Tfh Germinal Center Research Articles

Influenza vaccination stimulates maturation of the human T follicular helper cell response

The differentiation and specificity of human CD4+ T follicular helper cells (TFH cells) after influenza vaccination have been poorly defined. Here we profiled blood and draining lymph node (LN) samples from human volunteers for over 2 years after two influenza vaccines were administered 1 year apart to define the evolution of the CD4+ TFH cell response. The first vaccination induced an increase in the frequency of circulating TFH (cTFH) and LN TFH cells at week 1 postvaccination. This increase was transient for cTFH cells, whereas the LN TFH cells further expanded during week 2 and remained elevated in frequency for at least 3 months. We observed several distinct subsets of TFH cells in the LN, including pre-TFH cells, memory TFH cells, germinal center (GC) TFH cells and interleukin-10+ TFH cell subsets beginning at baseline and at all time points postvaccination. The shift toward a GC TFH cell phenotype occurred with faster kinetics after the second vaccine compared to the first vaccine. We identified several influenza-specific TFH cell clonal lineages, including multiple responses targeting internal influenza virus proteins, and found that each TFH cell state was attainable within a clonal lineage. Thus, human TFH cells form a durable and dynamic multitissue network.

Specialized Tfh cell subsets driving type-1 and type-2 humoral responses in lymphoid tissue

Effective antibody responses are essential to generate protective humoral immunity. Different inflammatory signals polarize T cells towards appropriate effector phenotypes during an infection or immunization. Th1 and Th2 cells have been associated with the polarization of humoral responses. However, T follicular helper cells (Tfh) have a unique ability to access the B cell follicle and support the germinal center (GC) responses by providing B cell help. We investigated the specialization of Tfh cells induced under type-1 and type-2 conditions. We first studied homogenous Tfh cell populations generated by adoptively transferred TCR-transgenic T cells in mice immunized with type-1 and type-2 adjuvants. Using a machine learning approach, we established a gene expression signature that discriminates Tfh cells polarized towards type-1 and type-2 response, defined as Tfh1 and Tfh2 cells. The distinct signatures of Tfh1 and Tfh2 cells were validated against datasets of Tfh cells induced following lymphocytic choriomeningitis virus (LCMV) or helminth infection. We generated single-cell and spatial transcriptomics datasets to dissect the heterogeneity of Tfh cells and their localization under the two immunizing conditions. Besides a distinct specialization of GC Tfh cells under the two immunizations and in different regions of the lymph nodes, we found a population of Gzmk+ Tfh cells specific for type-1 conditions. In human individuals, we could equally identify CMV-specific Tfh cells that expressed Gzmk. Our results show that Tfh cells acquire a specialized function under distinct types of immune responses and with particular properties within the B cell follicle and the GC.

Novel highly immunogenic DNA vaccine regimen potently activates lymph nodes in macaques.

Abstract Background We evaluated a novel DNA vaccine delivery by LION™ nanoparticles for its ability to induce innate and adaptive immune responses in Rhesus macaques. Methods CoV-2 Spike or HIV Env DNA were formulated with a cationic nanocarrier (Lipid InOrganic Nanoparticle; LION™) administered via the IM route. Chemokine/cytokine and vaccine-specific responses were monitored in blood and lymph nodes (LN) by proteomics, flow, antibody assays. Results DNA/LION™ induced strong cellular and higher humoral responses than DNA/Electroporation and induced rapid (4-24hr) transient innate responses. Flow analysis showed activation of T, B, myeloid incl. dendritic cells in blood and their trafficking to LN with a strong increase of Tfh, Germinal Center Tfh CD4 and CD8 cells. Cell subsets were activated by cytokine networks incl. CXCL11, IL-7 and IL-15. GC-B cell activation directly correlated with increase of FLT3L and CXCL12 and importantly with antibody levels. Conclusion DNA/LION™ led to robust induction of cellular and humoral responses against different immunogens, serving as promising platform of effective nucleic acid vaccine delivery. Transient cytokine responses, incl. IL-15, resulted in strong activation of draining lymph nodes, and contributed to shaping adaptive (cellular and humoral) immunity. Our data showed rapid and coordinated cytokine responses to the vaccine and highlight the important role of innate responses to vaccination in modulating adaptive immunity.

