Vagus nerve stimulation modulates distinct acetylcholine receptors on B cells and limits the germinal center response.

Acetylcholine is produced in the spleen in response to vagus nerve activation; however, the effects on antibody production have been largely unexplored. Here, we use a chronic vagus nerve stimulation (VNS) mouse model to study the effect of VNS on T-dependent B cell responses. We observed lower titers of high-affinity IgG and fewer antigen-specific germinal center (GC) B cells. GC B cells from chronic VNS mice exhibited altered mRNA and protein expression suggesting increased apoptosis and impaired plasma cell differentiation. Follicular dendritic cell (FDC) cluster dispersal and altered gene expression suggested poor function. The absence of acetylcholine-producing CD4+ T cells diminished these alterations. In vitro studies revealed that α7 and α9 nicotinic acetylcholine receptors (nAChRs) directly regulated B cell production of TNF, a cytokine crucial to FDC clustering. α4 nAChR inhibited coligation of CD19 to the B cell receptor, presumably decreasing B cell survival. Thus, VNS-induced GC impairment can be attributed to distinct effects of nAChRs on B cells.

Follicular dendritic cells restrict interleukin-4 availability in germinal centers and foster memory B cell generation.

B cells within germinal centers (GCs) enter cycles of antibody affinity maturation or exit the GC as memory cells or plasma cells. Here, we examined the contribution of interleukin (IL)-4 on B cell fate decisions in the GC. Single-cell RNA-sequencing identified a subset of light zone GC B cells expressing high IL-4 receptor-a (IL4Ra) and CD23 and lacking a Myc-associated signature. These cells could differentiate into pre-memory cells. B cell-specific deletion of IL4Ra or STAT6 favored the pre-memory cell trajectory, and provision of exogenous IL-4 in a wild-type context reduced pre-memory cell frequencies. IL-4 acted during antigen-specific interactions but also influenced bystander cells. Deletion of IL4Ra from follicular dendritic cells (FDCs) increased the availability of IL-4 in the GC, impaired the selection of affinity-matured B cells, and reduced memory cell generation. We propose that GC FDCs establish a niche that limits bystander IL-4 activity, focusing IL-4 action on B cells undergoing selection and enhancing memory cell differentiation.

Establishment of an ATLL cell line (YG-PLL) dependent on IL-2 and IL-4, which are replaced by OX40-ligand+ HK with poly-L-histidine and dermatan sulfate.

We established an IL-2 and IL-4 (IL2/4) - dependent adult T-cell leukemia/lymphoma (ATLL) cell line (YG-PLL) by adding poly-L-lysine (PLL) to the culture medium. YG-PLL originates from lymphoma cells and contains a defective HTLV-I proviral genome. Although YG-PLL cannot survive without IL-2/4, the follicular dendritic cell (FDC)-like cell line HK expressing OX40-ligand gene (OX40L+HK) inhibited their death in the presence of soluble neutral polymers. After the prevention of cell death, YG-PLL proliferated on OX40L+HK without IL2/4 in the presence of two kinds of positively or negatively charged polymers. In particular, dermatan sulfate and poly-L-histidine supported growth for more than 4 months. Therefore, the original lymphoma cells proliferated transiently in the presence of IL2/4, and their growth arrest was inhibited by the addition of PLL. Furthermore, YG-PLL lost IL2/4 dependency by the following 3-step procedure: preculture with IL2/4 and neutral polymers, 3-day culture with neutral polymer on OX40L+HK to inhibit cell death, and co-culture with OX40L+HK in the presence of the positively and negatively charged polymers. The extracellular environment made by soluble polymers plays a role in the growth of ATLL in vitro.

The Migration of Human Follicular Dendritic Cell-Like Cell Is Facilitated by Matrix Metalloproteinase 3 Expression That Is Mediated through TNFα-ERK1/2-AP1 Signaling.

