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. 2012 Nov 8;120(19):4093-103.
doi: 10.1182/blood-2012-01-403196. Epub 2012 Sep 12.

IFNγR signaling mediates alloreactive T-cell trafficking and GVHD

Jaebok Choi  1 , Edward D Ziga, Julie Ritchey, Lynne Collins, Julie L Prior, Matthew L Cooper, David Piwnica-Worms, John F DiPersio
Affiliations

IFNγR signaling mediates alloreactive T-cell trafficking and GVHD

Jaebok Choi et al. Blood. .

Abstract

The clinical goal of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is to minimize GVHD while maintaining GvL. Here, we show that interferon γ receptor-deficient (IFNγR(-/-)) allogeneic Tconv, which possess normal alloreactivity and cytotoxicity, induce significantly less GVHD than wild-type (WT) Tconv. This effect is mediated by altered trafficking of IFNγR(-/-) Tconv to GVHD target organs, especially the gastrointestinal (GI) tract. We show that the chemokine receptor CXCR3 is induced via IFNγR-mediated signaling and partially contributes to the trafficking of WT Tconv to GVHD target organs. Indeed, CXCR3(-/-) Tconv recapitulate the reduced GVHD potential of IFNγR(-/-) Tconv in a minor-mismatched GVHD model. Most importantly, IFNγR(-/-) (and CXCR3(-/-)) Tconv mediate a robust and beneficial GvL effect. In addition, we show that IFNγR(-/-) regulatory T cells (Tregs) are fully suppressive in vitro although defective in suppressor function in vivo and that WT Tregs suppress GVHD in vivo only when allogeneic Tconv produce interferon γ (IFNγ), suggesting that the IFNγR signaling pathway is the major mechanism for both Tregs and Tconv to migrate to GVHD target organs. Finally, pharmacologic inhibition of IFNγR signaling with inhibitors of JAK1/JAK2, which are mediators of IFNγR signaling, results in the decreased expression of CXCR3 and reduced GVHD and improved survival after allo-HSCT and this effect is mediated by altered trafficking of Tconv to GVHD target organs.

