Negative Regulator Of T-cell Activation Research Articles

Investigation of the Relationship between PTPN22 Polymorphisms and Lung Sarcoidosis: A cross sectional study

Objective: Sarcoidosis is a systemic granulomatous disease with an unknown etiology characterized by noncaseating granuloma formation. It most often affects the lungs. Protein tyrosine phosphatase non-receptor 22 (PTPN22) is a gene that acts as a negative regulator of T-cell activation. Polymorphisms of this gene are associated with multiple human autoimmune diseases. The aim of this study was to assess the relationship between PTPN22 polymorphisms and lung sarcoidosis in a selected population.
 Methods: The study included 64 patients with lung sarcoidosis and 30 healthy controls. Patients were genotyped to determine two single nucleotide polymorphisms (SNPs) using the polymerase chain reaction (PCR) method. Hardy–Weinberg equilibrium (HWE), an important tool for detecting genotyping errors, was tested. Direct counting was used to estimate genotype frequencies. Logistic regression analysis was performed to compare allele and genotype frequencies between the patient and control groups.
 Results: The results of the study showed no correlation between lung sarcoidosis and the tested two SNPs of the PTPN22 gene (rs2488457, rs1310182) (p>0.05). The categorization analysis according to the clinical features, laboratory, and radiographic characteristics showed no correlation between the tested polymorphism of PTPN22 and these characteristics (p>0.05). 
 Conclusion: The present study demonstrated that the studied two SNPs of the PTPN22 gene (rs2488457, rs1310182) were not correlated with lung sarcoidosis, suggesting that it might be different from other classic autoimmune disorders. There is a need for more studies to verify these results concerning lung sarcoidosis in other ethnic origins.

393-P: Acute Hypoglycemia Alters Serum Immune Mediators Associated with Macrophage and T-Cell Activation in Type 2 Diabetes

Objective: Hypoglycemia complicates intensive glycemia regimens in type 2 diabetes (T2D). Hyperglycemia induces macrophage priming and dysregulates T-cell activation/function. Immune cell response to acute hypoglycemia has not been investigated in T2D. Thus, immune mediators associated with macrophage/T-cell activation in response to insulin-induced hypoglycemia were studied. Methods: Hypoglycaemia (<2.2mmol/l; <40mg/dl) was achieved in T2D (n=23) and controls (n=23) by intravenous insulin infusion. Somalogic analysis of plasma proteins related to macrophage/T-cell activation was performed at baseline, hypoglycemia and post-hypoglycemia to 24 hours. Results: IL-4 and IL-13 (promotors of alternative macrophage activation (M2 macrophages)) decreased 2hours post-hypoglycemia in T2D (p<0.05). Co-stimulatory molecules of T-cell activation expressed in antigen presenting cells (APCs), CD80 and CD86, were decreased at 2h post-hypoglycemia (CD86 in T2D, p<0.05; CD80 in controls and T2D, p<0.05). Hypoglycemia did not affect ligands of co-stimulatory molecules, CTLA4 (ligand of CD80, expressed in APCs) and CD40LG (ligand of CD40, expressed in dendritic cells). The negative regulator of T-cell activation, IL-10, was decreased at 2h (p<0.001) and increased at 4h post-hypoglycemia (p<0.01). Elevation of CD80 (p<0.05) and CD86 (p<0.05), with increased T-cell proliferator IL-6 (p<0.05) was seen in T2D at 4h post-hypoglycemia. Positive correlations of IL10 with IL6, IL4, were observed at 2h post-hypoglycemia in T2D, suggesting a role for IL10 in altering immune cell activation mediators following hypoglycemia in T2D. No correlations with glucagon suggest changes are independent of glucose counter-regulation. Conclusion: Induced hypoglycemia is associated with decreases in macrophage and T-cell activation mediators (cytokines) in T2D. Thus, this study reveals a novel link between hypoglycemia and inefficient immune system activation. Disclosure A.S.Moin: None. T.Sathyapalan: Other Relationship; Novo Nordisk, Abbott Diabetes, Research Support; Abbott Diabetes, Novo Nordisk, Eli Lilly and Company. S.Atkin: None. A.E.Butler: None.

