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Resistance to the parasite Toxoplasma gondii is mediated by NK and T cell production of IFN-γ, but the failure to contract this response can lead to severe T cell–dependent immunopathology. Although the cytokines IL-10 and IL-27 prevent immune hyperactivity during toxoplasmosis, inhibitory receptors, expressed by NK and T cells, are also implicated in this process. The inhibitory receptor TIGIT is expressed on NK and T cells and competes with the costimulatory receptor CD226 for binding of the ligand CD155. During toxoplasmosis, the activation of NK and T cells is associated with increased expression of CD226 and TIGIT, whereas DCs express increased levels of CD155. To determine if the loss of TIGIT impacts NK and T cell activities, wild-type and TIGIT knockout mice were infected with T. gondii. During the acute stage of infection, wild-type and TIGIT knockout mice had comparable parasite burdens and similar NK and T cell responses. Likewise, during the chronic phase of this infection, the loss of TIGIT did not affect the magnitude or phenotype of the T cell response nor the ability to control pathogen load. These data suggest that during toxoplasmosis, despite upregulation of relevant ligands, TIGIT signaling does not limit NK and T cell activities. Thus, TIGIT-independent mechanisms dominate the restraint of the immune response during toxoplasmosis.

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint, which may be engaged by cells in the tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®, MK-3475) is a potent and highly selective humanized mAb of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. This blockade enhances the functional activity of T cells to facilitate tumor regression and ultimately immune rejection. Pembrolizumab binds to human and cynomolgus monkey PD-1 with picomolar affinity and blocks the binding of human and cynomolgus monkey PD-1 to PD-L1 and PD-L2 with comparable potency. Pembrolizumab binds both the C′D and FG loops of PD-1. Pembrolizumab overcomes human and cynomolgus monkey PD-L1–mediated immune suppression in T-cell cultures by enhancing IL2 production following staphylococcal enterotoxin B stimulation of healthy donor and cancer patient cells, and IFNγ production in human primary tumor histoculture. Ex vivo and in vitro studies with human and primate T cells show that pembrolizumab enhances antigen-specific T-cell IFNγ and IL2 production. Pembrolizumab does not mediate FcR or complement-driven effector function against PD-1–expressing cells. Pembrolizumab displays dose-dependent clearance and half-life in cynomolgus monkey pharmacokinetic and toxicokinetic studies typical for human IgG4 antibodies. In nonhuman primate toxicology studies, no findings of toxicologic significance were observed. The preclinical data for pembrolizumab are consistent with the clinical anticancer activity and safety that has been demonstrated in human clinical trials.

High-dose Interleukin-2 (IL-2) monotherapy induces complete responses in cancer patients but its use is limited by acute vascular toxicities including capillary leak syndrome and severe hypotension 1,2. IL-2 activates lymphocytes and NK-cells through the intermediate-affinity dimeric IL-2 receptor, IL-2Rβ/γ (CD122/CD132), while antigen activated T-cells and regulatory T-cells (Tregs) have increased sensitivity to IL-2 by expressing the high-affinity trimeric IL-2 receptor, IL-2Rα/β/γ (CD25/CD122/CD132)3. CD25-independent IL-2s (“non-α-IL-2s”) aim to increase the therapeutic efficacy of IL2 in cancer patients by avoiding Treg activation through selective binding to IL-2Rβ/γ 4. However, those molecules still are reported to induce fever and hypotension and have limited efficacy as a monotherapy or in combination with anti-PD-1 5,6. Here we show that a novel α/β-IL-2 agonist that was designed to preferentially bind to the IL-2Rα/β/γ receptor highly upregulated on antigen activated T-cells can greatly improve on the efficacy of IL-2 while avoiding the vascular toxicity commonly associated with IL-2 treatment. In syngeneic tumor models, this α/β-IL-2 agonist significantly reduced exhaustion of tumor infiltrating T cells compared to WT-IL-2 or a non-α-IL-2 leading to improved expansion of tumor antigen specific CD25+PD-1+CD8+ T cells systemically and in the tumor microenvironment. This resulted in complete responses and tumor immune memory with α/β-IL-2 monotherapy as well as improved outcomes in combination with anti-PD-1 therapy in PD-1 refractory syngeneic tumors. In contrast, WT-IL-2 reduced T cell exhaustion and drove antigen specific T cell responses to a lesser degree, resulting in reduced combinatorial efficacy with anti-PD-1, while the non-α-IL-2 failed to do either. Furthermore, the α/β-IL-2 agonist reduced intratumoral Tregs compared to treatment with WT-IL-2 or PBS improving the intratumoral CD8 to Treg ratio. In non-human primates and mice, WT-IL-2 and a non-α-IL-2 led to broad extravasation of lymphocytes and NK cells and activation of intra-pulmonal T cells resulting in systemic tissue inflammation and NK cell-mediated lethal capillary leak syndrome whereas the α/β-IL-2 agonist, which avoids binding the dimeric IL-2Rβ/γ expressed on NK cells, avoided systemic lymphocyte activation which facilitated continuous treatment without acute vascular toxicities. Overall, through selective engagement of CD25+ T cells, this α/β-IL-2 agonist demonstrated improved efficacy and tolerability of IL-2 in preclinical tumor models. Clinical trials with STK-012, a human α/β-IL-2 agonist, are in progress.1 Atkins, et al.; JCO 1999, 2 Dutcher, et al.; JITC 2014, 3 Liao, et al.; Immunity 2013, 4 Levin, et al.; Nature 2012, 5 Janku, et al.; Cancer Research 2021; 6 Diab, et al.; Cancer Disc. 2020 Citation Format: Martin Oft, Navneet Ratti, Sandro Vivona, Jan Emmerich, Romina Riener, Ievgen O. Koliesnik, Scott McCauley, Michele Bauer, Marie Semana, Deepti Rokkam, Bhargavi Jayaraman, Rene de Waal Malefyt, Paul-Joseph Aspuria, Michael Totagrande, Anita Mehta-Damani, Patrick J. Lupardus, Rob A. Kastelein. STK-012, an a/b-selective IL-2 activates tumor antigen specific CD25+ CD8 T cells to reject tumors without acute vascular toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1801.