Follicular dendritic cells are important stromal components of the germinal center (GC) and have pivotal roles in maintaining the GC microenvironment for high-affinity antibody production. Tumor necrosis factor-α (TNFα) is essential for the development and functions of follicular dendritic cells. Despite the importance of follicular dendritic cells in humoral immunity, their molecular control mechanisms have yet to be fully elucidated due to the lack of an adequate investigation system. Here, we have used a unique human primary follicular dendritic cell-like cell (FDCLC) to demonstrate that the migration of these cells is enhanced by TNFα-mediated metalloproteinase 3 (MMP3) expression. MMP3 was found to be highly expressed in normal human GCs and markedly upregulated in human primary FDCLCs by TNFα. TNFα induced ERK1/2 phosphorylation and the transcription of MMP3 through AP1. TNFα treatment increased FDCLC migration, and a knockdown of MMP3 significantly reduced the TNFα-induced migration of FDCLCs. Overall, we have newly identified a control mechanism for the expression of MMP3 in FDCLCs that modulates their migration and may indicate an important role in GC biology. Since GCs are observed in the lesions of autoimmune diseases and lymphomas, targeting the MMP3/TNFα-mediated migration of stromal cells in the B cell follicle may have great potential as a future therapeutic modality against aberrant GC-associated disorders.

Germinal Centre Shutdown.

Germinal Centres (GCs) are transient structures in secondary lymphoid organs, where affinity maturation of B cells takes place following an infection. While GCs are responsible for protective antibody responses, dysregulated GC reactions are associated with autoimmune disease and B cell lymphoma. Typically, 'normal' GCs persist for a limited period of time and eventually undergo shutdown. In this review, we focus on an important but unanswered question - what causes the natural termination of the GC reaction? In murine experiments, lack of antigen, absence or constitutive T cell help leads to premature termination of the GC reaction. Consequently, our present understanding is limited to the idea that GCs are terminated due to a decrease in antigen access or changes in the nature of T cell help. However, there is no direct evidence on which biological signals are primarily responsible for natural termination of GCs and a mechanistic understanding is clearly lacking. We discuss the present understanding of the GC shutdown, from factors impacting GC dynamics to changes in cellular interactions/dynamics during the GC lifetime. We also address potential missing links and remaining questions in GC biology, to facilitate further studies to promote a better understanding of GC shutdown in infection and immune dysregulation.

The magnitude of germinal center reactions is restricted by a fixed number of preexisting niches.

Antibody affinity maturation occurs in the germinal center (GC), a highly dynamic structure that arises upon antigen stimulation and recedes after infection is resolved. While the magnitude of the GC reaction is highly fluctuating and depends on antigens or pathological conditions, it is unclear whether GCs are assembled ad hoc in different locations or in preexisting niches within B cell follicles. We show that follicular dendritic cells (FDCs), the essential cellular components of the GC architecture, form a predetermined number of clusters. The total number of FDC clusters is the same on several different genetic backgrounds and is not altered by immunization or inflammatory conditions. In unimmunized and germ-free mice, a few FDC clusters contain GC B cells; in contrast, immunization or autoimmune milieu significantly increases the frequency of FDC clusters occupied by GC B cells. Excessive occupancy of GC niches by GC B cells after repeated immunizations or in autoimmune conditions suppresses subsequent antibody responses to new antigens. These data indicate that the magnitude of the GC reaction is restricted by a fixed number of permissive GC niches containing preassembled FDC clusters. This finding may help in the future design of vaccination strategies and in the modulation of antibody-mediated autoimmunity.

Follicular dendritic cell dysfunction contributes to impaired antigen-specific humoral responses in sepsis-surviving mice.

Sepsis survivors exhibit impaired responsiveness to antigen (Ag) challenge associated with increased mortality from infection. The contribution of follicular dendritic cells (FDCs) in the impaired humoral response in sepsis-surviving mice is investigated in this study. We demonstrated that mice subjected to sepsis from cecal ligation and puncture (CLP mice) have reduced NP-specific high-affinity class-switched Ig antibodies (Abs) compared with sham-operated control mice following immunization with the T cell-dependent Ag, NP-CGG. NP-specific germinal center (GC) B cells in CLP mice exhibited reduced TNF-α and AID mRNA expression compared with sham-operated mice. CLP mice showed a reduction in FDC clusters, a reduced binding of immune complexes on FDCs, and reduced mRNA expression of CR2, ICAM-1, VCAM-1, FcγRIIB, TNFR1, IKK2, and LTßR compared with sham-operated mice. Adoptive transfer studies showed that there was no B cell-intrinsic defect. In summary, our data suggest that the reduced Ag-specific Ab response in CLP mice is secondary to a disruption in FDC and GC B cell function.

