B cell-mediated regulatory mechanisms control tumor-promoting intestinal inflammation.

Mechanisms underlying tumor-promoting inflammatory processes in colitis-associated colorectal cancer (CAC) remain largely elusive. Here, we provide genetic evidence for distinct B cell-mediated immunoregulatory mechanisms that protect from chronic colitis versus CAC. We demonstrate an inherent capacity of interleukin-10 (IL-10)-producing B cells to differentiate into immunoglobulin A (IgA) plasma cells (PCs) upon Toll-like receptor (TLR) activation. Our data show that B cell-derived IL-10 is essential to limit pathogenic T helper type 1 (Th1)/Th17 T cell responses during chronic colitis, while IgA PCs derived from IL-10+ B cells are being implicated in restraining tumorigenesis during CAC. Formation of a tumor-protective intestinal environment was associated with clonal expansion of specific types of colonic IgA PCs and development of an altered microbiota that attenuated CAC. We thus propose that regulatory B cell-mediated immunomodulation entails temporal release of IL-10, which is superseded by the generation of specific IgA affecting the microbial community, thereby controlling chronic inflammation and tumorigenesis in a distinctive but interrelated manner.

Autoimmunity linked protein phosphatase PTPN22 as a target for cancer immunotherapy.

BACKGROUND: Cancer immunotherapy has evolved from interferon-alpha (IFNα) and interleukin-2 in the 1980s to CTLA-4 and PD-1/PD-L1 checkpoint inhibitors (CPIs), the latter highlighting the importance of enhancing T-cell functions. While the search for novel immunomodulatory pathways continues, combination therapies augmenting multiple pathways can also increase efficacy. The association of autoimmune-related adverse events with clinical efficacy following CPI treatment has been inferred and suggests that breaking tolerance thresholds associated with autoimmunity may affect host immune responses for effective cancer immunotherapy. RESULTS: Here, we show that loss of autoimmune associated PTPN22, a key desensitization node for multiple signaling pathways, including IFNα receptor (IFNAR) and T-cell receptor, can augment tumor responses. Implantation of syngeneic tumors in Ptpn22-/- mice led to expansion and activation of peripheral and intratumoral T cells and, in turn, spontaneous tumor regression as well as enhanced responses in combination with anti-PD-L1 treatment. Using genetically modified mice expressing a catalytically inactive PTPN22 or the autoimmunity-associated human single-nucleotide polymorphism variant, augmentation of antitumor immunity was dependent on PTPN22 phosphatase activity and partially on its adaptor functions. Further, antitumor responses were dependent on both CD4+ and CD8+T cells and, in part, IFNAR function. Finally, we demonstrate that the autoimmune susceptibility Ptpn22(C1858T) variant is associated with lower risk of developing non-melanoma skin cancers, improved overall survival and increased risk for development of hyperthyroidism or hypothyroidism following atezolizumab (anti-PD-L1) treatment. CONCLUSIONS: Together, these data suggest that inhibition of PTPN22 phosphatase activity may provide an effective therapeutic option for cancer immunotherapy and that exploring genetic variants that shift immune tolerance thresholds may serve as a paradigm for finding new cancer immunotherapy targets.

30 Years of Biotherapeutics Development-What Have We Learned?

Since the birth of biotechnology, hundreds of biotherapeutics have been developed and approved by the US Food and Drug Administration (FDA) for human use. These novel medicines not only bring significant benefit to patients but also represent precision tools to interrogate human disease biology. Accordingly, much has been learned from the successes and failures of hundreds of high-quality clinical trials. In this review, we discuss general and broadly applicable themes that have emerged from this collective experience. We base our discussion on insights gained from exploring some of the most important target classes, including interleukin-1 (IL-1), tumor necrosis factor α (TNF-α), IL-6, IL-12/23, IL-17, IL-4/13, IL-5, immunoglobulin E (IgE), integrins and B cells. We also describe current challenges and speculate about how emerging technological capabilities may enable the discovery and development of the next generation of biotherapeutics.

NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22.

