Figure 5.
Depletion of A2-specific hybridoma cells by A2Fc in vivo. (A) TKO mice were injected with 1 million PA2.1 cells per mouse intravenously followed by no treatment or A2Fc or A2Fc-LALAPG via intraperitoneal injection at 0.4 mg per mouse. The untreated group did not receive any placebo. Mice were bled before cell transfer and treatment (Pre) and then weekly thereafter. Mice were followed for survival, and serially collected serum specimens were tested by crossmatching with splenocytes from A2 transgenic mice. (B) Representative crossmatching on A2 transgenic splenocytes. Cells were stained with serum from a mouse prior to injection of PA2.1 cells (prebleed; purple) or serum from a mouse that carried PA2.1 cells for 1 week but received no HLA-Fc treatment (gray), followed by staining with phycoerythrin (PE)−conjugated anti-mouse IgG (mIgG). Gating on CD3+ T cells allowed detection of anti-A2 antibody, which could be quantified by the median channel shift (MCS) of fluorescent intensity relative to the prebleed specimen. (C) Levels of circulating anti-A2 antibody monitored over time in TKO recipients of PA2.1 cells that were untreated or treated with A2Fc or A2Fc-LALAPG, as measured by crossmatching against A2+ splenocytes, represented as the median channel shift. Data are from 5 to 6 mice per group from 3 experiments. Mean ± SD. (D) Survival of mice following PA2.1 cell infusion and treatment with A2Fc or A2Fc-LALAPG or no treatment. Data are from 6 mice per group from 3 experiments. (E) Multifocal tumors formed by hybridoma cells in the liver of representative untreated and A2Fc-LALAPG−treated mice (gross examination, upper panel; microscopic examination of hematoxylin and eosin−stained sections, middle and lower panels). (F) Immunogenicity of A2Fc and A2Fc-LALAPG. B6 mice were treated with the same dose of A2Fc or A2Fc-LALAPG via intraperitoneal injection as in (C), and anti-A2 levels in sera collected before and weekly after the treatment were measured by crossmatching against A2+ splenocytes. Median channel shifts are from 6 mice per group from 2 experiments. Mean ± SD; SDs for the A2Fc group were smaller than the symbols in the plot. ∗P < .05; ∗∗P < .01, compared with the A2Fc-treated group by Mann-Whitney test (C,F) and log-rank test (D).

Depletion of A2-specific hybridoma cells by A2Fc in vivo. (A) TKO mice were injected with 1 million PA2.1 cells per mouse intravenously followed by no treatment or A2Fc or A2Fc-LALAPG via intraperitoneal injection at 0.4 mg per mouse. The untreated group did not receive any placebo. Mice were bled before cell transfer and treatment (Pre) and then weekly thereafter. Mice were followed for survival, and serially collected serum specimens were tested by crossmatching with splenocytes from A2 transgenic mice. (B) Representative crossmatching on A2 transgenic splenocytes. Cells were stained with serum from a mouse prior to injection of PA2.1 cells (prebleed; purple) or serum from a mouse that carried PA2.1 cells for 1 week but received no HLA-Fc treatment (gray), followed by staining with phycoerythrin (PE)−conjugated anti-mouse IgG (mIgG). Gating on CD3+ T cells allowed detection of anti-A2 antibody, which could be quantified by the median channel shift (MCS) of fluorescent intensity relative to the prebleed specimen. (C) Levels of circulating anti-A2 antibody monitored over time in TKO recipients of PA2.1 cells that were untreated or treated with A2Fc or A2Fc-LALAPG, as measured by crossmatching against A2+ splenocytes, represented as the median channel shift. Data are from 5 to 6 mice per group from 3 experiments. Mean ± SD. (D) Survival of mice following PA2.1 cell infusion and treatment with A2Fc or A2Fc-LALAPG or no treatment. Data are from 6 mice per group from 3 experiments. (E) Multifocal tumors formed by hybridoma cells in the liver of representative untreated and A2Fc-LALAPG−treated mice (gross examination, upper panel; microscopic examination of hematoxylin and eosin−stained sections, middle and lower panels). (F) Immunogenicity of A2Fc and A2Fc-LALAPG. B6 mice were treated with the same dose of A2Fc or A2Fc-LALAPG via intraperitoneal injection as in (C), and anti-A2 levels in sera collected before and weekly after the treatment were measured by crossmatching against A2+ splenocytes. Median channel shifts are from 6 mice per group from 2 experiments. Mean ± SD; SDs for the A2Fc group were smaller than the symbols in the plot. ∗P < .05; ∗∗P < .01, compared with the A2Fc-treated group by Mann-Whitney test (C,F) and log-rank test (D).

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