Protein Amounts of the MYC Transcription Factor Determine Germinal Center B Cell Division Capacity.

High-affinity B cell selection in the germinal center (GC) is governed by signals delivered by follicular helper T (Tfh) cells to B cells. Selected B cells undergo clonal expansion and affinity maturation in the GC dark zone in direct proportion to the amount of antigen they capture and present to Tfh cells in the light zone. Here, we examined the mechanisms whereby Tfh cells program the number of GC B cell divisions. Gene expression analysis revealed that Tfh cells induce Myc expression in light-zone B cells in direct proportion to antigen capture. Conditional Myc haplo-insufficiency or overexpression combined with cell division tracking showed that MYC expression produces a metabolic reservoir in selected light-zone B cells that is proportional to the number of cell divisions in the dark zone. Thus, MYC constitutes the GC B cell division timer that when deregulated leads to emergence of B cell lymphoma.

Scavenging of soluble and immobilized CCL21 by ACKR4 regulates peripheral dendritic cell emigration.

Leukocyte homing driven by the chemokine CCL21 is pivotal for adaptive immunity because it controls dendritic cell (DC) and T cell migration through CCR7. ACKR4 scavenges CCL21 and has been shown to play an essential role in DC trafficking at the steady state and during immune responses to tumors and cutaneous inflammation. However, the mechanism by which ACKR4 regulates peripheral DC migration is unknown, and the extent to which it regulates CCL21 in steady-state skin and lymph nodes (LNs) is contested. Specifically, our previous findings that CCL21 levels are increased in LNs of ACKR4-deficient mice [I. Comerford et al., Blood 116, 4130-4140 (2010)] were refuted [M. H. Ulvmar et al., Nat. Immunol. 15, 623-630 (2014)], and no differences in CCL21 levels in steady-state skin of ACKR4-deficient mice were reported despite compromised CCR7-dependent DC egress in these animals [S. A. Bryce et al., J. Immunol. 196, 3341-3353 (2016)]. Here, we resolve these issues and reveal that two forms of CCL21, full-length immobilized and cleaved soluble CCL21, exist in steady-state barrier tissues, and both are regulated by ACKR4. Without ACKR4, extracellular CCL21 gradients in barrier sites are saturated and nonfunctional, DCs cannot home directly to lymphatic vessels, and excess soluble CCL21 from peripheral tissues pollutes downstream LNs. The results identify the mechanism by which ACKR4 controls DC migration in barrier tissues and reveal a complex mode of CCL21 regulation in vivo, which enhances understanding of functional chemokine gradient formation.

Anti-HIV-1 B cell responses are dependent on B cell precursor frequency and antigen-binding affinity.

The discovery that humans can produce potent broadly neutralizing antibodies (bNAbs) to several different epitopes on the HIV-1 spike has reinvigorated efforts to develop an antibody-based HIV-1 vaccine. Antibody cloning from single cells revealed that nearly all bNAbs show unusual features that could help explain why it has not been possible to elicit them by traditional vaccination and instead would require a sequence of different immunogens. This idea is supported by experiments with genetically modified immunoglobulin (Ig) knock-in mice. Sequential immunization with a series of specifically designed immunogens was required to shepherd the development of bNAbs. However, knock-in mice contain superphysiologic numbers of bNAb precursor-expressing B cells, and therefore how these results can be translated to a more physiologic setting remains to be determined. Here we make use of adoptive transfer experiments using knock-in B cells that carry a synthetic intermediate in the pathway to anti-HIV-1 bNAb development to examine how the relationship between B cell receptor affinity and precursor frequency affects germinal center (GC) B cell recruitment and clonal expansion. Immunization with soluble HIV-1 antigens can recruit bNAb precursor B cells to the GC when there are as few as 10 such cells per mouse. However, at low precursor frequencies, the extent of clonal expansion is directly proportional to the affinity of the antigen for the B cell receptor, and recruitment to GCs is variable and dependent on recirculation.

Early CCR6 expression on B cells modulates germinal centre kinetics and efficient antibody responses.

