Targeting Interleukin-2-Inducible T-Cell Kinase (ITK) Differentiates GVL and GVHD in Allo-HSCT

Abstract

Allogeneic hematopoietic stem cell transplantation is a potentially curative procedure for many malignant diseases. Donor T cells prevent disease recurrence via graft-versus-leukemia (GVL) effect. Donor T cells also contribute to graft-versus-host disease (GVHD), a debilitating and potentially fatal complication. Novel treatment strategies are needed which allow preservation of GVL effects without causing GVHD. Using murine models, we show that targeting IL-2-inducible T cell kinase (ITK) in donor T cells reduces GVHD while preserving GVL effects. Both CD8⁺ and CD4⁺ donor T cells from Itk^-/- mice produce less inflammatory cytokines and show decrease migration to GVHD target organs such as the liver and small intestine, while maintaining GVL efficacy against primary B-cell acute lymphoblastic leukemia (B-ALL). Itk^-/- T cells exhibit reduced expression of IRF4 and decreased JAK/STAT signaling activity but upregulating expression of Eomesodermin (Eomes) and preserve cytotoxicity, necessary for GVL effect. Transcriptome analysis indicates that ITK signaling controls chemokine receptor expression during alloactivation, which in turn affects the ability of donor T cells to migrate to GVHD target organs. Our data suggest that inhibiting ITK could be a therapeutic strategy to reduce GVHD while preserving the beneficial GVL effects following allo-HSCT treatment.

Keywords: Eomesodermin (EOMES); GVHD after blood transfusion; GvL; ITK deficiency; JAK-STAT signalling pathway; T cell.

Figure 1

Absence of ITK avoids GVHD while retaining GVL effects during allo-HSCT. 1 × 10⁶ purified CD4+ and 1 × 10⁶ CD8+ T cells (WT or Itk^-/-) were mixed at a 1:1 ratio, and transplanted along with 2 × 10⁵ B-ALL-luc cells into irradiated BALB/c mice. Host BALB/c mice were imaged using the IVIS imaging system 3 times a week. Group 1 received 10 × 10⁶ T cell depleted bone marrow only (labeled as _TCDBM). Group 2 received 10 × 10⁶ _TCDBM along with 2 × 10⁵ B-ALL-luc cells (_TCDBM+B-ALL ^luc+), Group 3 was transplanted with 1 × 10⁶ purified WT CD8⁺ and 1 × 10⁶ CD4⁺ T cells (1:1 ratio) along with 2 × 10⁵ B-ALL-luc+ cells 10 × 10⁶ (_TCDBM+B-ALL ^luc+ WT CD8+CD4). Group 4 received 1 × 10⁶ purified Itk^-/- CD8⁺ and 1 × 10⁶ CD4⁺ T cells (1:1 ratio) along with 2 × 10⁵ B-ALL-luc+ cells 10 × 10⁶ (_TCDBM+B-ALL^luc+ Itk^-/- CD8+CD4). (A) Recipient BALB/c mice were imaged using IVIS 3 times a week. (B) The mice were monitored for survival, (C) changes in body weight, and (D) clinical score for 65 days post BMT. (E) Quantified luciferase bioluminescence of leukemia cell growth. Statistical analysis for survival and clinical score was performed using log-rank test and two-way ANOVA, respectively. For weight changes and clinical score, one representative of 2 independent experiments is shown (n = 3 mice/group for BM alone; n = 5 experimental mice/group for all three groups. Survival is a combination of 2 experiments. P values presented with each group. Two-way ANOVA and students t-test were used for statistical analysis. Note: Controls are naïve mice used as negative control for bioluminescence (BLI).

