T-cell Influx Research Articles

Unveiling the Innate and Adaptive Immunity Interplay: Global Transcriptomic Profiling of the Host Immune Response in Candida albicans Endophthalmitis in a Murine Model.

Intraocular fungal infection poses a significant clinical challenge characterized by chronic inflammation along with vision impairment. Understanding the host defense pathways involved in fungal endophthalmitis will play a pivotal role in identifying adjuvant immunotherapy. Clinical isolates of Candida albicans (15,000 CFU/μL) were intravitreally injected in C57BL/6 mice followed by enucleation at 24 and 72 h postinfection. Histopathological analysis was performed to evaluate the retinal changes and the disease severity. RNA-seq analysis was conducted on homogenized eyeballs to assess the relevant gene profiles and their differentially expressed genes (DEGs). Pathway enrichment analysis was performed to further annotate the functions of the DEGs. Histopathological analysis demonstrated a higher disease severity with increased inflammatory cells at 72 hpi and transcriptome analysis revealed 27,717 DEGs, of which 1493 were significant (adj p value ≤0.05, FC ≥ 1.5). Among these, 924 were upregulated, and 569 were downregulated. Majority of the upregulated genes were associated with the inflammatory/host immune response and signal transduction and enriched in the T-cell signaling pathway, natural killer cell-mediated cytotoxicity, C-type receptor signaling pathway, and NOD-like receptor signaling pathway. Furthermore, inflammation-associated genes such as T-cell surface glycoprotein CD3, cathelicidin antimicrobial peptide, and lymphocyte cell-specific protein tyrosine kinase were enriched, while pathways such as MAPK, cAMP, and metabolic pathways were downregulated. Regulating the T-cell influx could be a potential strategy to modulate excessive inflammation in the retina and could potentially aid in better vision recovery in fungal endophthalmitis.

The CD47/TSP-1 axis: a promising avenue for ovarian cancer treatment and biomarker research

BackgroundOvarian cancer (OC) remains one of the most challenging and deadly malignancies facing women today. While PARP inhibitors (PARPis) have transformed the treatment landscape for women with advanced OC, many patients will relapse and the PARPi-resistant setting is an area of unmet medical need. Traditional immunotherapies targeting PD-1/PD-L1 have failed to show any benefit in OC. The CD47/TSP-1 axis may be relevant in OC. We aimed to describe changes in CD47 expression with platinum therapy and their relationship with immune features and prognosis.MethodsTumor and blood samples collected from OC patients in the CHIVA trial were assessed for CD47 and TSP-1 before and after neoadjuvant chemotherapy (NACT) and multiplex analysis was used to investigate immune markers. Considering the therapeutic relevance of targeting the CD47/TSP-1 axis, we used the CD47-derived TAX2 peptide to selectively antagonize it in a preclinical model of aggressive ovarian carcinoma.ResultsSignificant reductions in CD47 expression were observed post NACT. Tumor patients having the highest CD47 expression profile at baseline showed the greatest CD4+ and CD8+ T-cell influx post NACT and displayed a better prognosis. In addition, TSP-1 plasma levels decreased significantly under NACT, and high TSP-1 was associated with a worse prognosis. We demonstrated that TAX2 exhibited a selective and favorable biodistribution profile in mice, localizing at the tumor sites. Using a relevant peritoneal carcinomatosis model displaying PARPi resistance, we demonstrated that post-olaparib (post-PARPi) administration of TAX2 significantly reduced tumor burden and prolonged survival. Remarkably, TAX2 used sequentially was also able to increase animal survival even under treatment conditions allowing olaparib efficacy.ConclusionsOur study thus (1) proposes a CD47-based stratification of patients who may be most likely to benefit from postoperative immunotherapy, and (2) suggests that TAX2 is a potential alternative therapy for patients relapsing on PARP inhibitors.

Associations of T-cell fitness prior to B-cell maturation antigen (BCMA)–targeted chimeric antigen receptor T-cell (CART) and bispecific T-cell engager (BiTE) therapies and efficacy/toxicity in relapsed/refractory multiple myeloma (RRMM).

