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A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features., (© 2024. The Author(s).)

The nonpolymorphic major histocompatibility complex E (MHC-E) molecule is up-regulated on many cancer cells, thus contributing to immune evasion by engaging inhibitory NKG2A/CD94 receptors on NK cells and tumor-infiltrating T cells. To investigate whether MHC-E expression by cancer cells can be targeted for MHC-E-restricted T cell control, we immunized rhesus macaques (RM) with rhesus cytomegalovirus (RhCMV) vectors genetically programmed to elicit MHC-E-restricted CD8 + T cells and to express established tumor-associated antigens (TAAs) including prostatic acidic phosphatase (PAP), Wilms tumor-1 protein, or Mesothelin. T cell responses to all three tumor antigens were comparable to viral antigen-specific responses with respect to frequency, duration, phenotype, epitope density, and MHC restriction. Thus, CMV-vectored cancer vaccines can bypass central tolerance by eliciting T cells to noncanonical epitopes. We further demonstrate that PAP-specific, MHC-E-restricted CD8 + T cells from RhCMV/PAP-immunized RM respond to PAP-expressing HLA-E + prostate cancer cells, suggesting that the HLA-E/NKG2A immune checkpoint can be exploited for CD8 + T cell-based immunotherapies.

Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity, and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month time frame. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both prefusion and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sublineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants., Competing Interests: R.M., J.B., C.S.-F., I.B., F.M., K.C., N.S., G.L., C.S., E.C., E.A.D.J., J.R.D., N.C., C.H.-D., A.T., A.A., H.W.V., A.L., D.C., and L.Pi. are or were employees of Vir Biotechnology Inc. and may hold shares in Vir Biotechnology Inc. C.G. is an external scientific consultant to Humabs BioMed SA. The other authors declare no competing interests., (© 2022 The Authors.)

The recently emerged SARS-CoV-2 Omicron variant encodes 37 amino acid substitutions in the spike protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody-based therapeutics. Here we show that the Omicron RBD binds to human ACE2 with enhanced affinity, relative to the Wuhan-Hu-1 RBD, and binds to mouse ACE2. Marked reductions in neutralizing activity were observed against Omicron compared to the ancestral pseudovirus in plasma from convalescent individuals and from individuals who had been vaccinated against SARS-CoV-2, but this loss was less pronounced after a third dose of vaccine. Most monoclonal antibodies that are directed against the receptor-binding motif lost in vitro neutralizing activity against Omicron, with only 3 out of 29 monoclonal antibodies retaining unaltered potency, including the ACE2-mimicking S2K146 antibody 1 . Furthermore, a fraction of broadly neutralizing sarbecovirus monoclonal antibodies neutralized Omicron through recognition of antigenic sites outside the receptor-binding motif, including sotrovimab 2 , S2X259 3 and S2H97 4 . The magnitude of Omicron-mediated immune evasion marks a major antigenic shift in SARS-CoV-2. Broadly neutralizing monoclonal antibodies that recognize RBD epitopes that are conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

A successful HIV vaccine eliciting broadly neutralizing antibodies (bnAbs) must overcome the hurdle of being able to activate naive precursor B cells encoding features within their germline B cell receptors (BCR) that allow recognition of broadly neutralizing epitopes. Knowledge of whether bnAb precursor B cells are circulating at sufficient frequencies within individuals in communities heavily impacted by HIV may be important. Using a germline-targeting eOD-GT8 immunogen and high-throughput droplet-based single-cell BCR sequencing, we demonstrate that large numbers of paired BCR sequences from multiple donors can be efficiently screened to elucidate precursor frequencies of rare, naive VRC01-class B cells. Further, we analyzed IGHV1-2 allelic usage among three different cohorts; we find that IGHV1-2 alleles traditionally thought to be incompatible with VRC01-class responses are relatively common in various human populations and that germline variation within IGHV1-2 associates with gene usage frequencies in the naive BCR repertoire., (© 2021. The Author(s).)

The spillovers of betacoronaviruses in humans and the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight the need for broad coronavirus countermeasures. We describe five monoclonal antibodies (mAbs) cross-reacting with the stem helix of multiple betacoronavirus spike glycoproteins isolated from COVID-19 convalescent individuals. Using structural and functional studies, we show that the mAb with the greatest breadth (S2P6) neutralizes pseudotyped viruses from three different subgenera through the inhibition of membrane fusion, and we delineate the molecular basis for its cross-reactivity. S2P6 reduces viral burden in hamsters challenged with SARS-CoV-2 through viral neutralization and Fc-mediated effector functions. Stem helix antibodies are rare, oftentimes of narrow specificity, and can acquire neutralization breadth through somatic mutations. These data provide a framework for structure-guided design of pan-betacoronavirus vaccines eliciting broad protection.