The CC-chemokine receptor 6 (CCR6) can be detected on naive and activated B cells. Counterintuitively, its absence accelerates the appearance of germinal centres (GCs) and increases the production of low-affinity antibodies. The detailed mechanism of CCR6 function during the humoral response has remained elusive, but previously we identified a distinct CCR6high B-cell population in vivo early after antigenic challenge. In this study, we defined this population specifically as early, activated pre-GC B cells. In accordance, we show that CCR6 is upregulated rapidly within hours on the protein or mRNA level after activation in vitro. In addition, only activated B cells migrated specifically towards CCL20, the specific ligand for CCR6. Lack of CCR6 increased the dark zone/light zone ratio of GC and led to decreased antigen-specific IgG1 and IgG2a antibody generation in a B-cell intrinsic manner in mixed bone marrow chimeras. In contrast, antigen-specific IgM responses were normal. Hence, CCR6 negatively regulates entry of activated, antigen-specific pre-GC B cells into the GC reaction.

Enhanced protective responses to a serotype-independent pneumococcal vaccine when combined with an inactivated influenza vaccine.

Streptococcus pneumoniae and influenza are the world's foremost bacterial and viral respiratory pathogens. We have previously described a γ-irradiated influenza A virus (γ-FLU) vaccine that provides cross-protective immunity against heterosubtypic infections. More recently, we reported a novel non-adjuvanted γ-irradiated S pneumoniae (γ-PN) vaccine that elicits serotype-independent protection. Considering the clinical synergism of both pathogens, combination of a serotype-independent pneumococcal vaccine with a broad-spectrum influenza vaccine to protect against both infections would have a considerable clinical impact. In the present study, we co-immunized C57BL/6 mice intranasally (IN) with a mixture of γ-PN (whole inactivated cells) and γ-FLU (whole inactivated virions) and examined protective efficacy. Co-immunization enhanced γ-PN vaccine efficacy against virulent pneumococcal challenge, which was dependent on CD4+ T-cell responses. In contrast, vaccination with γ-PN alone, co-immunization enhanced pneumococcal-specific effector T-helper 17 cell (Th17) and Th1 memory cell, promoted development of CD4+ tissue-resident memory (TRM) cells and enhanced Pneumococcus-specific antibody responses. Furthermore, co-immunization elicited significant protection against lethal influenza challenge, as well as against co-infection with both influenza and S pneumoniae. This is the first report showing the synergistic effect of combining whole cell and whole virion vaccines to both S pneumoniae and influenza as a single vaccine to protect against individual and co-infection, without compromising pathogen-specific immunity.

Tailored immune responses: novel effector helper T cell subsets in protective immunity.

Differentiation of naïve CD4⁺ cells into functionally distinct effector helper T cell subsets, characterised by distinct "cytokine signatures," is a cardinal strategy employed by the mammalian immune system to efficiently deal with the rapidly evolving array of pathogenic microorganisms encountered by the host. Since the T(H)1/T(H)2 paradigm was first described by Mosmann and Coffman, research in the field of helper T cell biology has grown exponentially with seven functionally unique subsets having now been described. In this review, recent insights into the molecular mechanisms that govern differentiation and function of effector helper T cell subsets will be discussed in the context of microbial infections, with a focus on how these different helper T cell subsets orchestrate immune responses tailored to combat the nature of the pathogenic threat encountered.

Intranasal vaccination with γ-irradiated Streptococcus pneumoniae whole-cell vaccine provides serotype-independent protection mediated by B-cells and innate IL-17 responses.

Generating a pneumococcal vaccine that is serotype independent and cost effective remains a global challenge. γ-Irradiation has been used widely to sterilize biological products. It can also be utilized as an inactivation technique to generate whole-cell bacterial and viral vaccines with minimal impact on pathogen structure and antigenic determinants. In the present study, we utilized γ-irradiation to inactivate an un-encapsulated Streptococcus pneumoniae strain Rx1 with an unmarked deletion of the autolysin gene lytA and with the pneumolysin gene ply replaced with an allele encoding a non-toxic pneumolysoid (PdT) (designated γ-PN vaccine). Intranasal vaccination of C57BL/6 mice with γ-PN was shown to elicit serotype-independent protection in lethal challenge models of pneumococcal pneumonia and sepsis. Vaccine efficacy was shown to be reliant on B-cells and interleukin (IL)-17A responses. Interestingly, immunization promoted IL-17 production by innate cells not T helper 17 (Th17) cells. These data are the first to report the development of a non-adjuvanted intranasal γ-irradiated pneumococcal vaccine that generates effective serotype-independent protection, which is mediated by both humoral and innate IL-17 responses.