Figure 2

The regulatory function of ITK in GVHD is T cell-intrinsic. (A) Purified WT and Itk^-/- CD8⁺ T cells were examined for expression of CD44, CD122, and Eomes prior to transplantation. (B) Whole bone marrow cells isolated from C57Bl/6 WT (CD45.2) and Itk^-/- (CD45.1) mice were mixed in 1:3 ration WT: Itk ^-/-, and transplanted into irradiated Thy1.1 C57Bl/6 mice. 9-10 weeks later CD8⁺ T cells were sorted by CD45.2 and CD45.1 expression (donor T cells) and exclusion of Thy1.1 positive (host T cells). Isolated sorted T cells were examined for expression of CD44, CD122, and Eomes and transplanted into irradiated BALB/c mice. This experiment was repeated more than three times. (C) Irradiated BALB/c mice were divided in four different groups and transplanted with the sorted T cells described in (B) as follows: Group one was transplanted with 10 × 10⁶ _TCDBM alone (_TCDBM). Group two was transplanted with 10 × 10⁶ _TCDBM and 2 × 10⁵ B-ALL-luc, (_TCDBM+B-ALL^luc). Group three was transplanted with 10 × 10⁶ _TCDBM along with 1 × 10⁶ purified WT CD8⁺ T cells and 2 × 10⁵ B-ALL-luc (_TCDBM+B-ALL^luc+WT CD45.2). The fourth group was transplanted 10 × 10⁶ _TCDBM along with and 1 × 10⁶ purified Itk^-/- CD8⁺ T cells and 2 × 10⁵ B-ALL-luc (_TCDBM+B-ALL^luc+Itk^-/- CD45.1). These mice were monitored for leukemia cell growth using the IVIS system. (D) The mice were monitored for survival, (E) changes in body weight, and (F) clinical score for 47 days post BMT. For body weight changes and clinical score, one representative of 2 independent experiments is shown (n = 3 mice/group for BM alone; n = 5 experimental mice/group for all three groups). (G) Quantified luciferase bioluminescence of luciferase expressing B-ALL-luc cells. Statistical analysis for survival and clinical score was performed using log-rank test and two-way ANOVA, respectively. One representative experiment out of 2. Survival is a combination of 2 experiments, 3 mice per group of control _TCDBM, and 5 mice per group for all of the experimental groups. P value presented with each figure. Note: Controls are naïve mice used as negative control for BLI.

Figure 3

IMP T cells are not sufficient for GVL effect. (A, B) Purified WT and Itk^-/- CD8⁺ and CD4⁺ T cells were examined for expression of CD44, CD122, and Eomes by flow cytometry. (C) Purified WT or Itk^-/- T cells were transplanted into irradiated BALB/c mice, at day 7 purified T cells were sorted using H2K^d, CD45.1 and CD45.2 expression. Ex vivo purified CD8⁺T cells were used in cytotoxicity assay against primary leukemia target B-ALLluc+ cells at a 40:1, 20:1, or 5:1 ratio. (D) Purified T cells were examined for perforin by western blot. Quantitative analysis of perforin expression by western blot with data normalized against β–Actin. (E) Purified WT or Itk/Eomes DKO donor T cells were transplanted into irradiated BALB/c mice. On day 7 donor T cells were purified as described and used in an ex vivo cytotoxicity assay against B-ALL^luc-cells at 5:1, 20:1, and 40:1 ratio. (F) 1 × 10⁶ purified WT and Itk^-/- Itk/Eomes DKO CD8⁺ T cells and 1 × 10⁶ purified CD4⁺ T total of 2 × 10⁶ mixed CD4⁺ and CD8⁺ T cells were mixed and transplanted along with 2 × 10⁵ B-ALL-luc cells and 10 × 10⁶ _TCDBM into irradiated BALB/c mice. Host BALB/c mice were imaged using IVIS 3 times a week. Group one received 10 × 10⁶ _TCDBM alone as (_TCDBM). Group two received 10 × 10⁶ _TCDBM along with 2 × 10⁵ B-ALL-luc cells (_TCDBM+B-ALL^luc). Group three were transplanted with 10 × 10⁶ _TCDBM and 1 × 10⁶ purified WT CD8⁺ T cells +1 × 10⁶ CD4⁺ T cells and 2 × 10⁵ B-ALL-luc cells (_TCDBM+B-ALL^luc+WT CD8+CD4). Group four received 10 × 10⁶ T _TCDBM and 1 × 10⁶ purified CD8⁺ T cells +1 × 10⁶ CD4⁺ T cells from Itk/Eomes DKO along with 2 × 10⁵ B-ALL-luc cells (_TCDBM+B ALL^luc+ Itk^-/- EomesFF+CD4cre CD8+CD4). Group five received 10 × 10⁶ _TCDBM and 1 × 10⁶ CD8⁺ T cells +1 × 10⁶ CD4⁺ purified T cells Itk/Eomes DKO CD4⁺ T cells along with 2 × 10⁵ B-ALL-luc cells (_TCDBM+B-ALL^luc+ Itk^{-/- EomesFF+CD4cre-} CD8+CD4). (F) The mice were monitored for survival, (G) body weight changes, and (H) clinical score for 60 days post BMT. For weight changes and clinical score, one representative of 2 independent experiments is shown (n = 3 mice/group for BM alone; n = 5 experimental mice/group for all three group. The survival groups are a combination of all experiments. (I) Quantitated luciferase bioluminescence of tumor growth. Statistical analysis for survival and clinical score was performed using log- Two-way ANOVA were used for statistical analysis confirming by students t test, p values are presented. Note: Controls are naïve mice used as negative control for bioluminescence (BLI).

