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The presence of the HIV-1 reservoir, a group of immune cells that contain intact, integrated, and replication-competent proviruses, is a major challenge to cure HIV-1. HIV-1 reservoir cells are largely unaffected by the cytopathic effects of viruses, antiviral immune responses, or antiretroviral therapy (ART). The HIV-1 reservoir is seeded early during HIV-1 infection and augmented during active viral replication. CD4+ T cells are the primary target for HIV-1 infection, and recent studies suggest that memory T follicular helper cells within the lymph node, more precisely in the B cell follicle, harbor integrated provirus, which contribute to viral rebound upon ART discontinuation. The B cell follicle, more specifically the germinal center, possesses a unique environment because of its distinct property of being partly immune privileged, potentially allowing HIV-1-infected cells within the lymph nodes to be protected from CD8+ T cells. This modified immune response in the germinal center of the follicle is potentially explained by the exclusion of CD8+ T cells and the presence of T regulatory cells at the junction of the follicle and extrafollicular region. The proviral makeup of HIV-1-infected cells is similar in lymph nodes and blood, suggesting trafficking between these compartments. Little is known about the cell-to-cell interactions, microenvironment of HIV-1-infected cells in the follicle, and trafficking between the lymph node follicle and other body compartments. Applying a spatiotemporal approach that integrates genomics, transcriptomics, and proteomics to investigate the HIV-1 reservoir and its neighboring cells in the lymph node has promising potential for informing HIV-1 cure efforts., Competing Interests: The authors declare no conflict of interest.

Most mature B-cell malignancies originate from the malignant transformation of germinal center (GC) B cells. The GC reaction appears to have a role in malignant transformation, in which a major player of the GC reaction is BCL6, a key regulator of this process. We now demonstrate that BCL6 protein levels were dramatically decreased in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines and Burkitt's lymphoma cell lines. Notably, BCL6 degradation was significantly enhanced in the presence of both EBNA3C and FBXO11. Furthermore, the amino-terminal domain of EBNA3C, which contains residues 50-100, interacts directly with FBXO11. The expression of EBNA3C and FBXO11 resulted in a significant induction of cell proliferation. Furthermore, BCL6 protein expression levels were regulated by EBNA3C via the Skp Cullin Fbox (SCF) FBXO11 complex, which mediated its ubiquitylation, and knockdown of FBXO11 suppressed the transformation of lymphoblastoid cell lines. These data provide new insights into the function of EBNA3C in B-cell transformation during GC reaction and raise the possibility of developing new targeted therapies against EBV-associated cancers., Importance: The novel revelation in our study involves the suppression of BCL6 expression by the essential Epstein-Barr virus (EBV) antigen EBNA3C, shedding new light on our current comprehension of how EBV contributes to lymphomagenesis by impeding the germinal center reaction. It is crucial to note that while several EBV latent proteins are expressed in infected cells, the collaborative mechanisms among these proteins in regulating B-cell development or inducing B-cell lymphoma require additional investigation. Nonetheless, our findings carry significance for the development of emerging strategies aimed at addressing EBV-associated cancers., Competing Interests: The authors declare no conflict of interest.

Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Liao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Importance: The human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are etiologic agents of numerous B cell lymphomas. A hallmark of gammaherpesvirus infection is their ability to establish lifelong latency in B cells. However, the specific mechanisms that mediate chronic infection in B cells in vivo remain elusive. Cellular E3 ubiquitin ligases regulate numerous biological processes by catalyzing ubiquitylation and modifying protein location, function, or half-life. Many viruses hijack host ubiquitin ligases to evade antiviral host defense and promote viral fitness. Here, we used the murine gammaherpesvirus 68 in vivo system to demonstrate that the E3 ligase Cul4b is essential for this virus to establish latency in germinal center B cells. These findings highlight an essential role for this E3 ligase in promoting chronic gammaherpesvirus infection in vivo and suggest that targeted inhibition of E3 ligases may provide a novel and effective intervention strategy against gammaherpesvirus-associated diseases., Competing Interests: The authors declare no conflict of interest.

The germinal center (GC) plays a central role in the generation of antigen-specific B cells and antibodies. Tight regulation of the GC is essential due to the inherent risks of tumorigenesis and autoimmunity posed by inappropriate GC B cell processes. Gammaherpesviruses such as Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) utilize numerous armaments to drive infected naïve B cells, independent of antigen, through GC reactions to expand the latently infected B cell population and establish a stable latency reservoir. We previously demonstrated that the MHV68 microRNA (miRNA) mghv-miR-M1-7-5p represses host EWSR1 (Ewing sarcoma breakpoint region 1) to promote B cell infection. EWSR1 is a transcription and splicing regulator that is recognized for its involvement as a fusion protein in Ewing sarcoma. A function for EWSR1 in B cell responses has not been previously reported. Here, we demonstrate that 1) B cell-specific deletion of EWSR1 had no effect on generation of mature B cell subsets or basal immunoglobulin levels in naïve mice, 2) repression or ablation of EWSR1 in B cells promoted expansion of MHV68 latently infected GC B cells, and 3) B cell-specific deletion of EWSR1 during a normal immune response to nonviral antigen resulted in significantly elevated numbers of antigen-specific GC B cells, plasma cells, and circulating antibodies. Notably, EWSR1 deficiency did not affect the proliferation or survival of GC B cells but instead resulted in the generation of increased numbers of precursor GC B cells. Cumulatively, these findings demonstrate that EWSR1 is a negative regulator of B cell responses.

T follicular helper cell (TFH)-dependent antibody responses are critical for long-term immunity. Antibody responses are diminished in early life, limiting long-term protective immunity and allowing prolonged or recurrent infection, which may be important for viral lung infections that are highly prevalent in infancy. In a murine model using respiratory syncytial virus (RSV), we show that TFH and the high-affinity antibody production they promote are vital for preventing disease on RSV reinfection. Following a secondary RSV infection, TFH-deficient mice had significantly exacerbated disease characterized by delayed viral clearance, increased weight loss, and immunopathology. TFH generation in early life was compromised by heightened IL-2 and STAT5 signaling in differentiating naive T cells. Neutralization of IL-2 during early-life RSV infection resulted in a TFH-dependent increase in antibody-mediated immunity and was sufficient to limit disease severity upon reinfection. These data demonstrate the importance of TFH in protection against recurrent RSV infection and highlight a mechanism by which this is suppressed in early life., Competing Interests: Disclosures: H.T. Groves reported, "I am currently employed by the Wellcome Trust but I was not employed by the Wellcome Trust at the time of generating data for this research paper. I did not and do not have any role in the funding decisions behind the research included in the submitted work." J.S. Tregoning reported grants from MRC during the conduct of the study. No other disclosures were reported., (© 2021 Pyle et al.)

Epstein-Barr virus (EBV) is an oncogenic herpesvirus implicated in the pathogenesis of several malignant and non-malignant conditions. However, a number of fundamental aspects about the biology of EBV and the mechanism(s) by which this virus induces pathology remain unknown. One major obstacle has been the lack of a suitable animal model for EBV infection. In this study, using our recently established rabbit model of EBV infection, we examined the early events following primary EBV infection. We show that, both immunocompetent and immunosuppressed animals were readily susceptible to EBV infection. However, immunosuppressed animals showed marked splenomegaly and widespread infection. Following EBV infection, the virus primarily targeted naïve IgM + , CD20 + , CD21 + and CD79a + B cells. Infected cells expressed varying sets of viral latent/lytic gene products. Notably, co-expression of latent and lytic proteins in the same cell was not observed. Infected cells in type 0/1 latency (EBERs + ), were small and proliferating (Ki67 + ). By contrast, cells in type 2/3 latency (LMP1 + ), were large, non-proliferating (Ki-67 - ) and p53 + . Although infected B-cells were widely present in splenic follicles, they did not express germinal center marker, BCL-6. Taken together, this study shows for the first time, some of the early events following primary EBV infection., (© 2021. The Author(s).)

