Browsing by Author "Kelsoe, Garnett H"

B lymphocytes are multifunctional and play important roles in both innate and adaptive immunity. The diverse roles of B cells can be attributed to the various and distinct types of B cells as determined by their origin, developmental stage, antigen specificity, and function.

Evidence suggests that human innate-like B cells (i.e., marginal zone and/or B1-like B cells) develop during fetal life. However, the characteristics of human fetal B-lineage cells are less understood. Recent studies of fetal and human umbilical cord B cells indicated that CD27, a well-established marker of human memory B cells, may also be expressed on human B1-like B cells. Indeed, CD27+ B cells are present in patients with hyper-IgM 1 (HIGM1) syndrome who are unable to generate GCs or memory B cells. In order to define the origin of naïve CD27+IgD+ human B cells, I studied B-cell development in both fetal and adult tissues.

In human fetal liver, most CD19+ cells co-express CD10, a marker of human developing B cells. Some CD19+CD10+ B cells express CD27, and these fetal CD27+ cells are present in the pro-B, pre-B, and immature/transitional B-cell compartments. Lower frequencies of phenotypically identical cells are also identified in adult bone marrow. CD27+ pro-B, pre-B, and immature/transitional B cells express recombination activating gene-1, terminal deoxynucleotidyl transferase, and Vpre-B mRNA comparable to their CD27− counterparts. CD27+ and CD27− developing B cells show similar immunoglobulin heavy chain gene usage with low levels of mutations, suggesting that CD27+ developing B cells are distinct from mutated memory B cells. Despite these similarities, CD27+ developing B cells differ from CD27− developing B cells by their increased expression of LIN28B, a transcription factor associated with the fetal lymphoid lineages of mice. Furthermore, CD27+ pro-B cells efficiently generate IgM+IgD+ immature/transitional B cells in vitro. Our observations suggest that CD27 expression during B-cell development identifies a physiologic state or lineage for human B-cell development distinct from the memory B-cell compartment.

Regarding B-cell repertoire, due to the random recombination of immunoglobulin V, D, and J gene segments during B-cell development, B cells are highly diversified in their antigen specificity. Through their specific B-cell antigen receptors (BCRs), B cells recognize foreign (and self-) antigens, and present these antigens to cognate T cells to elicit/establish humoral responses, such as germinal centers, immunological memory, and long-lasting circulating antibodies. Some bacteria and viruses escape the host’s immune system by mimicking host antigens, as B cells that recognize shared epitopes on self- and foreign antigens may provide protection against such pathogens; however, these B cells are normally eliminated by tolerance mechanisms during development. The extent of tolerization manifest among human B cells that recognize both self- and foreign antigens is unknown. Here, I and my colleagues use an efficient single B-cell culture method and multiplexed antigen-binding assays to determine the specificity of about 2,300 clonal IgG antibodies produced by the progeny of single transitional and mature B cells. We show that in healthy individuals, half of the self-reactive B cells crossreact with foreign antigen, and that the frequencies of crossreactive B cells decrease by half between the transitional and mature B-cell stages, indicating that a substantial fraction of foreign specificities is lost by the second tolerance mechanisms. In SLE patients, who show defective peripheral tolerance, frequencies of crossreactive B cells are unchanged between the B-cell stages. The crossreactive, mature B cells in SLE patients show distinct reactivity to foreign antigens. We propose that activating forbidden B cells may be a good strategy for protection against host-mimicking pathogens if we can control tolerance.

Activated B cells can present antigen to T cells, as well as differentiate into memory B cells and plasma cells. Indeed, activated B cells express high levels of MHCII and are considered to be professional antigen presenting cells (APC), along with dendritic cells and macrophages. APC can be used to discover the epitopes targeted in T-cell responses; T cells are co-cultured with autologous APC in the presence of antigens and T-cell responses are evaluated. With numerous epitope candidates, mapping T-cell epitopes requires large numbers of APC; the availability of APC in blood is a limiting component and leukapheresis is often required. Since B cells can be expanded in vitro more easily than other APC, they represent a solution for the challenge of isolating adequate numbers of APC from blood in order to determine T-cell antigen specificity. I modified our single B-cell culture to support efficient activation and proliferation of both naïve and memory human B cells for the purpose of generating large numbers of autologous APC. Briefly, naïve or memory B cells recovered from blood are cultured with recombinant human IL-2, IL-4, IL-21, and BAFF on CD154+ feeder cells; this culture supports extensive B-cell proliferation, with approximately 103 fold increases following 8 days in culture, and 106 fold increases when cultures are split and cultured for 8 more days. The capacity for continued proliferation is stable for at least another week. In culture, a significant fraction of naïve B cells undergo isotype switching and terminally differentiate into plasmacytes. Culture-derived (CD) B cells are readily cryopreserved, and when recovered, retain their ability to proliferate and differentiate. Significantly, proliferating CD B cells express high levels of MHCII, CD80, and CD86. I have examined the APC function of CD B cells and found that they present both allo- and microbial antigens to autologous T cells with comparable efficiency to PBMC. Moreover, I am able to activate and expand antigen-specific memory B cells; these cultured cells are highly effective in presenting antigen to T cells. This culture method provides a platform for studying the BCR and TCR repertoires within a single individual.

