Cytotoxic T Lymphocytes Research Articles

Exploration of immunomodulatory mechanism of caprine Wharton’s jelly derived mesenchymal stem cells

The present study was aimed to explore the possible mechanisms by which caprine Wharton’s jelly-derived MSCs (WJ-MSCs) perform their immunomodulatory function. WJ-MSCs were isolated through explants culture and characterized as per ISCT criteria using culture behavior, expression of surface markers by PCR, FACS and immunocytochemical localization (ICC), trilineage differentiation potential etc. Secretory behavior for important biomolecules (IDO, TGFβ1, VEGF, IL6) was evaluated by ICC and western blot assay. Cell-to-cell communication was studied by culturing cells in cell–cell contact and trans-well system. The MSCs when co-cultured with activated Tc and Th cells, down-regulation of T cell cytokine as well as upregulation of immunomodulatory factors (VEGF A, IL10, IL6, IDO, iNOS, PTGS2, HGF, TGFβ, CXCL10, CXCL11) was noticed in both cell–cell contact and trans-well culture system which was significantly higher in cell–cell contact system. Trilineage differentiation of MSCs showed significant upregulation of MHC I (CAHI) and MHC II (CLA DRB3) molecules suggesting better clinical applications of MSCs without differentiation to avoid immune rejection. It can be concluded that WJ-MSCs perform their immunomodulation through the secretion of a battery of biomolecules and work in both cell–cell contact manner and through their secretome.

Proteinuria and albuminuria among a global primary CVD prevention cohort of PWH: prevalence and associated factors.

To determine baseline prevalence of proteinuria and albuminuria among REPRIEVE participants and evaluate associated risk factors. Cross sectional analysis of a baseline sample of participants from the REPRIEVE Trial. REPRIEVE is an international primary cardiovascular prevention RCT of pitavastatin calcium vs. placebo among PWH on antiretroviral therapy. A representative subset (2791 participants) had urine collected at study entry. Urine protein to creatinine ratios (uPCR) and albumin to creatinine ratios (uACR) were classified as normal, moderately increased and severely increased. These were dichotomized to Normal or Abnormal for log-binomial regression analysis. Demographic, cardiometabolic, and HIV-specific data were compared among those with normal versus abnormal results. Overall, median age 49 years, 41% female sex, 47% black or African American race, 36% had eGFR <90 mL/min/1.73 mm2. For uPCR, 27% had moderately or severely increased values. For uACR, 9% had moderately or severely increased values. In the fully adjusted model for proteinuria, female sex, older age, residence in sub-Saharan Africa or East Asia, lower BMI, lower CD4 cell count, and use of TDF were associated with abnormal values. In the fully adjusted model for albuminuria, a diagnosis of HTN was associated with abnormal values. Abnormal proteinuria and albuminuria remain common (27% and 9%) despite controlled HIV. Lower current CD4 count and TDF use were strongly associated with proteinuria. Certain modifiable comorbidities, including HTN and smoking, were associated with abnormal values. In PWH with preserved eGFR, urine measures identify subclinical kidney disease and afford the opportunity for intervention.

Abstract PR-07: Discovery and therapeutic potential of novel cryptic peptides in pancreatic cancer

