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Blood, Vol. 109, Issue 11, 4810-4815, June 1, 2007
Eradication of lymphoma by CD8 T cells following anti-CD40 monoclonal antibody therapy is critically dependent on CD27 costimulation
Blood French et al.
109: 4810
Supplemental materials for: French et al, Vol 109, Issue 11, 4810-4815
Files in this Data Supplement:
- Figure S1. TAN1-6 (anti-CD70) and AT113-2 (anti–4-1BBL) block the 4-1BBL/4-1BB and CD70/CD27 interactions, respectively, in vitro and during in vivo responses (PDF, 30 KB) -
(A-B) BALB/c splenocytes were activated for 48 hours with 1 µg/mL anti-CD3 to up-regulate the expression of CD27 and 4-1BB on T cells. (A) The binding of soluble CD70-Fc (sCD70) fusion protein (1.125 µg/mL) to the activated splenocytes was detected using the FITC-labelled anti–human Fc mAb (SB2H2, 10 µg/mL) in the presence of AT113-2 (solid line) or TAN1-6 (dashed line) (50 µg/mL). (B) The binding of soluble 4-1BBL-Fc (s4-1BBL) fusion protein (1.125 µg/mL) to the cells was detected using the FITC-labeled anti–human Fc mAb SB2H2 in the presence of TAN1-6 (solid line) or AT113-2 (dashed line) (50 µg/mL). (A-B) The filled histogram shows control staining with human IgG. Note that in panel B the dashed line overlays the outline of the filled histogram and therefore is not visible. To confirm that the mAbs were able to block responses in vivo, we investigated their effect on the endogenous CD8 response to ovalbumin (OVA) in C57BL/6 mice (Figure 3C). (C) The endogenous OVA-specific CD8 T-cell response in C57BL/6 mice was monitored following intraperitoneal administration of OVA (5 mg) and anti-CD40 (0.5 mg) in combination with control IgG (anti-A31 idiotype), anti-CD70 (TAN1-6), or anti–4-1BBL (AT113-2) mAb (0.5 mg of each). After 24 hours, the mice received a repeat injection of control, anti-CD70, or anti–4-1BBL mAb. Six days later, OVA-specific T cells in the peripheral blood were detected by staining with anti-CD8 and H-2 Kb SIINFEKL tetramer. Error bars represent mean ± SEM with 3 mice per group. As shown previously,14 the CD40-mediated expansion of endogenous OVA-specific CD8 T cells was almost completely inhibited by anti-CD70 mAb. In comparison, the anti–4-1BBL mAb resulted in a 50% reduction in the level of anti-CD40–induced OVA-specific T cells.
- Figure S2. The expression of CD70 and 4-BBL on BCL1 tumor cells (PDF, 37.2 KB) -
Expression was detected using biotinylated anti-CD70 (TAN1-6) or anti–4-1BBL (AT113-2).
- Figure S3. Anti-CD27 mAb stimulates the proliferation of OT-I T cells in vivo (PDF, 24.4 KB) -
One day after adoptive transfer of 4 × 106 CD8+ OT-I lymphocytes (day 0), the recipient C57BL/6 mice were immunized intravenously with 30 nmol OVA peptide (SIINFEKL). The animals also received 4 intravenous injections (on days 0, 1, 2, and 3; 250 µg per injection) of recombinant soluble CD70 or anti–mouse CD27 (or control mAb). For tracking of OVA-specific CD8+ T cells in vivo, blood samples were stained with PE H-2Kb OVA257–264 tetramer and APC anti-CD8 . Expansion of OVA-specific CD8+ T cells is shown on day 4, at the peak of the response.
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