The recent emergence of SARS-CoV-2 variants of concern 1-10 and the recurrent spillovers of coronaviruses 11,12 into the human population highlight the need for broadly neutralizing antibodies that are not affected by the ongoing antigenic drift and that can prevent or treat future zoonotic infections. Here we describe a human monoclonal antibody designated S2X259, which recognizes a highly conserved cryptic epitope of the receptor-binding domain and cross-reacts with spikes from all clades of sarbecovirus. S2X259 broadly neutralizes spike-mediated cell entry of SARS-CoV-2, including variants of concern (B.1.1.7, B.1.351, P.1, and B.1.427/B.1.429), as well as a wide spectrum of human and potentially zoonotic sarbecoviruses through inhibition of angiotensin-converting enzyme 2 (ACE2) binding to the receptor-binding domain. Furthermore, deep-mutational scanning and in vitro escape selection experiments demonstrate that S2X259 possesses an escape profile that is limited to a single substitution, G504D. We show that prophylactic and therapeutic administration of S2X259 protects Syrian hamsters (Mesocricetus auratus) against challenge with the prototypic SARS-CoV-2 and the B.1.351 variant of concern, which suggests that this monoclonal antibody is a promising candidate for the prevention and treatment of emergent variants and zoonotic infections. Our data reveal a key antigenic site that is targeted by broadly neutralizing antibodies and will guide the design of vaccines that are effective against all sarbecoviruses., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape 1-3 , have activity against diverse sarbecoviruses 4-7 , and be highly protective through viral neutralization 8-11 and effector functions 12,13 . Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E12 8 ) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The development of an effective human immunodeficiency virus (HIV-1) vaccine is a high global health priority. Soluble native-like HIV-1 envelope glycoprotein trimers (Env), including those based on the SOSIP design, have shown promise as vaccine candidates by inducing neutralizing antibody responses against the autologous virus in animal models. However, to overcome HIV-1's extreme diversity a vaccine needs to induce broadly neutralizing antibodies (bNAbs). Such bNAbs can protect non-human primates (NHPs) and humans from infection. The prototypic BG505 SOSIP.664 immunogen is based on the BG505 env sequence isolated from an HIV-1-infected infant from Kenya who developed a bNAb response. Studying bNAb development during natural HIV-1 infection can inform vaccine design, however, it is unclear to what extent vaccine-induced antibody responses to Env are comparable to those induced by natural infection. Here, we compared Env antibody responses in BG505 SOSIP-immunized NHPs with those in BG505 SHIV-infected NHPs, by analyzing monoclonal antibodies (mAbs). We observed three major differences between BG505 SOSIP immunization and BG505 SHIV infection. First, SHIV infection resulted in more clonal expansion and less antibody diversity compared to SOSIP immunization, likely because of higher and/or prolonged antigenic stimulation and increased antigen diversity during infection. Second, while we retrieved comparatively fewer neutralizing mAbs (NAbs) from SOSIP-immunized animals, these NAbs targeted more diverse epitopes compared to NAbs from SHIV-infected animals. However, none of the NAbs, either elicited by vaccination or infection, showed any breadth. Finally, SOSIP immunization elicited antibodies against the base of the trimer, while infection did not, consistent with the base being placed onto the virus membrane in the latter setting. Together these data provide new insights into the antibody response against BG505 Env during infection and immunization and limitations that need to be overcome to induce better responses after vaccination., Competing Interests: The authors have declared that no competing interests exist.

Efficient therapeutic options are needed to control the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has caused more than 922,000 fatalities as of 13 September 2020. We report the isolation and characterization of two ultrapotent SARS-CoV-2 human neutralizing antibodies (S2E12 and S2M11) that protect hamsters against SARS-CoV-2 challenge. Cryo-electron microscopy structures show that S2E12 and S2M11 competitively block angiotensin-converting enzyme 2 (ACE2) attachment and that S2M11 also locks the spike in a closed conformation by recognition of a quaternary epitope spanning two adjacent receptor-binding domains. Antibody cocktails that include S2M11, S2E12, or the previously identified S309 antibody broadly neutralize a panel of circulating SARS-CoV-2 isolates and activate effector functions. Our results pave the way to implement antibody cocktails for prophylaxis or therapy, circumventing or limiting the emergence of viral escape mutants., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. In a cohort of 647 SARS-CoV-2-infected subjects, we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics., Competing Interests: Declaration of Interests L.P., N.C., M. Beltramello, C.S.-F., D.P., L.E.R., F.Z., N.S., J.B., A.P., S. Jaconi, B.G., A.M., A.D.M., M.S.P., E.C., S.V.G., F.J., C.H.-D., M.S., D.H., G.S., K.F., H.W.V., A.L., and D.C. are employees of Vir Biotechnology Inc. and may hold shares in Vir Biotechnology Inc. D.C. is currently listed as an inventor on multiple patent applications, which disclose the subject matter described in this manuscript. The Veesler laboratory has received a sponsored research agreement from Vir Biotechnology Inc. The other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Conventional vaccine design has been based on trial-and-error approaches, which have been generally successful. However, there have been some major failures in vaccine development and we still do not have highly effective licensed vaccines for tuberculosis, HIV, respiratory syncytial virus, and other major infections of global significance. Approaches at rational vaccine design have been limited by our understanding of the immune response to vaccination at the molecular level. Tools now exist to undertake in-depth analysis using systems biology approaches, but to be fully realized, studies are required in humans with intensive blood and tissue sampling. Methods that support this intensive sampling need to be developed and validated as feasible. To this end, we describe here a detailed approach that was applied in a study of 15 healthy adults, who were immunized with hepatitis B vaccine. Sampling included ~350 mL of blood, 12 microbiome samples, and lymph node fine needle aspirates obtained over a ~7-month period, enabling comprehensive analysis of the immune response at the molecular level, including single cell and tissue sample analysis. Samples were collected for analysis of immune phenotyping, whole blood and single cell gene expression, proteomics, lipidomics, epigenetics, whole blood response to key immune stimuli, cytokine responses, in vitro T cell responses, antibody repertoire analysis and the microbiome. Data integration was undertaken using different approaches-NetworkAnalyst and DIABLO. Our results demonstrate that such intensive sampling studies are feasible in healthy adults, and data integration tools exist to analyze the vast amount of data generated from a multi-omics systems biology approach. This will provide the basis for a better understanding of vaccine-induced immunity and accelerate future rational vaccine design., (Copyright © 2020 Ben-Othman, Cai, Liu, Varankovich, He, Blimkie, Lee, Gill, Novotny, Aevermann, Drissler, Shannon, McCann, Marty, Bjornson, Edgar, Lin, Gladish, Maclsaac, Amenyogbe, Chan, Llibre, Collin, Landais, Le, Reiss, Koff, Havenar-Daughton, Heran, Sangha, Walt, Krajden, Crotty, Sok, Briney, Burton, Duffy, Foster, Mohn, Kobor, Tebbutt, Brinkman, Scheuermann, Hancock, Kollmann and Sadarangani.)