Here we report on a novel human IL-12 partial agonist (hIL-12v) that has diminished binding to IL-12Rb1. IL-12v is designed to more selectively engage antigen activated T-cells, which strongly upregulate IL-12Rb1 upon activation, and to reduce stimulation of NK cells or resting T cells, which express modest levels of IL-12Rb1. To explore anti-tumor efficacy and toxicity in mouse syngeneic tumor models, we generated a half-life extended mouse surrogate of the IL-12 partial agonist (mIL-12v Fc) and compared it to a similarly engineered half-life extended version of wild type mouse IL-12 (mIL-12wt Fc). At efficacious doses, systemic administration of mIL-12wt Fc induced significant weight loss and lethality characterized by early proinflammatory cytokine release and systemic NK cell activation. Conversely, mIL-12v Fc was well tolerated and avoided the robust and rapid NK cell activation and peripheral NK count decreases seen with mIL-12v Fc, suggestive of extravasation to tissues. Both mIL-12v Fc and mIL-12 WT Fc showed similar robust single-agent anti-tumor efficacy in syngeneic tumor models. Depletion of NK cells did not diminish anti-tumor efficacy. Efficacy for both molecules was characterized by CD8 T cell activation, myeloid cell reprograming and antigen presentation. Moreover, combination of mIL-12v Fc with systemic immunotherapies further enhanced anti-tumor activity without compromising tolerability. Overall, mIL-12v Fc retained anti-tumor efficacy without induction of severe toxicities compared to mIL-12wt Fc. These data suggest IL-12 partial agonists may represent a novel immunotherapy approach to maintain efficacy while avoiding classical toxicity associated with IL-12 therapy.1 Atkins, et al.; (1997) Clinical Cancer Research 3(3):409-172 Carson, et al. (1999) J Immunology 162 (8): 4943-4951. Citation Format: Ievgen Koliesnik, Jan Emmerich, Kim Q. Tran, Michele Bauer, Michael Totagrande, Bhargavi Jayaraman, Cindy Buffone, Priyanka Balasubrahmanyam, Deepti Rokkam, Rene de Waal Malefyt, Luis Zuniga, Heiko Greb, Navneet Ratti, Sandro Vivona, Martin Oft, Patrick J. Lupardus, Robert A. Kastelein. Novel IL-12 Partial Agonist For Cancer Immunotherapy Avoids NK-cell Mediated Toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1833.

Asthma is a disease of complex origin and multiple pathologies. There are currently very few biomarkers of proven utility in its diagnosis, management or response to treatment. Recent studies have identified multiple asthma phenotypes following biofluid analysis; however, such findings may be driven by the well-characterised alterations in immune cell populations in asthma. We present a study designed to identify cell type-specific gene signatures of severe allergic asthma in peripheral blood samples. Using transcriptomic profiling of four magnetically purified peripheral blood cell types, we identify significant gene expression changes in monocytes and NK cells but not T lymphocytes in severe asthmatics. Pathway analysis indicates dysfunction of immune cell regulation and bacterial suppression in the NK cells. These gene expression changes may be useful on their own as prognostic peripheral blood cell markers of severe asthma, but also may indicate novel cell pathways for therapeutic intervention.

Chimeric antigen receptor (CAR) T cells induce durable responses in patients with refractory hematological tumors. However, low CAR T cell activity, poor engraftment, or short in-patient persistence can lead to tumor progression or relapse. Furthermore, excessive CAR T cell expansion and activation can result in life-threatening cytokine release syndrome (CRS). Thus, in-patient control of the CAR T cell population is essential. Interleukin-2 (IL-2) is a critical cytokine for T cell proliferation and effector function, but its clinical use is limited by immune-mediated toxicity. Here, we report on an orthogonal IL-2 receptor and ligand system that enables specific in vivo control of CAR T cell expansion and activation, wherein an orthogonal human IL-2 (STK-009) selectively pairs with an orthogonal human IL-2Rβ (hoRb) expressed on CAR T cells. STK-009 expands hoRb-expressing CAR T cells in the presence and absence of tumor antigen and maintains the presence of stem cell memory T cells (T

Resistance to the parasite

Phenotypic and transcriptional profiling of regulatory T (T

Introduction: IL10 is a pleiotropic cytokine that is secreted as a homodimer and initiates signaling on various cell types by engaging two copies of a heterodimeric receptor complex consisting of an IL-10Rα subunit and a IL-10Rβ subunit. IL10 signaling through the natural ligand has both immunosuppressive and immunostimulatory effects and this pleiotropy makes it difficult to develop wild type IL-10 for therapeutic use. In clinical trials that have studied IL10 in cancer, anemia, thrombocytopenia and hemophagocytosis associated with macrophage activation led to dose interruption and dose reduction. To drive more selective cytokine signaling only on cells of interest, we have generated surrogate cytokine agonists that bind to IL10Rα and IL2Rγ, resulting in the selective activation of T cells over macrophages. We establish a novel means of bridging the IL10Rα and IL2Rγ receptors using a IL10Rα/IL2Rγ surrogate cytokine agonist that consists of a domain that binds to IL10Rα and a second domain that binds to IL2Rγ. Upon contact with a cell that allows bridging of IL10Rα and IL2Rγ, the surrogate cytokine agonist causes the functional association of IL10Rα and IL2Rγ, resulting in downstream signaling in select cell types. Experimental Procedures: Human IL-10Rα and IL-2Ry specific single domain VHHs were generated by camel immunization and screening of VHH libraries prepared from peripheral blood cells for binding. Seven IL-10Rα VHHs and six IL-2Ry VHHs were identified and coupled as IL10Rα/IL2Ry dual VHHs in an all by all matrix and in both amino-carboxy and carboxy-amino orientations, yielding 84 unique surrogate cytokine agonists. These surrogate cytokine agonists were screened on a (p)STAT3 assay on primary human cells and further tested in T cell and monocyte functional assays. Results: Several IL10Rα/IL2Rγ surrogate cytokine agonists were biologically active on primary human lymphoid cells, inducing (p)STAT3 signal in B cells, NK cells, CD4 and CD8 T cells with little to no (p)STAT3 signal in monocytes. These IL10Rα/IL2Rγ surrogate cytokine agonists were also functionally active in promoting cell survival and at inducing IFN-γ and Granzyme production by CD8 T cell blasts generated upon CD3/CD28 activation. Consistent with the lack of STAT3 signaling in monocytes, the IL10Rα/IL2Rγ surrogate cytokine agonists did not inhibit LPS induced secretion of IL1β and TNFα by monocytes, suggesting selectivity and a lack of immunosuppressive activities. Conclusions: Designing surrogate cytokine agonists that pair non-natural cytokine receptors provides the possibility of generating molecules that can decouple the pleiotropy of cytokines like IL10 by stimulating only the desired cell population. Here, we have generated IL10Rα/IL2Rγ surrogate cytokine agonists that are biologically active and signal with varying strengths in the lymphoid cells with little to no activity on monocytes, thus providing an opportunity to decouple the pleiotropy of IL10 for use in cancer therapy. Citation Format: Mahalakshmi Ramadass, Sandro Vivona, Deepti Rokkam, Heiko Greb, Anu Gangopadhyay, Romina Riener, Rene de Waal Malefyt, Patrick Lupardus, Martin Oft, Robert Kastelein. IL10/IL2 surrogate cytokine agonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5544.

