Host T-cell Research Articles

EXTH-18. ADAPTER CHIMERIC ANTIGEN RECEPTOR T CELL THERAPY TARGETING SSTR2 IN MENINGIOMA

Abstract Effective treatment options for meningioma patients beyond surgical resection and radiotherapy are limited. This study presents preclinical and translational evidence supporting the efficacy of an adaptor CAR-T cell system targeting Somatostatin Receptor (SSTR) 2 in otherwise untreatable meningiomas. Tissue microarray analysis revealed that SSTR2 expression was generally heterogeneous but high or intermediate in most World Health Organization (WHO) grade 2 (87.8%) and grade 3 (87.5%) meningiomas. The fluorescein-linked, SSTR2 antagonist peptide octofluo was used in conjunction with adaptor FITC (AdFITC) CAR-T cells to treat experimental meningiomas. In vitro, 10 nM octofluo induced tumor cell killing within 72 hours at effector-target cell ratios (E:T) as low as 1:100. This was observed in two human meningioma cell lines, Ben-Men-1 (CAR-T cells vs. untransduced T-cells [UTT]: 47.9% vs. 4.7%, P < 0.001) and IOMM-Lee (CAR-T cells vs. UTT: 40.2% vs. 4.9%, P < 0.001). In vivo, the AdFITC CAR-T cell system inhibited meningioma growth (P = 0.002) and prolonged survival (P = 0.003) in the IOMM-Lee orthotopic model compared to PBS. In a genetically engineered murine meningioma model, the AdFITC CAR-T cell system not only restricted tumor growth (P = 0.0018) and improved survival (P < 0.0001), but cured a substantial proportion of meningioma bearing mice. This treatment also induced the expansion of both CD8+ CAR-T cells (P = 0.0041) and a host T-cell response. Preliminary ex vivo killing assays, involving co-culture of the AdFITC CAR-T cell system with patient-derived meningioma tissue, showed specific lysis rates of 15-20% (ET 1:1) within 12 hours. Targeting SSTR2 with the AdFITC CAR-T cell system emerges as a promising therapeutic approach for meningioma, an early phase clinical trial is warranted.

MSC–extracellular vesicle microRNAs target host cell-entry receptors in COVID-19: in silico modeling for in vivo validation

BackgroundCoronavirus disease 2019 (COVID-19) has created a global pandemic with significant morbidity and mortality. SARS-CoV-2 primarily infects the lungs and is associated with various organ complications. Therapeutic approaches to combat COVID-19, including convalescent plasma and vaccination, have been developed. However, the high mutation rate of SARS-CoV-2 and its ability to inhibit host T-cell activity pose challenges for effective treatment. Mesenchymal stem cells (MSCs) and their extracellular vesicles (MSCs–EVs) have shown promise in COVID-19 therapy because of their immunomodulatory and regenerative properties. MicroRNAs (miRNAs) play crucial regulatory roles in various biological processes and can be manipulated for therapeutic purposes.ObjectiveWe aimed to investigate the role of lyophilized MSC–EVs and their microRNAs in targeting the receptors involved in SARS-CoV-2 entry into host cells as a strategy to limit infection. In silico microRNA prediction, structural predictions of the microRNA–mRNA duplex, and molecular docking with the Argonaut protein were performed.MethodsMale Syrian hamsters infected with SARS-CoV-2 were treated with human Wharton’s jelly-derived Mesenchymal Stem cell-derived lyophilized exosomes (Bioluga Company)via intraperitoneal injection, and viral shedding was assessed. The potential therapeutic effects of MSCs–EVs were measured via histopathology of lung tissues and PCR for microRNAs.ResultsThe results revealed strong binding potential between miRNA‒mRNA duplexes and the AGO protein via molecular docking. MSCs–EVs reduced inflammation markers and normalized blood indices via the suppression of viral entry by regulating ACE2 and TMPRSS2 expression. MSCs–EVs alleviated histopathological aberrations. They improved lung histology and reduced collagen fiber deposition in infected lungs.ConclusionWe demonstrated that MSCs–EVs are a potential therapeutic option for treating COVID-19 by preventing viral entry into host cells.

Genetic variants associated with immune-mediated liver injury from checkpoint inhibitors.