Besides antigen-specific responses to viral antigens, humoral immune response in virus infection can generate polyreactive and autoreactive antibodies. Dengue and Zika virus infections have been linked to antibody-mediated autoimmune disorders, including Guillain-Barré syndrome. A unique feature of flaviviruses is the secretion of nonstructural protein 1 (NS1) by infected cells. NS1 is highly immunogenic, and antibodies targeting NS1 can have both protective and pathogenic roles. In the present study, we investigated the humoral immune response to Zika virus NS1 and found NS1 to be an immunodominant viral antigen associated with the presence of autoreactive antibodies. Through single B cell cultures, we coupled binding assays and BCR sequencing, confirming the immunodominance of NS1. We demonstrate the presence of self-reactive clones in germinal centers after both infection and immunization, some of which present cross-reactivity with NS1. Sequence analysis of anti-NS1 B cell clones showed sequence features associated with pathogenic autoreactive antibodies. Our findings demonstrate NS1 immunodominance at the cellular level as well as a potential role for NS1 in ZIKV-associated autoimmune manifestations., Competing Interests: Disclosures: The authors declare no competing financial interests., (© 2021 Cavazzoni et al.)

The Epstein-Barr virus (EBV) is a well-adapted human virus, and its infection is exclusive to our species, generally beginning in the childhood and then persisting throughout the life of most of the affected adults. Although this infection generally remains asymptomatic, EBV can trigger life-threatening conditions under unclear circumstances. The EBV lifecycle is characterized by interactions with other viruses or bacteria, which increases the probability of awakening its pathobiont capacity. For instance, EBV infects B cells with the potential to alter the germinal center reaction (GCR)-an adaptive immune structure wherein mutagenic-driven processes take place. HIV- and Plasmodium falciparum -induced B cell hyperactivation also feeds the GCR. These agents, along with the B cell tropic KSHV, converge in the ontogeny of germinal center (GC) or post-GC lymphomas. EBV oral transmission facilitates interactions with local bacteria and HPV, thereby increasing the risk of periodontal diseases and head and neck carcinomas. It is less clear as to how EBV is localized in the stomach, but together with Helicobacter pylori , they are known to be responsible for gastric cancer. Perhaps this mechanism is reminiscent of the local inflammation that attracts different herpesviruses and enhances graft damage and chances of rejection in transplanted patients. In this review, we discussed the existing evidence suggestive of EBV possessing the potential to synergize or cooperate with these agents to trigger or worsen the disease.

Location of immune cells that form the germinal center reaction within secondary lymphoid tissues can be characterized using confocal microscopy. Here, we present an optimized immunofluorescence staining protocol to image germinal center structures in fixed/frozen spleen sections from ChAdOx1 nCoV-19 immunized mice. This protocol can be adapted to identify other cell types within secondary lymphoid tissues. For complete information on the generation and use of this protocol to examine immune responses to the COVID vaccine ChAdOx1 nCoV-19, please refer to Silva-Cayetano et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Moysi, Del Rio Estrada, Torres-Ruiz, Reyes-Terán, Koup and Petrovas.)

HIV-infected infants are at an increased risk of progressing rapidly to AIDS in the first weeks of life. Here, we evaluated immunological and virological parameters in 25 SIV-infected infant rhesus macaques to understand the factors influencing a rapid disease outcome. Infant macaques were infected with SIVmac251 and monitored for 10 to 17 weeks post-infection. SIV-infected infants were divided into either typical (TypP) or rapid (RP) progressor groups based on levels of plasma anti-SIV antibody and viral load, with RP infants having low SIV-specific antibodies and high viral loads. Following SIV infection, 11 out of 25 infant macaques exhibited an RP phenotype. Interestingly, TypP had lower levels of total CD4 T cells, similar reductions in CD4/CD8 ratios and elevated activation of CD8 T cells, as measured by the levels of HLA-DR, compared to RP. Differences between the two groups were identified in other immune cell populations, including a failure to expand activated memory (CD21-CD27+) B cells in peripheral blood in RP infant macaques, as well as reduced levels of germinal center (GC) B cells and T follicular helper (Tfh) cells in spleens (4- and 10-weeks post-SIV). Reduced B cell proliferation in splenic germinal GCs was associated with increased SIV+ cell density and follicular type 1 interferon (IFN)-induced immune activation. Further analyses determined that at 2-weeks post SIV infection TypP infants exhibited elevated levels of the GC-inducing chemokine CXCL13 in plasma, as well as significantly lower levels of viral envelope diversity compared to RP infants. Our findings provide evidence that early viral and immunologic events following SIV infection contributes to impairment of B cells, Tfh cells and germinal center formation, ultimately impeding the development of SIV-specific antibody responses in rapidly progressing infant macaques., Competing Interests: The authors have declared that no competing interests exist.

Enterovirus 71 (EV71) is a positive single-stranded RNA (ssRNA) virus from the enterovirus genus of Picornaviridae family and causes diseases ranged from the mild disease of hand, foot and mouth disease (HFMD) to the severe disease of neurological involvement in young children. TLR7 is an intracellular pattern recognition receptor (PRR) recognizing viral ssRNA. In this study, we investigated the role of TLR7 in EV71 infection in mouse pups (10-12 days old) and found that wild-type (WT) and TLR7 knock-out (TLR7KO) mice infected with EV71 showed similar limb paralysis at the onset and peak of the disease, comparable loss of motor neurons, and similar levels of antiviral molecules in the spinal cord. These results suggest that TLR7 is not the absolute PRR for EV71 in the spinal cord. Interestingly, TLR7KO mice infected with EV71 exhibited significantly delayed recovery from limb paralysis compared with WT mice. TLR7KO mice infected with EV71 showed significantly decreased levels of IgM and IgG2, important antibodies for antiviral humoral immunity. Furthermore, TLR7KO mice infected with EV71 showed a decrease of germinal center B cells in the spleen compared with WT mice. Altogether, our study suggests that TLR7 plays a critical role in anti-viral humoral immunity rather than in being a PRR in the spinal cord during EV71 infection in young mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lin, Lu, Chuang, Kuo, Lin, Li, Wang, Hsueh and Liao.)

