Combination Of IL-2 Research Articles

Isotretinoin promotes elimination of translation-competent HIV latent reservoirs in CD4T cells.

Development of novel therapeutic strategies that reactivate latent HIV and sensitize reactivated cells to apoptosis is crucial towards elimination of the latent viral reservoir. Among the clinically relevant latency reversing agents (LRA) under investigation, the γc-cytokine IL-15 and the superagonist N-803 have been shown to reactivate latent HIV ex vivo and in vivo. However, their clinical benefit can be hindered by IL-15 promoting survival of infected cells. We previously identified a small molecule, HODHBt, that sensitizes latently infected cells to death upon reactivation with γc-cytokines through a STAT-dependent pathway. In here, we aimed to identify and evaluate FDA-approved compounds that could also sensitize HIV-infected cells to apoptosis. Using the Connectivity Map (CMap), we identified the retinol derivative 13-cis-retinoic acid (Isotretinoin) causes similar transcriptional changes as HODHBt. Isotretinoin enhances IL-15-mediated latency reversal without inducing proliferation of memory CD4 T cells. Ex vivo analysis of PBMCs from ACTG A5325, where Isotretinoin was administered to ART-suppressed people with HIV, showed that Isotretinoin treatment enhances IL-15-mediated latency reversal. Furthermore, we showed that a combination of IL-15 with Isotretinoin promotes the reduction of translation-competent reservoirs ex vivo. Mechanistically, combination of IL-15 and Isotretinoin increases caspase-3 activation specifically in HIV-infected cells but not uninfected cells. Our results suggest that Isotretinoin can be a novel approach to target and eliminate translation-competent HIV reservoirs.

Improved antitumor effectiveness of oncolytic HSV-1 viruses engineered with IL-15/IL-15Rα complex combined with oncolytic HSV-1-aPD1 targets colon cancer

Oncolytic virotherapy is emerging as a promising therapeutic avenue for cancer treatment, harnessing both innate and tumor-specific immune responses for targeted tumor elimination. In this study, we present a novel oncolytic virus (oHSV1-IL15B) derived from herpes simplex virus-1 (HSV-1), armed with IL-15/IL-15Rα complex, with a focus on treating colon cancer combined with oncolytic HSV-1 expressing anti-PD-1 antibody (oHSV1-aPD1). Results from our study reveal that recombinant oHSV-1 virus equipped with IL-15/IL-15Rα complex exhibited significant anti-tumor effects in a murine CT26 colon adenocarcinoma model. Notably, oHSV1-IL15B combined with oHSV-1-aPD1 demonstrates superior tumor inhibition and prolonged overall survival compared to oHSV1-mock and monotherapy groups. Further exploration highlights the impact of oHSV1-IL15B, oHSV-1-aPD1 and combined group on antitumor capacity, revealing a substantial increase in CD8+ T and CD4+ T cell proportions of CT26-bearing BALB/c mice and promoting apoptosis in tumor tissue. The study emphasizes the pivotal role of cytotoxic CD8+T cells in oncolytic virotherapy, demonstrating that recombinant oHSV1-IL15B combined with oncolytic HSV-1-aPD1 induces a robust tumor-specific T cell response. RNA sequence analysis highlighted oHSV1-IL15B combined with oHSV1-aPD1 improved tumors immune microenvironment on immune response, antiviral response-related genes and apoptosis-related genes, which contributed to anti-tumor immunotherapy. The findings underscore the promising antitumor activity achieved through the combination of IL-15/IL-15Rα complex and anti-PD-1 antibody with oHSV-1. This research opens avenues for diverse therapeutic strategies, suggesting the potential of synergistically utilizing cytokines and anti-PD-1 antibody with oncolytic viruses to enhance immunotherapy for cancer management.

Parsimonious immune-response endotypes and global outcome in patients with traumatic brain injury

