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.

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.

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.

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.

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.

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.

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.

Atypical chemokine receptor 4 shapes activated B cell fate.

Activated B cells can initially differentiate into three functionally distinct fates-early plasmablasts (PBs), germinal center (GC) B cells, or early memory B cells-by mechanisms that remain poorly understood. Here, we identify atypical chemokine receptor 4 (ACKR4), a decoy receptor that binds and degrades CCR7 ligands CCL19/CCL21, as a regulator of early activated B cell differentiation. By restricting initial access to splenic interfollicular zones (IFZs), ACKR4 limits the early proliferation of activated B cells, reducing the numbers available for subsequent differentiation. Consequently, ACKR4 deficiency enhanced early PB and GC B cell responses in a CCL19/CCL21-dependent and B cell-intrinsic manner. Conversely, aberrant localization of ACKR4-deficient activated B cells to the IFZ was associated with their preferential commitment to the early PB linage. Our results reveal a regulatory mechanism of B cell trafficking via an atypical chemokine receptor that shapes activated B cell fate.

The microanatomic segregation of selection by apoptosis in the germinal center.

B cells undergo rapid cell division and affinity maturation in anatomically distinct sites in lymphoid organs called germinal centers (GCs). Homeostasis is maintained in part by B cell apoptosis. However, the precise contribution of apoptosis to GC biology and selection is not well defined. We developed apoptosis-indicator mice and used them to visualize, purify, and characterize dying GC B cells. Apoptosis is prevalent in the GC, with up to half of all GC B cells dying every 6 hours. Moreover, programmed cell death is differentially regulated in the light zone and the dark zone: Light-zone B cells die by default if they are not positively selected, whereas dark-zone cells die when their antigen receptors are damaged by activation-induced cytidine deaminase.

IL-17-producing γδ T cells switch migratory patterns between resting and activated states.

Interleukin 17-producing γδ T (γδT17) cells have unconventional trafficking characteristics, residing in mucocutaneous tissues but also homing into inflamed tissues via circulation. Despite being fundamental to γδT17-driven early protective immunity and exacerbation of autoimmunity and cancer, migratory cues controlling γδT17 cell positioning in barrier tissues and recruitment to inflammatory sites are still unclear. Here we show that γδT17 cells constitutively express chemokine receptors CCR6 and CCR2. While CCR6 recruits resting γδT17 cells to the dermis, CCR2 drives rapid γδT17 cell recruitment to inflamed tissues during autoimmunity, cancer and infection. Downregulation of CCR6 by IRF4 and BATF upon γδT17 activation is required for optimal recruitment of γδT17 cells to inflamed tissue by preventing their sequestration into uninflamed dermis. These findings establish a lymphocyte trafficking model whereby a hierarchy of homing signals is prioritized by dynamic receptor expression to drive both tissue surveillance and rapid recruitment of γδT17 cells to inflammatory lesions.

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.

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.

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.

CCR2 defines in vivo development and homing of IL-23-driven GM-CSF-producing Th17 cells.

IL-17-producing helper T (Th17) cells are critical for host defense against extracellular pathogens but also drive numerous autoimmune diseases. Th17 cells that differ in their inflammatory potential have been described including IL-10-producing Th17 cells that are weak inducers of inflammation and highly inflammatory, IL-23-driven, GM-CSF/IFNγ-producing Th17 cells. However, their distinct developmental requirements, functions and trafficking mechanisms in vivo remain poorly understood. Here we identify a temporally regulated IL-23-dependent switch from CCR6 to CCR2 usage by developing Th17 cells that is critical for pathogenic Th17 cell-driven inflammation in experimental autoimmune encephalomyelitis (EAE). This switch defines a unique in vivo cell surface signature (CCR6(-)CCR2(+)) of GM-CSF/IFNγ-producing Th17 cells in EAE and experimental persistent extracellular bacterial infection, and in humans. Using this signature, we identify an IL-23/IL-1/IFNγ/TNFα/T-bet/Eomesodermin-driven circuit driving GM-CSF/IFNγ-producing Th17 cell formation in vivo. Thus, our data identify a unique cell surface signature, trafficking mechanism and T-cell intrinsic regulators of GM-CSF/IFNγ-producing Th17 cells.

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.

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.

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.