Delayed CD4 +T-cell response in older adults is associated with reduced immunogenicity and reactogenicity after COVID-19 mRNA vaccination

Abstract While the newly developed COVID-19 mRNA vaccines are highly effective, characteristics of the immune response after the vaccination and its association with the adverse effects, especially for an older population, remain elusive. In this study, we recruited a total of 216 SARS-CoV-2-naïve healthy donors of various ages (23–81 years) who received two doses of the BNT162b2 vaccine and assessed age-associated differences in vaccine-induced T-cell responses and adverse effects over 3 months. We observed that older adults (≥65 years) had fewer vaccine-induced Spike-specific CD4 +T cells, including CXCR3 +cTfh and Th1 cells, after the first dose, which correlated with their lower peak IgG levels and fewer systemic adverse effects after the second dose, compared with younger adults. Moreover, Spike-specific Th1 cells in older adults expressed higher levels of PD-1, a negative regulator of T-cell activation, which was associated with low Spike-specific CD8 +T-cell response. These data indicated that delayed induction of CD4 +T-cell response in older adults is linked to impaired humoral and cellular immunity and reduced systemic reactogenicity. Therefore, enhancing CD4 +T-cell response following the first dose is key to improving vaccine efficacy in older adults. This work was supported by the Japan Agency for Medical Research and Development (grant numbers JP21gm5010005, JP20fk0108454, and JP223fa627009; Y.H.), iPS Cell Research Fund (Y.H.), the COVID-19 Private Fund to the Shinya Yamanaka Laboratory, CiRA, Kyoto University (Y.H.), Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (grant number 19K23862; N.J.), Kansai Economic Federation (KANKEIREN; Y.H.), Sumitomo-Mitsui Trust Bank Fund for Covid-19 Research, Kyoto University (Y.H.), and Takeda Science Foundation (Y.H.).

Discovery of Macrocycle-Based HPK1 Inhibitors for T-Cell-Based Immunotherapy.

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T-cell activation, and targeting HPK1 is considered a promising strategy for improving responses to antitumor immune therapies. The biggest challenge of HPK1 inhibitor design is to achieve a higher selectivity to GLK, an HPK1 homology protein as a positive regulator of T-cell activation. Herein, we report the design of a series of macrocycle-based HPK1 inhibitors via a conformational constraint strategy. The identified candidate compound 5i exhibited HPK1 inhibition with an IC50 value of 0.8 nM and 101.3-fold selectivity against GLK. Compound 5i also displayed good oral bioavailability (F = 27-49%) in mice and beagles and favorable metabolic stability (T1/2 > 186.4 min) in human liver microsomes. More importantly, compound 5i demonstrated a clear synergistic effect with anti-PD-1 in both MC38 (MSI) and CT26 (MSS) syngeneic tumor mouse models. These results showed that compound 5i has a great potential in immunotherapy.

Distinct Dynamics of Migratory Response to PD-1 and CTLA-4 Blockade Reveals New Mechanistic Insights for Potential T-Cell Reinvigoration following Immune Checkpoint Blockade.

Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), two clinically relevant targets for the immunotherapy of cancer, are negative regulators of T-cell activation and migration. Optimizing the therapeutic response to CTLA-4 and PD-1 blockade calls for a more comprehensive insight into the coordinated function of these immune regulators. Mathematical modeling can be used to elucidate nonlinear tumor-immune interactions and highlight the underlying mechanisms to tackle the problem. Here, we investigated and statistically characterized the dynamics of T-cell migration as a measure of the functional response to these pathways. We used a previously developed three-dimensional organotypic culture of patient-derived tumor spheroids treated with anti-CTLA-4 and anti-PD-1 antibodies for this purpose. Experiment-based dynamical modeling revealed the delayed kinetics of PD-1 activation, which originates from the distinct characteristics of PD-1 and CTLA-4 regulation, and followed through with the modification of their contributions to immune modulation. The simulation results show good agreement with the tumor cell reduction and active immune cell count in each experiment. Our findings demonstrate that while PD-1 activation provokes a more exhaustive intracellular cascade within a mature tumor environment, the time-delayed kinetics of PD-1 activation outweighs its preeminence at the individual cell level and consequently confers a functional dominance to the CTLA-4 checkpoint. The proposed model explains the distinct immunostimulatory pattern of PD-1 and CTLA-4 blockade based on mechanisms involved in the regulation of their expression and may be useful for planning effective treatment schemes targeting PD-1 and CTLA-4 functions.