Inflammasomes form as the result of the intracellular presence of danger-associated molecular patterns and mediate the release of active IL-1ß, which influences a variety of inflammatory responses. Excessive inflammasome activation results in severe inflammatory conditions, but physiological IL-1ß secretion is necessary for intestinal homeostasis. Here, we have described a mechanism of NLRP3 inflammasome regulation by tyrosine phosphorylation of NLRP3 at Tyr861. We demonstrated that protein tyrosine phosphatase non-receptor 22 (PTPN22), variants in which are associated with chronic inflammatory disorders, dephosphorylates NLRP3 upon inflammasome induction, allowing efficient NLRP3 activation and subsequent IL-1ß release. In murine models, PTPN22 deficiency resulted in pronounced colitis, increased NLRP3 phosphorylation, but reduced levels of mature IL-1ß. Conversely, patients with inflammatory bowel disease (IBD) that carried an autoimmunity-associated PTPN22 variant had increased IL-1ß levels. Together, our results identify tyrosine phosphorylation as an important regulatory mechanism for NLRP3 that prevents aberrant inflammasome activation.

Autoimmunity-associated protein tyrosine phosphatase PEP negatively regulates IFN-α receptor signaling.

The protein tyrosine phosphatase PTPN22(C1858T) allelic polymorphism is associated with increased susceptibility for development of systemic lupus erythematosus (SLE) and other autoimmune diseases. PTPN22 (also known as LYP) and its mouse orthologue PEP play important roles in antigen and Toll-like receptor signaling in immune cell functions. We demonstrate here that PEP also plays an important inhibitory role in interferon-α receptor (IFNAR) signaling in mice. PEP co-immunoprecipitates with components of the IFNAR signaling complex. Pep(-/-) hematopoietic progenitors demonstrate increased IFNAR signaling, increased IFN-inducible gene expression, and enhanced proliferation and activation compared to Pep(+/+) progenitors in response to IFN-α. In addition, Pep(-/-) mice treated with IFN-α display a profound defect in hematopoiesis, resulting in anemia, thrombocytopenia, and neutropenia when compared to IFN-α-treated Pep(+/+) mice. As SLE patients carrying the PTPN22(C1858T) risk variant have higher serum IFN-α activity, these data provide a molecular basis for how type I IFNs and PTPN22 may cooperate to contribute to lupus-associated cytopenias.

Accumulation of 4-1BBL+ B cells in the elderly induces the generation of granzyme-B+ CD8+ T cells with potential antitumor activity.

Although the accumulation of highly-differentiated and granzyme B (GrB)-expressing CD8(+)CD28(-) T cells has been associated with aging, the mechanism for their enrichment and contribution to immune function remains poorly understood. Here we report a novel B-cell subset expressing 4-1BBL, which increases with age in humans, rhesus macaques, and mice, and with immune reconstitution after chemotherapy and autologous progenitor cell transplantation. These cells (termed 4BL cells) induce GrB(+)CD8(+) T cells by presenting endogenous antigens and using the 4-1BBL/4-1BB axis. We found that the 4BL cells increase antitumor responses in old mice, which may explain in part the paradox of retarded tumor growth in the elderly. 4BL cell accumulation and its capacity to evoke the generation of GrB(+)CD8(+) T cells can be eliminated by inducing reconstitution of B cells in old mice, suggesting that the age-associated skewed cellular immune responses are reversible. We propose that 4BL cells and the 4-1BBL signaling pathway are useful targets for improved effectiveness of natural antitumor defenses and therapeutic immune manipulations in the elderly.

Anti-CD20 antibody promotes cancer escape via enrichment of tumor-evoked regulatory B cells expressing low levels of CD20 and CD137L.

The possible therapeutic benefits of B-cell depletion in combating tumoral immune escape have been debated. In support of this concept, metastasis of highly aggressive 4T1 breast cancer cells in mice can be abrogated by inactivation of tumor-evoked regulatory B cells (tBreg). Here, we report the unexpected finding that B-cell depletion by CD20 antibody will greatly enhance cancer progression and metastasis. Both murine and human tBregs express low levels of CD20 and, as such, anti-CD20 mostly enriches for these cells. In the 4T1 model of murine breast cancer, this effect of enriching for tBregs suggests that B-cell depletion by anti-CD20 may not be beneficial at all in some cancers. In contrast, we show that in vivo-targeted stimulation of B cells with CXCL13-coupled CpG oligonucleotides (CpG-ODN) can block cancer metastasis by inhibiting CD20(Low) tBregs. Mechanistic investigations suggested that CpG-ODN upregulates low surface levels of 4-1BBL on tBregs to elicit granzyme B-expressing cytolytic CD8(+) T cells, offering some explanative power for the effect. These findings underscore the immunotherapeutic importance of tBreg inactivation as a strategy to enhance cancer therapy by targeting both the regulatory and activating arms of the immune system in vivo.

Personalizing medicine for autoimmune and inflammatory diseases.