Distinct chemokine receptor axes regulate Th9 cell trafficking to allergic and autoimmune inflammatory sites.

Migration of Th cells to peripheral sites of inflammation is essential for execution of their effector function. The recently described Th9 subset characteristically produces IL-9 and has been implicated in both allergy and autoimmunity. Despite this, the migratory properties of Th9 cells remain enigmatic. In this study, we examined chemokine receptor usage by Th9 cells and demonstrate, in models of allergy and autoimmunity, that these cells express functional CCR3, CCR6, and CXCR3, chemokine receptors commonly associated with other, functionally opposed effector Th subsets. Most Th9 cells that express CCR3 also express CXCR3 and CCR6, and expression of these receptors appears to account for the recruitment of Th9 cells to disparate inflammatory sites. During allergic inflammation, Th9 cells use CCR3 and CCR6, but not CXCR3, to home to the peritoneal cavity, whereas Th9 homing to the CNS during experimental autoimmune encephalomyelitis involves CXCR3 and CCR6 but not CCR3. To our knowledge, these data provide the first insights into regulation of Th9 cell trafficking in allergy and autoimmunity.

Advances in understanding the pathogenesis of autoimmune disorders: focus on chemokines and lymphocyte trafficking.

Lymphocyte trafficking is a key step in the pathogenesis of various autoimmune diseases. Recruitment of autoreactive lymphocytes to inflamed tissues is a defining feature of numerous persistent organ-specific autoimmune conditions and various therapies are now used in several of these diseases which appear to specifically block lymphocyte migration. Thus, better understanding of the molecular events involved in homing of autoreactive pathogenic lymphocytes may present novel opportunities for pharmacological intervention in autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, type-1 diabetes and psoriasis. This review describes recent progress in understanding lymphocyte trafficking in autoimmunity, focusing on the involvement of the chemokine and chemokine receptor superfamily. Possible strategies to improve therapeutics for autoimmune diseases arising from these studies are discussed.

CCR6 supports migration and differentiation of a subset of DN1 early thymocyte progenitors but is not required for thymic nTreg development.

T-cell selection and development occurs as precursor cells journey through the thymus and interact with stromal cells residing in distinct microenvironments. Although the chemokines CCL19, CCL21, CCL25 and CXCL12 are known to have major roles in intrathymic migration of thymocytes and thymocyte precursors, the significance of other chemokines such as CCL20, which is also expressed in the thymus, is unknown. This is of particular interest given that the thymus is the location of development of the natural regulatory T-cell (nTreg) population and that the CCL20 receptor CCR6 has an important role in peripheral tolerance via control of Treg cell migration. However, whether the CCL20/CCR6 axis has a role in the formation or migration of nTregs in the thymus is unknown. In this study, we addressed this by analyzing expression of CCR6/CCL20 within the thymus and assessing their role in thymocyte development using Ccr6(-/-) mice. CCL20 is predominately expressed in the thymic medulla and CCR6 expression is restricted to nTregs and a subset of early thymocyte progenitor double-negative 1 (DN1) cells (CD4(-)CD8(-)CD25(-)CD44(+)CD117(+)). Ex vivo chemotaxis assays indicated that these two subsets were apparently the sole subsets of thymocytes responsive to CCL20. The data indicate that nTreg frequencies and localization are unperturbed by deletion of Ccr6. However, in Ccr6(-/-) thymi, reduced frequencies of DN2 and DN3 cells, the thymocyte progenitor subsets that follow the DN1 stage, were apparent. Together, these data indicate that CCR6 has a supplementary role in coordination of early thymocyte precursor migration events important for normal subsequent thymocyte precursor development, but is not required for normal nTreg development.

CCR6 is transiently upregulated on B cells after activation and modulates the germinal center reaction in the mouse.