The clinical features, liver histology, and genetic variants in 57 patients with moderate to severe immune-mediated liver injury from checkpoint inhibitors (ILICI) are presented. Between 2010 and 2022, 57 high-causality ILICI cases were enrolled in the Drug-Induced Liver Injury Network. HLA and selected candidate gene variants were tested for association with ILICI risk compared to the general population and other DILI controls. The 57 high-causality cases were attributed to pembrolizumab (16), ipilimumab (15), ipilimumab and nivolumab (13), and other immune checkpoint inhibitors (13) and occurred at a median of 72 days after the first infusion. Median age was 57.8 years, 66% male, and 89% were non-Hispanic Whites. At DILI onset, 53% had hepatocellular, 35% mixed, and 15% cholestatic, with younger patients more likely to have hepatocellular injury. The incidence of ANA, smooth muscle antibody, and elevated IgG levels was low (17%, 23%, and 0%), but corticosteroids were given to 86%. Microgranulomas and hepatic steatosis were seen in 54% and 46% of the 26 liver biopsies, respectively. The HLA alleles associated with autoimmune hepatitis were not over-represented, but 2 host immune response genes (EDIL3 and SAMA5A) and 3 other genes (GABRP, SMAD3, and SLCO1B1) were associated with ILICI (OR: 2.08-2.4, p<0.01). ILICI typically arises within 12 weeks of initiating immunotherapy and is self-limited in most cases. Genetic variants involved in host T-cell regulation and drug disposition were identified, implicating these pathways in the pathogenesis of ILICI. If validated, these findings could lead to improved diagnostic instruments and possible treatments for ILICI.

Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under physiological conditions. In recent years, Mertk has also been linked to pathophysiological roles in cancer, whereby, in several cancer types, including solid cancers and leukemia/lymphomas. Mertk contributes to oncogenic features of proliferation and cell survival as an oncogenic tyrosine kinase. In addition, Mertk expressed on macrophages, including tumor-associated macrophages, promotes immune evasion in cancer and is suggested to act akin to a myeloid checkpoint inhibitor that skews macrophages towards inhibitory phenotypes that suppress host T-cell anti-tumor immunity. In the present study, to better understand the post-translational regulation mechanisms controlling Mertk expression in monocytes/macrophages, we used a PMA-differentiated THP-1 cell model to interrogate the regulation of Mertk expression and developed a novel Mertk reporter cell line to study the intracellular trafficking of Mertk. We show that PMA treatment potently up-regulates Mertk as well as components of the ectodomain proteolytic processing platform ADAM17, whereas PMA differentially regulates the canonical Mertk ligands Gas6 and Pros1 (Gas6 is down-regulated and Pros1 is up-regulated). Under non-stimulated homeostatic conditions, Mertk in PMA-differentiated THP1 cells shows active constitutive proteolytic cleavage by the sequential activities of ADAM17 and the Presenilin/γ-secretase complex, indicating that Mertk is cleaved homeostatically by the combined sequential action of ADAM17 and γ-secretase, after which the cleaved intracellular fragment of Mertk is degraded in a proteasome-dependent mechanism. Using chimeric Flag-Mertk-EGFP-Myc reporter receptors, we confirm that inhibitors of γ-secretase and MG132, which inhibits the 26S proteasome, stabilize the intracellular fragment of Mertk without evidence of nuclear translocation. Finally, the treatment of cells with active γ-carboxylated Gas6, but not inactive Warfarin-treated non-γ-carboxylated Gas6, regulates a distinct proteolytic itinerary-involved receptor clearance and lysosomal proteolysis. Together, these results indicate that pleotropic and complex proteolytic activities regulate Mertk ectodomain cleavage as a homeostatic negative regulatory event to safeguard against the overactivation of Mertk.

Routine evaluation of HBV-specific T cell reactivity in chronic hepatitis B using a broad-spectrum T-cell epitope peptide library and ELISpot assay

BackgroundThe clinical routine test of HBV-specific T cell reactivity is still limited due to the high polymorphisms of human leukocyte antigens (HLA) in patient cohort and the lack of universal detection kit, thus the clinical implication remains disputed.MethodsA broad-spectrum peptide library, which consists of 103 functionally validated CD8+ T-cell epitopes spanning overall HBsAg, HBeAg, HBx and HBpol proteins and fits to the HLA polymorphisms of Chinese and Northeast Asian populations, was grouped into eight peptide pools and was used to establish an ELISpot assay for enumerating the reactive HBV-specific T cells in PBMCs. Totally 294 HBV-infected patients including 203 ones with chronic hepatitis B (CHB), 13 ones in acute resolved stage (R), 52 ones with liver cirrhosis (LC) and 26 ones with hepatocellular carcinoma (HCC) were detected, and 33 CHB patients were longitudinally monitored for 3 times with an interval of 3–5 months.ResultsThe numbers of reactive HBV-specific T cells were significantly correlated with ALT level, HBsAg level, and disease stage (R, CHB, LC and HCC), and R patients displayed the strongest HBV-specific T cell reactivity while CHB patients showed the weakest one. For 203 CHB patients, the numbers of reactive HBV-specific T cells presented a significantly declined trend when the serum viral DNA load, HBsAg, HBeAg or ALT level gradually increased, but only a very low negative correlation coefficient was defined (r = − 0.21, − 0.21, − 0.27, − 0.079, respectively). Different Nucleotide Analogs (NUCs) did not bring difference on HBV-specific T cell reactivity in the same duration of treatment. NUCs/pegIFN-α combination led to much more reactive HBV-specific T cells than NUCs monotherapy. The dynamic numbers of reactive HBV-specific T cells were obviously increasing in most CHB patients undergoing routine treatment, and the longitudinal trend possess a high predictive power for the hepatitis progression 6 or 12 months later.ConclusionThe presented method could be developed into an efficient reference method for the clinical evaluation of cellular immunity. The CHB patients presenting low reactivity of HBV-specific T cells have a worse prognosis for hepatitis progression and should be treated using pegIFN-α to improve host T-cell immunity.