7549 Background: While CART and BiTE have led to unprecedented responses in RRMM, some patients (pts) do not respond or have short-lived responses. Currently no predictive markers exist to identify these pts. This study explored pretreatment T-cell fitness and efficacy/toxicity in RRMM using a novel single-cell secretome analysis. We hypothesized that pretreatment Polyfunctional Strength Index (PSI) may predict efficacy to BCMA-directed T-cell therapies. Methods: We included 14 RRMM pts treated with idecabtagene vicleucel or teclistamab at Yale Cancer Center with ≥6 mos post-treatment follow-up. Peripheral blood prior to treatment was frozen and then PBMC’s thawed for analysis. PSI, a metric for T-cell fitness combining polyfunctional T-cells % with the intensity of secreted cytokines, was obtained using the IsoPlexis’ Single-Cell Secretome Platform. The overall PSI was an average of CD4+ and CD8+ PSIs. Response was assessed by the International Myeloma Working Group criteria and response duration was defined as time from response to disease progression. Responder (R) was defined as ≥very good partial response for ≥6 mos. Non-responder (NR) was defined as stable or progressive disease ≤3 mos. Cytokine release syndrome (CRS) and immune-effector cell associated neurotoxicity syndrome (ICANS) were graded using the American Society for Transplantation & Cellular Therapy system. Statistics were performed with Mann-Whitney U test using GraphPad PRISM v.9. Results: There were 7 pts in R group (2 BiTE & 5 CART) and 7 pts in NR group (3 BiTE & 4 CART). Median follow-up time was 13.5 mos(range, 7-27). Median age at treatment was 64 yrs in R and 63 yrs in NR. Extramedullary disease (EM) was present in 14.3%(n=1) in R and 71.4%(n=5) in NR. High-risk cytogenetics, defined as del17p, t(4;14), t(14;16), t(14;20), 1q gain/amplification and del1p, were seen in 42.9%(n=3) in R and 85.7%(n=6) in NR. Median prior lines of therapy was 6(range, 4-9) in R and 8(range, 4-10) in NR. CRS/ICANS occurred 85.7%(n=6) in R and 28.6%(n=2) in NR. Overall PSI was 184 in R and 91 in NR(p=0.1649). CD4+ PSI was 160 in R and 75 in NR(p=0.1649). CD8+ PSI was 207 in R and 108 in NR(p=0.1981). Overall PSI was 143 in CRS/ICANS and 130 in no CRS/ICANS(p=0.7546). Conclusions: Overall PSI, CD4+ PSI and CD8+ PSI were 1.9-2.1 times higher in R compared to NR and overall PSI was slightly higher in CRS/ICANS compared to no CRS/ICANS though the difference was not statistically significant. One limitation was a small sample size and thus testing PSI in a larger cohort might yield statistically significant results. The NR group had more high-risk cytogenetics and higher EM. One confounder could be that measuring peripheral T-cell fitness may not be sufficient to predict response in EM where spatial determinants of T-cell influx play a role.

CCL18 aggravates atherosclerosis by inducing CCR6-dependent T-cell influx and polarization.

The CC chemokine ligand 18 (CCL18) is a chemokine highly expressed in chronic inflammation in humans. Recent observations of elevated CCL18 plasma levels in patients with acute cardiovascular syndromes prompted an investigation into the role of CCL18 in the pathogenesis of human and mouse atherosclerosis. CCL18 was profoundly upregulated in ruptured human atherosclerotic plaque, particularly within macrophages. Repeated administration of CCL18 in Western-type diet-fed ApoE -/- mice or PCSK9mut-overexpressing wild type (WT) mice led to increased plaque burden, enriched in CD3+ T cells. In subsequent experimental and molecular modeling studies, we identified CCR6 as a functional receptor mediating CCL18 chemotaxis, intracellular Ca2+ flux, and downstream signaling in human Jurkat and mouse T cells. CCL18 failed to induce these effects in vitro in murine spleen T cells with CCR6 deficiency. The ability of CCR6 to act as CCL18 receptor was confirmed in vivo in an inflammation model, where subcutaneous CCL18 injection induced profound focal skin inflammation in WT but not in CCR6-/- mice. This inflammation featured edema and marked infiltration of various leukocyte subsets, including T cells with a Th17 signature, supporting CCR6's role as a Th17 chemotactic receptor. Notably, focal overexpression of CCL18 in plaques was associated with an increased presence of CCR6+ (T) cells. Our studies are the first to identify the CCL18/CCR6 axis as a regulator of immune responses in advanced murine and human atherosclerosis.

Global Transcriptomic Profiling of Innate and Adaptive Immunity During Aspergillus flavus Endophthalmitis in a Murine Model.

Fungal endophthalmitis is characterized by chronic inflammation leading to the partial or complete vision loss. Herein, we analyzed the transcriptomic landscape of Aspergillus flavus (A. flavus) endophthalmitis in C57BL/6 mice to understand the host-pathogen interactions. Endophthalmitis was induced by intravitreal injection of A. flavus spores in C57BL/6 mice and monitored for disease progression up to 72hours. The enucleated eyeballs were subjected to histopathological analysis and mRNA sequencing using the Illumina Nextseq 2000. Pathway enrichment analysis was performed to further annotate the functions of differentially expressed genes (DEGs) and validation of cytokines was performed in vitreous of patients with fungal endophthalmitis using multiplex ELISA. Transcriptomic landscape of A. flavus endophthalmitis revealed upregulated T-cell receptor signaling, PI3K-AKT, MAPK, NF-κB, JAK-STAT, and NOD like receptor signaling pathways. We observed significant increase in the T-cells during infection especially at 72 hours infection along with elevated expression levels of IL-6, IL-10, IL-12, IL-18, IL-19, IL-23, CCR3, and CCR7. Furthermore, host-immune response associated genes, such as T-cell interacting activating receptor, TNF receptor-associated factor 1, TLR1, TLR9, and bradykinin receptor beta 1, were enriched. Histopathological assessment validated the significant increase in inflammatory cells, especially T-cells at 72 hours post-infection along with increased disruption in the retinal architecture. Additionally, IL-6, IL-8, IL-17, TNF-α, and IL-1β were also significantly elevated, whereas IL-10 was downregulated in vitreous of patients with Aspergillus endophthalmitis. Regulating T-cell influx could be a potential strategy to modulate the excessive inflammation in the retina and potentially aid in better vision recovery in fungal endophthalmitis.

