T-cell Mediated Immune Response Research Articles

Improvement of the Nuclease Resistance and Immunostimulatory Activity of CpG Oligodeoxynucleotides by Conjugation to Sugar-Immobilized Gold Nanoparticles.

Adjuvants are essential substances for vaccines and immunotherapies that enhance antigen-specific immune responses. Single-stranded oligodeoxynucleotides containing an unmethylated CpG motif (CpG ODNs) are agonistic ligands for toll-like receptor 9 that initiate an innate immune response. They represent promising adjuvants for antiviral and antitumor immunotherapies; however, CpG ODNs have some limitations, such as poor nuclease resistance and low cell membrane permeability. Therefore, an effective formulation is needed to improve the nuclease resistance and immunostimulatory effects of CpG ODNs. Previously, we demonstrated the selective delivery of a small molecule toll-like receptor 7 ligand to immune cells through sugar-binding receptors using sugar-immobilized gold nanoparticles (SGNPs), which significantly enhanced the potency of the ligand. In this study, we examined SGNPs as carriers for partially phosphorothioated A-type CpG ODN (D35) and an entirely phosphorothioated B-type CpG ODN (K3) and evaluated the functionality of the sugar moiety on SGNPs immobilized with CpG ODN. SGNPs immobilized with D35 (D35-SGNPs) exhibited improved nuclease resistance and the in vitro and in vivo potency was significantly higher compared with that of unconjugated D35. Furthermore, the sugar structure on the GNPs was a significant factor in enhancing the cell internalization ability, and enhanced intracellular delivery of D35 resulted in improving the potencies of the A-type CpG ODN, D35. SGNPs immobilized with K3 (K3-SGNPs) exhibited significantly higher induction activities for both humoral and cellular immunity compared with unconjugated K3 and D35-SGNPs. On the other hand, sugar structure on K3-SGNPs did not affect the immunostimulatory effects. These results indicate that the sugar moiety on K3-SGNPs primarily functions as a hydrophilic dispersant for GNPs and the formulation of K3 to SGNPs contributes to improving the immunostimulatory activity of K3. Because our CpG ODN-SGNPs have superior induction activities for antigen-specific T-cell mediated immune responses, they may be effective adjuvants for vaccines and immunotherapies.

Abstract 2717: Bifunctional fusion protein PDL1sFv/MICAe for cancer immunotherapy

Abstract Many cancer cells upregulate the expression of programmed death ligand 1 (PD-L1) to surpass T-cell mediated immune response. Immune checkpoint blockers targeting PD-L1 or its receptor PD1 have been shown to suppress the inhibitory signals and enhance T cell function. As a new immunotherapy, immune checkpoint blockers have been used to treat multiple cancers. However, most clinically available blockers only engage with one type of immune cell: the T cells. Natural killer (NK) cells, on the other hand, play a significant role in anti-cancer immune responses. It will represent an advancement in anti-cancer immunotherapy if NK cells are engaged with immune checkpoint blockers. In this study, we designed a unique bifunctional fusion gene, PDL1sFv/MICAe, consisting of the variable regions of an anti-PD-L1 antibody and the extracellular domain of stress protein MICA and hypothesized that fusion protein PDL1sFv/MICAe will simultaneously bind to PD-L1 on tumor cells to block the inhibitory signal and extend T cells activity, and to NKG2D to activate NK Cells. cDNA sequence of fusion gene PDL1sFv/MICAe was designed based on the variable region of an established clinically used antibody, Atezolizumab, and the extracellular domain of MICA with linkers in between. After a computational confirmation of its structure and binding affinities, the fusion gene was synthesized and cloned into an expressing vector, and the fusion protein was produced for evaluation. Western blotting and fluorescent microscopic analyses confirmed that the protein binds to PD-L1 of human tumor cells, including MCF7 and MDAMB231, and NKG2D of KHYG1 cells. Preliminary results also show that the fusion protein enhances NK cell cytotoxicity against tumor cells. Studies on confirming the fusion protein’s activities on other tumor cells, NK cell IFN-γ production, T cell function, etc., are underway. In summary, fusion protein PDL1sFv/MICAe represents an improved version of the immune checkpoint blockers and could treat cancer patients quickly and effectively. Citation Format: Junyi Li, Yanzhang Wei. Bifunctional fusion protein PDL1sFv/MICAe for cancer immunotherapy [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 2717.

Long-term Exposure to Industrial Chemical Contamination Affects the Magnitude of Predator-induced Immunosuppression in a Free-living Passerine.

Industrial chemical contamination is known to have immuno-toxic effects on birds. It may also interfere with natural stressful conditions to further disrupt the immune responses, but these possible interactive effects are still poorly documented in free-living birds. Using the phytohaemagglutinin skin-swelling test, we assessed how the T-cell mediated immune response varied according to the perceived risk of predation in hybrid sparrows, Passer domesticus × Passer hispaniolensis, originating from two sites differentially impacted by industrial chemical contamination, in southern Tunisia. Results showed that T-cell mediated immune response decreased with increasing perceived risk of predation, but the extent of this predator-associated immunosuppression was weaker in birds from the contaminated site compared to those from the control site. The immune response of birds living in the contaminated site was so weak that it could not be further weakened by a predator-related stress. Overall, these results support the idea that chemical contamination interferes with natural environmental stressors, such as predators, thus entailing profound disruption of the immune responses, with possible deleterious repercussions on the ability of birds to cope with diseases.