Acellular scaffold-based approach for in situ genetic engineering of host T-cells in solid tumor immunotherapy

BackgroundTargeted T-cell therapy has emerged as a promising strategy for the treatment of hematological malignancies. However, its application to solid tumors presents significant challenges due to the limited accessibility and heterogeneity. Localized delivery of tumor-specific T-cells using biomaterials has shown promise, however, procedures required for genetic modification and generation of a sufficient number of tumor-specific T-cells ex vivo remain major obstacles due to cost and time constraints.MethodsPolyethylene glycol (PEG)-based three-dimensional (3D) scaffolds were developed and conjugated with positively charged poly-L-lysine (PLL) using carbamide chemistry for efficient loading of lentiviruses (LVs) carrying tumor antigen-specific T-cell receptors (TCRs). The physical and biological properties of the scaffold were extensively characterized. Further, the scaffold loaded with OVA-TCR LVs was implanted in B16F10 cells expressing ovalbumin (B16-OVA) tumor model to evaluate the anti-tumor response and the presence of transduced T-cells.ResultsOur findings demonstrate that the scaffolds do not induce any systemic inflammation upon subcutaneous implantation and effectively recruit T-cells to the site. In B16-OVA melanoma tumor-bearing mice, the scaffolds efficiently transduce host T-cells with OVA-specific TCRs. These genetically modified T-cells exhibit homing capability towards the tumor and secondary lymphoid organs, resulting in a significant reduction of tumor size and systemic increase in anti-tumor cytokines. Immune cell profiling revealed a significantly high percentage of transduced T-cells and a notable reduction in suppressor immune cells within the tumors of mice implanted with these scaffolds.ConclusionOur scaffold-based T-cell therapy presents an innovative in situ localized approach for programming T-cells to target solid tumors. This approach offers a viable alternative to in vitro manipulation of T-cells, circumventing the need for large-scale in vitro generation and culture of tumor-specific T-cells. It offers an off-the-shelf alternative that facilitates the use of host cells instead of allogeneic cells, thereby, overcoming a major hurdle.

Visualizing HIV-1 Assembly at the T-Cell Plasma Membrane Using Single-Molecule Localization Microscopy.

The 20-year revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and timely acquisition, allows the visualization of nanoscaled objects in cell biology. Currently, the use of a recent generation of super-resolution fluorescence microscope coupled with improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus particle or protein level. Here, we highlight the protocol for visualizing HIV-1 Gag assembly at the host T-cell plasma membrane using super-resolution light microscopy. Total internal reflection fluorescence microscopy (TIRF-M) coupled with single-molecule localization microscopy (SMLM) enables the detection and characterization of the assembly of viral proteins at the plasma membrane of infected host cells at the single protein level. Here, we describe the TIRF equipment, the T-cell culture for HIV-1, the sample preparation for single-molecule localization microscopies such as PALM and STORM, acquisition protocols, and Gag assembling cluster analysis.

Laboratory Evaluation of SARS-CoV-2 Specific T-Cell Immunity by ELISpot Interferon-γ Based Immunoassay

Cellular and humoral immune responses are fundamental for SARS-CoV-2 elimination, infection resolution, and protection against reinfection. Data on long-term SARS-CoV-2-specific T-cell responses are limited. We conducted this study to evaluate the presence of SARS-CoV-2 specific T lymphocytes using the ELISpot interferon-γ-based kit for outpatients with or without a history of SARS-CoV-2 infection. SARS-CoV-2 activated T-cell response was defined from peripheral blood mononuclear cells (PBMCs) of nine (n=9) outpatients- 6 women and 3 men, aged 32 to 73 years old, all from Varna city, Bulgaria. They were tested between November and December 2021. Eight of them – 88.9% were in contact with the COVID-19 virus from 1 to 20 months ago. The number of spots obtained provides a measure of the abundance of SARS-CoV-2 – sensitized effector T cells in the peripheral blood. A reactive T-cellular immune response was ob¬served 12 months after SARS-CoV-2 viral infection. In addition, IgG humoral immunity was analyzed and compared with the specific T-cell response in some participants. Compared with humoral immunity-re¬lated studies, those focusing on SARS-CoV-2-specific cellular immunity using the ElISpot immunoassay are relatively falling behind standardization. The SARS-CoV-2 specific ELISpot Interferon-γ immunoassay, described in our study, conducted with a small group of patients, can serve as an effective immunoassay to measure host T-cell responses and help further detailed understanding of pathogenetic mechanisms and persistence of cellular and humoral immunity against SARS-CoV-2.