Understanding B-lymphocyte fate specification during B-cell development and humoral responses is important for developing vaccines and tools for studying B cells.

In the first study, I elucidated major factors influencing the fate of 2F5 B-cell receptor (BCR) knockin B cells as they traverse immune tolerance checkpoints. 2F5 is an HIV-1 broadly neutralizing antibody (bnAb) whose neutralizing activity is linked to its ability to bind two autoantigens: host-derived viral membrane phospholipids and the ELDKWA epitope found in both the viral envelope (Env) protein and in the host enzyme, kynureninase (KYNU). The development of 2F5-like B cells is proscribed by immune tolerance controls, but it is unknown whether tolerizing counterselection is driven primarily by lipid- or by KYNU-specificity (or by both equally). It is also unclear whether BCR editing purges the 2F5 BCR from the peripheral repertoire of 2F5 knockin mice. Answering these questions is important for evaluating potential HIV-1 vaccine strategies.

I sampled thousands of B cells from before and after the first and second tolerance checkpoints in 2F5 BCR knockin mice. After culturing individual B cells in an in vitro system that supports B-cell proliferation and differentiation into Ab-secreting cells, I determined the specificities of the secreted clonal IgGs, and recovered the V(D)J rearrangements encoding these Abs. I found that in 2F5 knockin mice, nearly all (pre-tolerance) small pre-B cells express the knockin heavy chain and light chain (LC), and avidly bind Env, KYNU, and a model lipid, cardiolipin. In contrast, extensive LC editing completely purges the (post-tolerance) mature B cell compartment of Env- and KYNU-reactivity, although cardiolipin-reactivity remains mostly intact. Thus, I conclude that tolerization of 2F5 B cells is driven primarily by KYNU-reactivity, and occurs in large part due to LC editing. Remarkably, peripheral anergic B cells (B220+IgM-IgDhi) are enriched for Env- and KYNU-reactivity, and express a restricted repertoire that partially overlaps with that of mature B cells, indicating that peripheral fate determination is at least partly stochastic. The data support that activation of anergic B cells may be a viable route for HIV-1 vaccination.

In the second study, I tested whether transcriptional re-programming can force activated mature B cells to sustain B-cell identity and block terminal differentiation into plasma cells (PCs). Controlling B-cell terminal fate commitment has numerous potential applications in science and medicine, but the means to do so have remained elusive. PC differentiation is governed by a transcription factor network comprising Pax5, Bcl6, and Bach2 – which promote B-cell identity and antagonize PC differentiation – and IRF4 and Blimp1, which cooperate to extinguish B-cell identity and coordinate PC differentiation.

I generated mouse primary B cells harboring gain-of-function in Pax5, Bach2, or Bcl6, or loss-of-function in IRF4 or Blimp1, and then continuously stimulated these B cells with CD154 and IL-21 in vitro. I found that transgenic expression of Bach2 or Bcl6 prohibits PC commitment, maintains markers of B-cell identity, and endows B cells with extraordinary growth potential in response to T-cell help signals. Long-term Bach2-transgenic B cell lines have genetically stable BCRs, express high levels of MHCII and molecules for co-stimulation of T cells, and transduce intracellular signals when incubated with BCR ligands. Silencing the Bach2 transgene in an established cell line causes the cells to secrete large quantities of immunoglobulin. These results provide insight into molecular control over activated B-cell fate, and suggest that enforced expression of Bach2 in vivo may augment germinal center (GC) B cell or memory B cell (MBC) differentiation at the expense of PC commitment. Additionally, culturing Bach2-transgenic B cells has potential applications in monoclonal Ab production, BCR signaling studies, T-cell epitope-mapping studies, and more.