The role of programmed death receptor (PD-)1/PD-ligand (L)1 in periodontitis and cancer.

The programmed-death-ligand-1 (PD-L1) is an immune-modulating molecule that is constitutively expressed on various immune cells, different epithelial cells and a multitude of cancer cells. It is a costimulatory molecule that may impair T-cell mediated immune response. Ligation to the programmed-death-receptor (PD)-1, on activated T-cells and further triggering of the related signaling pathways can induce T-cells apoptosis or anergy. The upregulation of PD-L1 in various cancer types, including oral squamous cell carcinomas, was demonstrated and has been linked to immune escape of tumors and poor prognosis. A bidirectional relationship exists between the increased PD-L1 expression and periodontitis as well as the epithelial-mesenchymal transition (EMT), a process of interconversion of epithelial cells to mesenchymal cells that may induce immune escape of tumors. Interaction between exosomal PD-L1 and PD-1 on T-cells may cause immunosuppression by blocking the activation and proliferation of T-cells. The efficacy and importance of treatment with PD-1/PD-L1 checkpoint inhibitors and their prognostic influence on human cancers was demonstrated. Regarding PD-1/PD-L1 checkpoint inhibitors, resistances exist or may develop, basing on various factors. Further investigations of the underlying mechanisms will help to overcome the therapeutic limitations that result from resistances and to develop new strategies for the treatment of cancer.

The effects of primary and secondary bacterial exposure on the seahorse (Hippocampus erectus) immune response

Evolutionary adaptations in the Syngnathidae teleost family (seahorses, pipefish and seadragons) culminated in an array of spectacular morphologies, key immune gene losses, and the enigmatic male pregnancy. In seahorses, genome modifications associated with immunoglobulins, complement, and major histocompatibility complex (MHC II) pathway components raise questions concerning their immunological efficiency and the evolution of compensatory measures that may act in their place. In this investigation heat-killed bacteria (Vibrio aestuarianus and Tenacibaculum maritimum) were used in a two-phased experiment to assess the immune response dynamics of Hippocampus erectus. Gill transcriptomes from double and single-exposed individuals were analysed in order to determine the differentially expressed genes contributing to immune system responses towards immune priming. Double-exposed individuals exhibited a greater adaptive immune response when compared with single-exposed individuals, while single-exposed individuals, particularly with V. aestuarianus replicates, associated more with the innate branch of the immune system. T. maritimum double-exposed replicates exhibited the strongest immune reaction, likely due to their immunological naivety towards the bacterium, while there are also potential signs of innate trained immunity. MHC II upregulated expression was identified in selected V. aestuarianus-exposed seahorses, in the absence of other pathway constituents suggesting a possible alternative or non-classical MHC II immune function in seahorses. Gene Ontology (GO) enrichment analysis highlighted prominent angiogenesis activity following secondary exposure, which could be linked to an adaptive immune process in seahorses. This investigation highlights the prominent role of T-cell mediated adaptive immune responses in seahorses when exposed to sequential foreign bacteria exposures. If classical MHC II pathway function has been lost, innate trained immunity in syngnathids could be a potential compensatory mechanism.

Crude Tieguanyin oolong tea polysaccharides regulate intestinal immune and gut microflora in dextran sulfate sodium-induced mice colitis.

The global incidence and prevalence of inflammatory bowel diseases (IBDs) have been increasing. Epidemiological studies, clinical trials, and animal experiments have indicated a negative association between the consumption of tea and IBD. This study aims to investigate the protective effects of crude Tieguanyin oolong tea polysaccharides (CTPS) on experimental colitis, while also exploring the underlying mechanisms. The administration of CTPS significantly alleviated IBD in the mouse model, and was found to regulate T-cell mediated immune responses in the colon by modulating cytokine production associated with T cells. Furthermore, CTPS demonstrated a positive impact on the gut microbiota, reversing the increase in pathogenic Helicobacter and enhancing the relative abundances of beneficial bacteria such as Akkermansia, Lachnospiraceae, and Odoribacter. Oral administration of CTPS also led to an improvement in intestinal metabolism, specifically by increasing the levels of short-chain fatty acids. This study provides the first in vivo evidence of the protective effects of CTPS on colitis in mice. The effects are likely facilitated through the regulation of T cell-mediated responses and modulation of the gut microbiota, suggesting that CTPS may be a potential preventive and therapeutic approach for IBD. © 2023 Society of Chemical Industry.