Animal models of human antigen-specific B cell receptors (BCRs) generally depend on "inferred germline" sequences, and thus their relationship to authentic naive human B cell BCR sequences and affinities is unclear. Here, BCR sequences from authentic naive human VRC01-class B cells from healthy human donors were selected for the generation of three BCR knockin mice. The BCRs span the physiological range of affinities found in humans, and use three different light chains (VK3-20, VK1-5, and VK1-33) found among subclasses of naive human VRC01-class B cells and HIV broadly neutralizing antibodies (bnAbs). The germline-targeting HIV immunogen eOD-GT8 60mer is currently in clinical trial as a candidate bnAb vaccine priming immunogen. To attempt to model human immune responses to the eOD-GT8 60mer, we tested each authentic naive human VRC01-class BCR mouse model under rare human physiological B cell precursor frequency conditions. B cells with high (HuGL18 HL ) or medium (HuGL17 HL ) affinity BCRs were primed, recruited to germinal centers, and they affinity matured, and formed memory B cells. Precursor frequency and affinity interdependently influenced responses. Taken together, these experiments utilizing authentic naive human VRC01-class BCRs validate a central tenet of germline-targeting vaccine design and extend the overall concept of the reverse vaccinology approach to vaccine development., Competing Interests: Competing interest statement: W.R.S. is an inventor on a patent application submitted by IAVI and The Scripps Research Institute that covers the eOD-GT8 immunogen. W.R.S. is involved as a principal investigator of a human clinical trial involving eOD-GT8 60mer (G001). As part of that involvement, W.R.S. is privy to certain clinical trial data, and he was forbidden from sharing that data with the authors of this study during the course of this HuGL study, as is standard for blinded clinical trials. Any conclusions in this manuscript regarding relationships between HuGL mouse models and humans were made by the other authors, without input from W.R.S. related to any clinical trial activities., (Copyright © 2020 the Author(s). Published by PNAS.)

Antibody-dependent enhancement (ADE) of disease is a general concern for the development of vaccines and antibody therapies because the mechanisms that underlie antibody protection against any virus have a theoretical potential to amplify the infection or trigger harmful immunopathology. This possibility requires careful consideration at this critical point in the pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we review observations relevant to the risks of ADE of disease, and their potential implications for SARS-CoV-2 infection. At present, there are no known clinical findings, immunological assays or biomarkers that can differentiate any severe viral infection from immune-enhanced disease, whether by measuring antibodies, T cells or intrinsic host responses. In vitro systems and animal models do not predict the risk of ADE of disease, in part because protective and potentially detrimental antibody-mediated mechanisms are the same and designing animal models depends on understanding how antiviral host responses may become harmful in humans. The implications of our lack of knowledge are twofold. First, comprehensive studies are urgently needed to define clinical correlates of protective immunity against SARS-CoV-2. Second, because ADE of disease cannot be reliably predicted after either vaccination or treatment with antibodies-regardless of what virus is the causative agent-it will be essential to depend on careful analysis of safety in humans as immune interventions for COVID-19 move forward.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerged coronavirus that is responsible for the current pandemic of coronavirus disease 2019 (COVID-19), which has resulted in more than 3.7 million infections and 260,000 deaths as of 6 May 2020 1,2 . Vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. The SARS-CoV-2 spike (S) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. Here we describe several monoclonal antibodies that target the S glycoprotein of SARS-CoV-2, which we identified from memory B cells of an individual who was infected with severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003. One antibody (named S309) potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2, by engaging the receptor-binding domain of the S glycoprotein. Using cryo-electron microscopy and binding assays, we show that S309 recognizes an epitope containing a glycan that is conserved within the Sarbecovirus subgenus, without competing with receptor attachment. Antibody cocktails that include S309 in combination with other antibodies that we identified further enhanced SARS-CoV-2 neutralization, and may limit the emergence of neutralization-escape mutants. These results pave the way for using S309 and antibody cocktails containing S309 for prophylaxis in individuals at a high risk of exposure or as a post-exposure therapy to limit or treat severe disease.

