Granzyme B Research Articles

MTORC1 Inhibition Protects Human Regulatory T Cells From Granzyme-B-Induced Apoptosis.

Regulatory T cells (Tregs) have shown great promise as a means of cellular therapy in a multitude of allo- and auto-immune diseases—due in part to their immunosuppressive potency. Nevertheless, the clinical efficacy of human Tregs in patients has been limited by their poor in vivo homeostasis. To avert apoptosis, Tregs require stable antigenic (CD3ζ/T-cell-receptor-mediated), co-stimulatory (CD28-driven), and cytokine (IL-2-dependent) signaling. Notably, this sequence of signals supports an activated Treg phenotype that includes a high expression of granzymes, particularly granzyme B (GrB). Previously, we have shown that aside from the functional effects of GrB in lysing target cells to modulate allo-immunity, GrB can leak out of the intracellular lysosomal granules of host Tregs, initiating pro-apoptotic pathways. Here, we assessed the role of inhibiting mechanistic target of rapamycin complex 1 (mTORC1), a recently favored drug target in the transplant field, in regulating human Treg apoptosis via GrB. Using ex vivo models of human Treg culture and a humanized mouse model of human skin allotransplantation, we found that by inhibiting mTORC1 using rapamycin, intracytoplasmic expression and functionality of GrB diminished in host Tregs; lowering human Treg apoptosis by in part decreasing the phosphorylation of S6K and c-Jun. These findings support the already clinically validated effects of mTORC1 inhibition in patients, most notably their stabilization of Treg bioactivity and in vivo homeostasis.

Nodal T-zone lymphoma and T-zone hyperplasia in dogs.

T-zone lymphoma (TZL) is an indolent, nodal lymphoma that has been clinically characterized in detail in dogs, and T-zone hyperplasia (TZH) is a hyperplastic change in lymph nodes associated with antigen processing. In some cases, histopathological features of TZL and TZH are similar, and are difficult to differentiate by morphology alone. Since there have been few publications characterizing their immunohistochemical profiles, histological, immunohistochemical, and clonality examinations were performed using formalin-fixed paraffin-embedded samples of canine lymph nodes with TZL (14 cases) and canine lymph nodes with TZH associated with nonlymphocytic tumors (10 cases). Immunohistochemically, small- to medium-sized lymphocytes of TZL were immunopositive for CD3, CD5, and HLA-DR, and negative for CD45, FOXP3, and granzyme B (GRB) in all cases. Among these 14 cases, 11 were immunopositive for CD8 and 1 was CD20 positive. Paracortical lymphocytes in TZH were diffusely immunopositive for CD3, CD5, and CD45, with scattered immunopositivity for CD8, HLA-DR, FOXP3, and GRB, and negative for CD20 in all cases. A clonal TCR gene rearrangement was detected in 13/14 TZL and none of the TZH cases. The present study revealed that TZL is a clonal proliferation of monomorphic CD8+CD45-GRB- T cells, while TZH consists of an immunophenotypically heterogenous population of CD45+ T cells that are variably positive for CD8 and FOXP3. These results suggest that canine TZL is a clonal proliferation of naïve or premature cytotoxic T cells. Regarding TZH, variable immunopositivity for cytotoxic and regulatory T-cell antigens may reflect immune responses to a variety of regional neoplastic lesions.

Expression of the GZMB Gene Polymorphism, SNP rs8192917, in 990 Han Chinese Patients with Postoperative Keloids.

BackgroundA keloid is a pathological scar hyperplasia that is affected by genetic and environmental factors. Although the involvement of cytotoxic granzyme B in keloids has been recognized, there is almost no research on granzyme B (GZMB) gene polymorphisms and keloids. This study aimed to explore the relationship between genetic polymorphisms of GZMB and postsurgical keloid susceptibility in the Han Chinese population.Material/MethodsA total of 3078 participants, including 990 patients with postsurgical keloids and 2088 controls without postsurgical keloids, were enrolled. We selected 15 common DNA variants in the GZMB gene for analysis. Associations were analyzed in both single marker-based and haplotype-based methods. The Genotype-Tissue Expression database was used to examine the biological significance of the targeted single nucleotide polymorphisms (SNPs).ResultsSNP rs8192917 was found to be associated with the susceptibility of keloids (t statistic=4.82, P=1.47×10−6). An increased risk of keloids was significantly associated with the minor allele (C allele) of rs8192917 (odds ratio=1.33; 95% CI=1.18–1.49], P=1.47×10−6). In addition, a significant association was reported for genotypes of rs8192917 and clinical severity of keloids (χ2=10.61, P=0.03).ConclusionsThe results suggested there are significant associations between common genetic variants in GZMB and the susceptibility of postsurgical keloids in the Chinese Han population. These genetic polymorphisms were also related with the severity of postsurgical keloid symptoms in participants with keloids. The current study can contribute to future etiological and clinical research of keloids.

