Unveiling the immune system aging in single-cell resolution.

Development of high-affinity Abs in the germinal center (GC) is dependent on a specialized subset of T cells called "T follicular helper" (TFH) cells that help select Ag-specific B cells. A second T cell subset, T follicular regulatory (TFR) cells, can act as repressors of the GC and Ab response but can also provide a helper function for GC B cells in some contexts. Recent studies showed that, apart from their traditional helper role, TFH cells can also act as repressors of the Ab response, particularly for IgE responses. We review how both TFH and TFR cells express helper and repressor factors that coordinately regulate the Ab response and how the line between these two subsets is less clear than initially thought. Thus, TFH and TFR cells are interconnected and have "nonbinary" functions. However, many questions remain about how these critical cells control the Ab response.

Pathogen exposure elicits production of high-affinity antibodies stimulated by T follicular helper (Tfh) cells in the germinal center reaction. Tfh cells provide both costimulation and stimulatory cytokines to B cells to facilitate affinity maturation, class switch recombination, and plasma cell differentiation within the germinal center. Under normal circumstances, the germinal center reaction results in antibodies that precisely target foreign pathogens while limiting autoimmunity and excessive inflammation. In order to have this degree of control, the immune system ensures Tfh-mediated B-cell help is regulated locally in the germinal center. The recently identified T follicular regulatory (Tfr) cell subset can migrate to the germinal center and inhibit Tfh-mediated B-cell activation and antibody production. Although many aspects of Tfr cell biology are still unclear, recent data have begun to delineate the specialized roles of Tfr cells in controlling the germinal center reaction. Here we discuss the current understanding of Tfr-cell differentiation and function and how this knowledge is providing new insights into the dynamic regulation of germinal centers, and suggesting more efficacious vaccine strategies and ways to treat antibody-mediated diseases.

Ultraviolet B (UVB) light is a common environmental trigger that can induce flares in several autoimmune diseases, but the mechanisms that mediate UVB-induced photosensitivity remain poorly understood. We used mice which express an autoimmune-driving isoform of Foxp3, called FoxP3ΔE2 mice, to develop a model autoimmune cutaneous photosensitivity. In the steady-state, FoxP3ΔE2 mice have high serum titers of IgE that target skin-associated autoantigens, and increased numbers of IL4-producing T follicular helper cells that target skin-associated proteins (Keratin 14) in the absence of overt immune challenge. After skin exposure to UVB light, FoxP3ΔE2 mice had further elevated IgE, worsened skin inflammation, and increased IgE complex deposition that localized to the challenge site. IgE production in UVB-challenged mice correlated with increased frequencies of germinal center (GC) B cells, elevated percentages of IL-4+ T follicular helper cells, and numbers of basophils with high surface expression of IgE in the draining lymph nodes of UVB-challenged skin. We also observed a significant increase in IgE accumulation, increased numbers of Tug8+ basophil extracellular traps in UVB-challenged skin and increased frequencies of activated plasmacytoid dendritic cells (CD80+CD86+) in the skin and skin-draining lymph nodes of FoxP3ΔE2 mice when compared to wild-type littermates. Collectively, we found that T follicular regulatory cells in FoxP3ΔE2 mice fail to maintain self-tolerance in the GC, resulting in increased production of skin reactive IgE and worsened skin inflammation driven by basophil extracellular trap accumulation in UVB-damaged skin. Supported by grants from the NIH (F32 AI154787, R01 AI136475, R21 AI169418, and R21 AI152444)

T follicular helper (TFH) cells are critical for a germinal center response and a robust humoral response. Aging has a profound impact on the formation of a strong humoral/germinal center response, yet the impact of aging on TFH remains unclear. This study explores the effects of aging on TFH transcription factors, which could explain some of the age related germinal center defects. During influenza infection, young mice exhibit a very robust IgG response, which is significantly reduced in aged mice. Initial studies showed that there are no differences in the number of flu-specific TFH (CXCR5hi PD-1hi) during the course of the infection, as determined using MHC class II NP tetramer. To determine transcription factor expression and localization of TFH, we performed multicolor flow cytometry and confocal microscopy on spleens from these mice. There was no difference in BCL6 expression in young and aged flu-specific TFH. But, strikingly, flu-specific BCL6hi TFH from aged mice had increased co-expression of Foxp3, characteristic of T follicular regulatory cells, a critical controller of germinal center responses. We are currently exploring the contribution of whether the differences in TFH transcription factor expression are intrinsic to aged CD4+ T cells or are a result of the aged microenvironment. These results strongly suggest that aging has a dramatic impact on transcription factor expression in CD4 T cells, which can influence the establishment of a robust immune response.