The CC-chemokine receptor 6 (CCR6) is expressed constitutively at an intermediate level on naïve B cells and is upregulated after activation on pregerminal center (GC) B cells. We hypothesized that it could be involved in the events leading to GC reaction and high-affinity antibody production, and therefore investigated the potential role of CCR6 in B-cell differentiation in vivo. After antigenic challenge of CCR6-/- mice with the T-cell-dependent antigen nitrophenyl-keyhole limpet hemocyanin (NP-KLH), GC B-cell development was found to be accelerated and the number of GC had increased significantly compared with control mice, but the antibodies produced by CCR6-/- B cells were on average of lower affinity. We conclude from these data that the CCR6/CCL20 axis has an important role in regulating the kinetics and efficiency of the GC reaction.

An immune paradox: how can the same chemokine axis regulate both immune tolerance and activation?: CCR6/CCL20: a chemokine axis balancing immunological tolerance and inflammation in autoimmune disease.

Chemokines (chemotactic cytokines) drive and direct leukocyte traffic. New evidence suggests that the unusual CCR6/CCL20 chemokine receptor/ligand axis provides key homing signals for recently identified cells of the adaptive immune system, recruiting both pro-inflammatory and suppressive T cell subsets. Thus CCR6 and CCL20 have been recently implicated in various human pathologies, particularly in autoimmune disease. These studies have revealed that targeting CCR6/CCL20 can enhance or inhibit autoimmune disease depending on the cellular basis of pathogenesis and the cell subtype most affected through different CCR6/CCL20 manipulations. Here, we discuss the significance of this chemokine receptor/ligand axis in immune and inflammatory functions, consider the potential for targeting CCR6/CCL20 in human autoimmunity and propose that the shared evolutionary origins of pro-inflammatory and regulatory T cells may contribute to the reason why both immune activation and regulation might be controlled through the same chemokine pathway.

TFR Cells Express Functional CCR6 But It Is Dispensable for Their Development and Localization During Splenic Humoral Immune Responses.

Follicular T cells including T follicular helper (TFH) and T follicular regulatory (TFR) cells are essential in supporting and regulating the quality of antibody responses that develop in the germinal centre (GC). Follicular T cell migration during the propagation of antibody responses is largely attributed to the chemokine receptor CXCR5, however CXCR5 is reportedly redundant in migratory events prior to formation of the GC, and CXCR5-deficient TFH and TFR cells are still capable of localizing to GCs. Here we comprehensively assess chemokine receptor expression by follicular T cells during a model humoral immune response in the spleen. In addition to the known follicular T cell chemokine receptors Cxcr5 and Cxcr4, we show that follicular T cells express high levels of Ccr6, Ccr2 and Cxcr3 transcripts and we identify functional expression of CCR6 protein by both TFH and TFR cells. Notably, a greater proportion of TFR cells expressed CCR6 compared to TFH cells and gating on CCR6+CXCR5hiPD-1hi T cells strongly enriched for TFR cells. Examination of Ccr6-/- mice revealed that CCR6 is not essential for development of the GC response in the spleen, and mixed bone marrow chimera experiments found no evidence for an intrinsic requirement for CCR6 in TFR cell development or localisation during splenic humoral responses. These findings point towards multiple functionally redundant chemotactic signals regulating T cell localisation in the GC.

PI3Kδ drives the pathogenesis of experimental autoimmune encephalomyelitis by inhibiting effector T cell apoptosis and promoting Th17 differentiation.

The Class IA phosphoinositide 3-kinase delta (PI3Kδ) has been implicated in multiple signaling pathways involved in leukocyte activation and hence is an attractive target in many human autoimmune diseases, including multiple sclerosis (MS). Here, using mice expressing a catalytically inactive form of the PI3Kδ subunit p110δ, we show that signaling through PI3Kδ is required for full and sustained pathology of experimental autoimmune encephalomyelitis (EAE), a Th17-driven model of MS. In p110δ-inactivated mice, T cell activation and function during EAE was markedly reduced and fewer T cells were observed in the central nervous system (CNS). The decrease in T cell activation is unlikely to be due to defects in dendritic cell (DC) function, as p110δ-inactivated DCs migrate and present antigen normally. However, significant increases in the proportion of T cells undergoing apoptosis at early stages of EAE were evident in the absence of PI3Kδ activity. Furthermore, a profound defect in Th17 cellular responses during EAE was apparent in the absence of PI3Kδ activity while Th1 responses were less affected. A highly selective PI3Kδ inhibitor, IC87114, also had greater inhibitory effects on Th17 cell generation in vitro than it did on Th1 cell generation. Thus, PI3Kδ plays an important role in Th17 responses in EAE, suggesting that small molecule inhibitors of PI3Kδ may be useful therapeutics for treatment of MS and other autoimmune diseases.

