Aggregated mAb 36–71 requires B cells to induce early proliferation of CA30 T cells.

<p>(A) Adoptive transfer scheme designed to test early CA30 T cell proliferation after B cell depletion. (B) B220+ cell numbers at day 5. (C) CA30 T cell numbers at day 5. Data are representative of two independent experiments. Statistical differences were determined by a two-tailed Mann-Whitney <i>U</i> test with n = 5 mice per group. Data are representative of 2 independent experiments using n ≥ 5 mice per group.</p

Removing the Fc from deaggregated mAb 36–71 ablates early proliferation of CA30 T cells.

<p>(A) Adoptive transfer and immunization protocol using deaggregated intact mAb 36–71 (100 μg) or the molar equivalent of its Fab (66 μg) or F(ab’)₂ (73 μg) fragments, all deaggregated. (B) Representative CFSE histograms for CD4+ CD45.1+ (CA30) cells pregated in the CD4+, MHC II-, CD19-, CD8α-, F4/80- gate. (C) Graph of percentage of CD4+ CD45.1+ (CA30) cells with SEM in each cell cycle division gate as defined by the FlowJo CFSE proliferation algorithm. Data presented are from a single experiment with mice treated with deaggregated (n = 5), Fab (n = 5), or F(ab’)₂ (n = 5) mAb 36–71. Statistical differences were determined by a two-tailed Mann-Whitney <i>U</i> test (* = p<0.05, ** = p<0.005). Data are representative of 2 independent experiments using n > 4 mice per group.</p

Heat-aggregated and immune complexes of mAb 36–71 elicit an IgG anti-Id response without requiring adjuvant.

<p>(A) Adoptive transfer and immunization scheme. (B) Serum IgG anti-idiotypic antibodies directed against the Vκ of mAb 36–71 were quantified using DELFIA as described in Materials and Methods. Connected lines denote a single mouse. Data are representative of two independent experiments with n = 5 mice per group. (C) Table summarizing results from 2 experiments with n = 5 mice per group with positive titers at each time point by treatment. Both experiments had 3 mice with positive titers at d42 and 5 mice with positive titers at d63 in both the heat-aggregated and immune complex groups.</p

Immunogenicity of Isogenic IgG in Aggregates and Immune Complexes

<div><p>A paradox in monoclonal antibody (mAb) therapy is that despite the well-documented tolerogenic properties of deaggregated IgG, most therapeutic IgG mAb induce anti-mAb responses. To analyze CD4 T cell reactions against IgG in various physical states, we developed an adoptive transfer model using CD4+ T cells specific for a Vκ region-derived peptide in the hapten-specific IgG mAb 36–71. We found that heat-aggregated or immune complexes (IC) of mAb 36–71 elicited anti-idiotypic (anti-Id) antibodies, while the deaggregated form was tolerogenic. All 3 forms of mAb 36–71 induced proliferation of cognate CD4+ T cells, but the aggregated and immune complex forms drove more division cycles and induced T follicular helper cells (T_FH) development more effectively than did the deaggregated form. These responses occurred despite no adjuvant and no or only trace levels of endotoxin in the preparations. Physical analyses revealed large differences in micron- and nanometer-sized particles between the aggregated and IC forms. These differences may be functionally relevant, as CD4+ T cell proliferation to aggregated, but not IC mAb 36–71, was nearly ablated upon peritoneal injection of B cell-depleting antibody. Our results imply that, in addition to denatured aggregates, immune complexes formed <i>in vivo</i> between therapeutic mAb and their intended targets can be immunogenic.</p></div

Deaggregated mAb 36–71 suppresses a humoral anti-Id response against mAb 36–71.

<p>(A) Adoptive transfer and immunization scheme. Primary and secondary injections were as specified in the figure. Naïve mice were B6AF1 mice that received CA30 cells, but no primary or secondary injection. (B) Mean titration curves (above) and concentrations (below) of serum IgG anti-Id at day 21. Each point represents an experimental mouse (n = 5/group). Results representative of 2 independent experiments with n = 5 mice per group.</p

Aggregated and IC mAb 36–71 drive CA30 T cells through more division cycles than does the deaggregated form.

<p>(A) Adoptive transfer scheme. (B) Representative FACS plots of CFSE and CD45.1 staining in the CD4+, MHC II-, CD19-, CD8α-, F4/80- gate and representative CFSE histograms for CD4+ CD45.1+ (CA30) cells. (C) Graph of percentage or total numbers (D) of CD4+ CD45.1+ (CA30) cells with SEM in each cell cycle division gate as defined by the FlowJo CFSE proliferation algorithm. Data presented are from a single experiment with mice treated with deaggregated (n = 7), immune complex (n = 7), or heat aggregated (n = 4) mAb 36–71. Statistical differences were determined by a two-tailed Mann-Whitney <i>U</i> test (* = p<0.05, ** = p<0.005). Data are representative of 3 independent experiments using n ≥ 4 mice per group.</p