GC Reaction Research Articles

Divergent chemokine receptor expression and the consequence for human IgG4 Bcell responses.

IgG4 antibodies are unique to humans. IgG4 is associated with tolerance during immunotherapy in allergy, but also with pathology, as in pemphigus vulgaris and IgG4-related disease. Its induction is largely restricted to nonmicrobial antigens, and requires repeated or prolonged antigenic stimulation, for reasons poorly understood. An important aspect in generating high-affinity IgG antibodies is chemokine receptor-mediated migration of Bcells into appropriate niches, such as germinal centers. Here, we show that compared to IgG1 Bcells, circulating IgG4 Bcells express lower levels of CXCR3, CXCR4, CXCR5, CCR6, and CCR7, chemokine receptors involved in GC reactions and generation of long-lived plasma cells. This phenotype was recapitulated by in vitro priming of naive Bcells with an IgG4-inducing combination of TFH /TH2 cytokines. Consistent with these observations, we found a low abundance of IgG4 Bcells in secondary lymphoid tissues in vivo, and the IgG4 antibody response is substantially more short-lived compared to other IgG subclasses in patient groups undergoing CD20+ Bcell depletion therapy with rituximab. These results prompt the hypothesis that factors needed to form IgG4 Bcells restrain at the same time the induction of a robust migratory phenotype that could support a long-lived IgG4 antibody response.

A germinal center B cell-specific long non-coding RNA regulates the germinal center response

Abstract Long non-coding RNAs are a unique class of molecules involved in an exceptional variety of cellular processes, including transcriptional, translational, and epigenetic regulation. Here, we report the initial characterization of a lncRNA expressed specifically in the germinal center--organized sites of B cell proliferation, somatic hyper-mutation, and cellular differentiation that develop in response to antigenic challenge. The precise signals dictating how and when differentiated cells, such as memory B cells and long-lived plasma cells, exit the GC reaction remain incompletely understood. Thus, additional levels of molecular regulation are likely at work in the coordination of these diverse processes. We identify GCLnc1--a novel, nuclear-localized lncRNA--as a regulator of the GC reaction. As GCLnc1 is located adjacent to the murine Bcl6 locus, this lncRNA has the potential to modulate expression of key transcription factors controlling B cell identity and/or differentiation. Interestingly, forced over-expression of GCLnc1 in B cells led to upregulation of TFs associated with plasma cell identity, such as IRF4 and Blimp-1. When adoptively transferred in vivo, over-expression of GCLnc1 led to transduced B cells predominantly adopting a non-GC phenotype at the typical peak GC response, in addition to enhancing expression of IRF4 and Blimp-1. Genetic deletion of GCLnc1 led to reduced frequency of GC B cells, decreased Bcl6 expression, and dysregulated light zone/dark zone distribution in response to NP-KLH immunization. Collectively, we suggest GCLnc1 plays a key role in the integration of signaling events during the GC reaction, and may modulate the processes that drive GC exit.

Glycan Remodelling Controls Germinal Centre B cell Responses

Abstract Germinal centre (GC) B cells are routinely identified through distinct changes on their surface carbohydrates, called glycans; yet, whether these modifications play an essential role in GC reaction remain unknown. One striking change relates to the monosaccharide sialic acid. In mice, this change is mediated through downregulation of an enzyme called Cmah. As a consequence, GC B cells lose the preferred ligand for CD22, a member of the sialic acid-binding immunoglobulin-type lectins (Siglecs) and a co-inhibitory receptor of the B cell antigen receptor. Since CD22 regulates B cell function, we hypothesize that this specific glycan remodelling on B cells control the GC reaction by modulating the activity of CD22. To test our hypothesis, we developed a mouse model that constitutively expresses Cmah in B cells. Following immunization, we find that sustained expression of Cmah in B cells results in impaired generation of antigen-specific GC B cells. More significantly, this observed defect is dependent on CD22, highlighting that coordinated loss of preferred ligands for CD22 on B cells plays a critical function in the GC. Furthermore, antibody affinity maturation is significantly impaired in immunized mice harbouring B cells which lack either CD22 or its cis glycan ligands, further supporting the role of CD22 and its glycan ligands in the GC. Ongoing efforts are focused on defining the GC-specific nature of these observations and examining potential mechanism related to BCR signalling, GC B cell survival and exit to plasma cells, and B-T cell interactions.