CD169+ Subcapsular Macrophage Role in Antigen Adjuvant Activity.

Vaccines have played a pivotal role in improving public health, however, many infectious diseases lack an effective vaccine. Controlling the spread of infectious diseases requires continuing studies to develop new and improved vaccines. Our laboratory has been investigating the immune enhancing mechanisms of Toll-like receptor (TLR) ligand-based adjuvants, including the TLR2 ligand Neisseria meningitidis outer membrane protein, PorB. Adjuvant use of PorB increases costimulatory factors on antigen presenting cells (APC), increases antigen specific antibody production, and cytokine producing T cells. We have demonstrated that macrophage expression of MyD88 (required for TLR2 signaling) is an absolute requirement for the improved antibody response induced by PorB. Here-in, we specifically investigated the role of subcapsular CD169+ marginal zone macrophages in antibody production induced by the use of TLR-ligand based adjuvants (PorB and CpG) and non-TLR-ligand adjuvants (aluminum salts). CD169 knockout mice and mice treated with low dose clodronate treated animals (which only remove marginal zone macrophages), were used to investigate the role of these macrophages in adjuvant-dependent antibody production. In both sets of mice, total antigen specific immunoglobulins (IgGs) were diminished regardless of adjuvant used. However, the greatest reduction was seen with the use of TLR ligands as adjuvants. In addition, the effect of the absence of CD169+ macrophages on adjuvant induced antigen and antigen presenting cell trafficking to the lymph nodes was examined using immunofluorescence by determining the relative extent of antigen loading on dendritic cells (DCs) and antigen deposition on follicular dendritic cells (FDC). Interestingly, only vaccine preparations containing PorB had significant decreases in antigen deposition in lymphoid follicles and germinal centers in CD169 knockout mice or mice treated with low dose clodronate as compared to wildtype controls. Mice immunized with CpG containing preparations demonstrated decreased FDC networks in the mice treated with low dose clodronate. Conversely, alum containing preparations only demonstrated significant decreases in IgG in CD169 knockout mice. These studies stress that importance of subcapsular macrophages and their unique role in adjuvant-mediated antibody production, potentially due to an effect of these adjuvants on antigen trafficking to the lymph node and deposition on follicular dendritic cells.

A Novel Image Analysis Approach Reveals a Role for Complement Receptors 1 and 2 in Follicular Dendritic Cell Organization in Germinal Centers.

Follicular dendritic cells (FDCs) are rare and enigmatic cells that mainly reside in germinal centers (GCs). They are capable of capturing immune complexes, via their Fc (FcRs) and complement receptors (CRs) and storing them for long periods in non-degradative vesicles. Presentation of ICs on FDCs to B cells is believed to drive affinity maturation. CR1 and CR2 are expressed on B cells and FDCs. Cr2 knock out (KO) mice, lacking both receptors, have impaired antibody and GC responses. Utilizing a novel ImageJ macro to analyze confocal fluorescence microscopy images of spleen sections, we here investigate how FDCs in wild type (WT) and Cr2 KO mice behave during the first two weeks after immunization with sheep red blood cells (SRBC). Mice were immunized with SRBC i.v. and spleen and serum samples harvested at various time points. As expected, antibody and GC responses in Cr2 KO mice were impaired in comparison to WT mice. Fewer FDCs were identified in Cr2 KO mice, and these exhibited differential localization and organization in comparison to WT mice. WT FDCs were primarily located within GCs at the light zone/dark zone border. FDCs from WT but not Cr2 KO mice were actively dispersed in GCs, i.e. tended to move away from each other, presumably to increase their surface area for B cell interaction. FDCs from Cr2 KO mice were more often found on follicles outside of the GCs and those within the GCs were closer to the periphery in comparison to WT FDCs. Expression of CR1 and CR2, FcγRIIB, and FcµR increased in FDCs from WT mice during the course of immunization. The results suggest that decreased ability to capture ICs by FDCs lacking CR1 and CR2 may not be the only explanation for the impaired GC and antibody responses in Cr2 KO mice. Poor FDC organization in GCs and failure to increase receptor expression after immunization may further contribute to the inefficient immune responses observed.