Next, I generated a new knockin mouse harboring a doxycycline-inducible Bach2 transgene and a fluorescent reporter protein, and I used this mouse strain to determine how enforced expression of Bach2 influences the fate of B cells activated in vivo. Constitutive Bach2 expression had no impact on the total number of GC B cells or MBCs in the draining lymph node at 12 days post-immunization, but reduced local PC numbers by ~10-fold. Interestingly, B cells expressing transgenic Bach2 were ~2-fold enriched among GC B cells and MBCs relative to their frequency among mature follicular B cells. Additionally, populations of GC B and MBCs expressing transgenic Bach2 contained significantly higher frequencies of cells brightly stained by fluorescent antigen. As these antigen-bright cells are presumed to have the highest-affinity BCRs, which would normally pre-dispose them toward PC differentiation, I tentatively conclude that enforced expression of Bach2 prevents GC B cells from differentiating into PCs, and instead causes them to remain in GCs or join the MBC pool.

The inducible Bach2-knockin mouse also made it practical to attempt high-throughput isolation of long-lived, monoclonal, antigen-specific B cell lines. By isolating and culturing individual follicular and GC B cells constitutively expressing transgenic Bach2, I generated >20 long-lived cell lines, of which two expressed immunogen-specific BCRs. On withdrawing doxycycline and silencing the Bach2 transgene, all of the cell lines rapidly differentiated into PCs and secreted large quantities of Ig. Thus, inducible Bach2 mice may be an attractive tool for cloning antigen-specific B-cell lines.

Many human monoclonal antibodies that neutralize multiple clades of HIV-1 are polyreactive and bind avidly to mammalian autoantigens. Indeed, the generation of neutralizing antibodies to the 2F5 and 4E10 epitopes of HIV-1 gp41 in man may be proscribed by immune tolerance since mice expressing the VH and VL of 2F5 have an arrested B-cell development characteristic of central tolerance. This developmental blockade implies the presence of tolerizing autoantigens that mitigate effective humoral responses. I hypothesize that discreet human antigens are mimicked by the membrane-proximal external region (MPER) of HIV-1 gp41, and that such mimicry is a wide-spread strategy for HIV-1 to evade immune attacks to its vulnerable neutralizing epitopes.

In the first part of the study, I propose to identify autoantigens mimicked by the 2F5 and 4E10 epitopes. I used immunoprecipitation coupled with mass spectrometry as well as protein arrays to identify the self-antigens recognized by 2F5 and 4E10. The binding of antigens was confirmed using serological assays and targeted mutagenesis was used to map the binding epitope. We identified human kynureninase (KYNU) and splicing factor 3b subunit 3 (SF3B3) as the primary conserved, vertebrate self-antigens recognized by the 2F5 and 4E10 antibodies, respectively. 2F5 binds the H4 domain of KYNU which contains the complete 2F5 linear epitope (ELDKWA). 4E10 recognizes a conformational epitope of SF3B3 that is strongly dependent on hydrophobic interactions. Opossums carry a rare KYNU H4 domain that abolishes 2F5 binding, but retain all SF3B3 4E10 epitopes. Immunization of opossums with HIV-1 gp140 induced extraordinary titers of serum antibody to the 2F5 ELDKWA epitope but little or nothing to the 4E10 determinant.

Our identification of structural motif shared by vertebrates and HIV-1 provides direct evidence that immunological tolerance can impair humoral responses to HIV-1. In the second part of the project, I propose to study the mechanisms of immune tolerance to B cells expressing the 2F5 antibodies. To determine the B cell repertoire before and after tolerance checkpoints, I used the Nojima-Kitamura single B-cell culture that supports differentiation into IgG-secreting plasma cells, even autoreactive cells that are normally subject to tolerization in vivo. I found that the pre-tolerance compartment (small pre-B) from 2F5 KI mice are cells that express the 2F5 V(D)J rearrangements and bind HIV-1 gp41, KYNU, and cardiolipin. Mature, post-tolerance B cells from 2F5 KI mice, however, are purged of gp41- and KYNU-reactivity, but retain cardiolipin-binding, and sequence analysis revealed extensive light-chain editing. The anergic B cells in the post-tolerance compartment are enriched with self-reactivity to KYNU and maintain binding to HIV-1 gp41. Our results demonstrate that tolerance of the 2F5 epitope is driven by specific reactivity to KYNU, but not general cross-reactivity to cardiolipin. In addition, that the peripheral anergic B cells retain self-reactivity and binding to HIV-1 gp41 suggests a potential target for activation by immunizations.

