The HPK1 Inhibitor A-745 Verifies the Potential of Modulating T Cell Kinase Signaling for Immunotherapy.

Hematopoietic progenitor kinase 1 (HPK1) is an MAP4K family member within the Ste20-like serine/threonine branch of the kinome. HPK1 expression is limited to hematopoietic cells and has a predominant role as a negative regulator of T cell function. Because of the central/dominant role in negatively regulating T cell function, HPK1 has long been in the center of interest as a potential pharmacological target for immune therapy. The development of a small molecule HPK1 inhibitor remains challenging because of the need for high specificity relative to other kinases, including additional MAP4K family members, that are required for efficient immune cell activation. Here, we report the identification of the selective and potent HPK1 chemical probe, A-745. In unbiased cellular kinase-binding assays, A-745 demonstrates an excellent cellular selectivity binding profile within pharmacologically relevant concentrations. This HPK1 selectivity translates to an in vitro immune cell activation phenotype reminiscent of Hpk1-deficient and Hpk1-kinase-dead T cells, including augmented proliferation and cytokine production. The results from this work give a path forward for further developmental efforts to generate additional selective and potent small molecule HPK1 inhibitors with the pharmacological properties for immunotherapy.

Aire Enforces Immune Tolerance by Directing Autoreactive T Cells into the Regulatory T Cell Lineage.

The promiscuous expression of tissue-restricted antigens in the thymus, driven in part by autoimmune regulator (Aire), is critical for the protection of peripheral tissues from autoimmune attack. Aire-dependent processes are thought to promote both clonal deletion and the development of Foxp3(+) regulatory T (Treg) cells, suggesting that autoimmunity associated with Aire deficiency results from two failed tolerance mechanisms. Here, examination of autoimmune lesions in Aire(-/-) mice revealed an unexpected third possibility. We found that the predominant conventional T cell clonotypes infiltrating target lesions express antigen receptors that were preferentially expressed by Foxp3(+) Treg cells in Aire(+/+) mice. Thus, Aire enforces immune tolerance by ensuring that distinct autoreactive T cell specificities differentiate into the Treg cell lineage; dysregulation of this process results in the diversion of Treg cell-biased clonotypes into pathogenic conventional T cells.

Dendritic Cells Coordinate the Development and Homeostasis of Organ-Specific Regulatory T Cells.

Although antigen recognition mediated by the T cell receptor (TCR) influences many facets of Foxp3(+) regulatory T (Treg) cell biology, including development and function, the cell types that present antigen to Treg cells in vivo remain largely undefined. By tracking a clonal population of Aire-dependent, prostate-specific Treg cells in mice, we demonstrated an essential role for dendritic cells (DCs) in regulating organ-specific Treg cell biology. We have shown that the thymic development of prostate-specific Treg cells required antigen presentation by DCs. Moreover, Batf3-dependent CD8α(+) DCs were dispensable for the development of this clonotype and had negligible impact on the polyclonal Treg cell repertoire. In the periphery, CCR7-dependent migratory DCs coordinated the activation of organ-specific Treg cells in the prostate-draining lymph nodes. Our results demonstrate that the development and peripheral regulation of organ-specific Treg cells are dependent on antigen presentation by DCs, implicating DCs as key mediators of organ-specific immune tolerance.

Shaping the repertoire of tumor-infiltrating effector and regulatory T cells.

Many tumors express antigens that can be specifically or selectively recognized by T lymphocytes, suggesting that T-cell-mediated immunity may be harnessed for the immunotherapy of cancer. However, since tumors originate from normal cells and evolve within the context of self-tissues, the immune mechanisms that prevent the autoimmune attack of normal tissues function in parallel to restrict anti-tumor immunity. In particular, the purging of autoreactive T cells and the development of immune-suppressive regulatory T cells (Tregs) are thought to be major barriers impeding anti-tumor immune responses. Here, we discuss current understanding regarding the antigens recognized by tumor-infiltrating T-cell populations, the mechanisms that shape the repertoire of these cells, and the role of the transcription factor autoimmune regulator (Aire) in these processes. Further elucidation of these principles is likely to be critical for optimizing emerging cancer immunotherapies, and for the rational design of novel therapies exhibiting robust anti-tumor activity with limited toxicity.

