Tolerance to MHC class II disparate allografts through genetic modification of bone marrow

Abstract

Induction of molecular chimerism through genetic modification of bone marrow is a powerful tool for the induction of tolerance. Here, we demonstrate for the first time that expression of an allogeneic MHC class II gene in autologous bone marrow cells, resulting in a state of molecular chimerism, induces tolerance to MHC class II mismatched skin grafts, a stringent test of transplant tolerance. Reconstitution of recipients with syngeneic bone marrow transduced with retrovirus encoding H-2I-A(b) (I-A(b)) resulted the long-term expression of the retroviral gene product on the surface of MHC class II-expressing bone marrow-derived cell types. Mechanistically, tolerance was maintained by the presence of regulatory T cells, which prevented proliferation and cytokine production by alloreactive host T cells. Thus, the introduction of MHC class II genes into bone marrow-derived cells through genetic engineering results in tolerance. These results have the potential to extend the clinical applicability of molecular chimerism for tolerance induction.

Figure 1. Bicistronic retroviral vector encoding MHC class II confers cell surface expression of I-A^b, and transduces murine bone marrow

(A) Schematic of the bicistronic retroviral construct containing the I-A^b β gene fused to GFP, an internal ribosomal entry site and I-A^b α. Control virus encodes GFP alone (B) A20 cells were transduced with VSV-I-A^b (right panels) or mock transduced (left panels) and cell surface expression of the I-A^b α chain (upper panels) or the I-A^b/ peptide complex (lower panels) was measured by cell surface staining and flow cytometry. Mock transduced A20 cells were used as staining controls. (C) Bone marrow cells were harvested from mice treated 7 days prior with 5-FU, and transduced with VSV-GFP control virus (left panel) or VSV-I-A^b(right panel). 72 hours later, transduction was monitored via GFP fluorescence and flow cytometry. Shown is one representative of three independent experiments.

Figure 2. Long-term multi-lineage expression of retrovirally encoded proteins

(A) 4 weeks after reconstitution with bone marrow transduced with VSV-I-A^b (left column) or mock transduced (right column), B10.MBR mice were bled, and peripheral blood mononuclear cells (PBMC) were examined by cell surface staining with the lineage markers CD4, CD8, B220, CD11b and CD11c. Cells were then analyzed by flow cytometry. Shown is one representative of three independent experiments. (B) 11 weeks mice were reconstituted with bone marrow transduced with VSV-I-A^b, PBMC were harvested and stimulated with LPS. After 72 hours, cells were stained with antibodies specific for IA^b, IA^k, B220 and CD3 prior to analysis by flow cytometry. Shown are B220+ cells (upper panels) gated on transduced GFP+ cells (solid black line) or GFP- cells (solid gray line. Cells from naïve B10.MBR stimulated with LPS were used as a control (dashed line). Also shown are GFP+ (black line) and GFP- (gray line) CD3+ T cells (lower panel). (C) 24 weeks after bone marrow reconstitution, recipients of VSV-I-A^b (left panel) or mock (right panel) transduced bone marrow were bled, and GFP and IA^b expression in lymphocytes was analyzed by flow cytometry. Shown is one representative of three independent experiments.

Figure 3. Expression of retrovirally encoded MHC class II proteins induces specific tolerance to skin grafts

(A) 8-10 weeks after bone marrow reconstitution, B10.MBR recipients of VSV-I-A^b (triangles) or VSV-GFP (squares) transduced bone marrow received MHC class II mismatched B10.QBR skin grafts. (B) B10.MBR recipients of VSV-I-A^b (triangles) or VSV-GFP (diamonds) transduced bone marrow also received BALB/c skin grafts. Shown are the combined results of 3 independent experiments.

Figure 4. Expression of retrovirally encoded MHC class II prevents T cell proliferation in response to MHC class II mismatched splenocytes

B10.MBR mice were reconstituted with VSV-I-A^b (I-Ab) or control VSV-GFP (GFP) transduced bone marrow. Recipients were immunized with 10⁷ irradiated B10.QBR splenocytes and then sacrificed 10 days later. (A) Splenocytes were CFSE labeled and stimulated with irradiated syngeneic B10.MBR (dotted line), or allogeneic B10.QBR (solid line)or BALB/c dashed line. Proliferation was monitored by flow cytometry after 72 hours. (B). The results of the experiment in panel A expressed percent proliferation. VSV-I-A^b (white bars) or VSV-GFP (black bars). Shown is one representative experiment of two. Each experiment assayed 2-3 individual mice per group.

Figure 5. Expression of retrovirally encoded MHC class II prevents T cell cytokine production in response to MHC class II mismatched splenocytes

B10.MBR mice were reconstituted with VSV-I-A^b (white bars) or control VSV-GFP (black bars) transduced bone marrow. 8-10 weeks after reconstitution, mice were immunized with 10⁷ irradiated B10.QBR splenocytes and sacrificed 10 days later. (A) Splenocytes were re-stimulated with irradiated B10.QBR splenocytes and analyzed by ELISPOT for IL-2, IL-4 or IFN-γ. Data are presented as spots per 10⁶ T cells for each cytokine examined. Shown are the cumulative mean values and standard deviations obtained from three separate experiments. (B) Splenocytes were re-stimulated with B10.QBR or syngeneic B10.MBR splenocytes, and after 48 hours, supernatants were collected and analyzed for cytokine expression by Luminex assay. Background response to B10.MBR has been subtracted from the results shown. Shown are the cumulative mean values and standard deviations obtained from three separate experiments.

Figure 6. Tolerance induced by retrovirally encoded MHC class II is dependent on regulatory T cells

A. Frequency of CD4+CD25+ T cells in mice that received VSV-GFP (GFP) or VSV-I-A^b before and after administration of PC61. Immediately prior to injection with PC61, recipients of bone marrow transduced with VSV-GFP (GFP) or VSV-I-A^b were bled, and PBMC were examined by cell surface staining and flow cytometry for expression of CD4 and CD25. N=5 for all groups B. 4 weeks after bone marrow reconstitution, B10.MBR recipients of VSV-I-A^b transduced bone marrow received MHC class II mismatched B10.QBR skin grafts. 56 days after skin grafting, mice were treated with anti-CD25 antibody PC61 (0.125 mg/mouse administered every other day), circles. N=6 Control mice that did not receive antibody are represented by squares. N=8. Shown are combined results of three independent experiments.