The inflammatory response in patients with traumatic brain injury (TBI) offers opportunities for stratification and intervention. Previous unselected approaches to immunomodulation in patients with TBI have not improved patient outcomes. Serum and plasma samples from two prospective, multi-centre observational studies of patients with TBI were used to discover (Collaborative European NeuroTrauma Effectiveness Research [CENTER-TBI], Europe) and validate (Transforming Research and Clinical Knowledge in Traumatic Brain Injury [TRACK-TBI] Pilot, USA) individual variations in the immune response using a multiplex panel of 30 inflammatory mediators. Mediators that were associated with unfavourable outcomes (Glasgow outcome score-extended [GOS-E]≤4) were used for hierarchical clustering to identify patients with similar signatures. Two clusters were identified in both the discovery and validation cohorts, termed early-inflammatory and pauci-inflammatory. The early-inflammatory phenotype had higher concentrations of interleukin-6 (IL-6), IL-15, and monocyte chemoattractant protein 1 (MCP1). Patients with the early-inflammatory phenotype were older and more likely to have an unfavourable GOS-E at 6 months. There were no differences in the baseline injury severity scores between patients in each phenotype. A combined IL-15 and MCP1 signature identified patients with the early-inflammatory phenotype in both cohorts. Inflammatory processes mediated outcomes in older patients with moderate-severe TBI. Our findings offer a precision medicine approach for future clinical trials of immunomodulation in patients with TBI, by using inflammatory signatures to stratify patients. CENTER-TBI study was supported by the European Union 7th Framework Programme. TRACK-TBI is supported by the National Institute of Neurological Disorders and Stroke.

IL-15/IL-15Ra Synergies with IL-12 to Induce Functional CD8 T Cells and NK Cells During Chronic SHIV Infection.

Cytokines are key mediators of immune regulation, orchestrate communication between immune cells, and play a pivotal role in shaping the immune landscape during chronic infection and cancer. The therapeutic potential of IL-15/IL-15Rα and IL-12 has been explored individually in various immunotherapeutic strategies, though not as a combination. Therefore, we investigated whether the combination of IL-15/IL-15Rα and IL-12 treatment would enhance the potency and quality of either NK cells, SIV-specific CD8 T cells, or both, compared with single cytokine treatment. Our findings reveal that in vitro IL-15/IL-15Rα and IL-15/IL-15Rα plus IL-12 treatment results in an expansion of functional CD8 T cells and NK cells from uninfected and chronically infected macaques with simian/human immunodeficiency virus. Additionally, the cytokine combination significantly reduced CCR5 expression on total CD4 T cells, limiting the number of viral targets. This study supports the potential utilization of combined IL-15/IL-15Rα plus IL-12 treatment for chronic viral infections and cancer.

Type 2 Cytokine–Dependent Skin Barrier Regulation in Personalized 2-Dimensional and 3-Dimensional Skin Models of Atopic Dermatitis: A Pilot Study

Keratinocytes from healthy donors stimulated with type 2 cytokines are often used to experimentally study atopic dermatitis (AD) inflammatory responses. Due to potential intrinsic alterations, it seems favorable to use keratinocytes from AD patients. Keratinocytes isolated from hair follicles offer a non-invasive approach to investigate AD-derived keratinocytes. To evaluate whether such AD-derived keratinocytes are suitable to mimic AD inflammatory responses we compared hair follicle-derived keratinocytes from healthy donors and AD patients in a type 2 cytokine environment. Stimulation of AD-derived keratinocytes with IL-4 and IL-13 induced higher expression changes of AD-associated markers as compared to healthy keratinocytes. The combination of IL-4 and IL-13 generally induced highest expression changes, but IL-13 alone also induced significant changes of AD-specific markers. Similar to the 2D cultures, IL-4/IL-13 stimulation of 3D skin models generated with AD-derived keratinocytes modulated the expression of several AD-relevant factors. Whole transcriptome analysis revealed that IL-4 and IL-13 acted similarly on these 3D skin models. Histologically, IL-13 alone and in combination with IL-4 increased epidermal spongiosis, a histological hallmark of AD skin. Taken together, our pilot study suggests that hair follicle-derived keratinocytes from AD patients represent a useful model system to study AD-related inflammation in a personalized in vitro model.

Tumor-Localized Interleukin-2 and Interleukin-12 Combine with Radiation Therapy to Safely Potentiate Regression of Advanced Malignant Melanoma in Pet Dogs.

Cytokines IL2 and IL12 exhibit potent anticancer activity but suffer a narrow therapeutic window due to off-tumor immune cell activation. Engineering cytokines with the ability to bind and associate with tumor collagen after intratumoral injection potentiated response without toxicity in mice and was previously safe in pet dogs with sarcoma. Here, we sought to test the efficacy of this approach in dogs with advanced melanoma. This study examined 15 client-owned dogs with histologically or cytologically confirmed malignant melanoma that received a single 9-Gy fraction of radiotherapy, followed by six cycles of combined collagen-anchored IL2 and IL12 therapy every 2 weeks. Cytokine dosing followed a 3 + 3 dose escalation design, with the initial cytokine dose chosen from prior evaluation in canine sarcomas. No exclusion criteria for tumor stage or metastatic burden, age, weight, or neuter status were applied for this trial. Median survival regardless of the tumor stage or dose level was 256 days, and 10/13 (76.9%) dogs that completed treatment had CT-measured tumor regression at the treated lesion. In dogs with metastatic disease, 8/13 (61.5%) had partial responses across their combined lesions, which is evidence of locoregional response. Profiling by NanoString of treatment-resistant dogs revealed that B2m loss was predictive of poor response to this therapy. Collectively, these results confirm the ability of locally administered tumor-anchored cytokines to potentiate responses at regional disease sites when combined with radiation. This evidence supports the clinical translation of this approach and highlights the utility of comparative investigation in canine cancers.