Abstract B087: Mutant-p53 amplifies Cxcl1 expression from distal enhancers blunting immune checkpoint inhibition efficacy in pancreatic cancer

Abstract Pancreatic ductal Adenocarcinoma (PDAC) is an aggressive malignancy complicated by poor early diagnosis and a lack of response to traditional treatments. It is characterized by a desmoplastic stroma, a lack of infiltration and activation of T cells, and a low mutational burden. The genetic landscape of PDAC is defined by activating KRAS mutations (~90%) and p53 alterations (~70%), but the molecular switches perturbed by these genetic aberrations remain unclear. p53 missense mutations, unlike mutations resulting in the loss of p53, are considered to acquire tumor-supporting functions. But these novel functions remain uncharacterized. The ambiguity in the molecular mechanism of mutant-p53 is further exacerbated by the existence of various types of p53 mutations. The majority of p53 mutations are missense mutations in the DNA binding domain. The repertoire of transcription factors (TFs) it can interact with and the vast regulatory landscape of each TF—composed of gene promoters and distal enhancers—present obstacles in understanding the molecular mechanisms promoting PDAC. In this study, we examined how a common p53 missense mutation in PDAC plays a role in weakening the Immune checkpoint Inhibitors (ICIs) efficacy. Using cells derived from a genetically engineered mouse model (GEMM) of PDAC with activating KRAS mutation (KrasG12D/+) and a p53 missense mutation (p53R172H/-), we found that the PDAC tumorigenesis and resistance to ICIs are dependent on the mutant-p53. We used isogenic p53-null PDAC cells and the restoration of p53R172H in p53-null cells to demonstrate the role of p53R172H in controlling the expression of immunosuppressive chemokine genes such as Cxcl1. p53R172H deletion attenuated PDAC tumor growth, increased the influx of cytotoxic T-cells, and sensitized the tumor to ICIs. The p53R172H-mediated TME reprogramming was replicated by the deletion of the Cxcl1 gene, suggesting the anti-tumorigenic effect of p53R172H was mediated by the Cxcl1 gene. We probed the mechanism of Cxcl1 expression dependence on p53R172H. We found that in conjunction with NF-kB, p53R172H occupies the distal transcription regulatory elements (dTREs) of the Cxcl1 gene harboring NF-kB binding sites. Strikingly, deletion of the Cxcl1 dTREs in PDAC cells recapitulates the phenotypes of p53R172H deletion and Cxcl1 deletion in terms of tumor size, immune landscape of the TME, and ICI responsiveness. Furthermore, we examined the interplay between p53R172H and NF-kB and found that the p53R172H physically interacts with the NF-kB subunit RelA and facilitates its nuclear translocation. Overall, we characterize how a common p53 mutation in PDAC co-opts non-coding regulatory DNA to augment the expression of selective chemokine genes and establishes an immunosuppressive TME to shield the therapeutic benefits of ICIs. Citation Format: Dig B. Mahat, Heena Kumra, Emily Metcalf, Sarah Castro, Kim Nguyen1, Arundeep Singh, William W. Ho, Ivy Chen, Brandon Sullivan, Leon Yim, Enrico Moiso, Vikash Chauhan, Hernandez Moura Silva, Stefani Spranger, Rakesh Jain, Phillip A. Sharp. Mutant-p53 amplifies Cxcl1 expression from distal enhancers blunting immune checkpoint inhibition efficacy in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B087.

PNAd-expressing vessels characterize the dermis of CD3+ T-cell-mediated cutaneous diseases.

T-cell recruitment to skin tissues is essential for inflammation in different cutaneous diseases; however, the mechanisms by which these T cells access the skin remain unclear. High endothelial venules expressing peripheral node address in (PNAd), an L-selectin ligand, are located in secondary lymphoid organs and are responsible for increasing T-cell influx into the lymphoid tissues. They are also found in non-lymphoid tissues during inflammation. However, their presence in different common inflammatory cutaneous diseases and their correlation with T-cell infiltration remain unclear. Herein, we explored the mechanisms underlying the access of T cells to the skin by investigating the presence of PNAd-expressing vessels in different cutaneous diseases, and its correlation with T cells' presence. Skin sections of 43 patients with different diseases were subjected to immunohistochemical and immunofluorescence staining to examine the presence of PNAd-expressing vessels in the dermis. The correlation of the percentage of these vessels in the dermis of these patients with the severity/grade of CD3+ T-cell infiltration was assessed. PNAd-expressing vessels were commonly found in the skin of patients with different inflammatory diseases. A high percentage of these vessels in the dermis was associated with increased severity of CD3+ T-cell infiltration (P < 0.05). Additionally, CD3+ T cells were found both around the PNAd-expressing vessels and within the vessel lumen. PNAd-expressing vessels in cutaneous inflammatory diseases, characterized by CD3+ T-cell infiltration, could be a crucial entry point for T cells into the skin. Thus, selective targeting of these vessels could be beneficial in cutaneous inflammatory disease treatment.

Neutralizing Antibodies Impair the Oncolytic Efficacy of Reovirus but Permit Effective Combination with T cell-Based Immunotherapies.

Reovirus type 3 Dearing (Reo), manufactured for clinical application as pelareorep, is an attractive anticancer agent under evaluation in multiple phase 2 clinical trials for the treatment of solid tumors. It elicits its anticancer efficacy by inducing both oncolysis and intratumoral T-cell influx. Because most people have been preexposed to Reo, neutralizing antibodies (NAb) are prevalent in patients with cancer and might present a barrier to effective Reo therapy. Here, we tested serum of patients with cancer and healthy controls (n = 100) and confirmed that Reo NAbs are present in >80% of individuals. To investigate the effect of NAbs on both the oncolytic and the immunostimulatory efficacy of Reo, we established an experimental mouse model with Reo preexposure. The presence of preexposure-induced NAbs reduced Reo tumor infection and prevented Reo-mediated control of tumor growth after intratumoral Reo administration. In B cell-deficient mice, the lack of NAbs provided enhanced tumor growth control after Reo monotherapy, indicating that NAbs limit the oncolytic capacity of Reo. In immunocompetent mice, intratumoral T-cell influx was not affected by the presence of preexposure-induced NAbs and consequently, combinatorial immunotherapy strategies comprising Reo and T-cell engagers or checkpoint inhibitors remained effective in these settings, also after a clinically applied regimen of multiple intravenous pelareorep administrations. Altogether, our data indicate that NAbs hamper the oncolytic efficacy of Reo, but not its immunotherapeutic capacity. Given the high prevalence of seropositivity for Reo in patients with cancer, our data strongly advocate for the application of Reo as part of T cell-based immunotherapeutic strategies.