Forced expression of the non-coding RNA miR-17∼92 restores activation and function in CD28-deficient CD4+ T cells

CD28 provides the prototypical costimulatory signal required for productive T-cell activation. Known molecular consequences of CD28 costimulation are mostly based on studies of protein signaling molecules. The microRNA cluster miR-17∼92 is induced by Tcell receptor stimulation and further enhanced by combined CD28 costimulation. We demonstrate that transgenic miR-17∼92 cell-intrinsically largely overcomes defects caused by CD28 deficiency. Combining genetics, transcriptomics, bioinformatics, and biochemical miRNA:mRNA interaction maps we empirically validate miR-17∼92 target genes that include several negative regulators of Tcell activation. CD28-deficient Tcells exhibit derepressed miR-17∼92 target genes during activation. CRISPR/Cas9-mediated ablation of the miR-17∼92 targets Pten and Nrbp1 in naive CD28-/- CD4+ Tcells differentially increases proliferation and expression of the activation markers CD25 and CD44, respectively. Thus, we propose that miR-17∼92 constitutes a central mediator for Tcell activation, integrating signals by the TCR and CD28 costimulation by dampening multiple brakes that prevent Tcell activation.

Immune checkpoint inhibitors in advanced and recurrent/metastatic cervical cancer

Cervical cancer (CC) poses a serious threat to women’s health. Although many early-stage patients have a good prognosis, there are still a lack of effective therapies for advanced and recurrent/metastatic CC. In this context, immunotherapy and immune checkpoint inhibitors (ICIs) are particularly likely to play a role in the treatment of cervical tumors in a variety of disease settings. Some promising immune checkpoints include programmed cell death 1 (PD-1), programmed death ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4), which exert immunomodulatory effects as negative regulators of T-cell activation and suppress immune responses in cervical cancer through cancer cell immune evasion. Initial trials of ICIs for CC have shown encouraging results in terms of objective response rate (ORR), progression-free survival (PFS), and overall survival (OS), both monotherapy and combination strategies. Meanwhile, human papillomavirus, vaginal microecology and intestinal microenvironment play an important role in CC, which provides new treatment directions. This review analyzed a number of completed or ongoing clinical trials of ICIs in the treatment of advanced and recurrent/metastatic CC. And we also analyzed the important relationship between vaginal microecology and intestinal microecology with CC and their related immunotherapy prospects.

Comparative Analysis of the Antitumor Immune Profiles of Paired Radiotherapy-naive and Radiotherapy-treated Cervical Cancer Tissues.

This study aimed to elucidate the effect of radiotherapy on expression of immune response-related genes in cervical cancer tissues. Tumor tissues were obtained from 16 patients with cervical cancer before initiation of radiotherapy and after treatment with 10 Gy X-rays, delivered in five fractions. Expression of 730 immune response-related genes was assessed using an nCounter PanCancer Immune Profiling Panel (NanoString Technologies. Seattle, WA, USA). Of the 730 genes examined, 41 showed significant changes (fold change of >1.5 or <0.66) in expression in post-radiotherapy samples (28 up-regulated and 13 down-regulated). Analysis of immune cell type-specific genes suggested predominant upregulation of those related to innate immunity postradiotherapy. Interestingly, cytotoxic T-lymphocyte-associated protein (CTLA4), a key negative regulator of T-cell activation, was marked down-regulated in 93.7% of patients, with an average fold-change of 2.0. To our knowledge, this study is the first to show down-regulation of CTLA4 in clinical cervical cancer tissues after treatment with radiotherapy.

Insight Into Biological Targets and Molecular Mechanisms in the Treatment of Arsenic-Related Dermatitis With Vitamin A via Integrated in silico Approach

Exposure to arsenic (As), an inorganic poison, may lead to skin lesions, including dermatitis. Vitamin A (VA), a fat-soluble vitamin essential for mucous membrane integrity, plays a key role in skin protection. Although the beneficial actions of VA are known, the anti-As-related dermatitis effects of VA action remain unclear. Hence, in this study, we aimed to interpret and identify the core target genes and therapeutic mechanisms of VA action in the treatment of As-related dermatitis through integrated in silico approaches of network pharmacology and molecular docking. We integrated the key VA-biological target-signaling pathway-As-related dermatitis networks for identifying core drug targets and interaction pathways associated with VA action. The network pharmacology data indicated that VA may possess potential activity for treating As-related dermatitis through the effective regulation of core target genes. An enrichment analysis in biological processes further revealed multiple immunoregulation-associated functions, including interferon-gamma production and negative regulation of T-cell activation and production of molecular mediator of immune response. An enrichment analysis in molecular pathways mainly uncovered multiple biological signaling, including natural killer cell mediated cytotoxicity, autophagy, apoptosis, necroptosis, platelet activation involved in cell fate, and immunity regulations. Molecular docking study was used to identify docked well core target proteins with VA, including Jun, tumor protein p53 (TP53), mitogen-activated protein kinase-3 (MAPK3), MAPK1, and MAPK14. In conclusion, the potential use of VA may suppress the inflammatory stress and enhance the immunity against As-related dermatitis. In the future, VA might be useful in the treatment of dermatitis associated with As through multi-targets and multi-pathways in clinical practice.