Antigen-Specific Germinal Center Tfh cells are highly heterogenous in lymph nodes and Tfh1/Tfh17 cells are associated with viral load levels during chronic SHIV infection

Abstract HIV infection leads to alterations in HIV-specific CD4 T cells including increased expression of inhibitory receptors and skewing toward Tfh cell signature. Tfh cells have been shown to increase during chronic HIV infection. However, the heterogeneous nature of antigen-specific GC-Tfh cells and the relative contribution of specific GC-Tfh subset to viral persistence is not understood in HIV/SIV infection. Here, we characterized the phenotype and function of antigen-specific GC-Tfh cells based on the expression of chemokine receptors, Th1 (CXCR3), Th2 (CCR4), and Th17 (CCR6) along with activation-induced markers (AIM assay) CD25, OX40 and 41BB in the lymph nodes following chronic SHIV1157ipd3N4 infection in rhesus macaques. In SHIV naïve RM, only a small fraction of total GC-Tfh expressed CXCR3, CCR4, and CCR6. During chronic SHIV infection, there was an increase in GC-Tfh cells. Interestingly the majority of antigen-specific GC-Tfh cells expressed CXCR3 (Tfh1), while a significant proportion of them also expressed CCR4+ (Tfh2) and CCR6+ (Tfh17) cells. These cells expressed OX40, 41BB, and CD25 at higher levels than non-Tfh cells. Importantly, the expansion of GC-Tfh1 and GC-Tfh-17 cells is associated with high viremia. These data demonstrate that chronic SHIV infection promotes the expansion of antigen-specific GC-Tfh cells with heterogeneous populations, which are distinct from conventional GC-Tfh, and associated with higher viral RNA levels during chronic SHIV infection.

Anti-PD-1 chimeric antigen receptor T cells efficiently target SIV-infected CD4+ T cells in germinal centers.

Programmed cell death protein 1 (PD-1) is an immune checkpoint marker commonly expressed on memory T cells and enriched in latently HIV-infected CD4+ T cells. We engineered an anti-PD-1 chimeric antigen receptor (CAR) to assess the impact of PD-1 depletion on viral reservoirs and rebound dynamics in SIVmac239-infected rhesus macaques (RMs). Adoptive transfer of anti-PD-1 CAR T cells was done in 2 SIV-naive and 4 SIV-infected RMs on antiretroviral therapy (ART). In 3 of 6 RMs, anti-PD-1 CAR T cells expanded and persisted for up to 100 days concomitant with the depletion of PD-1+ memory T cells in blood and tissues, including lymph node CD4+ follicular helper T (TFH) cells. Loss of TFH cells was associated with depletion of detectable SIV RNA from the germinal center (GC). However, following CAR T infusion and ART interruption, there was a marked increase in SIV replication in extrafollicular portions of lymph nodes, a 2-log higher plasma viremia relative to controls, and accelerated disease progression associated with the depletion of CD8+ memory T cells. These data indicate anti-PD-1 CAR T cells depleted PD-1+ T cells, including GC TFH cells, and eradicated SIV from this immunological sanctuary.

Circulating Tfh cells are differentially modified by abatacept or TNF blockers, and predict treatment response in Rheumatoid Arthritis.