I-106 Early treatment initiation in children with vertical HIV infection influences HIV specific immune responses

Background: Despite successes in prevention of mother to child HIV transmission (MTCT), issues related to early diagnosis, ART initiation and adherence play a role in the disease course of HIV infected children. Early ART initiation in HIV infected infants leads to reduced size of HIV reservoirs and curtails clinical progression but role of the immune system in this process is not well understood. With early initiation of ART, HIV infected infants preserve their CD4+ T cells and in some instances remain seronegative for HIV due to limited exposure of B cells to HIV antigen. We are performing immunologic assessments in the HIV Exposed Pediatric Patients (HEPP) Project and discuss a group of children from Rome, Italy. The goal of the present study is to evaluate HIV-specific immunity of vertically HIV infected children in relation to timing of ART initiation. Methods: The study group consists of older vertically infected HIV+ children and adolescents who initiated ART at different ages after birth and have maintained durable viral control (plasma HIV-RNA <50 cp/mL). Immunologic studies were performed in 27 children (at ages 2.1–15.7 yrs) of whom 22 had started ART early, at age <6 mo [early treated (ET)] and 5 who started ART later, at age >2 yrs [late treated (LT)] with ET experiencing longer duration of ART (P < 0.05) at the time of study. ET children were also evaluated by HIV serology (Abbott Architect HIV Ag/Ab Combo Assay) to establish if they were seropositive (SP) or seronegative (SN) to HIV. Flow cytometry was performed using BD LSRFortessa for T cells and BD FACS Aria II for B cells. HIV-gag specific CD4+ and CD8+ T cells were investigated by flow cytometry in cryopreserved PBMC for the induction of intracellular cytokines (IL-2, IFN-g, TNF, IL-21) in CD40L+ CD4+ T cells and intracellular cytokines (IL-2, IFN-g, TNF) as well as Perforin, Granzyme B and CD107a in CD69+ CD8+ T cells following 12 hours stimulation with HIV gag PTE peptides. In addition, Env-specific CD4+ T cells were evaluated following 12 hours stimulation of PBMC with Env peptides. IgD+ and IgM+ B cell subsets were determined in gp140+ B cells. Moreover, env-specific and non-specific B cells were sorted to perform transcriptomic analysis with Fluidigm (Biomark). Differences between groups were determined by unpaired or Mann-Whitney t tests if the data were normally distributed or not, respectively. Results: In this group of HIV+ children, frequencies of HIV gag-specific CD4+ T cells and CD8+ T cells were similar between ET and LT but differed qualitatively. Gag stimulated CD4+ T cells of ET exhibited higher proportions of IL-21 and IFNg with enrichment of polyfunctional T cells defined by the simultaneous presence of 3 or more cytokines. Gag stimulated CD8+ T cells of ET exhibited higher frequencies of cytotoxic cells (CD107a+Perf+GrzB+). In ET, serologic evaluation of HIV Ab revealed 6/22 (27.3%) to be SN. The gp140-specific CD4+ T cell frequencies were similar in SN and SP but intracellular IL-21 expression was higher in SP. Frequencies of gp140+ CD19+ B cells were similar between SN and SP, but HIV-specific CD27+ memory B cells were predominantly IgM+ in SN while in SP, the gp140+CD27+ memory B cells were enriched in IgD-cells. Gene expression signatures in gp140+IgM+ cells revealed upregulation of genes involved in both plasma cell differentiation (PRDM1) and GC reaction (BCL6) only in SP after in-vitro stimulation. Conclusions: Time of ART initiation after birth has a long-term impact on the quality of the host HIV-specific immune responses in vertically infected HIV+ children with durable virus suppression. Earlier ART initiation is associated with preserved HIV gag-specific CD4+ and CD8+ T cells. Moreover, even in those who become seronegative for HIV, the HIV Env-specific CD4+ T cells and B cells persist, the latter demonstrating a blunted differentiation with enrichment of IgM+ B cells. ET children with consistent viral control are prime candidates for therapeutic approaches aimed at permanent remission of HIV. These studies are supported by ViiV Health Care grant to EPIICAL and NIH grant AI127347 to SP.

Dynamics of Virus-Specific Memory B Cells and Plasmablasts following Viral Infection of the Central Nervous System.