Abstract While neoantigen-directed therapies have primarily focused on tumor-specific somatic mutations, emerging evidence suggests that some malignancies aberrantly translate regions of the genome outside of annotated open reading frames (ORFs), leading to HLA-I presentation of cryptic peptides. Non-canonical HLA-I bound peptides (ncHLAp) can arise from aberrant translation of genomic elements like 5’ and 3’ untranslated regions (UTRs), long noncoding RNAs (lncRNAs), retained introns, and translation in alternative reading frames. Pancreatic cancer has a low-to-intermediate mutational burden; hence, efforts to broaden the landscape targetable antigens in this disease are greatly needed. Here, we leveraged twelve pancreatic cancer (PDAC) patient-derived organoids (PDOs) to purify and enrich the malignant compartment from low tumor cellularity tumor specimens. PDOs were subjected to extensive genomic (whole genome sequencing) and transcriptomic (RNA-sequencing) profiling to enable mutation calling and HLA typing. We then employed a personalized proteogenomic platform coupled with high-depth immunopeptidomics and empirically identified &amp;gt;90,000 unique PDAC HLA-I-bound peptides (HLAp). We detected HLAp arising from somatic mutations (both missense and frameshift) in a subset of PDAC patients; however, we did not detect any shared mutation-derived neoepitopes in our patient cohort. Moreover, we empirically identified &amp;gt;1,700 ncHLAp, primarily arising from translation of novel unannotated open reading frames (nuORFs), and a substantial proportion of ncHLAp were shared amongst PDAC patients with the appropriate HLA haplotype. We developed a highly stringent pipeline using immunopeptidomics and ribosome-sequencing to investigate translation of nuORFs across a range of healthy tissues, including healthy thymus, and found that &amp;gt;500 ncHLAp exhibit highly-specific cancer-restricted translation. We next investigated the immunogenicity of PDAC-restricted ncHLAp and mutation-derived HLAp using an ex vivo T cell priming and expansion platform. Here we demonstrate that a subset of both mutation-derived and PDAC-restricted ncHLAp harbor robust immunogenicity. ncHLAp-specific T cell receptors (TCRs) were identified from reactive cytotoxic T lymphocytes (CTLs) via single-cell TCR-sequencing, and antigen specificity was confirmed after TCR reconstruction and cloning. Finally, using a newly developed organoid: T cell co-culture platform, we demonstrate that ncHLAp-reactive TCR-redirected T cells exhibit robust cytotoxicity and tumoricidal activity in PDAC, underscoring the translational potential for this novel class of antigens. Collectively, we have shown that cryptic epitopes, arising from aberrant translation in PDAC, represent a particularly promising class of antigens for next generation immune-based therapies. Citation Format: Zackery A Ely, Zachary J Kulstad, Gurcan Gunaydin, Sudarsana Addepalli, Eva K Verzani, Jennifer G Abelin, Marta Casarrubios, Karl R Clauser, Xilin Wang, Isabelle Lippincott, Cedric Louvet, Tom Schmitt, Miles Agus, Kevin S Kapner, Connor J Hennessey, James Cleary, Sine R Hadrup, Susan Klaeger, Jennifer Su, Alex M Jaeger, Brian M Wolpin, Srivatsan Raghavan, Eric Smith, Philip D Greenberg, Andrew J Aguirre, Steven A Carr, Tyler Jacks, William A Freed-Pastor. Discovery and therapeutic potential of novel cryptic peptides in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-07.

Abstract B091: Spatial Transcriptomics Unveils Intraductal Papillary Mucinous Neoplasm Heterogeneity: From Novel Clusters to Immune Dynamics

Abstract Intraductal papillary mucinous neoplasms (IPMNs), a common precursor to pancreatic ductal adenocarcinoma, present as a spectrum of benign indolent behavior and progression to invasive carcinoma, which highlights the importance of identifying biomarkers of IPMN progression. This study utilized whole transcriptome spatial transcriptomics to define the molecular characteristics of Epithelial, Immune, and Fibroblast cell populations of the three distinct histological sub-types of IPMN to understand the dynamics between the cell types and identify biomarkers of neoplastic progression. Fifty-two surgically resected IPMN cores from 21 patients with resected IPMNs comprising 24 Gastric, 14 Intestinal, and 14 Pancreaticobiliary with a distribution of low-grade dysplasia (LGD), high-grade dysplasia (HGD), and Invasive carcinoma (IC) were analyzed. Epithelial, Immune, and Fibroblast components were analyzed using the GeoMx spatial profiler. Unsupervised hierarchical clustering of epithelial areas of interest (AOI) identified 3 distinct clusters demonstrating a spectrum of molecular phenotypes in IPMN that were correlated with the three histological subtypes. Comparing the gene expression profile based on the shared genes expressed on all three subtypes, the Intestinal type tends to have an overexpression of genes whereas the Pancreaticobiliary represents a repressed phenotype relative to the Gastric phenotype. Supervised clustering using Moffitt’s classifier to identify the Epithelial-Mesenchymal transition demonstrates the Pancreaticobiliary subtype to be more Basal-like versus the Gastric and Intestinal inclined towards the classical phenotype. Supervised clustering based on the degree of dysplasia within the epithelial compartments of each of these subtypes, demonstrated a spectrum of molecular heterogeneity and identified biomarkers specific to High-Grade dysplasia in the Gastric and Intestinal subtype. Immune AOI deconvolution analysis demonstrated that Tregs were associated with Pancreaticobiliary, Macrophage enrichment associated with the Intestinal phenotype, while there is enrichment of CD8 T-cell and CD4 T-cell gene signatures associated with the Gastric subtype. Fibroblast AOI analyses demonstrated heterogenous myCAF- iCAF signatures between the subtypes. In summary, Spatial whole transcriptome analysis of IPMN identifies molecular signatures in epithelial and immune compartments that differentiate between the three histological subtypes of IPMN and helps understand their progression from Low-grade dysplasia to High-grade dysplasia. Citation Format: Amaya Pankaj, Michael J Raabe, Yuhui Song, Bidish K Patel, Katherine Xu, Joshua R Kocher, Nicholas J Caldwell, Maria L Ganci, Linda T Nieman, Mari Mino-Kenudson, Vikram Deshpande, Nabeel Bardeesy, Carlos Fernandez-Del Castillo, Martin J Aryee, David T Ting, Yasmin G Hernandez-Barco. Spatial Transcriptomics Unveils Intraductal Papillary Mucinous Neoplasm Heterogeneity: From Novel Clusters to Immune Dynamics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B091.