CD4+CXCR5+PD-1hi follicular helper T (Tfh) cells dwell in the germinal centers (GCs) of lymphoid organs and participate in Rheumatoid Arthritis (RA) pathogenesis; the frequency of their circulating counterparts (cTfh-frequency) is expanded in RA and correlates with the pool of GC Tfh cells. Our objective was to study the effect of abatacept (ABT) or TNF blockers (TNFb) on the cTfh-frequency in RA. Peripheral blood was drawn from seropositive-longstanding RA patients chronically receiving csDMARDS (n = 45), TNFb (n = 59), or ABT (n = 34), and healthy controls (HC) (n = 137). Also, patients with an incomplete response to csDMARDS (n = 41) who initiated TNFb (n = 19) or ABT (n = 22), were studied at 0 and 12 months. The cTfh-frequency was examined by cytometry. As compared with HC, an increased cTfh-frequency was seen in seropositive-longstanding RA chronically receiving csDMARDs or TNFb but not ABT. After escalating from csDMARDs, the cTfh-frequency did not vary in patients who were given TNFb but decreased to HC levels in those given ABT. In the ABT group, the baseline cTfh-frequency was higher for patients who attained 12M remission (12Mr), vs those who remained active (12Ma): 0m cutoff for remission >0.38% (Sens. 92%, Sp. 90%), OR 25.3. Conversely, in the TNFb group, the baseline cTfh-frequency was lower for 12Mr vs 12Ma: 0m cutoff for non-remission >0.44% (Sens. 67%, Sp. 90%), OR 8.5. ABT but not TNFb, is able to curtail the cTfh-frequency in RA. A higher baseline cTfh-frequency predicts a good response to ABT but a poor response to TNFb.

A novel chimeric recombinant FliC-Pgp3 vaccine promotes immunoprotection against Chlamydia muridarum infection in mice

The Pgp3 subunit vaccine elicits immune protection against Chlamydia trachomatis infection, but additional adjuvants are still required to enhance its immunoprotective efficacy. Flagellin can selectively stimulate immunity and act as an adjuvant. In this research, the FliC-Pgp3 recombinant was successfully expressed and purified. Tri-immunization with the FliC-Pgp3 vaccine in Balb/C mice induced rapid and persistent germinal center B-cell response and Tfh differentiation, promoting a significantly higher IgG antibody titer compared to the Pgp3 group. FliC-Pgp3 immunization primarily induced Th1-type cellular immunity, leading to higher levels of IFN-γ, TNF-α, and IL-2 secreted by CD4+ T cells than in Pgp3-vaccinated mice. Chlamydia muridarum challenge results showed that FliC-Pgp3-vaccinated mice exhibited more rapid clearance of Chlamydia muridarum colonization in the lower genital tract, ensuring a lower hydrosalpinx rate and cumulative score. Histological analysis showed reduced dilation and inflammatory infiltration in the oviduct and uterine horn of FliC-Pgp3-vaccinated mice compared to the PBS and Pgp3 control. Importantly, tri-immunization with FliC-Pgp3 effectively activated CD4+ T cells and dendritic cells, as confirmed by the adoptive transfer, resulting in better immune protection in recipient mice. In summary, the novel FliC-Pgp3 chimeric is hoped to be a novel vaccine with improved immunoprotection against Chlamydia muridarum.