Human bocavirus 1 (HBoV1), a nonenveloped single-stranded DNA parvovirus, causes mild to life-threatening respiratory tract infections, acute otitis media, and encephalitis in young children. HBoV1 often persists in nasopharyngeal secretions for months, hampering diagnosis. It has also been shown to persist in pediatric palatine and adenoid tonsils, which suggests that lymphoid organs are reservoirs for virus spread; however, the tissue site and host cells remain unknown. Our aim was to determine, in healthy nonviremic children with preexisting HBoV1 immunity, the adenotonsillar persistence site(s), host cell types, and virus activity. We discovered that HBoV1 DNA persists in lymphoid germinal centers (GCs), but not in the corresponding tonsillar epithelium, and that the cell types harboring the virus are mainly naive, activated, and memory B cells and monocytes. Both viral DNA strands and both sides of the genome were detected, as well as infrequent mRNA. Moreover, we showed, in B-cell and monocyte cultures and ex vivo tonsillar B cells, that the cellular uptake of HBoV1 occurs via the Fc receptor (FcγRII) through antibody-dependent enhancement (ADE). This resulted in viral mRNA transcription, known to occur exclusively from double-stranded DNA in the nucleus, however, with no detectable productive replication. Confocal imaging with fluorescent virus-like particles moreover disclosed endocytosis. To which extent the active HBoV1 GC persistence has a role in chronic inflammation or B-cell maturation disturbances, and whether the virus can be reactivated, will be interesting topics for forthcoming studies. IMPORTANCE Human bocavirus 1 (HBoV1), a common pediatric respiratory pathogen, can persist in airway secretions for months hampering diagnosis. It also persists in tonsils, providing potential reservoirs for airway shedding, with the exact location, host cell types, and virus activity unknown. Our study provides new insights into tonsillar HBoV1 persistence. We observed HBoV1 persistence exclusively in germinal centers where immune maturation occurs, and the main host cells were B cells and monocytes. In cultured cell lines and primary tonsillar B cells, we showed the virus uptake to be significantly enhanced by HBoV1-specific antibodies, mediated by the cellular IgG receptor, leading to viral mRNA synthesis, but without detectable productive replication. Possible implications of such active viral persistence could be tonsillar inflammation, disturbances in immune maturation, reactivation, or cell death with release of virus DNA, explaining the long-lasting HBoV1 airway shedding., (Copyright © 2021 Xu et al.)

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) with CCR5- donor cells is the only treatment known to cure HIV-1 in patients with underlying malignancy. This is likely due to a donor cell-mediated graft-versus-host effect targeting HIV reservoirs. Allo-HSCT would not be an acceptable therapy for most people living with HIV due to the transplant-related side effects. Chimeric antigen receptor (CAR) immunotherapies specifically traffic to malignant lymphoid tissues (lymphomas) and, in some settings, are able to replace allo-HSCT. Here, we quantified the engraftment of HSC-derived, virus-directed CAR T cells within HIV reservoirs in a macaque model of HIV infection, using potentially novel IHC assays. HSC-derived CAR cells trafficked to and displayed multilineage engraftment within tissue-associated viral reservoirs, persisting for nearly 2 years in lymphoid germinal centers, the brain, and the gastrointestinal tract. Our findings demonstrate that HSC-derived CAR+ cells reside long-term and proliferate in numerous tissues relevant for HIV infection and cancer.

Gammaherpesviruses (GHVs) are DNA tumor viruses that establish lifelong, chronic infections in lymphocytes of humans and other mammals. GHV infections are associated with numerous cancers, especially in immunocompromised hosts. While it is known that GHVs utilize host germinal center (GC) B cell responses during latency establishment, an understanding of how viral gene products function in specific B cell subsets to regulate this process is incomplete. Using murine gammaherpesvirus 68 (MHV68) as a small-animal model to define mechanisms of GHV pathogenesis in vivo , we generated a virus in which the M2 gene was flanked by loxP sites (M2.loxP), enabling the use of Cre-lox technology to define M2 function in specific cell types in infection and disease. The M2 gene encodes a protein that is highly expressed in GC B cells that promotes plasma cell differentiation and viral reactivation. M2 was efficiently deleted in Cre-expressing cells, and the presence of loxP sites flanking M2 did not alter viral replication or latency in mice that do not express Cre. In contrast, M2.loxP MHV68 exhibited a deficit in latency establishment and reactivation that resembled M2-null virus, following intranasal (IN) infection of mice that express Cre in all B cells (CD19-Cre). Nearly identical phenotypes were observed for M2.loxP MHV68 in mice that express Cre in germinal center (GC) B cells (AID-Cre). However, colonization of neither draining lymph nodes after IN infection nor the spleen after intraperitoneal (IP) infection required M2, although the reactivation defect was retained. Together, these data confirm that M2 function is B cell-specific and demonstrate that M2 primarily functions in AID-expressing cells to facilitate MHV68 dissemination to distal latency reservoirs within the host and reactivation from latency. Our study reveals that a viral latency gene functions within a distinct subset of cells to facilitate host colonization. IMPORTANCE Gammaherpesviruses establish lifelong chronic infections in cells of the immune system that can lead to lymphomas and other diseases. To facilitate colonization of a host, gammaherpesviruses encode gene products that manipulate processes involved in cellular proliferation and differentiation. Whether and how these viral gene products function in specific cells of the immune system is poorly defined. We report here the use of a viral genetic system that allows for deletion of specific viral genes in discrete populations of cells. We employ this system in an in vivo model to demonstrate cell-type-specific requirements for a particular viral gene. Our findings reveal that a viral gene product can function in distinct cellular subsets to direct gammaherpesvirus pathogenesis., (Copyright © 2020 American Society for Microbiology.)

We present this case of human herpes virus 8-positive germinotropic lymphoproliferative disorder in a 20-year-old woman seen in the surgical oncology clinic for localised lymphadenopathy. This is the first case to be reported in the UK, and we discuss it along with a literature review including investigations and treatment options. This will demonstrate the importance of preoperative workup and multidisciplinary teamwork in deciding management plans and serve as a guide for future encounters of this rare condition in clinical practice., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

T Follicular Helper (Tfh) cells are an independent CD4 +  T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

One of the defining characteristics of the B cell receptor (BCR) is the extensive diversity in the repertoire of immunoglobulin genes that make up the BCR, resulting in broad range of specificity. Gammaherpesviruses are B lymphotropic viruses that establish life-long infection in B cells, and although the B cell receptor plays a central role in B cell biology, very little is known about the immunoglobulin repertoire of gammaherpesvirus infected cells. To begin to characterize the Ig genes expressed by murine gammaherpesvirus 68 (MHV68) infected cells, we utilized single cell sorting to sequence and clone the Ig variable regions of infected germinal center (GC) B cells and plasma cells. We show that MHV68 infection is biased towards cells that express the Igλ light chain along with a single heavy chain variable gene, IGHV10-1*01. This population arises through clonal expansion but is not viral antigen specific. Furthermore, we show that class-switching in MHV68 infected cells differs from that of uninfected cells. Fewer infected GC B cells are class-switched compared to uninfected GC B cells, while more infected plasma cells are class-switched compared to uninfected plasma cells. Additionally, although they are germinal center derived, the majority of class switched plasma cells display no somatic hypermutation regardless of infection status. Taken together, these data indicate that selection of infected B cells with a specific BCR, as well as virus mediated manipulation of class switching and somatic hypermutation, are critical aspects in establishing life-long gammaherpesvirus infection., Competing Interests: The authors have declared that no competing interests exist.

The gammaherpesviruses (γHVs), human Kaposi sarcoma-associated herpesvirus (KSHV), EBV, and murine γHV68 are prevalent infections associated with lymphocyte pathologies. After primary infection, EBV and γHV68 undergo latent expansion in germinal center (GC) B cells and persists in memory cells. The GC reaction evolves and selects antigen-specific B cells for memory development but whether γHV passively transients or manipulates this process in vivo is unknown. Using the γHV68 infection model, we analyzed the Ig repertoire of infected and uninfected GC cells from individual mice. We found that infected cells displayed the hallmarks of affinity maturation, hypermutation, and isotype switching but underwent clonal expansion. Strikingly, infected cells displayed distinct repertoire, not found in uninfected cells, with recurrent utilization of certain Ig heavy V segments including Ighv10-1 In a manner observed with KSHV, γHV68 infected cells also displayed lambda light chain bias. Thus, γHV68 subverts GC selection to expand in a specific B cell subset during the process that develops long-lived immunologic memory., (© 2020 Zelazowska et al.)

