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. 2012 Apr 15;188(8):3804-11.
doi: 10.4049/jimmunol.1102795. Epub 2012 Mar 14.

Profound depletion of host conventional dendritic cells, plasmacytoid dendritic cells, and B cells does not prevent graft-versus-host disease induction

Hongmei Li  1 , Anthony J Demetris, Jennifer McNiff, Catherine Matte-Martone, Hung Sheng Tan, David M Rothstein, Fadi G Lakkis, Warren D Shlomchik
Affiliations

Profound depletion of host conventional dendritic cells, plasmacytoid dendritic cells, and B cells does not prevent graft-versus-host disease induction

Hongmei Li et al. J Immunol. .

Abstract

The efficacy of allogeneic hematopoietic stem cell transplantation is limited by graft-versus-host disease (GVHD). Host hematopoietic APCs are important initiators of GVHD, making them logical targets for GVHD prevention. Conventional dendritic cells (DCs) are key APCs for T cell responses in other models of T cell immunity, and they are sufficient for GVHD induction. However, we report in this article that in two polyclonal GVHD models in which host hematopoietic APCs are essential, GVHD was not decreased when recipient conventional DCs were inducibly or constitutively deleted. Additional profound depletion of plasmacytoid DCs and B cells, with or without partial depletion of CD11b(+) cells, also did not ameliorate GVHD. These data indicate that, in contrast with pathogen models, there is a surprising redundancy as to which host cells can initiate GVHD. Alternatively, very low numbers of targeted APCs were sufficient. We hypothesize the difference in APC requirements in pathogen and GVHD models relates to the availability of target Ags. In antipathogen responses, specialized APCs are uniquely equipped to acquire and present exogenous Ags, whereas in GVHD, all host cells directly present alloantigens. These studies make it unlikely that reagent-based host APC depletion will prevent GVHD in the clinic.

