1. Generation of CD8+ T cell–mediated immunity against idiotype-negative lymphoma escapees
by Bindu Varghese, Adam Widman, James Do, Behnaz Taidi, Debra K. Czerwinski, John Timmerman, Shoshana Levy, and Ronald Levy
Blood
Volume 114(20):
November 12, 2009
©2009 by American Society of Hematology

2. Anti-A20 Id mAb binds A20 tumor cells and inhibits A20 tumor growth in vitro.
Anti-A20 Id mAb binds A20 tumor cells and inhibits A20 tumor growth in vitro. (A) A20 tumor cells were washed and stained with an AlexaFluor 647-labeled anti-A20 Id mAb or with an AlexaFluor 647-labeled isotype control mAb. (B) A20 tumor cells were stimulated with anti-A20 Id mAb (S) or an isotype control mAb (I) for 1 hour at 37°C. The proteins were lysed, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blotted, and probed for total tyrosine phosphorylation expression. (C) A20 cells or 38C13 cells were incubated in the presence of anti-A20 Id mAb. Cells were then pulsed with [3H]thymidine and harvested. Data are represented as mean ± SD of triplicate values.
Bindu Varghese et al. Blood 2009;114:
©2009 by American Society of Hematology

3. Anti-A20 Id mAb can inhibit A20 tumor cell growth in vivo.
Anti-A20 Id mAb can inhibit A20 tumor cell growth in vivo. (A) Balb/c mice were inoculated with 107 A20 tumor cells subcutaneously and then treated intraperitoneally with saline, anti-A20 Id mAb, or an isotype control mAb (anti-38C13 Id) 3 hours later. Numbers in parentheses indicate animals cured by therapy. (B) Balb/c mice were inoculated with 107 A20 tumor cells. Mice were then treated with anti-A20 Id mAb either 3 hours after tumor inoculation (tumor size, 0 cm2) or when tumors reached 0.5, 1.0, or 1.5 cm2 (as indicated by the arrow). Numbers in parentheses indicate animals cured by therapy. Each line represents a group consisting of 10 mice. (C) FcRKO mice were inoculated with 106 A20 tumor cells. Mice were then treated with anti-A20 Id mAb 3 hours after tumor inoculation. Each line represents a group consisting of 10 mice. (D) CD8−/− mice were inoculated with 106 A20 tumor cells. Mice were then treated with anti-A20 Id mAb 3 hours after tumor inoculation. Each line represents a group consisting of 10 mice.
Bindu Varghese et al. Blood 2009;114:
©2009 by American Society of Hematology

4. Anti-A20 Id mAb can inhibit A20 tumor cell growth in vivo, but ultimately Id-negative tumor cells escape.
Anti-A20 Id mAb can inhibit A20 tumor cell growth in vivo, but ultimately Id-negative tumor cells escape. (A) Escapee tumor cells from anti-A20 Id mAb-treated mice or A20 tumors derived from nontreated mice were stained for surface or intracellular IgG2a expression and analyzed by flow cytometry. Histograms are gated on live lymphocytes and are representative of 5 separate tumors. (B) Escapee tumor cells from anti-A20 Id mAb-treated mice or A20 tumors derived from nontreated mice were surface-stained with anti-κ light chain antibody or anti-A20 Id mAb. Graphs are gated on live lymphocytes and are representative of 5 separate tumors. (C) Escapee cells were plated for 4 days in the presence of either anti-A20 Id mAb or anti-38C13 Id mAb. Cells were then pulsed with [3H]thymidine for 12 hours and harvested. (D) Escapee tumor cells were cultured in vitro for 24 hours in complete media. Cells were then stimulated with anti-A20 Id mAb (S) or an isotype control mAb (I) for 1 hour at 37°C. The proteins were lysed, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blotted, and probed for total tyrosine phosphorylation expression. (E) Wild-type mice were inoculated with escapee tumor cells subcutaneously and then treated intraperitoneally with saline, anti-A20 Id mAb, or an isotype control mAb (anti-38C13 Id) 3 hours later. Each line represents a group consisting of 10 mice.
Bindu Varghese et al. Blood 2009;114:
©2009 by American Society of Hematology

