Tfh Cell Function Research Articles

Targeting TFH cells in human diseases and vaccination: rationale and practice.

The identification of CD4+ T cells localizing to B cell follicles has revolutionized the knowledge of how humoral immunity is generated. Follicular helper T (TFH) cells support germinal center (GC) formation and regulate clonal selection and differentiation of memory and antibody-secreting B cells, thus controlling antibody affinity maturation and memory. TFH cells are essential in sustaining protective antibody responses necessary for pathogen clearance in infection and vaccine-mediated protection. Conversely, aberrant and excessive TFH cell responses mediate and sustain pathogenic antibodies to autoantigens, alloantigens, and allergens, facilitate lymphomagenesis, and even harbor viral reservoirs. TFH cell generation and function are determined by T cell antigen receptor (TCR), costimulation, and cytokine signals, together with specific metabolic and survival mechanisms. Such regulation is crucial to understanding disease pathogenesis and informing the development of emerging therapies for disease or novel approaches to boost vaccine efficacy.

Iguratimod Restrains Circulating Follicular Helper T Cell Function by Inhibiting Glucose Metabolism via Hif1α-HK2 Axis in Rheumatoid Arthritis.

Iguratimod (IGU) is a novel disease modified anti-rheumatic drug, which has been found to act directly on B cells for inhibiting the production of antibodies in rheumatoid arthritis (RA) patients. Follicular helper T (Tfh) cells, a key T cell subsets in supporting B cell differentiation and antibody production, have been shown to play critical roles in RA. However, whether IGU can inhibit RA Tfh cells which further restrains B cell function remains unclear. Here, we aimed to explore the roles of IGU in regulating RA circulating Tfh (cTfh) cell function and investigate the potential mechanism associated with cell glucose metabolism. In our study, we found that IGU could act on RA-CD4+ T cells to reduce T cell-dependent antibody production. IGU decreased the percentage of RA cTfh cells and the expression of Tfh cell-related molecules and cytokines which were involved in B cell functions. Importantly, our data showed that IGU significantly restrained the cTfh cell function by inhibiting glucose metabolism, which relied on Hif1α-HK2 axis. In summary, we clarified a new target and mechanism of IGU by restraining RA cTfh cell function via inhibiting Hif1α-HK2-glucose metabolism axis. Our study demonstrates the potential application of IGU in the treatment of diseases related to abnormal metabolism and function of Tfh cells.

The Aryl Hydrocarbon Receptor Modulates T Follicular Helper Cell Responses to Influenza Virus Infection in Mice.

T follicular helper (Tfh) cells support Ab responses and are a critical component of adaptive immune responses to respiratory viral infections. Tfh cells are regulated by a network of signaling pathways that are controlled, in part, by transcription factors. The aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that modulates many aspects of adaptive immunity by binding a range of small molecules. However, the contribution of AHR signaling to Tfh cell differentiation and function is not known. In this article, we report that AHR activation by three different agonists reduced the frequency of Tfh cells during primary infection of C57BL/6 mice with influenza A virus (IAV). Further, using the high-affinity and AHR-specific agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin, we show that AHR activation reduced Tfh cell differentiation and T cell-dependent B cell responses. Using conditional AHR knockout mice, we demonstrated that alterations of Tfh cells and T cell-dependent B cell responses after AHR activation required the AHR in T cells. AHR activation reduced the number of T follicular regulatory (Tfr) cells; however, the ratio of Tfr to Tfh cells was amplified. These alterations to Tfh and Tfr cells during IAV infection corresponded with differences in expression of BCL6 and FOXP3 in CD4+ T cells and required the AHR to have a functional DNA-binding domain. Overall, these findings support that the AHR modulates Tfh cells during viral infection, which has broad-reaching consequences for understanding how environmental factors contribute to variation in immune defenses against infectious pathogens, such as influenza and severe acute respiratory syndrome coronavirus.

Adenosine deaminase-1 (ADA-1) induces maturation and skewing of cytokine and chemokine production in dendritic cells to improve adaptive immune responses.

