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Fibroblastic Niches Prime T Cell Alloimmunity Through Delta-like Notch Ligands

Jooho Chung et al. J Clin Invest.

Abstract

Alloimmune T cell responses induce graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (allo-BMT). Although Notch signaling mediated by Delta-like 1/4 (DLL1/4) Notch ligands has emerged as a major regulator of GVHD pathogenesis, little is known about the timing of essential Notch signals and the cellular source of Notch ligands after allo-BMT. Here, we have shown that critical DLL1/4-mediated Notch signals are delivered to donor T cells during a short 48-hour window after transplantation in a mouse allo-BMT model. Stromal, but not hematopoietic, cells were the essential source of Notch ligands during in vivo priming of alloreactive T cells. GVHD could be prevented by selective inactivation of Dll1 and Dll4 in subsets of fibroblastic stromal cells that were derived from chemokine Ccl19-expressing host cells, including fibroblastic reticular cells and follicular dendritic cells. However, neither T cell recruitment into secondary lymphoid organs nor initial T cell activation was affected by Dll1/4 loss. Thus, we have uncovered a pathogenic function for fibroblastic stromal cells in alloimmune reactivity that can be dissociated from their homeostatic functions. Our results reveal what we believe to be a previously unrecognized Notch-mediated immunopathogenic role for stromal cell niches in secondary lymphoid organs after allo-BMT and define a framework of early cellular and molecular interactions that regulate T cell alloimmunity.

Conflict of interest statement

Conflict of interest: M. Yan and C.W. Siebel are employed by Genentech.