CAR T Cells Targeting Membrane-Bound Hsp70 on Tumor Cells Mimic Hsp70-Primed NK Cells.

Strategies to boost anti-tumor immunity are urgently needed to treat therapy-resistant late-stage cancers, including colorectal cancers (CRCs). Cytokine stimulation and genetic modifications with chimeric antigen receptors (CAR) represent promising strategies to more specifically redirect anti-tumor activities of effector cells like natural killer (NK) and T cells. However, these approaches are critically dependent on tumor-specific antigens while circumventing the suppressive power of the solid tumor microenvironment and avoiding off-tumor toxicities. Previously, we have shown that the stress-inducible heat shock protein 70 (Hsp70) is frequently and specifically expressed on the cell surface of many different, highly aggressive tumors but not normal tissues. We could take advantage of tumors expressing Hsp70 on their membrane (‘mHsp70’) to attract and engage NK cells after in vitro stimulation with the 14-mer Hsp70 peptide TKDNNLLGRFELSG (TKD) plus low dose interleukin (IL)-2. However, a potential limitation of activated primary NK cells after adoptive transfer is their comparably short life span. T cells are typically long-lived but do not recognize mHsp70 on tumor cells, even after stimulation with TKD/IL-2. To combine the advantages of mHsp70-specificity with longevity, we constructed a CAR having specificity for mHsp70 and retrovirally transduced it into primary T cells. Co-culture of anti-Hsp70 CAR-transduced T cells with mHsp70-positive tumor cells stimulates their functional responsiveness. Herein, we demonstrated that human CRCs with a high mHsp70 expression similarly attract TKD/IL-2 stimulated NK cells and anti-Hsp70 CAR T cells, triggering the release of their lytic effector protein granzyme B (GrB) and the pro-inflammatory cytokine interferon (IFN)-γ, after 4 and 24 hours, respectively. In sum, stimulated NK cells and anti-Hsp70 CAR T cells demonstrated comparable anti-tumor effects, albeit with somewhat differing kinetics. These findings, together with the fact that mHsp70 is expressed on a large variety of different cancer entities, highlight the potential of TKD/IL-2 pre-stimulated NK, as well as anti-Hsp70 CAR T cells to provide a promising direction in the field of targeted, cell-based immunotherapies which can address significant unmet clinical needs in a wide range of cancer settings.

Ileum tissue single-cell mRNA sequencing elucidates the cellular architecture of pathophysiological changes associated with weaning in piglets

BackgroundIn mammals, transitioning from sole milk uptake to the intake of solid feed results in dramatic developmental changes in intestinal function and immunological status. In fact, weaning stress is often accompanied by intestinal inflammatory processes. To develop effective intervention strategies, it is necessary to characterize the developmental pattern and immune response that occurs on weaning, as we have done in this study for piglets.ResultsTo comprehensively delineate cell heterogeneity in ileum tissues and the underlying mechanisms in weaning-induced intestinal inflammation of piglets, we have analyzed the transcriptomes of 42,149 cells from ileum mucosa of normally suckling and post-weaned piglets. There were 31 cell subtypes including epithelial, stromal, and immune cells. A bifurcating trajectory was inferred to separate secretory and absorptive lineages. Integrated cross-species datasets showed well-conserved cellular architectures and transcription signatures between human and pig. Comparative analyses of cellular components, cell–cell communications, and molecular states revealed that T cell subpopulations were significantly altered in weaned piglets. We found that T helper (Th) 17 functional plasticity across changes in the cytokine milieu and the enrichment of granzyme B (GZMB)-expressing cytotoxic T cells potentially exacerbate mucosal inflammation via mitochondrial dysfunction in epithelial cells.ConclusionsOur work has elucidated the single-cell molecular characteristics of the piglet ileum before and after weaning. We have provided an atlas that portrays the landscape of the intestinal pathophysiological inflammatory process associated with weaning, finding a level of conservation between human and pig that support the use of piglets as a model for human infants.