5. Anti-A20 Id mAb + CpG combination therapy can cure large established A20 tumors.
Anti-A20 Id mAb + CpG combination therapy can cure large established A20 tumors. (A) Balb/c mice were inoculated with 107 A20 cells subcutaneously. Therapy began when tumors were 1 cm2 (day 0; as indicated by the arrows). Anti-A20 Id mAb was administered intraperitoneally on day 0, and CpG was administered intratumorally on days 2, 3, 4, 6, and 8 (P < .001). Numbers in parentheses indicate animals cured by therapy. (B) Photographs were taken of mice (on day 15) shown in panel A demonstrating that anti-A20 Id mAb + CpG combination therapy was superior to CpG or antibody alone in curing mice of large established tumors. The mice in each panel are representative of the group from which they are taken.
Bindu Varghese et al. Blood 2009;114:
©2009 by American Society of Hematology

6. Anti-A20 Id mAb + CpG combination therapy cures large established A20 tumors using a CD8-dependent, CD4-independent mechanism.
Anti-A20 Id mAb + CpG combination therapy cures large established A20 tumors using a CD8-dependent, CD4-independent mechanism. (A-B) CD8−/− mice were inoculated with 106 A20 tumor cells. When tumors reached 1 cm2 (day 0), mice were treated once intraperitoneally with anti-A20 Id mAb. CpG was given intratumorally on days 2, 3, 4, 6, and 8. Numbers in parentheses indicate animals cured by therapy. Each line in panel B represents a group consisting of 10 mice. (C-D) Balb/c mice were inoculated with 106 A20 tumor cells. Anti-CD4 mAb was administered on days −3, −2, −1, 7, 14, 28, and 35. When tumors reached 1 cm2 (day 0), mice were treated once intraperitoneally with anti-A20 Id mAb. CpG was given intratumorally on days 2, 3, 4, 6, and 8. Numbers in parentheses indicate animals cured by therapy. Each line in panel D represents a group consisting of 10 mice. (E) Splenocytes were isolated from naive mice or from tumor-bearing mice that were cured with anti-A20 Id mAb + CpG and had survived for 100 days. Splenocytes were used as effecter cells against 51Cr-labeled A20 target cells at the indicated ratio for 4 hours, and specific release of 51Cr was measured. Each line is representative of 4 mice.
Bindu Varghese et al. Blood 2009;114:
©2009 by American Society of Hematology

7. Anti-A20 Id + CpG combination therapy can mediate tumor destruction at local and distant tumor sites.
Anti-A20 Id + CpG combination therapy can mediate tumor destruction at local and distant tumor sites. (A) Balb/c mice were inoculated with 107 A20 cells subcutaneously on2 different locations on the abdomen. Therapy was administered when tumors were 1 cm2 (day 0). Anti-A20 Id mAb was administered intraperitoneally on day 0, and CpG was administered intratumorally on days 2, 3, 4, 6, and 8 in only a single tumor (P < .001). Each line is representative of a group consisting of 10 mice. (B) Photographs were taken of mice shown (on day 15) in panel A demonstrating that anti-A20 Id mAb + CpG combination therapy was superior to CpG or antibody alone in inhibiting the growth of both treated and untreated A20 tumors. ↑ points to the treated tumor.
Bindu Varghese et al. Blood 2009;114:
©2009 by American Society of Hematology

8. The anti-A20 Id mAb + CpG combination therapy prevents the emergence of Id-negative tumor escapees.
The anti-A20 Id mAb + CpG combination therapy prevents the emergence of Id-negative tumor escapees. Mice that were cured from anti-A20 Id mAb + CpG combination therapy or naive Balb/c mice were inoculated with 3 different tumor cell types subcutaneously on 3 separate locations of each mouse: 107 A20 cells, 107 escapee A20 cells, or 5 × 105 CT26. Each line is representative of a group consisting of 10 mice.
Bindu Varghese et al. Blood 2009;114:
©2009 by American Society of Hematology