Figures

Figure 1
Figure 1. An early pulse of Notch signaling is critical to drive pathogenic T cell alloreactivity after BMT.
(A) Dosing schedule of systemic neutralizing antibodies against DLL1 and DLL4 Notch ligands. (B) Survival, GVHD score, and weight of lethally irradiated (8.5 Gy) BALB/c mice transplanted with 5 × 106 T cell–depleted (TCD) B6 BM or 5 × 106 TCD B6 BM plus 5 × 106 allogeneic B6 splenocytes. Isotype control versus anti-DLL1/4 antibodies were injected i.p., as shown in A (n = 10 mice/group). (C) Intracellular cytokine production by donor CD4+ T cells after anti-CD3/CD28 restimulation on day 6 after transplantation (n = 5 mice/group). (D) Intracellular FoxP3 in donor CD4+ T cells on day 6 (n = 5 mice/group). *P < 0.05, **P < 0.01, ***P < 0.001, and NS = P > 0.05, by unpaired, 2-tailed Student’s t test with Sidak’s correction for multiple comparisons. Data are representative of at least 4 experiments; error bars indicate SD.
Figure 2
Figure 2. Host hematopoietic cells are dispensable as cellular sources of DLL1/4 Notch ligands in acute GVHD.
(A) Experimental strategy. BM chimeras were generated via transplantation of syngeneic B6-CD45.2+ poly(I:C)-induced TgMx1-Cre– littermate control or TgMx1-Cre+ Dll1Δ/Δ Dll4Δ/Δ BM into irradiated B6-CD45.1 recipients. After reestablishment of steady-state hematopoiesis 12 weeks later, BM chimeras were subjected to a second syngeneic or allogeneic transplant, with or without systemic anti-DLL1/4 blockade. (B) Quantification of Dll1 and Dll4 inactivation in sort-purified Gr1+CD11b+ blood myeloid cells from BM chimeras 12 weeks after transplantation (PCR). In this particular experiment, control BM chimeras were generated from poly(I:C)-induced TgMx1-Cre– Dll1fl/+ Dll4fl/+ donor mice. Each lane represents an individual mouse. (C) Donor chimerism (frequency of CD45.2+ donor cells) in the indicated splenic cell populations 12 weeks after transplantation. MΦ, macrophages; pDCs, plasmacytoid DCs. (D) Survival and weight loss of lethally irradiated (11 Gy) BM chimeras transplanted with 8 × 106 TCD BM plus 30 × 106 B6 splenocytes (syngeneic control) or 30 × 106 allogeneic BALB/c splenocytes (allo-BMT). Isotype control or anti-DLL1/4 antibodies were injected i.p. on days 0, 3, 7, and 10 (n = 10 mice/group). (E) Abundance of Dtx1 Notch target gene transcripts (qRT-PCR) in sort-purified donor CD4+ T cells and CD8+ cells on day 6 (n = 5 mice/group). *P < 0.05, **P < 0.01, and NS = P > 0.05, by unpaired, 2-tailed Student’s t test with Sidak’s correction for multiple comparisons. Data are representative of at least 2 experiments; error bars indicate SD.
Figure 3
Figure 3. Ccl19-Cre+ lineage–traced stromal cells are the critical cellular source of DLL1/4 Notch ligands during acute GVHD.
(A) LNs were collected on day 1.5 after transplantation from lethally irradiated TgCcl19-Cre+ ROSA26eYFP mice receiving allogeneic BALB/c splenocytes and enzymatically digested. (A, top) eYFP expression in LN-resident bulk fibroblastic stromal cells (PDPN+CD31) as well as subfractionated CD157+ FRCs, CD157 FRCs, and CD21+ FDCs. (A, middle) eYFP in LECs, BECs, PDPNCD31 stromal cells (DNs). (A, bottom) eYFP in macrophages, conventional DCs (cDCs), pDCs, and skin-derived Langerhans cells. Bars in histograms define gating for eYFP+ cells, and numbers indicate the percentage of gated eYFP+ cells within parental cell populations, as identified by flow cytometric analysis. Bar graph in A shows the mean percentage of eYFP expression in each indicated nonhematopoietic subset (n = 4 mice/group; error bars indicate SD). (B) Survival, GVHD score, and weight of lethally irradiated (12 Gy) littermate control TgCcl19-Cre– or TgCcl19-Cre+ Dll1Δ/Δ Dll4Δ/Δ mice that were transplanted with 10 × 106 TCD BM only or 10 × 106 TCD BM plus 20 × 106 allogeneic BALB/c splenocytes. Isotype control or anti-DLL1/4–neutralizing antibodies were injected i.p. on days 0, 3, 7, and 10 (n = 10 mice/group). (C) Intracellular cytokines in donor CD4+ cells after anti-CD3/CD28 restimulation on day 6 (n = 5 mice/group). (D) Relative abundance of Dtx1 and Hes1 Notch target gene transcripts in donor CD4+ T cells sort purified from TgCcl19-Cre– plus isotype control, TgCcl19-Cre– plus anti-DLL1/4, or TgCcl19-Cre+ Dll1Δ/Δ Dll4Δ/Δ recipient mice on day 2 after transplantation (n = 5 mice/group). *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed Student’s t test with Sidak’s correction for multiple comparisons. Data are representative of at least 5 experiments; error bars indicate SD.
Figure 4
Figure 4. Ccl19-Cre–mediated Dll1 and Dll4 inactivation preserves lymphocyte numbers and distribution in SLOs at steady state.
