Biophysical Properties of Self-Assembled Immune Signals Impact Signal Processing and the Nature of Regulatory Immune Function.

Outcomes during immunotherapy are impacted not only by the specific therapeutic signals and pharmacodynamics, but also by the biophysical forms in which signals are delivered. This integration is determinative in autoimmunity because the disease is caused by immune dysregulation and inflammation. Unfortunately, the links between nanomaterial design, biophysical properties, and immune regulation are poorly defined. Here we designed cationic peptide antigens with defined charge distributions and then used electrostatics to assemble these peptides into complexes with anionic regulatory cues. We first show complexes induce antigen-specific tolerance during myelin-driven autoimmunity. We next show the affinity between these immune cues is controlled by charge balance and that affinity confers distinct biophysical properties important in immunological processing, including antigen availability. The underlying binding affinities between the self-assembled signals influences inflammatory gene expression in dendritic cells and antigen-specific regulatory outcomes in self-reactive transgenic T cells. This granular understanding of nanomaterial-immune interactions contributes to a more rational immunotherapy design.

Self-Assembly as a Molecular Strategy to Improve Immunotherapy.

Immunotherapies harness an individual's immune system to battle diseases such as cancer and autoimmunity. During cancer, the immune system often fails to detect and destroy cancerous cells, whereas during autoimmune disease, the immune system mistakenly attacks self-tissue. Immunotherapies can help guide more effective responses in these settings, as evidenced by recent advances with monoclonal antibodies and adoptive cell therapies. However, despite the transformative gains of immunotherapies for patients, many therapies are not curative, work only for a small subset of patients, and lack specificity in distinguishing between healthy and diseased cells, which can cause severe side effects. From this perspective, self-assembled biomaterials are promising technologies that could help address some of the limitations facing immunotherapies. For example, self-assembly allows precision control over the combination and relative concentration of immune cues and directed cargo display densities. These capabilities support selectivity and potency that could decrease off-target effects and enable modular or personalized immunotherapies. The underlying forces driving self-assembly of most systems in aqueous solution result from hydrophobic interactions or charge polarity. In this Account, we highlight how these forces are being used to self-assemble immunotherapies for cancer and autoimmune disease.Hydrophobic interactions can create a range of intricate structures, including peptide nanofibers, nanogels, micelle-like particles, and in vivo assemblies with protein carriers. Certain nanofibers with hydrophobic domains uniquely benefit from the ability to elicit immune responses without additional stimulatory signals. This feature can reduce nonspecific inflammation but may also limit the nanofiber's application because of their inherent stimulatory properties. Micelle-like particles have been developed with the ability to incorporate a range of tumor-specific antigens for immunotherapies in mouse models of cancer. Key observations have revealed that both the total dose of antigen and display density of antigen per particle can impact immune response and efficacy of immunotherapies. These developments are promising benchmarks that could reveal design principles for engineering more specific and personalized immunotherapies.There has also been extensive work to develop platforms using electrostatic interactions to drive assembly of oppositely charged immune signals. These strategies benefit from the ability to tune biophysical interactions between components by altering the ratio of cationic to anionic charge during formulation, or the density of charge. Using a layer-by-layer assembly method, our lab developed hollow capsules composed entirely of immune signals for therapies in cancer and autoimmune disease models. This platform allowed for 100% of the immunotherapy to be composed of immune signals and completely prevents the onset of disease in a mouse model of multiple sclerosis. Layer-by-layer assembly has also been used to coat microneedle patches to target signals to immune cells in the dermal layer. As an alternative to layer-by-layer assembly, one step assembly can be achieved by mixing cationic and anionic components in solution. Additional approaches have created molecular structures that leverage hydrogen bonding for self-assembly. The creativity of engineered self-assembly has led to key insights that could benefit future immunotherapies and revealed aspects that require further study. The challenge now remains to utilize these insights to push development of new immunotherapeutics into clinical settings.

H3.3K4M destabilizes enhancer H3K4 methyltransferases MLL3/MLL4 and impairs adipose tissue development.

Histone 3 lysine 4 (H3K4) methyltransferases MLL3 and MLL4 (MLL3/4) are required for enhancer activation during cell differentiation, though the mechanism is incompletely understood. We have attempted to address this issue by generating two mouse lines: one expressing H3.3K4M, a lysine-4-to-methionine (K4M) mutation of histone H3.3 that inhibits H3K4 methylation, and the other carrying conditional double knockout of MLL3/4 enzymatic SET domain. Expression of H3.3K4M in lineage-specific precursor cells depletes H3K4 methylation and impairs adipose tissue and muscle development. Mechanistically, H3.3K4M prevents enhancer activation in adipogenesis by destabilizing MLL3/4 proteins but not other Set1-like H3K4 methyltransferases MLL1, MLL2, SET1A and SET1B. Notably, deletion of the enzymatic SET domain in lineage-specific precursor cells mimics H3.3K4M expression, destabilizes MLL3/4 proteins, and prevents adipose tissue and muscle development. Interestingly, destabilization of MLL3/4 by H3.3K4M in adipocytes does not affect adipose tissue maintenance and thermogenic function. Together, our findings indicate that expression of H3.3K4M, or deletion of the enzymatic SET domain, destabilizes enhancer H3K4 methyltransferases MLL3/4 and impairs adipose tissue and muscle development.

Tumor immunogenicity dictates reliance on TCF1 in CD8+ T cells for response to immunotherapy.