T-cell stimulating vaccines empower CD3 bispecific antibody therapy in solid tumors

CD3 bispecific antibody (CD3 bsAb) therapy is clinically approved for refractory hematological malignancies, but responses in solid tumors have been limited so far. One of the main hurdles in solid tumors is the lack of sufficient T-cell infiltrate. Here, we show that pre-treatment vaccination, even when composed of tumor-unrelated antigens, induces CXCR3-mediated T-cell influx in immunologically ‘cold’ tumor models in male mice. In the absence of CD3 bsAb, the infiltrate is confined to the tumor invasive margin, whereas subsequent CD3 bsAb administration induces infiltration of activated effector CD8 T cells into the tumor cell nests. This combination therapy installs a broadly inflamed Th1-type tumor microenvironment, resulting in effective tumor eradication. Multiple vaccination formulations, including synthetic long peptides and viruses, empower CD3 bsAb therapy. Our results imply that eliciting tumor infiltration with vaccine-induced tumor-(un)related T cells can greatly improve the efficacy of CD3 bsAbs in solid tumors.

FLT3L-dependent dendritic cells control tumor immunity by modulating Treg and NK cell homeostasis

FLT3-L-dependent classical dendritic cells (cDCs) recruit anti-tumor and tumor-protecting lymphocytes. We evaluate cancer growth in mice with low, normal, or high levels of cDCs. Paradoxically, both low or high numbers of cDCs improve survival in mice with melanoma. In low cDC context, tumors are restrained by the adaptive immune system through influx of effector Tcells and depletion of Tregs and NK cells. High cDC numbers favor the innate anti-tumor response, with massive recruitment of activated NK cells, despite high Treg infiltration. Anti CTLA-4 but not anti PD-1 therapy synergizes with FLT3-L therapy in the cDCHi but not in the cDCLo context. A combination of cDC boost and Treg depletion dramatically improves survival of tumor-bearing mice. Transcriptomic data confirm the paradoxical effect of cDC levels on survival in several human tumor types. cDCHi-TregLo state in such patients predicts best survival. Modulating cDC numbers via FLT3 signaling may have therapeutic potential in human cancer.

Xenogeneic equine stem cells activate anti-tumor adaptive immunity in a 4T1-based intraductal mouse model for triple-negative breast cancer: proof-of-principle.

Triple-negative breast cancer (TNBC) remains difficult to treat, especially due to ineffective immune responses. Current treatments mainly aim at a cytotoxic effect, whereas (stem) cell therapies are being investigated for their immune stimulatory capacities to initiate the anti-tumor immunity. Here, a thoroughly characterized, homogenous and non-tumorigenic mixture of equine mesenchymal stem cells (eMSCs) harvested from horse peripheral blood as innovative xenogeneic immunomodulators were tested in a 4T1-based intraductal mouse model for TNBC. The eMSCs significantly reduced 4T1 progression upon systemic injection, with induction of inflammatory mediators and T-cell influx in primary tumors, already after a single dose. These xenogeneic anti-cancer effects were not restricted to MSCs as systemic treatment with alternative equine epithelial stem cells (eEpSCs) mimicked the reported disease reduction. Mechanistically, effective eMSC treatment did not rely on the spleen as systemic entrapment site, whereas CD4+ and CD8α+ T-cell infiltration and activation were critical. These results show that eMSCs and potentially also other equine stem cell types can be a valuable TNBC treatment strategy for further (pre)clinical evaluation.

Intratumoral INT230-6 (cisplatin, vinblastine, shao) alone or with ipilimumab prolonged survival with favorable safety in adults with refractory sarcomas.

