A novel role for the semaphorin Sema4D in the induction of allo-responses

Abstract

Sema4D (CD100), a member of the neuro-semaphorin family of proteins, has recently been shown to play a role in modulating certain immune responses. We tested the requirement of Sema4D expression on T cells in the induction of T cell allo-immune responses. Sema4D-/- T cells showed reduced expansion in vitro upon stimulation with allo-geneic antigen presenting cells (APCs) when compared to wild-type (wt) T cells. Similar in vitro results were observed using anti-Sema4D mAbs. Further studies demonstrated that the reduced proliferation was not due to intrinsic T cell defects, and that the cytotoxic functions were preserved. After allo-geneic bone marrow transplant (BMT), recipients of Sema4D-/- T cells showed reduced mortality and graft-versus-host disease (GVHD) target organ damage. Allo-geneic dendritic cells (DCs) cocultured with Sema4D-/- responder T cells secreted less TNF-alpha and IL-12p70 compared to wt T cells. Similar reduction of DC function was observed with anti-Sema4D mAbs. Given the preservation of CTL function we evaluated graft-versus-leukemia (GVL) responses. When BALB/c recipient mice were challenged with the P815 murine mastocytoma cell line (H2(d)) the recipients of allo-geneic Sema4D-/- B6 T cells showed a significant improvement in tumor free survival when compared to syngeneic recipients, thus demonstrating preservation of GVL, albeit of a lesser magnitude than allo-geneic wt T cells. In summary, Sema4D plays a significant role in mediating in vitro and in vivo allo-geneic responses by modulating T cell-APC interactions.

FIGURE 1. Deficiency of Sema4D decreases cytokine production of Tcells and DCs and lowers T cell expansion in an allogeneic in vitro setting

BALB/c stimulators and T cells from either B6 wt (black bars, allogeneic), Sema4D −/− (grey bars, allogeneic), or BALB/c Tc (white bars, syngeneic) were used., Fig 1a-c, BALB/c splenocytes irradiated and co-cultured with either BALB/c, B6 wt, or Sema4D −/− Tcells at a 2/1 stimulator/responder ratio. (a)- proliferation (72 hours), (b)- IL-2 production (48 hours), (c)- IFNγ (48 hours). (d-e) TNFα and IL12p70 levels measured from supernatants in 18-24 hours co-cultures with non-irradiated CD11c+ BALB/c DCs and BALB/c T cells (white bar), B6 wt T cells (black bars), Sema4D −/− Tcells (gray bar) at a 2:1 ratio. *p<0.01 **p<0.02

FIGURE 2. Maintenance of CTL by Sema4D−/− T cells

B6 wt (allogeneic H2^b) or Sema4D−/− (allogeneic H2^b) CD90+ T cells cells were cultured in vitro for 5 days with irradiated BALB/c (H2^d) splenocytes (a) or transplanted into irradiated BALB/c mice (b) as described in materials and methods. Splenocytes were harvested from the cultures on day +5-6 in the in vitro priming experiments (a) or from recipients (n=5/group) on day +14 after BMT. CTL assays, normalized for total donor (H2^b) CD8⁺ cells, and used in a ⁵¹Cr-release assay. Cytotoxic T lymphocyte activity against allo targets (P815) in wt allo-control (□) vs. Sema4D−/− allo (▲) groups. Lysis in allogeneic groups was similar, whereas no significant lysis of syngeneic targets (EL-4) observed in either of wt (●) or Sema4D−/− (◇)groups . Data from one of two similar experiments shown.

FIGURE 3. Sema4D −/− Tcell recipients have improved survival and clinical GVHD scores

(a-f) BALB/c (allogeneic) or B6 (syngeneic) recipients were lethally irradiated (800cGy) and intravenously given 5.0×10⁶ B6 wt BM and either 5.0×10⁵ wt B6 or Sema4D −/− T cells. (a-c) On day +14 mice were humanely euthanized and (a) spleens of recipient animals were analyzed for total H2^b CD3+ Tcells (b) serum IFNγ levels (c) CD4+ and CD8+ donor derived (H2^b) chimerism. (d) survival (e) % weight loss and (f) GVHD scores compared to d+0. (d-f) B6 (solid line), Sema4D −/− Tcells (dashed line) and BALB/c (dotted line). # p<0.01 *p<0.05 **p<0.02 ***p<0.06. Results are representative of one of three similar experiments with a total of n =18 / allogeneic recipients of the control and sema4−/− donors.

FIGURE 4. Sema4D−/− T cell recipients have improved immune reconstitution and decreased GVHD target organ pathology

(a-e) BALB/c (allogeneic) or B6 (syngeneic) recipients were irradiated with 800cGy and intravenously given 5.0×10⁶ B6 wt BM and either 5.0×10⁵ wt B6 or Sema4D−/− T cells. n=4syn, n=6 allogeneic control, n=6 Sema4D−/− (a-c) Pathology scores of GVHD target organs on day +60 post BMT as described in materials and methods of (a) liver (b) small and large intestine, and (c) skin. (d) Splenic cellularity on d+60 post BMT. (e) Donor (H2^b) CD3+, CD4+ and CD8+ from recipient spleens on d+60. Syngeneic recipients (white bars), allogeneic B6 wt Tc (black bars) and allogeneic recipients of Sema4D −/− B6 Tc recipients (gray bars). One of two similar experiments. *p<0.03

FIGURE 5. Sema4D−/− GVL effects

BALB/c (syngeneic H2^d), B6 wt (allogeneic H2^b) or Sema4D−/− (allogeneic H2^b) recipients were irradiated with 800cGy and intravenously given 5.0×10⁶ BM cells from BALB/c (syngeneic) or wt B6 (allogeneic groups) mice, plus either 5.0×10⁵ wt B6 (allogeneic control), Sema4D−/− T cells (experimental) or BALB/c T cells (syngeneic control). 400 P815 tumor cells were added into the BM inoculum of all groups. A syngeneic [BALB/c→BALB/c] control group did not receive tumor. A) BLI images show progression of tumor growth systemically over 24 days n=4/group. Tumor burden in Sema4D−/− T cell recipients is similar to B6 wt T cell recipients. (B) Mean values ± SEM for photon flux/animal (n = 4 mice). (C and D) Tumor free survival n=6 syngeneic without tumor, n=10 syngeneic + P815, n=10 allo B6 wt T cell recipients + P815 and n=10 allo Sema4D−/− T cell recipients + 400 P815(C) or 1000 P815 (D). Improved tumor free survival in allogeneic T cell recipients compared to the syngeneic recipients in both tumor challenges. Sema4D−/− allogeneic T cell recipients had a similar GVL effect as wt T cell control allogeneic recipients. *p<0.01