A fundamental challenge in vaccinology is learning how to induce durable antibody responses. Live viral vaccines induce antibody responses that last a lifetime, but those induced with subunit vaccines wane rapidly. Studies in mice and humans have established that long-lived plasma cells (LLPCs) in the bone marrow (BM) are critical mediators of durable antibody responses. Here, we present data that adjuvanting an HIV-1 clade C 1086.C-derived gp140 immunogen (Env) with a novel synthetic Toll-like receptor (TLR)-7/8 agonist named 3M-052 formulated in poly(lactic- co -glycolic)acid or PLGA nanoparticles (NPs) or with alum, either alone or in combination with a TLR-4 agonist GLA, induces notably high and persistent (up to ~1 year) frequencies of Env-specific LLPCs in the BM and serum antibody responses in rhesus macaques. Up to 36 and 18% of Env-specific cells among total IgG-secreting BM-resident plasma cells were detected at peak and termination, respectively. In contrast, adjuvanting Env with alum or GLA in NP induced significantly lower (~<100-fold) LLPC and antibody responses. Immune responses induced by 3M-052 were also significantly higher than those induced by a combination of TLR-7/8 (R848) and TLR-4 (MPL) agonists. Adjuvanting Env with 3M-052 also induced robust activation of blood monocytes, strong plasmablast responses in blood, germinal center B cells, T follicular helper (T FH ) cells, and persistent Env-specific plasma cells in draining lymph nodes. Overall, these results demonstrate efficacy of 3M-052 in promoting high magnitude and durability of antibody responses via robust stimulation of innate immunity and BM-resident LLPCs., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Germinal centers (GC) are critically important for maturation of the antibody response and generation of memory B cells, processes that form the basis for long-term protection from pathogens. GCs only occur in lymphoid tissue, such as lymph nodes, and are not present in blood. Therefore, GC B cells and GC T follicular helper (T FH ) cells are not well-studied in humans under normal healthy conditions, due to the limited availability of healthy lymph node samples. We used a minimally invasive, routine clinical procedure, lymph node fine needle aspirations (LN FNAs), to obtain LN cells from healthy human subjects. This study of 73 LNs demonstrates that human LN FNAs are a safe and feasible technique for immunological research, and suggests benchmarks for human GC biology under noninflammatory conditions. The findings indicate that assessment of the GC response via LN FNAs will have application to the study of human vaccination, allergy, and autoimmune disease., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Vaccine induction of broadly neutralizing antibodies (bnAbs) to HIV remains a major challenge. Germline-targeting immunogens hold promise for initiating the induction of certain bnAb classes; yet for most bnAbs, a strong dependence on antibody heavy chain complementarity-determining region 3 (HCDR3) is a major barrier. Exploiting ultradeep human antibody sequencing data, we identified a diverse set of potential antibody precursors for a bnAb with dominant HCDR3 contacts. We then developed HIV envelope trimer-based immunogens that primed responses from rare bnAb-precursor B cells in a mouse model and bound a range of potential bnAb-precursor human naïve B cells in ex vivo screens. Our repertoire-guided germline-targeting approach provides a framework for priming the induction of many HIV bnAbs and could be applied to most HCDR3-dominant antibodies from other pathogens., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The first immunization in a protein prime-boost vaccination is likely to be critical for how the immune response unfolds. Using fine needle aspirates (FNAs) of draining lymph nodes (LNs), we tracked the kinetics of the primary immune response in rhesus monkeys immunized intramuscularly (IM) or subcutaneously (s.c.) with an eOD-GT8 60-mer nanoparticle immunogen to facilitate clinical trial design. Significant numbers of germinal center B (B GC ) cells and antigen-specific CD4 T cells were detectable in the draining LN as early as 7 days post-immunization and peaked near day 21. Strikingly, s.c. immunization results in 10-fold larger antigen-specific B GC cell responses compared to IM immunization. Lymphatic drainage studies revealed that s.c. immunization resulted in faster and more consistent axillary LN drainage than IM immunization. These data indicate robust antigen-specific germinal center responses can occur rapidly to a single immunization with a nanoparticle immunogen and vaccine drainage substantially impacts immune responses in local LNs., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

"Strep throat" is highly prevalent among children, yet it is unknown why only some children develop recurrent tonsillitis (RT), a common indication for tonsillectomy. To gain insights into this classic childhood disease, we performed phenotypic, genotypic, and functional studies on pediatric group A Streptococcus (GAS) RT and non-RT tonsils from two independent cohorts. GAS RT tonsils had smaller germinal centers, with an underrepresentation of GAS-specific CD4 + germinal center T follicular helper (GC-T FH ) cells. RT children exhibited reduced antibody responses to an important GAS virulence factor, streptococcal pyrogenic exotoxin A (SpeA). Risk and protective human leukocyte antigen (HLA) class II alleles for RT were identified. Lastly, SpeA induced granzyme B production in GC-T FH cells from RT tonsils with the capacity to kill B cells and the potential to hobble the germinal center response. These observations suggest that RT is a multifactorial disease and that contributors to RT susceptibility include HLA class II differences, aberrant SpeA-activated GC-T FH cells, and lower SpeA antibody titers., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Passive administration of HIV neutralizing antibodies (nAbs) can protect macaques from hard-to-neutralize (tier 2) chimeric simian-human immunodeficiency virus (SHIV) challenge. However, conditions for nAb-mediated protection after vaccination have not been established. Here, we selected groups of 6 rhesus macaques with either high or low serum nAb titers from a total of 78 animals immunized with recombinant native-like (SOSIP) Env trimers. Repeat intrarectal challenge with homologous tier 2 SHIV BG505 led to rapid infection in unimmunized and low-titer animals. High-titer animals, however, demonstrated protection that was gradually lost as nAb titers waned over time. An autologous serum ID 50 nAb titer of ∼1:500 afforded more than 90% protection from medium-dose SHIV infection. In contrast, antibody-dependent cellular cytotoxicity and T cell activity did not correlate with protection. Therefore, Env protein-based vaccination strategies can protect against hard-to-neutralize SHIV challenge in rhesus macaques by inducing tier 2 nAbs, provided appropriate neutralizing titers can be reached and maintained., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Phase 1 clinical studies will soon evaluate novel HIV-1 envelope immunogens targeting distinct 'germline' and memory B cell receptors to ultimately elicit HIV-1 broadly neutralizing antibodies (bNAbs). The National Institute of Allergy and Infectious Diseases (NIAID) recently convened a panel of US-based expert scientists, clinicians, sponsors and ethicists to discuss the role of sampling draining lymph nodes within preventive HIV vaccine trials. The meeting addressed the importance of evaluating germinal center (GC) responses following immunization to predict bNAb potency and breadth, and reviewed key aspects of this procedure within the clinical research setting, including informed consent, adverse event monitoring, study participant acceptability, medical expertise and training. We review highlights from the meeting and discuss the advantages and disadvantages of sampling lymph nodes by excisional biopsies compared to fine needle aspirations (FNA) in the context of prophylactic HIV vaccine trials., (Copyright © 2018.)