RhIL-7-hyFc, a long-acting interleukin-7, improves efficacy of CAR-T cell therapy in solid tumors

BackgroundChimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable remission in patients with B-cell malignancies. However, its efficacy in treating solid tumors remains limited. Here, we investigated a combination therapy...

Pretransplant, Th17 dominant alloreactivity in highly sensitized kidney transplant candidates.

Sensitization to donor human leukocyte antigen (HLA) molecules prior to transplantation is a significant risk factor for delayed access to transplantation and to long-term outcomes. Memory T cells and their cytokines play a pivotal role in shaping immune responses, thereby increasing the risk of allograft rejection among highly sensitized patients. This study aims to elucidate the precise contribution of different CD4+ memory T cell subsets to alloreactivity in highly sensitized (HS) kidney transplant recipients. Stimulation of peripheral blood mononuclear cells (PBMC) with various polyclonal stimulating agents to assess non-specific immune responses revealed that HS patients exhibit elevated immune reactivity even before kidney transplantation, compared to non-sensitized (NS) patients. HS patients' PBMC displayed higher frequencies of CD4+ T cells expressing IFNγ, IL4, IL6, IL17A, and TNFα and secreted relatively higher levels of IL17A and IL21 upon stimulation with PMA/ionomycin. Additionally, PBMC from HS patients stimulated with T cell stimulating agent phytohemagglutinin (PHA) exhibited elevated expression levels of IFNγ, IL4 and, IL21. On the other hand, stimulation with a combination of resiquimod (R848) and IL2 for the activation of memory B cells demonstrated higher expression of IL17A, TNFα and IL21, as determined by quantitative real-time PCR. A mixed leukocyte reaction (MLR) assay, employing third-party donor antigen presenting cells (APCs), was implemented to evaluate the direct alloreactive response. HS patients demonstrated notably higher frequencies of CD4+ T cells expressing IL4, IL6 and IL17A. Interestingly, APCs expressing recall HLA antigens triggered a stronger Th17 response compared to APCs lacking recall HLA antigens in sensitized patients. Furthermore, donor APCs induced higher activation of effector memory T cells in HS patients as compared to NS patients. These results provide an assessment of pretransplant alloreactive T cell subsets in highly sensitized patients and emphasize the significance of Th17 cells in alloimmune responses. These findings hold promise for the development of treatment strategies tailored to sensitized kidney transplant recipients, with potential clinical implications.

Abstract 1191: Personalized neoantigen DNA vaccine GNOS-PV02 and pembrolizumab as second-line treatment for advanced hepatocellular carcinoma