The Tfh/Tfr Pathway Is Pivotal in FVIII Inhibitor Development in Mice

The development of neutralizing anti-FVIII antibodies (inhibitors) is one major complication of FVIII protein replacement therapy in hemophilia A patients. Our previous study demonstrated that the induction of activated follicular helper T (Tfh) cells in FVIII null mice is critical for FVIII inhibitor development. Follicular regulatory T (Tfr) cells are a specialized subset of Foxp3 + T cells identified in the germinal center (GC) that can modulate Tfh cell activation of B cells and antibody development. However, the role of Tfr cells in controlling anti-FVIII inhibitor development in vivo is still unclear. Here, we explored the activation and induction of Tfr cells and their functions in anti-FVIII inhibitor development in FVIII-immunized mice. We first characterized the features of activated Tfr cells in the spleen of immunized FVIII null mice by flow cytometry analysis. Activated Tfr cells were defined as CD4 +Foxp3 +CD44 +CD62L -CXCR5 +PD-1 +. Tfr cells express a high level of transcription factor BCL6 and costimulatory receptor ICOS. Activated Tfr cells also express a high level of costimulatory receptor CTLA4, which is similar to non-Tfr effector Treg cells, but CD25 expression is low on Tfr cells. FVIII immunization increases the frequency and the absolute number of activated Tfr cells in the spleen of mice that produce inhibitors. The percentage of activated CXCR5 hiPD-1 hi Tfr cells within the CD4 population significantly increased in inhibitor-producing mice compared to saline-treated controls and non-inhibitor-producing mice (0.88 ± 0.2 %, 0.33 ± 0.16 %, and 0.36 ± 0.2%, respectively). The Tfh/Tfr ratio also significantly increased in FVIII inhibitor-producing mice, suggesting a preferential expansion of Tfh cells relative to Tfr cells in inhibitor-producing mice. Interestingly, similar to Tfh cells as we reported before, the emergence of Tfr cells correlated with titers of anti-FVIII inhibitors in FVIII immunized mice. This indicates that a functional relationship exists between Tfr cell activation and FVIII inhibitor production after FVIII immunization. Using Tfr deficient Foxp3-Cre Bcl6 fl/fl (BCL6FC) mice, we determined the role of Tfr cells in inhibitor generation in vivo. Loss of Tfr cells led to significantly decreased FVIII inhibitor levels, with FVIII inhibitor titers in BCL6FC mice markedly lower than in wild-type (WT) mice upon recombinant human full-length FVIII (rhFVIII, Kogenate 200 U/kg/week IV x4) immunization (24 ± 16 and 131 ± 114 BU/ml, respectively). However, the total anti-FVIII IgG titers were similar between the BCL6FC and WT groups, suggesting that Tfr cells help generate neutralizing anti-FVIII antibodies. FVIII immunization could still efficiently induce Tfh cell activation in BCL6FC mice. However, both GC Tfh cells and GC B cells were reduced in FVIII immunized BCL6FC mice, suggesting that Tfr cells promote FVIII-specific GC responses. To further investigate the regulatory function of Tfr cells on FVIII immune responses, mice with phosphatase Pten deficiency in Treg cells (Foxp3-Cre Pten fl/fl [PtenFC]), a model with augmented Treg and Tfr cells, were immunized with rhFVIII and anti-FVIII antibody titers were measured at 1 week after the 4 th weekly immunization. Of note, FVIII inhibitor titers (8.06 ± 8.6 BU/ml) were significantly lower than those in WT controls under the same immunization protocol. There were significant positive correlations between the inhibitor titers and total anti-FVIII IgG in the WT and PtenFC groups but not in the BCL6FC group after rhFVIII immunization. To confirm the important role of the Tfh/Tfr pathway in FVIII inhibitor development, we used CD4-Cre Bcl6 fl/fl (CD4-Bcl6-cKO) mice, a model with Bcl6 deficiency in all CD4 + T cells. As expected, none of the CD4-Bcl6-cKO mice developed detectable levels of FVIII inhibitors, demonstrating that the transcription repressor BCL6 is pivotal for FVIII inhibitor development. In conclusion, we found that FVIII immunization induces Tfr cell activation and expansion. Tfr cells have a dual function in regulating FVIII immune responses in mice, and the Tfh/Tfr pathway is essential for FVIII inhibitor development in hemophilia A mice.

IL-21-IgFc immunotherapy alters transcriptional landscape of lymph node cells leading to enhanced flu vaccine response in aging and SIV infection.