Transcriptome profiling of microdissected cortex and medulla unravels functional regionalization in the European sea bass Dicentrarchus labrax thymus

The thymus is a sophisticated primary lymphoid organ in jawed vertebrates, but knowledge on teleost thymus remains scarce. In this study, for the first time in the European sea bass, laser capture microdissection was leveraged to collect two thymic regions based on histological features, namely the cortex and the medulla. The two regions were then processed by RNAseq and in-depth functional transcriptome analyses with the aim of revealing differential gene expression patterns and gene sets enrichments, ultimately unraveling unique microenvironments imperative for the development of functional T cells.The sea bass cortex emerged as a hub of T cell commitment, somatic recombination, chromatin remodeling, cell cycle regulation, and presentation of self antigens from autophagy-, proteasome- or proteases-processed proteins. The cortex therefore accommodated extensive thymocyte proliferation and differentiation up to the checkpoint of positive selection.The medulla instead appeared as the center stage in autoimmune regulation by negative selection and deletion of autoreactive T cells, central tolerance mechanisms and extracellular matrix organization.Region-specific canonical markers of T and non-T lineage cells as well as signals for migration to/from, and trafficking within, the thymus were identified, shedding light on the highly coordinated and exquisitely complex bi-directional interactions among thymocytes and stromal components. Markers ascribable to thymic nurse cells and poorly characterized post-aire mTEC populations were found in the cortex and medulla, respectively. An in-depth data mining also exposed previously un-annotated genomic resources with differential signatures.Overall, our findings contribute to a broader understanding of the relationship between regional organization and function in the European sea bass thymus, and provide essential insights into the molecular mechanisms underlying T-cell mediated adaptive immune responses in teleosts.

Dual silencing of tumor-intrinsic VISTA and CTLA-4 stimulates T-cell mediated immune responses and inhibits MCF7 breast cancer development

BackgroundAs inhibitory immune checkpoint molecules, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and V-domain Ig suppressor of T-cell activation (VISTA) can be expressed in tumoral cells and facilitate immune evasion of tumoral cells. Herein, we studied the significance of tumor-intrinsic CTLA-4 and VISTA silencing in tumor development and inflammatory factors expression in a co-culture system with MCF7 and T-cells. MethodsMCF7 cells were transfected with 60 pmol of CTLA-siRNA, VISTA-siRNA, and dual VISTA-/CTLA-4-siRNA. The MTT assay was performed to study the effect of CTLA-4 and VISTA knockdown on the viability of MCF7 cells. Colony formation and wound-healing assays were performed to investigate the effect of CTLA-4 and VISTA silencing on the clonogenicity and migration of MCF7 cells. Flow cytometry was used to study the significance of CTLA-4 and VISTA knockdown on the apoptosis and cell cycle of MCF7 cells. Also, a co-culture system with MCF7 and T-cells was developed to study the expression levels of IL-2, IFN-γ, TNF-α, TGF-β, and IL-10 following CTLA-4 and VISTA knockdown. The expression levels of caspase3, Bax, Bcl2, and MMP-9 were also investigated using quantitative real-time PCR. Finally, the TCGA Breast Cancer and GSE45827 datasets were analyzed to study the potential prognostic values of VISTA and CTLA-4, their expression difference in luminal A breast cancer and non-tumoral tissues, and their correlation in luminal A breast cancer tissues. ResultsCombined knockdown of tumor-intrinsic VISTA and CTLA-4 is superior in upregulating IL-2, IFN-γ, and TNF-α, downregulating TGF-β and IL-10 in T lymphocytes. Also, the combined silencing arrests the cell cycle at the sub-G1 phase, decreases migration, inhibits clonogenicity, and reduces cell viability of MCF7 cells. This combined treatment upregulates caspase 9 and BAX and downregulates MMP-9 in MCF7 cells. Our in-silico results have demonstrated a significant positive correlation between CTLA-4 and VISTA in luminal A breast cancer. ConclusionThe additive effect of the combined knockdown of tumor-intrinsic VISTA and CTLA-4 can substantially upregulate pro-inflammatory factors, downregulate anti-inflammatory factors, and inhibit tumor development in MCF7 cells. The significant positive correlation between VISTA and CTLA-4 in luminal A breast cancer might support the idea that a network of inhibitory immune checkpoint molecules regulates anti-tumoral immune responses; thus, combinational immune checkpoint molecules blockade can be suggested.

A rare case of Disseminated Histoplasmosis with Hemophagocytic Lymphohistiocytosis mimicking a flare of Systemic Lupus Erythematosus in a middle-aged man: A Case Report

Abstract Introduction/Objective Disseminated histoplasmosis is a progressive granulomatous disease caused by Histoplasmosis capsulatum which is an intracellular dimorphic fungus endemic to Ohio and Mississippi River valleys in the United States. The progressive spread of the infection to organs is thought to be due to the failure of the activation of the T-cell mediated immune response. Hemophagocytic lymphohistiocytosis is a rare but potentially fatal condition in which histiocytes and lymphocytes build up in and damage organs and other blood cells. Diagnosing Disseminated Histoplasmosis with Hemophagocytic Lymphohistiocytosis in a patient with Systemic Lupus Erythematosus is diagnostically challenging due to the rare presentation. Hence, we are reporting this case to alert physicians to keep a high index of suspicion in such presentations. Methods/Case Report We present a 37-year-old man with a past medical history of Systemic Lupus Erythematosus complicated by Lupus Nephritis on immunosuppressive therapy who presented to the emergency department with hypotension and was admitted for acute kidney injury. Prior to the presentation, he had persistent fever, myalgias, cough, mild shortness of breath, and back pain. CT Chest shows “eggshell” calcification and further parasitology evaluation revealed a diagnosis of histoplasmosis using a blood sample where intracellular Histoplasma organisms were identified. A diagnosis of Hemophagocytic lymphohistiocytosis was made clinically after “Clinical and Testing Criteria” was evaluated. Despite further management, he developed coagulopathy and sepsis which led to his death. Results (if a Case Study enter NA) At autopsy, we found organomegaly of the liver, spleen, and kidneys. Microscopically, these enlarged organs show old fibrotic granulomas and granulomatous inflammation with suspected fungal organisms. Periodic Acid Schiff (Fungus) and Gomori's Methenamine Silver special stain confirm these fungal organisms to be consistent with Histoplasma species (3-5 micron budding yeasts). Conclusion This case report highlights that physicians should be aware of the diagnostic challenge that Disseminated Histoplasmosis with Hemophagocytic Lymphohistiocytosis could pose in patients with Systemic Lupus Erythematosus, especially those on immunosuppression. The clinical presentation could easily mimic a flare of Systemic Lupus Erythematosus, leading to more aggressive immunosuppression. Failure to recognize the infection promptly could lead to grievous complications and sometimes death.