Abstract Background: PD-1 inhibitors have modest efficacy as monotherapy in hepatocellular carcinoma (HCC). A personalized therapeutic cancer vaccine (PTCV) tailored against neoantigens identified in an individual’s tumor may enhance responses to PD-1 inhibitors through the induction of tumor-specific immunity. Here, we present results from a single-arm Phase Ib/2a trial evaluating a DNA plasmid (GNOS-PV02) encoding up to 40 neoantigens co-administered with plasmid-encoded IL-12 (pIL12) in combination with pembrolizumab (PEMBRO) in patients (pts) with advanced HCC.Methods: Pts were eligible for study therapy upon progression or intolerance with a 1L tyrosine kinase inhibitor (TKI). The PTCV [GNOS-PV02 (1mg) and pIL12 (0.34mg)] was administered intradermally via in vivo electroporation Q3w x 4 doses, and Q9w thereafter. PEMBRO was administered at 200mg IV Q3w. The primary endpoints were safety and immunogenicity. To evaluate the secondary endpoint of ORR per RECIST 1.1 by investigator review with a null hypothesis of an ORR of 16.9% (KN-240, Finn et al ASCO 2019), 36 pts were included to provide 80% power to reject the null hypothesis at the one-sided 0.10 level, assuming the true ORR rate of 33.1%. The data cut date was August 18, 2023.Results: Among the 36 enrolled pts who received at least one dose of treatment, there were no DLTs or treatment-related grade ≥3 events. The most common treatment-related adverse events were injection site reactions, observed in 41.6% of pts. ORR (mITT) per RECIST 1.1 was 30.6% (11/36; 9 confirmed and 2 unconfirmed) with 8.3% (3/36) of pts achieving a CR. This achieved statistical significance with a one-sided p-value = 0.031 (1-sided 90% CI 20.4%-100%) The mOS was 19.9 months. ctDNA changes correlated with radiographic responses and preceded them. A complete molecular response (100% ctDNA clearance) was detected in 7 pts including the 3 radiographic CRs, and 4 additional pts who continue to show durable tumor control (3PR, 1 SD). Immunological analyses confirmed the induction of neoantigen-specific T cell responses by IFNγ-ELISpot in 19/22 (86.4%) evaluable pts, and pts with a larger ELISpot response showed a trend towards longer OS. Multi-parametric cellular profiling and single-cell analysis revealed active, proliferative, and cytolytic vaccine-specific CD4+ and CD8+ effector T cells in the blood of immunized pts. In 14/14 (100%) of pts with paired pre- and on-treatment blood and tumor biopsies, we identified by TCRβ bulk sequencing expanded T cell clones in the peripheral blood that also trafficked into the tumor. Conclusions: Our results show that a PTCV plus PEMBRO is well tolerated and has clinical activity in pts with advanced HCC, and support the PTCV mechanism of action based on the induction of anti-tumor T cells in peripheral blood and tumor. A confirmatory phase 3 clinical study assessing OS is planned. Citation Format: Mark Yarchoan, Edward J. Gane, Thomas U. Marron, Renzo Perales-Linares, Jian Yan, Neil Cooch, Daniel Shu, Elana J. Fertig, Luciane T. Kagohara, Gabor Bartha, Josette Northcott, John Lyle, Sarah Rochestie, Joann Peters, Jason Connor, Elizabeth Jaffee, Alfredo Perales-Puchalt, David B. Weiner, Ildiko Csiki, Niranjan Y. Sardesai. Personalized neoantigen DNA vaccine GNOS-PV02 and pembrolizumab as second-line treatment for advanced hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1191.

Cytokine-Treated Natural Killer Cells Derived from Cord Blood Haematopoietic Stem Cells Exhibit Potential Cytotoxicity Against Cancer Cells

Background: Despite substantial efforts to leverage natural killer (NK) cells in cancer immunotherapy, challenges associated with limited cell numbers and sources persist. In this study, our objective is to differentiate NK cells from cord blood hematopoietic stem cells (CB-HSCs) and assess their anti-tumor effects. Methods: Cord blood samples were obtained from pregnant women undergoing normal delivery. Mononuclear cells (MNC) were isolated by gradient centrifugation. Subsequently, HSCs were isolated using the MACs cell separation kit. The isolated HSCs were cultured in NK cell differentiation and expansion media for 21 days and 7 days, respectively. The NK cells were examined for expression of activating markers, cytokine secretion and cytolytic effects by flow cytometry, ELISA and XTT tests, respectively. Results: High-purity HSCs and NK cells were obtained in this study. The CB-HSCs-derived NK cells exhibited significantly higher expression of NKG2D, NKp30, NKp44 and NKp46 receptors (at day 28 of treatment) after treatment by IL-15 and IL-2, compared to the early differentiated NK cells (day 21). NK cells derived from CB-HSCs treated with the combination of IL-15 and IL-2 could robustly lyse breast cancer MCF-7 and K562 cells. Also, the obtained NK cells were able to release higher amounts of TNF-α and IFN-γ cytokines in response to tumor cell experiences. Conclusions: Our findings showed that functionally active CB-HSCs-derived NK cells can be successfully generated ex vivo using a cytokine cocktail without a need for stroma for potential use in the cancer immunotherapy.

Co-expression of interleukin (IL)-17RA and IL-36γ in the hypothalamic paraventricular nucleus of the stress-induced hypertension rats