Lastly, we sought to determine whether the host mimicry by 4E10 and 2F5 epitopes is also present in other HIV-1 epitopes, including additional conserved neutralizing epitopes and more importantly, non-neutralizing epitopes. We used protein microarrays to assess autoreactivity of HIV-1 broadly neutralizing antibodies (bnAbs) and non-neutralizing antibodies (nnAbs) and found that as a class, bnAbs are more polyreactive and autoreactive than nnAbs. The poly- and autoreactive property is therefore not a result of chronic inflammation, but rather uniquely associated with neutralization, consistent with the role of heteroligation for HIV-1 neutralizing activity. In addition, mutation frequencies of bNAbs and nnAbs per se do not correlate with poly- and autoreactivity. Our results demonstrate that HIV-1 bnAbs are significantly more polyreactive and self-reactive than non-neutralizers, which may subject them to immunological tolerance control in vivo. Infrequent poly- or autoreactivity among nnAbs implies that their dominance in humoral responses is due to the absence of negative control by immune tolerance.

The results of this study indicate that mimicry of host antigens by HIV-1 is an effective mechanism to camouflage vulnerable neutralizing epitopes of HIV-1 and evade host immune responses. As a result, protective HIV-1 bnAbs are rare and often poly- or autoreactive, constituting a major hurdle that must be overcome to effectively elicit protective responses by an HIV-1 vaccine.

Chemokine receptors are members of the seven transmembrane receptor (7TMR) superfamily and are regulated by the G-protein coupled Receptor Kinase (GRK)/ b-arrestin system. CCL19 and CCL21 are endogenous agonists for the chemokine receptor CCR7. They are known to be equipotent in promoting Gi/o mediated calcium mobilization, chemotaxis and inhibition of adenylyl cyclase activity. Here we test the hypothesis that these ligands are biased agonists that differentially activate the G-protein coupled Receptor Kinase (GRK)/ b-arrestin system.

In order to test whether these ligands have distinct activity, murine T lymphocytes were used to compare the effects of CCL19 and CCL21 activation of CCR7 at endogenous expression levels. While each ligand stimulates similar chemotactic responses, we also find that CCR7 ligands lead to differential signaling. For instance, CCL19 is markedly more efficacious than CCL21 for the activation of ERK and JNK, but not AKT in these cells. Furthermore, ERK activation and chemotaxis are maintained as separate pathways, also distinguishable by their dependency upon PKC and PI3 kinase, respectively. Thus, CCL19 and CCL21 stimulate equal activation of PI3 kinase, AKT, and chemotaxis, but are in fact biased agonists leading to differential activation of MAP kinase in murine T lymphocytes.

To determine the mechanism of CCR7 ligand bias, we used HEK-293 cells expressing CCR7 to compare the proximate signaling events following CCL19 and CCL21 activation. We found striking differences in the activation of the GRK/ b-arrestin system. CCL19 leads to robust CCR7 phosphorylation and b-arrestin2 recruitment catalyzed by both GRK3 and GRK6 while CCL21 activates GRK6 alone. This differential GRK activation leads to distinct functional consequences. Only CCL19 leads to the recruitment of b-arrestin2-GFP into endocytic vesicles and classical receptor desensitization. In contrast, each agonist is fully capable of signaling to MAP kinase through b-arrestin2 in a GRK6 dependent fashion.

Therefore, CCR7 and its ligands represent a natural example of ligand bias whose mechanism involves differential GRK isoform utilization by CCL19 and CCL21 despite similar G-protein signaling. This study suggests that the GRK signatures of 7TMRs can determine the function of discrete pools of b-arrestin and thus guide its cellular effects.

Toll-like receptor (TLR) signaling pathways have been demonstrated to be important in many aspects of innate and adaptive immunity. Binding of TLRs by their respective ligands initiates signaling cascades which promote a number of cellular responses including, but not limited to, pathogen recognition, co-stimulation, cell maturation and activation and initiation of adaptive immune responses. While many roles for TLRs have been rigorously tested, the exact contributions of these receptors to the quality of B-cell immune responses remain unclear. Specifically, a role for intracellular TLR signaling in B-cell development is one area that remains largely uncharacterized. Endogenous TLRs that recognize viral and microbial DNAs and RNAs are also capable of recognizing self-nucleic acids. B cells, which express TLRs, undergo tolerizing mechanisms in the bone marrow (BM) and periphery to eliminate self-reactive clones. I hypothesize that endogenous TLR recognition of self-nucleic acids during B cell development promotes the elimination of these autoreactive cells from the B cell repertoire.