Most viral vaccines provide protection from infection through the generation of neutralizing antibodies (nAbs). The repertoire of B cells responding to immunization is the starting material from which nAbs eventually arise. Immunization strategies are increasingly targeting precise B cell specificities to mimic nAbs generated during natural infection, in an effort to maximize the potency of the vaccine-elicited Ab response. An understanding of the human B cell specificities capable of immunogen recognition can aid in immunogen design and inform decision-making for clinical advancement. Here, we review what is known about antigen-specific and epitope-specific naive B cell repertoires in humans and mice, and we consider the challenges for identifying and analyzing antigen-specific naive B cell repertoires. Finally, we provide a framework for further exploration, interpretation, utilization of the B cell repertoire to facilitate vaccine discovery., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Traditional vaccine development to prevent some of the worst current pandemic diseases has been unsuccessful so far. Germline-targeting immunogens have potential to prime protective antibodies (Abs) via more targeted immune responses. Success of germline-targeting vaccines in humans will depend on the composition of the human naive B cell repertoire, including the frequencies and affinities of epitope-specific B cells. However, the human naive B cell repertoire remains largely undefined. Assessment of antigen-specific human naive B cells among hundreds of millions of B cells from multiple donors may be used as pre-phase 1 ex vivo human testing to potentially forecast B cell and Ab responses to new vaccine designs. VRC01 is an HIV broadly neutralizing Ab (bnAb) against the envelope CD4-binding site (CD4bs). We characterized naive human B cells recognizing eOD-GT8, a germline-targeting HIV-1 vaccine candidate immunogen designed to prime VRC01-class Abs. Several distinct subclasses of VRC01-class naive B cells were identified, sharing sequence characteristics with inferred precursors of known bnAbs VRC01, VRC23, PCIN63, and N6. Multiple naive B cell clones exactly matched mature VRC01-class bnAb L-CDR3 sequences. Non-VRC01-class B cells were also characterized, revealing recurrent public light chain sequences. Unexpectedly, we also identified naive B cells related to the IOMA-class CD4bs bnAb. These different subclasses within the human repertoire had strong initial affinities ( K D ) to the immunogen, up to 13 nM, and represent encouraging indications that multiple independent pathways may exist for vaccine-elicited VRC01-class bnAb development in most individuals. The frequencies of these distinct eOD-GT8 B cell specificities give insights into antigen-specific compositional features of the human naive B cell repertoire and provide actionable information for vaccine design and advancement., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

B cells play a critical role in the immune response by producing antibodies, which display remarkable diversity. Here we describe a bioinformatic pipeline, BALDR (BCR Assignment of Lineage using De novo Reconstruction) that accurately reconstructs the paired heavy and light chain immunoglobulin gene sequences from Illumina single-cell RNA-seq data. BALDR was accurate for clonotype identification in human and rhesus macaque influenza vaccine and simian immunodeficiency virus vaccine induced vaccine-induced plasmablasts and naïve and antigen-specific memory B cells. BALDR enables matching of clonotype identity with single-cell transcriptional information in B cell lineages and will have broad application in the fields of vaccines, human immunodeficiency virus broadly neutralizing antibody development, and cancer.BALDR is available at https://github.com/BosingerLab/BALDR .