Low-Dose Staphylococcal Enterotoxin C2 Mutant Maintains Bone Homeostasis via Regulating Crosstalk between Bone Formation and Host T-Cell Effector Immunity.

Studies in recent years have highlighted an elaborate crosstalk between T cells and bone cells, suggesting that T cells may be alternative therapeutic targets for the maintenance of bone homeostasis. Here, it is reported that systemic administration of low-dose staphylococcal enterotoxin C2 (SEC2) 2M-118, a form of mutant superantigen, dramatically alleviates ovariectomy (OVX)-induced bone loss via modulating T cells. Specially, SEC2 2M-118 treatment increases trabecular bone mass significantly via promoting bone formation in OVX mice. These beneficial effects are largely diminished in T-cell-deficient nude mice and can be rescued by T-cell reconstruction. Neutralizing assays determine interferon gamma (IFN-γ)as the key factor that mediates the beneficial effects of SEC2 2M-118 on bone. Mechanistic studies demonstrate that IFN-γ stimulates Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling, leading to enhanced production of nitric oxide, which further activates p38 mitogen-activated protein kinase (MAPK) and Runt-related transcription factor 2 (Runx2) signaling and promotes osteogenic differentiation. IFN-γ also directly inhibits osteoclast differentiation, but this effect is counteracted by proabsorptive factors tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) secreted from IFN-γ-stimulated macrophages. Taken together, this work provides clues for developing innovative approaches which target T cells for the prevention and treatment of osteoporosis.

Role of T cells in cervical cancer.

Cervical cancer is an important health problem and it is considered the fourth most lethal women's cancer worldwide. The intertumoral T cell pool is exposed in a number of immunosuppressive pathways. Therefore, it is of interest to document the effect of cervical cancer on immune system, the role of T cells in the development and pathogenesis of cervical cancer. HPV is considered the most important risk factors for developing cervical cancer, HPV 16 and 18, the two most common oncogenic types which are high risk HPV cause 70% of cervical cancer cases. In the cervical mucosa, the proportion of CD4+ and CD8+ T cells is related to the severity of the lesions. Cervical cancer can be treated by immunotherapeutic vaccine which involves T cells. T cells play an important part in cervical cancer pathogenesis because HPV exploits several methods to avoid host T-cell immune surveillance. T-cell-based immunotherapy is important because it is selective and has therapeutic potential.

Abstract 1564: Novel combination of TRIP13 and Aurora kinase A inhibition demonstrated extensive DNA damage and immunogenic cell death in RB-deficient cancers

Abstract Many lethal cancer types have inactivation of the retinoblastoma (Rb) tumor suppressor pathway including cancers caused by human papillomavirus (HPV), which causes &amp;gt;5% of all cancers worldwide. There are no therapies that uniquely target Rb-deficient cancers. Biomarker-selected, molecular targeted therapy for Rb-deficient cancers represents both an unmet need and a translational knowledge gap. To address this gap, we recently published data that support a model in which Rb-deficient cancers, having high levels of Mad2, survive owing to TRIP13-mediated inhibition of Mad2 and Aurora A-mediated mitotic progression. The combination of Aurora A inhibition (alisertib) plus TRIP13 depletion caused extensive apoptosis in Rb-deficient, but not in Rb-proficient, cancer cells. Both Aurora A and TRIP13 are involved in the cell’s response to DNA damage and apoptosis that can lead to immunogenic cell death (ICD), prompting us to investigate the mechanism underlying the synthetic lethality of TRIP13 and Aurora A in Rb-deficient cancer cells. We transfected two HPV+ cell lines with TRIP13 or control siRNA and incubated with 100nM alisertib. We also generated two stable HPV+ cell lines with TRIP13KO. In both these systems, TRIP13 KD or KO alone did not cause apoptosis. Incubation with alisertib resulted in significant cell death, that validated our previous findings. We then tested the effect of the combination on DNA damage. Alisertib caused robust increases in γH2AX, pTIF1β (KAP1, TRIM28), and DNA PKcs, measured using immunoblotting, in both TRIP13 KD and KO HPV+ cell lines. We also observed enhanced γH2AX staining using confocal microscopy. ICD leads to activation of immune cells by the dying cancer cells in the tumor microenvironment involving the release of antigenic fragments that are engulfed, processed and presented by antigen presenting cells for the induction of antigen-specific T-cell responses. Studies have shown DNA damage stimulates the cGAS/STING pathway leading to ICD. In our system, we saw that TRIP13KO and KD in HPV+ cells treated with alisertib, resulted in marked activation of the cGAS/STING pathway and release of cytochrome C and IL1α in the cell supernatant, which are markers of pyroptosis. Additionally, we also observed gasdermin-E cleavage. The combination further led to increased cancer cell secretion of GM-CSF, IL6, and IL18 that can activate macrophages, NK cells and T-cells. The selective effect of the combination in Rb-deficient cancer cells allowed us to significantly reduce the concentration of alisertib. By sparing normal cells that express Rb, this combination can potentially reduce treatment toxicity in patients with Rb-deficient cancers. The ICD induced by this combination may lead to host T-cell engagement, thereby further enhancing tumor elimination. This work can shape future clinical trials that can change current treatment regime. Citation Format: Soma Ghosh, Tuhina Mazumdar, Lacin Yapindi, Pragya Sinha, Jagannadha Sastry, Faye M. Johnson. Novel combination of TRIP13 and Aurora kinase A inhibition demonstrated extensive DNA damage and immunogenic cell death in RB-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1564.