Investigating CTLA‐4 binding to generate novel small‐molecule inhibitor

The CTLA‐4 receptor may be found on the plasma membranes of T‐cells where it acts as a negative regulator of T‐cell activation. When CTLA‐4 receptors bind B7 ligands (CD80/86) found on antigen presenting cells, they activate signaling pathways which, in combination with signaling from T‐cell receptors bound to antigen‐carrying MHC proteins, decrease the inflammatory response. Regulatory T‐cells (Tregs) constitutively express CTLA‐4 on their membranes and have T‐cell receptors that recognize self‐antigens; this allows them to moderate the self‐tolerance of the immune system. Specifically, CTLA‐4 signaling in Tregs leads to the release of anti‐inflammatory cytokines which decrease the proliferation of helper and cytotoxic T‐cells and prevent monocytes, macrophage precursors, from differentiating into macrophages that would produce pro‐inflammatory cytokines. Unfortunately, it is frequently the case that Tregs recognizing cancerous cells accumulate within tumors. This leads to the inability of the body to form an immune reaction against the cancer. For this reason, cancer therapies which inhibit CTLA‐4 signaling are being developed. Current strategies to limit CTLA‐4 signaling have been successful in treating multiple forms of cancer but primarily rely on reducing CTLA‐4 interactions with B7 through monoclonal antibody therapy targeting the extracellular domain of CTLA‐4. Monoclonal antibodies are not particularly convenient as they must be administered through IV infusion and some formulations carry the risk of inducing serum‐sickness, in which the immune system develops a reaction against the foreign anti‐CTLA4 antibodies. However, instead of inhibiting CTLA‐4 binding to B7 with an antibody, it may be possible to inhibit the intracellular signaling of CTLA‐4 that occurs after this binding with a small molecule that is easier to administer. One of the main proteins that binds to and is activated by stimulated CTLA4 receptors is the phosphatase SHP2. SHP2 uses an SH2 domain at its N‐terminus to bind CTLA‐4 through the YVKM motif of the latter. Binding causes a conformational change in the SH2 domain which makes the phosphatase active site on an adjacent domain of SHP2 more accessible. This conformational change also causes SHP2 to unbind from CTLA‐4 so that a new protein can take its place to be activated. Activated SHP2 dephosphorylates many cytoplasmic proteins at their tyrosine residues leading to Treg activation and thus decreased inflammatory response.

Application of a patient-centered reverse translational systems-based approach to understand mechanisms of an adverse drug reaction of immune checkpoint inhibitors.

Immunotherapy became a key pillar of cancer therapeutics with the approvals of ipilimumab, nivolumab, and pembrolizumab, which inhibit either cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) or programmed death‐1 (PD‐1) that are negative regulators of T‐cell activation. However, boosting T‐cell activation is often accompanied by autoimmunity, leading to adverse drug reactions (ADRs), including high grade 3–4 colitis and its severe complications whose prevalence may reach 14% for combination checkpoint inhibitors. In this research, we investigated how mechanistic differences between anti‐CTLA‐4 (ipilimumab) and anti‐PD‐1 (nivolumab and pembrolizumab) affect colitis, a general class toxicity. The data analytical platform Molecular Health Effect was utilized to map population ADR data from the US Food and Drug Administration (FDA) Adverse Event Reporting System to chemical and biological databases for hypothesis generation regarding the underlying molecular mechanisms causing colitis. Disproportionality analysis was used to assess the statistical relevance between adverse events of interest and molecular causation. We verified that the anti‐CTLA‐4 drug is associated with an approximately three‐fold higher proportional reporting ratio associated with colitis than those of the anti‐PD‐1 drugs. The signal of the molecular mechanisms, including signaling pathways of inflammatory cytokines, was statistically insignificant to test the hypothesis that the severer rate of colitis associated with ipilimumab would be due to a greater magnitude of T‐cell activation as a result of earlier response of the anti‐CTLA‐4 drug in the immune response. This patient‐centered systems‐based approach provides an exploratory process to better understand drug pair adverse events at pathway and target levels through reverse translation from postmarket surveillance safety reports.

