MHC mismatch results in neural progenitor cell rejection following spinal cord transplantation in a model of viral-induced demyelination

Abstract

Transplantation of syngeneic neural progenitor cells (NPCs) into mice persistently infected with the JHM strain of mouse hepatitis virus (JHMV) results in enhanced differentiation into oligodendrocyte progenitor cells that is associated with remyelination, axonal sparing, and clinical improvement. Whether allogeneic NPCs are tolerated or induce immune-mediated rejection is controversial and poorly defined under neuroinflammatory demyelinating conditions. We have used the JHMV-induced demyelination model to evaluate the antigenicity of transplanted allogeneic NPCs within the central nervous system (CNS) of mice with established immune-mediated demyelination. Cultured NPCs constitutively expressed the costimulatory molecules CD80/CD86, and IFN-γ treatment induced expression of MHC class I and II antigens. Injection of allogeneic C57BL/6 NPCs (H-2b background) led to a delayed type hypersensitivity response in BALB/c (H-2d background) mice associated with T-cell proliferation and IFN-γ secretion following coculture with allogeneic NPCs. Transplantation of MHC-mismatched NPCs into JHMV-infected mice resulted in increased transcripts encoding the T-cell chemoattractant chemokines CXCL9 and CXCL10 that correlated with increased T-cell infiltration that was associated with NPC rejection. Treatment of MHC-mismatched mice with T-cell subset-specific depleting antibodies increased survival of allogeneic NPCs without affecting commitment to an oligodendrocyte lineage. Collectively, these results show that allogeneic NPCs are antigenic, and T-cells contribute to rejection following transplantation into an inflamed CNS suggesting that immunomodulatory treatments may be necessary to prolong survival of allogeneic cells.

Figure 1. GFP-NPCs express MHC antigens and are antigenic

Cultured GFP-NPCs were treated with IFN-γ (100U/ml) for defined periods of time and MHC class I and II as well as CD80/86 determined by flow cytometry. (A) Representative flow analysis for MHC class I and II shown and (B) quantification of both class I and II induction. Data represents three independent experiments and data is shown as average+SD. (C) Representative histogram for CD80/86 staining show that IFN-γ does not increase expression of co-stimulatory antigens. (D) Dual expression of MHC class II and CD80/86 on IFN-γ-treated (24 h) GFP-NPCs. (E) Balb/c mice sensitized with subcutaneous injection of C57BL/6 splenocytes were challenged with either GFP-NPCs or C57BL/6 splenocytes and footpad swelling determined at 24 and 48 h post-injection. Footpad swelling in experimental mice was compared to unsensitized but footpad challenged mice. Data presented represents three independent experiments and data is presented as average+SD; * p<0.05, # p<0.01, ** p<0.001.

Figure 2. NPCs express MHC class and II in response to treatment with conditioned media from antigen sensitized T cells

Conditioned media (CM) was collected following exposure of T cells isolated from JHMV-immunized mice with defined CD4+ (M133-147) and CD8+ (S510-518) viral peptides for 48 h. Cultured GFP-NPCs were treated with either CD4 CM, CD8 CM, IFN-γ (100U/ml) or control media (untreated) for 18, 24, or 42 h and expression of MHC class I and II determined by flow cytometry. (A) Representative flow analysis at one time point (42 h) and (B) quantification of MHC I and II at 18, 24, and 42 h. Statistics are calculated for MHC expression on GFP-NPCs following treatment with CD4 or CD8 CM relative to treatment with IFN-γ; *p<0.05, #p<0.01. (C) Representative histogram of MHC class I and II expression at 18, 24, and 42 h. (D) The MFI for MHC I and II expression on GFP-NPCs following incubation with CD4 or CD8 CM was determined and divided by the MFI for MHC I and II following IFN-γ treatment to determine the fold increase in MFI.