Humoral responses within the central nervous system (CNS) are common to many neurotropic viral infections, with antibody (Ab)-secreting cells (ASC) contributing to local protection. However, a role for virus-specific memory B cells (Bmem) within the CNS is poorly explored due to lack of robust phenotypic or functional identification in mice. This study takes advantage of the progeny of mice expressing tamoxifen-inducible Cre recombinase (Cre-ERT2) under the Aicda promoter crossed with Rosa26-loxP-tdTomato reporter mice (AIDCre-Rosa26tdTomato) to monitor B cells having undergone activation-induced cytidine deaminase (AID)-mediated somatic hypermutation (SHM) following neurotropic coronavirus infection. AID detection via tdTomato expression allowed tracking of virus-specific ASC and Bmem in priming and effector sites throughout infection. In draining lymph nodes, tdTomato-positive (tdTomato+) ASC were most prevalent prior to germinal center (GC) formation, but total tdTomato+ B cells only peaked with robust GC formation at day 14 p.i. Moreover, their proportion of Bmem dominated over the proportion of ASC throughout infection. In the CNS, tdTomato+ cells started emerging at day 14 p.i. While they initially comprised mainly Bmem, the proportions of ASC and Bmem became similar as tdTomato+ B cells increased throughout viral persistence. Delayed tamoxifen treatment demonstrated ongoing CNS recruitment of tdTomato+ B cells, mainly ASC, primed late during GC reactions. Overall, the data support the idea that virus-induced B cells exhibiting SHM require peripheral GC formation to emerge in the CNS. Ongoing GC reactions and regional signals further regulate dynamics within the CNS, with preferential maintenance of tdTomato+ B cells in spinal cords relative to that in brains during viral persistence.IMPORTANCE The prevalence and role of antigen-specific Bmem in the CNS during viral encephalomyelitis is largely undefined. A lack of reliable markers identifying murine Bmem has made it difficult to assess their contribution to local antiviral protection via antigen presentation or conversion to ASC. Using reporter mice infected with neurotropic coronavirus to track virus-specific Bmem and ASC, this report demonstrates that both subsets only emerge in the CNS following peripheral GC formation and subsequently prevail. While early GC reactions supported preferential Bmem accumulation in the CNS, late GC reactions favored ASC accumulation, although Bmem outnumbered ASC in draining lymph nodes throughout infection. Importantly, virus-specific B cells undergoing sustained GC selection were continually recruited to the persistently infected CNS. Elucidating the factors governing temporal events within GCs, as well as regional CNS cues during viral persistence, will aid intervention to modulate CNS humoral responses in the context of infection and associated autoimmune pathologies.

Germinal center humoral autoimmunity independently mediates progression of allograft vasculopathy

The development of humoral autoimmunity following organ transplantation is increasingly recognised, but of uncertain significance. We examine whether autoimmunity contributes independently to allograft rejection.In a MHC class II-mismatched murine model of chronic humoral rejection, we report that effector antinuclear autoantibody responses were initiated upon graft-versus-host allorecognition of recipient B cells by donor CD4 T-cells transferred within heart allografts. Consequently, grafts were rejected more rapidly, and with markedly augmented autoantibody responses, upon transplantation of hearts from donors previously primed against recipient. Nevertheless, rejection was dependent upon recipient T follicular helper (TFH) cell differentiation and provision of cognate (peptide-specific) help for maintenance as long-lived GC reactions, which diversified to encompass responses against vimentin autoantigen. Heart grafts transplanted into stable donor/recipient mixed haematopoietic chimeras, or from parental strain donors into F1 recipients (neither of which can trigger host adaptive alloimmune responses), nevertheless provoked GC autoimmunity and were rejected chronically, with rejection similarly dependent upon host TFH cell differentiation.Thus, autoantibody responses contribute independently of host adaptive alloimmunity to graft rejection, but require host TFH cell differentiation to maintain long-lived GC responses. The demonstration that one population of helper CD4 T-cells initiates humoral autoimmunity, but that a second population of TFH cells is required for its maintenance as a GC reaction, has important implications for how autoimmune-related phenomena manifest.

T-bet in Tfh cells: Now you see me, now you don't.

In this issue, Fang et al. (https://doi.org/10.1084/jem.20180927) report on a subset of T follicular helper (Tfh) cells that transiently expresses T-bet yet continues to produce IFN-γ at late stages of GC reactions following immunization. They find other genes uniquely expressed in this IFN-γ-producing Tfh subset, such as NKG2D, that can be used to better distinguish these functionally distinct Tfh cells.

T5: Yin and Yang regulation of GC reaction

American Journal of Reproductive ImmunologyVolume 80, Issue S1 p. 24-24 ABSTRACTS T5: Yin and Yang regulation of GC reaction First published: 26 June 2018 https://doi.org/10.1111/aji.04_12981Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume80, IssueS1Special Issue: American-Sino Joint Meeting of Reproductive Immunology (ASRI-CSI 2018), June 28-1 July, 2018 Shanghai, ChinaJune 2018Pages 24-24 RelatedInformation

Comparison of immune cells from germinal centers specific to protein or lipid antigens

Abstract Anti-lipid IgG antibodies can participate in the physiopathology of some autoimmune diseases. In our research group, we developed a mouse model of Lupus by the administration of liposomes bearing non-bilayer phospholipid arrangements NPA induced by some drugs like chloropromazine CPZ. The mice of this model produce anti-NPA IgG antibodies; these antibodies are mainly formed via germinal center GC. However, the cells that participate in a GC reaction against lipidic antigens are unknown; therefore, we study these cells in the mouse model of Lupus induced by NPA and compared them with a GC reaction against ovalbumin OVA, because the cells that participate in a GC reaction against protein antigens are well known. We studied germinal center B cells, plasma cells, dendritic cells, follicular dendritic cells FDC, T follicular helper cells TFH, T follicular regulatory cells TFR and macrophages from the groups administered with OVA or NPA and compared them with mice without administration. Then we extracted the spleen and the mesenteric lymph nodes and perform flow cytometry and H&E staining at days 5, 10, 15 and 30 postadministration. Histologically, germinal centers were found in both groups at day 5. The mice administered with NPA, present an increase of germinal center B cells 15 days post-administration and a bigger size of the secondary lymphoid organs, the mice administrated with OVA present an increase of these cells and bigger lymph organs at day 10. The higher percentage of plasma cells was found 30 days after the administration of OVA; while dendritic cells and TFH cells presented similar percentages in both groups at all times. For both antigens, the FDC increase at day 5 and 15, the macrophages from day 10 to 15 and the TFR cells increase at day 15.

