Brentuximab Vedotin-Driven Microtubule Disruption Results in Endoplasmic Reticulum Stress Leading to Immunogenic Cell Death and Antitumor Immunity.

Brentuximab vedotin, a CD30-directed antibody-drug conjugate (ADC), is approved for clinical use in multiple CD30-expressing lymphomas. The cytotoxic payload component of brentuximab vedotin is monomethyl auristatin E (MMAE), a highly potent microtubule-disrupting agent. Preclinical results provided here demonstrate that treatment of cancer cells with brentuximab vedotin or free MMAE leads to a catastrophic disruption of the microtubule network eliciting a robust endoplasmic reticulum (ER) stress response that culminates in the induction of the classic hallmarks of immunogenic cell death (ICD). In accordance with the induction of ICD, brentuximab vedotin-killed lymphoma cells drove innate immune cell activation in vitro and in vivo. In the "gold-standard" test of ICD, vaccination of mice with brentuximab vedotin or free MMAE-killed tumor cells protected animals from tumor rechallenge; in addition, T cells transferred from previously vaccinated animals slowed tumor growth in immunodeficient mice. Immunity acquired from killed tumor cell vaccination was further amplified by the addition of PD-1 blockade. In a humanized model of CD30+ B-cell tumors, treatment with brentuximab vedotin drove the expansion and recruitment of autologous Epstein-Barr virus-reactive CD8+ T cells potentiating the activity of anti-PD-1 therapy. Together, these data support the ability of brentuximab vedotin and MMAE to drive ICD in tumor cells resulting in the activation of antigen-presenting cells and augmented T-cell immunity. These data provide a strong rationale for the clinical combination of brentuximab vedotin and other MMAE-based ADCs with checkpoint inhibitors.

Nonfucosylation of an anti-TIGIT antibody enhances FcγR engagement, driving innate immune activation and antitumor activity.

TIGIT is an immune checkpoint receptor expressed on activated and memory T cells, immunosuppressive T regulatory cells, and natural killer (NK) cells. TIGIT has emerged as an attractive target for antitumor therapies, due to its proposed immunosuppressive effects on lymphocyte function and T cell activation. We generated an anti-TIGIT monoclonal antibody (mAb) that binds with high affinity to human, non-human primate, and murine TIGIT and through multiple experimental methodologies demonstrated that checkpoint blockade alone is insufficient for antitumor activity. Generating anti-TIGIT mAbs with various Fc backbones we show that muting the Fc-Fcγ receptor (FcγR) interaction failed to drive antitumor activity, while mAbs with Fc functional backbones demonstrate substantial antitumor activity, mediated through activation of antigen-presenting cells (APCs), T cell priming, and NK-mediated depletion of suppressive Tregs and exhausted T cells. Further, nonfucosylation of the Fc backbone resulted in enhanced immune responses and antitumor activity relative to the intact IgG1 backbone. The improved activity correlated with the biased FcγR interaction profile of the nonfucosylated anti-TIGIT mAb, which supports that FcγRIIIa binding with decreased FcγRIIb binding favorably activates APCs and enhances tumor-specific CD8+ T cell responses. The anti-TIGIT mAbs with intact FcγR interacting backbones also demonstrated synergistic enhancement of other standard antitumor treatments, including anti-PD-1 treatment and a model monomethyl auristatin E antibody-drug conjugate. These findings highlight the importance of the anti-TIGIT mAb's Fc backbone to its antitumor activity and the extent to which this activity can be enhanced through nonfucosylation of the backbone.

γδ T Cells Shape Preimmune Peripheral B Cell Populations.

We previously reported that selective ablation of certain γδ T cell subsets, rather than removal of all γδ T cells, strongly affects serum Ab levels in nonimmunized mice. This type of manipulation also changed T cells, including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4(+) and Vγ6(+) γδ T cells (B6.TCR-Vγ4(-/-)/6(-/-)), we observed expanded Vγ1(+) cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αß T cells as well as spontaneous germinal center formation in the spleen, and elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4(-/-)/6(-/-) mice and other peripheral B cell populations were diminished, most of all splenic marginal zone (MZ) B cells. However, relative frequencies and absolute numbers of Ab-producing cells, as well as serum levels of Abs, IL-4, and BAFF, were increased. Cell transfers confirmed that these changes are directly dependent on the altered γδ T cells in this strain and on their enhanced potential of producing IL-4. Further evidence suggests the possibility of direct interactions between γδ T cells and B cells in the splenic MZ. Taken together, these data demonstrate the capability of γδ T cells of modulating size and productivity of preimmune peripheral B cell populations.