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Figures

Figure 1
Figure 1
IFNγR−/− Tconvs do not cause life-threatening GVHD. (A) Effect of IFNγR on GVHD and survival (a pool of 4 independent experiments). Allo-HSCT (B6 [H-2b] → Balb/c]H-2d, CD45.2+]) was performed as follows. T cell–depleted bone marrow cells (TCD BMs; 5 × 106; CD45.1+ B6) and 5 × 105 Tconvs (CD45.2+ B6, either WT or IFNγR−/−) were injected into lethally irradiated (925cGy) Balb/c recipient mice. XRT: irradiation control, BM: TCD BM only, WT: TCD BM + WT Tconv, IFNγR−/−: TCD BM + IFNγR−/−Tconv, IFNγ−/−: TCD BM + IFNγ−/− Tconv. (B-D) One hundred percent donor chimerism is achieved in the IFNγR group. Peripheral blood was analyzed at day 30 after allo-HSCT. The IFNγR group shows higher CD3+ T cells and B220+ B cells in peripheral blood at day 30 after HSCT (a pool of 3 independent experiments) and better weight maintenance compared with the WT group (n = 4; one representative of 4 independent experiments). (E) Tissue sections of skin, liver, and intestine were graded by a veterinary pathologist in blinded fashion on day 20 after allo-HSCT for acute GVHD according to the Lerner grading system (see “Methods” for details).
Figure 2
Figure 2
IFNγR−/− T cells respond normally to allogeneic APCs both in vitro and in vivo. (A) IFNγR−/− Tconvs respond normally to allogeneic APC. Tconvs were labeled with CFSE and mixed lymphocyte reactions (MLRs) were performed in which 2 different strains of APC, Balb/c (H-2d) and third party SWR (H-2q) were used as stimulators. Data represent the pool of 2 independent experiments. (B) BLI was performed. The click beetle red luciferase gene (CBRluc)–expressing Tconvs (4 × 106 cells) were transplanted into Balb/c recipients. Photon flux (photons/s) was measured from the dorsal and the ventral view with a region of interest drawn over the entire body of each mouse. P values at days 10 and 31 were .1165 or higher. (C) IFNγR−/− Tconvs (bottom panel) express similar levels of GZMB, compared with WT Tconv (top panel) after 3 days of activation using anti-CD3/CD28 antibody-coated beads.
Figure 3
Figure 3
IFNγR is required for trafficking of Tconvs to GVHD target organs and CXCR3 expression. (A) In vivo BLI was performed to specifically track CBRluc-transduced Tconvs (2 or 4 × 106 cells) after allo-HSCT. BLI images of 1 dissected representative mouse from WT (left) and IFNγR−/− (right) T cell recipients at day 31 after allo-HSCT (top panels). Spleens and GI tracts were separated from the body cavities. White arrows indicate LNs. Ratio of signal intensities (photons/s/cm2/sr) from spleen and GI tract and the rest of body were compared in the bottom panel. Data represent the pool of 2 independent experiments. (B) WT Tconvs express IFNγR (CD119) and CXCR3 before (top panels) and after (bottom panels) activation by anti-CD3/CD28 antibody-coated beads. The expressions of IFNγR (CD119) and CXCR3 are correlated in WT Tconv. (C) IFNγR−/− Tconv (both CD4+ and CD8+ T cells; CD4− T cells are CD8+ T cells right panels) express CXCR3 significantly less than WT Tconvs (left panels) after activation (bottom panels). Mean and SD of activated WT Tconvs are as follows (n = 8). CD8+CXCR3+: 42.7% ± 5.5%, CD8+CXCR3−: 5.2% ± 1.3%, CD4+CXCR3+: 41.2% ± 5.0%, CD4+CXCR3−: 11.0% ± 4.7%. Mean and standard deviation of activated IFNγR−/− Tconv are as follows (n = 8). CD8+CXCR3+: 24.1% ± 8.2%, CD8+CXCR3−: 21.0% ± 3.3%, CD4+CXCR3+: 5.5% ± 1.7%, and CD4+CXCR3−: 49.3% ± 4.8%. (D) IFNγ−/− Tconv (both CD4+ and CD8+ T cells; CD4− T cells are CD8+ T cells) express CXCR3 significantly less than WT Tconvs (C) after activation (bottom panels) but up-regulate IFNγR expression similar to activated WT Tconvs (left panels). Mean and standard deviation of activated IFNγ−/− Tconv are as follows (n = 9). CD8+CXCR3+: 23.4% ± 5.3%, CD8+CXCR3−: 24.6% ± 4.9%, CD4+CXCR3+: 5.6% ± 2.6%, and CD4+CXCR3−: 46.4% ± 6.9%. (E) IFNγR−/− Tconvs were retrovirally transduced with either the CBRluc-GFP gene (IFNγR−/−) or the CXCR3-ires-CBRluc-GFP gene (CXCR3+ IFNγR−/−). WT Tconvs were also transduced with the CBRluc-GFP gene. Transduced cells were purified using the Reflection cell sorter (iCyt; GFP+ WT Tconvs and GFP+ IFNγR−/− Tconvs) or AutoMACS (CXCR3+ IFNγR−/− Tconvs; all cells > 96% pure) and transplanted (2 × 106) into recipient mice along with TCD BM (5 × 106). Shown is percent survival after allo-HSCT (5 × 106 TCD BM + 2 × 106 Tconv). Data represent a pool of 3 independent experiments.
Figure 4
Figure 4