Abstract Dendritic cells (DCs) are critical mediators of antigen-specific immunity through their ability to present antigen and drive T cell differentiation into different effector subsets such as follicular helper T cells (TFH). TFH cells provide both physical and cytokine-mediated stimuli to B cells resulting in somatic hypermutation and class-switching of B cell receptors. Molecules that target TFH cells, such as adenosine deaminase-1 (ADA-1), would improve humoral immunity. We have previously demonstrated that co-delivery of plasmid-encoded adenosine deaminase with an HIV-1 envelope and SARS-CoV-2 DNA vaccines in vivo, enhanced both humoral and cellular responses. However, the mechanism by which ADA-1 is acting as an adjuvant remains to be elucidated. To this end, we treated monocyte-derived DCs from healthy human donors in vitro with recombinant ADA-1 protein and evaluated the expression of maturation markers, cytokines and chemokines. ADA-1-treated DCs had a significantly increased expression of CD40 and CD86 as well as HLA-DR compared to their unstimulated, immature counterparts. The level of co-stimulatory marker and HLA-DR expression on ADA-1-treated DCs was similar to that on LPS/IFN-γ-treated DCs, indicating ADA-1-mediated DC maturation. ADA-1-treated DCs also exhibited a significant increase in IL-6, IL-1β and CXCL13 expression. IL-6 is a key pro-TFH cytokine and IL-1β and CXCL13 may play a role in TFH cell differentiation, function, and proliferation. Ongoing studies are aimed to drive ADA-1 overexpression in DCs and evaluate effects on antigen presentation. Overall, elucidating the mechanism of ADA-1’s adjuvanticity will allow for its progression as a clinical adjuvant. This work was supported by funding to Dr. Elias Haddad from NIH 5RO1AI106482-01A and 1U19 AI128910-01.

Tfh cells with NLRP3 inflammasome activation are essential for high-affinity antibody generation, germinal centre formation and autoimmunity

ObjectiveNLRP3 inflammasome regulates T cell responses. This study examined the roles of NLRP3 inflammasome activation in the regulation of T follicular helper (Tfh) cells during humoral response to T dependent...

MiR-138 Reduces the Dysfunction of T Follicular Helper Cells in Osteosarcoma via the PI3K/Akt/mTOR Pathway by Targeting PDK1.

Objective To study the effect of miR-138 on the function of osteosarcoma (OS) T follicular helper cells (Tfh cells) and its mechanism. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from patients with osteosarcoma (OS group) and healthy volunteers (control group). CD4+CXCR5+ Tfh cells and CD9+ B cells were sorted by flow cytometry. qRT-PCR was used to detect the expression of miR-138 and PDK1 in the peripheral blood and CD4+CXCR5+ Tfh cells. Flow cytometry was employed to detect the proportion of CD4+CXCR5+ Tfh cells in CD4+ T cells, the level of CD40L in CD4+CXCR5+ Tfh cells, and the expression of CD27 and CD38 in B cells. Western blot was used to determine the protein expression of PDK1, PI3K, p-Akt, Akt, p-mTOR, and mTOR. In addition, dual-luciferase reporter assay was performed to verify the relationship between miR-138 and PDK1. ELISA method was used to determine the levels of IgM, IgG, IL-10, and IL-21. Results Compared with that of the control group, the expression of miR-138 in PBMC and CD4+CXCR5+ Tfh cells of the OS group was lower; overexpression of miR-138 could promote the maturation of Tfh cells and immature B cells. The results of the dual-luciferase report experiment showed that miR-138 can target and negatively regulate PDK1, and PDK1 can reverse the effect of miR-138 on the function of Tfh cells and immature B cells. Conclusion miR-138 inhibits the PI3K/Akt/mTOR pathway by targeting and negatively regulating PDK1 to alleviate the dysfunction of T follicular helper cells in OS.

Identification of Antigen Presenting Cell Subsets Supporting Human Tfh Differentiation.