γδ T cells affect IL-4 production and B-cell tolerance.

γδ T cells can influence specific antibody responses. Here, we report that mice deficient in individual γδ T-cell subsets have altered levels of serum antibodies, including all major subclasses, sometimes regardless of the presence of αß T cells. One strain with a partial γδ deficiency that increases IgE antibodies also displayed increases in IL-4-producing T cells (both residual γδ T cells and αß T cells) and in systemic IL-4 levels. Its B cells expressed IL-4-regulated inhibitory receptors (CD5, CD22, and CD32) at diminished levels, whereas IL-4-inducible IL-4 receptor α and MHCII were increased. They also showed signs of activation and spontaneously formed germinal centers. These mice displayed IgE-dependent features found in hyper-IgE syndrome and developed antichromatin, antinuclear, and anticytoplasmic autoantibodies. In contrast, mice deficient in all γδ T cells had nearly unchanged Ig levels and did not develop autoantibodies. Removing IL-4 abrogated the increases in IgE, antichromatin antibodies, and autoantibodies in the partially γδ-deficient mice. Our data suggest that γδ T cells, controlled by their own cross-talk, affect IL-4 production, B-cell activation, and B-cell tolerance.

Antigen-specific suppression of humoral immunity by anergic Ars/A1 B cells.

Autoreactive anergic B lymphocytes are considered to be dangerous because of their potential for activation and recruitment into autoimmune responses. However, they persist for days and constitute ∼5% of the B cell pool. We assessed their functional potential in the Ars/A1 transgene model, where anergic B cells express a dual-reactive Ag receptor that binds, in addition to a self-Ag, the hapten p-azophenylarsonate (Ars). When Ars/A1 B cells were transferred into adoptive recipients that were immunized with foreign proteins covalently conjugated with Ars, endogenous IgG immune responses to both were selectively and severely diminished, and the development of T helper cells was impaired. Approximately 95% inhibition of the anti-Ars response was attained with ∼4000 transferred Ars/A1 B cells through redundant mechanisms, one of which depended on their expression of MHC class II but not upon secretion of IL-10 or IgM. This Ag-specific suppressive activity implicates the autoreactive anergic B cell as an enforcer of immunological tolerance to self-Ags.

Self-tolerance checkpoints in CD4 T cells specific for a peptide derived from the B cell antigen receptor.

Linked recognition of Ag by B and T lymphocytes is ensured in part by a state of tolerance acquired by CD4 T cells to germline-encoded sequences within the B cell Ag receptor (BCR). We sought to determine how such tolerance is attained when a peptide from the BCR variable (V) region is expressed by small numbers of B cells as it is in the physiological state. Mixed bone marrow (BM) chimeras were generated using donor BM from mice with B cells that expressed a transgene (Tg)-encoded κ L chain and BM from TCR Tg mice in which the CD4 T cells (CA30) were specific for a Vκ peptide encoded by the κTg. In chimeras where few B cells express the κTg, many CA30 cells were deleted in the thymus. However, a substantial fraction survived to the CD4 single-positive stage. Among single-positive CA30 thymocytes, few reached maturity and migrated to the periphery. Maturation was strongly associated with, and likely promoted by, expression of an endogenous TCR α-chain. CD4(+) CA30 cells that reached peripheral lymphoid tissues were Ag-experienced and anergic, and some developed into regulatory cells. These findings reveal several checkpoints and mechanisms that enforce a state of self-tolerance in developing T cells specific for BCR V region sequences, thus ensuring that T cell help to B cells occurs through linked recognition of foreign Ag.

Aborted germinal center reactions and B cell memory by follicular T cells specific for a B cell receptor V region peptide.

A fundamental problem in immunoregulation is how CD4(+) T cells react to immunogenic peptides derived from the V region of the BCR that are created by somatic mechanisms, presented in MHC II, and amplified to abundance by B cell clonal expansion during immunity. BCR neo Ags open a potentially dangerous avenue of T cell help in violation of the principle of linked Ag recognition. To analyze this issue, we developed a murine adoptive transfer model using paired donor B cells and CD4 T cells specific for a BCR-derived peptide. BCR peptide-specific T cells aborted ongoing germinal center reactions and impeded the secondary immune response. Instead, they induced the B cells to differentiate into short-lived extrafollicular plasmablasts that secreted modest quantities of Ig. These results uncover an immunoregulatory process that restricts the memory pathway to B cells that communicate with CD4 T cells via exogenous foreign Ag.