How precursor frequencies and antigen affinities impact interclonal B cell competition is a particularly relevant issue for candidate germline-targeting HIV vaccine designs because of the in vivo rarity of naive B cells that recognize broadly neutralizing epitopes. Knowing the frequencies and affinities of HIV-specific VRC01-class naive human B cells, we transferred B cells with germline VRC01 B cell receptors into congenic recipients to elucidate the roles of precursor frequency, antigen affinity, and avidity on B cell responses following immunization. All three factors were interdependently limiting for competitive success of VRC01-class B cells. In physiological high-affinity conditions using a multivalent immunogen, rare VRC01-class B cells successfully competed in germinal centers (GC), underwent extensive somatic hypermutation, and differentiated into memory B cells. The data reveal dominant influences of precursor frequency, affinity, and avidity for interclonal GC competition and indicate that germline-targeting immunogens can overcome these challenges with high-affinity multimeric designs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Elicitation of broadly neutralizing antibodies (bnAbs) is a primary HIV vaccine goal. Native-like trimers mimicking virion-associated spikes present nearly all bnAb epitopes and are therefore promising vaccine antigens. However, first generation native-like trimers expose epitopes for non-neutralizing antibodies (non-nAbs), which may hinder bnAb induction. We here employ computational and structure-guided design to develop improved native-like trimers that reduce exposure of non-nAb epitopes in the V3-loop and trimer base, minimize both CD4 reactivity and CD4-induced non-nAb epitope exposure, and increase thermal stability while maintaining bnAb antigenicity. In rabbit immunizations with native-like trimers of the 327c isolate, improved trimers suppress elicitation of V3-directed and tier-1 neutralizing antibodies and induce robust autologous tier-2 neutralization, unlike a first-generation trimer. The improved native-like trimers from diverse HIV isolates, and the design methods, have promise to assist in the development of a HIV vaccine.

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells.

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.)

Our previous work has shown that antigens adjuvanted with ligands specific for Toll-like receptor 4 (TLR4) and TLR7/8 encapsulated in poly(lactic-co-glycolic) acid (PLGA)-based nanoparticles (NPs) induce robust and durable immune responses in mice and macaques. We investigated the efficacy of these NP adjuvants in inducing protective immunity against simian immunodeficiency virus (SIV). Rhesus macaques (RMs) were immunized with NPs containing TLR4 and TLR7/8 agonists mixed with soluble recombinant SIVmac239-derived envelope (Env) gp140 and Gag p55 (protein) or with virus-like particles (VLPs) containing SIVmac239 Env and Gag. NP-adjuvanted vaccines induced robust innate responses, antigen-specific antibody responses of a greater magnitude and persistence, and enhanced plasmablast responses compared to those achieved with alum-adjuvanted vaccines. NP-adjuvanted vaccines induced antigen-specific, long-lived plasma cells (LLPCs), which persisted in the bone marrow for several months after vaccination. NP-adjuvanted vaccines induced immune responses that were associated with enhanced protection against repeated low-dose, intravaginal challenges with heterologous SIVsmE660 in animals that carried TRIM5α restrictive alleles. The protection induced by immunization with protein-NP correlated with the prechallenge titers of Env-specific IgG antibodies in serum and vaginal secretions. However, no such correlate was apparent for immunization with VLP-NP or alum as the adjuvant. Transcriptional profiling of peripheral blood mononuclear cells isolated within the first few hours to days after primary vaccination revealed that NP-adjuvanted vaccines induced a molecular signature similar to that induced by the live attenuated yellow fever viral vaccine. This systems approach identified early blood transcriptional signatures that correlate with Env-specific antibody responses in vaginal secretions and protection against infection. These results demonstrate the adjuvanticity of the NP adjuvant in inducing persistent and protective antibody responses against SIV in RMs with implications for the design of vaccines against human immunodeficiency virus (HIV)., Importance: The results of the RV144 HIV vaccine trial, which demonstrated a rapid waning of protective immunity with time, have underscored the need to develop strategies to enhance the durability of protective immune responses. Our recent work in mice has highlighted the capacity of nanoparticle-encapsulated TLR ligands (NP) to induce potent and durable antibody responses that last a lifetime in mice. In the present study, we evaluated the ability of these NP adjuvants to promote robust and durable protective immune responses against SIV in nonhuman primates. Our results demonstrate that immunization of rhesus macaques with NP adjuvants mixed with soluble SIV Env or a virus-like particle form of Env (VLP) induces potent and durable Env-specific antibody responses in the serum and in vaginal secretions. These responses were superior to those induced by alum adjuvant, and they resulted in enhanced protection against a low-dose intravaginal challenge with a heterologous strain of SIV in animals with TRIM5a restrictive alleles. These results highlight the potential for such NP TLR L adjuvants in promoting robust and durable antibody responses against HIV in the next generation of HIV immunogens currently being developed., (Copyright © 2017 American Society for Microbiology.)

The generation of HIV bnAbs may be one of the greatest feats of the human immune system and our best hope of finally creating an HIV vaccine. The striking amount of somatic hypermutation in HIV bnAbs led to the hypothesis that T follicular helper (Tfh) cells and germinal centers (GC) play a critical role in the ability of the immune system to generate these uncommon antibodies. In this review, we first summarize what is known about the immunological process of HIV bnAb development, the challenges of eliciting bnAbs via immunizations, and the putative central roles of Tfh cells and GC in the generation of HIV bnAbs. Next, we explore factors that have impeded our understanding of the GC and Tfh-cell processes involved in bnAb generation, including the difficulty of quantifying antigen-specific GC Tfh cells and the difficulty of tracking GC in human and non-human primate vaccine studies. Finally, we discuss antibody immunodominance pertaining to neutralizing antibody generation and the GC response, propose models to explain the negative effects of immunodominance on neutralizing antibody generation, and consider means of optimizing Tfh and GC responses to potentially overcome these problems., (© 2017 The Authors. Immunological Reviews published by John Wiley & Sons Ltd.)

