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Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co‐primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported. We report 5-year results from the phase III KEYNOTE-042 study (ClinicalTrials.gov identifier: NCT02220894 ). Eligible patients with locally advanced/metastatic non–small-cell lung cancer (NSCLC) without EGFR/ALK alterations and with programmed death ligand-1 (PD-L1) tumor proportion score (TPS) ≥ 1% received pembrolizumab 200 mg once every 3 weeks for 35 cycles or chemotherapy (carboplatin + paclitaxel or pemetrexed) for 4-6 cycles with optional maintenance pemetrexed. Primary end points were overall survival (OS) in PD-L1 TPS ≥ 50%, ≥ 20%, and ≥ 1% groups. Patients who completed 35 cycles of pembrolizumab with ≥ stable disease could begin second-course pembrolizumab upon progression. One thousand two hundred seventy‐four patients were randomly assigned (pembrolizumab, n = 637; chemotherapy, n = 637). Median follow-up time was 61.1 (range, 50.0-76.3) months. OS outcomes favored pembrolizumab ( v chemotherapy) regardless of PD-L1 TPS (hazard ratio [95% CI] for TPS ≥ 50%, 0.68 [0.57 to 0.81]; TPS ≥ 20%, 0.75 [0.64 to 0.87]; TPS ≥ 1%, 0.79 [0.70 to 0.89]), with estimated 5-year OS rates with pembrolizumab of 21.9%, 19.4%, and 16.6%, respectively. No new toxicities were identified. Objective response rate was 84.3% among 102 patients who completed 35 cycles of pembrolizumab and 15.2% among 33 patients who received second-course pembrolizumab. First-line pembrolizumab monotherapy continued to show durable clinical benefit versus chemotherapy after 5 years of follow-up in PD-L1–positive, locally advanced/metastatic NSCLC without EGFR/ALK alterations and remains a standard of care.

BackgroundPrimary analysis of KEYNOTE-042 (NCT02220894), a global, randomized, phase 3 trial, showed that pembrolizumab significantly improved OS versus platinum-based chemotherapy in patients with locally advanced or metastatic non–small-cell lung cancer (NSCLC) without sensitizing EGFR/ALK alterations and with PD-L1 tumor proportion score (TPS) ≥50%, ≥20%, and ≥1% with fewer treatment-related AEs than chemotherapy. We report an updated analysis with ~5 years of follow-up.MethodsEligible adults were randomized 1:1 to receive pembrolizumab 200 mg Q3W for 35 cycles or investigator’s choice of chemotherapy (carboplatin + paclitaxel or pemetrexed) Q3W for 4–6 cycles with optional maintenance pemetrexed (nonsquamous only). Primary endpoints were OS in patients with PD-L1 TPS ≥50%, ≥20%, and ≥1%; secondary endpoints included PFS and ORR per RECIST v1.1 by central review, and safety (secondary). Eligible patients randomized to pembrolizumab who completed 35 cycles with SD or better or stopped treatment after confirmed CR could begin a second course of pembrolizumab at the time of progression.Results1274 patients were randomized to pembrolizumab or chemotherapy (n = 637 each). Median (range) time from randomization to data cutoff (Apr 28, 2021) was 61.1 (50.0–76.3) months. OS outcomes favored the pembrolizumab group (vs chemotherapy alone) regardless of PD-L1 TPS (HR [95% CI] for TPS ≥50%, 0.68 [0.57–0.81]; TPS ≥20%, 0.75 [0.64–0.87]; TPS ≥1%, 0.79 [0.70–0.89]), with estimated 5-year OS rates (95% CI) of 21.9% (17.3%–26.9%), 19.4% (15.6%–23.4%) and 16.6% (13.7%–19.6%), respectively, in the pembrolizumab group (table 1). Median duration of response (DOR) was 28.1 vs 10.8 months in PD-L1 TPS ≥50% group, 27.7 vs 10.8 months in PD-L1 TPS ≥20% group and, 26.5 vs 8.4 months in PD-L1 TPS ≥1% for pembrolizumab group vs chemotherapy. Treatment-related grade 3–5 AEs occurred in 120 patients (18.9%) in the pembrolizumab group and 257 (41.8%) in the chemotherapy group. Among 102 patients who completed 35 cycles of pembrolizumab: ORR was 84.3%; estimated 4-year OS rate after completion of 35 cycles of pembrolizumab (ie, approximately 6 years after randomization) was 61.8%. Among 33 patients who received second-course pembrolizumab, ORR was 15.2%.Abstract 363 Table 1Key efficacy outcomes in the KEYNOTE-042 ITT populationConclusionsWith 5 years of follow-up, first-line pembrolizumab monotherapy continued to show substantial clinical benefit with higher 5-year OS rates, and durable response over chemotherapy in patients with PD-L1–positive, locally advanced/metastatic NSCLC without EGFR/ALK alterations. First-line pembrolizumab remains a standard of care in patients with PD-L1 TPS ≥1%, as underscored by these long-term results.AcknowledgementsMedical writing assistance was provided by Kathleen Estes, PhD, of ICON plc (North Wales, PA, USA). This assistance was funded by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.Trial RegistrationClinicaltrialsgov, NCT02220894Ethics ApprovalThe protocol and all amendments were approved by the appropriate ethics committee at each center, the study was conducted in accordance with the standards of Good Clinical Practice and in compliance with the Declaration of Helsinki. Patients provided written informed consent before enrollment.