Introduction: Heterodimerization of the intermediate affinity Interleukin-2 Receptors (IL-2R), IL-2Rβ and IL-2Rγ, initiates a signaling cascade in T and NK cells that ultimately results in proliferation and production of Interferon-gamma (IFN-γ). Binding of IL-2 in a dimeric complex with intermediate affinity or in the high affinity trimeric complex, which also includes IL-2Rα and is predominantly expressed on antigen activated T cells and Tregs leads to trans-phosphorylation of signaling motifs on the IL-2Rβ and IL-2Rγ intracellular domains by the associated JAK kinases. The function of the wild type IL-2 is to bring the receptor chains in close proximity to produce an optimal level of phosphorylation and activation of associated STAT transcription factors. Here we established a way to bring IL-2Rβ and IL-2Rγ chains together in a cytokine-independent manner that results in functional activation. Experimental Procedures: Human IL-2Rβ and IL-2Rγ specific heavy chain single domain antibodies (VHH) were generated by camel immunization and screening of VHH libraries prepared from peripheral blood cells for binding. Ten IL-2Rβ VHH segments and six IL-2Rγ VHH segments, all with low nanomolar affinity, were identified and coupled as IL-2Rβ/IL-2Rγ VHH dimers in all possible combinations in both amino-carboxy and carboxy-amino orientations yielding 120 different proteins that were tested in human T and NK cell functional assays. Results: IL-2Rβ/IL-2Rγ VHH dimers were biologically active and induced pSTAT-5 phosphorylation in the IL-2 dependent NK cell line NKL at various levels. The biological activity of these IL-2Rβ/IL-2Rγ VHH dimers was further confirmed on primary cells. IL-2Rβ/IL-2Rγ VHH dimers induced pSTAT5 phosphorylation on NK cells isolated from human peripheral blood and culture with IL-2Rβ/IL-2Rγ VHH dimers resulted in proliferation and production of high but varied levels of IFN-γ. The activity of IL-2Rβ/IL-2Rγ VHH dimers on primary T cells was also examined. Similar to NK cells, IL-2Rβ/IL-2Rγ VHH dimers induced pSTAT5 phosphorylation, proliferation and IFN-γ production by CD4 positive and CD8 positive T cell blasts generated after CD3/CD28 activation of human PBMC. Conclusions: In conclusion, we have generated a series of functional IL-2Rβ/IL-2Rγ VHH dimers representing surrogate cytokine agonists each with unique signaling strengths that will now be further analyzed for potential therapeutic application in tumor immunology and auto-immunity Citation Format: Rene de Waal Malefyt, PJ Aspuria, Sandro Vivona, Priyanka Balasubrahmanyam, Cindy Buffone, Mahalakshmi Ramadass, Romina Riener, Martin Oft, Patrick Lupardus, Robert Kastelein. IL-2Rβ/IL-2R γsynthetic cytokines induce activation of human T and NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4225.

LAG3 is an important regulator of T cell homeostasis and studies in mouse tumor models have demonstrated that simultaneously antagonizing LAG3 and PD1 can augment tumor-specific T cell responses and induce tumor rejection. The combined use of LAG3 antagonist antibodies with established anti-PD1 therapies is currently being evaluated in human clinical trials. A functional assay for human LAG3 was developed by co-culture of a Jurkat T-cell lymphoma line overexpressing LAG3 with a Raji B-cell lymphoma line in the presence of staphylococcal enterotoxins. Reversal of LAG3 repression was measured as an increase in IL-2 production or NFAT activation in response to treatment with MK-4280, an anti-human LAG3 antagonist antibody. Changes in cytokines, chemokines, and other mRNA transcripts were in agreement with published in vitro and in vivo models for LAG3 biology which highlights the physiological relevance of the Jurkat functional assay. Additional engineering of PD1 and PDL1 components into the LAG3 assay resulted in a bi-functional assay that is capable of inducing a 10-fold response to individual antibodies blocking either PD1 or LAG3. Importantly, when MK-4280 and pembrolizumab were combined to block both pathways, a synergistic 50-fold increase in response was observed.

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint, which may be engaged by cells in the tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®, MK-3475) is a potent and highly selective humanized mAb of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. This blockade enhances the functional activity of T cells to facilitate tumor regression and ultimately immune rejection. Pembrolizumab binds to human and cynomolgus monkey PD-1 with picomolar affinity and blocks the binding of human and cynomolgus monkey PD-1 to PD-L1 and PD-L2 with comparable potency. Pembrolizumab binds both the C'D and FG loops of PD-1. Pembrolizumab overcomes human and cynomolgus monkey PD-L1-mediated immune suppression in T-cell cultures by enhancing IL2 production following staphylococcal enterotoxin B stimulation of healthy donor and cancer patient cells, and IFNγ production in human primary tumor histoculture.

Interleukin-2 (IL-2) is a potent stimulator of T and NK cell proliferation, survival, and cytotoxic function. High dose IL-2 induces complete responses as a single agent in certain cancers. Its use is limited due to toxicities such as severe hypotension and vascular leak syndrome (VLS). A better understanding of the mechanisms of IL-2 efficacy and toxicity and development of more selective therapies are needed to improve the use of IL-2. Next generation IL-2 therapeutics have been engineered to bias activity towards the dimeric form of the IL-2 receptor, which consists of IL-2Rβ (CD122) and IL-2Rγ (CD132), and away from the high affinity trimeric form of the receptor, which also includes IL-2Rα (CD25). This approach is designed to stimulate NK cells and naïve effector T-cells, which express the dimeric, and not the trimeric, form of the receptor. However, this approach largely misses antigen-activated and tumor-specific T-cells, which have high expression of both CD25 and CD122. In addition, this approach expands peripheral NK cells, which have been shown in mice to cause VLS (1-3).To specifically stimulate antigen-activated CD25+ effector T cells in cancer patients and avoid systemic NK and naïve T cell activation, we have developed a PEGylated, CD25/CD122-selective IL-2 mutein (STK-012) and its mouse surrogate (STK-014). Here we present efficacy and safety of STK-014 compared to wild type IL-2 (wtIL-2) and a CD122/CD132 biased IL-2 (βγIL-2). We also present safety of STK-012 in non-human primates (NHP). STK-012/STK-014 selectively induced STAT5 phosphorylation and proliferation in antigen activated T cells but not in NK cells and naïve T cells. In mice, STK-014 showed reduced toxicity compared to wtIL-2 and βγIL-2. In particular, wtIL-2 and βγIL-2 induced VLS while STK-014 did not. Moreover, STK-014 demonstrated complete responses both as single agent and in combination with a PD-1 antibody in syngeneic tumor models. In general, STK-014 demonstrated improved efficacy compared to wtIL-2 and βγIL-2. STK-014 dramatically increased intratumoral T cells and intratumoral cytotoxic activity compared wtIL-2 and βγIL-2 while avoiding T cell activation in the spleen. STK-014 treatment drastically increased the CD8+ T cell to Treg ratio within the tumor when compared to control tumors (25-fold), but also in comparison to wtIL-2 (2.75-fold) and to βγIL-2 (5.2-fold).In NHP, STK-012 was well tolerated at doses supra-efficacious in mice. Weekly dosing of STK-012 led to continuous elevated serum concentrations, demonstrating selectivity for CD25/122+ T cells. STK-012 selectively induced memory T cell expansion, including CD28+ CD95+ CD8 T cells. In summary, STK-012 avoids IL-2 mediated toxicity and may enable the specific expansion of antigen activated memory T cells in cancer patients, leading to durable tumor response. 1 Peace DJ and Cheever MA. JEM, 1989.2 Gatley MK et al. JI, 1988.3 Assier E et al. JI; 2004. Citation Format: Jan Emmerich, Michele Bauer, Marie Semana, Bhargavi Jayaraman, Sandro Vivona, Scott McCauley, Romina Riener, Rene De Waal Malefyt, Paul-Joseph Aspuria, Deepti Rokkam, Patrick Lupardus, Rob Kastelein, Martin Oft. STK-012, an alpha/beta selective IL-2 mutein for the activation of the antigen-activated T cells in solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1744.