Aging people living with HIV (PWH) frequently manifest impaired antibody (Ab) responses to seasonal flu vaccination which has been attributed to ongoing inflammation and immune activation. We have recently reported a similar scenario in old simian immunodeficiency virus (SIV) infected rhesus macaques (RM) with controlled viremia and have been able to compensate for this deficiency by immunotherapy with interleukin (IL)-21-IgFc. To understand the underlying mechanisms of IL-21-induced immunomodulation leading to enhanced flu vaccine response in aging and SIV, we have investigated draining lymph node (LN) cells of IL-21-treated and -untreated animals at postvaccination. We observed IL-21-induced proliferation of flu-specific LN memory CD4 T cells, expansion of B cells expressing IL-21 receptor (IL-21R), and modest expansion of T follicular helper cells (Tfh) co-expressing T-cell immunoreceptor with Ig and ITIM domains (TIGIT) and DNAX accessory molecule (DNAM-1). Transcriptional analysis of LN cells of IL-21-treated animals revealed significant inhibition of germinal center (GC) Tfh and B-cell interferon signaling pathways along with enhanced B-cell development and antigen presentation pathways. We conclude that IL-21 treatment at the time of flu vaccination in aging SIV-infected animals modulates the inductive LN GC activity, to reverse SIV-associated LN Tfh and B-cell dysfunction. IL-21 is a potential candidate molecule for immunotherapy to enhance flu vaccine responses in aging PWH who have deficient antibody responses.

Intradermal but not intramuscular modified vaccinia Ankara immunizations protect against intravaginal tier2 simian-human immunodeficiency virus challenges in female macaques

Route of immunization can markedly influence the quality of immune response. Here, we show that intradermal (ID) but not intramuscular (IM) modified vaccinia Ankara (MVA) vaccinations provide protection from acquisition of intravaginal tier2 simian-human immunodeficiency virus (SHIV) challenges in female macaques. Both routes of vaccination induce comparable levels of serum IgG with neutralizing and non-neutralizing activities. The protection in MVA-ID group correlates positively with serum neutralizing and antibody-dependent phagocytic activities, and envelope-specific vaginal IgA; while the limited protection in MVA-IM group correlates only with serum neutralizing activity. MVA-ID immunizations induce greater germinal center Tfh and B cell responses, reduced the ratio of Th1 to Tfh cells in blood and showed lower activation of intermediate monocytes and inflammasome compared to MVA-IM immunizations. This lower innate activation correlates negatively with induction of Tfh responses. These data demonstrate that the MVA-ID vaccinations protect against intravaginal SHIV challenges by modulating the innate and T helper responses.

Heterogeneous Tfh cell populations that develop during enteric helminth infection predict the quality of type 2 protective response

T follicular helper (Tfh) cells are an important component of germinal center (GC)-mediated humoral immunity. Yet, how a chronic type 1 versus protective type 2 helminth infection modulates Tfh-GC responses remains poorly understood. Here, we employ the helminth Trichuris muris model and demonstrate that Tfh cell phenotypes and GC are differentially regulated in acute versus chronic infection. The latter failed to induce Tfh-GC B cell responses, with Tfh cells expressing Τ-bet and interferon-γ. In contrast, interleukin-4-producing Tfh cells dominate responses to an acute, resolving infection. Heightened expression and increased chromatin accessibility of T helper (Th)1- and Th2 cell-associated genes are observed in chronic and acute induced Tfh cells, respectively. Blockade of the Th1 cell response by T-cell-intrinsic T-bet deletion promoted Tfh cell expansion during chronic infection, pointing to a correlation between a robust Tfh cell response and protective immunity to parasites. Finally, blockade of Tfh-GC interactions impaired type 2 immunity, revealing the critical protective role of GC-dependent Th2-like Tfh cell responses during acute infection. Collectively, these results provide new insights into the protective roles of Tfh-GC responses and identify distinct transcriptional and epigenetic features of Tfh cells that emerge during resolving or chronic T. muris infection.

Spatiotemporal resolution of germinal center Tfh cell differentiation and divergence from central memory CD4+ T cell fate

Follicular helper T (Tfh) cells are essential for germinal center (GC) B cell responses. However, it is not clear which PD-1+CXCR5+Bcl6+CD4+ T cells will differentiate into PD-1hiCXCR5hiBcl6hi GC-Tfh cells and how GC-Tfh cell differentiation is regulated. Here, we report that the sustained Tigit expression in PD-1+CXCR5+CD4+ T cells marks the precursor Tfh (pre-Tfh) to GC-Tfh transition, whereas Tigit–PD-1+CXCR5+CD4+ T cells upregulate IL-7Rα to become CXCR5+CD4+ T memory cells with or without CCR7. We demonstrate that pre-Tfh cells undergo substantial further differentiation at the transcriptome and chromatin accessibility levels to become GC-Tfh cells. The transcription factor c-Maf appears critical in governing the pre-Tfh to GC-Tfh transition, and we identify Plekho1 as a stage-specific downstream factor regulating the GC-Tfh competitive fitness. In summary, our work identifies an important marker and regulatory mechanism of PD-1+CXCR5+CD4+ T cells during their developmental choice between memory T cell fate and GC-Tfh cell differentiation.