Abstract B043: Synergy of Subasumstat and anti-CD40 improves survival by augmenting tumor macrophage infiltration

Abstract Background: Identification of effective immunomodulatory strategies remains a major unmet need in pancreatic ductal adenocarcinoma (PDAC). Agonistic anti-CD40 antibody (aCD40) exhibits direct cytotoxicity on tumor cells and augments antigen presentation. Subasumstat(SUB), a sumoylation inhibitor, augments innate and adaptive immune responses by increasing interferon signaling. We hypothesized that SUB+aCD40 combination would promote an effective antitumor immune response in an aggressive orthotopic model of PDAC. Methods: 2000 KPC 46 cells were implanted in the pancreas of four groups of F1 hybrid (initial survival, CD8+ T-cell depletion, and clodronate liposome depletion) and nude mice, enrolled when tumors had reached 3-5mm. The vehicle (veh) group received 80ul SUB vehicle (q48 hrs) and 100 ug blank IgG (qwk); SUB only group received 15mg/kg q48h; aCD40 only group received 100ug qwk; SUB+aCD40 group received 15mg/kg SUB q48h and 100 ug aCD40 qwk. Single cell RNA sequencing (scRNASeq) was conducted on n=2 tumors from each group. Immunohistochemistry (IHC) was performed on veh and SUB+aCD40 tumors for macrophage infiltration using F4/80. Flow cytometry (FC) was employed for the presence of lymphoid and myeloid markers in veh, SUB+aCD40, and clodronate depleted SUB+aCD40 (SUB+aCD40+clod) groups. Results: Mice receiving SUB+aCD40 had a significantly improved median survival over those receiving monotherapy or vehicle (24.5d vs. SUB:15.5d, aCD40:20d, veh: 19d). scRNASeq revealed increased tumor infiltrating CD8+ T-cell and myeloid populations when comparing veh and monotherapy to SUB+aCD40. F4/80 IHC confirmed increased macrophage infiltration in SUB+aCD40 vs veh (p = 0.01). The survival advantage conferred by SUB+aCD40 was preserved in both CD8+ T-cell depleted mice (p=0.025) and nude mice (p=0.037), while clodronate eliminated the treatment survival advantage (p=0.39). FC showed increased tumor macrophage/monocyte infiltration in SUB+aCD40 vs veh (veh:11.9%, SUB+aCD40:22.5%, p=0.03) and reduction in SUB+aCD40+clod (12.6%, p=0.01). CD8+ T-cell CD69 expression decreased in the SUB+aCD40+clod group (SUB+aCD40:19%, SUB+aCD40+clod:6%, p&amp;lt; 0.01). CD8 T-cell infiltration (SUB+aCD40:4.95%, SUB+aCD40+clod:3.85%, p=0.27) and CD4/CD8 T-cell ratio (veh: 1.26, SUB+aCD40:0.52, SUB+aCD40+clod:0.69, p=0.09) were not affected by clodronate. Conclusion: SUB+aCD40 therapy confers a survival advantage in the KPC46 model of PDAC in a T-cell independent manner by increasing macrophage/monocyte infiltration. SUB+aCD40 promotes CD8+ T-cell infiltration and activation, but they do not contribute to survival advantage, possibly due to exhaustion. Future work will include addition of therapeutics to mitigate/reverse T-cell exhaustion to harness both innate and adaptive immunity in treating PDAC. Citation Format: Asimina Courelli, Kevin Li, James Lee, Herve Tiriac, Yuan Chen, Andrew Lowy. Synergy of Subasumstat and anti-CD40 improves survival by augmenting tumor macrophage infiltration [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B043.