CAR T cell therapy (CAR) has demonstrated remarkable clinical efficacy in hematological malignancies. However, barriers such as poor T cell effector function, lack of proliferation, and limited persistence prevent CARs from reaching their full curative potential. IL-2 is a potent stimulator of T cell proliferation, survival, and cytotoxic function, making it an attractive cytokine to support CARs. However, therapeutic use of IL-2 is limited by systemic toxicity due its promiscuous activation of undesired immune cell populations. To facilitate selective expansion and activation of CARs we have developed a human orthogonal ligand/receptor system consisting of a pegylated IL-2 mutein (STK-009) that does not significantly activate the wild type IL-2 receptor and a mutated IL-2 Receptor Beta (hoRb) that is fully activated by STK-009, but does not respond to the native IL-2 ligand. This system enables in vivo IL-2 signaling in CARs engineered to express hoRb while avoiding signaling bystander T cells and NK cells. Here, we demonstrate the ability of the STK-009/hoRb receptor pair to selectively enhance the anti-tumor efficacy of hoRb expressing CD19 CARs (SYNCAR-001) in preclinical lymphoma mouse models at will. We also demonstrate, in non-human primates (NHP), that STK-009 is selective for hoRb expressing cells. SYNCAR-001 + STK-009 can lead to complete responses in subcutaneous Raji mouse models, even with SYNCAR-001 dosed at sub-efficacious levels (400,000 CAR-Ts/mouse). Subcutaneous dosing of STK-009 expands SYNCAR-001 systemically and drives infiltration of SYNCAR-001 into tumors. UMAP analysis of STK-009 treated SYNCAR-001 reveal a small PD1+LAG3+ subpopulation during tumor rejection which disappeared once tumors are controlled. The majority of T cells showed no exhaustion markers. Further, an IL-7R+ population arises and remains after tumor control, indicating long term memory development. Nanostring analysis confirms upregulation of IL-7R and other critical genes involved in cytotoxic activity and persistence in SYNCAR-001 when treated with STK-009. These data demonstrate that STK-009 treatment expands SYNCAR-001 and delivers a gene signature indicative of enhanced T cell fitness and activity. Subcutaneous administration of STK-009 in NHP shows no evidence of toxicity or physiological IL-2 mediated activity on immune cells, including Teffs, Tregs, and NK cells. Pharmacokinetic analysis of STK-009 shows stable exposure with minimal clearance, demonstrating the selectivity of STK-009. These findings validate an orthogonal platform that selectively drives potent T cell effector functions of engineered cells without the toxicities mediated by NK cells or non-tumor specific T cells associated with high dose IL-2 therapy. These results demonstrate the ability of this orthogonal platform to improve the efficacy and durability of CARs. Citation Format: Paul-Joseph P. Aspuria, Sandro Vivona, Michele Bauer, Marie Semana, Navneet Ratti, Scott McCauley, Romina Riener, Rene de Waal Malefyt, Deepti Rokkam, Jan Emmerich, Patrick J. Lupardus, Rob A. Kastelein, Martin Oft. OrthoCARs: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance CAR T cell antitumor efficacy by driving T cell expansion and fitness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1512.

Geha and collaborators show that IL-23 released by keratinocytes in response to TLR4 ligands stimulates skin DCs to produce IL-23, driving CD4 T cell IL-22 secretion, which causes epidermal thickening., Atopic dermatitis (AD) is a Th2-dominated inflammatory skin disease characterized by epidermal thickening. Serum levels of IL-22, a cytokine known to induce keratinocyte proliferation, are elevated in AD, and Th22 cells infiltrate AD skin lesions. We show that application of antigen to mouse skin subjected to tape stripping, a surrogate for scratching, induces an IL-22 response that drives epidermal hyperplasia and keratinocyte proliferation in a mouse model of skin inflammation that shares many features of AD. DC-derived IL-23 is known to act on CD4+ T cells to induce IL-22 production. However, the mechanisms that drive IL-23 production by skin DCs in response to cutaneous sensitization are not well understood. We demonstrate that IL-23 released by keratinocytes in response to endogenous TLR4 ligands causes skin DCs, which selectively express IL-23R, to up-regulate their endogenous IL-23 production and drive an IL-22 response in naive CD4+ T cells that mediates epidermal thickening. We also show that IL-23 is released in human skin after scratching and polarizes human skin DCs to drive an IL-22 response, supporting the utility of IL-23 and IL-22 blockade in AD.