Deconstructing T-B interactions by manipulation of IRF4 to understand regulation of T follicular helper cells.

Abstract Germinal center (GC) derived high affinity antibody is important for protecting against infectious disease. Interactions between T follicular helper (Tfh) cells and GC B cells are requisite for affinity maturation. While it is clear that bi-directional signals are important for the generation of Tfh and GC B cells, a full understanding of the signaling dynamics that lead to efficient T and B cell differentiation is not known. Studying these B cell-derived signals and their role in Tfh cell commitment, maintenance, and function during GC reactions will lead to an improved understanding of this tightly regulated system. The transcription factor IRF4 is necessary and sufficient for generating GC B and plasma cell fates through graded expression. We have shown previously through gene ontology analysis that IRF4 targets include genes involved in T and B cell interaction. This raises the possibility that beyond cell-specific control of B cell fates, IRF4-regulated genes in B cells may facilitate interactions between B and T cells. We hypothesize that IRF4 controls a gene program in B cells necessary to generate efficient T cell activation and Tfh cell differentiation. Using in-vitro co-culture, we assessed changes in T and B cell activation and functionality by flow cytometry. We found that Irf4−/− B cells processed antigen poorly and led to quantitatively and qualitatively reduced T cell activation compared to Irf4+/+ B cells. This indicates a major role for IRF4 in controlling a B cell gene program necessary for efficient T cell activation. We are currently investigating the nature of these signals through in-vitro and in-vivo approaches in order to deconstruct the molecular nature of T-B interactions during the GC program.

Generation of memory B cells and their reactivation.

The successful establishment of humoral memory response depends on at least two layers of defense. Pre-existing protective antibodies secreted by long-lived plasma cells act as a first line of defense against reinfection ("constitutive humoral memory"). Previously, a second line of defense in which pathogen-experienced memory B cells are rapidly reactivated to produce antibodies ("reactive humoral memory"), was considered as simply a back-up system for the first line (particularly for re-infection with homologous viruses). However, in the case of re-infection with similar but different strains of viruses, or in response to viral escape mutants, the reactive humoral memory plays a crucial role. Here, we review recent progress in our understanding of how memory B cells are generated in the pre-GC stage and during the GC reaction, and how these memory B cells are robustly reactivated with the help of memory Tfh cells to generate the secondary antibody response. In addition, we discuss how these advances may be relevant to the quest for a vaccine that can induce broadly reactive antibodies against influenza and HIV.

Activated Circulating T Follicular Helper Cells with Increased Function during Chronic Graft Versus Host Disease after Allogeneic Stem Cell Transplantation