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infections in the majority of adults worldwide. Chronic gammaherpesvirus infection has been implicated in both lymphomagenesis and, somewhat controversially, autoimmune disease development. Pathogenesis is largely associated with the unique ability of gammaherpesviruses to usurp B cell differentiation, specifically, the germinal center response, to establish long-term latency in memory B cells. The host tyrosine phosphatase SHP1 is known as a brake on immune cell activation and is downregulated in several gammaherpesvirus-driven malignancies. However, here we demonstrate that B cell- but not T cell-intrinsic SHP1 expression supports the gammaherpesvirus-driven germinal center response and the establishment of viral latency. Furthermore, B cell-intrinsic SHP1 deficiency cooperated with gammaherpesvirus infection to increase the levels of double-stranded DNA-reactive antibodies at the peak of viral latency. Thus, in spite of decreased SHP1 levels in gammaherpesvirus-driven B cell lymphomas, B cell-intrinsic SHP1 expression plays a proviral role during the establishment of chronic infection, suggesting that the gammaherpesvirus-SHP1 interaction is more nuanced and is modified by the stage of infection and pathogenesis. IMPORTANCE Gammaherpesviruses establish lifelong infection in a majority of adults worldwide and are associated with a number of malignancies, including B cell lymphomas. These viruses infect naive B cells and manipulate B cell differentiation to achieve a lifelong infection of memory B cells. The germinal center stage of B cell differentiation is important as both an amplifier of the viral latent reservoir and the target of malignant transformation. In this study, we demonstrate that expression of tyrosine phosphatase SHP1, a negative regulator that normally limits the activation and proliferation of hematopoietic cells, enhances the gammaherpesvirus-driven germinal center response and the establishment of chronic infection. The results of this study uncover an intriguing beneficial interaction between gammaherpesviruses that are presumed to profit from B cell activation and a cellular phosphatase that is traditionally perceived to be a negative regulator of the same processes., (Copyright © 2019 American Society for Microbiology.)

As we learn more about the HIV latent reservoir, we continue to discover that the viral reservoir is more complicated than just a pool of infected resting memory CD4+ T cells in peripheral blood. Evidence increasingly points to both certain tissues and certain types of cells as potential viral reservoirs. T follicular helper cells (T FH ) are prime targets of HIV infection-this creates a sanctuary for infected cells because CD8+ T cells generally do not enter lymph node follicles unless they express CXCR5, and are not as effective at killing infected CD4+ T cells as peripheral CD8+ T cells. In this review, we summarize the current state of research on T FH cell infection in peripheral lymphoid tissues and focus on the question of whether CD8+ T cell exclusion from B cell follicles is responsible, at least in part, for establishing secondary lymphoid tissue B cell follicles as an anatomic site of HIV transcription and replication., (Copyright © 2019 Beck, Veenhuis and Blankson.)

The pleiotropic cytokine IL-6 plays an integral role not only in innate inflammatory responses but also in the activation and differentiation of lymphocyte subsets. In this study, by using a conditional knockout (cKO) model with selective IL-6 receptor deletion in T cells (IL-6R-cKO), we demonstrated that T cell-specific IL-6R signaling is essential for viral control during persistent lymphocytic choriomeningitis virus clone 13 infection. Strikingly, we observed that in contrast to previous studies with ubiquitous IL-6 deletion or blockade, specific IL-6R deletion in T cells did not affect T follicular helper (Tfh) cell accumulation unless IL-6R-deficient T cells were competing with wild-type cells in mixed bone marrow chimeras. In contrast, Tfh cells from IL-6R-cKO-infected mice exhibited reduced ICOS expression in both chimeric and nonchimeric settings, and this sole identifiable Tfh defect was associated with reduced germinal centers, compromised Ig switch and low avidity of lymphocytic choriomeningitis virus-specific Abs despite intact IL-6R expression in B cells. We posit that IL-6R cis- signaling is absolutely required for appropriate ICOS expression in Tfh cells and provides a competitive advantage for Tfh accumulation, enabling generation of optimal B cell and Ab responses, and ultimately viral control during in vivo chronic infection., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Following infection, inflammatory cues upregulate core transcriptional programs to establish pathogen-specific protection. In viral infections, T follicular helper (TFH) cells express the prototypical T helper 1 transcription factor T-bet. Several studies have demonstrated essential but conflicting roles for T-bet in TFH biology. Understanding the basis of this controversy is crucial, as modulation of T-bet expression instructs TFH differentiation and ultimately protective antibody responses. Comparing influenza and LCMV viral infections, we demonstrate that the role of T-bet is contingent on the environmental setting of TFH differentiation, IL-2 signaling, and T cell competition. Furthermore, we demonstrate that T-bet expression by either TFH or GC B cells independently drives antibody isotype class switching. Specifically, T cell-specific loss of T-bet promotes IgG1, whereas B cell-specific loss of T-bet inhibits IgG2a/c switching. Combined, this work highlights that the context-dependent induction of T-bet instructs the development of protective, neutralizing antibodies following viral infection or vaccination., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Persistent central nervous system (CNS) inflammation, as seen in chronic infections or inflammatory demyelinating diseases such as Multiple Sclerosis (MS), results in the accumulation of various B cell subsets in the CNS, including naïve, activated, memory B cells (Bmem), and antibody secreting cells (ASC). However, factors driving heterogeneous B cell subset accumulation and antibody (Ab) production in the CNS compartment, including the contribution of ectopic lymphoid follicles (ELF), during chronic CNS inflammation remain unclear and is a major gap in our understanding of neuroinflammation. We sought to address this gap using the Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) model of progressive MS. In this model, injection of the virus into susceptible mouse strains results in a persistent infection associated with demyelination and progressive disability. During chronic infection, the predominant B cell phenotypes accumulating in the CNS were isotype-switched B cells, including Bmem and ASC with naïve/early activated and transitional B cells present at low frequencies. B cell accumulation in the CNS during chronic TMEV-IDD coincided with intrathecal Ab synthesis in the cerebrospinal fluid (CSF). Mature and isotype-switched B cells predominately localized to the meninges and perivascular space, with IgG isotype-switched B cells frequently accumulating in the parenchymal space. Both mature and isotype-switched B cells and T cells occupied meningeal and perivascular spaces, with minimal evidence for spatial organization typical of ELF mimicking secondary lymphoid organs (SLO). Moreover, immunohistological analysis of immune cell aggregates revealed a lack of SLO-like ELF features, such as cell proliferation, cell death, and germinal center B cell markers. Nonetheless, flow cytometric assessment of B cells within the CNS showed enhanced expression of activation markers, including moderate upregulation of GL7 and expression of the costimulatory molecule CD80. B cell-related chemokines and trophic factors, including APRIL, BAFF, CXCL9, CXCL10, CCL19, and CXCL13, were elevated in the CNS. These results indicate that localization of heterogeneous B cell populations, including activated and isotype-switched B cell phenotypes, to the CNS and intrathecal Ab (ItAb) synthesis can occur independently of SLO-like follicles during chronic inflammatory demyelinating disease.