CD4+ T follicular helper (Tfh) cells are essential for initiating and regulating efficient humoral responses in secondary lymphoid organs. Tfh polarization and differentiation is driven by multiple stimuli delivered by antigen presenting cells (APCs). APCs represent a complex population of immune cells, comprising several subpopulations (dendritic cells and macrophages) that are distinguished by their phenotype, ontogeny, and functions. In order to better identify and understand the role of the different APC subsets in human Tfh biology, we have used in vitro assays based on the co-culture of APCs and T cells. This chapter describes two complementary protocols. The first protocol describes an assay to study the capacity of APCs to drive Tfh polarization from naive CD4+ T cells. The second protocol is designed to address the role of APCs in modulating effector functions of mature Tfh cells.

Therapeutic Modulation of T Follicular Helper Cells by Low-Dose IL-2 Treatment.

T follicular helper (Tfh) cells are a subset of specialized CD4+ T cell residing in B cell follicles and confer essential support for germinal center responses, which lead to the generation of long-lived humoral immunity. A great deal of evidence from the past 15years indicate that excessive differentiation and dysregulated function of Tfh cells often promote autoimmunity by inducing autoantibody production. Interleukin-2 was identified as a major suppressor to inhibit Tfh differentiation. Therefore, IL-2 treatment was applied in suppressing Tfh function in mouse models and more recently in a clinical trial of patients with systemic lupus erythematosus. Here we describe a protocol for low-dose IL-2 treatment in a murine immunization model and on the assessment of the suppression of Tfh response using flow cytometry.

Critical regulation of follicular helper T cell differentiation and function by Gα13 signaling

GPCR-Gα protein-mediated signal transduction contributes to spatiotemporal interactions between immune cells to fine-tune and facilitate the process of inflammation and host protection. Beyond this, however, how Gα proteins contribute to the helper T cell subset differentiation and adaptive response have been underappreciated. Here, we found that Gα13 signaling in T cells plays a crucial role in inducing follicular helper T (Tfh) cell differentiation in vivo. T cell-specific Gα13-deficient mice have diminished Tfh cell responses in a cell-intrinsic manner in response to immunization, lymphocytic choriomeningitis virus infection, and allergen challenges. Moreover, Gα13-deficient Tfh cells express reduced levels of Bcl-6 and CXCR5 and are functionally impaired in their ability to adhere to and stimulate B cells. Mechanistically, Gα13-deficient Tfh cells harbor defective Rho-ROCK2 activation, and Rho agonist treatment recuperates Tfh cell differentiation and expression of Bcl-6 and CXCR5 in Tfh cells of T cell-specific Gα13-deficient mice. Conversely, ROCK inhibitor treatment hampers Tfh cell differentiation in wild-type mice. These findings unveil a crucial regulatory role of Gα13-Rho-ROCK axis in optimal Tfh cell differentiation and function, which might be a promising target for pharmacologic intervention in vaccine development as well as antibody-mediated immune disorders.

Attenuate ICOSL and IL-27 in Aire-overexpressing DC2.4 cells suppress TFH cell differentiation

Autoimmune regulator (Aire) is a transcription factor that plays a pivotal role in the maintenance of immune tolerance. However, little is known about its roles in peripheral immune tolerance. Aire is predominantly expressed in dendritic cells (DCs) in the periphery. DCs with higher inducible costimulatory ligand (ICOSL) expression and interleukin (IL)-27 production have been reported highly suggesting its roles in inducing follicular helper T cells (TFH). Here we use Aire-overexpressing DC2.4 cells in a coculture system composed of naïve CD4+ T cells to test whether Aire in DCs affects TFH cell differentiation. We found that the frequency of TFH cells and its specific cytokine IL-21 were decreased in CD4+ T lymphocytes after cocultured with Aire overexpressed DC2.4 cells. In activated DCs, ICOSL expression and IL-27 production were significantly suppressed by Aire. Furthermore, addition of recombinant ICOSL or IL-27 in the coculture system enhanced TFH cell differentiation and IL-21 expression. These results revealed that Aire plays an indispensable role in the repression of dendritic cells on the differentiation and function of TFH cells by inhibiting ICOSL and IL-27 expression.

Transcription factor c-Rel mediates communication between commensal bacteria and mucosal lymphocytes.