Objectives: The up-regulation of proinflammatory cytokines in the hypothalamic paraventricular nucleus (PVN) is well demonstrated to be involved in the development of neurogenic hypertension, including stress-induced hypertension (SIH). IL-17A has been found to be increased in the PVN of several hypertensive animal models and has been shown to play a key role in the development of hypertension. Although IL-36γ was found to be expressed in spinal neurons, its role in hypertension remains elusive. Here, we investigated the co-expression of IL-17 receptor A (IL-17RA) and IL-36γ in the PVN cells of SIH rats. Methods: The electric foot shock combined with buzzer noise stressors were used to make hypertensive rat model. The immunochemical staining or immunofluorescence staining was used to reveal cells as requested. The Western blot was used to detect the related protein levels. Results and Conclusion: In the PVN of the SIH rats, the number of CD3+CD4+ T cells was significantly increased by the immunochemical staining. Additionally, the protein levels of RORγt and IL-17A were significantly upregulated by Western blot, confirming the infiltration of CD4+ T cells and differentiation into Th17 cells in the PVN of SIH rats. Immunofluorescence staining revealed abundant expression of IL-17RA in PVN neurons, with relatively less expression in astrocytes or microglia. Furthermore, IL-36γ positive cells and protein expression of IL-36R were significantly increased. Notably, this study demonstrates for the first time that most IL-36γ cells were strongly colocalized with IL-17RA positive cells in the PVN of SIH rats, and the colocalized cells were significantly higher in SIH rats. This suggests that IL-17A secreted by infiltrated Th17 cells may stimulate PVN neurons to produce IL-36γ via IL-17RA, indicating that the combination of IL-17 and IL-36γ might produce strong pro-inflammatory effects in the PVN of SIH rats.

Efficient In Vitro Plasma Cell Differentiation by B Cell Receptor Activation and Cytokine Stimulation.

Plasma cells (PCs) constitute a small proportion of B cells, limiting their biochemical characterization. An in vitro culture system that reliably generates PCs could provide an alternative method to obtain PCs for further analysis and manipulation. To date, most in vitro PC differentiation methods rely on B cell receptor (BCR)-independent stimulants, including TLR ligands, CD40L, and cytokines. However, these methods do not fully recapitulate the natural T cell-dependent PC differentiation process, in which BCR activation is the initial events. In this study, we established an efficient in vitro PC differentiation method incorporating BCR stimulation. Naïve B cells were first stimulated with anti-IgM and anti-CD40 Abs, followed by stimulation with various cytokines. By screening cytokines known to participate in PC differentiation in vivo, we identified that the combination of IL-4 and IL-5 induced the most efficient PC differentiation. The in vitro generated PCs highly expressed PC-associated surface markers and regulatory genes. Additionally, they secreted high amounts of IgM and IgG Abs. Moreover, retroviral transduction of B cells resulted in efficient target gene expression in PCs. Our new method closely mimics natural PC differentiation and effectively generates a large quantity of PCs for various applications, including elucidating the molecular mechanisms underlying PC differentiation.

LFA-1 regulated by IL-2/STAT5 pathway boosts antitumor function of intratumoral CD8+ T cells for improving anti-PD-1 antibody therapy

ABSTRACT Anti-PD-1 antibody therapy has achieved success in tumor treatment; however, the duration of its clinical benefits are typically short. The functional state of intratumoral CD8+ T cells substantially affects the efficacy of anti-PD-1 antibody therapy. Understanding how intratumoral CD8+ T cells change will contribute to the improvement in anti-PD-1 antibody therapy. In this study, we found that tumor growth was not arrested after the late administration of anti-PD-1 antibody and that the antitumor function of CD8+ T cells decreased with tumor progression. The results of the RNA sequencing of CD8+ T cells infiltrating the tumor site on days 7 and 14 showed that the cell adhesion molecule Lymphocyte Function-associated Antigen-1 (LFA-1) participates in regulating the antitumor function of CD8+ T cells and that decreased LFA-1 expression in intratumoral CD8+ T cells is associated with tumor progression. By analyzing the Gene Expression Omnibus (GEO) database and our results, we found that the antitumor function of intratumoral CD8+ T cells with high LFA-1 expression was stronger. The formation of immune synapses is impaired in Itgal-si CD8+ T cells, resulting in decreased anti-tumor function. LFA-1 expression in intratumoral CD8+ T cells is regulated by the IL-2/STAT5 pathway. The combination of IL-2 and anti-PD-1 antibody effectively enhanced LFA-1 expression and the antitumor function of intratumoral CD8+ T cells. The adoptive transfer of OT-1 T cells overexpressing LFA-1, STAT5A, or STAT5B resulted in higher antitumor function, deferred tumor growth, and prolonged survival. These findings indicate that LFA-1-mediated immune synapse acts as a regulator of the antitumor function of intratumoral CD8+ T cells, which can be applied to improve anti-PD-1 antibody therapy.

