Axel Kallies

Foxp3+ follicular regulatory T cells control the germinal center response

Follicular helper (TFH) cells provide crucial signals to germinal center B cells undergoing somatic hypermutation and selection that results in affinity maturation. Tight control of TFH numbers maintains self tolerance. We describe a population of Foxp3+Blimp-1+CD4+ T cells constituting 10–25% of the CXCR5highPD-1highCD4+ T cells found in the germinal center after immunization with protein antigens. These follicular regulatory T (TFR) cells share phenotypic characteristics with TFH and conventional Foxp3+ regulatory T (Treg) cells yet are distinct from both. Similar to TFH cells, TFR cell development depends on Bcl-6, SLAM-associated protein (SAP), CD28 and B cells; however, TFR cells originate from thymic-derived Foxp3+ precursors, not naive or TFH cells. TFR cells are suppressive in vitro and limit TFH cell and germinal center B cell numbers in vivo. In the absence of TFR cells, an outgrowth of non–antigen-specific B cells in germinal centers leads to fewer antigen-specific cells. Thus, the TFH differentiation pathway is co-opted by Treg cells to control the germinal center response.

IL-21 regulates germinal center B cell differentiation and proliferation through a B cell–intrinsic mechanism

Germinal centers (GCs) are sites of B cell proliferation, somatic hypermutation, and selection of variants with improved affinity for antigen. Long-lived memory B cells and plasma cells are also generated in GCs, although how B cell differentiation in GCs is regulated is unclear. IL-21, secreted by T follicular helper cells, is important for adaptive immune responses, although there are conflicting reports on its target cells and mode of action in vivo. We show that the absence of IL-21 signaling profoundly affects the B cell response to protein antigen, reducing splenic and bone marrow plasma cell formation and GC persistence and function, influencing their proliferation, transition into memory B cells, and affinity maturation. Using bone marrow chimeras, we show that these activities are primarily a result of CD3-expressing cells producing IL-21 that acts directly on B cells. Molecularly, IL-21 maintains expression of Bcl-6 in GC B cells. The absence of IL-21 or IL-21 receptor does not abrogate the appearance of T cells in GCs or the appearance of CD4 T cells with a follicular helper phenotype. IL-21 thus controls fate choices of GC B cells directly.

Plasma Cell Ontogeny Defined by Quantitative Changes in Blimp-1 Expression

Plasma cells comprise a population of terminally differentiated B cells that are dependent on the transcriptional regulator B lymphocyte–induced maturation protein 1 (Blimp-1) for their development. We have introduced a gfp reporter into the Blimp-1 locus and shown that heterozygous mice express the green fluorescent protein in all antibody-secreting cells (ASCs) in vivo and in vitro. In vitro, these cells display considerable heterogeneity in surface phenotype, immunoglobulin secretion rate, and Blimp-1 expression levels. Importantly, analysis of in vivo ASCs induced by immunization reveals a developmental pathway in which increasing levels of Blimp-1 expression define developmental stages of plasma cell differentiation that have many phenotypic and molecular correlates. Thus, maturation from transient plasmablast to long-lived ASCs in bone marrow is predicated on quantitative increases in Blimp-1 expression.

Blimp-1 Transcription Factor Is Required for the Differentiation of Effector CD8+ T Cells and Memory Responses

In response to viral infection, naive CD8(+) T cells proliferate and differentiate into cytotoxic and cytokine-producing effector cells. Here we showed that the transcription factor Blimp-1, a crucial regulator of plasma cell differentiation, was required for CD8(+) T cells to differentiate into functional killer T cells in response to influenza virus. Blimp-1 was not essential for the generation of memory T cells but was crucial for their efficient recall response upon reinfection. Antigen-specific Blimp-1-deficient CD8(+) T cells failed to appropriately regulate the transcriptional program essential for killer T cell responses and showed impaired migration to the site of infection. This study identifies Blimp-1 as a master regulator of the terminal differentiation of CD8(+) effector T cells and uncovers a conservation of the pathways that regulate the terminal differentiation of T and B cells.

The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells

Regulatory T cells (Treg cells) are required for peripheral tolerance. Evidence indicates that Treg cells can adopt specialized differentiation programs in the periphery that are controlled by transcription factors usually associated with helper T cell differentiation. Here we demonstrate that expression of the transcription factor Blimp-1 defined a population of Treg cells that localized mainly to mucosal sites and produced IL-10. Blimp-1 was required for IL-10 production by these cells and for their tissue homeostasis. We provide evidence that the transcription factor IRF4, but not the transcription factor T-bet, was essential for Blimp-1 expression and for the differentiation of all effector Treg cells. Thus, our study defines a differentiation pathway that leads to the acquisition of Treg cell effector functions and requires both IRF4 and Blimp-1.