Figure 3. Transplanted allogeneic GFP-NPCs are rejected following intraspinal injection into JHMV-infected mice

(A, B) Representative coronal spinal cord sections obtained at defined locations rostral and caudal to the transplantation site from JHMV-infected mice receiving either syngeneic (A) or allogeneic (B) NPCs. Experimental mice were sacrificed at either 8 or 21 days p.t. and migration/survival of transplanted cells evaluated by visualization of GFP-expression from transplanted cells. (C) At day 21 p.t. migration/survival of transplanted was cells enumerated. Dual-positive DAPI and GFP NPCs were counted in coronal sections 9mm rostral and 6mm caudal to transplant site at 3mm intervals from allogeneically transplanted (n=6) and syngeneically transplanted (n=7) mice. Arrow indicates transplantation site. Increased numbers (* p<0.05; # p<0.01) of GFP-NPCs were present within the spinal cords of syngeneic recipients compared to allogeneic. (D) GFP expression (determined by quantitative RT-PCR) is increased (** p<0.001) within the spinal cords of mice following syngeneic versus allogeneic transplant. The fold change of GFP expression was determined at d21 p.t. relative to d1 p.t. in syngeneically transplanted (day 1, n=3, d21: n=4) and allogeneically transplanted (day 1, n=3, d21: n=3) mice.

Figure 4. Increased CD4+ T cells in spinal cord of allogeneically transplanted mice

A) Flow cytometric analysis was performed on mononuclear cells isolated from spinal cord (9mm rostral and caudal to transplant site was used) of mice 8 days following allogeneic transplantation of NPCs or sham transplantation. Two spinal cords were pooled for each cell isolation. Cells were stained with PE-conjugated CD4 and PE-Cy7-conjugated CD8. The number of CD4+ and CD8+ T cells was determined in each NPC transplanted and sham transplanted group and normalized to mg of isolated spinal cord. B,C) CXCL9, CXCL10, and IFN-γ mRNA from spinal cords of mice allogeneically transplanted with GFP-NPC (n=9) or sham transplanted (n=7) at day 1 p.t. (B) and day 8 p.t. (C) was analyzed by qRT-PCR. C_t values were normalized to GAPDH and the ratio of each mRNA from allogeneic transplant to sham transplant at 8 p.t. was determined (ΔΔC_t). ΔC_t for allogeneic transplant compared to ΔC_t for sham transplant was used for calculating statistical significance, *p<0.05. Standard error is presented as ΔΔC_t.

Figure 5. Allogeneic GFP-NPCs elicit T cell response

(A) Representative flow cytometric analysis revealing >99% purity of FACS sorted CD4+ and CD8+ T cells from spleens of mice transplanted with allogeneic NPCs at day 21 p.t. (B) Purified T cells from (A) were co-cultured with GFP-NPCs and splenocytes treated with either medium or IFN-γ (100U/ml) and proliferation determined by EF670 dye expression. Statistical significance was determined by comparing the number of dividing T cells from Balb/c mice at day 21 p.t. with the number of dividing T cells from naive non-transplanted Balb/c mice (* p<0.05, # p<0.01). (C) IFN-γ production from T cells isolated from spleens of mice receiving either allogeneic NPCs or naïve mice was determined by ELISPOT following exposure to GFP-NPCs or C57BL/6 splenocytes. (D) Footpad swelling was determined at 24 and 48 h post GFP-NPC footpad injection (d21 p.t.) in naive Balb/c mice and Balb/c mice that received intraspinal transplant of GFP-NPCs.

Figure 6. Depleting T cells increases survival of allogeneic GFP-NPCs within the spinal cords of JHMV-infected mice

JHMV infected mice transplanted with either syngeneic and allogeneic GFP-NPCs were treated with anti-CD4 (GK1.5, n=5), anti-CD8 (Ly2.2, n=3), or isogenic IgG control (n=4) at d -1, 1, 3, 5, 12, and 19 p.t. and mice sacrificed at d21 p.t. Presence of NPCs was determined by immunofluorescence imaging of GFP expression. (A) Representative flow analysis of the spleen (top panel) and spinal cord (bottom panel) reveals the presence of CD4+ and CD8+ T cells following anti-CD4 or anti-CD8 treatment. (B) Representative spinal cord sections from transplanted mice treated with either CD4 or CD8 depleting antibodies showing the presence of GFP-NPCs. (C) Quantification of GFP-NPCs in transplanted mice treated with T cell depleting antibodies and numbers of GFP-NPCs determined along the length of the spinal cord both rostral and caudal to implantation site. Depletion of either CD4+ or CD8+ T cell subsets increases survival (* p<0.05, # p<0.01, ** p<0.001) in mice transplanted with allogeneic NPCs compared to mice transplanted with syngeneic NPCs. (D) Representative immunofluorescence 40× images showing PDGFRα+ (red) GFP-NPCs (green), with DAPI stained nuclei (blue), at day 21 p.t. of syngeneically transplanted mice without antibody treatment or allogeneically transplanted mice with anti-CD4 or anti-CD8 treatment.