(A) Absolute numbers of CD4+ T cells, CD8+ T cells, and B cells in spleens and LNs of B6 littermate control TgCcl19-Cre– or TgCcl19-Cre+ Dll1Δ/Δ Dll4Δ/Δ mice at steady state (n = 5 mice/group). (B and C) CD62L and CD44 expression in CD4+ (B) and CD8+ (C) T cells from spleens and LNs of TgCcl19-Cre– or TgCcl19-Cre+ Dll1Δ/Δ Dll4Δ/Δ mice at steady state. (D) Abundance of Il7 transcripts (qRT-PCR) in sort-purified PDPN+CD31 fibroblastic stromal cells from TgCcl19-Cre– or TgCcl19-Cre+ Dll1Δ/Δ Dll4Δ/Δ mice (n = 5 mice/group). NS = P > 0.05, by unpaired, 2-tailed Student’s t test. Data are representative of at least 3 experiments; error bars indicate SD.
Figure 5
Figure 5. Ccl19-Cre–mediated Dll1 and Dll4 inactivation does not impair T cell recruitment or proliferation in SLOs after irradiation, but selectively affects Notch target gene transcripts.
(AE) Absolute numbers (A), proliferation (CFSE dilution) on day 2.5 (B) versus day 6 after transplantation (C), and expression of activation markers. Bars in histograms define gating for proliferated CFSElow and unproliferated CFSEhi cells, and numbers indicate the percentage of gated cells among parental cell populations, as identified by flow cytometric analysis. Bar graphs represent the mean percentage of proliferated (CFSElow) cells in each population (n=5 mice/group); error bars indicate SD. (D and E) by donor-derived CD4+ and CD8+ T cells after transplantation into lethally irradiated (12 Gy) littermate control TgCcl19-Cre– or TgCcl19-Cre+ Dll1Δ/Δ Dll4Δ/Δ B6 recipient mice. Donor cells were isolated on day 2.5 or day 6 after transplantation (n = 5 mice/group). (F) Abundance of the indicated transcripts (qRT-PCR) in sort-purified donor-derived CFSEdiluted BALB/c CD4+ T cells on day 2 after transplantation into irradiated B6 hosts treated with or without anti-DLL1/4 antibodies. *P < 0.05, **P < 0.01, NS = P > 0.05, by unpaired, 2-tailed Student’s t test. Data are representative of at least 3 experiments; error bars indicate SD. MFI, mean fluorescence intensity.
Figure 6
Figure 6. Irradiation changes the LN ultrastructure and increases the relative density of Ccl19-Cre+ stromal cells.
(A) Total cellularity, absolute numbers of CD45+ cells, and absolute numbers and frequencies of eYFP+ cells in LNs of unirradiated or lethally irradiated (12 Gy) TgCcl19-Cre+ ROSA26eYFP reporter mice receiving allogeneic BALB/c splenocytes (n = 6 mice/group). **P < 0.01, ***P < 0.001, NS = P > 0.05, by unpaired, 2-tailed Student’s t test. (B) Immunofluorescence microscopy images of LN cryosections from TgCcl19-Cre+ ROSA26eYFP mice stained for GFP. Cryosections were from unirradiated or lethally irradiated (12 Gy) mice receiving no T cells, syngeneic B6 CD4+ T cells, or allogeneic BALB/c CD4+ T cells. (C and D) Immunofluorescence microscopy images of LN cryosections from TgCcl19-Cre+ ROSA26eYFP mice stained for B220 and CD3 (C, top panel); eYFP, CD11b, and CD169 (C, bottom panel); eYFP and PDPN (D, top panel); and eYFP and CD35 (D, bottom panel). Cryosections were from unirradiated or lethally irradiated (12 Gy) mice receiving allogeneic BALB/c CD4+ T cells on day 1.5 after transplantation. Data are representative of 2 experiments.
Figure 7
Figure 7. DLL4 expression within different LN nonhematopoietic cell populations after allogeneic BMT.
(A) LN stromal cell subsets. TgCcl19-Cre+ Dll1Δ/Δ Dll4Δ/Δ and TgCcl19-Cre– littermate control mice were lethally irradiated before infusion of allogeneic BALB/c splenocytes. LNs were collected on day 1.5 after transplantation and enzymatically digested. Dot plots show representative flow cytometric analysis of CD45Ter119 nonhematopoietic stromal cells. (B) DLL4 expression in LN-resident nonhematopoietic cell subsets. Roman numerals refer to the cell populations shown in A. (C) MFI comparing DLL4 expression in stromal cell subsets from TgCcl19-Cre– versus TgCcl19-Cre+ Dll1Δ/Δ Dll4Δ/Δ mice as well as isotype staining in controls. **P < 0.01 and ***P < 0.001, by unpaired 2-tailed Student’s t test with Sidak’s correction for multiple comparisons. (D) Representative flow cytometric plots of PDPN+CD31CD21/35 FRCs showing DLL4 expression in the CD157hi subset. Data are representative of at least 3 experiments.
Figure 8
Figure 8. Fibroblastic niches in spleen express DLL1/4 Notch ligands and localize next to alloreactive T cells.
(A and B) Immunofluorescence microscopy of splenic cryosections from TgCcl19-Cre+ Dll1+/+ Dll4+/+ ROSA26eYFP and TgCcl19-Cre+ Dll1Δ/Δ Dll4Δ/Δ ROSA26eYFP mice stained for GFP, CD35, and DLL4 (A) or GFP, CD157, and DLL4 (B). (CE) Immunofluorescence microscopy of splenic cryosections from lethally irradiated (8.5 Gy) BALB/c mice transplanted with CMTMR-labeled alloantigen-specific CD4+ 4C TCR–transgenic cells and pulsed with EdU 12 hours prior to organ collection to label proliferating cells. Cryosections were incubated with Alexa Fluor 488 picolyl azide to reveal EdU, along with anti-DLL4 (C), anti-CD35 (D), or anti-CD157 (E). Organs were collected on day 1.5 after transplantation. Data are representative of at least 2 experiments.

Comment in

  • T cells take directions from supporting cast in graft-versus-host disease

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