T Follicular helper (Tfh) cells promote germinal center (GC) B cell responses to develop effective humoral immunity against pathogens. However, dysregulated Tfh cells can also trigger autoantibody production and the development of autoimmune diseases. We report here that Tsc1, a regulator for mTOR signaling, plays differential roles in Tfh cell/GC B cell responses in the steady state and in immune responses to antigen immunization. In the steady state, Tsc1 in T cells intrinsically suppresses spontaneous GC-Tfh cell differentiation and subsequent GC-B cell formation and autoantibody production. In immune responses to antigen immunization, Tsc1 in T cells is required for efficient GC-Tfh cell expansion, GC-B cell induction, and antigen-specific antibody responses, at least in part via promoting GC-Tfh cell mitochondrial integrity and survival. Interestingly, in mixed bone marrow chimeric mice reconstituted with both wild-type and T cell-specific Tsc1-deficient bone marrow cells, Tsc1 deficiency leads to enhanced GC-Tfh cell differentiation of wild-type CD4 T cells and increased accumulation of wild-type T regulatory cells and T follicular regulatory cells. Such bystander GC-Tfh cell differentiation suggests a potential mechanism that could trigger self-reactive GC-Tfh cell/GC responses and autoimmunity via neighboring GC-Tfh cells.

Description T follicular helper (Tfh) cells play a crucial role in humoral immunity by fine-tuning the quality of neutralizing antibodies (nAbs). However, they are mostly restricted to tissue sites, making them challenging to study in the context of vaccination. In this study, we used spleens, lymph nodes, and tonsils collected from humans (n = 30, ages 2-58 years) to comprehensively profile the diversity of Tfh ex vivo using flow cytometry and single cell transcriptomics. Multimodal analyses revealed that Tfh were enriched and were more clonal in young children, but their cytokines responses to ex vivo stimulation and polarized phenotypes (Tfh1, Tfh2, and Tfh17 associated) increased with age. Next, we used the tonsil immune organoid system to dissect Tfh responses to ex vivo vaccination with influenza antigens. Single cell analyses revealed early proliferation and heightened Tfh activation in young children compared to adults. Frequencies of activated Tfh1 strongly correlated with nAbs in both groups. Depleting Tfh in organoids reduced germinal center (GC) B cell frequencies but did not alter the expansion of antigen-specific B cells. While overall antibody magnitude remained unchanged, nAbs were attenuated with Tfh depletion in adults but remained unchanged in children. These findings suggest that Tfh play distinct functional roles in GCs in children versus adults. Ongoing investigations are focused on understanding how Tfh regulate affinity maturation in GCs in different age groups. Funding Sources Supported by NIH/NIAID 1R01AI173023; NIH/NIAID 1U01AI180164; Wellcome LEAP (Wellcome Trust, UK); Bill and Melinda Gates Foundation Topic Categories Computational and Systems Immunology (COMP)