Transforming growth factor (TGF)-β has been implicated in regulation of the immune system, including autoimmunity. We have found that TGF-β is readily produced by T cells following immunization with self-peptide epitopes that downregulate autoimmune responses in type 1 diabetes (T1D) prone nonobese diabetic (NOD) mice. These include multiple peptide epitopes derived from the islet β-cell antigens GAD65 (GAD65 p202-221, GAD65 p217-236), GAD67 (GAD67 p210-229, GAD67 p225-244), IGRP (IGRP p123-145, IGRP p195-214) and insulin B-chain (Ins. B:9-23) that protected NOD mice from T1D. Immunization of NOD mice with the self-MHC class II I-Ag7 β-chain-derived peptide, I-Aβg7 p54-76 also induced large amounts of TGF-β and also protected these mice from diabetes development. These results indicate that peptides derived from disease related self-antigens and MHC class II molecules primarily induce TGF-β producing regulatory Th3 and Tr1-like cells. TGF-β produced by these cells could enhance the differentiation of induced regulatory iTreg and iTreg17 cells to prevent induction and progression of autoimmune diseases. We therefore suggest that peripheral immune tolerance could be induced and maintained by immunization with self-peptides that induce TGF-β producing T cells.

The present study was undertaken to determine whether supplementation with polymethoxylated flavones (PMFs) could ameliorate the fructose-induced hypertriglyceridemia and other metabolic abnormalities associated with insulin resistance (IR) in hamsters. Following feeding with the fructose diet, hamsters were supplemented orally with PMF-L or PMF-H (62.5 and 125 mg/kg/day) for 4 weeks. Both PMF-treated groups showed a statistically significant (p

Tumor necrosis factor (TNF) is a multipotential cytokine that interacts with a wide variety of cells through two distinct receptors, referred to as the p60 and p80 receptors. Why there are two distinct receptors for the same ligand and whether these receptors mediate their signal independently or synergistically is not known. We examined the role of these two receptors in the ligand-dependent activation of a transcriptional factor, NF-kappaB, an early response (5-15 min) to TNF in human myeloid ML-1a cells. By using receptor type-specific antibodies, these cells were found to express almost equal amounts of both receptors. TNF-dependent activation of NF-kappaB could be blocked partially by both anti-p60 and anti-p80, suggesting that TNF mediates its effect independently through the p60 and p80 receptors. In comparison, the activation of NF-kappaB by lymphotoxin (LT), which shares receptors with TNF, was completely blocked by anti-p60, whereas anti-p80 had no effect. Anti-p60 but not anti-p80 by itself was found to activate NF-kappaB in a dose-dependent manner, but on a molar basis anti-p60 was found to be 100 times less potent than TNF. Interestingly, even though anti-p80 by itself was inactive, it potentiated the effect of anti-p60 synergistically, suggesting an interaction between the two types of TNF receptor. Thus, overall these results demonstrate that the two forms of TNF receptors could mediate their signal in both an independent and synergistic manner and that TNF mediates its signal through both forms of receptors, whereas LT mediates its signal through the p60 receptor.