The NF-κB transcription factor c-Rel plays a crucial role in promoting and regulating immune responses and inflammation. However, the function of c-Rel in modulating the mucosal immune system is poorly understood. T follicular helper (Tfh) cells and IgA production in gut-associated lymphoid tissues (GALT) such as Peyer's patches (PPs) are important for maintaining the intestinal homeostasis. Here, c-Rel was identified as an essential factor regulating intestinal IgA generation and function of Tfh cells. Genetic deletion of c-Rel resulted in the aberrant formation of germinal centers (GCs) in PPs, significantly reduced IgA generation and defective Tfh cell differentiation. Supporting these findings, the Ag-specific IgA response to Citrobacter rodentium was strongly impaired in c-Rel-deficient mice. Interestingly, an excessive expansion of segmented filamentous bacteria (SFB) was observed in the small intestine of animals lacking c-Rel. Yet, the production of IL-17A, IgA, and IL-21, which are induced by SFB, was impaired due to the lack of transcriptional control by c-Rel. Collectively, the transcriptional activity of c-Rel regulates Tfh cell function and IgA production in the gut, thus preserving the intestinal homeostasis.

Molecular regulation and dysregulation of T follicular helper cells - learning from inborn errors of immunity.

The production of high-affinity antibodies is a key feature of the vertebrate immune system. Antibodies neutralize and clear pathogens, thereby protecting against infectious diseases. However, dysregulated production of antibodies can cause immune pathologies, such as autoimmunity and immune deficiency. Long-lived humoral immunity depends on B-cell help provided by T follicular helper (Tfh) cells, which support the differentiation of antigen (Ag)-specific B cells into memory and plasma cells. Tfh cells are generated from naïve CD4+ T cells following the receipt of inputs from various cell surface receptors, and can undergo further differentiation into subsets with specialised effector functions to induce and maintain serological memory. While genetically modified mice have provided great understanding of the requirements for generating Tfh cells, it is critical that requirements for human Tfh cell generation and function are also established. Key insights into the molecular requirements for human Tfh cells have been elucidated from the systematic analysis of humans with monogenic germline variants that cause inborn errors of immunity characterised by impaired humoral immunity following infection or vaccination or immune dysregulation and autoimmunity. In this review we will discuss how studying rare 'experiments of nature' has enabled discovery of non-redundant molecules and pathways necessary for Tfh cell generation, differentiation, regulation and function in humans, and how these findings inform us about basic and clinical immunology.

The PD-1/PD-L1 Checkpoint in Normal Germinal Centers and Diffuse Large B-Cell Lymphomas.

Simple SummaryThe PD-1/PD-L1 axis is not only involved in anti-tumour immune evasion of germinal center (GC)-derived diffuse large B cell lymphomas (DLBCL), but also inherently in the fine-tuned regulation of normal GC reactions during humoral immune responses. This checkpoint axis modulates crosstalks between B and T cells that allow positive selection for survival and proliferation. Malignant DLBCL cells may deceive and take advantage of these mechanisms to establish an immunosuppressive microenvironment. This review delves into PD-1/PD-L1 role in the complex inter-cellular interactions from normal GC reactions to DLBCL progression, in order to highlight vulnerabilities that could be targeted by promising combination immunotherapies.Besides a recognized role of PD-1/PD-L1 checkpoint in anti-tumour immune evasion, there is accumulating evidence that PD-1/PD-L1 interactions between B and T cells also play an important role in normal germinal center (GC) reactions. Even when smaller in number, T follicular helper cells (TFH) and regulatory T (TFR) or B (Breg) cells are involved in positive selection of GC B cells and may result critical in the lymphoma microenvironment. Here, we discuss a role of PD-1/PD-L1 during tumour evolution in diffuse large B cell lymphoma (DLBCL), a paradigm of GC-derived lymphomagenesis. We depict a progression model, in two phases, where malignant B cells take advantage of positive selection signals derived from correct antigen-presentation and PD-1/PD-L1 inter-cellular crosstalks to survive and initiate tumour expansion. Later, a constant pressure for the accumulation of genetic/epigenetic alterations facilitates that DLBCL cells exhibit higher PD-L1 levels and capacity to secrete IL-10, resembling Breg-like features. As a result, a complex immunosuppressive microenvironment is established where DLBCL cells sustain proliferation and survival by impairing regulatory control of TFR cells and limiting IL-21-mediated anti-tumour functions of TFH cells and maximize the use of PD-1/PD-L1 signaling to escape from CD8+ cytotoxic activity. Integration of these molecular and cellular addictions into a framework may contribute to the better understanding of the lymphoma microenvironment and contribute to the rationale for novel PD-1/PD-L1-based combinational immunotherapies in DLBCL.