Generating tier 2 HIV-neutralizing antibody (nAb) responses by immunization remains a challenging problem, and the immunological barriers to induction of such responses with Env immunogens remain unclear. Here, some rhesus monkeys developed autologous tier 2 nAbs upon HIV Env trimer immunization (SOSIP.v5.2) whereas others did not. This was not because HIV Env trimers were immunologically silent because all monkeys made similar ELISA-binding antibody responses; the key difference was nAb versus non-nAb responses. We explored the immunological barriers to HIV nAb responses by combining a suite of techniques, including longitudinal lymph node fine needle aspirates. Unexpectedly, nAb development best correlated with booster immunization GC B cell magnitude and Tfh characteristics of the Env-specific CD4 T cells. Notably, these factors distinguished between successful and unsuccessful antibody responses because GC B cell frequencies and stoichiometry to GC Tfh cells correlated with nAb development, but did not correlate with total Env Ab binding titers., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Detection of Ag-specific CD4(+) T cells is central to the study of many human infectious diseases, vaccines, and autoimmune diseases. However, such cells are generally rare and heterogeneous in their cytokine profiles. Identification of Ag-specific germinal center (GC) T follicular helper (Tfh) cells by cytokine production has been particularly problematic. The function of a GC Tfh cell is to selectively help adjacent GC B cells via cognate interaction; thus, GC Tfh cells may be stingy cytokine producers, fundamentally different from Th1 or Th17 cells in the quantities of cytokines produced. Conventional identification of Ag-specific cells by intracellular cytokine staining relies on the ability of the CD4(+) T cell to generate substantial amounts of cytokine. To address this problem, we have developed a cytokine-independent activation-induced marker (AIM) methodology to identify Ag-specific GC Tfh cells in human lymphoid tissue. Whereas Group A Streptococcus-specific GC Tfh cells produced minimal detectable cytokines by intracellular cytokine staining, the AIM method identified 85-fold more Ag-specific GC Tfh cells. Intriguingly, these GC Tfh cells consistently expressed programmed death ligand 1 upon activation. AIM also detected non-Tfh cells in lymphoid tissue. As such, we applied AIM for identification of rare Ag-specific CD4(+) T cells in human peripheral blood. Dengue, tuberculosis, and pertussis vaccine-specific CD4(+) T cells were readily detectable by AIM. In summary, cytokine assays missed 98% of Ag-specific human GC Tfh cells, reflecting the biology of these cells, which could instead be sensitively identified by coexpression of TCR-dependent activation markers., (Copyright © 2016 by The American Association of Immunologists, Inc.)

A range of current candidate AIDS vaccine regimens are focused on generating protective HIV-neutralizing Ab responses. Many of these efforts rely on the rhesus macaque animal model. Understanding how protective Ab responses develop and how to increase their efficacy are both major knowledge gaps. Germinal centers (GCs) are the engines of Ab affinity maturation. GC T follicular helper (Tfh) CD4 T cells are required for GCs. Studying vaccine-specific GC Tfh cells after protein immunizations has been challenging, as Ag-specific GC Tfh cells are difficult to identify by conventional intracellular cytokine staining. Cytokine production by GC Tfh cells may be intrinsically limited in comparison with other Th effector cells, as the biological role of a GC Tfh cell is to provide help to individual B cells within the GC, rather than secreting large amounts of cytokines bathing a tissue. To test this idea, we developed a cytokine-independent method to identify Ag-specific GC Tfh cells. RNA sequencing was performed using TCR-stimulated GC Tfh cells to identify candidate markers. Validation experiments determined CD25 (IL-2Rα) and OX40 to be highly upregulated activation-induced markers (AIM) on the surface of GC Tfh cells after stimulation. In comparison with intracellular cytokine staining, the AIM assay identified >10-fold more Ag-specific GC Tfh cells in HIV Env protein-immunized macaques (BG505 SOSIP). CD4 T cells in blood were also studied. In summary, AIM demonstrates that Ag-specific GC Tfh cells are intrinsically stingy producers of cytokines, which is likely an essential part of their biological function., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. Using deep mutational scanning and multitarget optimization, we developed a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens., (Copyright © 2016, American Association for the Advancement of Science.)

Significantly higher levels of plasma CXCL13 [chemokine (C-X-C motif) ligand 13] were associated with the generation of broadly neutralizing antibodies (bnAbs) against HIV in a large longitudinal cohort of HIV-infected individuals. Germinal centers (GCs) perform the remarkable task of optimizing B-cell Ab responses. GCs are required for almost all B-cell receptor affinity maturation and will be a critical parameter to monitor if HIV bnAbs are to be induced by vaccination. However, lymphoid tissue is rarely available from immunized humans, making the monitoring of GC activity by direct assessment of GC B cells and germinal center CD4(+) T follicular helper (GC Tfh) cells problematic. The CXCL13-CXCR5 [chemokine (C-X-C motif) receptor 5] chemokine axis plays a central role in organizing both B-cell follicles and GCs. Because GC Tfh cells can produce CXCL13, we explored the potential use of CXCL13 as a blood biomarker to indicate GC activity. In a series of studies, we found that plasma CXCL13 levels correlated with GC activity in draining lymph nodes of immunized mice, immunized macaques, and HIV-infected humans. Furthermore, plasma CXCL13 levels in immunized humans correlated with the magnitude of Ab responses and the frequency of ICOS(+) (inducible T-cell costimulator) Tfh-like cells in blood. Together, these findings support the potential use of CXCL13 as a plasma biomarker of GC activity in human vaccine trials and other clinical settings.