Development of a highly cytotoxic, clinical-grade virus-specific T cell product for adoptive T cell therapy

At present, recipients of allogeneic hematopoietic stem-cells are still suffering from recurrent infections after transplantation. Infusion of virus-specific T cells (VST) post-transplant reportedly fights several viruses without increasing the risk of de novo graft-versus-host disease. This study targeted cytomegalovirus (CMV) for the development of an innovative approach for generating a very specific VST product following Good Manufacturing Practices (GMP) guidelines.We used a sterile disposable compartment named the Leukoreduction System Chamber (LRS-chamber) from the apheresis platelet donation kit as the starting material, which has demonstrated high levels of T cells. Using a combination of IL-2 and IL-7 we could improve expansion of CMV-specific T cells. Moreover, by developing and establishing a new product protocol, we were able to stimulate VST proliferation and favors T cell effector memory profile. The expanded VST were enriched in a closed automated system, creating a highly pure anti-CMV product, which was pre-clinically tested for specificity in vitro and for persistence, biodistribution, and toxicity in vivo using NOD scid mice. Our results demonstrated very specific VST, able to secrete high amounts of interferon only in the presence of cells infected by the human CMV strain (AD169), and innocuous to cells partially HLA compatible without viral infection.

Abstract A006: Understanding the Potential of Human IL-12 and Chimeric IL-15 Combination for Cancer Immunotherapy

Abstract Autologous cell-based therapy is a preferred approach for Cancer immunotherapy. However, generating potent NK cells and antigen experienced T cells has been a challenge. To address this issue, we have developed a strategy of using the combination of IL-12 and IL-15 to activate CD4+ T cells, CD8+ T cells and NK cells. IL-12 and IL-15 possess distinct roles but both are potent inflammatory cytokines. IL-12 is a vital promoter of Th1 differentiation via activation of signal transducer and activator of transcription 4 (STAT4), while IL-15 is essential for developing innate immune response as well as contributing to the maintenance of memory CD8+ T cells. Substantial in vivo and in vitro data have been reported to support the role of IL-15 as a T cell growth factor. These activities of IL-12 and IL-15 make them ideal for combinatorial immunotherapy of cancer. However, the short half-life (~ 1hr) and poor bioavailability limits the therapeutic use of IL-15. To overcome these limitations, we have designed chimeric IL-15-IgG2 (Indian Patent Application No. 201721010096A) with an objective of augmenting the generation of memory T cells as well as reactivating them during the antigenic challenge. Our stable chimeric IL-15 has a half-life of >40 Hrs. In humans, IL-12 is needed for DCs to provoke rapid IFN-g production by NK cells, IL-15 seems essential for the NK cell proliferation. In the present study, we are testing the potential of chimeric IL-15 individually and in combination with native hIL-12 as an activator of CD4+ T cells, CD8+ T cells and NK cells. The combination of both cytokines shows potent release of IFN-g as well as activation of the T-cell subsets and NK cells. In our future studies we will use these pre-activated cells for adoptive transfer in cancer immunotherapy. Detailed findings will be reported during the conference. Citation Format: Priya Chandramohan Sharma, Digna Patel, Sarat K Dalai. Understanding the Potential of Human IL-12 and Chimeric IL-15 Combination for Cancer Immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A006.

Correlation between salivary cytokine profiles and white spot lesions in adolescent patients receiving clear aligner orthodontic treatment

BackgroundTo explore the relationship between changes in salivary cytokine levels and the occurrence of white spot lesions in adolescents receiving clear aligner orthodontic treatment and investigate the predictive value of various factors for lesion occurrence.MethodsWe retrospectively analyzed sixthy eight adolescent in the permanent dentition period, who received clear aligner orthodontics in our hospital were randomly divided into two groups according to the occurrence or aggravation of white spot lesions after treatment. The general condition of the oral cavity was analyzed, saliva was collected, and inflammation-related cytokines with varying transcription levels between groups were screened by transcriptome analysis. The expression levels of inflammatory cytokines in the saliva of the patients in the two groups were measured, and the risk factors for white spot lesions were screened by correlation analysis and binary logistic regression analysis. The value of the independent and combined application of risk factors for predicting the occurrence of white spot lesions in adolescent patients after invisible orthodontic treatment was analyzed by receiver operating characteristic (ROC) curve analysis.ResultsTranscriptome and GO and KEGG pathway analyses showed that there were differences in the transcription levels of inflammatory cytokines such as CXCL1, CXCL2, CXCL8, CCL3, CCL4, IL-1β and IL-2 between groups. The levels of CXCL8, CCL3, CCL4, IL-1β and IL-2 in the saliva of patients with white spot lesions were significantly higher in patients after invisible orthodontic treatment (P < 0.05). Correlation analysis and binary logistic regression analysis showed that elevated levels of CXCL8, IL-1β and IL-2 were independent risk factors for the occurrence of white spot lesions (P < 0.05). CXCL8 had the highest independent predictive value for the occurrence of white spot lesions (AUC = 0.773, P < 0.05), and the combination of IL-1β and IL-2 was also of high value in predicting the occurrence of white spot lesions.ConclusionAfter invisible orthodontic treatment, the oral microenvironment, including inflammatory cytokine levels, in adolescent patients changes; in particular, the levels of inflammatory cytokines such as CXCLs and ILs change. CXCL8 expression is significantly associated with the occurrence of white spot lesions and is an important potential target for the prevention and treatment of white spot lesions in the future.