Therapies that target molecular pathways do not provide uniform benefits for all patients at present. New transformative therapies for autoimmune and inflammatory diseases require greater molecular understanding of patient subsets and the ability to personalize targeted therapies for each subset.

Toso regulates the balance between apoptotic and nonapoptotic death receptor signaling by facilitating RIP1 ubiquitination.

The regulation of cellular survival and apoptosis is of critical importance for the immune system to maintain immune homeostasis and to establish tolerance. Here, we demonstrate that the immune specific cell surface molecule Toso exhibits antiapoptotic effects on death receptor signaling by a novel regulatory mechanism involving the adaptor kinase RIP1. The antiapoptotic function of Toso depends on RIP1 ubiquitination and involves the recruitment of the death adaptor FADD to a Toso/RIP1 protein complex. In response to CD95L and TNFα, Toso promotes the activation of MAPK and NF-κB signaling pathways. Because of this relative augmentation of survival versus apoptotic signals, Toso raises the threshold for death receptor-mediated apoptosis. Our analysis of Toso-deficient mice revealed that Toso is essential for TNFα-mediated liver damage. Furthermore, the antiapoptotic function of Toso could be blocked by a Toso-specific monoclonal antibody, opening up new therapeutic prospects for the treatment of immune disorders and hematologic malignancies.

Restricted TCR-alpha CDR3 diversity disadvantages natural regulatory T cell development in the B6.2.16 beta-chain transgenic mouse.

To date, analysis of mice expressing TCR-beta transgenes derived from CD4(+) T cell clones has demonstrated equivalent or higher TCR diversity in naturally occurring regulatory CD4(+) T cells (Tregs) versus conventional CD4(+) T cells (Tcons). However, TCR-alpha-chain diversity in these mice may be influenced by the inherent bias toward the CD4(+) lineage in the selected repertoires. We wished to determine whether the choice of TCR-beta-chain influences the relative diversity of the Treg and Tcon repertoires, examining as a model the B6.2.16beta-transgenic mouse, in which the fixed beta-chain is derived from a CD8(+) T cell clone. B6.2.16beta Treg thymocytes showed significantly lower TRAV17 (AV9) CDR3 sequence diversity than both syngeneic Tcon thymocytes, and Treg and Tcon thymocytes from wild-type C57BL/6 (B6) mice. The ratio of single-positive CD4(+)/single-positive CD8(+) thymocytes in B6.2.16beta mice was similar to that in B6, yet both the proportional frequency and absolute number of CD4(+)Foxp3(+) cells was significantly lower in the thymi and peripheral lymph nodes of B6.2.16beta mice. Furthermore, B6 + B6.2.16beta-->B6 mixed bone marrow chimeras revealed that the transgenic beta-chain disadvantaged Treg development in a competitive environment. These data underline the importance of the beta-chain in assessments of Treg alpha-chain diversity and provide further support for the notion that interclonal competition for entry into the Treg lineage is a significant factor in determining the composition of this lineage.

B-cell targeted therapies in human autoimmune diseases: an updated perspective.

The advent of therapies that specifically target the B-lymphocyte lineage in human disease has rejuvenated interest in the mechanistic biology by which B cells mediate autoimmunity. B cells have a multitude of effector functions including production of self-reactive antibodies, ability to present antigen to T lymphocytes in the context of costimulation, involvement in generation and maintenance of neo-organogenesis at sites of disease, and opposing function through production of both immunostimulatory and immunomodulatory cytokines. In this review, we first discuss the role of B cells in driving autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, and Sjögren's syndrome, and discuss how studies in these diseases have revealed differentially important roles for the multiple B-cell effector functions. These data reveal the complex and interrelated roles of B cells working in concert with other components of the innate and adaptive immune system to drive pathogenesis. We then focus on data from mouse and human in which B cells in the setting of disease have been targeted with drugs directed against CD20, CD22, and the BAFF (B-cell activating factor belonging to the tumor necrosis factor family)/APRIL (a proliferation inducing ligand) pathways. Pre-clinical studies in animal models in addition to and clinical trials targeting B cells have added further to the understanding of the differential roles B cells play in disease both through demonstration of clinical efficacy in the context of B-cell depletion or modulation, and also by failure of B-cell targeting in some diseases and disease patient subgroups. Moving forward, it will be imperative to apply these lessons to new interventional trials to ensure better targeting of the B-cell lineage and concomitantly better selection of patients most likely to benefit from these therapies.

Autoimmune-associated PTPN22 R620W variation reduces phosphorylation of lymphoid phosphatase on an inhibitory tyrosine residue.