Cd70 Deficiency Impairs CD4 + and CD8 + T-Cell Immune Surveillance in Bcl6-Driven Diffuse Large B-Cell Lymphomas

Anti-tumor immune responses to lymphoid malignancies are modulated by co-stimulatory and co-inhibitory pathways. Our group previously characterized the recurrent genetic alterations in primary diffuse large B-cell lymphomas (DLBCL) and identified frequent inactivating mutations or copy loss of the CD70 co-stimulatory ligand in these tumors. CD70 co-stimulation of CD27 + T cells induces antigen-dependent T-cell expansion and immune surveillance of normal and malignant B cells. Given the frequent combination of CD70 and BCL6 alterations in our human DLBCL series, we investigated the consequences of CD70deficiency ( Cd70 -/-) in a murine model of BCL6-driven lymphomagenesis ( Bcl6 tg/+). Cd70 -/-; Bcl6 tg/+ animals developed predominantly splenic DLBCLs with earlier onset and increased penetrance when compared to Bcl6 tg/+ mice (p<.01). Hence, to elucidate potential differences in T-cell mediated anti-tumor responses in Cd70 -/-; Bcl6 tg/+ versus Bcl6 tg/+ mice (and wild-type (WT) and Cd70 -/- animals) and to characterize the concurrent processes of B-cell transformation and immune evasion over time, we performed single-cell RNA sequencing of splenic cell suspensions from animals euthanized for symptoms and additional asymptomatic mice at pre-determined timepoints (6, 14 and 18 months). We applied unsupervised clustering and defined the major B- and T-cell types based on canonical lineage markers. Additionally, we reconstructed the splenic T-cell receptor (TCR) and B-cell receptor (BCR) sequences with the TRUST4 algorithm to assess their diversity and clonal expansion. TRUST4 analyses revealed clonally expanded B cells that were primarily found in specific clusters. These clusters also had an increased number of aberrant cells identified with an orthogonal approach that detects copy number alterations, InferCNV. Certain of these clusters were more abundant in aged asymptomatic Bcl6 tg/+ and Cd70 -/-; Bcl6 tg/+ animals than in symptomatic mice. Aberrant cells in these clusters had a memory-like IgM + phenotype ( Nt5e, Zeb2, Cd38, Cd44, Itgb1) and included two discrete populations: 1) previously described age-associated/autoimmune-like cells ( Tbx21, Cd11c, Cd11b) and 2) newly appreciated atypical memory cells . Additional clusters that were predominantly found in symptomatic animals included aberrant cells with increased expression of cycling genes and downregulation of lineage-defining markers and MHC II (p<.001). We interpret the aberrant memory-like B-cells in asymptomatic Cd70 -/-; Bcl6 tg/+ and Bcl6 tg/+ animals to be precursors of the ones detected in symptomatic mice. Importantly, malignant clonal expansion and splenomegaly occurred at earlier timepoints and to a greater extent in Cd70 -/-; Bcl6 tg/+ than Bcl6 tg/+ mice (p<.01). To further characterize potential anti-tumor immune responses, we examined splenic CD8 + and CD4 + T-cell subsets in tumor-bearing cohorts ( Bcl6 tg/+ and Cd70 -/-; Bcl6 tg/+) and control animals (WT and Cd70 -/-). In the tumor-bearing cohorts, we detected selective expansion of CD8 + cytotoxic T cells (CTLs) with a spectrum of activation and exhaustion markers. CD8 + CTL expansion was significantly greater in Bcl6 tg/+ mice than Cd70 -/-; Bcl6 tg/+ animals. We also identified selective expansion of a major CD4 + CTL compartment ( GzmB, Prf1, Eomes) in the tumor-bearing cohorts that persisted significantly longer in Bcl6 tg/+ than Cd70 -/-; Bcl6 tg/+ mice. In the tumor-bearing cohorts at 18 months, there was significantly greater clonal expansion of CD4 + CTLs, in comparison to CD8 + CTLs, highlighting the likely importance of this understudied cytotoxic CD4 + T-cell subset. In this regard, the downregulation of MHC II on the malignant B cells of symptomatic mice likely favored CD4 + CTL immune escape and accelerated the development of overt lymphoma. Taken together, our data indicate that genetic perturbation of CD70/CD27 co-stimulation limits the development of an effective T-cell mediated immune response to Bcl6 tg/+-driven DLBCL and highlights the unanticipated role of CD4 + CTLs and MHC II down-regulation in the attempted development and subsequent loss of an anti-tumor immune response. Additionally, we identified previously unappreciated precursor populations of murine DLBCL that warrant further investigation in human tumors.