Abstract B058: Rewiring CD8+ T cell responses to PD-1 immune checkpoint blockade in PDAC via the inhibitory receptor PSGL-1

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, poorly immunogenic cancer and non-responsive to all currently available treatments, including immune checkpoint blockade (ICB) therapies. As survival rates remain low (13%), there is an urgent need for new therapeutic options. A hallmark of PDAC is the limited infiltration of anti-tumor cytotoxic CD8+ T cells. Analysis of single-cell RNA-sequencing data of PDAC tumors from treatment naïve patients reveals high expression of P-selectin glycoprotein ligand 1 (PSGL-1) on tumor-infiltrating CD8+ T cells. PSGL-1 is an intrinsic negative regulator of CD8+ T cells that promotes the development of functional exhaustion. We therefore hypothesized that PSGL-1-mediated immune suppression is a critical barrier to effective anti-tumor immunity in PDAC. To test this, we used preclinical models of orthotopic injection PDAC tumor cells into pancreata, recapitulating patient responses to PDAC with limited T cell infiltrates and uncontrolled tumor growth in C57BL/6 (wildtype) control mice and compared anti-tumor immune responses in PSGL-1KO (C57BL/6 background) mice. At 28 days post-injection of KPC.4662 PDAC tumor cells, tumor burden in PSGL-1KO mice was reduced by &amp;gt;50% compared to controls, with 45% of PSGL-1KO animals exhibiting &amp;gt;70% reduction. Total immune infiltration (CD45+) was 2-fold greater in tumors from PSGL-1KO mice, with significant increases in infiltrating CD4+ and CD8+ T cells. Detailed analyses revealed that infiltrating CD8+ T cells from PSGL-1KO mice were less terminally differentiated towards exhaustion. PSGL-1KO PD-1+ CD8+ T cells expressed less TOX expression and fewer cells co-expressed CD38 and CD101 while more expressed CX3CR1 compared to controls. Intratumoral PSGL-1KO CD8+ T cells also retained greater functionality as well as indicated by inflammatory cytokine production (IFNg, TNFa) and Granzyme B expression. Moreover, PSGL-1KO mice showed protection against metastatic disease to the lungs. We find that protection by PSGL-1KO mice is T cell dependent as transient depletion of CD4+ and CD8+ T cells prior to tumor cell implantation results in the loss of PDAC tumor growth inhibition by PSGL-1KO mice. Less-differentiated, proliferation-competent precursors of exhausted CD8+ T cells (TPEX) are essential to PD-1 ICB responsiveness in patients and preclinical models. Given that PSGL-1-deficiency limits terminal differentiation of CD8+ T cells in PDAC, we hypothesized that PSGL-1-deficiency would promote responsiveness to PD-1 ICB, which is ineffective as a monotherapy in PDAC patients and mouse models. While therapeutic treatment with anti-PD-1 had no effect on the tumor growth in the control animals, PD-1 ICB resulted in tumor ablation in 85% of PSGL-1KO animals, demonstrating a profound synergistic effect of PD-1 inhibition in the absence of PSGL-1. From these studies, we conclude that PSGL- 1 is a critical inhibitor anti-tumor T cell immunity in PDAC and represents a novel ICB target with high translational potential for promoting immune responses to PDAC tumors. Citation Format: Jennifer L Hope, Yijuan Zhang, Hannah A. F. Hetrick, Evelyn S. Sanchez Hernandez, Gabriele Romano, Sreeja Roy, Michelle Lin, Ashley B Palete, Swetha Maganti, Dennis C Otero, Katelyn T Byrne, Cosimo Commisso, Linda M Bradley. Rewiring CD8T cell responses to PD-1 immune checkpoint blockade in PDAC via the inhibitory receptor PSGL-1 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B058.

Transformable self-delivered supramolecular nanomaterials combined with anti-PD-1 antibodies alleviate tumor immunosuppression to treat breast cancer with bone metastasis

Breast cancer is the most common malignant tumor that threatens women’s life and health, and metastasis often occurs in the advanced stage of breast cancer, leading to pathological bone destruction and seriously reducing patient quality of life. In this study, we coupled chlorin e6 (Ce6) with mono-(6-amino-6-deoxy)-beta-cyclodextrin (β-CD) to form Ce6-CD, and combined ferrocene with the FFVLG3C peptide and PEG chains to form the triblock molecule Fc-pep-PEG. In addition, the IDO-1 inhibitor NLG919 was loaded with Ce6-CD and Fc-pep-PEG to construct the supramolecular nanoparticle NLG919@Ce6-CD/Fc-pep-PEG (NLG919@CF). After laser irradiation, Ce6 produced robust reactive oxidative species to induce tumor cell apoptosis. Simultaneously, ferrocene became charged, and Fc-pep-PEG dissociated from the spherical nanoparticles, enabling their transformation into nanofibers, which increased both the retention effect and the induction of ferroptosis. The released NLG919 reduced the number of regulatory T cells (Tregs) and restored the function of cytotoxic T lymphocytes (CTLs) by inhibiting the activity of IDO-1. Moreover, combined administration with an anti-PD-1 antibody further relieved immune suppression in the tumor microenvironment. This article presents a new strategy for the clinical treatment of breast cancer with bone metastasis and osteolysis.Graphical

Targeting GProtein-Coupled Receptors in Immuno-Oncological Therapies.