11568 Background: Sarcomas are a rare and diverse group of solid tumors from mesenchymal cells; chemotherapy provides limited benefit for metastatic disease. INT230-6 is a novel formulation of cisplatin (CIS), vinblastine (VIN) and a tissue dispersion enhancer (SHAO) designed for intratumoral (IT) delivery. The drug diffuses into cancer cells, causes apoptosis and recruits dendritic and T-cells to the tumor. Adding ipilimumab (IPI) appears to improve INT230-6 responses in models and clinically. The study evaluated INT230-6 for safety and efficacy alone and with intravenous (IV) IPI. Previously reported results showed INT230-6 alone induced tumor regression, T-cell influx and abscopal effects in uninjected lesions1,2. Methods: IT-01 is an open-label phase1/2 study in adults with locally advanced, unresectable or metastatic solid tumors, including sarcoma. INT230-6 dose was set by tumor diameter or volume. INT230-6 was dosed IT Q2W for up to 5 doses alone or with IPI at 3mg/kg Q3 weeks for 4 doses. Maintenance INT230-6 dosing was Q9W. Results: The study enrolled 29 sarcoma patients with 11 subtypes (mainly leiomyosarcoma, liposarcoma, pleomorphic, chondrosarcoma and chordoma). The maximum INT230-6 dose at a single visit was 175 mL (87.5 mg of CIS, 17.5 mg VIN) in 1 or more tumors, an amount that exceeds an IV dose of VIN or CIS. PK analysis shows that &gt;95% of VIN stays in the tumor. The &gt;20% treatment-related adverse events (TRAEs) in evaluable monotherapy patients (n=15) were localized pain (80%), nausea (40%), fatigue (33%), decreased appetite (27%), and vomiting (20%). The &gt;20% TRAEs in evaluable IPI/INT230-6 patients (n=14) were fatigue (39%), localized pain (39%), nausea (31%), pruritus (23%), rash (23%) and vomiting. G3 TRAEs occurred in 20% and 7% of patients in the INT230-6 and combination arms respectively with no grade 4 or 5 events in either arm. RECIST metrics are confounded by IT injections due to the large volume of highly retained INT230-6 and the influx of immune infiltrates. Analysis of median overall survival (mOS) for INT230-6 alone (n=15) was 649 days. For INT230-6 dosed at a volume/total tumor burden (TTB) ratio of ≥40%, the mOS was 715 days. The mOS of the combination has not been reached with over 1 year of median follow-up. OS data compare favorably to a synthetic control (mOS of 205 days) based on historical data3. The hazard ratios of INT230-6 alone or with IPI for OS to the synthetic control were 0.446 and 0.270, p-values &lt;0.01. Conclusions: INT230-6 dosed IT alone or with IPI was well-tolerated in diverse sarcomas. INT230-6 use was associated with immune infiltration and favorable mOS as compared to a synthetic control, particularly when ≥40% of the TTB was injected. A randomized phase 3 trial vs. SOC in selected sarcoma subtypes with an OS endpoint is planned.

Effect of intratumoral INT230-6 on tumor necrosis and promotion of a systemic immune response: Results from a multicenter phase 1/2 study of solid tumors with and without pembrolizumab (PEM) [Intensity IT-01; Merck KEYNOTE-A10

2520 Background: INT230-6 is a new product with a unique dual anti-cancer mechanism. The drug is comprised of cisplatin (CIS) and vinblastine (VIN) co-formulated with an amphiphilic molecule that enables drug dispersion throughout a tumor and passive diffusion into cancer cells following intratumoral (IT) delivery. A neoadjuvant study in breast cancer confirms that a single injection can kill 95% of an injected tumor and recruit TILs. Methods: INT230-6 treatments are Q2W for up to 5 treatments followed by maintenance dosing every 9 weeks. Dose is set by the tumor’s longest diameter or volume. One arm received INT230-6 plus PEM 200mg IV Q3W. Biopsies from injected tumor are taken pretreatment and Day 28 for immunohistochemistry (IHC) analysis. Results: Sixty-two subjects received INT230-6 alone (median age 61, with 4 prior treatments), and 21 INT230-6 + PEM (median age 70, with 3 prior treatments). To these subjects over 575 image guided INT230-6 IT injections were given (320 to visceral tumors such as lung, liver, pancreas). Doses ranged from 0.14 to 175mL (87.5 mg of CIS, 17.5 mg VIN - higher than typical IV doses). Pharmacokinetic data shows &gt; 95% of the INT230-6 active agents remain in the tumor. The most common ( &gt; 25%) adverse events (AEs) related to INT230-6 were localized pain (58%), nausea (40%), and fatigue (29%). The most common AEs attributed to the PEM combination were nausea (62%), localized pain (57%), vomiting (57%), decreased appetite (43%), fatigue (43%) and constipation (29%). The incidence of grade 3 AEs for the INT230-6 arm was 11% and for the PEM combination was 14%. There were no related grade 4 or 5 AEs in the INT230-6 arm; and one grade 4 neutrophil count decrease was seen on the PEM combination. There were no dose limiting adverse events. No patient discontinued therapy due to toxicities related to either drug or injection procedure. The monotherapy arm enrolled patients from 17 tumor types; while the PEM combo recruited primarily pancreatic, CRC, triple negative breast, or bile duct cancer. IHC results confirm a marked reduction in proliferating tumor cells with influx of CD4 and CD8 T-cells. Seven of the INT230-6 monotherapy patients had non injected tumor shrinkage in 9 visceral/deep lesions. Estimated median overall survival (mOS) was over 1 year for both arms. Conclusions: In this clinical trial, deep and superficial tumor injections into patients with widely metastatic disease was feasible and well tolerated. Biopsies confirm the dual anti-cancer mechanism, and study patients live longer than would be expected for these refractory populations. INT230-6’s rapid tumor killing and immune activation properties may offer an alternative to control refractory patients (even those that are chemotherapy refractory) and the product is moving into randomized controlled registration trials. Clinical trial information: NCT03058289.

GALLANT-1: Galectin-3 (Gal-3) inhibitor GB1211 plus atezolizumab (atezo) in patients with non–small cell lung cancer (NSCLC)—A randomized, double-blind trial.