IFNγR is required for Tregs to function appropriately in vivo. (A) Tregs also up-regulate both CXCR3 and IFNγR when activated (right panels). Mean and standard deviation of activated Tregs are as follows (n = 3). IFNγR+CXCR3−: 16.8% ± 7.0%, IFNγR-CXCR3−: 1.39% ± 1.2%, IFNγR+CXCR3+: 79.1% ± 6.1%, and IFNγR-CXCR3+: 2.7% ± 1.8%. (B) IFNγR Tregs were equally suppressive as WT Tregs in in vitro MLR assays ([3H]-thymidine incorporation was measured). Tregs were serial-diluted. The experiment was performed in triplicate or quadruplicate. Shown is 1 representative of 3 independent experiments with similar results. (C) IFNγR−/− Tregs do not suppress GVHD (B6 → Balb/c). TCD BM (B6, 5 × 106; CD45.1+) were injected into lethally irradiated Balb/c mice, followed by DLI of 2 × 106 pan T (B6, CD45.2+) and 1.5 × 106 Tregs (B6, CD45.2+). Data represent the pool of 2 independent experiments. (D) WT Tregs suppress GVHD induced by WT Tconvs but not by IFNγ−/− Tconv. Both Tregs and Tconvs (5 × 105 cells each) were injected along with TCD BM (5 × 106 cells) at day 0. Data represent the pool of 2 independent experiments.
Figure 5
Figure 5
IFNγR−/− and CXCR3−/− Tconvs mediate a robust GvL/GvT. (A) Systemic leukemia model. Photon flux was measured with a region of interest drawn over the entire body of each mouse. Actual images of 1 representative mouse from each group are shown in bottom panels. Data represent the pool of 2 independent experiments. n = 10 each group (n = 6 in BM only group). (B) Solid tumor model. Photon flux was measured with a region of interest drawn over the entire body of each mouse. Actual images of 1 representative mouse from each group are shown in bottom panels. Data represent the pool of 2 independent experiments. n = 10 each group (n = 6 in BM only group). (C) Effect of CXCR3 on GVHD and survival. Data represent the pool of 2 independent experiments (n = 10).
Figure 6
Figure 6
JAK1/JAK2 inhibitors block the IFNγR-CXCR3 axis both in mouse and human T cells and mitigates GVHD. (A) Shown are WT pan T cells (both CD4+ and CD8+ T cells; CD4− T cells are CD8+ T cells) after drug treatment. (B) Tissue sections of skin, liver, and intestine were graded by a veterinary pathologist in blinded fashion on day 21 after allo-HSCT for acute GVHD according to the Lerner grading system (see “Methods” for details). (C) Effect of INCB018424 on GVHD. Allo-HSCT (B6 (H-2b) → Balb/c (H-2d) was performed as follows. Five × 106 T cell–depleted bone marrow cells (TCD BM) and 5 × 105 Tconvs were injected into lethally irradiated (925cGy) Balb/c recipient mice. INCB018424 was injected intraperitoneally into recipients daily from day 0 through 20 (D0: 100 μg twice daily for the first 7 days and 100 μg once daily for the following 14 days) or day 3 through 23 (D3: 100 μg twice daily for the first 4 days and 100 μg once daily for the following 17 days). (D) In vivo BLI was performed to specifically track T cells (0.5 × 106 cells) obtained from FVB-Tg(CAG-luc,-GFP)L2G85Chco/J mice (H-2q) after allo-HSCT. FVB/NJ mice (H-2q) were used as TCD BM donors (5 × 106 cells) and Balb/c as recipients. INCB018424 or 10% DMSO was administered twice a day from days 3 to 6 and once daily from days 7 to 23. BLI images of dissected mice (n = 10 each) at day 31 after allo-HSCT were analyzed. Spleens, livers, and GI tracts were separated from the body cavities. Photon flux (photons/s) was measured from whole body (right panel) and the ratio of signal intensities (photons/s/cm2/sr) from spleen, liver, and GI tract and the rest of body were compared (left panel). (E-G) INCB018424 does not inhibit donor engraftment and reduce neither platelet (PLT) counts nor white blood cell (WBC) counts. Day 21 after allo-HSCT, BM+PBS (n = 2), BM+INCB (n = 5), PBS (n = 4), INCB (D0; n = 18), and INCB (D3; n = 15). (H) Shown are human pan T cells 5 days after drug treatment in the presence of anti-CD3/CD28 antibody coated beads (cell:bead = 1:1). Mean and SD of activated human T cells are as follows (n = 2); 0μM of INCB018424: CD8+CXCR3+: 6.0% ± 2.7%, CD8+CXCR3−: 6.3% ± 2.8%, CD4+CXCR3+: 49.6% ± 1.6%, CD4+CXCR3−: 38.1% ± 1.5%. 0.2μM of INCB018424: CD8+CXCR3+: 0.4% ± 0.2%, CD8+CXCR3−: 8.7% ± 0.3%, CD4+CXCR3+: 10.2% ± 4.2%, and CD4+CXCR3−: 80.8% ± 3.7%.
Figure 7
Figure 7
Model for the role of IFNγR signaling in T-cell trafficking and GVHD. Naive Tconvs and Tregs become activated by host APCs because of MHC mismatch. (1) Activated Tconv secrete IFNγ (2) that will initiate the IFNγR signaling, which in turn up-regulates CXCR3 and other unknown mediators of T-cell trafficking on both Tconvs and Tregs. (3) CXCR3+ T cells migrate to the CXCR3 ligands-expressing GVHD target organs (4) where activated Tconv are destructive but suppressed in the presence of activated Tregs. (5) When Tconvs fail to secrete IFNγ (IFNγ−/− Tconv), Tregs will not activate IFNγR signaling, thereby failing to migrate to the same sites of inflammation as IFNγ−/− Tconv. This will result in more severe GVHD than WT Tconv. Likewise, when Tregs are defective in up-regulating CXCR3 because of the lack of IFNγR, the same result will be observed. Tn: naive Tconv, Tact: activated Tconv, nTreg: naive Tregs, aTreg: activated Tregs, M: MHC molecules, T: T-cell receptors, R: IFNγR, 3: CXCR3, γ: IFNγ.
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