Jane Seagal

Germinal centers (GCs) represent the main sites for the generation of high-affinity, class-switched antibodies during T cell-dependent antibody responses. To study gene function specifically in GC B cells, we generated Cgamma1-cre mice in which the expression of Cre recombinase is induced by transcription of the Ig gamma1 constant region gene segment (Cgamma1). In these mice, Cre-mediated recombination at the fas, Igbeta, IgH, and Rosa26 loci occurred in GC B cells as early as 4 days after immunization with T cell-dependent antigens and involved >85% of GC B cells at the peak of the GC reaction. Less than 2% of IgM(+) B cells showed Cre-mediated recombination. These cells carried few Ig somatic mutations, expressed germ-line Cgamma1- and activation-induced cytidine deaminase-specific transcripts and likely include GC B cell founders and/or plasma cell precursors. Cre-mediated recombination involved most IgG1, but also a fraction of IgG3-, IgG2a-, IgG2b-, and IgA-expressing GC and post-GC B cells. This result indicates that a GC B cell can transcribe more than one downstream C(H) gene before undergoing class switch recombination. The efficient induction of Cre expression in GC B cells makes the Cgamma1-cre allele a powerful tool for the genetic analysis of these cells, as well as, in combination with a suitable marker for Cre-mediated recombination, the tracking of class-switched memory B and plasma cells in vivo. To expedite the genetic analysis of GC B cells, we have established Cgamma1-cre F(1) embryonic stem cells, allowing further rounds of gene targeting and the cloning of compound mutants by tetraploid embryo complementation.

While immune checkpoint blockade leads to potent antitumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in inflammation of multiple organs, including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA4 antibody (anti-CTLA4 DVD, where CTLA4 is defined as cytotoxic T lymphocyte-associated antigen-4) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA4-binding domain. Upon reaching the tumor, the outer domain was cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA4 blockade. Anti-CTLA4 DVD markedly reduced multiorgan immune toxicity by preserving tissue-resident Tregs in Rag 1-/- mice that received naive donor CD4+ T cells from WT C57BL/6j mice. Moreover, anti-CTLA4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2-bearing C57BL/6j mice. Treg depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA4 without the FcγR-binding portion (anti-CTLA4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrate an approach to anti-CTLA4 blockade that depletes tumor-infiltrating, but not tissue-resident, Tregs, preserving antitumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA4 DVD provides an avenue for uncoupling antitumor efficacy from immunotherapy-induced toxicities.