T follicular helper cells (TFH) interact with B cells in the germinal center to support B cell proliferation, differentiation and immunoglobulin class switch. Previous studies have demonstrated that donor B cells contribute to the immune pathology of chronic GVHD (cGVHD) and studies in murine models of cGVHD have suggested that TFH play a central role in this process. Although TFH are located primarily within germinal centers of secondary lymphoid organs, TFH can also be found in the peripheral blood and are identified as a subset of memory CD4 T cells expressing CXCR5. Circulating TFH (cTFH) share functional and phenotypic characteristics of TFH including the ability to interact with B cells and provide helper signals. To examine the potential role of cTFH in the pathogenesis of cGVHD we monitored the reconstitution of cTFH (CD4+CD45RA-CXCR5+ T cells) in fresh blood samples obtained at different times after allogeneic HSCT. Recovery of cTFH mirrored the recovery of CD4 T cells and absolute numbers of cTFH gradually increased over time, reaching a plateau at 24 months post-HSCT in the range of normal values. Within the CD4 T cell population, the frequency of cTFH was low initially, but reached normal levels 6-9 months post-HSCT.Detailed phenotypic and functional analysis of cTFH was undertaken in a cohort of 66 adult patients (>9 months post-HSCT): 16 had no cGVHD, 12 had resolved cGVHD, and 38 had active cGVHD (mild = 15, moderate = 14, severe = 9). Results were compared to 22 healthy donors (HD). Overall, the frequency of cTFH was significantly reduced in HSCT patients compared to HD (median: 9.87 vs. 13.65 % of CD4+ T-cells, respectively, p =0.0003). cTFH frequency was lower in active cGVHD compared to no cGVHD patients (median: 9.44 vs. 11.65 % of CD4+ T-cells, p = 0.029); while cTFH in patients with resolved cGVHD was similar to patients without cGVHD (Figure 1). CXCL13 chemokine facilitates T-B interaction, thus promoting the GC reaction. CXCL13 levels were increased in active cGVHD compared to no cGVHD (137.7 pg/mL vs. 33.74 pg/mL, p < 10-4) suggesting that trafficking of cTFH to lymphoid organs was promoted in cGVHD. cTFH activation results in increased surface expression of ICOS and PD1 and increased proliferation. ICOShi PD1hi cTFH were increased in active cGVHD compared to no cGVHD (2.035 % vs 1.065 %, p = 0.016) in association with high proliferative activity (Ki67) (3.38 % vs. 2.31 % respectively, p = 0.0086). Functional cTFH subsets are also characterized by expression of CXCR3 and CCR6 as follows: Th1 (CXCR3+CCR6-), Th2 (CXCR3-CCR6-), Th17 (CXCR3-CCR6+). Within these subsets, Th2 and Th17 cTFH provide greater levels of B cell help measured by in vitro co-cultureassays (plasmablast generation and IgG production). Although not statistically significant, Th1 cTFH frequency was decreased during active cGVHD (33.95 vs 37.6 for no cGVHD) and Th2 and Th17 cTFH frequencies were increased in the moderate-severe cGVHD group (31.1 and 30.5, respectively) compared to no cGVHD (27.05 and 24.45, respectively). Within active cGVHD patients, the frequencies of Th1 and Th17 were associated with clinical grade of cGVHD (Th1: 40, 34, 19, p=0.008; Th17: 21, 26, 37, p=0.02; for mild, moderate, severe, respectively). We used the ratio (Th2+Th17)/Th1 cTFH to normalize B cell help capacity, and found a higher ratio in severe cGVHD compared to no cGVHD (3.59 vs 1.36, p = 0.0006). To measure cTFH functional activity, cTFH and naive B cells were purified from patient samples by cell sorting and co-cultured for 6 days. cTFH from patients with active cGVHD induced greater plasmablast generation (CD27hi CD38hi B cells) compared to cTFH from non-active cGVHD (Fig. 2). Increased functional activity of cTFH was confirmed when patient cTFH were purified and cocultured with naive B cells from a normal allogeneic donor (Fig. 2). In patients with cGVHD, there was also a significant correlation between the proliferative activity of cTFH and the generation of CD27hi CD38hi B cells (r = 0.48, p = 0.0029).These results indicate that active cGVHD is characterized by phenotypic and functional abnormalities of cTFH. In cGVHD, cTFH show high levels of activation, proliferation and switch toward Th2 and Th17 phenotype. These changes enhance cTFH function, promoting GC reactions and B cell differentiation. These data support the involvement of cTFH in the immune pathology of human cGVHD, suggesting the investigation of novel therapies targeting the GC reaction. [Display omitted] DisclosuresArmand:Merck: Consultancy, Research Funding; Infinity Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Research Funding. Antin:Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Complexity of the human memory B-cell compartment is determined by the versatility of clonal diversification in germinal centers

Our knowledge about the clonal composition and intraclonal diversity of the human memory B-cell compartment and the relationship between memory B-cell subsets is still limited, although these are central issues for our understanding of adaptive immunity. We performed a deep sequencing analysis of rearranged immunoglobulin (Ig) heavy chain genes from biological replicates, covering more than 100,000 memory B lymphocytes from two healthy adults. We reveal a highly similar B-cell receptor repertoire among the four main human IgM(+) and IgG(+) memory B-cell subsets. Strikingly, in both donors, 45% of sequences could be assigned to expanded clones, demonstrating that the human memory B-cell compartment is characterized by many, often very large, B-cell clones. Twenty percent of the clones consisted of class switched and IgM(+)(IgD(+)) members, a feature that correlated significantly with clone size. Hence, we provide strong evidence that the vast majority of Ig mutated B cells--including IgM(+)IgD(+)CD27(+) B cells--are post-germinal center (GC) memory B cells. Clone members showed high intraclonal sequence diversity and high intraclonal versatility in Ig class and IgG subclass composition, with particular patterns of memory B-cell clone generation in GC reactions. In conclusion, GC produce amazingly large, complex, and diverse memory B-cell clones, equipping the human immune system with a versatile and highly diverse compartment of IgM(+)(IgD(+)) and class-switched memory B cells.

Negative Regulation of Humoral Immunity Due to Interplay between the SLAMF1, SLAMF5, and SLAMF6 Receptors.