Single-cell sequencing of human white adipose tissue identifies new cell states in health and obesity.

White adipose tissue (WAT) is an essential regulator of energy storage and systemic metabolic homeostasis. Regulatory networks consisting of immune and structural cells are necessary to maintain WAT metabolism, which can become impaired during obesity in mammals. Using single-cell transcriptomics and flow cytometry, we unveil a large-scale comprehensive cellular census of the stromal vascular fraction of healthy lean and obese human WAT. We report new subsets and developmental trajectories of adipose-resident innate lymphoid cells, dendritic cells and monocyte-derived macrophage populations that accumulate in obese WAT. Analysis of cell-cell ligand-receptor interactions and obesity-enriched signaling pathways revealed a switch from immunoregulatory mechanisms in lean WAT to inflammatory networks in obese WAT. These results provide a detailed and unbiased cellular landscape of homeostatic and inflammatory circuits in healthy human WAT.

Gremlin 1+ fibroblastic niche maintains dendritic cell homeostasis in lymphoid tissues.

Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.

Affinity Selection in Germinal Centers: Cautionary Tales and New Opportunities.

Our current quantitative knowledge of the kinetics of antibody-mediated immunity is partly based on idealized experiments throughout the last decades. However, new experimental techniques often render contradictory quantitative outcomes that shake previously uncontroversial assumptions. This has been the case in the field of T-cell receptors, where recent techniques for measuring the 2-dimensional rate constants of T-cell receptor-ligand interactions exposed results contradictory to those obtained with techniques measuring 3-dimensional interactions. Recently, we have developed a mathematical framework to rationalize those discrepancies, focusing on the proper fine-grained description of the underlying kinetic steps involved in the immune synapse. In this perspective article, we apply this approach to unveil potential blind spots in the case of B-cell receptors (BCR) and to rethink the interactions between B cells and follicular dendritic cells (FDC) during the germinal center (GC) reaction. Also, we elaborate on the concept of "catch bonds" and on the recent observations that B-cell synapses retract and pull antigen generating a "retracting force", and propose some testable predictions that can lead to future research.

Rate of Immune Complex Cycling in Follicular Dendritic Cells Determines the Extent of Protecting Antigen Integrity and Availability to Germinal Center B Cells.

Follicular dendritic cells (FDCs) retain immune complexes (ICs) for prolonged time periods and are important for germinal center (GC) reactions. ICs undergo periodic cycling in FDCs, a mechanism supporting an extended half-life of Ag. Based on experimental data, we estimated that the average residence time of PE-ICs on FDC surface and interior were 21 and 36 min, respectively. GC simulations show that Ag cycling might impact GC dynamics because of redistribution of Ag on the FDC surface and by protecting Ag from degradation. Ag protection and influence on GC dynamics varied with Ag cycling time and total Ag concentration. Simulations predict that blocking Ag cycling terminates the GC reaction and decreases plasma cell production. Considering that cycling of Ag could be a target for the modulation of GC reactions, our findings highlight the importance of understanding the mechanism and regulation of IC cycling in FDCs.

Comprehensive Flow Cytometry Profiling of the Immune System in COVID-19 Convalescent Individuals.

SARS-CoV-2 has infected more than 200 million people worldwide, with more than 4 million associated deaths. Although more than 80% of infected people develop asymptomatic or mild COVID-19, SARS-CoV-2 can induce a profound dysregulation of the immune system. Therefore, it is important to investigate whether clinically recovered individuals present immune sequelae. The potential presence of a long-term dysregulation of the immune system could constitute a risk factor for re-infection and the development of other pathologies. Here, we performed a deep analysis of the immune system in 35 COVID-19 recovered individuals previously infected with SARS-CoV-2 compared to 16 healthy donors, by flow cytometry. Samples from COVID-19 individuals were analysed from 12 days to 305 days post-infection. We observed that, 10 months post-infection, recovered COVID-19 patients presented alterations in the values of some T-cell, B-cell, and innate cell subsets compared to healthy controls. Moreover, we found in recovered COVID-19 individuals increased levels of circulating follicular helper type 1 (cTfh1), plasmablast/plasma cells, and follicular dendritic cells (foDC), which could indicate that the Tfh-B-foDC axis might be functional to produce specific immunoglobulins 10 months post-infection. The presence of this axis and the immune system alterations could constitute prognosis markers and could play an important role in potential re-infection or the presence of long-term symptoms in some individuals.