Expression of the latent membrane protein-1 (LMP1) of Epstein-Barr virus (EBV) was investigated in 153 cases of EBV+ classic Hodgkin lymphoma (cHL); 120 cases were pediatric patients (< 14 years of age) from Iraq, and 33 cases were adult patients from Italy. We describe for the first time the presence of LMP1 protein in EBV-encoded RNA (EBER)-negative follicular dendritic cells (FDCs) of reactive germinal centers (GC) associated with EBV+ cHL. Presence of LMP1+ GCs was independent of geographic region and age of patients. Variable numbers of reactive GCs were present in 22.2% of cases (34 of 153), whereas LMP1 staining of FDCs was present in about a third of cases (10 of 34) with reactive GC. Most cases with LMP1+ GC were mixed-cellularity (MC) subtype, but some nodular sclerosis (NS) was also present. GC cells with LMP1+ FDCs were surrounded by numerous EBV-infected cells which were positive for EBER, LMP1, and CD30. Double immunolocalization analysis revealed that LMP1 was associated with CD63, an exosomal marker, and with CD21. The possibility is discussed that peri-follicular EBV-infected cells release LMP1 protein, perhaps through exosomes, and that the protein is then captured by FDCs and is presented to EBER-negative GC B cells.

T-follicular helper (Tfh) cells, co-expressing PD-1 and TIGIT, serve as a major cell reservoir for HIV-1 and are responsible for active and persistent HIV-1 transcription after prolonged antiretroviral therapy (ART). However, the precise mechanisms regulating HIV-1 transcription in lymph nodes (LNs) remain unclear. In the present study, we investigated the potential role of immune checkpoint (IC)/IC-Ligand (IC-L) interactions on HIV-1 transcription in LN-microenvironment. We show that PD-L1 (PD-1-ligand) and CD155 (TIGIT-ligand) are predominantly co-expressed on LN migratory (CD1chighCCR7+CD127+) dendritic cells (DCs), that locate predominantly in extra-follicular areas in ART treated individuals. We demonstrate that TCR-mediated HIV production is suppressed in vitro in the presence of recombinant PD-L1 or CD155 and, more importantly, when LN migratory DCs are co-cultured with PD-1+/Tfh cells. These results indicate that LN migratory DCs expressing IC-Ls may more efficiently restrict HIV-1 transcription in the extra-follicular areas and explain the persistence of HIV transcription in PD-1+/Tfh cells after prolonged ART within germinal centers., Competing Interests: The authors declare no competing interests.

African buffaloes ( Syncerus caffer ) are the principal "carrier" hosts of foot-and-mouth disease virus (FMDV). Currently, the epithelia and lymphoid germinal centers of the oropharynx have been identified as sites for FMDV persistence. We carried out studies in FMDV SAT1 persistently infected buffaloes to characterize the diversity of viruses in oropharyngeal epithelia, germinal centers, probang samples (oropharyngeal scrapings), and tonsil swabs to determine if sufficient virus variation is generated during persistence for immune escape. Most sequencing reads of the VP1 coding region of the SAT1 virus inoculum clustered around 2 subpopulations differing by 22 single-nucleotide variants of intermediate frequency. Similarly, most sequences from oropharynx tissue clustered into two subpopulations, albeit with different proportions, depending on the day postinfection (dpi). There was a significant difference between the populations of viruses in the inoculum and in lymphoid tissue taken at 35 dpi. Thereafter, until 400 dpi, no significant variation was detected in the viral populations in samples from individual animals, germinal centers, and epithelial tissues. Deep sequencing of virus from probang or tonsil swab samples harvested prior to postmortem showed less within-sample variability of VP1 than that of tissue sample sequences analyzed at the same time. Importantly, there was no significant difference in the ability of sera collected between 14 and 400 dpi to neutralize the inoculum or viruses isolated at later time points in the study from the same animal. Therefore, based on this study, there is no evidence of escape from antibody neutralization contributing to FMDV persistent infection in African buffalo. IMPORTANCE Foot-and-mouth disease virus (FMDV) is a highly contagious virus of cloven-hoofed animals and is recognized as the most important constraint to international trade in animals and animal products. African buffaloes ( Syncerus caffer ) are efficient carriers of FMDV, and it has been proposed that new virus variants are produced in buffalo during the prolonged carriage after acute infection, which may spread to cause disease in livestock populations. Here, we show that despite an accumulation of low-frequency sequence variants over time, there is no evidence of significant antigenic variation leading to immune escape. Therefore, carrier buffalo are unlikely to be a major source of new virus variants., (Copyright © 2019 American Society for Microbiology.)

Purpose of Review: This review aims to summarize the recent findings on germinal center B-cell reaction and Tfh cells in HIV-1 infection, with particular emphasis on the spatial organization of the germinal center, follicular cell regulation, and cellular alterations resulting from HIV infection., Recent Findings: HIV-specific bNAbs are generated by iterative cycles of B-cell maturation supported by GC environment. Recent observations underline that germinal center structural alterations at the earliest stages of HIV infection could impact Tfh cell and germinal center B-cell homeostasis, thus preventing the rise of efficient humoral immunity. Moreover, despite ART treatment, HIV-derived antigens persist, particularly in follicular CD4+ T cells. Antigenic persistence and variability lead to unregulated chronic stimulation. In this context, regulation of the germinal center appears of special interest. In addition to follicular T-regulatory cells (Tfr), new potent regulators of germinal center reaction, such as follicular CD8 T and NK cells have been recently identified., Summary: Altogether these new data provide a better understanding on how HIV infection severely impacts germinal center reaction. Here we propose several therapeutic approaches to promote the bNAb development in HIV-infected patients by improving the preservation of germinal center architecture and its regulation.

Conventional immunization strategies will likely be insufficient for the development of a broadly neutralizing antibody (bnAb) vaccine for HIV or other difficult pathogens because of the immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. We found that two independent methods of slow delivery immunization of rhesus monkeys (RMs) resulted in more robust T follicular helper (T FH ) cell responses and GC B cells with improved Env-binding, tracked by longitudinal fine needle aspirates. Improved GCs correlated with the development of >20-fold higher titers of autologous nAbs. Using a new RM genomic immunoglobulin locus reference, we identified differential IgV gene use between immunization modalities. Ab mapping demonstrated targeting of immunodominant non-neutralizing epitopes by conventional bolus-immunized animals, whereas slow delivery-immunized animals targeted a more diverse set of epitopes. Thus, alternative immunization strategies can enhance nAb development by altering GCs and modulating the immunodominance of non-neutralizing epitopes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Seasonally recurrent influenza virus infections are a significant cause of global morbidity and mortality. In murine models, primary influenza infection in the respiratory tract elicits potent humoral responses concentrated in the draining mediastinal lymph node and the spleen. In addition to immunity within secondary lymphoid organs (SLO), pulmonary infection is also associated with formation of ectopic inducible bronchus-associated tissues (iBALT) in the lung. These structures display a lymphoid organization, but their function and protective benefits remain unclear. Here we examined the phenotype, transcriptional profile and antigen specificity of B cell populations forming iBALT in influenza infected mice. We show that the cellular composition of iBALT was comparable to SLO, containing populations of follicular dendritic cells (FDC), T-follicular helper (Tfh) cells, and germinal center (GC)-like B cells with classical dark- and light-zone polarization. Transcriptional profiles of GC B cells in iBALT and SLO were conserved regardless of anatomical localization. The architecture of iBALT was pleiomorphic and less structurally defined than SLO. Nevertheless, we show that GC-like structures within iBALT serve as a distinct niche that independently support the maturation and selection of B cells primarily targeted against the influenza virus nucleoprotein. Our findings suggest that iBALT, which are positioned at the frontline of the lung mucosa, drive long-lived, and unique GC reactions that contribute to the diversity of the humoral response targeting influenza.