ETCTN 10476: A randomized phase 2 study of combination atezolizumab and varlilumab (CDX-1127) with or without addition of cobimetinib in previously treated unresectable biliary tract cancer.

TPS639 Background: The combination of the MEK inhibitor, Cobimetinib, with the PD-L1 antagonist, Atezolizumab, significantly improved progression-free survival (PFS) compared to Atezolizumab monotherapy in patients with advanced biliary tract cancer (BTC) following first-line chemotherapy (NCT03201458). Interrogation of biospecimens from this trial and parallel pre-clinical work showed that the addition of MEK inhibition enhanced tumor immunogenicity but simultaneously had detrimental effects on host T-cell activation and priming. The addition of immune co-stimulants rescues T-cell function in the setting of systemic MEK inhibition in vivo. Unlike other immune co-stimulatory markers, CD27 is one of the most positively differentially expressed receptors in the TME post-treatment with combined MEK inhibition and PD-L1 blockade. We hypothesize that addition of CD27 immune agonism to the combination of PD-L1 and MEK inhibition can improve immunotherapy outcomes in patients with BTC. Methods: We are conducting an open-label, randomized ph 2 trial evaluating a PD-L1 inhibitor (atezolizumab) in combination with a CD27 immune agonist (CDX-1127 [Varlilumab]) with or without addition of a MEK inhibitor (Cobimetinib) in patients with unresectable, previously treated, BTC. Key inclusion criteria include: adults with pathologically-confirmed BTC, s/p 1-2 lines of systemic therapy (including any FDA-approved targeted therapies) in the metastatic setting, measurable disease, ECOG performance status ≤1, adequate baseline organ and marrow function. Patients who have received gem-cis-durvalumab, and/or prior FGFR2/IDH1 targeted therapy are eligible for enrollment. The study is planned for 64 evaluable subjects (32 subjects per treatment arm) randomized in a 1:1 ratio, stratified by site of BTC location, to either Atezolizumab + CDX-1127 or Atezolizumab + CDX-1127 + Cobimetinib. During the 28-day treatment cycles, all patients receive Atezolizumab (840mg flat dose) and CDX-1127 (3mg/kg) infusions on D1 and D15. For those randomized to the triplet regimen arm, the additional Cobimetinib will be dosed orally at 60mg daily on days 1-21. Overall response rate and PFS are co-primary endpoints. A clinically meaningful improvement in ORR warranting further study is 20%. The primary correlative outcome is treatment-related changes in CD8+ infiltrating T cells. After an initial safety lead-in, a planned interim efficacy analysis will take place following enrollment of the first 18 patients in each treatment arm. Currently, 14 participants (n=6 [triplet arm], n=8 [doublet arm]), have been enrolled &amp; treated on protocol. This study is sponsored by the NCI Cancer Therapy Evaluation Program and is open nationally at designated NCI Experimental Therapeutics Clinical Trials Network sites. Clinical trial information: NCT04941287 .

CD30.CAR-Modified Epstein-Barr Virus-Specific T Cells (CD30.CAR EBVSTs) Provide a Safe and Effective Off-the-Shelf Therapy for Patients with CD30-Positive Lymphoma