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Figures

Figure 1
Figure 1. DT depletes cDCs in CD11c-DTR→B6 BM chimeras
(A, B) CD11c-DTR+/−→B6 CD45.1 and WT→B6 CD45.1 BM chimeras were treated a single 100ng dose of DT i.p and cohorts were sacrificed daily for 4 days to quantitate spleen and LN live lineage-CD45.2+CD11c+ cells. (A) Representative flow cytometry, gating on live/lineage- cells. (B) Numbers of live lineage-CD11c+ cells per spleen (left panel) and in cervical, axillary, inguinal and mesenteric LNs combined (right panel) in DT-treated CD11c-DTR+/− →B6 chimeras. To determine whether DT-treatment depleted cDCs beyond what is achieved with radiation, CD11c-DTR+/−→B6 CD45.1 BM chimeras were injected with DT or PBS followed by 450cGy × 2 several hours later. Splenic CD11c+ cells were enumerated 1 and 2 days after treatment. (C) Representative flow cytometry, gating on live/lineage- cells. (D) Numbers of splenic live lineage-CD11c+ cells. P<0.0120 comparing number of splenic cDCs with or without DT treatment at day +1 and day +2.
Figure 2
Figure 2. Depletion of host CD11c+ cells does not diminish CD8-mediated GVHD in the C3H.SW→B6 strain pairing
CD11c-DTR→B6 (CD45.1) chimeras (“CD11c+”) or control wt→wt chimeras (“CD11c-”) mice were injected with DT (100ng/mouse i.p.) on days -1, 0, +1, +3, +5 and +7. On day 0 chimeras were reirradiated (450cGy × 2) and reconstituted with 107 T cell-depleted C3H.SW BM cells, with or without 2×106 C3H.SW CD8 cells. An additional group of CD11c+ chimeras were transplanted but not injected with DT. (A) Weight loss. P<0.02 comparing any CD8 recipient group with its BM alone control beginning on day +25; P>0.13 comparing any CD8 recipient group to any other CD8 recipient group beginning on day +11. (B) Incidence of skin disease. P<0.04 comparing any CD8 recipient group with its BM alone control; P>0.93 comparing any CD8 recipient group to any other CD8 recipient group. (C) Organ pathology scores. * P≤0.05 compared to its BM alone control; P>0.29 comparing any CD8 recipient group to any other CD8 recipient group. Data are representative of 3 independent experiments.
Figure 3
Figure 3. Failure of cDC depletion to decrease GVHD is not due to donor T cell activation by donor APCs
Mice were transplanted as in Figure 2 except donor BM was C3H.SW β2M-/-. All mice were treated with 0.2mg anti-NK1.1 (PK136) on days -2, -1 and +7 as described (5, 8). (A) Weight loss. P<0.03 comparing CD11c+ and CD11c- DT-treated CD8 recipient groups with their BM alone controls beginning on day +27; P<0.044 comparing the CD11c+ no DT group with its BM alone control beginning on day +35. P>0.135 comparing any CD8 recipient group to any other CD8 recipient group beginning on day +15. (B) Organ pathology scores. Skin and ear GVHD were not significant, consistent with the reduced cutaneous GVHD we previously observed when donor BM is β2M-/- (5). P<0.013 comparing any CD8 recipient group with its BM alone control; P>0.23 comparing any CD8 recipient group to any other CD8 recipient group except for P=0.035 comparing colon scores in CD11c+ and CD11c- DT-treated CD8 recipients. Data are from 1 experiment.
Figure 4
Figure 4. Depletion of host CD11c+ cells does not diminish CD4-mediated GVHD in the B6bm12→(B6→B6bm12) model
CD11c-DTR→B6bm12 chimeras (CD11c+) or control wt→B6bm12 chimeras (CD11c-) mice were injected with DT (4ng/gm i.p.) on days -1, 0, +1, +3, +5 and +7. On day 0, chimeras were reirradiated (450cGy × 2) and reconstituted with T cell-depleted B6bm12 BM with or without B6bm12 splenocytes containing 106 CD4 cells. An additional CD11c+ group was transplanted without DT injection. (A) Weight loss. P≤0.0003 comparing any CD4 recipient group with its BM alone control on days +7 and +9; P>0.06 comparing any CD4 recipient group to any other CD4 recipient group at all days except day +26. (B) Serum IFN-γ levels measured by Luminex at day +7 (3 BM alone and 7 spleen cell recipients for each group). P=0.0083 comparing any CD4 recipient group to its BM alone control; P>0.7 comparing any CD4 recipient group to any other CD4 recipient group. (C) Liver pathology scores from one of two experiments. P≤0.0029 comparing any CD4 recipient group to its BM alone control; P<0.027 comparing DT-treated CD11c+ and CD11c- CD4-recipients and DT-treated and untreated CD11c+ CD4 recipients.
Figure 5
Figure 5. DT treatment of CD11c/b-DTR→B6 (Tg+) chimeras with B cell depletion, with or without pDC depletion, does not decrease GVHD
All BM chimeric hosts (Tg+ and Tg-) were DT-treated (4ng/gm i.p.) every other day from day -5 to day +5. All Tg+ chimeras received 0.25mg of anti-CD20 two weeks prior to transplantation as previously described (28). One group of Tg+ chimeras also received 0.5mg BST2 (27) i.p on days -2 and -1 to deplete pDCs (Supplemental Figure 2A). Mice were otherwise transplanted as in Figure 3. DT treatment and B cell and pDC depletion did not diminish weight loss (A) or incidence of clinical skin disease (B). P≤0.036 comparing weight change in any CD8 recipient to its BM alone control, beginning on day +20. P≥0.28 comparing weights of Tg+ + BST2 vs Tg- CD8 recipients beginning on day +13. P=0.042 and P=0.024 comparing incidence of skin disease in Tg+ CD8 recipients with or without BST2 treatment to the combined BM alone controls. P=0.09 comparing Tg- CD8 recipients and BM alone controls. Survival was inferior in Tg+ CD8 recipients, with or without pDC depletion, as compared to Tg- CD8 recipients (C; P<0.027). (D) Histopathology scores. P<0.05 comparing skin or ear scores in any CD8 recipient group versus its BM alone control. P=0.014 and P=0.083 comparing skin and ear scores (respectively) of Tg+ + BST2 to Tg- CD8 recipients. Data combined from 2 experiments, terminated on days +29 and +39; the Tg+ BM alone group was only present in the experiment which ended on day +29.
Figure 6
Figure 6. DT treatment of CD11c/b-DTR→B6bm12 (Tg+) chimeras with B cell depletion and pDC depletion does not decrease GVHD
CD11c/b-DTR→B6bm12 chimeras (Tg+) or control wt→B6bm12 chimeras (Tg-) mice were injected with DT (4ng/gm i.p.) every other day from day -5 to day +5. All Tg+ mice received 0.25mg of anti-CD20 on day -14 and with 0.5mg of BST2 i.p. on days -2 and -1 to deplete B cells and pDCs, respectively. On day 0, chimeras were reirradiated (450cGy × 2) and reconstituted with T cell-depleted B6bm12 BM with or without B6bm12 splenocytes containing 106 CD4 cells. (A) Weight change. P<0.03 comparing weights of Tg+ or WT→B6bm12 (WT) spleen cell recipients versus their BM alone controls from days +5 to +27. P>0.05 comparing Tg+ and WT spleen recipients on days +5, +7, +12 and +14. P<0.0001 comparing serum IFN-γ levels (B) at day +7 in Tg+ or Tg- spleen cell recipients versus BM alone controls (4 Tg+ and WT BM alone controls; 10 Tg+ and WT spleen cell recipients). (C) Liver histopathology scores. P=0.353 comparing spleen cell recipient groups. P=0.14 and 0.04 comparing Tg+ and WT spleen cell recipients to their BM alone controls, respectively. P=0.03 comparing Tg+ and WT spleen cell recipients. P=0.095 comparing Tg+ and WT BM alone groups. Data combined from 2 experiments.
Figure 7
Figure 7. GVHD is not reduced in CD11c:DTA recipients
(A) Shown are staining of representative Tg- and CD11c:DTA splenocytes for CD11c+MHCII+ (left panel) and BST2+B220+ pDCs (right panel). (B-D) CD11c:DTA or control CD11c-cre or ROSA26stop/flox/stop DT mice were irradiated (475cGy × 2) and reconstituted with 107 T cell-depleted C3H.SW BM with no T cells or with 2×106 C3H.SW CD8 cells. CD11c:DTA mice were also depleted of B cells and pDCs as described in Figure 5. (B) Weight change. P<0.006 comparing CD11c:DTA or control CD8 recipients with their BM alone controls from day +22 onward. (C) Survival. P<0.0001 comparing CD8 recipient groups. (D) Histopathology scores. P≤0.008 comparing skin, ear and colon scores in CD11c:DTA CD8 recipients as compared to the CD11c:DTA BM alone controls. P<0.0003 comparing skin and colon scores and P=0.053 comparing ear scores in control CD8 recipients and BM alone controls. P≥0.15 comparing all histopathology scores in CD11c:DTA and control CD8 recipients. Liver GVHD could not be evaluated due to a high baseline level of inflammation in the CD11:DT BM alone group (not shown). Data combined from two experiments with similar results.
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