TLRs are also known to be integral in the subsequent triggering of the adaptive immune response. A series of controversial studies has attempted to determine whether TLR signaling is required for antibody responses to thymus-dependent protein antigens. Initial reports indicated that MyD88 was required for antibody responses to a native protein antigen. However, later studies using haptenated protein antigens did not confirm a requirement for TLR signaling in adjuvant-enhanced antibody responses. In an effort to resolve these discordant results, it was suggested that haptenated protein antigens, unlike native proteins, are fundamentally distinct in that they do not require MyD88 signaling. I hypothesize that antibody and germinal center (GC) responses to native and haptenated proteins are independent of MyD88 signaling. I further propose that the unique immunogenicity ascribed to haptenated proteins is simply immunodominance of hapten, a phenomena observed and described almost 100 years ago.

Deficiency in the global adaptor protein for TLR signaling, myeloid differentiation primary response protein 88 (MyD88), through which all TLRs signal (with the exception of TLR 3), effectively silences the TLR pathway. Utilizing mice deficient in MyD88, I show that TLR signaling through MyD88 mediates central tolerance of B cells. Specifically, crossing the MyD88 deficiency onto a 3H9 autoreactive heavy-chain knock-in rescues the immature (imm) and transitional 1(T1) B cell compartments in the BM. This finding demonstrates the requirement for MyD88 signaling in the removal of autoreactive B cell clones at the first tolerance checkpoint.

I also find that MyD88 signaling is not required for antibody, GC or memory responses to native or haptenated proteins. Additionally, affinity maturation, determined by BCR mutation frequency in GC was comparable between MyD88 deficient and sufficient mice. Both MyD88 deficient and sufficient mice were able to elicit secondary immune responses to native and haptenated proteins. Furthermore, I demonstrate that the MyD88 independent immunogenicity attributed to haptenated protein is a misinterpretation of the established concept of immunodominance- haptenated proteins elicited hapten-specific responses that were approx. 20- to 100-fold greater than to the carrier. Regardless of MyD88 signaling, native proteins elicited significantly less serum Ab than their haptenated forms.

I conclude that TLR signaling through MyD88 mediates tolerance during B cell development in the BM, but is not required for B cell immune responses.

Natural IgM (nIgM) is constitutively present in the serum, where it aids in the early control of viral and bacterial expansions. nIgM also plays a significant role in the prevention of autoimmune disease by promoting the clearance of cellular debris. However, the cells that maintain high titers of nIgM in the circulation had not yet been identified. Several studies have linked serum nIgM with the presence of fetal-lineage B cells, and others have detected IgM secretion directly by B1a cells in various tissues. Nevertheless, a substantial contribution of undifferentiated B1 cells to nIgM titers is doubtful, as the ability to produce large quantities of antibody (Ab) is a function of the phenotype and morphology of differentiated plasma cells (PCs). No direct evidence exists to support the claim that a B1-cell population directly produces the bulk of circulating nIgM. The source of nIgM thus remained uncertain and unstudied.

In the first part of this study, I identified the primary source of nIgM. Using enzyme-linked immunosorbent spot (ELISPOT) assay, I determined that the majority of IgM Ab-secreting cells (ASCs) in naïve mice reside in the bone marrow (BM). Flow cytometric analysis of BM cells stained for intracellular IgM revealed that nIgM ASCs express IgM and the PC marker CD138 on their surface, but not the B1a cell marker CD5. By spinning these cells onto slides and staining them, following isolation by fluorescence-activated cell sorting (FACS), I found that they exhibit the typical morphological characteristics of terminally differentiated PCs. Transfer experiments demonstrated that BM nIgM PCs arise from a progenitor in the peritoneal cavity (PerC), but not isolated PerC B1a, B1b, or B2 cells. Immunoglobulin (Ig) gene sequence analysis and examination of B1-8i mice, which carry an Ig knockin that prohibits fetal B-cell development, indicated that nIgM PCs differentiate from fetal-lineage B cells. BrdU uptake experiments showed that the nIgM ASC compartment contains a substantial fraction of long-lived plasma cells (LLPCs). Finally, I demonstrated that nIgM PCs occupy a survival niche distinct from that used by IgG PCs.

In the second part of this dissertation, I characterized the unique survival niche of nIgM LLPCs, which maintain constitutive high titers of nIgM in the serum. By using genetically deficient or Ab-depleted mice, I found that neither T cells, type 2 innate lymphoid cells, nor mast cells, the three major hematopoietic producers of IL-5, were required for nIgM PC survival in the BM. However, IgM PCs associate strongly with IL-5-expressing BM stromal cells, which support their survival in vitro when stimulated. In vivo neutralization of IL-5 revealed that, like individual survival factors for IgG PCs, IL-5 is not the sole supporter of IgM PCs, but is likely one of several redundant molecules that together ensure uninterrupted signaling. Thus, the long-lived nIgM PC niche is not composed of hematopoietic sources of IL-5, but a stromal cell microenvironment that provides multiple redundant survival signals.