Overcoming lung cancer immunotherapy resistance by combining nontoxic variants of IL-12 and IL-2.

Engineered cytokine-based approaches for immunotherapy of cancer are poised to enter the clinic, with IL-12 being at the forefront. However, little is known about potential mechanisms of resistance to cytokine therapies. We found that orthotopic murine lung tumors were resistant to systemically delivered IL-12 fused to murine serum albumin (MSA, IL12-MSA) because of low IL-12 receptor (IL-12R) expression on tumor-reactive CD8+ T cells. IL2-MSA increased binding of IL12-MSA by tumor-reactive CD8+ T cells, and combined administration of IL12-MSA and IL2-MSA led to enhanced tumor-reactive CD8+ T cell effector differentiation, decreased numbers of tumor-infiltrating CD4+ regulatory T cells, and increased survival of lung tumor-bearing mice. Predictably, the combination of IL-2 and IL-12 at therapeutic doses led to significant dose-limiting toxicity. Administering IL-12 and IL-2 analogs with preferential binding to cells expressing Il12rb1 and CD25, respectively, led to a significant extension of survival in mice with lung tumors while abrogating dose-limiting toxicity. These findings suggest that IL-12 and IL-2 represent a rational approach to combination cytokine therapy whose dose-limiting toxicity can be overcome with engineered cytokine variants.

IL-2/αIL-2 Complexes Massively Expand Systemic Tumor-Specific T Cells and Enhance Abscopal Responses to Radiation and αPD-1

Early clinical trials have provided evidence for RT-induced systemic effects in conjunction with αPD1 or IL-2 in metastatic patients, but strong abscopal responses are clinically rare. We investigated the effect of a triple combination of CD122-directed IL-2/αIL-2 complexes (IL-2c), hRT, and αPD1 on tumor-specific CD8+ T cell differentiation and the abscopal effect vs. the respective dual treatments. Mice bearing bilateral tumors were treated with two fractions of 8 Gy (C51 colon carcinoma model) or 12 Gy (B16 melanoma model); αPD1 was given weekly; IL-2c was given for five consecutive days. Dependence of the therapeutic effect on CD8+ T cells and T cells expressing the chemokine receptor CXCR3 was assessed using depleting or blocking antibodies. Differentiation stages of tumor-specific CD8+ T cells in various compartments were determined flow cytometrically using MHC-I tetramers and appropriate antibodies. Anti-tumoral effects of blood-derived and tumor-derived T cells were assessed in adoptive T cell transfer experiments. The abscopal effect was significantly stronger in triple-treated mice compared to mice treated with RT/αPD1 (C51 model: p < 0.01; B16 model: p < 0.05), RT/IL-2c (C51 model: p < 0.01; B16 model: p < 0.001) or αPD1/IL-2c (C51 model: p < 0.0001, B16 model: p < 0.01). Triple therapy improved survival and resulted in complete cures of 3/12 mice in the C51 model and 2/12 mice in the B16 model. These anti-tumor effects were associated with dramatic expansion of tumor-specific CD8+ T cells. Undifferentiated stem-like (TCF1+TIM3-PD1+) and effector-like (CD101-TIM3+TCF1-PD1+) but not terminally differentiated (CD101+TIM3+TCF1-PD1+) exhausted cells particularly strongly increased. Moreover, IL-2c induced CXCR3 mainly on non-terminally differentiated CD8+ T cells. Both CD8+ (C51 model: p < 0.0001; B16 model: p < 0.01) and CXCR3+ (C51 model: p < 0.0001) T cells were crucial for the RT-induced abscopal effect upon RT/αPD1/IL-2c treatment. Finally, we found that peripheral blood from triple-treated mice is an effective source of T cells for adoptive T cell transfer. RT/αPD1/IL-2c triple treatment resulted in superior local and systemic expansion of tumor-specific CD8+ T cells with stem- and effector-like phenotypes. Also, IL-2c strongly increased CXCR3+CD8+ T cells that were associated with pronounced abscopal responses in models with an established metastasis resistant to αPD1/IL-2c and only transiently responding to RT/αPD1 or RT/IL-2c. Therefore, such triple combinations appear promising for clinical evaluation in metastatic patients.