The magnitude of the germinal center B cell and T follicular helper cell response predicts long-lasting antibody titers to plague vaccination.

The development of a safe and effective vaccine against Yersinia pestis, the causative organism for plague disease, remains an important global health priority. Studies have demonstrated effective immune-based protection against plague challenge that is induced by plague antigen subunit vaccination in an aqueous alhydrogel formulation; however, whether these candidate vaccines in this formulation and presentation, induce long-lasting immunological memory in the form of durable cellular and antibody recall responses has not been fully demonstrated. In this study, we analyzed germinal center T follicular helper and germinal center B cell responses following F1V and F1 + V plague subunit immunization of mice with vaccines formulated in various adjuvants. Our data demonstrate that recombinant plague protein immunization formulated with IL-2/GM-CSF cytokines bound to alhydrogel adjuvant drive an increase in the magnitude of the germinal center T follicular helper and germinal center B cell responses following primary immunization, compared to vaccines formulated with Alhydrogel adjuvant alone. In contrast, plague protein subunit immunization combined with CpG ODN bound to alhydrogel increased the magnitude and duration of the germinal center Tfh and B cell responses following booster immunization. Importantly, enhanced germinal center Tfh and B cell responses correlated with long-lasting and high F1V-specific antibody titers and more robust antibody recall responses to F1V re-exposure. These findings indicate that vaccine formulations that drive enhancement of the germinal center Tfh and B cell responses are critical for inducing durable plague-specific humoral immunity.

Identification of CD4+ T cells with T follicular helper cell characteristics in the pig

T follicular helper (Tfh) cells provide help to germinal center B cells for affinity maturation, class switch and memory formation. Despite these important functions, this subset has not been studied in detail in pigs due to a lack of species-specific antibodies. We investigated putative Tfh cells from lymphoid tissues and blood of healthy pigs by using cross-reactive antibodies for inducible T-cell costimulator (ICOS) and B-cell lymphoma 6 (Bcl-6). In lymph nodes, we identified a CD4+ T cell population with an ICOS+Bcl-6+CD8α+ phenotype, reminiscent of human and murine germinal center Tfh cells. Within blood-derived CD4+ T cells, sorted ICOShiCD25- and ICOSdimCD25dim cells were able to induce the differentiation of CD21+IgM+ B cells into Ig-secreting plasmablasts. Compared to naïve CD4+ T cells, these two phenotypes were 3- to 7-fold enriched for cells expressing the Tfh-related transcripts CD28, CD40LG, IL6R and MAF, as identified by single-cell RNA sequencing. These results provide a first characterization of Tfh cells in swine and confirm their ability to provide B-cell help.

Transcriptome and TCR Repertoire Measurements of CXCR3+ T Follicular Helper Cells Within HIV-Infected Human Lymph Nodes.