CAR T cell therapy has demonstrated remarkable clinical efficacy against relapsed and refractory hematological malignancies, such as B cell non-Hodgkin lymphoma (NHL) and acute lymphoblastic leukemia (ALL) (Neelapu et al. NEJM, 2017; Schuster et al. NEJM, 2018; Turtle et al. Sci Trans Med, 2016). Despite these advances, prominent barriers including poor T cell effector function, lack of proliferation, and limited CAR T cell persistence prevent CAR T cell therapies from reaching their full curative potential (Srivastava and Riddell, Journal of Immunology, 2019). Interleukin-2 (IL-2) is a potent stimulator of CD4 and CD8 T cell proliferation, survival, and cytotoxic function, thereby making it an attractive molecule to support CAR T cell therapy. However, therapeutic use of IL-2 is limited by systemic toxicity due its promiscuous activation of undesired immune cell populations, including non-tumor reactive T cells and NK cells (Rosenberg et al. Journal of Immunology, 2014). To facilitate selective ex vivo and in vivo expansion of engineered T cells we have developed a human orthogonal (ortho) ligand/receptor system consisting of a pegylated, IL-2 mutein (STK- 009) that does not significantly activate the wild type receptor and a mutated IL-2 Receptor Beta (orthoIL-2Rβ) that does not significantly respond to its native ligand, wild type IL-2. This system enables in vivo IL-2 signaling in engineered cells that express the orthoIL-2Rβ while avoiding signaling in bystander T cells and NK cells. Here, we demonstrate the ability of the STK-009/orthoIL-2Rb ligand/receptor pair to selectively potentiate human orthoIL-2Rb (hoRb) expressing CD19 CAR T cells (CD19 orthoCAR T cells) in vitro and in vivo. We also demonstrate that STK-009 is selective for the orthogonal IL-2Rb and in a non-human primate model does not potentiate wild type T or NK cells and shows no evidence of toxicity. The STK-009/CD19 orthoCAR T platform was evaluated in a disseminated Raji mouse model of aggressive lymphoma. Subcutaneous administration of STK-009 dramatically expanded the CD19 orthoCAR T cells possessing a clinically favorable TSCM and TEMRA immunophenotype and significant antitumor efficacy was observed even at doses of CAR T cells typically regarded as sub-efficacious. When STK-009 dosing was stopped after complete tumor responses, CD19 orthoCAR T cells contracted as expected. Subsequent redosing of STK-009 in these tumor free mice re-expanded CD19 orthoCAR T cell levels demonstrating the on-demand control of the STK-009/orthoCAR T cell platform. Given the deep and durable responses we observed in the disseminated Raji model, we subsequently invested investigated the efficacy of the STK-009/orthoCAR T cell platform in a subcutaneous Raji model of lymphoma characteristically resistant to CAR T cell therapy. No significant anti-tumor effect was observed in mice treated with either CAR T cells alone or the combination of high dose wild-type IL-2 and CAR T cells. The subcutaneous administration of STK-009 in combination with a sub-efficacious dose of CD19 orthoCAR T cells demonstrated significant expansion of the CD19 orthoCAR T cells with the clinically favorable TSCM and TEMRA immunophenotype and potent anti-tumor efficacy in this subcutaneous lymphoma model, demonstrating the selective potentiation of the CD19 orthoCAR T cells in response to STK-009. The toxicity of STK-009 was evaluated in a non-human primate dose-escalation study. Subcutaneous administration STK-009 at anticipated therapeutic doses showed no evidence of toxicity or biological effect on immune cells expressing the wild-type IL-2 receptor. Pharmacokinetic analysis of STK-009 in this study showed stable exposure with minimal clearance, demonstrating the selectivity of STK-009. These findings validate an orthogonal platform that selectively drives potent T cell effector functions of engineered cells without the toxicities mediated by NK cells or non-tumor specific T cells associated with high dose IL-2 therapy. These results demonstrate the ability of this orthogonal platform to improve the efficacy and durability of CAR T cell therapies. Disclosures Aspuria: Synthekine: Current Employment. Bauer:Synthekine: Current Employment. vivona:Synthekine: Current Employment. de Waal Malefyt:Synthekine: Current Employment. Kastelein:Synthekine: Current Employment. Oft:Synthekine: Current Employment. Emmerich:Synthekine: Current Employment. Rokkam:Synthekine: Current Employment. Kauder:Synthekine: Current Employment. McCauley:Synthekine: Current Employment. Riener:Synthekine: Current Employment. Verma:Synthekine: Current Employment.

T-cell activation requires co-stimulatory signals that are provided by the interaction between Immuno-Modulatory Receptor (IMR) receptors and ligands expressed on the T cells and APC or tumor cells. Hence, an antigen (Ag)-specific human T-cell clone system was developed. It has been applied not only to study the biology of IMRs, but also to characterize the different biologics targeting those molecules as single agents or in combination to test improved efficacy. Data shown will include the establishment of alloreactive human CD4+ T-cell clones, FACS profiling IMRs of T-cell clones, engineering of different antigen presenting cells transfected with IMR ligands, and application of T-cell clones in characterization of anti-PD1 and anti-Lag3 antibodies Citation Format: Sophie (Ying) Li, Sabine Le Saux, Robert Shields, Rene de Waal Malefyt. Antigen-specific human CD4+ T cell clones as tools to characterize immunomodulatory receptor antibodies for immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A96.

Recent studies have revealed IL-33 as a key factor in promoting antiviral T-cell responses. However, it is less clear as to how IL-33 regulates innate immunity. In this study, we infected wild-type (WT) and IL-33(-/-) mice with lymphocytic choriomeningitis virus and demonstrated an essential role of infection-induced IL-33 expression for robust innate IFN-γ production in the liver. We first show that IL-33 deficiency resulted in a marked reduction in the number of IFN-γ(+) γδ T and NK cells, but an increase in that of IL-17(+) γδ T cells at 16 h postinfection. Recombinant IL-33 (rIL-33) treatment could reverse such deficiency via increasing IFN-γ-producing γδ T and NK cells, and inhibiting IL-17(+) γδ T cells. We also found that rIL-33-induced type 2 innate lymphoid cells were not involved in T-cell responses and liver injury, since the adoptive transfer of type 2 innate lymphoid cells neither affected the IFN-γ and TNF-α production in T cells, nor liver transferase levels in lymphocytic choriomeningitis virus infected mice. Interestingly, we found that while IL-33 was not required for costimulatory molecule expression, it was critical for DC proliferation and cytokine production. Together, this study highlights an essential role of IL-33 in regulating innate IFN-γ-production and DC function during viral hepatitis.

Summary Interleukin-22 (IL-22)-producing group 3 innate lymphoid cells (ILC3) maintains gut homeostasis but can also promote inflammatory bowel disease (IBD). The regulation of ILC3-dependent colitis remains to be elucidated. Here we show that Foxp3+ regulatory T cells (Treg cells) prevented ILC3-mediated colitis in an IL-10-independent manner. Treg cells inhibited IL-23 and IL-1β production from intestinal-resident CX3CR1+ macrophages but not CD103+ dendritic cells. Moreover, Treg cells restrained ILC3 production of IL-22 through suppression of CX3CR1+ macrophage production of IL-23 and IL-1β. This suppression was contact dependent and was mediated by latent activation gene-3 (LAG-3)—an immune checkpoint receptor—expressed on Treg cells. Engagement of LAG-3 on MHC class II drove profound immunosuppression of CX3CR1+ tissue-resident macrophages. Our study reveals that the health of the intestinal mucosa is maintained by an axis driven by Treg cells communication with resident macrophages that withhold inflammatory stimuli required for ILC3 function.

Ischemia-reperfusion (IR) injury to the small intestine following clamping of the superior mesenteric artery results in an intense local inflammatory response that is characterized by villous damage and neutrophil infiltration. IL-17A, a cytokine produced by a variety of cells in response to inflammatory cytokines released following tissue injury, has been implicated in IR injury. Using Il17a−/−, Il23r−/−, and Rorc−/− mice and administration of anti–IL-17A and anti–IL-23 neutralizing Abs to wild-type mice, we demonstrate that intestinal IR injury depends on IL-17A and that IL-17A is downstream of the binding of autoantibody to ischemia-conditioned tissues and subsequent complement activation. Using bone marrow chimeras, we demonstrate that the IL-17A required for intestinal IR injury is derived from hematopoietic cells. Finally, by transferring autoantibody-rich sera into Rag2γc−/− and Rag2−/− mice, we demonstrate that innate lymphoid cells are the main producers of IL-17A in intestinal IR injury. We propose that local production of IL-17A by innate lymphoid cells is crucial for the development of intestinal IR injury and may provide a therapeutic target for clinical exploitation.