In the final part of my study, I identified and characterized the precursor of nIgM PCs, which I found in the first project to be resident in the PerC, but not a B1a, B1b, or B2 cell. By transferring PerC cells sorted based on expression of CD19, CD5, and CD11b, I found that only the CD19+CD5+CD11b- population contained cells capable of differentiating into nIgM PCs. Transfer of decreasing numbers of unfractionated PerC cells into Rag1 knockouts revealed an order-of-magnitude drop in the rate of serum IgM reconstitution between stochastically sampled pools of 106 and 3x105 PerC cells, suggesting that the CD19+CD5+CD11b- compartment comprises two cell types, and that interaction between the two necessary for nIgM-PC differentiation. By transferring neonatal liver, I determined that the early hematopoietic environment is required for nIgM PC precursors to develop. Using mice carrying a mutation that disturbs cKit expression, I also found that cKit appears to be required at a critical point near birth for the proper development of nIgM PC precursors.

The collective results of these studies demonstrate that nIgM is the product of BM-resident PCs, which differentiate from a PerC B cell precursor distinct from B1a cells, and survive long-term in a unique survival niche created by stromal cells. My work creates a new paradigm by which to understand nIgM, B1 cell, and PC biology.

Normally, neutrophil pools are maintained by "steady-state" granulopoiesis. Infections and inflammation, however, trigger neutrophilias that are supported by a hematopoietic program of accelerated granulopoiesis known as "emergency" granulopoiesis. Steady-state and emergency granulopoiesis are thought to depend on distinct members of the CCAAT enhancer binding protein (C/EBP) family of transcription factors, yet the extracellular cues that determine these developmental pathways are unclear. I hypothesize that inflammation elicits IL-1 which acts directly on hematopoietic progenitor cells for the induction of emergency granulopoiesis. Indeed, IL-1RI-/- mice fail to mount reactive neutrophilias in response to adjuvant-induced inflammation. Analysis of this specific impairment revealed an unanticipated role for IL-1RI in supporting increased proliferation by granulocyte/macrophage progenitors (GMP) and, surprisingly, more primitive multipotent progenitors (MPP) and hematopoietic stem cells (HSC). Whereas IL-1 drives HSC proliferation directly in vitro, inflammation induces comparable rates of proliferation in IL-1RI deficient and -sufficient HSC, MPP, and GMP in mixed chimeric mice. Thus, IL-1RI signals play a necessary, but indirect role in the support of alum-induced neutrophilias by expanding both pluripotent and myeloid progenitor compartments to accelerate granulopoiesis.

The lack of alum-induced neutrophilia in IL-1RI-/- mice is due to defective mobilization of bone marrow (BM) neutrophils and impaired proliferation of hematopoietic stem and progenitor cells (HSPC). Coincident defects in neutrophil mobilization and HSPC proliferation suggest that the trigger for emergency granulopoiesis might be the exhaustion of neutrophil compartments rather than inflammatory inductions of growth factors. Consistent with this hypothesis, non-inflammatory reductions in BM neutrophil numbers elicit granulopoietic responses similar to those induced by adjuvant. Alum mobilizes BM neutrophils via G-CSF, but increased HSPC proliferation results from a density-dependent mechanism that is only partially dependent on G-CSF. Notably, C/EBPβ, thought to be necessary for enhanced generative capacity of BM, is dispensable for increased proliferation of HSPC, but plays a role in the terminal differentiation of neutrophils. These observations indicate that the draining of BM neutrophil pools is sufficient to activate a latent, homeostatic mechanism of accelerated granulopoiesis. I propose a common model for the regulation of neutrophil production that explains both steady-state and emergency granulopoiesis through negative feedback.

The HIV-1 envelope (Env) glycoprotein trimer expressed on the virion surface is the target for both non-neutralizing and neutralizing antibodies (nAbs). In both infection and vaccination, the dominant neutralizing antibody responses are strain-specific, however, during chronic infection, antibodies that can potently neutralize genetically diverse HIV-1 isolates have been identified. These antibodies have been termed broadly neutralizing antibodies (bnAbs). BnAbs target conserved sites on the HIV-1 Env glycoprotein trimer including the membrane proximal external region, the gp120-gp41 interface, the CD4 binding site, V1V2-loop plus glycans, and the V3 loop plus glycans. Passive infusion studies with broadly neutralizing antibodies (bnAbs) of various specificities have been shown to be protective as well as control viral load.