FOXP3 Isoforms and Human Regulatory T Cells

Background: &#x0D; Regulatory T-cells (Tregs) are critical to maintaining immune tolerance, thus prevent autoimmunity and allergic diseases. The master transcription factor FOXP3, expressed in humans as two isoforms through mRNA alternative splicing, controls the development and function of Tregs. However, the functions of the two isoforms remain unclear. We hypothesized that the oligonucleotides, developed by the lab, would efficiently shift FOXP3 to its shorter isoform, lacking exon 2 (FOXP3 ΔE2), which enables us to study how the FOXP3 ΔE2 isoform affects Treg function. &#x0D; &#x0D; Methods: &#x0D; Human peripheral blood mononuclear cells (PBMC) were obtained from healthy donors and cultured in the presence of 1 µM isoform-shifting oligonucleotides for three weeks. Expression of the FOXP3 isoforms, CD25, CTLA-4, CD40L, was examined through flow cytometry. Another goal was to study how the FOXP3 ΔE2 isoform shift affects the Treg response to inflammatory cytokines. &#x0D; &#x0D; Results: &#x0D; The oligonucleotides are highly effective in shifting the FOXP3 to the FOXP3 ΔE2 isoform. The majority of Tregs express only the FOXP3 ΔE2 isoform after two weeks of culturing in 1 µM oligo and had reduced expression of CD25, which plays a role in Treg differentiation, function, and homeostasis. Tregs display reduced expression of CTLA-4, a negative regulator of T-cell activation, but elevated CD40L, which costimulates and regulates the immune response. The FOXP3 ΔE2 isoform shift also sensitizes the Tregs to proinflammatory cytokine stimulation, allowing for increased IL-4, IL-17A, and IFN-γ production. &#x0D; &#x0D; Conclusion: &#x0D; Our studies demonstrate that FOXP3 ΔE2 Tregs are less stable because of the lower CD25 expression and less suppressive as determined by lower CTLA-4 but higher CD40L expression. In the inflammatory environment, the FOXP3 ΔE2 Tregs also produce more inflammatory cytokines, which further reduces their regulatory function. This study establishes an understanding of FOXP3ΔE2 Tregs’ role in autoimmunity and provides a potential novel target to treating autoimmune diseases. &#x0D; &#x0D; Acknowledgements: &#x0D; This study was funded, in part, with support from the Immunology and Infectious Diseases Training Program Grant funded, in part by T32 AI 060519 from the National Institutes of Health to RS and NIH AI159804 to BZ.

Ubiquitination in T-Cell Activation and Checkpoint Inhibition: New Avenues for Targeted Cancer Immunotherapy.

The advent of T-cell-based immunotherapy has remarkably transformed cancer patient treatment. Despite their success, the currently approved immunotherapeutic protocols still encounter limitations, cause toxicity, and give disparate patient outcomes. Thus, a deeper understanding of the molecular mechanisms of T-cell activation and inhibition is much needed to rationally expand targets and possibilities to improve immunotherapies. Protein ubiquitination downstream of immune signaling pathways is essential to fine-tune virtually all immune responses, in particular, the positive and negative regulation of T-cell activation. Numerous studies have demonstrated that deregulation of ubiquitin-dependent pathways can significantly alter T-cell activation and enhance antitumor responses. Consequently, researchers in academia and industry are actively developing technologies to selectively exploit ubiquitin-related enzymes for cancer therapeutics. In this review, we discuss the molecular and functional roles of ubiquitination in key T-cell activation and checkpoint inhibitory pathways to highlight the vast possibilities that targeting ubiquitination offers for advancing T-cell-based immunotherapies.

JNK Pathway-Associated Phosphatase as a Serum Marker for Disease Activity and Treatment Outcome of Juvenile Idiopathic Arthritis.