Introduction Off-the-shelf allogeneic T-cell therapies face the major challenges of graft-versus-host disease (GVHD) and graft rejection mediated by host and recipient alloreactive T-cells respectively. To address GVHD, we are using Epstein-Barr Virus-specific T cells (EBVSTs), which are virus specific rather than allo-specific and have not produced GVHD in over 300 allogeneic recipients. To prevent graft rejection, we modified the EBVSTs with a chimeric antigen receptor targeting CD30, an antigen that is upregulated on allo-activated T-cells, which will consequently become targets for CD30.CAR EBVSTs. Hence, CD30.CAR EBVSTs can kill both CD30+ lymphoma cells and alloreactive T-cells through their CAR, and may be boosted in vivo in EBV+ recipients via their native TCR. We are evaluating this approach in a Phase I/II trial (NCT04288726) in patients with CD30-positive lymphoma. Methods We generated a bank of seven CD30.CAR EBVST lines by stimulating peripheral blood mononuclear cells (PBMC) from healthy donors with overlapping EBV peptide mixtures (pepmixes) followed by retroviral transduction with a second generation CD30.CAR containing a CD28 costimulatory endodomain. We have treated 14 patients (median age 36, range 22-53) with relapsed or refractory Hodgkin's lymphoma. Patients had undergone a median of 5 (range 3-6) prior lines of treatment. Escalating doses of 4 × 107 (DL1), 1 × 108 (DL2) or 4 × 108 (DL3) CD30.CAR EBVSTs were infused after lymphodepletion with cyclophosphamide and fludarabine. Blood drawn pre- and post- infusion was processed to isolate serum/plasma and PBMC for analysis. The frequency of CD30.CAR EBVSTs was measured by real time qPCR for the transgene. Changes in the frequency of T-cells responding to EBV or tumor-associated antigens (epitope spreading) were measured by IFNɣ ELISpots. Clinical responses were assessed by diagnostic PET/CT scans performed 4-8 weeks post infusion. Results CD30.CAR EBVSTs were well tolerated, with no GVHD and only two patients having reversible grade 4 cytopenia. Four patients at DL3 had grade 1 cytokine release syndrome (CRS), but all resolved without treatment. Of the 14 patients treated, 13 have been evaluated for responses per Lugano criteria; 4 had partial responses (PR) and 5/10 patients at DL2 and DL3 had complete responses (CR) giving an overall objective response rate (ORR) of 69.2%. Despite these remarkable responses, qPCR for the CD30.CAR transgene showed near background levels by week 1 post infusion. Possible explanations are that CD30.CAR EBVSTs; (1) Are eliminated by alloreactive T-cells, but persist sufficiently to eliminate tumors. (2) Can reactivate endogenous tumor-specific T-cells that are responsible for the tumor responses. (3) Rapidly eliminate tumors and are then lost due to being intrinsically short-lived. (4) Become tissue resident at the tumor site. We think explanation #1 cannot be a major contributor to the lack of circulating CD30CAR T cells since patient 10, who had bulky disease received 3 infusions from the same donor line and responded after each infusion as shown in Figure 1; an alloreactive response would be anticipated to be amplified after each infusion. Similarly, two additional patients receiving second infusions both achieved CR. While we cannot exclude explanation #2, we did not detect epitope spreading in patient PBMCs that would be expected if responses after infusion of CD30.CAR EBVSTs were primarily attributable to reactivation of host anti-tumor cells. We are therefore pursuing explanations #3 and #4 by analyzing tumor biopsy samples to determine the presence of CD30.CAR EBVSTs at tumor sites and characterize their phenotype for functionality. Conclusion We have shown CD30.CAR EBVSTs can be a safe and effective treatment for CD30+ lymphomas, and may avert GVHD and immediate rejection even after multiple infusions. We now seek to improve the durability of responses and test whether CD30.CAR EBVST can be used as a platform for other "off-the-shelf" CAR-T cell therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Modulation of Host-Parasite Interactions with Small Molecules Targeting Schistosoma mansoni microRNAs.

Parasites use different strategies of communication with their hosts. One communication channel that has been studied in recent years is the use of vesicle microRNAs to influence the host immune system by trematodes. sma-microRNA-10, secreted from Schistosoma mansoni, has been shown to influence the fate of host T-cells through manipulation of the NF-κB pathway. We have identified low molecular weight tool compounds that can interfere with this microRNA-mediated manipulation of the host immune system. We used a fragment-based screening approach by means of nuclear magnetic resonance (NMR) to identify binders to the precursor of the parasite sma-microRNA-10 present in their extracellular vesicles. The small fragments identified were used to select larger molecules. These molecules were shown to counteract the inhibition of NF-κB activity by sma-microRNA-10 in cell-based assays.

Hepatocytes infected with hepatitis C virus change immunological features in the liver microenvironment.