Contrasting Features of T-Cell Expansion in Classical Hodgkin Lymphoma: Tumor Microenvironment Vs. Peripheral Blood and before Vs. during Anti-PD1 Treatment

Introduction The Tumor Microenvironment (TME) in classical Hodgkin Lymphoma (HL) contains abundant CD4+ and CD8+ T-cells and only few Hodgkin-Reed-Sternberg cells (HRSC). Despite their low abundance, HRSC comprise the neoplastic cell population that intensively interacts with cells of the TME. Understanding these interactions is crucial to the further development of immune checkpoint blockade (ICB) based treatment options. Clinical trials have shown high efficacy of ICB with anti-PD1 antibodies in relapsed HL and more recently promising results of anti-PD1 antibodies in combination with conventional chemotherapy were reported in the first-line setting. (NIVAHL trial, NCT03004833) In solid cancers, anti-PD1 ICB was shown to revert tumor-induced exhaustion of CD4+ and CD8+ T-cells, thereby enabling T-cell activation and a tumor-directed immune response. Since HL differs in many aspects from non-lymphoid tumors due to e.g. loss of HLA-expression, the exact mechanisms of action of ICB in HL is not fully understood and T-cell expansion after anti-PD1 first-line treatment not yet studied. Methods To characterize T-cell activation at different timepoints and to investigate a possible T-cell mediated immune response in HL, we analyzed T-cell Receptor (TCR) repertoires of the NIVAHL study cohort before (tissue: n=90; blood: n=9) and during anti-PD1 treatment (tissue: n=4; blood: n=9). The final cohort comprised additional TCR repertoires of in-house tissue biopsies of treatment-naïve HL (n=18), relapsed HL after chemotherapy (HL-R; n=18)) and publicly available TCR repertoires of the blood of treatment-naïve HL (n=11), HL-R (n=20) and HL-R patients during anti-PD1 treatment (n=20). (Cader et al. Nat Med. 2020 Sep;26(9):1468-1479) TCR repertoires of in-house reactive lymph nodes (n=8, healthy control) and breast cancer (BC; n=6, positive control) patients served as controls for T-cell activation in the tissue, publicly available TCR repertoires of CMV- (n=22, healthy control) and CMV+ otherwise healthy (n=9, positive control) people as controls in the blood. (De Neuter et al. Genes Immun. 2019 Mar;20(3):255-260) TCR repertoires were sequenced by Adaptive Biotechnologies. For each sample, TCR sequences with the same amino acid sequence were aggregated by the sum of their counts. We computed three measures to describe T-cell expansion: debiased Simpson's Clonality (dSC), Percentage of Singletons (PoS), and clonal expansion. Singletons have recently been defined as TCR sequences detected only once in a given sample. Clonal expansion is computed as the percentage of TCR sequences that increase their frequency in a patient from one timepoint to a following by ≥ 2. Results In tissue biopsies, treatment-naïve HL showed a significantly lower dSC compared to treatment-naïve BC and significantly higher dSC compared to RLN from healthy controls. No significant differences could be observed comparing HL with different treatments (during anti-PD1 vs. after chemotherapy) and treatment-naïve HL vs. HL during anti-PD1 treatment. HL and HL-R biopsies showed a significantly lower clonal expansion of Non-Singletons than BC tumors. In peripheral blood, healthy controls, treatment-naïve HL and HL patients during anti-PD1 treatment showed significantly lower dSC in their CD4+ T-cells compared to CMV+ controls and also compared to HL-R patients. We identified significantly higher dSC in CD8+ T-cells compared to CD4+ T-cells in the blood of HL patients before and during any treatment and in relapsed HL. During anti-PD1 treatment, CD8+ T-cells showed a significantly higher clonal expansion of Non-Singletons than CD4+ T-cells in HL-R and a similar but not significant trend in HL. All observed differences in dSC were significant for the PoS too, but as expected by their respective definitions with an opposing pattern (i.e. dSC high = PoS low). Discussion We did not observe features of intratumoral T-cell expansion in primary HL samples, early during anti-PD1 treatment or at relapse after chemotherapy, suggesting that within the HL tissue, T-cell expansion is hampered. However, patterns of T-cell expansion differed between TME and the peripheral blood, where we observed features of CD8+ T-cell clonal expansion, already prior to any treatment. In summary, our findings suggest a possible anti-tumor immune response of CD8+ T-cells in the peripheral blood of HL patients, that is not found in the TME.