Juvenile idiopathic arthritis (JIA) is a heterogeneous autoimmune disease characterized by arthritis of unknown etiology. JNK pathway-associated phosphatase (JKAP) is reported to be a negative regulator of T-cell activation, but its clinical role in JIA is unknown. This study aimed to investigate the correlation of JKAP with disease activity and treatment response to a tumor necrosis factor (TNF) inhibitor, etanercept (ETN), in JIA patients. Totally, 104 JIA patients (6.9 ± 2.7 years old) and 100 age- and sex-matched healthy controls (HCs) (7.2 ± 2.4 years old) were enrolled, and their serum samples were collected for measuring JKAP by enzyme-linked immunoassay. In JIA patients, after 24-week ETN treatment, clinical response was assessed based on the American College of Rheumatology pediatric criteria (ACRpedi) 50 criteria. Results showed that JKAP levels were significantly lower in JIA patients compared with HCs, and of good value in differentiating JIA patients from HCs. Among JIA patients, higher JKAP levels were associated with lower disease activity indexes, including C-reactive protein, number of joints with active arthritis, physician's global assessment of disease activity, and the present history of disease-modifying antirheumatic drugs; higher baseline JKAP levels were correlated with worse ACRpedi 50 response to ETN at week 24, and was also an independent predictive factor for worse ACRpedi 50 response to ETN. Thus, it may be inappropriate to use ETN for JIA patients with higher JKAP levels. In conclusion, serum JKAP is a potential biomarker for JIA activity and treatment response to a TNF inhibitor.

Intratumoral Anti-CTLA-4 Blockade Can Potentiate Radiation-Induced Anti-Tumor Immunity In Breast Cancer

Monoclonal antibodies (mAb) that target CTLA-4, a negative regulator of T-cell activation, have significantly extended survival of patients with solid malignancies. However, the number of responders remain low. We have previously shown in the 4T1 mouse breast tumor model that resistance to anti-CTLA-4 therapy can be overcome by concurrent local radiotherapy (RT) (Demaria et al Clin Cancer Res 2005). Multiple effects of RT on the tumor microenvironment contributed to its ability to sensitize tumors to anti-CTLA-4 treatment, including enhanced homing of effector T cells to the tumor and formation of stable immune synapses between CTL and tumor cells (Matsumura et al J Immunol 2008; Ruocco et al J Clin Invest 2012). Our prior work, done exclusively via systemic intraperitoneal CTLA-4 treatment, support the hypothesis that RT generates an in situ vaccine while CTLA-4 expands primed T cells. Here we test whether intratumoral delivery can elicit equally robust responses when combined with fractionated RT. BALB/c mice were inoculated s.c. with 4T1 cells and thirteen days later, when tumors became palpable, randomly assigned to one of 6 treatment groups (N = 6-8 mice/group): control, RT alone, i.p. anti-CTLA-4 alone, i.t. anti-CTLA-4 alone, RT+ i.p. anti-CTLA-4 or RT+ i.t. anti-CTLA-4. RT was given in 3 fractions of 8 Gy on days 13,14 and 15 post-tumor inoculation. Anti-CTLA-4 mAb (Clone 9H10, 200 ug/mouse) was given on days 15, 18 and 21. Animals were followed for tumor growth and survival. In parallel experiments (N = 5 mice/group), mice were euthanized on day 22 for immunophenotyping of tumor-infiltrating lymphocytes and ex vivo IFNg production by tumor-specific CD8 T-cells in the draining lymph nodes. Treatment of 4T1 tumor-bearing mice with anti-CTLA-4 checkpoint blockade alone, whether administered intraperitoneal or intratumoral, was unable to exert any local control on tumor progression nor improve overall survival. As expected, focal RT significantly delayed tumor growth (Control vs RT, p<0.05) but had marginal effects on survival (median 35 days in control vs 40 days in RT grp, p = 0.301). The combination of RT and CTLA-4 blockade elicited the best responses, with i.p.-treated mice displaying a slight advantage vs i.p.-treated in both tumor inhibition (RT+i.t. CTLA-4 vs RT+i.p. CTLA4, p<0.05) and median survival (44.5 days in RT+i.t. CTLA-4 vs 42 days RT+i.p. CTLA4). Overall these results support rational combination of intratumoral anti-CTLA-4 with local RT. Ongoing work will further optimize the therapeutic response to combination treatment, including the addition of PD-1 blockade.