Hepatitis C virus (HCV) infection is remarkably efficient in establishing viral persistence, leading to the development of liver cirrhosis and hepatocellular carcinoma (HCC). Direct-acting antiviral agents (DAAs) are promising HCV therapies to clear the virus. However, recent reports indicate potential increased risk of HCC development among HCV patients with cirrhosis following DAA therapy. CD8+ T-cells participate in controlling HCV infection. However, in chronic hepatitis C patients, severe CD4+ and CD8+ T-cell dysfunctions have been observed. This suggests that HCV may employ mechanisms to counteract or suppress the host T-cell responses. The primary site of viral replication is within hepatocytes where infection can trigger the expression of costimulatory molecules and the secretion of immunoregulatory cytokines. Numerous studies indicate that HCV infection in hepatocytes impairs antiviral host immunity by modulating the expression of immunoregulatory molecules. Hepatocytes expressing whole HCV proteins upregulate the ligands of programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and transforming growth factor β (TGF-β) synthesis compared to those in hepatocytes in the absence of the HCV genome. Importantly, HCV-infected hepatocytes are capable of inducing regulatory CD4+ T-cells, releasing exosomes displaying TGF-β on exosome surfaces, and generating follicular regulatory T-cells. Recent studies report that the expression profile of exosome microRNAs provides biomarkers of HCV infection and HCV-related chronic liver diseases. A better understanding of the immunoregulatory mechanisms and identification of biomarkers associated with HCV infection will provide insight into designing vaccine against HCV to bypass HCV-induced immune dysregulation and prevent development of HCV-associated chronic liver diseases.

Identification of two Sel1-like proteins in SPI-19 of Salmonella enterica serovar Pullorum that can mediate bacterial infection through T3SS

The type VI secretion system (T6SS) encoded by Salmonella Pathogenicity Island 19 (SPI-19) has been confirmed to be involved in bacterial infection or colonization in hosts and in the inhibition of the host T-cell immune response. However, deletion of the core genes (clpV, vgrG, and hcp2) encoding the T6SS apparatus does not affect the phenotypes caused by SPI-19-encoded T6SS. As Salmonella infection in host cells and survival in chickens are closely associated with the type III secretion system (T3SS), RNA-Seq was performed, and the results revealed that most T3SS genes were downregulated in the C79–13ΔSPI-19 mutant. To identify the SPI-19 genes involved in regulating T3SS genes expression, we constructed mutants of genes encoding potential regulators (RS09140 and RS09275) or proteins with Sel1-like motifs (RS09150 and RS09155) and analyzed their associated phenotypes. Deletion of RS09150 and RS09155 caused the decreased bacterial infection in avian cells and bacterial colonization in chicken organs. In addition, qRT-PCR results revealed that both mutants showed decreased expression levels of regulatory genes of T3SS. The present findings demonstrate that the two Sel1-like proteins RS09150 and RS09155 in S. Pullorum SPI-19 contribute to bacterial infection in chickens by mediating the expression of T3SS genes, indicating a potential crosstalk between SPI-19 and T3SS in Salmonella.

Time-course of host cell transcription during the HTLV-1 transcriptional burst.

The human T-cell leukemia virus type 1 (HTLV-1) transactivator protein Tax has pleiotropic functions in the host cell affecting cell-cycle regulation, DNA damage response pathways and apoptosis. These actions of Tax have been implicated in the persistence and pathogenesis of HTLV-1-infected cells. It is now known that tax expression occurs in transcriptional bursts of the proviral plus-strand, but the effects of the burst on host transcription are not fully understood. We carried out RNA sequencing of two naturally-infected T-cell clones transduced with a Tax-responsive Timer protein, which undergoes a time-dependent shift in fluorescence emission, to study transcriptional changes during successive phases of the HTLV-1 plus-strand burst. We found that the transcriptional regulation of genes involved in the NF-κB pathway, cell-cycle regulation, DNA damage response and apoptosis inhibition were immediate effects accompanying the plus-strand burst, and are limited to the duration of the burst. The results distinguish between the immediate and delayed effects of HTLV-1 reactivation on host transcription, and between clone-specific effects and those observed in both clones. The major transcriptional changes in the infected host T-cells observed here, including NF-κB, are transient, suggesting that these pathways are not persistently activated at high levels in HTLV-1-infected cells. The two clones diverged strongly in their expression of genes regulating the cell cycle. Up-regulation of senescence markers was a delayed effect of the proviral plus-strand burst and the up-regulation of some pro-apoptotic genes outlasted the burst. We found that activation of the aryl hydrocarbon receptor (AhR) pathway enhanced and prolonged the proviral burst, but did not increase the rate of reactivation. Our results also suggest that sustained plus-strand expression is detrimental to the survival of infected cells.

US Severe Acute Respiratory Syndrome Coronavirus 2 Epsilon Variant: Highly Transmissible but With an Adjusted Muted Host T-Cell Response.