Metallophilic macrophages hold a strategic position within the thymic tissue and play a considerable function in thymic physiology. The development and positioning of these cells within thymic tissue are regulated by complex molecular mechanisms involving different cytokine/chemokine axes. Herein, we studied the role of XCL1 signaling in these processes. We show that in the XCL1-deficient thymus numerous metallophilic macrophages are aberrantly positioned in the thymic cortex, instead of their normal location in the cortico-medullary zone. Still, these cells retain their normal appearance: very large size with prominent, ramifying cytoplasmic prolongations. This shows that XCL1 signaling is not involved in morphological development, but rather in correct positioning of metallophilic macrophages within the thymic tissue. In contrast to thymic metallophilic macrophages, the positioning of splenic marginal metallophilic macrophages is not affected by XCL1-deficiency. Anat Rec, 297:1472–1477, 2014. © 2014 Wiley Periodicals, Inc.

Interleukin-22 (IL-22)-producing group 3 innate lymphoid cells (ILC3) maintains gut homeostasis but can also promote inflammatory bowel disease (IBD). The regulation of ILC3-dependent colitis remains to be elucidated. Here we show that Foxp3

CD4 + T cells have been shown to be essential for vaccine-induced protection against Helicobacter pylori infection in mice. Less is known about the relative contributions of individual cell subpopulations, such as T h 1 and T h 17 cells, and their associated cytokines. The aim of the present study was to find immune correlates to vaccine-induced protection and further study their role in protection against H. pylori infection. Immunized and unimmunized mice were challenged with H. pylori , and immune responses were compared. Vaccine-induced protection was assessed by measuring H. pylori colonization in the stomach. Gastric gene expression of T h 1- and/or T h 17-associated cytokines was analyzed by quantitative PCR, and contributions of individual cytokines to protection were evaluated by antibody-mediated in vivo neutralization. By analyzing immunized and unimmunized mice, a significant inverse correlation between the levels of interleukin-12p40 (IL-12p40), tumor necrosis factor alpha (TNF), gamma interferon (IFN-γ), and IL-17 gene expression and the number of H. pylori bacteria in the stomachs of individual animals after challenge could be demonstrated. In a kinetic study, upregulation of TNF, IFN-γ, and IL-17 coincided with vaccine-induced protection at 7 days after H. pylori challenge and was sustained for at least 21 days. In vivo neutralization of these cytokines during the effector phase of the immune response revealed a significant role for IL-17, but not for IFN-γ or TNF, in vaccine-induced protection. In conclusion, although both T h 1- and T h 17-associated gene expression in the stomach correlate with vaccine-induced protection against H. pylori infection, our study indicates that mainly T h 17 effector mechanisms are of critical importance to protection.

Aire regulates medullary epithelial cell production of XCL1, a chemoattractant for XCR1-expressing thymic DCs whose presence in the medulla contributes to the generation of T reg cells., Dendritic cells (DCs) in the thymus (tDCs) are predominantly accumulated in the medulla and contribute to the establishment of self-tolerance. However, how the medullary accumulation of tDCs is regulated and involved in self-tolerance is unclear. We show that the chemokine receptor XCR1 is expressed by tDCs, whereas medullary thymic epithelial cells (mTECs) express the ligand XCL1. XCL1-deficient mice are defective in the medullary accumulation of tDCs and the thymic generation of naturally occurring regulatory T cells (nT reg cells). Thymocytes from XCL1-deficient mice elicit dacryoadenitis in nude mice. mTEC expression of XCL1, tDC medullary accumulation, and nT reg cell generation are diminished in Aire-deficient mice. These results indicate that the XCL1-mediated medullary accumulation of tDCs contributes to nT reg cell development and is regulated by Aire.

Improving clinical outcomes using immunotherapeutics in cancer treatments will depend on a variety of factors including the composition and phenotype of effector T cell in tumors, the inhibitory factors from myeloid, tumor and stromal compartments of the tumor and selection of efficacious combination of therapeutic immunomodulatory agents that either neutralize immune checkpoint inhibitors or activate T cell costimulatory pathways. To address the issue of selecting optimal combinations of immune modulating agents we developed an in-vitro culture system for allo-stimulating and expanding tumor infiltrating lymphocytes (TILS) from tumor digests with an MHC Class II alloantigen expressing cell line. We selected allo-specific CD4 and CD8 clones from expanded TILS with an exhausted phenotype confirmed by cell surface expression of PD-1, LAG3 and TIGIT. T cell clones were incubated with the alloantigen expressing cell lines transduced with different combinations of immodulatory ligands and compared the effect of single agent versus combinations of checkpoint inhibitory monoclonal antibodies on T cell proliferation and cytokine production. Taqman and flow cytometry analysis was performed on several clones following allo-stimulation. A decrease in TIL clonality with repeated allo-stimulation was confirmed by TcR beta chain sequencing. In-vitro IFN gamma and Granzyme B production was enhanced with anti-PD-1 antibodies in combination with either anti-LAG3 or anti-TIGIT antibodies. This in-vitro system has the potential of screening combinations of immodulatory antibodies in a variety of TILS from different tumor types and can improve our understanding of new checkpoint inhibitor combinations. Citation Format: Robert L. Shields, Sabine Le Saux, Sophie Li, Manjiri Sathe, Terri McClanahan, Rene De Waal Malefyt. Tumor derived T cell clones for evaluation of check point inhibitor therapeutics [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A33.

Th17 cells have been named after their signature cytokine IL-17 and accumulating evidence indicates their involvement in the induction and progression of inflammatory diseases. In addition to IL-17 single-producing T cells, IL-17/IFN-γ double-positive T cells are found in significantly elevated numbers in inflamed tissues or blood from patients with chronic inflammatory disorders. Because IFN-γ is the classical Th1-associated cytokine, the origin and roles of these subsets remain elusive. In this paper, we show that not only IL-17+/IFN-γ+ but also IFN-γ+ (IL-17−) cells arise under Th17-inducing condition and have distinct properties from the Th1 lineage. In fact, these populations displayed characteristics reminiscent to IL-17 single-producing cells, including production of IL-22, CCL20, and induction of antimicrobial gene expression from epithelial cells. Live sorted IL-17+ and Th17–IFN-γ+ cells retained expression of IL-17 or IFN-γ after culture, respectively, whereas the IL-17+/IFN-γ+ population was less stable and could also become IL-17 or IFN-γ single-producing cells. Interestingly, these Th17 subsets became “Th1-like” cells in the presence of IL-12. These results provide novel insights into the relationship and functionality of the Th17 and Th1 subsets and have direct implications for the analysis and relevance of IL-17 and/or IFN-γ–producing T cells present in patients’ peripheral blood and inflamed tissues.