Human immunodeficiency virus (HIV) infection is characterized by dysfunctional B cell responses. Here we show that chronic simian immunodeficiency virus (SIV) infection is characterized by an expansion of either lymph node germinal center (GC) B cells that co-express Bcl6, Ki-67 and IL-21R and correlate with expanded T follicular helper (Tfh) cells or B cells that lack Bcl6, Ki-67 and IL-21R but express high levels of anti-apoptotic Bcl2 that negatively correlate with Tfh cells. The lack of Tfh cells likely contributes to persistence of dysfunctional non-proliferating B cells during chronic infection. These findings have implications for protective immunity in HIV-infected individuals who harbour low frequencies of Tfh cells., (© 2018 Uniformed Services University. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

We developed a set of rabbit antisera to characterize infections by the macaque RV2 rhadinovirus homologs of KSHV. We analyzed tissues from rhesus and pig-tailed macaques naturally infected with rhesus rhadinovirus (RRV) or Macaca nemestrina rhadinovirus 2 (MneRV2). Our study demonstrates that RV2 rhadinoviruses have a tropism for epithelial cells, lymphocytes and gonadal germ cells in vivo. We observed latent infections in both undifferentiated and differentiated epithelial cells with expression of the latency marker, LANA. Expression of the early (ORF59) and late (glycoprotein B) lytic markers were detected in highly differentiated cells in epithelial ducts in oral, renal, dermal and gastric mucosal tissue as well as differentiated germ cells in male and female gonads. Our data provides evidence that epithelial and germ cell differentiation in vivo induces rhadinovirus reactivation and suggests that infected epithelial and germ cells play a role in transmission and dissemination of RV2 rhadinovirus infections in vivo., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Germinal centers (GCs) are organized lymphoid tissue microstructures where B cells proliferate and differentiate into memory B cells and plasma cells. A few distinctive subsets of highly specialized T cells gain access to the GCs by expressing the B cell zone-homing C-X-C chemokine receptor type 5 (CXCR5) while losing the T cell zone-homing chemokine receptor CCR7. Help from T cells is critical to induce B cell proliferation and somatic hyper mutation and to limit GC reactions. CD4 + T follicular helper (T FH ) cells required for the formation of GCs and for the generation of long-lived, high-affinity B cells. Regulatory CD4 + (T FR ) and CD8 + T cells co-localize with T FH cells and keep their expansion in check, thus limiting GC reactions. A cytotoxic CXCR5 pos CD8 + T cell subset has been described in GCs in humans: although low in number, GC CD8 + T cells can expand rapidly during certain viral infections. Because these subsets find their home in secondary lymphoid tissues (lymph nodes and spleen) that are difficult to obtain in humans, GC-homing T cells have been extensively studied in mice. Nevertheless, significant limitations in using this model, such as evolutionary divergences between mice and humans and the lack of an optimal mouse model for certain human diseases, have prompted investigators to characterize GC-homing T cells in macaques instead. This review will focus on discoveries made in macaques, particularly in the non-human primate models of simian immunodeficiency virus and simian-human immunodeficiency virus infection. Indeed, experimental studies in these models have allowed researchers to gain insight into the relative role of follicular T cell subsets in HIV progression, virus persistence, and specific B cell responses induced by HIV vaccines. These discoveries have prompted the testing of novel approaches aimed to manipulate follicular T cells to increase the efficacy of HIV vaccines and to eliminate HIV reservoirs.

The production of high-affinity and broadly neutralizing antibodies plays a key role in the defense against pathogens. These antibody responses require effective germinal center (GC) reaction within anatomical niches of GCs, where follicular helper T (Tfh) cells provide cognate help to B cells for T cell-dependent antibody responses. Emerging evidences indicate that GC reaction in normal state and perhaps establishment of latent Tfh cell reservoir in HIV/SIV infection are tightly regulated by epigenetic histone modifications, which are responsible for activating or silencing chromatin. A better understanding of the mechanisms behind GC responses at cellular and molecular levels thus provides necessary knowledge for vaccination and immunotherapy. In this review, we discussed the epigenetic regulation of GC responses, especially for GC B and Tfh cell under normal state or HIV/SIV infection.

T follicular helper (Tfh) cells have the important function of providing B-cell help for the induction of antigen-specific antibody production. As such, it is important to determine the factors that regulate the development, differentiation and function of Tfh cells. This review highlights some of the recent advances in our understanding of Tfh cell migration, Tfh cell memory and the origins and fate of circulating Tfh cells in the blood, that have been revealed from studies in humans and mice., (© 2017 John Wiley & Sons Ltd.)

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that has been shown to be essential for the differentiation and function of various immune cells. Earlier in vitro studies showed that mTOR signalling regulates B-cell biology by supporting their activation and proliferation. However, how mTOR signalling temporally regulates in vivo germinal centre B (GCB) cell development and differentiation into short-lived plasma cells, long-lived plasma cells and memory cells is still not well understood. In this study, we used a combined conditional/inducible knock-out system to investigate the temporal regulation of mTOR complex 1 (mTORC1) in the GCB cell response to acute lymphocytic choriomeningitis virus infection by deleting Raptor, a main component of mTORC1, specifically in B cells in pre- and late GC phase. Early Raptor deficiency strongly inhibited GCB cell proliferation and differentiation and plasma cell differentiation. Nevertheless, late GC Raptor deficiency caused only decreases in the size of memory B cells and long-lived plasma cells through poor maintenance of GCB cells, but it did not change their differentiation. Collectively, our data revealed that mTORC1 signalling supports GCB cell responses at both early and late GC phases during viral infection but does not regulate GCB cell differentiation into memory B cells and plasma cells at the late GC stage., (© 2017 John Wiley & Sons Ltd.)

Lymph nodes play a central role in the development of adaptive immunity against pathogens and particularly the generation of antigen-specific B cell responses in specialized areas called germinal centers (GCs). Lymph node (LN) pathology was recognized as an important consequence of human immunodeficiency virus (HIV) infection since the beginning of the HIV epidemic. Investigation into the structural and functional alterations induced by HIV and Simian immunodeficiency virus (SIV) has further cemented the central role that lymphoid tissue plays in HIV/SIV pathogenesis. The coexistence of constant local inflammation, altered tissue architecture, and relative exclusion of virus-specific CD8 T cells from the GCs creates a unique environment for the virus evolution and establishment of viral reservoir in specific GC cells, namely T follicular helper CD4 T cells (Tfh). A better understanding of the biology of immune cells in HIV-infected lymph nodes is a prerequisite to attaining the ultimate goal of complete viral eradication.