Whereas the SLAMF-associated protein (SAP) is involved in differentiation of T follicular helper (Tfh) cells and antibody responses, the precise requirements of SLAMF receptors in humoral immune responses are incompletely understood. By analyzing mice with targeted disruptions of the Slamf1, Slamf5, and Slamf6 genes, we found that both T-dependent and T-independent antibody responses were twofold higher compared to those in single knockout mice. These data suggest a suppressive synergy of SLAMF1, SLAMF5, and SLAMF6 in humoral immunity, which contrasts the decreased antibody responses resulting from a defective GC reaction in the absence of the adapter SAP. In adoptive co-transfer assays, both [Slamf1 + 5 + 6]−/− B and T cells were capable of inducing enhanced antibody responses, but more pronounced enhancement was observed after adoptive transfer of [Slamf1 + 5 + 6]−/− B cells compared to that of [Slamf1 + 5 + 6]−/− T cells. In support of [Slamf1 + 5 + 6]−/− B cell intrinsic activity, [Slamf1 + 5 + 6]−/− mice also mounted significantly higher antibody responses to T-independent type 2 antigen. Furthermore, treatment of mice with anti-SLAMF6 monoclonal antibody results in severe inhibition of the development of Tfh cells and GC B cells, confirming a suppressive effect of SLAMF6. Taken together, these results establish SLAMF1, SLAMF5, and SLAMF6 as important negative regulators of humoral immune response, consistent with the notion that SLAM family receptors have dual functions in immune responses.

ICOS is required for the generation of both central and effector CD4(+) memory T-cell populations following acute bacterial infection.

Interactions between ICOS and ICOS ligand (ICOSL) are essential for the development of T follicular helper (Tfh) cells and thus the formation and maintenance of GC reactions. Given the conflicting reports on the requirement of other CD4+ T‐cell populations for ICOS signals, we have employed a range of in vivo approaches to dissect requirements for ICOS signals in mice during an endogenous CD4+ T‐cell response and contrasted this with CD28 signals. Genetic absence of ICOSL only modestly reduced the total number of antigen‐specific CD4+ T cells at the peak of the primary response, but resulted in a severely diminished number of both T central memory and T effector memory cells. Treatment with blocking anti‐ICOS mAb during the primary response recapitulated these effects and caused a more substantial reduction than blocking CD28 signals with CTLA4Ig. During the memory phase of the response further signals through ICOS or CD28 were not required for survival. However, upon secondary challenge only Tfh cell expansion remained heavily ICOS‐dependent, while CD28 signals were required for optimal expansion of all subsets. These data demonstrate the importance of ICOS signals specifically for memory CD4+ T‐cell formation, while highlighting the potential of therapeutically targeting this pathway.

Targeted EZH2 Inhibitors in Diffuse Large B-Cell Lymphoma (DLBCL): Immunohistochemical and Mutational Profiles of Patients May Determine Candidates for Treatment