The advent of cancer immunotherapy based on PD-1 and CTLA-4 immune checkpoint blockade (ICB) has revolutionized cancer treatment. However, many cancers do not respond to ICB, highlighting the urgent need for additional approaches to achieve durable cancer remission. The large family of G protein-coupled receptors (GPCRs) is the target of more than 30% of all approved drugs, but GPCRs have been underexploited in cancer immunotherapy. In this review, we discuss the central role of GPCRs in immune cell migration and function and describe how single-cell transcriptomic studies are illuminating the complexity of the human tumor immune GPCRome. These receptors include multiple GPCRs expressed in CD8 Tcells that are activated by inflammatory mediators, protons, neurotransmitters, and metabolites that accumulate in the tumor microenvironment, thereby promoting Tcell dysfunction. We also discuss new opportunities to target GPCRs as a multimodal approach to enhance the response to ICB for a myriad of human malignancies.

Extracellular Silica Nanomatrices Promote In Vitro Maturation of Anti-tumor Dendritic Cells via Activation of Focal Adhesion Kinase.

The efficacy of dendritic cell (DC)-based cancer vaccines is critically determined by the functionalities of in vitro maturated DCs. The maturation of DCs typically relies on chemicals that are cytotoxic or hinder the ability of DCs to efficiently activate the antigen-specific cytotoxic T-lymphocytes (CTLs) against tumors. Herein, the maturation chemicals are replaced with extracellular silica nanomatrices, fabricated by glancing angle deposition, to promote in vitro maturation of murine bone marrow-derived DCs (mBMDCs). The extracellular nanomatrices composed of silica nanozigzags (NZs) enable the generation of mature mBMDCs with upregulated levels of co-stimulatory molecules, C-C chemokine receptor type-7, X-C motif chemokine recetpor-1, DC-specific ICAM-3 grabbing nonintegrin, and enhanced endocytic capacity. The in vitro maturation is partially governed by focal adhesion kinase (FAK) that is mechanically activated in the curved cell adhesions formed at the DC-NZ interfaces. The NZ-maturated mBMDCs can prime the antigen-specific CTLs into programmed cell death protein-1 (PD-1)lowCD44high memory phenotypes in vitro and suppress the growth of tumors in vivo. Meanwhile, the NZ-mediated beneficial effects are also observed in human monocyte-derived DCs. This work demonstrates that the silica NZs promote the anti-tumor capacity of in vitro maturated DCs via the mechanoactivation of FAK, supporting the potential of silica NZs being a promising biomaterial for cancer immunotherapy.

Androgen Deprivation Therapy Drives a Distinct Immune Phenotype in Localized Prostate Cancer.

Androgen deprivation therapy (ADT) remains the backbone of prostate cancer treatment. Beyond suppression of testosterone and tumor cell growth, emerging evidence suggests ADT also modulates the immune tumor microenvironment (TME). However, a more precise understanding of the timing and intricacies of these immunological shifts is needed. Here we analyzed 49 primary prostate cancers, comparing those surgically removed either without treatment or following treatment with degarelix at 4, 7, and 14 days pre-surgery. Utilizing next-generation DNA and RNA sequencing, and multiplexed immunofluorescence, we examined alterations in immune phenotypes in the presence or absence of ADT. Our findings reveal that ADT rapidly transforms the typically bland prostate TME into an inflamed environment within days. Notably, we observed an increase in activated CD8 T-cells along with an increase in suppressive regulatory T-cells (Tregs). We also found an expansion of the myeloid compartment, particularly pro-inflammatory M1-like tumor-associated macrophages. Intriguingly, discernable changes which have not previously been described also occurred in tumor cells, including upregulation of antigen presentation by MHC class I and II and, unexpectedly, a decrease in the "don't eat me" signal CD47. These observations underscore the critical role of timing and disease context in order to optimize the therapeutic efficacy of immune modulators combined with androgen ablation, for which the presurgical neoadjuvant setting may be ideal. Our findings warrant future prospective validation, which is currently underway.

Ultrasound frequency-controlled microbubble dynamics in brain vessels regulate the enrichment of inflammatory pathways in the blood-brain barrier

Microbubble-enhanced ultrasound provides a noninvasive physical method to locally overcome major obstacles to the accumulation of blood-borne therapeutics in the brain, posed by the blood-brain barrier (BBB). However, due to the highly nonlinear and coupled behavior of microbubble dynamics in brain vessels, the impact of microbubble resonant effects on BBB signaling and function remains undefined. Here, combined theoretical and prospective experimental investigations reveal that microbubble resonant effects in brain capillaries can control the enrichment of inflammatory pathways that are sensitive to wall shear stress and promote differential expression of a range of transcripts in the BBB, supporting the notion that microbubble dynamics exerted mechanical stress can be used to establish molecular, in addition to spatial, therapeutic windows to target brain diseases. Consistent with these findings, a robust increase in cytotoxic T-cell accumulation in brain tumors was observed, demonstrating the functional relevance and potential clinical significance of the observed immuno-mechano-biological responses.