Organ-specific regulatory T cells of thymic origin are expanded in murine prostate tumors.

Little is known about the relative contributions of self-specific regulatory T cells (Tregs) of thymic origin and induced Tregs generated extrathymically to the pool of tumor-infiltrating Tregs. We have recently demonstrated that thymic-derived Tregs reactive to a prostate-associated self antigen are highly and recurrently enriched within oncogene-driven murine prostate cancers.

Aire-dependent thymic development of tumor-associated regulatory T cells.

Despite considerable interest in the modulation of tumor-associated Foxp3(+) regulatory T cells (T(regs)) for therapeutic benefit, little is known about the developmental origins of these cells and the nature of the antigens that they recognize. We identified an endogenous population of antigen-specific T(regs) (termed MJ23 T(regs)) found recurrently enriched in the tumors of mice with oncogene-driven prostate cancer. MJ23 T(regs) were not reactive to a tumor-specific antigen but instead recognized a prostate-associated antigen that was present in tumor-free mice. MJ23 T(regs) underwent autoimmune regulator (Aire)-dependent thymic development in both male and female mice. Thus, Aire-mediated expression of peripheral tissue antigens drives the thymic development of a subset of organ-specific T(regs), which are likely coopted by tumors developing within the associated organ.

Basic principles of tumor-associated regulatory T cell biology.

Due to the critical role of forkhead box (Fox)p3(+) regulatory T cells (Tregs) in the regulation of immunity and the enrichment of Tregs within many human tumors, several emerging therapeutic strategies for cancer involve the depletion or modulation of Tregs, with the aim of eliciting enhanced antitumor immune responses. Here, we review recent advances in understanding of the fundamental biology of Tregs, and discuss the implications of these findings for current models of tumor-associated Treg biology. In particular, we discuss the context-dependent functional diversity of Tregs, the developmental origins of these cells, and the nature of the antigens that they recognize within the tumor environment. In addition, we highlight critical areas of focus for future research.

ELPylated anti-human TNF therapeutic single-domain antibodies for prevention of lethal septic shock.

Tumour necrosis factor (TNF) is a major pro-inflammatory cytokine involved in multiple inflammatory diseases. The detrimental activity of TNF can be blocked by various antagonists, and commercial therapeutics based upon this principle have been approved for treatment of diseases including rheumatoid arthritis, Crohn's disease and psoriasis. In a search for new, improved anti-inflammatory therapeutics we have designed a single-domain monoclonal antibody (V(H) H), which recognizes TNF. The antibody component (TNF-V(H) H) is based upon an anti-human TNF Camelidae heavy-chain monoclonal antibody, which was linked to an elastin-like polypeptide (ELP). We demonstrate that ELP fusion to the TNF-V(H) H enhances accumulation of the fusion protein during biomanufacturing in transgenic tobacco plants. With this study, we show for the first time that this plant-derived anti-human TNF-V(H) H antibody was biologically active in vivo. Therefore, therapeutic application of TNF-V(H) H-ELP fusion protein was tested in humanized TNF mice and was shown to be effective in preventing death caused by septic shock. The in vivo persistence of the ELPylated antibody was ∼24 fold longer than that of non-ELPylated TNF-V(H) H.

Essential role of neutrophil mobilization in concanavalin A-induced hepatitis is based on classic IL-6 signaling but not on IL-6 trans-signaling.