▪DLBCL is the most common lymphoid malignancy, accounting for 30-40% of all Non Hodgkin Lymphomas. Gene expression profiling has identified two main subtypes: Germinal Center B-Cell like (GCB) and Activated B-Cell like (ABC). EZH2 plays an essential role in epigenetic regulation of DLBCL by specifically mono-, bi- and tri-methylating histone H3 lysine 27 (H3K27me1/-me2/-me3). Recurrent somatic heterozygous gain-of-function mutations of EZH2 have been identified in DLBCL, mostly affecting tyrosine 641 (Y641), inducing increased H3K27me3. Novel EZH2 inhibitors are currently being tested in phase 1 and 2 clinical trials in patients with and without EZH2 Y641 mutations, but no study has examined which patients would most benefit from this treatment.We studied a cohort of 100 patients with DLBCL with available biopsies (96 at diagnosis and 4 at relapse) and developed an immunohistochemical (IHC) assay based on antibodies specifically targeting EZH2, H3K27me3 or H3K27me2. Eighty-six biopsies (12 EZH2 Y641 mutant and 74 WT EZH2) were usable for IHC analysis. Biopsies were scored based on staining intensity and percentage of tumor cells stained, and a me3/me2 score (log of me3 to me2 ratio) was calculated for each patient. Sanger sequencing of EZH2was performed on all patients, GCB/ABC signature was determined by DASL technology based on the expression of 19 genes, and t(14;18) translocation was detected by karyotyping and FISH. The cohort was also extended to 15 patients with EZH2 Y641 mutations and 17 WT EZH2 patients for Next Generation Sequencing (NGS) analysis of a panel of 34 genes involved in lymphomagenesis.Among our cohort, 45 patients were ABC, 50 were GCB, and 5 were unclassified. Sanger sequencing identified 14 patients with EZH2 Y641 mutations (12 GCB, 1 ABC, 1 unclassified). The t(14;18) translocation was more frequent in patients with EZH2 Y641 mutations (9/14, 64%) (p<10-4). Three distinct IHC profiles emerged based on me3/me2 score: a me3-high/me2-low profile (me3/me2 score>0, n=12/86), a me3-low/me2-high profile (me3/me2 score<0, n=41/86) and an intermediate profile (me3/me2 score=0, n=33/86). Patients with EZH2 Y641 mutations mostly exhibit me3/me2 score>0 profiles (n=7/12), whereas patients with WT EZH2 are split between intermediate (n=29/74) and me3/me2 score<0 profiles (n=40/74) (p<10-5). Survival analysis was performed on patients with biopsies at diagnosis treated with Rituximab. ABC subtype is associated with both inferior OS and PFS within our cohort (p=0.03); among ABC patients, low EZH2 IHC expression is associated with superior OS (p=0.035) and PFS (p=0.02). No correlation was found between prognosis and IHC profile.All EZH2 mutations were confirmed by NGS along with their Variable Allele Frequency (VAF). Among GCB EZH2 Y641 mutant patients, a majority of clonal EZH2 mutations (n=11/14) and a minority of subclonal EZH2 mutations (n=3/14) were identified by comparing VAFs of EZH2 and of well-known DLBCL/Follicular Lymphoma driver mutations (including TNFRSF14,CREBBP and MYD88: figure).Among the 86 patients, a tendency toward a correlation between me3/me2 score and EZH2 VAF exists (p=0.09, r=0.51) and of the 5 patients with EZH2 Y641 mutations presenting a me3/me2 score ≤0, 3 exhibit a VAF inferior to the median. Two also present a subclonal EZH2mutation. These findings could potentially explain their unexpected IHC profile, and possibly decrease their response to EZH2 inhibitor treatment.Furthermore, 5 WT EZH2 patients present a me3/me2 score>0; 4 are of the ABC subtype, suggesting an EZH2 mutation bypass in ABC patients. NGS analysis also revealed remarkably similar mutational profiles for 2 of these patients, most notably mutations in PRDM1 and PIM1, potentially responsible for EZH2 upregulation by maintaining B cells in GC reaction and possibly justifying EZH2 inhibitor treatment.EZH2 inhibitors are currently being tested in clinical trials in DLBCL as novel and promising weapons in clinicians’ therapeutic arsenal. This study has shown that IHC and mutational profiles can identify patients most likely to benefit from EZH2 inhibitor treatment by highlighting their in vivo hyper-H3K27me3 status, pinpointing associated activating mutations, and determining EZH2mutation clonality. As such, analyzing these parameters could maximize EZH2 inhibitor benefit and potentially serve to grant access to patients who would otherwise not have been considered. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Csnk2β, the Regulatory Subunit of Protein Kinase CK2, modulates Peripheral B Cell Development Repressing Notch2 Signaling and Promoting a Proper B-Cell Receptor Signal Transmission

Background. Serine-threonine protein kinase CK2 has been recently involved in the pathogenesis of B-cell tumors, such as B acute lymphoblastic leukemia, B chronic lymphocytic leukemia, mantle cell lymphoma and multiple myeloma. CK2 acts through a “non-oncogene” addiction mechanism to propel tumor growth, protecting from apoptosis by a phosphorylation-dependent “shielding” mechanism of pro-survival molecules and stimulating oncogenic kinases by helping folding and enzymatic activity. In addition, CK2 has been shown to enhance the transactivation potential of several transcription factors, such as STAT3, NF-κB and c-Myc. The existing data on CK2 function in B cell tumors suggest that this kinase might act as a “hub” downstream signals from surface membrane molecules, like the B-cell (BCR), growth factor and cytokine receptors, as well as from cell-intrinsic pathways – like proteotoxic and DNA-damage-related stress cascades.Aims and methods. To gain insights into the role of CK2 in B-lymphopoiesis and, consequently, in B-cell tumors, we generated CK2β conditional knockout (KO) mice in B-cells by crossing Csnk2β-Flox/Flox mice with CD19-CRE transgenic mice.Results. CK2 kinase activity was decreased in Csnk2β KO B cells. In the bone marrow (BM), Csnk2β KO mice displayed a reduction of B-cells, especially of the B220high IgMint-high recirculating population of transitional and follicular (FO) B cells. Pro-B and pre-B-cell progenitors were slightly reduced in number. In peripheral blood, lymph-nodes, spleen and peritoneal cavity the number of B-cells was markedly reduced. Csnk2β KO mice had lower levels of all the immunoglobulin classes in the serum. The splenic IgDlow IgMhigh B-cell subset was increased whereas the IgDhigh IgMint-low population was decreased. An imbalance between the amount of FO and marginal zone (MZ) B-cells was found with an absolute reduction of FO B cells by approximately 2-folds and an increase of MZ B-cells and MZB cell precursors by up to three folds, on average. Histological and immunofluorescence (IF) analysis revealed a change of size/shape of spleen follicles and a significant expansion of the inter-follicular, marginal zone areas, which appeared to invade the follicle with larger cells. In vitro class-switch recombination assays demonstrated impairment in IgG1 and IgG3 class-switch and a marked reduction of the generation of antibody-producing cells. Anti-IgM stimulation was uncoupled to Ca++ mobilization, indicating a disrupted transmission of the signal from the BCR to the release of Ca++ stores in the endoplasmic reticulum. In vivo sheep red blood cells (SRBC) treatment (T-cell dependent response) showed a conserved up-regulation of GC markers, such as CD38, GL7 and PNA. Nonetheless, the architecture of the reactive follicles was found markedly changed. The analysis of FO, GC and MZ-associated genes showed normal levels of Bcl6, elevated levels of Lrf mRNA and, more significantly, a marked up-regulation of Notch2 target genes, such as Hes1 and Deltex1, in Csnk2β KO B cells. In vivo Notch2 blockage with neutralizing antibodies markedly reduced the MZB cell number in Csnk2β KO mice, indicating a Notch2-dependent MZB expansion associated with Csnk2β loss. High throughput RNAseq analysis was also performed and revealed significant alteration in FOB and MZB-regulating pathways.Conclusions. Here, we found that the β subunit of protein kinase CK2 is a novel regulator of peripheral B cell differentiation. CK2β sustains a proper BCR signal, controls the GC reaction and negatively regulates Notch2 signaling, acting as a master regulator of follicular/marginal zone architecture and terminal homeostasis of FOB and MZB cells. On one side our data enrich the knowledge on the mechanisms regulating B cell development, on the other side they inform about the potential mechanisms altered by CK2 during B-cell tumorigenesis. DisclosuresNo relevant conflicts of interest to declare.