Follicular-helper T cells (TFH) are an essential arm of the adaptive immune system. Although TFH were first discovered through their ability to contribute to antibody affinity maturation through co-stimulatory interactions with B cells, new light has been shed on their ability to remain a complex and functionally plastic cell type. Due to a lack sample availability, however, many studies have been limited to characterizing TFH in mice or non-canonical tissue types, such as peripheral blood. Such constraints have resulted in a limited, and sometimes contradictory, understanding of this fundamental cell type. One subset of TFH receiving attention in chronic infection are CXCR3-expressing TFH cells (CXCR3+TFH) due to their abnormal accumulation in secondary lymphoid tissues. Their function and clonal relationship with other TFH subsets in lymphoid tissues during infection, however, remains largely unclear. We thus systematically investigated this and other subsets of TFH within untreated HIV-infected human lymph nodes using Mass CyTOF and a combination of RNA and TCR repertoire sequencing. We show an inflation of the CXCR3+TFH compartment during HIV infection that correlates with a lower HIV burden. Deeper analysis into this population revealed a functional shift of CXCR3+TFH away from germinal center TFH (GC-TFH), including the altered expression of several important transcription factors and cytokines. CXCR3+TFH also upregulated cell migration transcriptional programs and were clonally related to peripheral TFH populations. In combination, these data suggest that CXCR3+TFH have a greater tendency to enter circulation than their CXCR3- counterparts, potentially functioning through distinct modalities that may lead to enhanced defense.

Understanding the temporal requirement for T follicular helper cells for germinal center output of long-lived plasma cells and B memory cells.

Abstract T follicular helper cells (Tfh) play a critical role in supplying help to B cells for the selection of high-affinity B cells in germinal centers (GC), that give rise to memory B cells (Bmem) and long-lived plasma cells (LLPCs). The products of the GC reaction appear to be temporally distinct, with Bmem cells produced early and LLPCs late. The temporal requirements for Tfh help are not well understood and we still do not know when and for how long this help is critical. In the total absence of help supplied by Tfh cells, both GCs and LLPCs are greatly diminished. Our previous work (Schrock, Leddon et al 2019, PNAS) suggested that sustained GC Tfh were critical for LLPC generation, but interestingly not for Bmem generation. We hypothesize that a temporal requirement for GC Tfh regulates LLPC versus Bmem GC output. To test this, we have established a number of models that allow for deletion of Tfh at different times post OVA/CFA immunization. To specifically target Tfh cells, CD4 iCRE Bcl6fl/fl mice were used for time-dependent deletion of Bcl6 upon tamoxifen administration at different stages of the GC reaction. Tfh and GC output are determined using multicolor Flow Cytometry panels, BM B cell Elispot for LLPCs and a Bmem Elispot. With these systems in place, we aim to pinpoint the temporal requirement for Tfh for both Bmem and LLPCs, speculating that timing of Tfh help will differ for these two arms of the GC response. Understanding this temporal requirement of Tfh help within the GC can not only help us to better design future vaccines that promote LLPCs, but also guide the development of drugs to modulate Tfh longevity in autoimmune disorders, where aberrant Tfh development or maintenance contribute to pathology.

Tet2-mediated programing balances T follicular helper cell and T helper 1 cell differentiation.

Abstract The differentiation of CD4+ T cell subsets in response to infection has been studied extensively, however the epigenetic programs that regulate these processes remain poorly understood. Active demethylation by tet methylcytosine dioxygenases of CpG dinucleotides within DNA is a key component of epigenetic programing that promotes lineage specific gene expression and contributes to cellular differentiation and function. Here we report that Tet2 acts to restrict the differentiation of T follicular helper (Tfh) cells in CD4+ T cells responding to viral infection. Using an adoptive transfer model of virus-specific CD4+ cells we found that Tet2-deficient CD4+ T cells skew away from the Th1 lineage and instead preferentially differentiate into highly functional germinal center (GC) Tfh cells that provide enhanced help for B cell responses. We found that the impact of Tet2-mediated programing on CD4+ T cell differentiation is cell intrinsic and the shift in lineage differentiation occurs as early as 2 days post infection. Using genome-wide expression, DNA methylation and transcription factor binding analyses, we found that Tet2 coordinates with multiple transcription factors to mediate the demethylation and expression of their target genes following activation. Our findings establish Tet2 as an important regulator of Tfh cell differentiation and reveal pathways that could be targeted to enhance GC responses against infectious disease. Supported by grants from NIH (R01 AI137238 to J.S.H., T32 AI055434 to L.M.S., and T32 AI138945 to A.B.