Low-dose Interleukin-2 For Psoriasis Therapy Based on the Regulation of Th17/Treg Cell Balance in Peripheral Blood

The imbalance between regulatory T (Treg) cells and efficient T cells plays an important role in psoriasis. Low-dose interleukin (IL)-2 can preferentially activate Treg cells and ameliorate the imbalance of Treg/efficient T cells. This study focused on the status of circulating CD4+ T subsets and the clinical efficacy of low-dose IL-2 therapies in psoriasis. This retrospective study included peripheral blood samples obtained from 45 psoriatic patients and 40 healthy controls. The 45 psoriatic patients received three cycles of subcutaneous low-dose IL-2 treatment (0.5 million IU/day for 2 weeks) combined with conventional therapies. Inflammatory indices, CD4+ T-lymphocyte subsets, and cytokines were measured in all patients before and after treatment. The percentage of Treg cells was dramatically decreased in the psoriasis group compared to the healthy group, and the percentage of Treg cells negatively correlated with the disease indices and the Psoriasis Area and Severity Index (PASI) (P < 0.001). The Th17/Treg ratio was significantly increased in the psoriasis group compared to the healthy group, and the Th17/Treg ratio positively correlated with disease indices and PASI (P < 0.001). Low-dose IL-2 treatment significantly amplified the percentage of Treg cells and restored the Th17 and Treg immune balance in psoriasis (P < 0.001). Low-dose IL-2 combination therapy effectively improved the clinical manifestations of psoriasis but decreased the inflammatory indicators of the disease activity, with no apparent side effects. Thus, low-dose IL-2 provides a new strategy for the treatment of psoriasis.

Tracking MRD and neoantigen targets using a tumor-informed liquid biopsy platform in HCC patients treated with personalized cancer vaccine and pembrolizumab.

e15062 Background: ctDNA analysis has enabled non-invasive detection and monitoring of potentially actionable mutations, and can identify therapeutic response/resistance prior to confirmation by radiographic imaging. Little is known about ctDNA in the neoantigen-targeted personalized cancer DNA vaccine setting, potentially due to the limited sensitivity of current ctDNA assays. We used an ultra-sensitive, tumor-informed ctDNA platform to longitudinally track tumor neoantigen targets and monitor molecular residual disease (MRD) in advanced HCC patients (pts) being treated with a DNA personalized therapeutic cancer vaccine (GNOS-PV02). Methods: Pts with unresectable or metastatic HCC with progression on, or intolerance to, first-line therapy with TKI were enrolled into the Phase 1b/2a GT-30 study [NCT04251117]. GNOS-PV02 was designed based on whole exome/transcriptome tumor sequencing. Pts were treated with GNOS-PV02 (1mg; Q3W x 4, Q9W) and plasmid-encoded IL-12 (0.3mg; Q3W x 4, Q9W) in combination with pembrolizumab (200mg; Q3W). Response was evaluated by RECIST 1.1 at baseline (bl) and Q9W. Over 100 prospective bl and on-treatment (ot) plasma samples were collected and analyzed using NeXT Personal, a tumor-informed ctDNA assay that leverages whole genome sequencing of tumor/normal samples to generate personalized liquid biopsy panels. Each panel included personalized neoantigen targets, up to 1,800 selected variants for ultra-sensitive detection of MRD, and a fixed set of 2,100 known clinically actionable and resistance loci for detection of variants emerging under therapeutic pressure. Results: ctDNA was detected across a broad dynamic range (3-100,000 PPM; minimum limit of detection = 2.5 PPM) with frequent positive detections below 100 PPM. Bl ctDNA was detected in 100% (12/12) of patients. Ot changes in ctDNA relative to bl correlated with disease status. ctDNA tracking of a patient with a target liver lesion reduction of 36% at W9 deepening to -59% at W54 by RECIST1.1 showed ctDNA clearance of all liver-specific neoantigen targets between W12 to w21. New adrenal lesions were observed on W18 by MRI and retrospective ctDNA analysis revealed an increase of adrenal-specific ctDNA measures by W9. These data indicate ctDNA can be used to track tumor neoantigens and provide important data supporting immune pressure-induced tumor escape. Conclusions: Highly sensitive tracking of MRD and neo-antigenic variants over the course of therapy was achieved using a single assay. We show that ctDNA can sensitively monitor disease status non-invasively, potentially leading to accurate clinical outcome prediction. Additionally, the ease of sample handling, analysis, and rapid availability of data could enable the use of ctDNA monitoring to allow real-time dynamic personalized cancer immunotherapy.