The development of stabilized recombinant HIV envelope trimers that mimic the virion surface molecule has increased enthusiasm for a neutralizing antibody (nAb)-based HIV vaccine. However, there is limited experience with recombinant trimers as immunogens in nonhuman primates, which are typically used as a model for humans. Here, we tested multiple immunogens and immunization strategies head-to-head to determine their impact on the quantity, quality, and kinetics of autologous tier 2 nAb development. A bilateral, adjuvanted, subcutaneous immunization protocol induced reproducible tier 2 nAb responses after only two immunizations 8 weeks apart, and these were further enhanced by a third immunization with BG505 SOSIP trimer. We identified immunogens that minimized non-neutralizing V3 responses and demonstrated that continuous immunogen delivery could enhance nAb responses. nAb responses were strongly associated with germinal center reactions, as assessed by lymph node fine needle aspiration. This study provides a framework for preclinical and clinical vaccine studies targeting nAb elicitation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Follicular helper CD4 T cells, T FH , residing in B-cell follicles within secondary lymphoid tissues, are readily infected by AIDS viruses and are a major source of persistent virus despite relative control of viral replication. This persistence is due at least in part to a relative exclusion of effective antiviral CD8 T cells from B-cell follicles. To determine whether CD8 T cells could be engineered to enter B-cell follicles, we genetically modified unselected CD8 T cells to express CXC chemokine receptor 5 (CXCR5), the chemokine receptor implicated in cellular entry into B-cell follicles. Engineered CD8 T cells expressing human CXCR5 (CD8 hCXCR5 ) exhibited ligand-specific signaling and chemotaxis in vitro Six infected rhesus macaques were infused with differentially fluorescent dye-labeled autologous CD8 hCXCR5 and untransduced CD8 T cells and necropsied 48 h later. Flow cytometry of both spleen and lymph node samples revealed higher frequencies of CD8 hCXCR5 than untransduced cells, consistent with preferential trafficking to B-cell follicle-containing tissues. Confocal fluorescence microscopy of thin-sectioned lymphoid tissues demonstrated strong preferential localization of CD8 hCXCR5 T cells within B-cell follicles with only rare cells in extrafollicular locations. CD8 hCXCR5 T cells were present throughout the follicles with some observed near infected T FH In contrast, untransduced CD8 T cells were found in the extrafollicular T-cell zone. Our ability to direct localization of unselected CD8 T cells into B-cell follicles using CXCR5 expression provides a strategy to place highly effective virus-specific CD8 T cells into these AIDS virus sanctuaries and potentially suppress residual viral replication. IMPORTANCE AIDS virus persistence in individuals under effective drug therapy or those who spontaneously control viremia remains an obstacle to definitive treatment. Infected follicular helper CD4 T cells, T FH , present inside B-cell follicles represent a major source of this residual virus. While effective CD8 T-cell responses can control viral replication in conjunction with drug therapy or in rare cases spontaneously, most antiviral CD8 T cells do not enter B-cell follicles, and those that do fail to robustly control viral replication in the T FH population. Thus, these sites are a sanctuary and a reservoir for replicating AIDS viruses. Here, we demonstrate that engineering unselected CD8 T cells to express CXCR5, a chemokine receptor on T FH associated with B-cell follicle localization, redirects them into B-cell follicles. These proof of principle results open a pathway for directing engineered antiviral T cells into these viral sanctuaries to help eliminate this source of persistent virus., (Copyright © 2017 American Society for Microbiology.)

Virus-specific memory B cells (B mem ) play a crucial role in protecting against variant viruses. The ability to recognize these variant viruses, defined as antibody breadth, is achieved in B mem populations by two very different pathways, germline-encoded cross-reactivity and affinity-driven, somatic evolution in germinal centers (GCs) for conserved viral epitopes. The latter class of broadly-reactive B mem cells are not cross-reactive per se, but bind epitopes crucial for viral fitness. Although these conserved epitopes are often weakly immunogenic, the GC reaction is surprisingly permissive for the continued survival/proliferation of B cells that bind with low affinity or react to cryptic epitopes, increasing their chance of memory recruitment. In this review, we discuss the adaptive strategies of B-cell memory to viral antigenic variations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Epstein-Barr virus (EBV) latently infects normal B cells and contributes to the development of certain human lymphomas. Newly infected B cells support a highly transforming form (type III) of viral latency; however, long-term EBV infection in immunocompetent hosts is limited to B cells with a more restricted form of latency (type I) in which most viral gene expression is silenced by promoter DNA methylation. How EBV converts latency type is unclear, although it is known that type I latency is associated with a germinal center (GC) B cell phenotype, and type III latency with an activated B cell (ABC) phenotype. In this study, we have examined whether expression of TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells. We found that TET2 expression is inhibited in normal GC cells and GC type lymphomas. In contrast, TET2 is expressed in normal naive B cells and ABC type lymphomas. We also demonstrate that GC type cell lines have increased 5mC levels and reduced 5hmC levels in comparison to those of ABC type lines. Finally, we show that TET2 promotes the ability of the EBV transcription factor EBNA2 to convert EBV-infected cells from type I to type III latency. These findings demonstrate that TET2 expression is repressed in GC cells independent of EBV infection and suggest that TET2 promotes type III EBV latency in B cells with an ABC or naive phenotype by enhancing EBNA2 activation of methylated EBV promoters. IMPORTANCE EBV establishes several different types of viral latency in B cells. However, cellular factors that determine whether EBV enters the highly transforming type III latency, versus the more restricted type I latency, have not been well characterized. Here we show that TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells by enhancing the ability of the viral transcription factor EBNA2 to activate methylated viral promoters that are expressed in type III (but not type I) latency. Furthermore, we demonstrate that (independent of EBV) TET2 is turned off in normal and malignant germinal center (GC) B cells but expressed in other B cell types. Thus, restricted TET2 expression in GC cells may promote type I EBV latency., (Copyright © 2017 American Society for Microbiology.)

Autophagy is important in a variety of cellular and pathophysiological situations; however, its role in immune responses remains elusive. Here, we show that among B cells, germinal center (GC) cells exhibited the highest rate of autophagy during viral infection. In contrast to mechanistic target of rapamycin complex 1-dependent canonical autophagy, GC B cell autophagy occurred predominantly through a noncanonical pathway. B cell stimulation was sufficient to down-regulate canonical autophagy transiently while triggering noncanonical autophagy. Genetic ablation of WD repeat domain, phosphoinositide-interacting protein 2 in B cells alone enhanced this noncanonical autophagy, resulting in changes of mitochondrial homeostasis and alterations in GC and antibody-secreting cells. Thus, B cell activation prompts a temporal switch from canonical to noncanonical autophagy that is important in controlling B cell differentiation and fate., (Copyright © 2017, American Association for the Advancement of Science.)

Current HIV vaccines are poor inducers of neutralizing antibodies (nAbs). A recent study in Cell Reports used serial fine-needle aspirates from rhesus macaque lymph nodes following HIV-1 surface envelope glycoprotein (Env) trimer immunization, generating a substantial production of HIV-1 nAbs. A remarkable correlation was found between antibody titers and a high frequency and ratio of germinal center B and T follicular helper (T FH ) lymphocytes., (Copyright © 2016. Published by Elsevier Ltd.)