TPS9152 Background: Gal-3 is a protein that binds specifically to N-acetylglucosamine-expressing carbohydrates, which are upregulated on key tumorigenic cell surface proteins. Gal-3 is widely over-expressed in the tumor microenvironment and is generally linked to poor outcomes. Gal-3 regulates immune cell function of T cells and macrophages, and promotes neovascularization and fibrosis [Peng Cancer Res 2008; Markowska J Biol Chem 2011; Kouo Cancer Immunol Res 2015]. Gal-3 sequesters interferon gamma, reduces T-cell influx, and contributes to tumor cell evasion of the immune system via LAG-3 activation [Chen PNAS 2009; Gordon-Alonso Nat Commun 2017]. Gal-3 has been identified as a marker of resistance to checkpoint inhibitors (CPIs); patients with stage IV NSCLC with high Gal-3 levels (&gt; 70% Gal-3 immunohistochemical staining) have been shown to be resistant to the CPI pembrolizumab [Capalbo Int J Mol Sci 2019]. Animal data indicate synergy between CPI therapy and Gal-3 inhibition [Vuong Cancer Res 2019; Zhang FEBS Open Bio 2021]. Thus, inhibiting Gal-3 together with CPI-based immunotherapy may enhance tumor-specific immune responses, and overcome CPI resistance. Methods: GALLANT-1 (NCT05240131) is a 3-part, placebo-controlled phase Ib/IIa trial that will investigate safety and efficacy of GB1211 (a Gal-3 inhibitor) + atezo vs placebo + atezo in patients with advanced NSCLC. Part A will include 8–12 patients and study safety and tolerability of 200 mg and 400 mg GB1211 twice-daily + atezo (open-label). Primary endpoint is number of adverse events (AEs) after 12 weeks’ treatment and will determine the dosage for Part B. Part B will include 75–94 patients, and is a randomized, double-blind study of GB1211 + atezo or placebo + atezo. Primary endpoints are safety (number of AEs) and efficacy (percentage change from baseline in the sum of longest diameter of target lesions after 12 weeks’ treatment). Part C is an expansion study including patients from Parts A and B, with safety and efficacy assessments. Eligibility criteria: advanced or metastatic stage IIIB or IV NSCLC adenocarcinoma; measurable disease per RECIST v1.1; expression of programmed death ligand-1 on ≥50% of tumor cells; eligible for 1200 mg atezo every 3 weeks. Exclusion criteria: symptomatic, untreated, or actively progressing central nervous system metastases; prior systemic chemotherapy for treatment of recurrent advanced or metastatic disease, except if part of neoadjuvant/adjuvant therapy; prior treatment with immune CPIs and/or GB1211; presence of EGFR mutation and ALK, ROS1, and RET alterations; treatment with antineoplastic or systemic immunotherapeutic agents prior to first GB1211 dose; severe infectious disease &lt; 4 weeks prior to first GB1211 dose; active hepatitis B or C, HIV, or COVID-19. The study is being initiated; updated enrollment status will be presented at the meeting. Clinical trial information: NCT05240131.

Clinical Significance of PD-1 and PD-L1 Expression and Ongoing Interaction in the Tumor Microenvironment in Diffuse Large B Cell Lymphoma (DLBCL) Treated with R-CHOP

Background: PD-1 blockade has demonstrated antitumor activity in diffuse large B-cell lymphoma (DLBCL). In order to select patients who are more likely to respond to PD-1 blockade, predictive biomarkers are being intensively pursued. PD-1 and PD-L1 expression in DLBCL treated with R-CHOP as standard-of-care has been examined in several studies. However, only overall PD-1+ cells but not T-cell specific PD-1 expression have been quantitated, and results of the prognostic significance of PD-L1 expression have been inconsistent in DLBCL patients. Whether ongoing PD-1/PD-L1 interaction in tumors contributes to the adverse prognostic effect of PD-1 and PD-L1 expression is unknown.Patients and Methods: Tissue microarray (TMA) sections ofdiagnostic samples from 414 patients with de novo DLBCL treated with R-CHOP were fluorescently stained with CD3 (T cell marker), PD-1, and PD-L1. Positive cells and PD-1/PD-L1 interaction were quantitatively assessed by the Automated Quantitative Analysis (AQUA) algorithms. To characterize PD-1/PD-L1 expression in each sample, CD3+, PD-1+, PD-L1+, and PD-1+CD3+ (double positive, i.e., PD-1+ T) cell numbers were divided by total nucleated cells (DAPI+) in the representative tumor areas. Ongoing PD-1/PD-L1 interactions were evaluated by the proximity of PD-1+ T cells to the PD-L1+ compartments and proportion of PD-1+ T cells co-localized with PD-L1, generating an interaction score for each sample. Biomarker expression and interaction scores were correlated with patient survival.Results: CD3+, PD-1+, PD-L1+, and PD-1+CD3+ cells were identified in 90%, 30%, 53%, and 18% of the cohort, respectively. The mean percentages of CD3+, PD-1+, PD-L1+, and PD-1+CD3+ cells in DLBCL tissues were 12.1%, 0.44%, 3.6%, and 0.19%, respectively. Activated B-cell-like DLBCL had a significantly higher mean percentage of PD-L1+ cells, but not PD-1+CD3+ cells, compared to germinal center B-cell-like DLBCL (p=0.0012). Three prognostic tiers were identified. Firstly, absence to very low percentage (0-3.5%) of tumor-infiltrating CD3+ T cells in the tissues was associated with poorer overall survival (OS) of DLBCL patients (p=0.028). CD3high DLBCL (>3.5% of all cells were CD3+) was associated with significantly higher percentages of PD-L1+ and PD-1+ cells (p<0.0001 and p=0.005, respectively). Secondly, in CD3high DLBCL, presence of PD-1+ T cells correlated with significantly poorer OS and progression-free survival (PFS) of patients (p=0.028 and p=0.0099, respectively). In contrast, presence/absence of PD-1+ non-T cells was not prognostic. Presence of PD-1+ T cells in CD3high DLBCL was associated with a higher mean percentage of PD-L1+ cells (p=0.019). However, PD-1/PD-L1 interaction scores did not add to the prognostic effect. This may suggest that phenotype of PD-1/PD-L1 interaction is not a biomarker to specify the immunosuppressive role of the PD-1/PD-L1 axis. Lastly, in CD3high DLBCL cases with PD-1−negative T cells, PD-L1 positivity correlated with significantly poorer OS and PFS of patients (p=0.0034 and p=0.0059, respectively), although PD-L1+ was not associated with non-T PD-1 expression either, suggesting PD-1−independent function of PD-L1. In the overall cohort, presence of PD-1+ T cells was associated with a trend of poorer PFS (p=0.079), whereas PD-L1 expression was not prognostic.Conclusions: PD-1 expression in T cells and lack of tumor-infiltrating CD3+ T cells are associated with significantly poorer survival of DLBCL patients. In addition, our data suggest that PD-1 can be a prognostic marker independent of PD-1/PD-L1 co-localization, and that PD-L1 has other PD-1-independent inhibitory functions. These results demonstrate the importance of both T-cell influx and antitumor function in prolonging the survival of DLBCL patients, and provide rationales for PD-1 blockade and immunotherapies activating T cell-mediated immune responses in DLBCL. DisclosuresTran:Genoptix, Inc: Employment. Adams:Navigate BioPhama Services, Inc: Employment. Roscoe:Navigate BioPhama Services, Inc: Employment. Hsi:Seattle Genetics: Consultancy, Honoraria, Speakers Bureau; Abbvie: Research Funding; Eli Lilly and Co.: Research Funding; Cellerant Therapeutics: Research Funding. Piris:Kura Oncology: Research Funding. Winter:Glaxo-Smith-Kline: Research Funding; Merck: Research Funding. Vaupel:Genoptix Laboratory: Employment. Dakappagari:Genoptix, Inc: Employment.