B Cell Speed and B-FDC Contacts in Germinal Centers Determine Plasma Cell Output via Swiprosin-1/EFhd2.

Plasma cells secreting affinity-matured antibodies develop in germinal centers (GCs), where B cells migrate persistently and directionally over defined periods of time. How modes of GC B cell migration influence plasma cell development remained unclear. Through genetic deletion of the F-actin bundling protein Swiprosin-1/EF-hand domain family member 2 (EFhd2) and by two-photon microscopy, we show that EFhd2 restrains B cell speed in GCs and hapten-specific plasma cell output. Modeling the GC reaction reveals that increasing GC B cell speed promotes plasma cell generation. Lack of EFhd2 also reduces contacts of GC B cells with follicular dendritic cells in vivo. Computational modeling uncovers that both GC output and antibody affinity depend quantitatively on contacts of GC B cells with follicular dendritic cells when B cells migrate more persistently. Collectively, our data explain how GC B cells integrate speed and persistence of cell migration with B cell receptor affinity.

An Agonistic Anti-CD137 Antibody Disrupts Lymphoid Follicle Structure and T-Cell-Dependent Antibody Responses.

CD137 is a costimulatory receptor expressed on natural killer cells, T cells, and subsets of dendritic cells. An agonistic monoclonal antibody (mAb) against CD137 has been used to reduce tumor burden or reverse autoimmunity in animal models and clinical trials. Here, we show that mice treated with an agonistic anti-CD137 mAb have reduced numbers of germinal center (GC) B cells and follicular dendritic cells (FDCs) in lymphoid tissues, which impair antibody responses to multiple T-cell-dependent antigens, including infectious virus, viral proteins, and conjugated haptens. These effects are not due to enhanced apoptosis or impaired proliferation of B cells but instead correlate with changes in lymphoid follicle structure and GC B cell dispersal and are mediated by CD137 signaling in CD4+ and CD8+ T cells. Our experiments in mice suggest that agonistic anti-CD137 mAbs used in cancer and autoimmunity therapy may impair long-term antibody and B cell memory responses.

Toll-Like Receptor Ligand Based Adjuvant, PorB, Increases Antigen Deposition on Germinal Center Follicular Dendritic Cells While Enhancing the Follicular Dendritic Cells Network.

Vaccines are arguably one of the greatest advancements in modern medicine. Subunit vaccines comprise the majority of current preparations and consist of two main components-antigen and adjuvant. The antigen is a small molecule against which the vaccine induces an immune response to provide protection via the immunostimulatory ability of the adjuvant. Our laboratory has investigated the adjuvant properties of Toll-like receptor (TLR) ligand-based adjuvants, especially the outer membrane protein from Neisseria mengingitidis, PorB. In this current study we used PorB, along with CpG, an intracellular TLR9 agonist, and a non-TLR adjuvant, aluminum salts (Alum), to further investigate cellular mechanisms of adjuvanticity, focusing on the fate of intact antigen in the germinal center and association with follicular dendritic cells (FDCs). FDCs are located in the B cell light zone of the germinal center and are imperative for affinity maturation. They are stromal cells that retain whole intact antigen allowing recognition by the B cell receptor of the germinal center B cells. Our studies demonstrate that TLR ligands, but not Alum, increase the FDC network, while PorB and Alum increased colocalization of FDC and the model soluble antigen, ovalbumin (OVA). As PorB is the only adjuvant tested that induces both a higher number of FDCs and increased deposition of antigen on FDCs, it has the greatest ability to increase FDC-antigen interaction, essential for induction of B cell affinity maturation. These studies demonstrate a further mechanism and potential superiority of PorB as an adjuvant and its influence on antibody production.

B cell zone reticular cell microenvironments shape CXCL13 gradient formation.