Figure 4

ITK deficiency results in reduced cytokine production. (A) Serum from several GVHD patients was isolated and examined for inflammatory cytokine production (IL-33, IL1α, IFN-γ and TNF-α, IL1β and IL-17A) as determined by ELISA. (B, C) 1 × 10⁶ purified WT or Itk^-/- CD8⁺ T or CD4⁺ T cells were separately transplanted with into irradiated BALB/c mice. At day 7 post allo-HSCT, recipient BALB/c were euthanized and serum cytokines (IL-33, IL1α, IFN-γ, and TNF-α and IL-17A) were measured by ELISA. (D) Intracellular IFN-γ and TNF-α expression by donor CD8⁺ and CD4⁺ T cells after stimulation with anti-CD3/anti-CD28 as determined by flow cytometry. (E, F) Combined data from 3 independent experiments is shown for each experiment shown in figures. (F) Flow cytometry analysis of purified WT and Itk^-/- T cells that were mixed at a 1:1 ratio for transplantation into irradiated BALB/c mice. At day 7 donor T cells were gated for expression of H-2K^b, CD45.1, and CD45.2 and intracellular expression of IFN-γ and TNF-α was analyzed by flow cytometry after stimulation with anti-CD3/anti-CD28. Combined data from four independent experiments is shown, and the p value for each experiment is shown. (G) Purified WT or Itk^-/- donor CD8⁺ and CD4⁺ T cells were transplanted into irradiated BALB/c. At day 7 donor cells were analyzed for donor T cell proliferation by examining BrdU incorporation by flow cytometry. (H) Purified WT and Itk^-/- donor T cells were mixed at a 1:1 WT: Itk^-/- ratio and transplanted into irradiated BALB/c mice, at day 7 splenic donor T cells were gated for the expression of H-2K^b, CD45.1, and CD45.2 and analyzed for BrDU incorporation. (I) Purified WT and Itk^-/-T cells were stimulated with CD3 and CD28 overnight examined for the expression and phosphorylation of IRF4, JAK1/2 and STAT3 by western blot. For statistical analysis we used two-way ANOVA and student’s t test, p values are presented.