Early appearance of germinal center–derived memory B cells and plasma cells in blood after primary immunization

Immunization with a T cell–dependent antigen elicits production of specific memory B cells and antibody-secreting cells (ASCs). The kinetic and developmental relationships between these populations and the phenotypic forms they and their precursors may take remain unclear. Therefore, we examined the early stages of a primary immune response, focusing on the appearance of antigen-specific B cells in blood. Within 1 wk, antigen-specific B cells appear in the blood with either a memory phenotype or as immunoglobulin (Ig)G1 ASCs expressing blimp-1. The memory cells have mutated VH genes; respond to the chemokine CXCL13 but not CXCL12, suggesting recirculation to secondary lymphoid organs; uniformly express B220; show limited differentiation potential unless stimulated by antigen; and develop independently of blimp-1 expression. The antigen-specific IgG1 ASCs in blood show affinity maturation paralleling that of bone marrow ASCs, raising the possibility that this compartment is established directly by blood-borne ASCs. We find no evidence for a blimp-1–expressing preplasma memory compartment, suggesting germinal center output is restricted to ASCs and B220+ memory B cells, and this is sufficient to account for the process of affinity maturation.

The development and fate of follicular helper T cells defined by an IL-21 reporter mouse

Germinal centers require CD4+ follicular helper T cells (TFH cells), whose hallmark is expression of the transcriptional repressor Bcl-6, the chemokine receptor CXCR5 and interleukin 21 (IL-21). To track the development and fate of TFH cells, we generated an IL-21 reporter mouse by introducing sequence encoding green fluorescent protein (GFP) into the Il21 locus; these mice had expression of IL-21–GFP in CD4+CXCR5+PD-1+ TFH cells. IL-21–GFP+ TFH cells were multifunctional helper cells that coexpressed several cytokines, including interferon-γ (IFN-γ), IL-2 and IL-4. TFH cells proliferated and gave rise to transferrable memory cells with plasticity, which differentiated after recall into conventional effector helper T cells and TFH cells. Thus, we demonstrated that TFH cells were not terminally differentiated but instead retained the flexibility to be recruited into other helper T cell subsets and nonlymphoid tissues.

Mouse CD8α+ DCs and human BDCA3+ DCs are major producers of IFN-λ in response to poly IC

In humans and mice, CD8α+ conventional dendritic cells are the primary source of interferon-λ released in response to the adjuvant and Toll-like receptor 3 agonist poly IC.

The transcriptional regulators IRF4, BATF and IL-33 orchestrate development and maintenance of adipose tissue-resident regulatory T cells

Foxp3+ regulatory T (Treg) cells in visceral adipose tissue (VAT-Treg cells) are functionally specialized tissue-resident cells that prevent obesity-associated inflammation and preserve insulin sensitivity and glucose tolerance. Their development depends on the transcription factor PPAR-γ; however, the environmental cues required for their differentiation are unknown. Here we show that interleukin 33 (IL-33) signaling through the IL-33 receptor ST2 and myeloid differentiation factor MyD88 is essential for development and maintenance of VAT-Treg cells and sustains their transcriptional signature. Furthermore, the transcriptional regulators BATF and IRF4 were necessary for VAT-Treg differentiation through direct regulation of ST2 and PPAR-γ expression. IL-33 administration induced vigorous population expansion of VAT-Treg cells, which tightly correlated with improvements in metabolic parameters in obese mice. Human omental adipose tissue Treg cells also showed high ST2 expression, suggesting an evolutionarily conserved requirement for IL-33 in VAT-Treg cell homeostasis.

Identification of Bcl-6-dependent follicular helper NKT cells that provide cognate help for B cell responses

Lipid antigens trigger help from natural killer T cells (NKT cells) for B cells, and direct conjugation of lipid agonists to antigen profoundly augments antibody responses. Here we show that in vivo, NKT cells engaged in stable and prolonged cognate interactions with B cells and induced the formation of early germinal centers. Mouse and human NKT cells formed CXCR5+PD-1hi follicular helper NKT cells (NKTFH cells), and this process required expression of the transcriptional repressor Bcl-6, signaling via the coreceptor CD28 and interaction with B cells. NKTFH cells provided direct cognate help to antigen-specific B cells that was dependent on interleukin 21 (IL-21). Unlike T cell–dependent germinal centers, those driven by NKTFH cells did not generate long-lived plasma cells. Our results demonstrate the existence of a Bcl-6-dependent subset of NKT cells specialized in providing help to B cells.