Clinical relevance of T follicular helper cells in systemic lupus erythematosus

ABSTRACT Introduction T helper cells regulate a variety of immune responses and are involved in the pathogenesis of infection, allergy and autoimmune diseases. T follicular helper (Tfh) cells, which induce B cell maturation, play an important role in the production of the extremely diverse autoantibodies found in systemic lupus erythematosus (SLE). Area covered We provide an overview of the plasticity and diversity of Tfh cells in humans and their involvement in the pathology and pathogenesis of SLE. Our review outlines the potential of Tfh cells as a therapeutic target for SLE. Expert opinion Tfh cells are involved in the pathogenesis of SLE based on their plasticity and diversity. Tfh cell differentiation and function are variably regulated by cytokines (IL-12, interferons, IL-2, etc), co-stimulatory molecules (ICOS, CD40L, OX40, etc), and intracellular signals (JAK-STAT, etc). Elucidation of the mechanisms underlying Tfh cell differentiation and function may lead to the development of new therapies for SLE.

Selenium-GPX4 axis protects follicular helper T cells from ferroptosis.

Follicular helper T (TFH) cells are a specialized subset of CD4+ T cells that essentially support germinal center responses where high-affinity and long-lived humoral immunity is generated. The regulation of TFH cell survival remains unclear. Here we report that TFH cells show intensified lipid peroxidation and altered mitochondrial morphology, resembling the features of ferroptosis, a form of programmed cell death that is driven by iron-dependent accumulation of lipid peroxidation. Glutathione peroxidase 4 (GPX4) is the major lipid peroxidation scavenger and is necessary for TFH cell survival. The deletion of GPX4 in T cells selectively abrogated TFH cells and germinal center responses in immunized mice. Selenium supplementation enhanced GPX4 expression in T cells, increased TFH cell numbers and promoted antibody responses in immunized mice and young adults after influenza vaccination. Our findings reveal the central role of the selenium-GPX4-ferroptosis axis in regulating TFH homeostasis, which can be targeted to enhance TFH cell function in infection and following vaccination.

The disturbance and clinical significance of B cell and circulating follicular helper T cell subsets in children with primary nephrotic syndrome

The immunopathogenesis of primary nephrotic syndrome is unclear. Here, we examined the frequency, subsets and molecular function of circulating B cells and follicular helper T cells by flow cytometry and explored the correlation between certain subsets and clinical disease indices in new-onset patients, relapsing patients and healthy controls. We found an increase in the proportions of CD86+ activated cells and CD19+CD138+ plasma cells in the patients at onset and patients in relapse. However, the increased percentage of CD27+ memory cells was observed in only the relapse group. Furthermore, the ICOS MFI was elevated in TFH cells and their three subsets in new-onset patients, whereas the expression of OX40 was increased in TFH cells, TFH17 cells and TFH2 cells in the relapse group. Additionally, the increased frequency of CD19+CD138+ plasma cells was positively associated with urea nitrogen in the new-onset groups. Notably, the positive correlation between CD86+ activated B cells and CD19+CD138+ plasma cells was obtained in only the new-onset group and HC group; however, the increased CD27+ memory cell percentage was positively correlated with 24-h urinary protein in only the relapse group. Our results indicate that CD19+CD138+ plasma cells may be a key factor for the onset of PNS, whereas CD27+ memory cells may play a more important role in the relapse of this disease. Furthermore, the functions of TFH cells are also diverse because the expression of vital molecules changes during the different disease phases.

Follicular Helper T Cells Remodel the Immune Microenvironment of Pancreatic Cancer via Secreting CXCL13 and IL-21.