Role of lamin A/C on dendritic cell function in antiviral immunity

Dendritic cells (DCs) play a crucial role in orchestrating immune responses, particularly in promoting IFNγ-producing-CD8 cytotoxic T lymphocytes (CTLs) and IFNγ-producing-CD4 T helper 1 (Th1) cells, which are essential for defending against viral infections. Additionally, the nuclear envelope protein lamin A/C has been implicated in T cell immunity. Nevertheless, the intricate interplay between innate and adaptive immunity in response to viral infections, particularly the role of lamin A/C in DC functions within this context, remains poorly understood. In this study, we demonstrate that mice lacking lamin A/C in myeloid LysM promoter-expressing cells exhibit a reduced capacity to induce Th1 and CD8 CTL responses, leading to impaired clearance of acute primary Vaccinia virus (VACV) infection. Remarkably, in vitro-generated granulocyte macrophage colony-stimulating factor bone marrow-derived DCs (GM-CSF BMDCs) show high levels of lamin A/C. Lamin A/C absence on GM-CSF BMDCs does not affect the expression of costimulatory molecules on the cell membrane but it reduces the cellular ability to form immunological synapses with naïve CD4 T cells. Lamin A/C deletion induces alterations in NFκB nuclear localization, thereby influencing NF-κB-dependent transcription. Furthermore, lamin A/C ablation modifies the gene accessibility of BMDCs, predisposing these cells to mount a less effective antiviral response upon TLR stimulation. This study highlights the critical role of DCs in interacting with CD4 T cells during antiviral responses and proposes some mechanisms through which lamin A/C may modulate DC function via gene accessibility and transcriptional regulation.

Abnormal Immune Profile in Individuals with Kabuki Syndrome.

To analyze the lymphocyte subsets in individuals with Kabuki syndrome for better characterizing the immunological phenotype of this rare congenital disorder. We characterized the immunological profile including B-, T- and natural killer-cell subsets in a series (N = 18) of individuals with Kabuki syndrome. All 18 individuals underwent genetic analysis: 15 had a variant in KMT2D and 3 a variant in KDM6A. Eleven of the 18 individuals (61%) had recurrent infections and 9 (50%) respiratory infections. Three (17%) had autoimmune diseases. On immunological analysis, 6 (33%) had CD4 T-cell lymphopenia, which was preferentially associated with the KMT2D truncating variant (5/9 individuals). Eight of 18 individuals (44%) had a humoral deficiency and eight (44%) had B lymphopenia. We found abnormal distributions of T-cell subsets, especially a frequent decrease in recent thymic emigrant CD4 + naive T-cell count in 13/16 individuals (81%). The immunological features of Kabuki syndrome showed variable immune disorders with CD4 + T-cell deficiency in one third of cases, which had not been previously reported. In particular, we found a reduction in recent thymic emigrant naïve CD4 + T-cell count in 13 of 16 individuals, representing a novel finding that had not previously been reported.

Tumor-associated neutrophils upregulate Nectin2 expression, creating the immunosuppressive microenvironment in pancreatic ductal adenocarcinoma

BackgroundTumor-associated neutrophils (TANs) constitute an abundant component among tumor-infiltrating immune cells and have recently emerged as a critical player in pancreatic ductal adenocarcinoma (PDAC) progression. This study aimed to elucidate the pro-tumor mechanisms of TAN and identify a novel target for effective immunotherapy against PDAC.MethodsMicroarray and cytokine array analyses were performed to identify the mechanisms underlying the function of TANs. Human and mouse TANs were obtained from differentiated HL-60 cells and orthotopically transplanted PDAC tumors, respectively. The interactions of TANs with cancer and cytotoxic T-cells were evaluated through in vitro co-culture and in vivo orthotopic or subcutaneous models. Single-cell transcriptomes from patients with PDAC were analyzed to validate the cellular findings.ResultsIncreased neutrophil infiltration in the tumor microenvironment was associated with poor survival in patients with PDAC. TANs secreted abundant amounts of chemokine ligand 5 (CCL5), subsequently enhancing cancer cell migration and invasion. TANs subpopulations negatively correlated with cytotoxic CD8+ T-cell infiltration in PDAC and promoted T-cell dysfunction. TANs upregulated the membranous expression of Nectin2, which contributed to CD8+ T-cell exhaustion. Blocking Nectin2 improved CD8+ T-cell function and suppressed tumor progression in the mouse model. Single-cell analysis of human PDAC revealed two immunosuppressive TANs phenotypes: Nectin2+ TANs and OLR1+ TANs. Endoplasmic reticulum stress regulated the protumor activities in TANs.ConclusionsTANs enhance PDAC progression by secreting CCL5 and upregulating Nectin2. Targeting the immune checkpoint Nectin2 could represent a novel strategy to enhance immunotherapy efficacy in PDAC.

