Kras-Deficient T Cells Attenuate Graft-versus-Host Disease but Retain Graft-versus-Leukemia Activity

Abstract

Acute graft-versus-host disease (aGVHD) is one major serious complication that is induced by alloreactive donor T cells recognizing host Ags and limits the success of allogeneic hematopoietic stem cell transplantation. In the current studies, we identified a critical role of Kras in regulating alloreactive T cell function during aGVHD. Kras deletion in donor T cells dramatically reduced aGVHD mortality and severity in an MHC-mismatched allogeneic hematopoietic stem cell transplantation mouse model but largely maintained the antitumor capacity. Kras-deficient CD4 and CD8 T cells exhibited impaired TCR-induced activation of the ERK pathway. Kras deficiency altered TCR-induced gene expression profiles, including the reduced expression of various inflammatory cytokines and chemokines. Moreover, Kras deficiency inhibited IL-6-mediated Th17 cell differentiation and impaired IL-6-induced ERK activation and gene expression in CD4 T cells. These findings support Kras as a novel and effective therapeutic target for aGVHD.

FIGURE 1.

Attenuation of aGVHD by Kras-deficient donor T cells. Lethally irradiated BALB/c mice were transplanted with Rag1-deficient BM alone or plus T cells from VavCreKras^fl/+ (control) or VavCreKras^fl/fl (Kras^−/−) C57BL/6 mice. (A) Marked reduction of aGVHD lethality in the allo-HSCT recipients received Kras-deficient donor T cells. Kaplan-Meier survival analysis of the recipients in each group was performed. (B) Reduction of aGVHD pathology scores in the allo-HSCT recipients received Kras-deficient donor T cells. (C-D) Reduced damages in the colons and lungs of the allo-HSCT recipients received Kras-deficient T cells. Colon and lung sections from the recipients received control T cells or Kras-deficient T cells at four weeks after allo-HSCT were stained with H&E (C). Histological GVHD scores of colons and lungs in all the recipients of each group were graded according to Lerner grading system (D). Data shown are representative of 4 independent experiments with a combined total of 20 mice in each group (A) or are obtained from or representative of 10 recipients in each group (B-D).

FIGURE 2.

Kras-deficient donor T cells preserving GVT effects. Lethally irradiated BALB/c mice were infused with B lymphoma cells (A20^luc) plus Rag1-deficient BM alone or Rag1-deficient BM with CD4⁺ and CD8⁺ T cell from VavCreKras^fl/+ (control) or VavCreKras^fl/fl (Kras^−/−) C57BL/6 mice. (A) In vivo bioluminescence imaging of lymphoma burden in each mouse on the indicated day. (B) Kaplan-Meier survival plots of the indicated mice. Data shown are representative of two independent experiments with a total of 8 mice in each group.

FIGURE 3.

Kras deficiency alters TCR-induced gene expression, including reduced expression of various inflammatory cytokines and chemokines. (A) Activation of Kras following TCR engagement. Mature splenic CD4 and CD8 T cells from wild-type mice were stimulated with anti-CD3. Kras-GTP or total Kras proteins in cell lysates were detected by Raf-RBD agarose bead pull-down and subsequent Western blotting with anti-Kras (upper) or direct Western blotting with anti-Kras (lower), respectively. (B-G) Kras deficiency alters TCR-induced gene expression. CD4 and CD8 T cells from control or Kras^−/− C57BL/6 mice together with Rag1-deficient BM were transplanted into lethally irrradiated BALB/c recipients. Seven days after transplantation, donor CD4 and CD8 T cells were sorted from the splenocytes of the recipients, restimulated with anti-CD3 and subjected to RNA-seq analysis. Volcano plots of differentially expressed genes in Kras-deficient relative to control CD4 (B) or CD8 (E) T cells. Red dots represent differentially expressed genes between Kras-deficient and corresponding control T cells with an adjusted p value < 0.05. Differential expression of cytokine, chemokine, chemokine receptor and cytotoxic effector genes in Kras-deficient relative to control CD4 (C) and CD8 (F) T cells. Each column represents an individual sample and each row represents a single gene. Expression values greater than mean are shown in red and values less than mean are shown in blue. Intensity of color corresponds to relative level of expression. Comparative GSEA of MAPK cascade-regulated or AP-1 target genes in Kras-deficient relative to control CD4 (D) and CD8 (G) T cells. Data shown are representative of 2 independent experiments (A) or obtained from 3 mice of each genotype (B-G).

FIGURE 4.

Reduced proliferation of Kras-deficient donor T cells in the MLR and impaired abilities of mutant T cells to produce inflammatory cytokines. (A) Reduced proliferation of Kras-deficient T cells in the MLR. CD4 and CD8 T cells from VavCre Kras^fl/+ (control) or VavCreKras^fl/fl (Kras^−/−) mice were stimulated with PMA plus inomycin (PMA + inonomycin), anti-CD3 plus anti-CD28 (anti-CD3 + anti-CD28) or irradiated splenocytes from BALB/c mice. Proliferative responses were determined by [³H] thymidine incorporation. (B-E) Impaired abilities of Kras-deficient T cells to produce inflammatory cytokines. CD4 and CD8 T cells from control or Kras^−/− C57BL/6 mice together with Rag1-deficient BM were transplanted into lethally irrradiated BALB/c recipients. Splenocytes were harvested from the recipients at day 7 after transplantation. Following in vitro anti-CD3/CD28 restimulation, intracellular staining of IFNγ, IL-17a and TNFα in donor-derived H2K^b CD4 T cells (B-D) and IFNγ in donor-derived H2K^b CD8 T cells (E) was performed. Data shown are representative of 4 independent experiments (A) or representative of (left and middle panels) or obtained from (right panels) 5 independent experiements with a combined total of 15 control or 14 Kras^−/− mice (B), 2 independent experiements with a combined total of 5 mice in each group (C) or 3 independent experiments with a combined total of 10 mice in each group (D-E). Each dot represents one mouse.