Broadly neutralizing antibodies (bnAbs) are thought to be a critical component of a protective HIV vaccine. However, designing vaccines immunogens able to elicit bnAbs has proven unsuccessful to date. Understanding the correlates and immunological mechanisms leading to the development of bnAb responses during natural HIV infection is thus critical to the design of a protective vaccine. The IAVI Protocol C program investigates a large longitudinal cohort of primary HIV-1 infection in Eastern and South Africa. Development of neutralization was evaluated in 439 donors using a 6 cross-clade pseudo-virus panel predictive of neutralization breadth on larger panels. About 15% of individuals developed bnAb responses, essentially between year 2 and year 4 of infection. Statistical analyses revealed no influence of gender, age or geographical origin on the development of neutralization breadth. However, cross-clade neutralization strongly correlated with high viral load as well as with low CD4 T cell counts, subtype-C infection and HLA-A*03(-) genotype. A correlation with high overall plasma IgG levels and anti-Env IgG binding titers was also found. The latter appeared not associated with higher affinity, suggesting a greater diversity of the anti-Env responses in broad neutralizers. Broadly neutralizing activity targeting glycan-dependent epitopes, largely the N332-glycan epitope region, was detected in nearly half of the broad neutralizers while CD4bs and gp41-MPER bnAb responses were only detected in very few individuals. Together the findings suggest that both viral and host factors are critical for the development of bnAbs and that the HIV Env N332-glycan supersite may be a favorable target for vaccine design.

We have investigated the dynamics of germinal center (GC) formation in lymphoid tissues following acute SIV infection. SIV induces a marked follicular hyperplasia, associated with an aberrant accumulation of nonproliferating T follicular helper cells within GCs, but with an abundance of cells producing IL-21, demonstrating that the mechanisms involved for these two events appear independent. IL-21-stimulated T follicular helper cells are considered a critical element for GC formation, a physiological process that seems dysregulated and excessive during HIV/SIV infection, contributing to lymphoid pathogenesis. However, the data suggest that the kinetics by which such GCs are formed may be an important predictor of the host-pathogen equilibrium, as early GC hyperplasia was associated with better control of viral replication. In contrast, monkeys undergoing fast disease progression upon infection exhibited an involution of GCs without local IL-21 production in GCs. These results provide important clues regarding GC-related hyperimmune responses in the context of disease progression within various individuals during HIV/SIV infection and may open novel therapeutic avenues to limit lymphoid dysfunction, postinfection., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The vast majority of currently licensed human vaccines work on the basis of long-term protective antibody responses. It is now conceivable that an antibody-dependent HIV vaccine might be possible, given the discovery of HIV broadly neutralizing antibodies (bnAbs) in some HIV-infected individuals. However, these antibodies are difficult to develop and have characteristics indicative of a high degree of affinity maturation in germinal centers (GCs). CD4⁺ T follicular helper (Tfh) cells are specialized for B cell help and necessary for GCs. Therefore, the development of HIV bnAbs might depend on Tfh cells. Here, we identified in normal individuals a subpopulation of circulating memory PD-1⁺CXCR5⁺CD4⁺ T cells that are resting memory cells most related to bona fide GC Tfh cells by gene expression profile, cytokine profile, and functional properties. Importantly, the frequency of these cells correlated with the development of bnAbs against HIV in a large cohort of HIV⁺ individuals., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Generating long-term humoral immunity is a crucial component of successful vaccines and requires interactions between T cells and B cells in germinal centers (GC). In GCs, a specialized subset of CD4+ helper T cells, called T follicular helper cells (Tfh), provide help to B cells; this help directs the magnitude and quality of the antibody response. Tfh cell help influences B cell survival, proliferation, somatic hypermutation, class switch recombination, and differentiation. Sustained contact between Tfh cells and B cells is necessary for the provision of help to B cells. SAP (Signaling lymphocytic activation molecule (SLAM)-associated protein, encoded by Sh2d1a) regulates the duration of T:B cell interactions and is required for long-term humoral immunity in animal models and in humans. SAP binds to SLAM family receptors and mediates signaling that affects cell adhesion, cytokine secretion, and TCR signaling strength. Therefore, the modulation of SAP and SLAM family receptor expression represents a major axis by which the quality and duration of an antibody response is controlled after vaccination., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Invariant natural killer T (iNKT) cells have the ability to rapidly secret cytokines in response to diverse stimuli, and therefore influence numerous immune reactions. Although IFN-γ and IL-4 are thought to dominate iNKT cytokine production, a distinct subset of iNKT cells, expressing RORγt and producing IL-17, has now been identified in both mice and humans. Although a role in pathogen and allergic responses has been assigned to the RORγt(+) iNKT subset, factors controlling their development and function remain illusive. Here, we demonstrate that RORγt(+) iNKT-cell differentiation obeys transforming growth factor-β (TGF-β) signaling control, different from that described for conventional iNKT cells. We reveal that TGF-β signaling, and particularly its SMAD4-dependent pathway, is required for both the survival of RORγt(+) iNKT cells during their development and IL-17 production at the periphery. Moreover, constitutive TGF-β signaling in RORγt(+) iNKT cells drives higher peripheral numbers and increased tissue distribution. Finally, we found that SMAD4-dependent TGF-β signaling is mandatory for the peripheral expansion of the RORγt(+) iNKT cells responding to inflammatory signals. Thus, this work demonstrates that both the development and responsiveness of the newly described IL-17-producing iNKT cell subset is under the control of a dedicated TGF-β signaling pathway.