Langerhans cells promote early germinal center formation in response to Leishmania-derived cutaneous antigens.

Efficient formation of early GCs depends on the close interaction between GC B cells and antigen-primed CD4(+) follicular helper T cells (TFH ). A tight and stable formation of TFH /B cell conjugates is required for cytokine-driven immunoglobulin class switching and somatic hypermutation of GC B cells. Recently, it has been shown that the formation of TFH /B cell conjugates is crucial for B-cell differentiation and class switch following infection with Leishmania major parasites. However, the subtype of DCs responsible for TFH -cell priming against dermal antigens is thus far unknown. Utilizing a transgenic C57BL/6 mouse model designed to trigger the ablation of Langerin(+) DC subsets in vivo, we show that the functionality of TFH /B cell conjugates is disturbed after depletion of Langerhans cells (LCs): LC-depleted mice show a reduction in somatic hypermutation in B cells isolated from TFH /B cell conjugates and markedly reduced GC reactions within skin-draining lymph nodes. In conclusion, this study reveals an indispensable role for LCs in promoting GC B-cell differentiation following cutaneous infection with Leishmania major parasites. We propose that LCs are key regulators of GC formation and therefore have broader implications for the development of allergies and autoimmunity as well as for future vaccination strategies.

Follicular regulatory T cells control humoral autoimmunity via NFAT2-regulated CXCR5 expression

Maturation of high-affinity B lymphocytes is precisely controlled during the germinal center reaction. This is dependent on CD4(+)CXCR5(+) follicular helper T cells (TFH) and inhibited by CD4(+)CXCR5(+)Foxp3(+) follicular regulatory T cells (TFR). Because NFAT2 was found to be highly expressed and activated in follicular T cells, we addressed its function herein. Unexpectedly, ablation of NFAT2 in T cells caused an augmented GC reaction upon immunization. Consistently, however, TFR cells were clearly reduced in the follicular T cell population due to impaired homing to B cell follicles. This was TFR-intrinsic because only in these cells NFAT2 was essential to up-regulate CXCR5. The physiological relevance for humoral (auto-)immunity was corroborated by exacerbated lupuslike disease in the presence of NFAT2-deficient TFR cells.

CCR6 is transiently upregulated on B cells after activation and modulates the germinal center reaction in the mouse

The CC-chemokine receptor 6 (CCR6) is expressed constitutively at an intermediate level on naïve B cells and is upregulated after activation on pregerminal center (GC) B cells. We hypothesized that it could be involved in the events leading to GC reaction and high-affinity antibody production, and therefore investigated the potential role of CCR6 in B-cell differentiation in vivo. After antigenic challenge of CCR6-/- mice with the T-cell-dependent antigen nitrophenyl-keyhole limpet hemocyanin (NP-KLH), GC B-cell development was found to be accelerated and the number of GC had increased significantly compared with control mice, but the antibodies produced by CCR6-/- B cells were on average of lower affinity. We conclude from these data that the CCR6/CCL20 axis has an important role in regulating the kinetics and efficiency of the GC reaction.