A missense C1858T single nucleotide polymorphism in the PTPN22 gene recently emerged as a major risk factor for human autoimmunity. PTPN22 encodes the lymphoid tyrosine phosphatase (LYP), which forms a complex with the kinase Csk and is a critical negative regulator of signaling through the T cell receptor. The C1858T single nucleotide polymorphism results in the LYP-R620W variation within the LYP-Csk interaction motif. LYP-W620 exhibits a greatly reduced interaction with Csk and is a gain-of-function inhibitor of signaling. Here we show that LYP constitutively interacts with its substrate Lck in a Csk-dependent manner. T cell receptor-induced phosphorylation of LYP by Lck on an inhibitory tyrosine residue releases tonic inhibition of signaling by LYP. The R620W variation disrupts the interaction between Lck and LYP, leading to reduced phosphorylation of LYP, which ultimately contributes to gain-of-function inhibition of T cell signaling.

Subcellular compartmentalization of FADD as a new level of regulation in death receptor signaling.

Fas-associated protein with death domain (FADD) is an essential adaptor protein in death receptor-mediated signal transduction. During apoptotic signaling, FADD functions in the cytoplasm, where it couples activated receptors with initiator caspase-8. However, in resting cells, FADD is predominantly stored in the nucleus. In this study, we examined the modalities of FADD intracellular trafficking. We demonstrate that, upon CD95 activation, FADD redistributes from the nucleus to the cytoplasm. This inducible nuclear-cytoplasmic translocation of FADD is independent of CD95 internalization, formation of the death-inducing signaling complex, and caspase-8 activation. In contrast to nuclear export of FADD, its subsequent recruitment and accumulation at endosomes containing internalized CD95 requires a caspase-8-dependent feedback loop. These data indicate the existence of differential pathways directing FADD nuclear export and cytoplasmic trafficking, and identify subcellular compartmentalization of FADD as a novel regulatory mechanism in death receptor signaling.

A specificity map for the PDZ domain family.

PDZ domains are protein-protein interaction modules that recognize specific C-terminal sequences to assemble protein complexes in multicellular organisms. By scanning billions of random peptides, we accurately map binding specificity for approximately half of the over 330 PDZ domains in the human and Caenorhabditis elegans proteomes. The domains recognize features of the last seven ligand positions, and we find 16 distinct specificity classes conserved from worm to human, significantly extending the canonical two-class system based on position -2. Thus, most PDZ domains are not promiscuous, but rather are fine-tuned for specific interactions. Specificity profiling of 91 point mutants of a model PDZ domain reveals that the binding site is highly robust, as all mutants were able to recognize C-terminal peptides. However, many mutations altered specificity for ligand positions both close and far from the mutated position, suggesting that binding specificity can evolve rapidly under mutational pressure. Our specificity map enables the prediction and prioritization of natural protein interactions, which can be used to guide PDZ domain cell biology experiments. Using this approach, we predicted and validated several viral ligands for the PDZ domains of the SCRIB polarity protein. These findings indicate that many viruses produce PDZ ligands that disrupt host protein complexes for their own benefit, and that highly pathogenic strains target PDZ domains involved in cell polarity and growth.

A biosynthetic pathway for anandamide.

The endocannabinoid arachidonoyl ethanolamine (anandamide) is a lipid transmitter synthesized and released "on demand" by neurons in the brain. Anandamide is also generated by macrophages where its endotoxin (LPS)-induced synthesis has been implicated in the hypotension of septic shock and advanced liver cirrhosis. Anandamide can be generated from its membrane precursor, N-arachidonoyl phosphatidylethanolamine (NAPE) through cleavage by a phospholipase D (NAPE-PLD). Here we document a biosynthetic pathway for anandamide in mouse brain and RAW264.7 macrophages that involves the phospholipase C (PLC)-catalyzed cleavage of NAPE to generate a lipid, phosphoanandamide, which is subsequently dephosphorylated by phosphatases, including PTPN22, previously described as a protein tyrosine phosphatase. Bacterial endotoxin (LPS)-induced synthesis of anandamide in macrophages is mediated exclusively by the PLC/phosphatase pathway, which is up-regulated by LPS, whereas NAPE-PLD is down-regulated by LPS and functions as a salvage pathway of anandamide synthesis when the PLC/phosphatase pathway is compromised. Both PTPN22 and endocannabinoids have been implicated in autoimmune diseases, suggesting that the PLC/phosphatase pathway of anandamide synthesis may be a pharmacotherapeutic target.