Human immunodeficiency virus (HIV)- and simian immunodeficiency virus (SIV)-specific CD8 + T cells are typically largely excluded from lymphoid B cell follicles, where HIV- and SIV-producing cells are most highly concentrated, indicating that B cell follicles are somewhat of an immunoprivileged site. To gain insights into virus-specific follicular CD8 + T cells, we determined the location and phenotype of follicular SIV-specific CD8 + T cells in situ, the local relationship of these cells to Foxp3 + cells, and the effects of CD8 depletion on levels of follicular SIV-producing cells in chronically SIV-infected rhesus macaques. We found that follicular SIV-specific CD8 + T cells were able to migrate throughout follicular areas, including germinal centers. Many expressed PD-1, indicating that they may have been exhausted. A small subset was in direct contact with and likely inhibited by Foxp3 + cells, and a few were themselves Foxp3 + In addition, subsets of follicular SIV-specific CD8 + T cells expressed low to medium levels of perforin, and subsets were activated and proliferating. Importantly, after CD8 depletion, the number of SIV-producing cells increased in B cell follicles and extrafollicular areas, suggesting that follicular and extrafollicular CD8 + T cells have a suppressive effect on SIV replication. Taken together, these results suggest that during chronic SIV infection, despite high levels of exhaustion and likely inhibition by Foxp3 + cells, a subset of follicular SIV-specific CD8 + T cells are functional and suppress viral replication in vivo These findings support HIV cure strategies that augment functional follicular virus-specific CD8 + T cells to enhance viral control., Importance: HIV- and SIV-specific CD8 + T cells are typically largely excluded from lymphoid B cell follicles, where virus-producing cells are most highly concentrated, suggesting that B cell follicles are somewhat of an immunoprivileged site where virus-specific CD8 + T cells are not able to clear all follicular HIV- and SIV-producing cells. To gain insights into follicular CD8 + T cell function, we characterized follicular virus-specific CD8 + T cells in situ by using an SIV-infected rhesus macaque model of HIV. We found that subsets of follicular SIV-specific CD8 + T cells are able to migrate throughout the follicle, are likely inhibited by Foxp3 + cells, and are likely exhausted but that, nonetheless, subsets are likely functional, as they express markers consistent with effector function and show signs of suppressing viral replication in vivo These findings support HIV cure strategies that increase the frequency of functional follicular virus-specific CD8 + T cells., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

During unresolved infections, some viruses escape immunological control and establish a persistant reservoir in certain cell types, such as human immunodeficiency virus (HIV), which persists in follicular helper T cells (TFH cells), and Epstein-Barr virus (EBV), which persists in B cells. Here we identified a specialized group of cytotoxic T cells (TC cells) that expressed the chemokine receptor CXCR5, selectively entered B cell follicles and eradicated infected TFH cells and B cells. The differentiation of these cells, which we have called 'follicular cytotoxic T cells' (TFC cells), required the transcription factors Bcl6, E2A and TCF-1 but was inhibited by the transcriptional regulators Blimp1, Id2 and Id3. Blimp1 and E2A directly regulated Cxcr5 expression and, together with Bcl6 and TCF-1, formed a transcriptional circuit that guided TFC cell development. The identification of TFC cells has far-reaching implications for the development of strategies to control infections that target B cells and TFH cells and to treat B cell-derived malignancies.

Considerable data implicate the thymus as the main site of autosensitization to the acetylcholine receptor in myasthenia gravis (MG), a B-cell-mediated autoimmune disease affecting the neuromuscular junction. We recently demonstrated an active Epstein-Barr virus (EBV) infection in the thymus of MG patients, suggesting that EBV might contribute to the onset or maintenance of the autoimmune response within MG thymus, because of its ability to activate and immortalize autoreactive B cells. EBV has been reported to elicit and modulate Toll-like receptor (TLR) 7- and TLR9-mediated innate immune responses, which are known to favor B-cell dysfunction and autoimmunity. Aim of this study was to investigate whether EBV infection is associated with altered expression of TLR7 and TLR9 in MG thymus. By real-time PCR, we found that TLR7 and TLR9 mRNA levels were significantly higher in EBV-positive MG compared to EBV-negative normal thymuses. By confocal microscopy, high expression levels of TLR7 and TLR9 proteins were observed in B cells and plasma cells of MG thymic germinal centers (GCs) and lymphoid infiltrates, where the two receptors co-localized with EBV antigens. An increased frequency of Ki67-positive proliferating B cells was found in MG thymuses, where we also detected proliferating cells expressing TLR7, TLR9 and EBV antigens, thus supporting the idea that EBV-associated TLR7/9 signaling may promote abnormal B-cell activation and proliferation. Along with B cells and plasma cells, thymic epithelium, plasmacytoid dendritic cells and macrophages exhibited enhanced TLR7 and TLR9 expression in MG thymus; TLR7 was also increased in thymic myeloid dendritic cells and its transcriptional levels positively correlated with those of interferon (IFN)-β. We suggested that TLR7/9 signaling may be involved in antiviral type I IFN production and long-term inflammation in EBV-infected MG thymuses. Our overall findings indicate that EBV-driven TLR7- and TLR9-mediated innate immune responses may participate in the intra-thymic pathogenesis of MG., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Despite the immune reconstitution promoted by combined antiretroviral therapy (cART), lymphomas still represent the most common type of cancer in HIV-infected individuals. Cofactors related to immunodeficiency such as oncogenic viruses, chronic antigenic stimulation, and cytokine overproduction are thought to be the main drivers of HIV lymphomagenesis, although the current scenario does not convincingly explain the still-high incidence of lymphomas and the occurrence of peculiar lymphoma histotypes in HIV-infected patients under cART. Recent findings are challenging the current view of a mainly indirect role of HIV in lymphoma development and support the possibility that HIV may directly contribute to lymphomagenesis. In fact, mechanisms other than immune suppression involve biologic effects mediated by HIV products that are secreted and accumulate in lymphoid tissues, mainly within lymph node germinal centers. Notably, HIV-infected patients with lymphomas, but not those not affected by these tumors, were recently shown to carry HIV p17 protein variants with enhanced B-cell clonogenic activity. HIV p17 protein variants were characterized by the presence of distinct insertions at the C-terminal region of the protein responsible for a structural destabilization and the acquisition of novel biologic properties. These data are changing the current paradigm assuming that HIV is only indirectly related to lymphomagenesis. Furthermore, these recent findings are consistent with a role of HIV as a critical microenvironmental factor promoting lymphoma development and pave the way for further studies that may lead to the design of more effective strategies for an early identification and improved control of lymphomas in the HIV setting., (© 2016 by The American Society of Hematology.)

Unlabelled: Gammaherpesviruses are ubiquitous pathogens that are associated with the development of B cell lymphomas. Gammaherpesviruses employ multiple mechanisms to transiently stimulate a broad, polyclonal germinal center reaction, an inherently mutagenic stage of B cell differentiation that is thought to be the primary target of malignant transformation in virus-driven lymphomagenesis. We found that this gammaherpesvirus-driven germinal center expansion was exaggerated and lost its transient nature in the absence of interferon-regulatory factor 1 (IRF-1), a transcription factor with antiviral and tumor suppressor functions. Uncontrolled and persistent expansion of germinal center B cells led to pathological changes in the spleens of chronically infected IRF-1-deficient animals. Additionally, we found decreased IRF-1 expression in cases of human posttransplant lymphoproliferative disorder, a malignant condition associated with gammaherpesvirus infection. The results of our study define an unappreciated role for IRF-1 in B cell biology and provide insight into the potential mechanism of gammaherpesvirus-driven lymphomagenesis., Importance: Gammaherpesviruses establish lifelong infection in most adults and are associated with B cell lymphomas. While the infection is asymptomatic in many hosts, it is critical to identify individuals who may be at an increased risk of virus-induced cancer. Such identification is currently impossible, as the host risk factors that predispose individuals toward viral lymphomagenesis are poorly understood. The current study identifies interferon-regulatory factor 1 (IRF-1) to be one of such candidate host factors. Specifically, we found that IRF-1 enforces long-term suppression of an inherently mutagenic stage of B cell differentiation that gammaherpesviruses are thought to target for transformation. Correspondingly, in the absence of IRF-1, chronic gammaherpesvirus infection induced pathological changes in the spleens of infected animals. Further, we found decreased IRF-1 expression in human gammaherpesvirus-induced B cell malignancies., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)