Naturally occurring regulatory T cells (Treg) express high levels of glucocorticoid-induced tumour necrosis factor receptor (GITR). However, studies of the role of GITR in Treg biology has been complicated by the observation that upon activation effector CD4(+) T (Teff) cells also express the receptor. Here, we dissect the contribution of GITR-induced signaling networks in the expansion and function of FoxP3(+) Treg. We demonstrate that a high-affinity soluble Fc-GITR-L dimer, in conjugation with alphaCD3, specifically enhances in vitro proliferation of Treg, which retain their phenotypic markers (CD25 and FoxP3) and their suppressor function, while minimally affecting Teff cells. Furthermore, Fc-GITR-L does not impair Teff susceptibility to suppression, as judged by cocultures employing GITR-deficient and GITR-sufficient CD4(+) T-cell subsets. Notably, this expansion of Treg could also be seen in vivo, by injecting FoxP3-IRES-GFP mice with Fc-GITR-L even in the absence of antigenic stimulation. In order to test the efficacy of these findings therapeutically, we made use of a C3H/HeJ hemophilia B-prone mouse model. The use of liver-targeted human coagulation factor IX (hF.IX) gene therapy in this model has been shown to induce liver toxicity and the subsequent failure of hF.IX expression. Interestingly, injection of Fc-GITR-L into the hemophilia-prone mice that were undergoing liver-targeted hF.IX gene therapy increased the expression of F.IX and reduced the anticoagulation factors. We conclude that GITR engagement enhances Treg proliferation both in vitro and in vivo and that Fc-GITR-L may be a useful tool for in vivo tolerance induction.

T(H)17 cells are the newest member of the T(H) cell family and are characterized by their ability to produce specific cytokines such as IL-17, IL-22, IL-17F, and CCL20. In this review, conditions for the differentiation of T(H)17 cells are defined in both murine and human systems, with discussion of T(H)17-specific cytokines and transcription factors. Functionally, T(H)17 cells contribute to host defense as a new effector T(H) cell subset with a role in protection against extracellular bacteria through activities on immune and nonimmune cells. Their activities, however, are also pivotal in the development of autoimmune diseases under pathologic conditions. T(H)17 cells are also beginning to be associated with the development and pathophysiology of allergic diseases, such as allergic contact dermatitis, atopic dermatitis, and asthma. Lymphoid tissue inducer-like cells and natural killer-like cells, termed RORgammat(+)NKp46(+) or NK-22 cells, might also play a role in allergic diseases because of their propensity to produce IL-17 and IL-22.

Prostaglandins, particularly prostaglandin E2 (PGE2), play an important role during inflammation. This is exemplified by the clinical use of cyclooxygenase 2 inhibitors, which interfere with PGE2 synthesis, as effective antiinflammatory drugs. Here, we show that PGE2 directly promotes differentiation and proinflammatory functions of human and murine IL-17–producing T helper (Th17) cells. In human purified naive T cells, PGE2 acts via prostaglandin receptor EP2- and EP4-mediated signaling and cyclic AMP pathways to up-regulate IL-23 and IL-1 receptor expression. Furthermore, PGE2 synergizes with IL-1β and IL-23 to drive retinoic acid receptor–related orphan receptor (ROR)-γt, IL-17, IL-17F, CCL20, and CCR6 expression, which is consistent with the reported Th17 phenotype. While enhancing Th17 cytokine expression mainly through EP2, PGE2 differentially regulates interferon (IFN)-γ production and inhibits production of the antiinflammatory cytokine IL-10 in Th17 cells predominantly through EP4. Furthermore, PGE2 is required for IL-17 production in the presence of antigen-presenting cells. Hence, the combination of inflammatory cytokines and noncytokine immunomodulators, such as PGE2, during differentiation and activation determines the ultimate phenotype of Th17 cells. These findings, together with the altered IL-12/IL-23 balance induced by PGE2 in dendritic cells, further highlight the crucial role of the inflammatory microenvironment in Th17 cell development and regulation.

IL-10 is a potent immunomodulatory cytokine that affects innate and acquired immune responses. The immunological consequences of IL-10 production during pulmonary tuberculosis (TB) are currently unknown, although IL-10 has been implicated in reactivation TB in humans and with TB disease in mice. Using Mycobacterium tuberculosis-susceptible CBA/J mice, we show that blocking the action of IL-10 in vivo during chronic infection stabilized the pulmonary bacterial load and improved survival. Furthermore, this beneficial outcome was highly associated with the recruitment of T cells to the lungs and enhanced T cell IFN-γ production. Our results indicate that IL-10 promotes TB disease progression. These findings have important diagnostic and/or therapeutic implications for the prevention of reactivation TB in humans.

Naive B cells can alter the effector function of their Ig molecule by isotype switching, thereby allowing them to secrete not only IgM, but also the switched isotypes IgG, IgA, and IgE. Different isotypes are elicited in response to specific pathogens. Similarly, dysregulated production of switched isotypes underlies the development of various diseases, such as autoimmunity and immunodeficiency. Thus, it is important to characterize mediators controlling isotype switching, as well as their contribution to the overall B cell response. Isotype switching in human naive B cells can be induced by CD40L together with IL-4, IL-10, IL-13, and/or TGF-β. Recently, IL-21 was identified as a switch factor for IgG1 and IgG3. However, the effect of IL-21 on switching to IgA, as well as the interplay between IL-21 and other switch factors, remains unknown. We found that IL-4 and IL-21 individually induced CD40L-stimulated human naive B cells to undergo switching to IgG, with IL-4 predominantly inducing IgG1+ cells and IL-21 inducing IgG3. Culture of naive B cells with CD40L and IL-21, but not IL-4, also yielded IgA+ cells. Combining IL-4 and IL-21 had divergent effects on isotype switching. Specifically, while IL-4 and IL-21 synergistically increased the generation of IgG1+ cells from CD40L-stimulated B cells, IL-4 concomitantly abolished IL-21-induced switching to IgA. Our findings demonstrate the dynamic interplay between IL-4 and IL-21 in regulating the production of IgG subclasses and IgA, and suggest temporal roles for these cytokines in humoral immune responses to specific pathogens.

mAb: Monoclonal antibody; STAT: Signal transducer and activator of transcription.Approximately 8 years ago, the research efforts of the immunology group in Paolo Alto (CA, USA) was focused on the c...

Pulmonary arterial remodeling characterized by increased vascular smooth muscle density is a common lesion seen in pulmonary arterial hypertension (PAH), a deadly condition. Clinical correlation studies have suggested an immune pathogenesis of pulmonary arterial remodeling, but experimental proof has been lacking. We show that immunization and prolonged intermittent challenge via the airways with either of two different soluble antigens induced severe muscularization in small- to medium-sized pulmonary arteries. Depletion of CD4+ T cells, antigen-specific T helper type 2 (Th2) response, or the pathogenic Th2 cytokine interleukin 13 significantly ameliorated pulmonary arterial muscularization. The severity of pulmonary arterial muscularization was associated with increased numbers of epithelial cells and macrophages that expressed a smooth muscle cell mitogen, resistin-like molecule α, but surprisingly, there was no correlation with pulmonary hypertension. Our data are the first to provide experimental proof that the adaptive immune response to a soluble antigen is sufficient to cause severe pulmonary arterial muscularization, and support the clinical observations in pediatric patients and in companion animals that muscularization represents one of several injurious events to the pulmonary artery that may collectively contribute to PAH.