Neutrophil depleted mice are protected from concanavalin A-mediated hepatitis, showing that neutrophils are critical for cellular liver damage. Interleukin-6 has pro- and anti-inflammatory properties and mediates neutrophil recruitment in diseases such as rheumatoid arthritis. In classic signaling, interleukin-6 binds to the membrane-bound interleukin-6-receptor and initiates signaling via gp130. In interleukin-6 trans-signaling, the agonistic soluble interleukin-6-receptor can form a soluble interleukin-6/interleukin-6-receptor complex and stimulate cells which only express gp130 but no interleukin-6-receptor. Interleukin-6 trans-signaling was shown to be important for liver regeneration and development of liver adenomas. Here, we show that blocking classic interleukin-6 signaling but not interleukin-6 trans-signaling reduced concanavalin A-induced liver damage in mice, with reduced liver STAT3 phosphorylation and liver neutrophil accumulation. However, the level of neutrophil-attracting chemokine KC is only reduced by inhibition of interleukin-6 trans-signaling. Analysis of circulating neutrophils after concanavalin A challenge revealed that classic interleukin-6 signaling is required for the mobilization of blood neutrophils. Reduced neutrophil infiltration was accompanied by increased levels of hepatoprotective monocyte chemoattractant protein-1 and reduced level of hepatodestructive interleukin-4. Abrogated classic interleukin-6 signaling in concanavalin A-mediated hepatitis exhibited liver-protective effects indicating that interleukin-6 classic but not interleukin-6 trans-signaling is responsible for liver damage. Classic interleukin-6 signaling is required to mount an efficient neutrophilia during concanavalin A-induced immune response, which might have clinical implications in the regard that blocking global interleukin-6 signaling pathways is a treatment option in different chronic inflammatory diseases.

Role of IL-6 trans-signaling in CCl4induced liver damage.

Interleukin-6 (IL-6) plays an important role in liver regeneration and protection against liver damage. In addition to IL-6 classic signaling via membrane bound receptor (mIL-6R), IL-6 signaling can also be mediated by soluble IL-6R (sIL-6R) thereby activating cells that do not express membrane bound IL-6R. This process has been named trans-signaling. IL-6 trans-signaling has been demonstrated to operate during liver regeneration. We have developed methods to specifically block or mimic IL-6 trans-signaling. A soluble gp130 protein (sgp130Fc) exclusively inhibits IL-6 trans-signaling whereas an IL-6/sIL-6R fusion protein (Hyper-IL-6) mimics IL-6 trans-signaling. Using these tools we investigate the role of IL-6 trans-signaling in CCl4 induced liver damage. Blockade of IL-6 trans-signaling during CCl4 induced liver damage led to higher liver damage, although induction of Cyp4502E1 and thus bioactivation of CCl4 was unchanged. Depletion of neutrophils resulted in reduced liver transaminase levels irrespective of IL-6 trans-signaling blockade. Furthermore, IL-6 trans-signaling was important for refilling of hepatocyte glycogen stores, which were depleted 24 h after CCl4 treatment. We conclude that IL-6 trans-signaling via the soluble IL-6R is important for the physiologic response of the liver to CCl4 induced chemical damage.

Impact of interleukin-6 classic- and trans-signaling on liver damage and regeneration.

Interleukin-6 (IL-6) has been suggested to play a pivotal role in liver regeneration. IL-6 on target cells activates a receptor complex consisting of the IL-6 receptor (IL-6R) and the signal transducing receptor subunit gp130. Not all cells in the body express the IL-6R on the cell surface. IL-6 can signal via two different pathways: classical signaling via the membrane bound IL-6R and IL-6 trans-signaling via a naturally occurring soluble IL-6R (sIL-6R). This second pathway widens the scope of IL-6 signaling since also cells expressing no membrane bound IL-6R can be stimulated by the trans-signal pathway. Mimicking IL-6 trans-signaling via a designer molecule, Hyper-IL-6 has been shown to accelerate liver regeneration. Another designer molecule, sgp130Fc, specifically blocks IL-6 trans-signaling. Using these proteins we investigated the contribution of IL-6 classic- and trans-signaling in the liver. Here we review the role of IL-6 signaling in response to liver damage and during liver regeneration.