Changes in immune subsets during chemotherapy as prognosis biomarkers for multiple myeloma patients by longitudinal monitoring.

Multiple myeloma (MM) is a malignancy of plasma cells accompanied by immune dysfunction. This study aimed to provide a comprehensive and dynamic characterization of the peripheral immune environment in MM patients and find its diagnostic and prognostic values for therapy. The peripheral immune profiles of MM inpatients and healthy controls were assessed by flow cytometry. A longitudinal study of immune subsets was observed during cycles of chemotherapy. The diagnostic and prognostic models were established based on immune subsets by the absolute shrinkage and selection operator (LASSO) and multivariate regression. MM patients possessed an impeded immune landscape, including reduced activation of B cells, increased effective T cells and regulatory T cells (Tregs), augmented CD16 expression on monocytes and dendritic cell percentages, decreased CD56dimCD16+ natural killer cells (NKs), and amplified CD56bright and HLA-DR+ natural killer T cells (NKTs). Chemotherapy has different dynamic effects on specific cells, of which 2 cycles is the key turning point. NKT, dendritic cells, naïve Tc and Th cells, HLA-DR+ Tc cells, CD56dim NKTs, CD16++ monocytes, and CD25+ B cells could have the diagnostic value, and a prognostic model including neutrophils, naïve Tc cells, CD56brightCD16dim NKs, and CD16+ dendritic cells was established with acceptable accuracy. Our data showed dynamic and abnormal peripheral immune profiles in MM patients, which had prognostic values and could provide the basis for clinical therapy.

Antiviral capacity of the early CD8 T-cell response is predictive of natural control of SIV infection: Learning in vivo dynamics using ex vivo data.

While most individuals suffer progressive disease following HIV infection, a small fraction spontaneously controls the infection. Although CD8 T-cells have been implicated in this natural control, their mechanistic roles are yet to be established. Here, we combined mathematical modeling and analysis of previously published data from 16 SIV-infected macaques, of which 12 were natural controllers, to elucidate the role of CD8 T-cells in natural control. For each macaque, we considered, in addition to the canonical in vivo plasma viral load and SIV DNA data, longitudinal ex vivo measurements of the virus suppressive capacity of CD8 T-cells. Available mathematical models do not allow analysis of such combined in vivo-ex vivo datasets. We explicitly modeled the ex vivo assay, derived analytical approximations that link the ex vivo measurements with the in vivo effector function of CD8-T cells, and integrated them with an in vivo model of virus dynamics, thus developing a new learning framework that enabled the analysis. Our model fit the data well and estimated the recruitment rate and/or maximal killing rate of CD8 T-cells to be up to 2-fold higher in controllers than non-controllers (p = 0.013). Importantly, the cumulative suppressive capacity of CD8 T-cells over the first 4-6 weeks of infection was associated with virus control (Spearman's ρ = -0.51; p = 0.05). Thus, our analysis identified the early cumulative suppressive capacity of CD8 T-cells as a predictor of natural control. Furthermore, simulating a large virtual population, our model quantified the minimum capacity of this early CD8 T-cell response necessary for long-term control. Our study presents new, quantitative insights into the role of CD8 T-cells in the natural control of HIV infection and has implications for remission strategies.

Dendritic Cells Pulsed with HAM/TSP Exosomes Sensitize CD4 T Cells to Enhance HTLV-1 Infection, Induce Helper T-Cell Polarization, and Decrease Cytotoxic T-Cell Response.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive demyelinating disease of the spinal cord due to chronic inflammation. Hallmarks of disease pathology include dysfunctional anti-viral responses and the infiltration of HTLV-1-infected CD4+ T cells and HTLV-1-specific CD8+ T cells in the central nervous system. HAM/TSP individuals exhibit CD4+ and CD8+ T cells with elevated co-expression of multiple inhibitory immune checkpoint proteins (ICPs), but ICP blockade strategies can only partially restore CD8+ T-cell effector function. Exosomes, small extracellular vesicles, can enhance the spread of viral infections and blunt anti-viral responses. Here, we evaluated the impact of exosomes isolated from HTLV-1-infected cells and HAM/TSP patient sera on dendritic cell (DC) and T-cell phenotypes and function. We observed that exosomes derived from HTLV-infected cell lines (OSP2) elicit proinflammatory cytokine responses in DCs, promote helper CD4+ T-cell polarization, and suppress CD8+ T-cell effector function. Furthermore, exosomes from individuals with HAM/TSP stimulate CD4+ T-cell polarization, marked by increased Th1 and regulatory T-cell differentiation. We conclude that exosomes in the setting of HAM/TSP are detrimental to DC and T-cell function and may contribute to the progression of pathology with HTLV-1 infection.