Role of Protein Tyrosine Phosphatase (PTPN22) Gene [C1858T] Functional Variant in Genetic Susceptibility of Psoriatic Arthritis in Kuwaiti Arabs

Background: Psoriatic arthritis (PsA) is a chronic, systemic inflammatory arthritic disease characterized by joint inflammation that is associated with cutaneous psoriasis, and can lead to pain, swelling, or stiffness in one or more joints. It results from a complex interplay between genetic, immunologic and environmental factors. A functional variant [C1858T] in the protein tyrosine phosphatase (PTPN22) gene, which encoded Arg620Trp in the lymphoid protein tyrosine phosphatase (LYP) has been shown to be a negative regulator of T-cell activation. Objective: The objective of this study was to investigate an association between PTPN22 gene [C1858T] functional variant and PsA in Kuwaiti patients. Methods: We have investigated the association of PTPN22 gene functional variant in 102 Kuwaiti patients with psoriatic arthritis and compared it to that in 214 healthy controls. The genotypes for the PTPN22 gene [C1858T] variant were determined by using a PCR-RFLP method and confirmed by DNA sequence analysis. Results: The frequency of homozygous variant genotype (TT) was found to be significantly higher in PsA patients compared to that in the controls (p &lt;0.0001). Collectively, the variant genotype was detected in homozygous and heterozygous combinations in 30% patients (p &lt;0.0001) compared to 16% in the controls. The frequency of variant genotype was found to be highest in the early-onset PsA patients (age &gt;25-34y). No correlation was detected between the variant genotype (TT) and gender in the Kuwaiti PsA patients. Conclusion: Our data show a significant association of PTPN22 gene functional variant [C1958T] with PsA in Kuwaiti patients and highlight its role in determining the genetic susceptibility along with other factors.

Abstract A55: Uncovering the mechanism of Trib1 in cancer immunotherapy

Abstract T cells provide a fundamental role in cell-mediated immunity, but during chronic antigen exposure such as in the case of cancer, T cells begin to lose effector function and undergo exhaustion. Our goal is to find a target within T cells that could be manipulated to prolong T-cell function and reduce tumor growth. Initial experiments focused on the pseudokinase Trib1, as data suggest that Trib1 acts as a negative regulator of T-cell activation. T cells isolated from mice in which Trib1 was specifically ablated within the T-cell compartment produced more IL-2 compared to wild-type T cells. T cells lacking Trib1 also proliferated more than wild-type T cells. In mice lacking Trib1 in grafted tumor models, tumor growth was reduced. These observations suggest that Trib1 restricts T-cell activation and function. Trib1 is a pseudokinase that is thought to act as a scaffolding molecule to facilitate protein interactions. To understand the mechanism by which Trib1 regulates T-cell function, we performed a proteomic screen to identify interacting partners. The screen was performed in Jurkat cells, an immortalized human T-cell line, expressing a FLAG/strep tagged Trib1. A FLAG pulldown was performed to isolate Trib1, and interacting partners were identified by mass spectrometry. The screen revealed two interacting partners: RFWD2, an E3 ubiquitin ligase also known as COP1, and DET1, a binding partner of COP1. These results provide evidence that Trib1 interacts with COP1 in T cells and suggest that this interaction might be important in the regulation of T-cell function and the regression of tumor growth. Note:This abstract was not presented at the conference. Citation Format: Franklin Iheanacho, Sarah J. Stein, Kelly S. Rome, Martha S. Jordan, Warren S. Pear. Uncovering the mechanism of Trib1 in cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A55.

Abstract A56: Releasing the brake on T-cell activation through inhibition of HPK1

Abstract Introduction: Loss of immune surveillance is required for cancer cell growth and metastasis; tumors co-opt suppressive mechanisms to evade detection by the immune system. The immune system is equipped with multiple feedback mechanisms to limit inflammation to prevent autoimmunity; tumors activate these pathways to escape immunity. HPK1 (hematopoietic progenitor kinase 1) is a negative regulator of T-cell activation; the kinase activity limits T-cell signaling and tumoricidal cytokine production. Recent literature has shown that inactivation of the kinase function of HPK1 prevents tumor progression in murine tumor models (1). Experimental Procedures: We used lentiviral delivered shRNAs and CRISPR/Cas9 technology to delete HPK1 in Jurkat cells and primary human T cells, respectively. Jurkat T cells (wt/KO) were activated with anti-CD3 antibody and cell lysates were assessed by Western blot to examine signaling events downstream of the T-cell receptor (TCR). Primary human T cells or CRISPR/Cas9 KO T cells were activated in vitro using anti-CD3/anti-CD28 antibodies in the presence or absence of prostaglandin E2 (PGE2). Cytokines were quantified by ELISA. Results: We present novel findings demonstrating that the role of HPK1 is conserved in human Jurkat cells as well as in primary human T cells. Loss of HPK1 enhanced T-cell receptor signaling in Jurkat cells. In CRISPR/Cas9 KO HPK1 primary human T cells, TCR activation resulted in enhanced cytokine secretion and proliferation concomitant to a decrease in pSLP76, the target molecule phosphorylated by HPK1. In addition, HPK1 KO cells were rescued from PGE2-mediated suppression. A screening and assessment of HPK-1 inhibitor compounds that enhance T-cell function is ongoing. Conclusions: In summary, HPK1 function is conserved in human T cells and inhibitors of HPK1 could enhance antitumor immunity through increased T-cell function, overcoming suppressive signals in the tumor microenvironment and broadening the response to check point inhibitors for cancer immunotherapy. Reference 1. Hernandez S, Qing J et al. The kinase activity of hematopoietic progenitor kinase 1 is essential for the regulation of T cell function. Cell Rep 2018;25:80-94. Citation Format: Neil Grimster, Lisa Drew, Stephen Fawell, Deanna A Mele, Gayathri Bommakanti, Minhui Shen, Kevin Xu, Kun Song, Rob Ziegler, Jason Kettle, Adelphe Mfuh, Jason Sheilds. Releasing the brake on T-cell activation through inhibition of HPK1 [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A56.