Presence of Protease Inhibitor 9 and Granzyme B in Healthy and Pathological Human Corneas.

Simple SummaryDetailed knowledge of the structure and properties of the human cornea is a prerequisite not only for the treatment of various corneal diseases but also for successful corneal transplantation and its long-term survival after grafting. Using various cell and molecular biology approaches, we found in cornea the protease inhibitor 9. This protein, known to be present in other human tissues but not yet reported in cornea, is directly involved in the immune response after transplantation. Together with its inhibitor (granzyme B), we localized this protein, especially in the superficial and inner cornea layers. This localization indicates that protease inhibitor 9 protein may be involved in protecting the cornea from external damage, but also in protection against immune cells inducing corneal graft rejection. Furthermore, we have shown on pathological corneal samples from corneal melting and herpes virus keratitis that the increased expression of both proteins is linked to these diseases. These experiments and their results represent an important contribution to the basic research of cornea biological properties with direct overlap into clinical practice.The aim of this study was to find out whether protease inhibitor 9 (PI-9) and granzyme B (GrB) molecules that contribute to immune response and the immunological privilege of various tissues are expressed in healthy and pathological human corneas. Using cryosections, cell imprints of control corneoscleral discs, we showed that PI-9 was expressed particularly in the endothelium, the superficial and suprabasal epithelium of healthy corneas, limbus, and conjunctiva. GrB was localized in healthy corneal and conjunctival epithelium, while the endothelium showed weak immunostaining. The expression of PI-6 and GrB was confirmed by qRT-PCR. Increased expression levels of the PI-9 and GrB genes were determined when the corneas were cultured with proinflammatory cytokines. Fluorescent and enzymatic immunohistochemistry of pathological corneal explants (corneal melting and herpes virus keratitis) showed pronounced PI-9, GrB, human leucocyte antigen (HLA)-DR, and leukocyte-common antigen (CD45) signals localized in multicellular stromal infiltrates and inflammatory cells scattered in the corneal stroma. We conclude that increased expression of the PI-9 and GrB proteins under pathological conditions and their upregulation in an inflammatory environment indicate their participation in immune response of the cornea during the inflammatory process.

Glycogen synthase kinase (GSK-3) synergizes with PD-1/PDL1 blockade to generate super-armed CD8 killers against tumors

Abstract Immune checkpoint blockade (ICB) of negative co-receptors on T-cells such as programmed cell death-1 (PD-1) is promising for the treatment of cancer. Despite success, the poor prognosis for most patients highlights the need for novel clinical interventions. We have shown that the kinase, glycogen synthase kinase-3 (GSK-3) negatively regulates T-cell activation due to altered PD-1 and LAG-3 expression (Taylor et al., 2016 Immunity; Rudd et al., 2019 Cell Reports). GSK-3 inhibition (GSK-3i) is as effective as anti-PD-1 in controlling the growth of melanoma (Taylor et al., 2017 Can Res; Krueger and Rudd, Immunity 2017; Stelle et al., 2021 iScience). GSK-3i increases Tbet (Tbx21) transcription, which inhibits PD-1/LAG-3 transcription, while increasing granzyme B (GZMB) and interferon gamma (IFNγ). Here, we show that Gsk3−/− mice and small molecule inhibitors (SMIs) synergize with anti-PD-1 to eliminate melanomas that are resistant to anti-PD-1 monotherapy. Transcriptomic profiling showed that GSK-3 × PD-1 cooperativity was characterized by a specific increase in a family of different granzymes (7/12 GZM genes out of a data base of 20,500 potential genes). Some GZMs have been characterized and others not, but as a family, this increased armory of GZMs in CD8+ T-cells is expected to greatly enhance tumor killing. Overall, our data shows the PD-1 × GSK-3 synergy in limiting tumor growth is due to a specific set of cytolysis mediators needed for tumor killing.

