stimulation; resultant 24 h supernatants were then tested for cytokine content by Multiplex assay. (b) % Foxp3 expression was measured using IC flow on splenocytes at day7 (c) After co-stimulation, CD4⁺ and CD8⁺ T cells from recipients in each cohort were evaluated by intra-cellular flow cytometry for IFN-γ and IL-4 production. * indicates P<0.05; n = 5 per cohort.</p

Characterization of rat Th2 cells.

<p>CD4⁺ T cells were isolated and expanded using anti-CD3 and anti-CD28 co-stimulation in the presence of rhIL-2, rrIL-4, and rhIL-7 either with rapamycin (“Th2.R”) or without rapamycin (“Th2”) for 3 days. (a) Expanded Th2 and Th2.R cells were subjected to repeat co-stimulation, and the 24 h supernatant was tested for cytokine content using multiplex bead array. (b) After repeat co-stimulation, Th2 and Th2.R cells were evaluated for intra-cellular expression of IFN-γ and IL-4 by flow cytometry. (c) On day 3, cells were harvested and intra-cellular flow cytometry was performed to evaluate Foxp3 transcription factor expression. Results are a summary of n = 10 cultures.</p

Host Th2.R cell infusion prolongs cardiac allograft survival.

<p>Rat allogeneic cardiac transplants was performed and assigned to one of five treatment cohorts, including: no drug and no cell therapy (“rejection control”); 28-day daily cyclosporine A therapy [“CSA(28);” engraftment control]; Th2.R adoptive cell therapy alone on the day of transplant [“Th2.R(d0)”]; 18-day, short-course CSA therapy alone [“CSA(18)”; experimental control]; or a combination of short-course CSA plus Th2.R cell therapy [“CSA(18)+Th2.R(d0)”]. (a) At day 28 post-transplant, recipients were euthanized and spleen, inguinal lymph nodes, and mesenteric lymph nodes were harvested. The frequency of activated CD4⁺ and CD8⁺ T cells in each cohort at each organ site was then determined by flow cytometry (percent of CD4⁺ or CD8⁺ T cells that co-expressed CD25 in the absence of Foxp3 expression; results are mean ± SEM of 3 evaluated per cohort; *, indicates p≤0.05; **, indicates p≤0.005). Cells harvested at day 28 post-transplant were co-stimulated (b) or allo-stimulated (c & d), and the 24 h supernatants were tested for cytokine content by multiplex assay.</p