Preconditioning of the tumor microenvironment with oncolytic reovirus converts CD3-bispecific antibody treatment into effective immunotherapy

BackgroundT-cell-engaging CD3-bispecific antibodies (CD3-bsAbs) are promising modalities for cancer immunotherapy. Although this therapy has reached clinical practice for hematological malignancies, the absence of sufficient infiltrating T cells is a major...

Densities of decidual high endothelial venules correlate with T-cell influx in healthy pregnancies and idiopathic recurrent pregnancy losses.

Do high endothelial venules (HEVs) appear in the uterus of healthy and pathological pregnancies? Our study reveals that HEVs are present in the non-pregnant endometrium and decidua parietalis (decP) but decline upon placentation in decidua basalis (decB) and are less abundant in decidual tissues from idiopathic, recurrent pregnancy losses (RPLs). RPL is associated with a compromised decidual vascular phenotype. Endometrial (n = 29) and first trimester decidual (n = 86, 6-12th week of gestation) tissue samples obtained from endometrial biopsies or elective pregnancy terminations were used to determine the number of HEVs and T cells. In addition, quantification of HEVs and immune cells was performed in a cohort of decidual tissues from RPL (n = 25). Position and frequency of HEVs were determined in non-pregnant endometrial as well as decidual tissue sections using immunofluorescence (IF) staining with antibodies against E-selectin, intercellular adhesion molecule, von Willebrand factor, ephrin receptor B4, CD34 and a carbohydrate epitope specific to HEVs (MECA-79). Immune cell distribution and characterization was determined by antibodies recognizing CD45 and CD3 by IF staining- and flow cytometry-based analyses. Antibodies against c-c motif chemokine ligand 21 (CCL21) and lymphotoxin-beta were used in IF staining and Western blot analyses of decidual tissues. Functional HEVs are found in high numbers in the secretory endometrium and decP but decline in numbers upon placentation in decB (P ≤ 0.001). Decidua parietalis tissues contain higher levels of the HEV-maintaining factor lymphotoxin beta and decP-associated HEVs also express CCL21 (P ≤ 0.05), a potent T-cell chemoattractant. Moreover, there is a positive correlation between the numbers of decidual HEVs and the abundance of CD3+ cells in decidual tissue sections (P ≤ 0.001). In-depth analysis of a RPL tissue collection revealed a decreased decB (P ≤ 0.01) and decP (P ≤ 0.01) HEV density as well as reduced numbers of T cells in decB (P ≤ 0.05) and decP (P ≤ .001) sections when compared with age-matched healthy control samples. Using receiver-operating characteristics analyses, we found significant predictive values for the ratios of CD3/CD45 (P < 0.001) and HEVs/total vessels (P < 0.001) for the occurrence of RPL. Analyses were performed in first trimester decidual tissues from elective terminations of pregnancy or non-pregnant endometrium samples from patients diagnosed with non-endometrial pathologies including cervical polyps, ovarian cysts and myomas. First trimester decidual tissues may include pregnancies which potentially would have developed placental disorders later in gestation. In addition, our cohort of non-pregnant endometrium may not reflect the endometrial vascular phenotype of healthy women. Finally, determination of immune cell distributions in the patient cohorts studied may be influenced by the different modes of tissue derivation. Pregnancy terminations were performed by surgical aspiration, endometrial tissues were obtained by biopsies and RPL tissues were collected after spontaneous loss of pregnancy. In this study, we propose an inherent mechanism by which the endometrium and in particular the decidua control T-cell recruitment. By demonstrating reduced HEV densities and numbers of T cells in decB and decP tissues of RPL samples we further support previous findings reporting an altered vascular phenotype in early pregnancy loss. Altogether, the findings provide important information to further decipher the etiologies of unexplained RPL. This study was supported by the Austrian Science Fund (P31470 B30 to M.K.) and by the Austrian National Bank (17613ONB to J.P.). There are no competing interests to declare. N/A.