APOBEC3B expression in 293T viral producer cells drives mutations in chimeric antigen receptors and reduces CAR T cell efficacy

Chimeric antigen receptor (CAR) Tcells are a clinically approved therapy for blood cancers. To produce clinical-grade CAR Tcells, a retroviral or lentiviral vector is used to deliver the CAR and associated genes to patient Tcells. Apolipoprotein B editing enzyme, catalytic polypeptide 3 (APOBEC3) enzymes are known to be upregulated after transfection and retroviral infection and to deaminate cytidine to uracil in nucleic acids, resulting in cytidine-to-thymine mutations in DNA. Here, we hypothesized that APOBEC3 enzymes, induced during the production of CAR Tcells, impact the efficacy of the resulting CAR Tcells. We demonstrated that APOBEC3 family member APOBEC3B was upregulated at the RNA and protein levels after transfection of HEK293T cells with plasmids to make lentivirus, and that APOBEC3 signature mutations were present in the CAR construct. APOBEC3B overexpression in HEK293T cells led to further mutations in the resulting CAR Tcells, and significantly decreased CAR Tcell killing. APOBEC3B knockout in HEK293T cells led to reduced mutations in the CAR construct and significantly increased in CAR Tcell killing. These results suggest that generation of CAR-expressing viruses from producer cell lines deficient in genome-modifying proteins such as APOBEC3B could enhance the quality of CAR Tcell production.

Breast Cancer Cell Destruction Using Individually Encapsulated Cytotoxic T Lymphocytes in Polyelectrolyte Layer Coated with Dual Nanoparticles in Combination with Magnetic Exposure and Laser Irradiation

ABSTRACTCell encapsulation can protect cells from the host immune system. It is well‐known that cytotoxic T lymphocytes (CTLs) can destroy cancer cells. In this study, we individually encapsulated CTLs in a cationic polyelectrolyte in combination with two types of nanomaterials: magnetic nanoparticles (MNPs) and gold nanorods (GNRs) conjugated to anti‐human epidermal growth factor receptor‐2 (HER2) antibodies referred to as CTLs/mMNPs/PAH/GNRs@HER2. When MCF‐7 breast cancer cells were co‐cultured with CTLs/mMNPs/PAH/GNRs@HER2 and subjected to a magnetic field and laser irradiation, the viability of MCF‐7 cells was significantly decreased to 42.98 ± 0.57% in comparison to MCF‐7 cells without any treatment or MCF‐7 cells co‐cultured with CTLs (51.52 ± 0.57%). This can be a novel strategy for cancer treatment using encapsulated CTLs in polyelectrolytes and dual nanomaterials in combination with magnetic exposure and laser irradiation.

Distinct Variations in Gene Expression and Cell Composition across Lichen Planus Subtypes.

Lichen planus (LP) is a highly prevalent inflammatory skin disease. While various clinical subtypes have been defined, detailed comparisons of these variants are lacking. This study aimed to elucidate differences in gene expression and cellular composition across LP subtypes. Lesional skin biopsies from 28 LP patients (classical, oral, genital, and lichen planopilaris) and seven non-diseased skin controls (NDC) were analyzed. Gene expression profiling of 730 inflammation-related genes was conducted using NanoString. Immune cell compositions were assessed by multiplex immunohistochemistry. Gene expression profiles revealed unique inflammatory signatures for each LP subtype. Lichen planopilaris exhibited the most divergence, with downregulated gene expression and upregulation of complement pathway genes (C5-7), along with elevated M2 macrophages. Oral and genital LP demonstrated similar profiles with strong upregulation of TNF-related and Toll-like receptor-associated genes. Oral LP showed the highest upregulation of cytotoxicity-associated genes, as well as high numbers of CD8+ IL-17A+ (Tc17) cells (8.02%). Interferon gene signatures were strongly upregulated in oral and classical LP. The study highlights distinct differences in inflammatory gene expression and cell composition across LP subtypes, emphasizing the need for tailored therapeutic approaches.