T follicular helper (TFH) cells are fundamental in germinal center (GC) maturation and selection of antigen-specific B cells within secondary lymphoid organs. GC-resident TFH cells have been fully characterized in human immunodeficiency virus (HIV) infection. However, the role of GC TFH cells in GC B cell responses following various simian immunodeficiency virus (SIV) vaccine regimens in rhesus macaques (RMs) has not been fully investigated. We characterized GC TFH cells of RMs over the course of a mucosal/systemic vaccination regimen to elucidate GC formation and SIV humoral response generation. Animals were mucosally primed twice with replicating adenovirus type 5 host range mutant (Ad5hr)-SIV recombinants and systemically boosted with ALVAC-SIVM766Gag/Pro/gp120-TM and SIVM766&CG7V gD-gp120 proteins formulated in alum hydroxide (ALVAC/Env) or DNA encoding SIVenv/SIVGag/rhesus interleukin 12 (IL-12) plus SIVM766&CG7V gD-gp120 proteins formulated in alum phosphate (DNA&Env). Lymph nodes were biopsied in macaque subgroups prevaccination and at day 3, 7, or 14 after the 2nd Ad5hr-SIV prime and the 2nd vector/Env boost. Evaluations of GC TFH and GC B cell dynamics including correlation analyses supported a significant role for early GC TFH cells in providing B cell help during initial phases of GC formation. GC TFH responses at day 3 post-mucosal priming were consistent with generation of Env-specific memory B cells in GCs and elicitation of prolonged Env-specific humoral immunity in the rectal mucosa. GC Env-specific memory B cell responses elicited early post-systemic boosting correlated significantly with decreased viremia postinfection. Our results highlight the importance of early GC TFH cell responses for robust GC maturation and generation of long-lasting SIV-specific humoral responses at mucosal and systemic sites. Further investigation of GC TFH cell dynamics should facilitate development of an efficacious HIV vaccine.IMPORTANCE The modest HIV protection observed in the human RV144 vaccine trial associated antibody responses with vaccine efficacy. T follicular helper (TFH) cells are CD4+ T cells that select antibody secreting cells with high antigenic affinity in germinal centers (GCs) within secondary lymphoid organs. To evaluate the role of TFH cells in eliciting prolonged virus-specific humoral responses, we vaccinated rhesus macaques with a combined mucosal prime/systemic boost regimen followed by repeated low-dose intrarectal challenges with SIV, mimicking human exposure to HIV-1. Although the vaccine regimen did not prevent SIV infection, decreased viremia was observed in the immunized macaques. Importantly, vaccine-induced TFH responses elicited at day 3 postimmunization and robust GC maturation were strongly associated. Further, early TFH-dependent SIV-specific B cell responses were also correlated with decreased viremia. Our findings highlight the contribution of early vaccine-induced GC TFH responses to elicitation of SIV-specific humoral immunity and implicate their participation in SIV control.

The aging immune system responds poorly to infection and vaccination, producing reduced numbers of germinal centers, low affinity antibody titers, and reduced B cell memory. Prolonged contacts between activated B cells and CD4+ T cells are crucial to germinal center formation and T follicular helper cell (Tfh) differentiation, but it is unclear how aging impacts the quality of this interaction. Peptide immunization confirmed that aged mice have less expansion of antigen-specific germinal center B cells and reduced antibody titers. Furthermore, aging was associated with decreased CD4+ T conventional cells and accumulated Tfh cells and regulatory T cells, even in naïve mice. Despite increased numbers, our results indicate that aged Tfh have decreased metabolic function, which may have repercussions on germinal center responses. To determine the contribution of cell-extrinsic influences on antigen-specific Tfh induction, we adoptively transferred young, antigen-specific B and CD4+ naïve T cells into aged hosts prior to peptide immunization, which resulted in reduced antigen-specific germinal center B cell and Tfh expansion and reduced antibody titers when compared to young hosts. The results from these experiments emphasize the importance of the lymphoid microenvironment in germinal center initiation and maintenance. We hypothesize that age-associated Tfh accumulation may impair antigen-specific Tfh induction and subsequent germinal center responses. Current studies use live-cell 2-photon microscopy to track the impact of age on lymphocyte motility and Tfh:B interactions, in order to determine the relative contributions of aged APCs, non-specific aged Tfh, and aged regulatory T cells to germinal center deficiency. Research supported by the Mayo Clinic Robert and Arlene Kogod Center on Aging Innovation Award UL1R002377