Thus, one component of a protective HIV-1 vaccine will likely require the induction of broad and potent neutralizing antibodies, however, it is not understood how such a response would be elicited through vaccination as these antibodies are rare and restricted by immune tolerance mechanisms due to their unusual features such as extensive somatic hypermutation, presence of a long HCDR3, glycan-reactivity, and poly- and or auto-reactivity. Due to the counter selection of B cells bearing antibodies with these characteristics by the host immune system, bnAb precursor B cells are rare in the B cell repertoire and would likely be difficult to engage through vaccination. As such, vaccinations using HIV-1 Env have induced dominant strain-specific antibody responses, but not broadly neutralizing antibody responses. To efficiently engage and expand bnAb-precursor B cells, production of stable homogeneous immunogens that selectively express bnAb epitopes may be necessary.

Following the Introduction (Chapter 1), Methods (Chapter 2), Chapter 3 of this dissertation describes antibody reagents that are used to characterize recombinantly-produced HIV-1 Env glycoproteins. Chapters 4 and 5 present data relating to the ontogeny of vaccine-induced antibody responses to the first and second variable loops of the Env glycoprotein plus glycan (V1V2-glycan epitope) while Chapters 6 and 7 present data relating to the ontogeny of vaccine-induced antibody responses to the third variable loop plus glycan (V3-glycan epitope).

Chapter 4 of this dissertation describes a study of the antibody responses in rhesus macaques following immunization with a synthetic glycopeptide mimic of the epitope bound by antibodies that target the first and second variable loops plus glycan (V1V2-glycan bnAb epitope). This V1V2 glycopeptide induced robust plasma antibody binding responses to HIV Env that contained the same V1V2 loop sequence as the immunogen and binding of plasma antibodies to HIV Env was dependent on a lysine at position 169 (K169). Dependency on K169 is common for antibodies that target the V1V2 loop, such as the strain-specific antibodies CH58 and CH59, that recognize only a peptidic epitope of the V1V2 loop, but also for V1V2-glycan bnAbs such as CH01 that recognize both the V1V2 loop peptide backbone in addition to glycans at positions N156 and N160). Even though binding of vaccine-induced antibodies was dependent on the presence of K169, no glycan-reactive responses were detected, thus suggesting the presence of strain-specific, peptide-reactive CH58 and CH59-like antibodies.

Moreover, the heavy and light chain sequences of vaccine-induced antibodies were isolated and majority of Env-reactive antibodies paired with the rhesus Vλ3-17 lambda chain gene segment. It has been previously demonstrated that the rhesus Vλ3-17 lambda chain gene segment is the rhesus ortholog to the human Vλ3-10 gene as used by the strain-specific vaccine-induced human antibody, CH59. This is due to a germline-encoded glutamic acid and aspartic acid motif in the antibody HCDR2 loop (ED motif). The ED motif forms a salt bridge with the HIV-1 Env K169 and thus generates germline-encoded HIV-1 Env reactivity. In summary, immunization with the V1V2 glycopeptide minimal immunogen that was designed to target and expand rare V1V2-glycan reactive precursors elicited a dominant antibody response that solely recognized peptide, but not glycan. Thus, these results demonstrate that despite vaccinating with the V1V2-glycopeptide that preferentially expressed the V1V2-glycan epitope, V1V2 antibody responses that recognized only peptide were profoundly immunodominant. As V1V2-glycan bnAbs typically have long HCDR3 loops and long HCDR3-bearing B cells are rare in the peripheral B cell repertoire, the observed results could be due to lack of B cells that recognize the V1V2-glycan epitope. From this work, however, it is unclear if the observed results were due to a deficiency of these types of B cells in the periphery or due to instability of the vaccine immunogen in vivo.

Chapter 5 describes the characterization and immunization of a humanized mouse model bearing the CH01 V1V2-glycan bnAb unmutated common ancestor antibody; i.e. the B cells in these mice bear the CH01 antibody heavy and light chain sequences that were predicted to be germline-encoded prior to somatic hypermutation (germline-reverted). With a long HCDR3 of 24 amino acids, B cells bearing the germline-reverted CH01 heavy and light chain were not deleted in the bone marrow, and peripheral B cell development was comparable to the background strain, C57BL/6, demonstrating that B cells bearing CH01-like antibodies could be responsive to an appropriately designed vaccine immunogen.