Chronic obstructive pulmonary diseases (COPD) may increase air pollution-related mortality. The relationship of immune mechanisms to mortality caused by fine particulates in healthy and COPD populations is incompletely understood. The objective of this study was to determine whether fine particulates from a single biomass fuel alter stress and inflammation biomarkers in people with COPD. Healthy and COPD subjects were exposed to smoke in a controlled indoor setting. Immune responses were quantified by measuring cell surface marker expression with flow-cytometric analysis and mRNA levels with quantitative reverse transcriptase-polymerase chain reactions in whole blood before and after exposure. Preexposure COPD subjects had more leukocytes, mainly CD14(+) monocytes and neutrophils, but fewer CD3(+) T cells. Fifty-seven of 186 genes were differentially expressed between healthy and COPD subjects' peripheral blood mononuclear cells (PBMCs). Of these, only nuclear factor (NF)-kappa B1, TIMP-1, TIMP-2, and Duffy genes were up-regulated in COPD subjects. At 4 hours post smoke exposure, monocyte levels decreased only in healthy subjects. Fifteen genes, particular to inflammation, immune response, and cell-to-cell signaling, were differentially expressed in COPD subjects, versus 4 genes in healthy subjects. The authors observed significant differences in subjects' PBMCs, which may elucidate the adverse effects of air pollution particulates on people with COPD.

Eosinophilic esophagitis (EoE) is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP), an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC), we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf), inactive EoE (

Differentiation of B cells into Ig-secreting cells (ISC) is critical for the generation of protective humoral immune responses. Because of the important role played by secreted Ig in host protection against infection, it is necessary to identify molecules that control B cell differentiation. Recently, IL-21 was reported to generate ISC from activated human B cells. In this study, we examined the effects of IL-21 on the differentiation of all human mature B cell subsets—neonatal, transitional, naive, germinal center, IgM-memory, and isotype-switched memory cells—into ISC and compared its efficacy to that of IL-10, a well-known mediator of human B cell differentiation. IL-21 rapidly induced the generation of ISC and the secretion of vast quantities IgM, IgG and IgA from all of these B cell subsets. Its effect exceeded that of IL-10 by up to 100-fold, highlighting the potency of IL-21 as a B cell differentiation factor. Strikingly, IL-4 suppressed the stimulatory effects of IL-21 on naive B cells by reducing the expression of B-lymphocyte induced maturation protein-1 (Blimp-1). In contrast, memory B cells were resistant to the inhibitory effects of IL-4. Finally, the ability of human tonsillar CD4+CXCR5+CCR7− T follicular helper (TFH) cells, known to be a rich source of IL-21, to induce the differentiation of autologous B cells into ISC was mediated by the production of IL-21. These findings suggest that IL-21 produced by TFH cells during the primary as well as the subsequent responses to T cell-dependent Ag makes a major contribution to eliciting and maintaining long-lived humoral immunity.

T(H)-17 cells are a distinct lineage of proinflammatory T helper cells that are essential for autoimmune disease. In mice, commitment to the T(H)-17 lineage is dependent on transforming growth factor-beta and interleukin 6 (IL-6). Here we demonstrate that IL-23 and IL-1beta induced the development of human T(H)-17 cells expressing IL-17A, IL-17F, IL-22, IL-26, interferon-gamma, the chemokine CCL20 and transcription factor RORgammat. In situ, T(H)-17 cells were identified by expression of the IL-23 receptor and the memory T cell marker CD45RO. Psoriatic skin lesions contained IL-23-producing dendritic cells and were enriched in the cytokines produced by human T(H)-17 cells that promote the production of antimicrobial peptides in human keratinocytes. Our data collectively indicate that human and mouse T(H)-17 cells require distinct factors during differentiation and that human T(H)-17 cells may regulate innate immunity in epithelial cells.

Dendritic cells (DCs) are professional antigen-presenting cells that have the ability to sense infection and tissue stress, sample and present antigen to T lymphocytes, and induce different forms of immunity and tolerance. The functional versatility of DCs depends on their remarkable ability to translate collectively the information from both the invading microbes and their resident tissue microenvironments and then make an appropriate immune response. Recent progress in understanding TLR biology has illuminated the mechanisms by which DCs link innate and adaptive antimicrobial immune responses. However, how tissue microenvironments shape the function of DCs has remained elusive. Recent studies of TSLP (thymic stromal lymphopoietin), an epithelial cell–derived cytokine that strongly activates DCs, provide evidence at a molecular level that epithelial cells/tissue microenvironments directly communicate with DCs. We review recent progress on how TSLP expressed within thymus and peripheral lymphoid and nonlymphoid tissues regulates DC-mediated central tolerance, peripheral T cell homeostasis, and inflammatory Th2 responses.

Meeting abstracts LAG-3 has been shown to act as an inhibitory molecule involved in the regulation of T cell activation, proliferation and homeostasis. Exhausted T cell populations that evolve in the tumor microenvironment or during chronic viral infections show coordinated expression of LAG-3 and

Background: Thymic stromal lymphopoietin (TSLP) is an attractive therapeutic target for the treatment of allergic diseases, and plasma TSLP is a potential patient selection marker in the development of therapeutic agents. Results: We developed and validated an ultrasensitive electrochemiluminescence assay for measurement of TSLP in plasma with a lower limit of quantitation of 0.12 pg/ml, which allowed the quantitation of TSLP in approximately 90% of human plasma samples tested. The assay demonstrated excellent performance characteristics, including precision, accuracy, sensitivity and dilution linearity. Stability and biological variability of TSLP in plasma were also assessed for clinical sample analysis and data interpretation. Conclusion: The validated TSLP assay enables assessment of circulating TSLP as a patient selection marker in the development of therapeutics to treat atopic diseases.

Immunoglobulin E (IgE)-mediated late-phase reactions can be induced in atopic humans by intradermal injection of relevant allergens or anti-IgE antibodies. The histology of these reactions resembles that of naturally occurring atopic dermatitis. Strikingly similar responses can be induced in dogs, suggesting that a canine model could prove valuable for preclinical investigation of drugs targeting late-phase reactions. This study was designed to characterize the cellular, cytokine and chemokine responses after intradermal anti-IgE injection in untreated and prednisolone-treated dogs. Normal beagles were untreated or treated with prednisolone before intradermal injection of polyclonal rabbit anti-canine IgE or normal rabbit IgG. Biopsies were taken before injection and 6, 24 and 48 hr after injection. Samples were evaluated by histological and immunohistochemical staining, as well as by real-time quantitative polymerase chain reaction analysis. Dermal eosinophil and neutrophil numbers increased dramatically within 6 hr after injection of rabbit anti-canine IgE, and remained moderately elevated at 48 hr. The numbers of CD1c(+) and CD3(+) mononuclear cells were also increased at 6 hr. The real-time quantitative polymerase chain reaction demonstrated marked increases in mRNA expression for interleukin-13 (IL-13), CCL2, CCL5 and CCL17. Levels of mRNA for IL-2, IL-4, IL-6 and IFN-gamma did not change within the limits of detection. Prednisolone administration suppressed the influx of neutrophils, eosinophils, CD1c(+) and CD3(+) cells, as well as expression of IL-13, CCL2, CCL5 and CCL17. These data document the cytokine and chemokine responses to anti-IgE injection in canine skin, and they demonstrate the ability of the model to characterize the anti-inflammatory effects of a known therapeutic agent.