The role of receptor internalization in CD95 signaling.

Activation of the cell surface CD95 receptor triggers a cascade of signaling events, including assembly of the death-inducing signaling complex (DISC), that culminate in cellular apoptosis. In this study, we demonstrate a general requirement of receptor internalization for CD95 ligand-mediated DISC amplification, caspase activation and apoptosis in type I cells. Recruitment of DISC components to the activated receptor predominantly occurs after the receptor has moved into an endosomal compartment and blockade of CD95 internalization impairs DISC formation and apoptosis. In contrast, CD95 ligand stimulation of cells unable to internalize CD95 results in activation of proliferative Erk and NF-kappaB signaling pathways. Hence, the subcellular localization and internalization pathways of CD95 play important roles in controlling activation of distinct signaling cascades to determine divergent cellular fates.

Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy.

B cell immunotherapy has emerged as a mainstay in the treatment of lymphomas and autoimmune diseases. Although the microenvironment has recently been demonstrated to play critical roles in B cell homeostasis, its contribution to immunotherapy is unknown. To analyze the in vivo factors that regulate mechanisms involved in B cell immunotherapy, we used a murine model for human CD20 (hCD20) expression in which treatment of hCD20(+) mice with anti-hCD20 mAbs mimics B cell depletion observed in humans. We demonstrate in this study that factors derived from the microenvironment, including signals from the B cell-activating factor belonging to the TNF family/BLyS survival factor, integrin-regulated homeostasis, and circulatory dynamics of B cells define distinct in vivo mechanism(s) and sensitivities of cells in anti-hCD20 mAb-directed therapies. These findings provide new insights into the mechanisms of immunotherapy and define new opportunities in the treatment of cancers and autoimmune diseases.

PEST domain-enriched tyrosine phosphatase (PEP) regulation of effector/memory T cells.

Protein tyrosine kinases and phosphatases cooperate to regulate normal immune cell function. We examined the role of PEST domain-enriched tyrosine phosphatase (PEP) in regulating T cell antigen-receptor function during thymocyte development and peripheral T cell differentiation. Although normal naïve T cell functions were retained in pep-deficient mice, effector/memory T cells demonstrated enhanced activation of Lck. In turn, this resulted in increased expansion and function of the effector/memory T cell pool, which was also associated with spontaneous development of germinal centers and elevated serum antibody levels. These results revealed a central role for PEP in negatively regulating specific aspects of T cell development and function.

B cell defects in SLP65/BLNK-deficient mice can be partially corrected by the absence of CD22, an inhibitory coreceptor for BCR signaling.

CD22 is an inhibitory coreceptor for B cell receptor (BCR) signaling. The inhibition is most likely mediated by activation of SHP-1. We found that SLP65/BLNK reaches maximal tyrosine-phosphorylation at earlier time points in CD22(-/-) than in wild type B cells upon BCR cross-linking, suggesting that SLP65/BLNK is a substrate of SHP-1. However, in contrast to the defective Ca(2+) mobilization of SLP65/BLNK(-/-) B cells, there was a clear Ca(2+) response in SLP65/BLNKxCD22 double-deficient B cells. This implies that SLP65/BLNK is not the sole target of SHP-1 in the regulation of the Ca(2+) signaling strength. While SLP65(-/-) mice show several blocks of B cell differentiation, in SLP65/BLNK x CD22 double-deficient mice the maturation block of B cells in the spleen was partially rescued. However, the proliferative responses of B cells from both SLP65/BLNK(-/-) and double-deficient mice were defective after IgM- or CD40-stimulation. These results show that SLP65/BLNK is not absolutely essential for Ca(2+) induction in B cells, because the deficiency of this adapter can be by-passed by the additional deletion of an inhibitory receptor. Furthermore, these experiments suggest that B cell maturation in the spleen is directly dependent on the strength of BCR-derived Ca(2+) signals.

T cell receptor signaling precedes immunological synapse formation.

The area of contact between a T cell and an antigen-presenting cell (APC) is known as the immunological synapse. Although its exact function is unknown, one model suggests that it allows for T cell receptor (TCR) clustering and for sustained signaling in T cells for many hours. Here we demonstrate that TCR-mediated tyrosine kinase signaling in naïve T cells occurred primarily at the periphery of the synapse and was largely abated before mature immunological synapses had formed. These data suggest that many hours of TCR signaling are not required for T cell activation. These observations challenge current ideas about the role of immunological synapses in T cell activation.