Dissolving microneedles-based programmed delivery system for enhanced chemo-immunotherapy of melanoma

Immune checkpoint blockade, especially the programmed cell death ligand 1 (PD-L1) blockade, has revolutionized the treatment of melanoma. However, PD-1/PD-L1 monotherapy leads to unsatisfactory therapeutic outcomes. The immunotherapy of melanoma could be improved by adding doxorubicin (DOX), which triggers immunogenic cell death (ICD) effect to activate anti-tumor immunity. Additionally, microneedles, especially dissolving microneedles (dMNs), can further enhance outcomes of chemo-immunotherapy due to the physical adjuvant effect of dMNs. Herein, we developed the dMNs-based programmed delivery system that incorporated pH-sensitive and melanoma-targeting liposomes to co-deliver DOX and siPD-L1, achieving enhanced chemo-immunotherapy of melanoma (si/DOX@LRGD dMNs). The incorporated si/DOX@LRGD LPs demonstrated uniform particle size, pH-sensitive drug release, high in vitro cytotoxicity and targeting ability. Besides, si/DOX@LRGD LPs effectively downregulated the expression of PD-L1, induced tumor cell apoptosis and triggered ICD effect. The si/DOX@LRGD LPs also showed deep penetration (approximately 80 μm) in 3D tumor spheroids. Moreover, si/DOX@LRGD dMNs dissolved rapidly into the skin and had sufficient mechanical strength to penetrate skin, reaching a depth of approximately 260 μm in mice skin. In mice model of melanoma tumor, si/DOX@LRGD dMNs exhibited better anti-tumor efficacy than monotherapy by dMNs and tail intravenous injection at the same dose. This was due to the higher cytotoxic CD8+ T cells and the secreted cytotoxic cytokine IFN-γ evoked by si/DOX@LRGD dMNs, thereby eliciting strong T-cell mediated immune response and resulted in enhanced anti-tumor effects. In conclusion, these findings suggested that si/DOX@LRGD dMNs provided a promising and effective strategy for enhanced chemo-immunotherapy of melanoma.

IMMU-11. PRECLINICAL HIGH-RISK PEDIATRIC BRAIN TUMOR MODELS FOR IMMUNOTHERAPY: HURDLES AND THE WAY FORWARD

Abstract Despite recent therapeutic advancements in the treatment of pediatric brain tumors, high-risk brain tumors (pHRBT) remain the leading cause of cancer-related deaths in children. In recent years, immunotherapy has become a viable treatment option for several cancers including brain tumors. However, several factors have limited the use of immunotherapy in the treatment of pHRBT. For example, an "immunologically cold" tumor environment has been predominantly implicated in the failure of checkpoint inhibitors (ICIs) as monotherapy in pHRBT. Nevertheless, priming the effects of ICIs with epigenetic modulators through a process called “viral mimicry” that induces the expression of human endogenous retroviruses has the potential to enhance immunotherapy by sparking a T-cell mediated immune response. However, the inadequate understanding of the limitations of the preclinical models has prevented the development of efficient immunotherapy strategies for pHRBT. Although several studies and reviews have compiled some limitations of the available models, a hands-on experience using preclinical models for ICIs testing has not been fully communicated. Here, we share our bedside to bench experience with syngeneic and humanized mouse models of DIPG, ATRT, and medulloblastoma using ICIs therapy in conjunction with an epigenetic alteration. We will present and discuss the limitations of mouse models for the development of immunotherapies for pHRBT. For instance, our results indicated that the baseline levels of MHC-I expression in patients’ DIPG tumors were comparable to matched normal tissue. However, these levels were significantly lower in mouse DIPG tumors. To compensate for this difference, we treated mice with IFN-γ in combination with an epigenetic modulator. Surprisingly, the addition of INF-γ had a negative effect on animal overall survival compared to the treatment with the epigenetic modulator alone. Together, our experience demonstrated the importance of suitability assessment of current models for developing and translating successful immunotherapy strategies to treat pHRBT.

Molecular features and outcomes for patients receiving elranatamab for relapsed or refractory multiple myeloma in MagnetisMM-1.

e20021 Background: Multiple myeloma (MM) is a plasma cell dyscrasia that expresses B-cell maturation antigen (BCMA). Elranatamab (PF-06863135), a BCMA x CD3 bispecific antibody, redirects the T-cell mediated immune response against MM. MagnetisMM-1 (NCT03269136) is the Phase 1 study evaluating safety, pharmacokinetics (PK), pharmacodynamics, and efficacy of elranatamab for patients (pts) with relapsed or refractory MM. Methods: Elranatamab was given subcutaneously at doses 80–1000 µg/kg weekly or every 2 weeks. Treatment-emergent adverse events (TEAEs) were graded by National Cancer Institute Common Terminology Criteria for Adverse Events (v4.03), whereas cytokine release syndrome (CRS) was assessed by criteria of American Society for Transplantation and Cellular Therapy. PK and cytokines were analyzed over time. Bone marrow aspirates were used for cytogenetic analysis, whole exome sequencing, and minimal residual disease (MRD). Clinical response and MRD (with sensitivity of 1×10-5 by next generation sequencing) were assessed by International Myeloma Working Group criteria. Results: 55 pts received elranatamab at efficacious doses ≥215 μg/kg as of September 30, 2022. Pts had median age of 64 years (range 42-80), median of 5 prior regimens (range 2-14), 91% were triple-class refractory, 29% had high cytogenetic risk, and 24% received prior BCMA-targeted therapy. The most common TEAEs included CRS, injection site reaction, neutropenia, anemia, lymphopenia, and thrombocytopenia. Premedication and 1-step priming mitigated CRS. Elranatamab showed linear PK. Genetic alterations potentially relevant to molecular pathogenesis of MM were identified and included functional mutations in KRAS, NRAS, and TP53. With median follow-up of 12.0 months (range 0.3-32.3), objective response rate (ORR) was 64% (95% CI 50-75%) with 56% of pts (31/55) achieving very good partial response (VGPR) or better and 38% of pts (21/55) achieving complete response (CR) or better. Among 13 pts with prior BCMA-targeted therapy, 54% (7/13) achieved response including 46% (6/13) with VGPR or better. All 13 MRD-evaluable pts with confirmed CR or better and dominant clone sequence at baseline achieved MRD negativity, including 2 patients with ongoing stringent CR beyond 2 years. Median duration of response (DOR) was 17.1 months (n = 35; 95% CI 11.1-NE). Median progression-free survival (PFS) was 11.8 months (n = 55; 95% CI 6.0-19.1), and median overall survival (OS) was 21.2 months (n = 55; 95% CI 10.9-NE). Conclusions: Elranatamab induced durable clinical and molecular responses with an acceptable safety profile for pts with relapsed or refractory MM. The ORR was 64% with more than half of these pts (38%) achieving CR or better, and 100% of evaluable pts achieved MRD negativity. These results, along with emerging evidence for both PFS and OS, support the clinical activity of elranatamab for pts with MM. Clinical trial information: NCT03269136 .