Letters from: [ Pratibha Chaturvedi and Rinee Mukherjee ][1] [ Fred Lenherr ][1] “Surfing the Net” can be confusing if one does not know what to look for or where to look. The recent article by Floyd E. Bloom ([15 Nov., p. 1104][2]) will be helpful to neuroscientists and provides information for anyone with specific or general questions about neuroscience. For researchers interested in neurodegenerative diseases, the catalog of sites at can be useful. This site provides a list of World Wide Web sites on many subjects, including a disease-associated site at . This site has listings of other sites on various diseases, including Alzheimer's, at . One site at has listings for other neurodegenerative diseases, including Parkinson's, Huntington's, and multiple sclerosis. # {#article-title-2} Bloom's superb article omitted one Web site that allows full-text searching on a database of more than 110,000 Web pages, all of which have been prequalified as relevant to neuroscience. The site allows easy access to an estimated 5000 megabytes of actual Web page content, including text, images, and movies. English and several foreign languages can be used, or a special query syntax can be employed by those wishing to master it. The URL for this site is . The service is free, noncommercial, and open to all. The only request is that users limit the total number of URLs requested for any single query to 200. There is no limit on the total number of different queries one may submit. [1]: /lookup/doi/10.1126/science.275.5302.909c [2]: /lookup/volpage/275/1104

SUMMARY Dengue type 2 virus (DV)-induced suppressor cylokine (SF) binds to macrophages to transmit the suppressor signal to recruit the second subpopulation of suppressor T cells. The present study was undertaken to identify and purify the receptor for SF(SF-R) on macrophages. The binding of 125I-SF to macrophages was saturable and reversible. Scatchard analysis showed the presence of both high (54 000/cell) and low (178 × 106/cell) affinity receptor sites. The binding of 125I-SF to macrophages was inhibited by pretreatment of macrophages with anti-SF antiseruin but not by a heterologous antiserum. Normal mouse peritoneal macrophage membrane was solubilized with Triton-X-100 and the components separated by low pressure liquid chromatography (LPLC) to purify SF-R. The presence of SF binding moiety (SF-R) was screened at each step of purification. The purified SF-R resolved into two bands of 45–50 kD mol. wt on SDS-PAGE. 125I-SF + SF-R complex run on SDS-PAGE showed a single band at about 55–60 kD mol. wt by autoradiography. Anti-SF-R anti serum reacted with SF-R in a Western blot test; the reaction was abolished by pretreatment of the blots with proteinase K, but not by pretreatment with periodic acid. SF-R was composed of two polypeptide chains (α and β) which were obtained in pure form by high performance liquid chromatography (HPLC) of dithiothreitol- and iodoacetamide-treated SF-R. Only the β chain bound SF.

We have previously shown that exogenous CLIP (class II associated invariant chain peptide) downregulated MHC class II expression on antigen presenting cells (APC) and modulated T cell mediated immune responses. The present study was undertaken to investigate the mechanism of uptake of exogenously added CLIP peptide by APC. We found that exogenous CLIP is rapidly internalized by APC and it co-localize with MHC class II in intracellular compartments including early-, late-endosomes and lysosomes. We suggest that exogenous CLIP acts as an in vivo regulator of immune response by internalization and passage through the intracellular compartments where it interferes in peptide loading and recycling of MHC class II molecules to the APC surface. Therefore, exogenous CLIP regulates immune responses by modulation of antigen presentation by the APC.

We have established a novel CD4 and CD8 double-positive CD25+ T regulatory (Treg) clone, MT-5B, from lymph nodes of type 1 diabetes prone non-obese diabetic (NOD) mice immunized with CFA. CFA has previously been shown to prevent the onset of diabetes by inducing Treg cells. In vitro, clone MT-5B was anergic to a panel of antigen stimulations and exerted an immunosuppressive effect in antigen-non-specific and cell contact-independent manners. In vivo, clone MT-5B blocked the adoptive transfer of diabetes. Proteomics and immunoadsorption studies identified the suppressive proteins secreted by clone MT-5B as granzyme B (GrB) and perforin (PFN). GrB-mediated immune suppression was PFN dependent. Removal of GrB or PFN from the culture supernatant (SN) of MT-5B cells or pre-incubation of MT-5B cells with ethyleneglycol-bis(aminoethylether)-tetraacetic acid which blocks PFN activity reduced the immunosuppressive effect in vitro. Pre-incubation of diabetogenic splenocytes from NOD mice with MT-5B SN impaired their ability to transfer disease by inducing T cell apoptosis, and removal of GrB from MT-5B SN by immunoadsorption decreased the effector function of MT-5B SN on diabetogenic splenocytes. Immunization of NOD mice with CFA increased the expression of GrB+ CD4 T cells, indicating that these cells are present in vivo. In conclusion, we describe a novel mechanism of cell contact-independent immune suppression in which Treg cells maintain immune homeostasis by secreting GrB/PFN.