Stem-like CD8+ T cells are regulated by T cell factor 1 (TCF1) and are considered requisite for immune checkpoint blockade (ICB) response. However, recent findings indicate that reliance on TCF1+CD8+ T cells for ICB efficacy may differ across tumor contexts. We find that TCF1 is essential for optimal priming of tumor antigen-specific CD8+ T cells and ICB response in poorly immunogenic tumors that accumulate TOX+ dysfunctional T cells, but is dispensable for T cell priming and therapy response in highly immunogenic tumors that efficiently expand transitory effectors. Importantly, improving T cell priming by vaccination or by enhancing antigen presentation on tumors rescues the defective responses of TCF1-deficient CD8+ T cells upon ICB in poorly immunogenic tumors. Our study highlights TCF1's role during the early stages of anti-tumor CD8+ T cell responses with important implications for guiding optimal therapeutic interventions in cancers with low TCF1+CD8+ T cells and low-neo-antigen expression.

Self-Assembly of Immune Signals to Program Innate Immunity through Rational Adjuvant Design.

Recent clinical studies show activating multiple innate immune pathways drives robust responses in infection and cancer. Biomaterials offer useful features to deliver multiple cargos, but add translational complexity and intrinsic immune signatures that complicate rational design. Here a modular adjuvant platform is created using self-assembly to build nanostructured capsules comprised entirely of antigens and multiple classes of toll-like receptor agonists (TLRas). These assemblies sequester TLR to endolysosomes, allowing programmable control over the relative signaling levels transduced through these receptors. Strikingly, this combinatorial control of innate signaling can generate divergent antigen-specific responses against a particular antigen. These assemblies drive reorganization of lymph node stroma to a pro-immune microenvironment, expanding antigen-specific T cells. Excitingly, assemblies built from antigen and multiple TLRas enhance T cell function and antitumor efficacy compared to ad-mixed formulations or capsules with a single TLRa. Finally, capsules built from a clinically relevant human melanoma antigen and up to three TLRa classes enable simultaneous control of signal transduction across each pathway. This creates a facile adjuvant design platform to tailor signaling for vaccines and immunotherapies without using carrier components. The modular nature supports precision juxtaposition of antigen with agonists relevant for several innate receptor families, such as toll, STING, NOD, and RIG.

Exploiting Rational Assembly to Map Distinct Roles of Regulatory Cues during Autoimmune Therapy.

Autoimmune diseases like multiple sclerosis (MS), type 1 diabetes, and lupus occur when the immune system attacks host tissue. Immunotherapies that promote selective tolerance without suppressing normal immune function are of tremendous interest. Here, nanotechnology was used for rational assembly of peptides and modulatory immune cues into immune complexes. Complexes containing self-peptides and regulatory nucleic acids reverse established paralysis in a preclinical MS model. Importantly, mice responding to immunotherapy maintain healthy, antigen-specific B and T cell responses during a foreign antigen challenge. A therapeutic library isolating specific components reveals that regulatory nucleic acids suppress inflammatory genes in innate immune cells, while disease-matched peptide sequences control specificity of tolerance. Distinct gene expression profiles in cells and animals are associated with the immune signals administered in particulate and soluble forms, highlighting the impact of biophysical presentation of signals. This work provides insight into the rational manipulation of immune signaling to drive tolerance.

Depletion of Nsd2-mediated histone H3K36 methylation impairs adipose tissue development and function.

The epigenetic mechanisms regulating adipose tissue development and function are poorly understood. In this study, we show that depletion of histone H3K36 methylation by H3.3K36M in preadipocytes inhibits adipogenesis by increasing H3K27me3 to prevent the induction of C/EBPα and other targets of the master adipogenic transcription factor peroxisome proliferator-activated receptor-γ (PPARγ). Depleting H3K36 methyltransferase Nsd2, but not Nsd1 or Setd2, phenocopies the effects of H3.3K36M on adipogenesis and PPARγ target expression. Consistently, expression of H3.3K36M in progenitor cells impairs brown adipose tissue (BAT) and muscle development in mice. In contrast, depletion of histone H3K36 methylation by H3.3K36M in adipocytes in vivo does not affect adipose tissue weight, but leads to profound whitening of BAT and insulin resistance in white adipose tissue (WAT). These mice are resistant to high fat diet-induced WAT expansion and show severe lipodystrophy. Together, these results suggest a critical role of Nsd2-mediated H3K36 methylation in adipose tissue development and function.

Histone H3 lysine 4 methyltransferase KMT2D.

Histone-lysine N-methyltransferase 2D (KMT2D), also known as MLL4 and MLL2 in humans and Mll4 in mice, belongs to a family of mammalian histone H3 lysine 4 (H3K4) methyltransferases. It is a large protein over 5500 amino acids in size and is partially functionally redundant with KMT2C. KMT2D is widely expressed in adult tissues and is essential for early embryonic development. The C-terminal SET domain is responsible for its H3K4 methyltransferase activity and is necessary for maintaining KMT2D protein stability in cells. KMT2D associates with WRAD (WDR5, RbBP5, ASH2L, and DPY30), NCOA6, PTIP, PA1, and H3K27 demethylase UTX in one protein complex. It acts as a scaffold protein within the complex and is responsible for maintaining the stability of UTX. KMT2D is a major mammalian H3K4 mono-methyltransferase and co-localizes with lineage determining transcription factors on transcriptional enhancers. It is required for the binding of histone H3K27 acetyltransferases CBP and p300 on enhancers, enhancer activation and cell-type specific gene expression during differentiation. KMT2D plays critical roles in regulating development, differentiation, metabolism, and tumor suppression. It is frequently mutated in developmental diseases, such as Kabuki syndrome and congenital heart disease, and various forms of cancer. Further understanding of the mechanism through which KMT2D regulates gene expression will reveal why KMT2D mutations are so harmful and may help generate novel therapeutic approaches.