Kidney-intrinsic factors determine the severity of ischemia/reperfusion injury in a mouse model of delayed graft function

Delayed graft function due to transplant ischemia/reperfusion injury adversely affects up to 50% of deceased-donor kidney transplant recipients. However, key factors contributing to the severity of ischemia/reperfusion injury remain unclear. Here, using a clinically relevant mouse model of delayed graft function, we demonstrated that donor genetic background and kidney-intrinsic MyD88/Trif-dependent innate immunity were key determinants of delayed graft function. Functional deterioration of kidney grafts directly corresponded with the duration of cold ischemia time. The graft dysfunction became irreversible after cold ischemia time exceeded six hours. When cold ischemia time reached four hours, kidney grafts displayed histological features reflective of delayed graft function seen in clinical kidney transplantation. Notably, kidneys of B6 mice exhibited significantly more severe histological and functional impairment than kidneys of C3H or BALB/c mice, regardless of recipient strains or alloreactivities. Furthermore, allografts of B6 mice also showed an upregulation of IL-6, neutrophil gelatinase-associated lipocalin, and endoplasmic reticulum stress genes, as well as an increased influx of host neutrophils and memory CD8 T-cells. In contrast, donor MyD88/Trif deficiency inhibited neutrophil influx and decreased the expression of IL-6 and endoplasmic reticulum stress genes, along with improved graft function and prolonged allograft survival. Thus, kidney-intrinsic factors involving genetic characteristics and innate immunity serve as critical determinants of the severity of delayed graft function. This preclinical murine model allows for further investigations of the mechanisms underlying delayed graft function.

Beneficial Effect of Mildly Pasteurized Whey Protein on Intestinal Integrity and Innate Defense in Preterm and Near-Term Piglets.

Background. The human digestive tract is structurally mature at birth, yet maturation of gut functions such as digestion and mucosal barrier continues for the next 1–2 years. Human milk and infant milk formulas (IMF) seem to impact maturation of these gut functions differently, which is at least partially related to high temperature processing of IMF causing loss of bioactive proteins and formation of advanced glycation end products (AGEs). Both loss of protein bioactivity and formation of AGEs depend on heating temperature and time. The aim of this study was to investigate the impact of mildly pasteurized whey protein concentrate (MP-WPC) compared to extensively heated WPC (EH-WPC) on gut maturation in a piglet model hypersensitive to enteral nutrition. Methods. WPC was obtained by cold filtration and mildly pasteurized (73 °C, 30 s) or extensively heat treated (73 °C, 30 s + 80 °C, 6 min). Preterm (~90% gestation) and near-term piglets (~96% gestation) received enteral nutrition based on MP-WPC or EH-WPC for five days. Macroscopic and histologic lesions in the gastro-intestinal tract were evaluated and intestinal responses were further assessed by RT-qPCR, immunohistochemistry and enzyme activity analysis. Results. A diet based on MP-WPC limited epithelial intestinal damage and improved colonic integrity compared to EH-WPC. MP-WPC dampened colonic IL1-β, IL-8 and TNF-α expression and lowered T-cell influx in both preterm and near-term piglets. Anti-microbial defense as measured by neutrophil influx in the colon was only observed in near-term piglets, correlated with histological damage and was reduced by MP-WPC. Moreover, MP-WPC stimulated iALP activity in the colonic epithelium and increased differentiation into enteroendocrine cells compared to EH-WPC. Conclusions. Compared to extensively heated WPC, a formula based on mildly pasteurized WPC limits gut inflammation and stimulates gut maturation in preterm and near-term piglets and might therefore also be beneficial for preterm and (near) term infants.

Clonality, Antigen Recognition, and Suppression of CD8+ T Cells Differentially Affect Prognosis of Breast Cancer Subtypes.

In breast cancer, response rates to immune therapies are generally low and differ significantly across molecular subtypes, urging a better understanding of immunogenicity and immune evasion. We interrogated large gene-expression data sets including 867 node-negative, treatment-naïve breast cancer patients (microarray data) and 347 breast cancer patients (whole-genome sequencing and transcriptome data) according to parameters of T cells as well as immune microenvironment in relation to patient survival. We developed a 109-immune gene signature that captures abundance of CD8 tumor-infiltrating lymphocytes (TIL) and is prognostic in basal-like, her2, and luminal B breast cancer, but not in luminal A or normal-like breast cancer. Basal-like and her2 are characterized by highest CD8 TIL abundance, highest T-cell clonality, highest frequencies of memory T cells, and highest antigenicity, yet only the former shows highest expression level of immune and metabolic checkpoints and highest frequency of myeloid suppressor cells. Also, luminal B shows a high antigenicity and T-cell clonality, yet a low abundance of CD8 TILs. In contrast, luminal A and normal-like both show a low antigenicity, and notably, a low and high abundance of CD8 TILs, respectively, which associates with T-cell influx parameters, such as expression of adhesion molecules. Collectively, our data argue that not only CD8 T-cell presence itself, but rather T-cell clonality, T-cell subset distribution, coinhibition, and antigen presentation reflect occurrence of a CD8 T-cell response in breast cancer subtypes, which have been aborted by distinct T-cell-suppressive mechanisms, providing a rationale for subtype-specific combination immune therapies.