Somatic hypermutation as a generator of antinuclear antibodies in a murine model of systemic autoimmunity.

Systemic lupus erythematosus (SLE) is characterized by high-avidity IgG antinuclear antibodies (ANAs) that are almost certainly products of T cell-dependent immune responses. Whether critical amino acids in the third complementarity-determining region (CDR3) of the ANA originate from V(D)J recombination or somatic hypermutation (SHM) is not known. We studied a mouse model of SLE in which all somatic mutations within ANA V regions, including those in CDR3, could be unequivocally identified. Mutation reversion analyses revealed that ANA arose predominantly from nonautoreactive B cells that diversified immunoglobulin genes via SHM. The resolution afforded by this model allowed us to demonstrate that one ANA clone was generated by SHM after a V(H) gene replacement event. Mutations producing arginine substitutions were frequent and arose largely (66%) from base changes in just two codons: AGC and AGT. These codons are abundant in the repertoires of mouse and human V genes. Our findings reveal the predominant role of SHM in the development of ANA and underscore the importance of self-tolerance checkpoints at the postmutational stage of B cell differentiation.

Developmental, molecular, and genetic dissection of INa in vivo in embryonic zebrafish sensory neurons.

The presence of multiple Nav1 isotypes within a neuron and the lack of specific blockers hamper identification of the in vivo roles of sodium current (INa) components, especially during embryonic stages. To identify the functional properties of INa components in vivo in developing neurons, we took a molecular genetic approach. Embryonic zebrafish Rohon-Beard (RB) mechanosensory neurons express two different sodium channel isotypes: Nav1.1 and Nav1.6. To examine the properties of Nav1.1- and Nav1.6-encoded currents in RB cells at different developmental stages, we eliminated the contribution of Nav1.6 and Nav1.1 channels, respectively, using an antisense morpholino (MO) approach. MOs were injected into one-cell stage embryos, and RB sodium currents were recorded using patch-clamp techniques in both conventional whole cell mode as well from nucleated patches. Only a subset of RB cells appeared to be affected by the Nav1.1MO. Overall, the effect of the Nav1.1MO was a small 25% average reduction in current amplitude. Further, Nav1.1MO effects were most pronounced in RB cells of younger embryos. In contrast, the effects of the Nav1.6 MO were observed in all cells and increased as development proceeded. These results indicated that developmental upregulation of RB INa entailed an increase in the number of functional Nav1.6 channels. In addition, analysis of voltage-dependent steady-state activation and inactivation parameters revealed that specific functional properties of channels were also developmentally regulated. Finally, analysis of macho mutants indicated that developmental upregulation of INa was absent in RB cells. These results indicate that MOs are a useful tool for the molecular dissection and analysis of ion channel function in vivo.

Activation and tolerance in CD4(+) T cells reactive to an immunoglobulin variable region.

Antibody diversity creates an immunoregulatory challenge for T cells that must cooperate with B cells, yet discriminate between self and nonself. To examine the consequences of T cell reactions to the B cell receptor (BCR), we generated a transgenic (Tg) line of mice expressing a T cell receptor (TCR) specific for a kappa variable region peptide in monoclonal antibody (mAb) 36-71. The kappa epitope was originally generated by a pair of somatic mutations that arose naturally during an immune response. By crossing this TCR Tg mouse with mice expressing the kappa chain of mAb 36-71, we found that kappa-specific T cells were centrally deleted in thymi of progeny that inherited the kappaTg. Maternally derived kappaTg antibody also induced central deletion. In marked contrast, adoptive transfer of TCR Tg T cells into kappaTg recipients resulted in T and B cell activation, lymphadenopathy, splenomegaly, and the production of IgG antichromatin antibodies by day 14. In most recipients, autoantibody levels increased with time, Tg T cells persisted for months, and a state of lupus nephritis developed. Despite this, Tg T cells appeared to be tolerant as assessed by severely diminished proliferative responses to the Vkappa peptide. These results reveal the importance of attaining central and peripheral T cell tolerance to BCR V regions. They suggest that nondeletional forms of T tolerance in BCR-reactive T cells may be insufficient to preclude helper activity for chromatin-reactive B cells.