GC Tfh Cell Metabolism and Survival of HIV-1 Infection

Abstract CD4 T cells are the primary targets for HIV-1 infection and their loss is a hallmark of HIV-1 disease. However, T follicular helper (Tfh) cells, a distinct subset of CD4 T cells that are specialized in helping B cells form germinal centers (GCs) for the generation of high-affinity class-switched antibodies, undergo expansion in people living with HIV-1 (PLHIV). Indeed, Tfh cells with latent HIV-1 are enriched in PLHIV, whether the PLHIV are on antiretroviral therapy (ART) or not. This shows that Tfh cells are preferentially infected by HIV-1 and are able to survive infection. Through total RNAseq of tonsillar subsets of T cells, including Tfh cells, our lab was able to find a sharp upregulation of an inhibitor of apoptosis family member gene (BIRC5) and a distinct metabolic gene profile in Tfhs compared to the other subsets. We further were able to find and confirm that (1) BIRC5 was highly expressed in human tonsil Tfh cells and the expression was upregulated upon HIV-1 infection, (2) human tonsil Tfh cells had high levels of Fatty Acid Oxidation related genes, but low levels of glycolytic genes, when compared with non-Tfh CD4 T cells, and (3) inhibition of FaO and BIRC5 led to changes in the survival of Tfh cells following HIV-1 infection. Our results show that Tfh survival of HIV-1 is associated with both the metabolic profile and expression of BIRC5 due to their ability to inhibit virus-induced direct and bystander apoptosis. Supported by grants from the NIH T32: Immunology and Infectious Disease Training Program

ICOS Expression Is Required for Maintenance but Not the Formation of Germinal Centers in the Spleen in Response to Plasmodium yoelii Infection.

Inducible T cell costimulator (ICOS) plays a key role in the differentiation and maintenance of follicular helper T (Tfh) cells and, thus, germinal center (GC) formation. Previously, our laboratory showed in a Plasmodium chabaudi infection model that Icos-/- mice were significantly impaired in their ability to form GCs despite persistent infection and, thus, a continued antigen (Ag) load. Here, we show that the resolution of primary infection with Plasmodium yoelii was delayed in Icos-/- mice. This phenotype was associated with a reduction in the accumulation of Tfh-like and GC Tfh cells and an early deficiency in Ag-specific antibody (Ab) production. However, Icos-/- mice could form GCs, although they were less frequent in number than in wild-type (WT) mice. Nonetheless, the Ag-specific Abs from Icos-/- mice lacked signs of affinity maturation, suggesting functional defects associated with these GCs. Eventually, these GC structures dissipated more rapidly in Icos-/- mice than in WT mice. Moreover, the ability of Icos-/- mice to form these GC structures is not reliant on the high Ag loads associated with P. yoelii infections, as GC formation was preserved in Icos-/- mice treated with atovaquone. Finally, mice were unable to form secondary GCs in the absence of ICOS after rechallenge. Overall, these data demonstrate the necessity of ICOS in the maintenance of Tfh cells, the formation and maintenance of sufficient numbers of functioning GCs, and the ability to generate new GC structures after reinfection with P. yoelii.

Early expansion of CD38+ICOS+ GC Tfh in draining lymph nodes during influenza vaccination immune response

SummaryT follicular helper (Tfh) cells provide critical help to B cells during the germinal center (GC) reaction to facilitate generation of protective humoral immunity. Accessing the human lymph node (LN) to study the commitment of CD4 T cells to GC Tfh cell differentiation during in vivo vaccine responses is difficult. We used ultrasound guided fine needle biopsy to monitor recall responses in axillary LNs to seasonal influenza vaccination in healthy volunteers. Specific expansion of GC cell subsets occurred exclusively within draining LNs five days postvaccination. Draining LN GC Tfh and precursor-Tfh cells express higher levels of CD38, ICOS, and Ki67, indicating they were significantly more activated, motile, and proliferating, compared to contralateral LN cells. These observations provide insight into the early expansion phase of the human Tfh lineage within LNs during a vaccine induced memory response and highlights early LN immune responses may not be reflected in the periphery.