Structural analysis of cancer-relevant TCR-CD3 and peptide-MHC complexes by cryoEM

The recognition of antigenic peptide-MHC (pMHC) molecules by T-cell receptors (TCR) initiates the T-cell mediated immune response. Structural characterization is key for understanding the specificity of TCR-pMHC interactions and informing the development of therapeutics. Despite the rapid rise of single particle cryoelectron microscopy (cryoEM), x-ray crystallography has remained the preferred method for structure determination of TCR-pMHC complexes. Here, we report cryoEM structures of two distinct full-length α/β TCR-CD3 complexes bound to their pMHC ligand, the cancer-testis antigen HLA-A2/MAGEA4 (230–239). We also determined cryoEM structures of pMHCs containing MAGEA4 (230–239) peptide and the closely related MAGEA8 (232–241) peptide in the absence of TCR, which provided a structural explanation for the MAGEA4 preference displayed by the TCRs. These findings provide insights into the TCR recognition of a clinically relevant cancer antigen and demonstrate the utility of cryoEM for high-resolution structural analysis of TCR-pMHC interactions.

Abstract 690: Modification of mRNA-encoded HPV16 antigens to include endolysosomal trafficking domains drives cross-presentation and results in superior in vivo and ex vivo antigen-specific responses

Abstract Many cancer immunotherapies require the strong activation of the T-cell mediated adaptive immune response to elicit their full anti-tumor potential. Antigen processing and presentation is a complex, multistep process that can be significantly enhanced via antigen modification, leading to better antigen-specific immune responses. Using mRNA, we can express exogenous antigens inside the cell and facilitate the subcellular localization of these antigens to enhance antigen processing and presentation. In this work, we introduced endolysosomal trafficking domains from CD1d molecules to drive subcellular localization of antigenic proteins to the late endosomes (LE) to enhance class I and class II antigen loading. CD1d are molecules that naturally traffic between the membrane and the late endosomes, where antigens are processed and loaded to both class II and class I molecules in a TAP-independent manner. We compared the immunogenicity of a novel endolysosomal trafficking domain, human CD1d, coupled to a chimeric HPV16 E6 and E7 (HPV16E6E7-hCD1d) antigen to antigen designs intended to be either a) secreted (Sec-HPV16E6E7), b) retained in the endoplasmic reticulum (ER) by an ER retention signal KDEL (HPV16E6E7-KDEL) or c) unmodified antigen sequence (HPV16E6E7). To ensure intracellular translation and expression, all antigens were encoded and delivered as mRNAs. The immunogenicity of each antigen design was assessed in HPV16 positive cervical intraepithelial neoplasia (CIN) donor derived PBMCs and healthy controls. Using confocal microscopy, we observed that the hCD1d trafficking domain directed the translated E6E7 protein into late endosomes, which was not observed for any of the other antigen designs. Importantly, antigen localization to late endosomes resulted in significantly improved immunogenicity in vitro as well as in vivo. We observed a consistent increase in numbers of activated antigen-specific T cells across multiple CIN donor PBMCs treated with HPV16E6E7-hCD1d compared to the other 3 designs, as measured by Interferon gamma secretion, surface marker expression, and T-cell proliferation. To assess the generalizability of this approach, we also compared immunogenicity in human donor PBMCs using the human cytomegalovirus antigen pp65, and observed similar results. Lastly, the immunogenicity of each antigen design was tested in vivo by intramuscular injection of nanoparticle formulated mRNAs in C56/Bl6 mice, and inclusion of the murine analogue (mCD1d) resulted in significantly increased generation of HPV16-specific T cell responses. Based on these findings, we are developing a novel mRNA-based cancer therapeutic that includes the addition of hCD1d trafficking domain in the HPV16-specific antigen component. Citation Format: Weiqun Liu, Daniel Fernandez, Colin J. McKinlay, Daniel O. Frimannsson, Meredith L. Leong, W. Martin Kast, Samuel Deutsch, Ole Audun W. Haabeth. Modification of mRNA-encoded HPV16 antigens to include endolysosomal trafficking domains drives cross-presentation and results in superior in vivo and ex vivo antigen-specific responses [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 690.