Mice bearing only the germline reverted CH01 heavy chain were then immunized with HIV-1 Env. Immunization of these mice expanded B cell populations that were dependent on the presence of the V1V2-loop glycan N160. Furthermore, immunization of these mice elicited neutralization against difficult to neutralize (tier 2) HIV-1 isolates. Following immunization, B cells bearing antibodies whose binding to HIV-1 Env was N160 glycan-dependent were isolated, and heavy and light chain sequences were recovered. Analysis of the recovered heavy and light chains revealed that 18 different murine variable-kappa chain genes paired with the knocked in CH01 UCA and these pairings conferred N160 dependence. However, the knocked CH01 UCA heavy chain sequences were largely unmutated. These data suggest that long HCDR3-bearing B cells that are specific for the V1V2-glycan epitope (dependent on the presence of glycan at N160) are not deleted in the bone marrow and as such, vaccine-elicitation of antibodies targeting the V1V2-glycan epitope in wild-type animals should be feasible. Moreover, these data suggest that a critical first step for the elicitation of V1V2-glycan targeting antibody responses will be expansion of the B cells from which these responses derive, but additional antigenic diversity will likely be required to induce the full neutralization breadth and potency observed by V1V2-glycan bnAbs isolated from HIV-1 infected donors.

Chapter 6 details the characterization of a stable synthetic glycopeptide that was designed to mimic the conformation of the peptide component of the HIV-1 V3-loop plus glycans, termed “Man9-V3.” Broadly neutralizing antibodies that recognize this epitope, termed “V3-glycan bnAbs” bound to Man9-V3 glycopeptide and bound with affinities comparable to those observed for native-like gp140 Env trimers. Moreover, both fluorophore-labeled Man9-V3 and native-like trimers similarly bound to bnAb memory B cells, and by flow sorting, members of a V3-glycan bnAb clonal lineage from an HIV-1-infected individual were isolated. Thus, these data suggest that Man9-V3 glycopeptide is a structural mimic of the HIV-1 Env epitope bound by V3-glycan bnAbs and is a candidate immunogen to initiate V3-glycan bnAb lineage maturation.

In Chapter 7, the immunogenicity of Man9-V3 glycopeptide was tested in rhesus macaques. Using Man9-V3 as an immunogen, V3-glycan antibody responses were elicited. Combining flow sorting with next generation sequencing of immunoglobulin genes, the ontogeny of a vaccine-elicited V3-glycan antibody lineage, termed DH717 was studied. This lineage targeted the base of the HIV-1 V3-loop in addition to the V3-loop N301 and N332 glycans. Neutralization however, was limited to Env pseudoviruses bearing only high-mannose glycans as these antibodies could not neutralize viruses bearing native glycoforms. The structure of the most broad and potent member of the DH717 lineage, DH717.1 was determined using X-ray crystallography. This revealed that the rhesus DH717.1 V3-glycan antibody and 2G12, a human V3-glycan bnAb isolated from a HIV-1 infected donor, showed remarkable similarity in accommodation of high-mannose glycans. Specifically, DH717.1 and 2G12 antibodies accommodate terminal branches of high-mannose glycans through the formation of a binding pocket comprised of the HCDR1 and HCDR2 loops. Furthermore, DH717, like 2G12, bound the yeast Candida albicans in a glycan-dependent manner.

With regards to the ontogeny of the V3-glycan DH717 lineage, next generation sequencing at pre-immunization time points revealed the DH717 lineage to be present prior to vaccination and to be mutated with regards to the computationally inferred DH717 germline sequence. While the already mutated pre-vaccination DH717 lineage member bound to both the yeast Candida albicans, and to Man9-V3 glycopeptide, the DH717 unmutated common ancestor bearing a computationally inferred germline sequence bound only to Candida albicans, and not Man9-V3. It was only after acquiring somatic mutations prior to immunization did the lineage acquire the ability to bind to Man9-V3, suggesting a role for high-mannose-bearing environmental antigens for priming such responses. After further acquisition of somatic mutations following vaccination did the lineage acquire the ability to bind the V3-glycan bnAb epitope as presented on a stabilized, native-like soluble recombinant HIV-1 Env trimer. This suggests that vaccination with a synthetic glycopeptide affinity matured a pre-existing, yeast-reactive B cell lineage to the HIV-1 V3-glycan bnAb epitope.

Together, the studies described in Chapters 4-7 suggest that vaccine-induction of V1V2- and V3-glycan bnAbs may be feasible, and that to do so, stable glycopeptides that mimic bnAb epitopes will be needed to select for and expand the precursors for the desired glycan-bnAb response.