FIGURE 5.

Reduced production of inflammatory cytokines by Kras-deficient donor T cells in vivo. CD4 and CD8 T cells from VavCre Kra^fl/+ (control) or VavCreKras^fl/fl (Kras^−/−) C57BL/6 mice together with Rag1-deficient BM were transplanted into lethally irrradiated BALB/c recipients. Fourteen or twenty-eight days after transplantation, the recipients were analyzed. (A) Detection of IFNγ⁺ or TNFα⁺CD4⁺ T cells and TNFα⁺CD8⁺ T cells in the lungs of the recipients 14 days post transplantation. (B) Detection of IFNγ⁺ or TNFα⁺ CD4 T cells in the livers of the recipients 14 days post transplantation. (C) Detection of IFNγ⁺ CD4 T cells in the guts of the recipients 28 days post transplantation. Numbers indicate percentages of IFNγ⁺ or TNFα⁺ cells in the gated donor CD4 or CD8 T cell population as indicated. Data shown are representative of (upper and middle panels) or obtained from (lower panels) 8 or 6 (A), 6 or 9 (B) and 5 (C) recipients in each group.

FIGURE 6.

Impaired activation of the Ras/ERK pathway by TCR ligation in Kras-deficient CD4 and CD8 T cells. (A) Normal TCR-induced Ca²⁺ flux in Kras-deficient CD4 and CD8 T cells. Splenocytes from VavCreKras^fl/+ (control) or VavCreKras^fl/fl (−/−) mice were labeled with Indo-1 and stained with anti-CD8 and anti-CD4 antibodies. The cells were stimulated with anti-CD3 and Ca²⁺ flux was measured in CD4 and CD8 T cells by flow cytometry analysis. (B, C) Normal TCR-induced activation of p38 and JNK in Kras-deficient CD4 and CD8 T cells. CD4 (B) and CD8 (C) T cells isolated from control or Kras^−/− mice were stimulated with anti-CD3 antibodies and cell lysates were subjected to direct Western blot analysis with the indicated antibodies. (D, E) Impaired TCR-induced activation of the Raf/MEK/ERK pathway in Kras-deficient CD8 but not CD4 T cells in vitro. CD4 (D) and CD8 (E) T cells isolated from control or Kras^−/− mice were stimulated with anti-CD3 and cell lysates were subjected to direct Western blot analysis with the indicated antibodies. (F) Impaired TCR-induced activation of AP-1 in Kras-deficient CD8 but not CD4 T cells in vitro. CD4 and CD8 T cells isolated from control or Kras^−/− mice were stimulated with anti-CD3 and cell lysates were subjected to AP-1 and NF-kB gel mobility shift analysis. (G, H) Impaired activation of the ERK in Kras-deficient CD4 T cells in ex vivo. CD4 and CD8 T cells from control or Kras^−/− C57BL/6 mice together with Rag1-deficient BM were transplanted into lethally irrradiated BALB/c recipients. Fourteen days after transplantation, splenocytes from the recipients were restimulated with anti-CD3 and phosphorylation of ERK1/2 within the gated CD4 T cells was measured by intracellular staining and flow cytometry. The number beneath each band in the western blot indicates the relative intensity of the corresponding band. Data shown are representative of 2 (A), 4 (B-F) or 3 (G) independent experiments or obtained from 3 recipients in each group (H).

FIGURE 7.

Kras deficiency impairs IL-6-induced Th17 cell differentiation, ERK pathway activation and gene expression. (A-D) Impaired Th17 but not Th1, Treg or Th2 differentiation of Kras-deficient CD4 T cells. Naïve CD4 T cells isolated from Kras-deficient and control mice were cultured under the polarizing conditions for IFNγ-producing Th1 (A), Foxp3⁺ Treg (B), IL4-producing Th2 (C) or IL17A-producing Th17 (D) cells. The percentages of IFNγ, Foxp3⁺, IL-4⁺ or IL-17A⁺ cells in CD4 T cells were determined by flow cytometry. (E) Impaired AP-1 activation by IL-6 in Kras-deficient CD4 T cells. CD4 T cells from Kras-deficient and control mice were stimulated with IL-6. Cell lysates were subjected to AP-1 gel mobility shift analysis and direct Western blot analysis with anti-β-actin antibodies. (F) Volcano plots of differentially expressed genes in IL-6-activated Kras-deficient relative to control CD4 T cells. Blue dots represent differentially expressed genes between Kras-deficient and control T cells with an adjusted p value < 0.05. (G) Comparative GSEA of IL-6-induced or IL-6-suppressed genes in Kras-deficient relative to control CD4 T cells. Data shown are representative or obtained from 2 independent experiments with 2 mice of each genotype in each experiment (A-D), representative of 2 independent experiments (E) and obtained from CD4 T cells isolated from 3 Kras-deficient and 4 control mice (F, G).