CXCR5+CD8+ T Cells Shape Antibody Responses In Vivo Following Protein Immunisation and Peripheral Viral Infection.

Crosstalk between T and B cells is crucial for generating high-affinity, class-switched antibody responses. The roles of CD4+ T cells in this process have been well-characterised. In contrast, regulation of antibody responses by CD8+ T cells is significantly less defined. CD8+ T cells are principally recognised for eliciting cytotoxic responses in peripheral tissues and forming protective memory. However, recent findings have identified a novel population of effector CD8+ T cells that co-opt a differentiation program characteristic of CD4+ T follicular helper (Tfh) cells, upregulate the chemokine receptor CXCR5 and localise to B cell follicles. While it has been shown that CXCR5+CD8+ T cells mediate the removal of viral reservoirs in the context of follicular-trophic viral infections and maintain the response to chronic insults by virtue of progenitor/stem-like properties, it is not known if CXCR5+CD8+ T cells arise during acute peripheral challenges in the absence of follicular infection and whether they influence B cell responses in vivo in these settings. Using the ovalbumin-specific T cell receptor transgenic (OT-I) system in an adoptive transfer-immunisation/infection model, this study demonstrates that CXCR5+CD8+ T cells arise in response to protein immunisation and peripheral viral infection, displaying a follicular-homing phenotype, expression of cell surface molecules associated with Tfh cells and limited cytotoxic potential. Furthermore, studies assessing the B cell response in the presence of OT-I or Cxcr5-/- OT-I cells revealed that CXCR5+CD8+ T cells shape the antibody response to protein immunisation and peripheral viral infection, promoting class switching to IgG2c in responding B cells. Overall, the results highlight a novel contribution of CD8+ T cells to antibody responses, expanding the functionality of the adaptive immune system.

ACKR4 restrains antitumor immunity by regulating CCL21.

Current immunotherapies involving CD8+ T cell responses show remarkable promise, but their efficacy in many solid tumors is limited, in part due to the low frequency of tumor-specific T cells in the tumor microenvironment (TME). Here, we identified a role for host atypical chemokine receptor 4 (ACKR4) in controlling intratumor T cell accumulation and activation. In the absence of ACKR4, an increase in intratumor CD8+ T cells inhibited tumor growth, and nonhematopoietic ACKR4 expression was critical. We show that ACKR4 inhibited CD103+ dendritic cell retention in tumors through regulation of the intratumor abundance of CCL21. In addition, preclinical studies indicate that ACKR4 and CCL21 are potential therapeutic targets to enhance responsiveness to immune checkpoint blockade or T cell costimulation.

Anti-idiotypic antibodies elicit anti-HIV-1-specific B cell responses.

Human anti-HIV-1 broadly neutralizing antibodies (bNAbs) protect against infection in animal models. However, bNAbs have not been elicited by vaccination in diverse wild-type animals or humans, in part because B cells expressing the precursors of these antibodies do not recognize most HIV-1 envelopes (Envs). Immunogens have been designed that activate these B cell precursors in vivo, but they also activate competing off-target responses. Here we report on a complementary approach to expand specific B cells using an anti-idiotypic antibody, iv8, that selects for naive human B cells expressing immunoglobulin light chains with 5-amino acid complementarity determining region 3s, a key feature of anti-CD4 binding site (CD4bs)-specific VRC01-class antibodies. In mice, iv8 induced target cells to expand and mature in the context of a polyclonal immune system and produced serologic responses targeting the CD4bs on Env. In summary, the results demonstrate that an anti-idiotypic antibody can specifically recognize and expand rare B cells that express VRC01-class antibodies against HIV-1.