Through the formation of concentration gradients, morphogens drive graded responses to extracellular signals, thereby fine-tuning cell behaviors in complex tissues. Here we show that the chemokine CXCL13 forms both soluble and immobilized gradients. Specifically, CXCL13+ follicular reticular cells form a small-world network of guidance structures, with computer simulations and optimization analysis predicting that immobilized gradients created by this network promote B cell trafficking. Consistent with this prediction, imaging analysis show that CXCL13 binds to extracellular matrix components in situ, constraining its diffusion. CXCL13 solubilization requires the protease cathepsin B that cleaves CXCL13 into a stable product. Mice lacking cathepsin B display aberrant follicular architecture, a phenotype associated with effective B cell homing to but not within lymph nodes. Our data thus suggest that reticular cells of the B cell zone generate microenvironments that shape both immobilized and soluble CXCL13 gradients.

Remodeling of light and dark zone follicular dendritic cells governs germinal center responses.

Efficient generation of germinal center (GC) responses requires directed movement of B cells between distinct microenvironments underpinned by specialized B cell-interacting reticular cells (BRCs). How BRCs are reprogrammed to cater to the developing GC remains unclear, and studying this process is largely hindered by incomplete resolution of the cellular composition of the B cell follicle. Here we used genetic targeting of Cxcl13-expressing cells to define the molecular identity of the BRC landscape. Single-cell transcriptomic analysis revealed that BRC subset specification was predetermined in the primary B cell follicle. Further topological remodeling of light and dark zone follicular dendritic cells required CXCL12-dependent crosstalk with B cells and dictated GC output by retaining B cells in the follicle and steering their interaction with follicular helper T cells. Together, our results reveal that poised BRC-defined microenvironments establish a feed-forward system that determines the efficacy of the GC reaction.

HIV-1-Specific Chimeric Antigen Receptor T Cells Fail To Recognize and Eliminate the Follicular Dendritic Cell HIV Reservoir In Vitro.

The major obstacle to a cure for HIV infection is the persistence of replication-competent viral reservoirs during antiretroviral therapy. HIV-specific chimeric antigen receptor (CAR) T cells have been developed to target latently infected CD4+ T cells that express virus either spontaneously or after intentional latency reversal. Whether HIV-specific CAR-T cells can recognize and eliminate the follicular dendritic cell (FDC) reservoir of HIV-bound immune complexes (ICs) is unknown. We created HIV-specific CAR-T cells using human peripheral blood mononuclear cells (PBMCs) and a CAR construct that enables the expression of CD4 (domains 1 and 2) and the carbohydrate recognition domain of mannose binding lectin (MBL) to target native HIV Env (CD4-MBL CAR). We assessed CAR-T cell cytotoxicity using a carboxyfluorescein succinimidyl ester (CFSE) release assay and evaluated CAR-T cell activation through interferon gamma (IFN-γ) production and CD107a membrane accumulation by flow cytometry. CD4-MBL CAR-T cells displayed potent lytic and functional responses to Env-expressing cell lines and HIV-infected CD4+ T cells but were ineffective at targeting FDC bearing HIV-ICs. CD4-MBL CAR-T cells were unresponsive to cell-free HIV or concentrated, immobilized HIV-ICs in cell-free experiments. Blocking intercellular adhesion molecule-1 (ICAM-1) inhibited the cytolytic response of CD4-MBL CAR-T cells to Env-expressing cell lines and HIV-infected CD4+ T cells, suggesting that factors such as adhesion molecules are necessary for the stabilization of the CAR-Env interaction to elicit a cytotoxic response. Thus, CD4-MBL CAR-T cells are unable to eliminate the FDC-associated HIV reservoir, and alternative strategies to eradicate this reservoir must be sought.IMPORTANCE Efforts to cure HIV infection have focused primarily on the elimination of latently infected CD4+ T cells. Few studies have addressed the unique reservoir of infectious HIV that exists on follicular dendritic cells (FDCs), persists in vivo during antiretroviral therapy, and likely contributes to viral rebound upon cessation of antiretroviral therapy. We assessed the efficacy of a novel HIV-specific chimeric antigen receptor (CAR) T cell to target both HIV-infected CD4+ T cells and the FDC reservoir in vitro Although CAR-T cells eliminated CD4+ T cells that express HIV, they did not respond to or eliminate FDC bound to HIV. These findings reveal a fundamental limitation to CAR-T cell therapy to eradicate HIV.