Browsing by Subject "Tolerance"

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.

Plants commonly produce multiple, seemingly redundant defenses, but the reasons for this are poorly understood. The specificity of defenses to particular herbivores could drive investment in multiple defenses. Alternatively, genetic correlations between defenses could lead to their joint expression, even if possessing both defenses is non-adaptive. Plants may produce multiple defenses if putative resistance traits do not reduce damage, forcing plants to rely on tolerance of damage instead. Furthermore, resource shortages caused by herbivore damage could lead to compensatory changes in expression and selection on non-defense traits, such as floral traits. Natural selection could favor producing multiple defenses if synergism between defenses increases the benefits or decrease the costs of producing multiple defenses. Non-linear relationships between the costs and benefits of defense trait investment could also favor multiple defenses.

Passiflora incarnata (`maypop') is a perennial vine native to the southeast United States that produces both direct, physical traits (leaf toughness and trichomes) and rewards thought to function in indirect defense (extrafloral nectar in a defense mutualism with ants), along with tolerance of herbivore damage. I performed two year-long common garden experiments with clonal replicates of plants originating from two populations. I measured plant fitness, herbivore damage, and defense traits. I ran a genotypic selection analysis to determine if manipulating herbivore damage through a pesticide exclusion treatment presence mediated selection on floral traits, and if herbivore damage led to plastic changes in floral trait expression. To evaluate the role of selection in maintaining multiple defenses, I estimated fitness surfaces for pairwise combinations of defense traits and evaluated where the fitness optima were on each surface.

I found that resistance traits did not reduce herbivore damage, but plants demonstrated specific tolerance to different classes of herbivore damage. Tolerance was negatively correlated with resistance, raising the possibility that tolerance of herbivore damage instead of resistance may be the key defense in this plant, and that production of the two type of defense is constrained by underlying genetic architecture. Plants with higher levels of generalist beetle damage flowered earlier and produced proportionally more male flowers. I found linear selection for both earlier flowering and a lower proportion of male flowers in the herbivore exclusion treatment. I found that selection favored investment in multiple resistance traits. However, for two tolerance traits or one resistance and one tolerance trait, investment in only one trait was favored.

These results highlight the possibility of several mechanisms selecting for the expression of multiple traits, including non-defense traits. Resistance traits may have a non-defensive primary function in this plant, and tolerance may instead be a key defense strategy. These results also emphasize the need to consider the type of trait--resistance or tolerance--when making broad predictions about their joint expression.

Tolerance is considered foundational for a multicultural society to defuse tensions over race, religion, and sexuality. However, critics of tolerance point out that its reliance on the consent of the majority to extend equal rights to a minority, along with its liberal method of individualizing prejudice, does not result in equality. This project historicizes tolerance by examining the trajectory of its adoption by leftist political parties in Mexico to address concerns over sexual identity and difference. It demonstrates that the embrace of tolerance was not only a political strategy for electoral gain, but also a method to maintain a masculinist party. By endorsing a policy of tolerance through the expansion of the principle of private life, leftist parties claimed solidarity with the feminist and sexual liberation movement rather than engage with their criticisms of the heterosexism of leftist militancy.

Issues of sexuality, particularly homosexual and reproductive rights, were in an uneasy, if not antagonistic, relationship with the revolutionary politics of left-wing organizations such as the Mexican Communist Party (PCM) since their foundation. However, between 1976 and 1981, leftist parties shifted their stances. Adopting a policy of tolerance, party leaders hoped to reconcile the growing lesbian, gay and feminist movements with their rank and file because these social movements provided the potential votes that could launch the Left out of electoral obscurity. Revolution in the Sheets traces the limits and outcomes of this strategy. Tolerance did little to stem homophobia or sexism among leftists in Mexico. Furthermore, militants rejected the tolerance policy because sexual politics were the primary outlet for rank and file leftists to dispute intra-party tensions, vocalize intimate grievances, and distinguish themselves from one another for political gain. In the end, the shift to tolerance – a defining feature of the conflicts over the cultural turns that marked the last decades of the twentieth century – was a contingent product of intimate feuds, electoral strategy, and interpersonal relationships.