The innate and T-cell mediated immune response during acute and chronic gammaherpesvirus infection.

Immediately after entry into host cells, viruses are sensed by the innate immune system, leading to the activation of innate antiviral effector mechanisms including the type I interferon (IFN) response and natural killer (NK) cells. This innate immune response helps to shape an effective adaptive T cell immune response mediated by cytotoxic T cells and CD4+ T helper cells and is also critical for the maintenance of protective T cells during chronic infection. The human gammaherpesvirus Epstein-Barr virus (EBV) is a highly prevalent lymphotropic oncovirus that establishes chronic lifelong infections in the vast majority of the adult population. Although acute EBV infection is controlled in an immunocompetent host, chronic EBV infection can lead to severe complications in immunosuppressed patients. Given that EBV is strictly host-specific, its murine homolog murid herpesvirus 4 or MHV68 is a widely used model to obtain in vivo insights into the interaction between gammaherpesviruses and their host. Despite the fact that EBV and MHV68 have developed strategies to evade the innate and adaptive immune response, innate antiviral effector mechanisms still play a vital role in not only controlling the acute infection but also shaping an efficient long-lasting adaptive immune response. Here, we summarize the current knowledge about the innate immune response mediated by the type I IFN system and NK cells, and the adaptive T cell-mediated response during EBV and MHV68 infection. Investigating the fine-tuned interplay between the innate immune and T cell response will provide valuable insights which may be exploited to design better therapeutic strategies to vanquish chronic herpesviral infection.

Cerebellar presence of immune cells in patients with neuro-coeliac disease

Although various neurodegenerative disorders have been associated with coeliac disease (CD), the underlying neuropathological link between these brain and gut diseases remains unclear. We postulated that the neuronal damage sporadically observed in CD patients is immune-mediated. Our aim was to determine if the loss of neurons, especially Purkinje cells, coincides with microglia activation and T- and B-cell infiltration in the cerebellum of patients with CD and a concomitant idiopathic neurological disease affecting the cerebellum (NeuroCD). Post-mortem cerebellar tissue was collected of validated NeuroCD cases. Gender- and age-matched genetic spinocerebellar ataxia (SCA) controls and non-neurological controls (NNC) were selected based on clinical reports and pathological findings. Cerebellar tissue of seventeen patients was included (6 NeuroCD, 5 SCA, 6 NNC). In SCA cases we found that the Purkinje cell layer was 58.6% reduced in comparison with NNC. In NeuroCD cases this reduction was even more prominent with a median reduction of 81.3% compared to NNC. Marked increased numbers of both CD3+ and CD8+ cells were observed in the NeuroCD but not in SCA patients. This coincided with significantly more microglial reactivity in NeuroCD patients. These findings demonstrate that the massive loss of Purkinje cells in the cerebellum of neuro CD patients is accompanied by local innate and T-cell mediated immune responses.

Neutrophil extracellular traps in tumor progression and immunotherapy.

Tumor immunity is a growing field of research that involves immune cells within the tumor microenvironment. Neutrophil extracellular traps (NETs) are neutrophil-derived extracellular web-like chromatin structures that are composed of histones and granule proteins. Initially discovered as the predominant host defense against pathogens, NETs have attracted increasing attention due to they have also been tightly associated with tumor. Excessive NET formation has been linked to increased tumor growth, metastasis, and drug resistance. Moreover, through direct and/or indirect effects on immune cells, an abnormal increase in NETs benefits immune exclusion and inhibits T-cell mediated antitumor immune responses. In this review, we summarize the recent but rapid progress in understanding the pivotal roles of NETs in tumor and anti-tumor immunity, highlighting the most relevant challenges in the field. We believe that NETs may be a promising therapeutic target for tumor immunotherapy.

IR780/Gemcitabine-conjugated metal-phenolic network enhanced photodynamic cancer therapy

Photodynamic therapy (PDT) is a clinically approved cancer treatment that uses energy of light to generate active substances that cause damage to the cancer. Photosensitizers are employed to absorb light and generate toxic reactive oxygen species (ROS) to damage biomolecules like DNA. At the same time, some chemotherapy drugs like nucleotide analogues can provide mechanism-guided promotion in the treatment efficacy of PDT. However, the photosensitizer and chemotherapy drugs used in PDT is usually organic molecules, which suffers from bad solubility, fast clearance, and acute toxicity. To achieve targeted treatment, a reasonable delivery system is necessary. Therefore, we reported a metal-phenolic network where IR780 and gemcitabine were coupled chemically to overcome these shortcomings. The enhanced PDT effects can be realized by the promoted cell death both in vitro and in vivo. Moreover, the synergistic therapy also induced T-cell mediated anti-tumor immune response, which was significant for the inhibition of distant tumor growth. This work expanded the biomedical application of metal-phenolic materials and contribute to the wider application of photodynamic cancer therapy.