After a 14 year tenure, my predecessor, Carleton Stewart, transferred the position of Editor-in-Chief of the Journal of Leukocyte Biology (JLB) to me [1]. That was 15 years ago. In point of fact, we are both indebted to the founders of the Society, who initiated JLB as the Journal of the Reticuloendothelial Society in 1964. I am particularly grateful to Carleton Stewart for initiating the invaluable pyramid-structured review system that remains in use today. Among the most notable features of JLB, our use of a panel of selected expert section editors ensures the identification of fair, knowledgeable, and conscientious reviewers—a feature that sets us apart from other journals of our size and scope. I am honored to note that our current editorial board includes five of the original members who have served JLB along with me for all these years: Craig Reynolds, Alan Kaplan, Alan Landay, Philip Murphy, and Wayne Smith. I am even more indebted to Carleton Stewart as well as to Council Members, Carol Nacy and Monte Meltzer, who, in 1984 had the foresight to change the name of the society and the journal to cover the field of “Leukocyte Biology.” This has provided a crucial focus leading to progressive improvement of JLB, because it broadened the scope of the journal to cover biological and molecular aspects of leukocytes in host defense, chronic disease, restoration of homeostasis, and tissue repair. Consequently, JLB then began publishing papers focused on leukocyte receptors, signal transduction, gene regulation; cell development, growth, differentiation, and function; extracellular mediators and effector molecules; pathogenesis and host defense; and translational and clinical reports. Concomitant with my appointment, Craig Reynolds was selected as Associate Editor. He has invited outstanding leukocyte biologists to publish scholarly reviews in JLB, which has contributed to marked improvement in our impact factor, from 2.7 to ∼4.5. This increase in citation rate has been accompanied by a fourfold increase in manuscripts submitted per year, from fewer than 200 to the current number of about 750. The acceptance rate has also increased, from 25% in 1995 to about 37% at the present time. Consequently, this has led to an increase in the number of journal pages from 1700 to over 3200 annually. This increase can also be attributed to the inclusion of 3 to 5 sets of invited meeting overviews each year. These shorter reviews based on meeting presentations have proven to be quite popular and have citation rates approximately midway between those of the lengthier reviews and original research papers. I would also like to thank Associate Editor, Helene Rosenberg, for arranging and carrying out published interviews with the corresponding authors of Pivotal Advance manuscripts and, more recently, for assistance with the winners of the Dolph Adams Awards. As I reread my editorial as the incoming editor from 14 years ago [2], I am grateful that I have not made too many enemies, and likewise to have survived my time as Editor-in-Chief without any major disasters. We have been very fortunate to have made the profound change from paper to online submission and review with the invaluable assistance of Managing Editor, Amy Huter-Imming. The journal is now published online as well as in print, a feature which has been handled admirably by the FASEB Office of Publications. These developments have enabled us to manage the increase in manuscript submissions with ever greater speed and efficiency. I am happy that, thanks to our team of editors, we have been succeeded in slowly changing the scientific community’s perception of JLB to an appreciation of it as a commendable repository for scientific achievements. More changes are in store in the future, as JLB will be required to find its place in competition with the increasing number of newly minted online journals and to comply with government stipulations and requirements for open access. I am therefore delighted that the Council of the Society for Leukocyte Biology has chosen one of our able and experienced section editors, Luis Montaner, to become Editor-in-Chief. With the help of my many colleagues and coworkers, I am pleased to have been able to facilitate scientific communication and to strengthen the Society during my tenure. I am confident that Luis Montaner will further improve the record and strength of JLB. Finally, I would like to extend my most sincere and heartfelt thanks to all JLB authors, reviewers, and readers who have contributed to the ongoing success of our journal.

We previously reported that Cd3e‐deficient mice adoptively transferred with CD4+ T cells generate high numbers of T follicular helper (Tfh) cells, which go on to induce a strong B‐cell and germinal center (GC) reaction. Here, we show that in this system, GC B cells display an altered distribution between the dark and light zones, and express low levels of activation‐induced cytidine deaminase. Furthermore, GC B cells from Cd3e–/– mice accumulate fewer somatic mutations as compared with GC B cells from wild‐type mice, and exhibit impaired affinity maturation and reduced differentiation into long‐lived plasma cells. Reconstitution of Cd3e–/– mice with regulatory T (Treg) cells restored Tfh‐cell numbers, GC B‐cell numbers and B‐cell distribution within dark and light zones, and the rate of antibody somatic mutations. Tfh‐cell numbers and GC B‐cell numbers and dynamics were also restored by pre‐reconstitution of Cd3e–/– mice with Cxcr5–/– Treg cells or non‐regulatory, memory CD4+ T cells. Taken together, these findings underline the importance of a quantitatively regulated Tfh‐cell response for an efficient and long‐lasting serological response.