Simple SummaryThe immunosuppressive microenvironment is closely related to the poor prognosis of patients with PDAC. Tfh cells play an anti-tumor function in various malignant solid tumors; however, the role of Tfh cells in PDAC has not been determined. In this study, we aimed to explore the function of Tfh cells in PDAC, and revealed a novel immunosuppressive mechanism mediated by Tfh cells. Tfh cells promoted the formation of an immunoactive tumor microenvironment by secreting CXCL13 and IL-21, and the high infiltration of Tfh cells correlated with better patient prognosis. However, the anti-tumor function of Tfh cells was inhibited by the PD-L1/PD-1 signaling pathway. Neoadjuvant chemotherapy could further reverse the function of Tfh cells. Our results provided new strategies to remodel the immunoactive tumor microenvironment of PDAC.Immunosuppression is an important factor for the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Follicular helper T cells (Tfh cells) play an anti-tumor role in various malignant solid tumors and predict better patient prognosis. In the present study, we aimed to determine the immunosuppressive mechanism associated with Tfh cells and explore a new strategy to improve the tumor microenvironment of PDAC. Flow cytometry was used to detect the infiltration and proportion of Tfh cells in tumor tissues and peripheral blood from patients with PDAC. The spatial correlations of Tfh cells with related immune cells were evaluated using immunofluorescence. The function of Tfh cells was examined using in vitro and in vivo model systems. The high infiltration of Tfh cells predicted better prognosis in patients with PDAC. Tfh cells recruited CD8+ T cells and B cells by secreting C-X-C motif chemokine ligand 13 (CXCL13), and promoted the maturation of B cells into antibody-producing plasma cells by secreting interleukin 21 (IL-21), thereby promoting the formation of an immunoactive tumor microenvironment. The function of Tfh cells was inhibited by the programmed cell death 1 ligand 1 (PD-L1)/programmed cell death 1 (PD-1) signaling pathway in PDAC, which could be reversed using neoadjuvant chemotherapy. Treatment with recombinant CXCL13, IL-21 and Tfh cells alleviated tumor growth and enhanced the infiltration of CD8+ T cells and B cells, as well as B cell maturation in a PDAC mouse model. Our results revealed the important role of Tfh cells in mediating anti-tumor cellular immunity and humoral immunity in PDAC via secreting CXCL13 and IL-21 and determined a novel mechanism of immunosuppression in PDAC.

Retinoic Acid Receptor-Related Orphan Nuclear Receptor γt Licenses the Differentiation and Function of a Unique Subset of Follicular Helper T Cells in Response to Immunogenic Self-DNA in Systemic Lupus Erythematosus.

Accumulating studies have identified self-DNA as driving IgG anti-double-stranded DNA (anti-dsDNA) in lupus, though the underpinning mechanisms of this process remain largely undefined. Here, we explored the activity of transcription factor retinoic acid receptor-related orphan nuclear receptor γt (RORγt) in the differentiation and function of self-DNA-specific follicular helper T (Tfh) cells in lupus. B6, TCRα-/- , CD4-/- , RORγtfl/fl CD4Cre, RORγt+/+ CD4Cre, Bcl-6fl/fl CD4Cre, Bcl-6+/+ CD4Cre, IL-17-/- , and ICOS-/- mice were immunized with normal self-DNA, immunogenic self-DNA, and pathogen DNA to induce the production of Tfh cells and IgG anti-dsDNA. Tfh cells with or without interleukin-17 (IL-17) were evaluated for their role in supporting the generation of IgG. NSG mice were reconstituted with immune cells and circulating DNA from human subjects for translational studies. IL-17-positive Tfh cells were analyzed for their correlation with IgG anti-dsDNA levels as well as their response to circulating self-DNA in lupus patients. Unlike normal self-DNA, immunogenic self-DNA and pathogen DNA efficiently induced IgG responses. Immunogenic self-DNA induced IgG in a CD4+ T cell-dependent manner, which was abrogated by RORγt deficiency. In contrast, RORγt was not required for the generation of pathogen DNA-induced IgG. Further analyses identified RORγt as essential for the differentiation and function of Tfh cells in response to immunogenic self-DNA, assigning IL-17 as a feature cytokine. These IL-17-positive Tfh cells functioned independent of inducible costimulator (ICOS), critically supporting IgG generation. Targeting immunogenic self-DNA-specific Tfh cells by RORγ knockdown and IL-17 blockade ameliorated IgG response and lupus nephritis in a humanized mouse model. The presence of IL-17-positive Tfh cells was associated with IgG anti-dsDNA levels and were expanded by circulating immunogenic self-DNA in lupus patients. Immunogenic self-DNA instructs ICOS-dispensable IL-17-positive Tfh cells via RORγt to produce an IgG anti-dsDNA response. As such, IL-17-positive Tfh cells are a promising therapeutic target for lupus patients.