Figure 5

ITK differentially regulates gene expression in T cells during GVHD. WT and Itk^-/- CD8⁺ T cells were FACS sorted then transplanted into irradiated BALB/c mice. At day 7 post-transplant, donor T cells were sort-isolated (based on expression of H-2K^b, CD3 and CD8) from host spleen. Sorted donor T cells were subjected to RNA sequencing. (A) Principal component analysis of genes with ≥2-fold change in any pairs of group combinations, with false discovery rate (FDR) ≤ 0.05. WT-Pre and Itk^-/–Pre denotes cells prior to transfer, and WT-Spl, and Itk^-/–Spl denotes cells isolated from the spleen (Spl) of the recipients post-transfer. (B) Venn diagram of genes that are ≥2-fold up- or down- regulated in the indicated comparisons, with FDR (P) ≤ 0.05. (C) Heat map of differentially expressed genes listed as (1) WT pre. (2) ITK-/- pre, (3) WT post spleen, and (4) ITK-/- post spleen. (E) Differentially expressed genes were enriched for pathway analysis comparing WT and Itk^-/-. (D) WT and Itk^-/- CD8⁺ T cells were FACS sorted then transplanted into irradiated BALB/c mice. At day 7 post-transplant, donor T cells were sort-isolated (based on expression of H-2K^b, CD3 and CD8) from host spleen and small intestine (Gut). Total RNA was isolated from sorted donor T cells were and qPCR was performed.

Figure 6

ITK signaling is required for T cell migration to the GVHD target tissues. (A) Irradiated BALB/c mice were allo-HSCT-transplanted and injected with FACS-sorted WT and Itk^-/- CD8⁺ T and CD4⁺ T cells mixed at a 1:1 ratio. FACS analysis of sorted T cells pre-transplant shown. (B) At day 7 post-BMT, the spleen, liver, and small intestine (SI) were examined for donor WT and Itk^-/- T cells. The ratio of WT: Itk^-/- CD8⁺ and CD4⁺ T cells in the organs was determined. (C) At day 7 post-allo-HSCT, small intestines were examined by H&E staining. (D) Irradiated BALB/c mice were BM-transplanted and injected with CD8⁺ T CD4⁺ T cells from luciferase-expressing WT or Itk^-/- mice. (E) On Day 7 post-allo-HSCT, donor T cells were isolated and examined for the expression of Aplnr, Cxcr5, Accr2, CCL12, CCL2, CCL5, CCr9, Ackr4, and Cmtm4 using q-RTPCR. P values were calculated using 2-way ANOVA and Student’s t test, p values are listed. (F) Irradiated BALB/c mice were transplanted with C57Bl/6-derived BM and FACS-sorted WT or Itk^-/- 1 × 10⁶ CD8⁺ T cells, and challenged subcutaneously with 2 × 10⁵luciferase-expressing B-All luc cells. Recipient animals were monitored for weight changes. Group one of recipient mice was transplanted with 10 × 10⁶ _TCD BM. The second group of recipient mice was transplanted with 10 × 10⁶ _TCDBM and 2 × 10⁵ primary B-ALL luc+ cells(_TCDBM+B-ALL^luc). The third group of recipient mice was transplanted with 10 × 10⁶ _TCDBM along with 1 × 10⁶ T cell from WT mice along with 2 × 10⁵ B-ALL-luc+ cells (_TCDBM+B-ALL^luc+WT CD8). The fourth group of recipient mice was transplanted with 10 × 10⁶ _TCDBM and 1 × 10⁶ T cell from Itk ^-/- mice along with 2 × 10⁵ B-ALL-luc+ cells. (_TCDBM+B-ALL^luc+ Itk ^-/- CD8). Representative bioluminescence images of leukemia cell-bearing mice on days 9, 16, 30, 38, and 47 are shown. Note: Controls are naïve mice used as negative control for bioluminescence (BLI). (G) Animals were monitored for survival over 47 days, (H) for changes in weight loss, (I) and for clinical score. (J) Recipient mice were monitored for leukemia cell growth using the IVIS imaging system and quantified data is shown. For weight changes and leukemia cell growth, one representative of 2 independent experiments is shown (n = 3 mice/group for control, n = 5 mice for WT, and n = 5 mice for Itk^-/-). Survival groups were combined from both experiments. P values were calculated using two-way ANOVA and Student’s t test, p values are listed.