Germinal centers (GCs) are confined anatomic regions where rapidly proliferating B cells undergo somatic mutation and selection and eventual differentiation into memory B cells or long-lived plasma cells. GCs are also the origin of malignancy, namely follicular lymphoma (FL), GC B cell-diffuse large B cell lymphoma (GCB-DLBCL), and Burkitt lymphoma (BL). GC B cell lymphomas maintain their GC transcriptional signatures and sustain many features of the GC microenvironment, including CD4+ T follicular helper (Tfh) cells. Tfh cells are essential for the formation and maintenance of GCs, providing critical helper signals such as CD40L. Large-scale sequencing efforts have led to new insights about the tightly regulated selection mechanisms that are commonly targeted during GC B cell lymphomagenesis. For instance, HVEM, a frequently mutated surface molecule in GC-derived lymphomas, engages the inhibitory receptor BTLA on Tfh cells and loss of HVEM leads to exaggerated T cell help. Here, we review current understanding of how Tfh cells contribute to the selection of GC B cells, with a particular emphasis on how Tfh cell signals may contribute to lymphomagenesis. The possibility of targeting Tfh cells for the treatment of GC-derived lymphomas is discussed.

Weakened germinal center responses by the aged immune system result in diminished immunity against pathogens and reduced efficacy of vaccines. Prolonged contacts between activated B cells and CD4+ T cells are crucial to germinal center formation and T follicular helper cell (Tfh) differentiation, but it is unclear how aging impacts the quality of this interaction. Peptide immunization confirmed that aged mice have decreased expansion of antigen-specific germinal center B cells and reduced antibody titers. Furthermore, aging was associated with accumulated Tfh cells, even in naïve mice. Despite increased numbers, aged Tfh had reduced expression of master transcription factor BCL6 and increased expression of the ectonucleotidase CD39. Invitro activation revealed that proliferative capacity was maintained in aged CD4+ T cells, but not the costimulatory molecule CD40L. When activated invitro by aged antigen-presenting cells, young CD4+ naïve T cells generated reduced numbers of activated cells with upregulated CD40L. To determine the contribution of cell-extrinsic influences on antigen-specific Tfh induction, young, antigen-specific B and CD4+ T cells were adoptively transferred into aged hosts prior to peptide immunization. Transferred cells had reduced expansion and differentiation into germinal center B cell and Tfh and reduced antigen-specific antibody titers when compared to young hosts. Young CD4+ T cells transferred aged hosts differentiated into Tfh cells with reduced PD-1 and BCL6 expression, and increased CD39 expression, though they maintained their mitochondrial capacity. These results highlight the role of the lymphoid microenvironment in modulating CD4+ T cell differentiation, which contributes to impaired establishment and maintenance of germinal centers.

BackgroundFractalkine/CX3CL1, a surface chemokine, binds to CX3CR1 expressed by different lymphocyte subsets. Since CX3CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX3CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood.Methodology/Principal FindingsWe demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX3CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX3CR1 but only germinal centre B cells were attracted by soluble CX3CL1 in a transwell assay. CX3CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX3CR1+ germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX3CL1. ELISA assay showed that soluble CX3CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX3CL1 did not attract spleen B cells from wild type mice. OVA immunized CX3CR1−/− or CX3CL1−/− mice showed significantly decreased specific IgG production compared to wild type mice.Conclusion/SignificanceWe propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX3CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.

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.

T follicular helper (T(FH)) cells are a specialized subset of effector T cells that provide help to and thereby select high-affinity B cells in germinal centers (GCs). To examine the dynamic behavior of T(FH) cells in GCs in mice, we used two-photon microscopy in combination with a photoactivatable fluorescent reporter. Unlike GC B cells, which are clonally restricted, T(FH) cells distributed among all GCs in lymph nodes and continually emigrated into the follicle and neighboring GCs. Moreover, newly activated T(FH) cells invaded preexisting GCs, where they contributed to B cell selection and plasmablast differentiation. Our data suggest that the dynamic exchange of T(FH) cells between GCs ensures maximal diversification of T cell help and that their ability to enter ongoing GCs accommodates antigenic variation during the immune response.