In Situ Characterization of Human Lymphoid Tissue Immune Cells by Multispectral Confocal Imaging and Quantitative Image Analysis; Implications for HIV Reservoir Characterization.

CD4 T cells are key mediators of adaptive immune responses during infection and vaccination. Within secondary lymphoid organs, helper CD4 T cells, particularly those residing in germinal centers known as follicular helper T cells (Tfh), provide critical help to B-cells to promote their survival, isotype switching and selection of high affinity memory B-cells. On the other hand, the important role of Tfh cells for the maintenance of HIV reservoir is well documented. Thus, interrogating and better understanding the tissue specific micro-environment and immune subsets that contribute to optimal Tfh cell differentiation and function is important for designing successful prevention and cure strategies. Here, we describe the development and optimization of eight multispectral confocal microscopy immunofluorescence panels designed for in depth characterization and immune-profiling of relevant immune cells in formalin-fixed paraffin-embedded human lymphoid tissue samples. We provide a comprehensive library of antibodies to use for the characterization of CD4+ T-cells -including Tfh and regulatory T-cells- as well as CD8 T-cells, B-cells, macrophages and dendritic cells and discuss how the resulting multispectral confocal datasets can be quantitatively dissected using the HistoCytometry pipeline to collect information about relative frequencies and immune cell spatial distributions. Cells harboring actively transcribed virus are analyzed using an in-situ hybridization assay for the characterization of HIV mRNA positive cells in combination with additional protein markers (multispectral RNAscope). The application of this methodology to lymphoid tissues offers a means to interrogate multiple relevant immune cell targets simultaneously at increased resolution in a reproducible manner to guide CD4 T-cell studies in infection and vaccination.

Supraphysiological estradiol promotes human T follicular helper cell differentiation and favours humoural immunity during in vitro fertilization.

During pregnancy, humoural immunity is essential for protection against many extracellular pathogens; however, autoimmune diseases may be induced or aggravated. T follicular helper (Tfh) cells contribute to humoural immunity. The aim of this study was to test whether Tfh cell function can be manipulated via hormones. Seventy‐four women who underwent in vitro fertilization were recruited and divided into four groups: menstrual period (MP), controlled ovarian hyperstimulation (COH), embryo transfer (ET) and pregnant after embryo transfer (P). A flow cytometry analysis was performed to identify Tfh cells in peripheral blood mononuclear cells (PBMCs). Bioinformatics analysis revealed a possible pathway between Tfh and B cells. Enzyme‐linked immunosorbent assays were used to detect interleukin (IL)‐21 and IL‐6. The quantitative polymerase chain reaction was performed to quantify BCL‐6, BACH2, XBP‐1, IRF‐4 and G protein‐coupled (GP)ER‐1 mRNA expression. Compared with the MP group, the COH, ET and P groups showed more Tfh and B cells, as well as higher IL‐21, IL‐6, BCL‐6 and BACH2 expression. Furthermore, Tfh cell frequency in PBMCs, as well as serum IL‐21 and IL‐6 levels, were all positively correlated with serum estradiol (E2) levels; the B cell percentage also correlated positively with Tfh cells in PBMCs. Combined with the bioinformatics analysis, XBP‐1, IRF‐4 and GPER‐1 expression was related to E2 levels, both in vivo and in vitro. We speculate that E2 augments Tfh cells and favours humoural immunity. This study indicates that Tfh cell regulation may be a novel target in maintaining the maternal‐foetal immune balance.