BackgroundThe multiple mutations comprising the epsilon variant demonstrate the independent convergent evolution of severe acute respiratory syndrome coronavirus (SARS-CoV-2), with its spike protein mutation L452R present in the delta (L452R), kappa (L452R), and lambda (L452Q) variants.MethodsCoronavirus disease 2019 (COVID-19) variants were detected in 1017 patients using whole-genome sequencing and were assessed for outcome and severity. The mechanistic effects of the epsilon versus non-epsilon variants were investigated using a multiomic approach including cellular response assays and paired cell and host transcriptomic and proteomic profiling.ResultsWe found that patients carrying the epsilon variant had increased mortality risk but not increased hospitalizations (P < .02). Cells infected with live epsilon compared with non-epsilon virus displayed increased sensitivity to neutralization antibodies in all patients but a slightly protective response in vaccinated individuals (P < .001). That the epsilon SARS-CoV-2 variant is more infectious but less virulent is supported mechanistically in the down-regulation of viral processing pathways seen by multiomic analyses. Importantly, this paired transcriptomics and proteomic profiling of host cellular response to live virus revealed an altered leukocyte response and metabolic messenger RNA processing with the epsilon variant. To ascertain host response to SARS-CoV-2 infection, primary COVID-19–positive nasopharyngeal samples were transcriptomically profiled and revealed a differential innate immune response (P < .001) and an adjusted T-cell response in patients carrying the epsilon variant (P < .002). In fact, patients infected with SARS-CoV-2 and those vaccinated with the BNT162b2 vaccine have comparable CD4+/CD8+ T-cell immune responses to the epsilon variant (P < .05).ConclusionsWhile the epsilon variant is more infectious, by altering viral processing, we showed that patients with COVID-19 have adapted their innate immune response to this fitter variant. A protective T-cell response molecular signature is generated by this more transmissible variant in both vaccinated and unvaccinated patients.

Neoantigen: A Promising Target for the Immunotherapy of Colorectal Cancer

At present, there are various treatment strategies for colorectal cancer, including surgery, chemotherapy, radiotherapy, and targeted therapy. In recent years, with the continuous development of immunotherapy, immune checkpoint inhibitors (ICIs) can significantly improve the treatment of advanced colorectal cancer patients with high levels of microsatellite instability. In addition to ICIs, neoantigens, as a class of tumor-specific antigens (TSA), are regarded as new immunotherapy targets for many cancer species and are being explored for antitumor therapy. Immunotherapy strategies based on neoantigens include tumor vaccines and adoptive cell therapy (ACT). These methods aim to eliminate tumor cells by enhancing the immune response of host T-cells to neoantigens. In addition, for MSS colorectal cancer, such “cold tumors” with low mutation rates and stable microsatellites are not sensitive to ICIs, whereas neoantigens could provide a promising immunotherapeutic avenue. In this review, we summarized the current status of colorectal cancer neoantigen prediction and current clinical trials of neoantigens and discussed the difficulties and limitations of neoantigens-based therapies for the treatment of CRC.

CTIM-26. PHASE I/II STUDY OF THE COMBINATION OF PEMBROLIZUMAB (MK-3475) AND LASER INTERSTITIAL THERMAL THERAPY (LITT) IN RECURRENT GLIOBLASTOMA

Abstract BACKGROUND The blood brain barrier (BBB) remains a potential barrier to central nervous system (CNS) penetration of novel immunotherapies in recurrent glioblastoma (rGBM). Laser interstitial thermal therapy (LITT) was recently demonstrated to induce BBB disruption. When combined with anti-PD1 blockade, LITT may therefore potentiate host T-cell mediated cytotoxicity. This phase I/II study aims to evaluate the safety and efficacy of combining LITT and the PD-1 inhibitor pembrolizumab for rGBM. METHODS Phase I treated eligible bevacizumab-naïve high grade glioma patients with LITT and the anti-PD1 inhibitor pembrolizumab at 3 dose levels (100, 150, and 200 mg IV Q3W; 3 patients at each level), while phase II was restricted to rGBM patients only with the recommended phase II dose (RP2D) of pembrolizumab. Phase II was initially designed to randomize rGBM to either pembrolizumab or pembrolizumab+LITT but was later amended to receive only pembrolizumab+LITT after 16 patients were randomized (10 pembrolizumab+LITT arm, 6 pembrolizumab-alone arm). Progression-free survival (PFS) and overall survival (OS) were evaluated using the Kaplan Meier method, and adverse events (AE) documented. RESULTS Phase I enrolled 9 patients (7 rGBM and 2 anaplastic astrocytomas, 33% IDH-mut, 44% MGMTp-methylated) with no dose limiting toxicities (DLT), prompting selection of 200mg Q3W as the RP2D. Phase II interim analysis included 34 rGBM patients (9% IDH-mut, 50% MGMTp-methylated; 6 receiving pembrolizumab alone and 28 pembrolizumab+LITT) plus two Phase I rGBM patients who received RP2D. On per-protocol analysis, pembrolizumab+LITT cohort had improved PFS (median PFS 10.5 months vs 2.1 months) and OS (median OS 11.4 months vs 5.2 months) than pembrolizumab alone. A single treatment-related grade 3 AE was noted (respiratory infection). CONCLUSION: LITT may be safely combined with pembrolizumab for rGBM with promising clinical outcomes on interim analysis. Enrollment for Phase II, correlative studies, and continued AE documentation are ongoing. Clinical Trial ID NCT02311582.