Antibodies are produced across multiple isotypes with distinct properties that coordinate initial antigen clearance and confer long-term antigen-specific immune protection. Here, we interrogate the molecular programs of isotype-specific murine plasma cells (PC) following helper T cell-dependent immunization and within established steady-state immunity. We developed a single-cell–indexed and targeted molecular strategy to dissect conserved and divergent components of the rapid effector phase of antigen-specific IgM + versus inflammation-modulating programs dictated by type 1 IgG2a/b + PC differentiation. During antibody affinity maturation, the germinal center (GC) cycle imparts separable programs for post-GC type 2 inhibitory IgG1 + and type 1 inflammatory IgG2a/b + PC to direct long-term cellular function. In the steady state, two subsets of IgM + and separate IgG2b + PC programs clearly segregate from splenic type 3 IgA + PC programs that emphasize mucosal barrier protection. These diverse isotype-specific molecular pathways of PC differentiation control complementary modules of antigen clearance and immune protection that could be selectively targeted for immunotherapeutic applications and vaccine design.
Adaptive B cell immunity to environmental antigens must be regulated by multiple CD4 T cell dependent tolerance mechanisms. Using integrated single cell strategies, we demonstrate that acute PD-1 blockade induces extensive and selective local anti-inflammatory IgG1 plasma cell (PC) differentiation. Expansion of pre-existing IgG1 germinal center (GC) B cell and enhanced GC programming without memory B cell involvement reveals an isotype-specific GC checkpoint that blocks steady-state IgG1 antibody maturation. While there was no adjuvant impact on immunization, acute PD-1 checkpoint blockade exaggerates anti-commensal IgG1 antibody production, alters microbiome composition and exerts its action in a CD4 T cell dependent manner. These findings reveal a PD-1 controlled adaptive B cell tolerance checkpoint that selectively constrains maturation of pre-existing anti-inflammatory antibodies to prevent over-reaction to steady-state foreign antigens.
Understanding how follicular helper T cells (TFH) regulate the specialization, maturation, and differentiation of adaptive B cell immunity is crucial for developing durable high-affinity immune protection. Using indexed-single cell molecular strategies, we reveal a skewed intra-clonal assortment of higher affinity TCR and the distinct molecular programming of the localized TFH compartment compared to emigrant conventional effector TH (ETH) cells. We find a temporal shift in BCR class switch which permits identification of inflammatory and anti-inflammatory modules of transcriptional programming that subspecialize TFH function before and during the germinal center (GC) reaction. Late collapse of this local primary GC reaction reveals a persistent post-GC TFH population which discloses a putative memory TFH program. These studies define specialized antigen-specific TFH transcriptional programs that progressively direct class-specific evolution of high-affinity B cell immunity and uncover the transcriptional program of a memory TFH population as the regulators of antigen recall.
Summary Adaptive T and B lymphocytes expand, respond, and persist across a multitude of separable cell differentiation states. Small compartments of these cells present defined cell surface phenotype, but express potentially divergent immune functions. Here, we use high resolution flow cytometry to provide direct access to rare lymphocyte subpopulations for evaluation of steady-state or reactive transcriptional programs. We sort and index single cells by phenotype in 384-well format for quantification of targeted gene amplification through RNA sequencing (single cell qtSEQ). For complete details on the use and execution of this profile, please refer to Dufaud et al. (2021) .
Antibodies are produced across multiple isotypes with distinct properties that coordinate initial antigen clearance and confer long-term antigen-specific immune protection. Here, we interrogate the molecular programs of isotype-specific murine plasma cells (PC) following helper T cell dependent immunization and within established steady-state immunity. Using integrated single cell strategies, we reveal conserved and divergent components of the rapid effector phase of antigen-specific IgM+ versus inflammation modulating programs dictated by IgG2a/b+ PC differentiation. During antibody affinity maturation, the germinal center (GC) cycle imparts separable programs for post-GC inhibitory IgG1+ and inflammatory IgG2a/b+ PC to direct long-term cellular function. In the steady-state, two subsets of IgM+ and separate IgG2b+ PC programs clearly segregate from splenic IgA+ PC programs that emphasize mucosal barrier protection. These diverse isotype-specific molecular pathways of PC differentiation control complementary modules of antigen clearance and immune protection that could be selectively targeted for immunotherapeutic applications and vaccine design.
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