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) has been identified as a novel CD8+ T cell-specific autoantigen in NOD mice. This study was undertaken to identify MHC class II-specific CD4+ T cell epitopes of IGRP. Peptides named P1, P2, P3, P4, P5, P6, and P7 were synthesized by aligning the IGRP protein amino acid sequence with peptide-binding motifs of the NOD MHC class II (I-Ag7) molecule. Peptides P1, P2, P3, and P7 were immunogenic and induced both spontaneous and primed responses. IGRP peptides P1-, P2-, P3-, and P7-induced responses were inhibited by the addition of anti-MHC class II (I-Ag7) Ab, confirming that the response is indeed I-Ag7 restricted. Experiments using purified CD4+ and CD8+ T cells from IGRP peptide-primed mice also showed a predominant CD4+ T cell response with no significant activation of CD8+ T cells. T cells from P1-, P3-, and P7-primed mice secreted both IFN-γ and IL-10 cytokines, whereas P2-primed cells secreted only IFN-γ. Peptides P3 and P7 prevented the development of spontaneous diabetes and delayed adoptive transfer of diabetes. Peptides P1 and P2 delayed the onset of diabetes in both these models. In summary, we have identified two I-Ag7-restricted CD4+ T cell epitopes of IGRP that can modulate and prevent the development of diabetes in NOD mice. These results provide the first evidence on the role of IGRP-specific, MHC class II-restricted CD4+ T cells in disease protection and may help in the development of novel therapies for type 1 diabetes.

NOD mice have a relative deficiency of CD4 + CD25 + regulatory T cells that could result in an inability to maintain peripheral tolerance. The aim of this study was to induce the generation of CD4 + CD25 + regulatory T cells in response to autoantigens to prevent type 1 diabetes (T1D). We found that immunization of NOD mice with insulin B-chain peptide B:9-23 followed by 72 h in vitro culture with B:9-23 peptide induces generation of CD4 + CD25 + regulatory T cells. Route of immunization has a critical role in the generation of these cells. Non-autoimmune mice BALB/c, C57BL/6 and NOR did not show up regulation of CD4 + CD25 + regulatory T cells. These cells secreted large amounts of TGF-β and TNF-α with little or no IFN-γ and IL-10. Adoptive transfer of these CD4 + CD25 + regulatory T cells into NOD-SCID mice completely prevented the adoptive transfer of disease by diabetogenic T cells. Although, non-self antigenic OVA (323-339) peptide immunization and in vitro culture with OVA (323-339) peptide does result in up regulation of CD4 + CD25 + T cells, these cells did not prevent transfer of diabetes. Our study for the first time identified the generation of antigen-specific CD4 + CD25 + regulatory T cells specifically in response to immunization with B:9-23 peptide in NOD mice that are capable of blocking adoptive transfer of diabetes. Our results suggest the possibility of using autoantigens to induce antigen-specific regulatory T cells to prevent and regulate autoimmune diabetes.

Oral administration of protein can induce antigen-specific immune hyporesponsiveness. However, the utility of oral tolerance to autoantigens in the treatment of autoimmune diseases may be limited when candidate autoantigens cannot be produced by conventional systems in quantities sufficient for clinical studies. Plants may be ideally suited for this purpose, as they can synthesize, glycosylate and assemble mammalian proteins to provide huge quantities of relatively low cost soluble proteins. Furthermore, edible transgenic plants could provide a simple and direct method of autoantigen delivery for oral tolerance. Therefore, the aim of this study was to determine whether a transgenic plant expression system was capable of synthesizing the diabetes-associated autoantigen, glutamic acid decarboxylase (GAD) in an immunogenic form and whether the oral administration of an autoantigen expressed by a plant could directly induce protective immune responses in a mouse model of diabetes. We show that a GAD-expressing transgenic plant, given as a dietary supplement, inhibits the development of diabetes in the non-obese diabetic (NOD) mouse.