Progression of acute-to-chronic atopic dermatitis is associated with quantitative rather than qualitative changes in cytokine responses

Although multiple studies have assessed molecular changes in chronic atopic dermatitis (AD) lesions, little is known about the transition from acute to chronic disease stages, and the factors and mechanisms that shape chronic inflammatory activity. We sought to assess the global transcriptome changes that characterize the progression from acute to chronic stages of AD. We analyzed transcriptome changes in paired nonlesional skin, acute and chronic AD lesions from 11 patients and 38 healthy controls by RNA-sequencing, and conducted invivo and histological assays to evaluate findings. Our data demonstrate that approximately 74% of the genes dysregulated in acute lesions remain or are further dysregulated in chronic lesions, whereas only 34% of the genes dysregulated in chronic lesions are altered already in the acute stage. Nonlesional AD skin exhibited enrichment of TNF, TH1, TH2, and TH17 response genes. Acute lesions showed marked dendritic-cell signatures and a prominent enrichment of TH1, TH2, and TH17 responses, along with increased IL-36 and thymic stromal lymphopoietin expression, which were further heightened in chronic lesions. In addition, genes involved in skin barrier repair, keratinocyte proliferation, wound healing, and negative regulation of T-cell activation showed a significant dysregulation in the chronic versus acute comparison. Furthermore, our data show progressive changes in vasculature and maturation of dendritic-cell subsets with chronicity, with FOXK1 acting as immune regulator. Our results show that the changes accompanying the transition from nonlesional to acute to chronic inflammation in AD are quantitative rather than qualitative, with chronic AD having heightened TH2, TH1, TH17, and IL36 responses and skin barrier repair mechanisms. These findings provide novel insights and highlight underappreciated pathways in AD pathogenesis that may be amenable to therapeutic targeting.

Cytotoxic T-Lymphocyte-Associated Antigen 4 (CTLA-4)- and Programmed Death 1 (PD-1)-Mediated Regulation of Monofunctional and Dual Functional CD4+ and CD8+ T-Cell Responses in a Chronic Helminth Infection.

Chronic helminth infections are known to be associated with the modulation of antigen-specific T-cell responses. Strongyloides stercoralis infection is characterized by the downmodulation of antigen-specific Th1 and Th17 responses and the upregulation of Th2 and Th9 responses. Immune homeostasis is partially maintained by negative regulators of T-cell activation, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1), which dampen effector responses during chronic infections. However, their roles in S. stercoralis infection are yet to be defined. Therefore, we sought to determine the role of CTLA-4 and PD-1 in regulating CD4+ and CD8+ T-cell responses and examined the frequencies of monofunctional and dual functional Th1/T cytotoxic type 1 (Tc1), Th17/Tc17, Th2/Tc2, and Th9/Tc9 cells in S. stercoralis infection in 15 infected individuals stimulated with parasite antigen following CTLA-4 or PD-1 blockade. Our data reveal that CTLA-4 or PD-1 blockade results in significantly enhanced frequencies of monofunctional and dual functional Th1/Tc1 and Th17/Tc17 cells and, in contrast, diminishes the frequencies of monofunctional and dual functional Th2/Tc2 and Th9/Tc9 cells with parasite antigen stimulation in whole-blood cultures. Thus, we demonstrate that CTLA-4 and PD-1 limit the induction of particular T-cell subsets in S. stercoralis infection, which suggests the importance of CTLA-4 and PD-1 in immune modulation in a chronic helminth infection.