Cytotoxic T Lymphocytes Expressing GPR56 are Up-regulated in the Peripheral Blood of Patients with Active Rheumatoid Arthritis and Reflect Disease Progression

ABSTRACT Objective This study aims to elucidate the changes in the percentage of GPR56 and/or granzyme B (GZMB) positive cells in rheumatoid arthritis (RA) CD4 and CD8 T lymphocytes, and to explore their clinical value in diagnosing and reflecting the progression of RA. Methods The percentages of GPR56 and/or GZMB positive cells were analyzed in peripheral blood (PB) and spleen T cells in a collagen-induced arthritis (CIA) model established in DBA/1 mice. The percentages of GPR56+ and/or GZMB+ cells were further analyzed in PBs from RA patients and healthy controls. Correlation analysis was performed between clinical indicators and GPR56+, GZMB+, and GPR56+ GZMB+ T cells. Receiver operating characteristic (ROC) curves were used to evaluate the value of GPR56 and GZMB in differentiating active and stable remitting RA. Results GPR56+ levels were increased in CD4 and CD8 T cells in the PB of CIA mice. The percentages of GPR56+ and GZMB+ cells were increased in both CD4 and CD8 T cell subsets in patients with active RA. GPR56+, GZMB+, and GPR56+ GZMB+ cells were positively correlated with rheumatoid factor and DAS28. ROC analysis revealed that AUCs for GPR56+, GZMB+, and GPR56+ GZMB+ cell percentages to distinguish active RA from stable remission RA were 0.7106, 0.6941, 0.7024, with cut-off values of 16.35, 16.40, 14.80 in CD4 + T cells, and 0.8031, 0.8086, 0.8196 with cut-off values 60.25, 62.15, 40.15 in CD8 + T cells, respectively. Conclusions GPR56+ and/or GZMB+ T cells are up-regulated in patients with active RA and reflect their condition. The detection of GPR56 and GZMB is helpful for RA disease assessment.

Identification of Key Genes and Pathways in Peripheral Blood Mononuclear Cells of Type 1 Diabetes Mellitus by Integrated Bioinformatics Analysis.

Background The onset and progression of type 1 diabetes mellitus (T1DM) is closely related to autoimmunity. Effective monitoring of the immune system and developing targeted therapies are frontier fields in T1DM treatment. Currently, the most available tissue that reflects the immune system is peripheral blood mononuclear cells (PBMCs). Thus, the aim of this study was to identify key PBMC biomarkers of T1DM.Methods Common differentially expressed genes (DEGs) were screened from the Gene Expression Omnibus (GEO) datasets GSE9006, GSE72377, and GSE55098, and PBMC mRNA expression in T1DM patients was compared with that in healthy participants by GEO2R. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein-protein interaction (PPI) network analyses of DEGs were performed using the Cytoscape, DAVID, and STRING databases. The vital hub genes were validated by reverse transcription-polymerase chain reaction using clinical samples. The disease-gene-drug interaction network was built using the Comparative Toxicogenomics Database (CTD) and Drug Gene Interaction Database (DGIdb).Results We found that various biological functions or pathways related to the immune system and glucose metabolism changed in PBMCs from T1DM patients. In the PPI network, the DEGs of module 1 were significantly enriched in processes including inflammatory and immune responses and in pathways of proteoglycans in cancer. Moreover, we focused on four vital hub genes, namely, chitinase-3-like protein 1 (CHI3L1), C-X-C motif chemokine ligand 1 (CXCL1), matrix metallopeptidase 9 (MMP9), and granzyme B (GZMB), and confirmed them in clinical PBMC samples. Furthermore, the disease-gene-drug interaction network revealed the potential of key genes as reference markers in T1DM.Conclusion These results provide new insight into T1DM pathogenesis and novel biomarkers that could be widely representative reference indicators or potential therapeutic targets for clinical applications.

Rasmussen\u2019s encephalitis is characterized by relatively lower production of IFN-\u03b2 and activated cytotoxic T cell upon herpes viruses infection

BackgroundThe etiology of Rasmussen's encephalitis (RE), a rare chronic neurological disorder characterized by CD8+ T cell infiltration and unihemispheric brain atrophy, is still unknown. Various human herpes viruses (HHVs) have been detected in RE brain, but their contribution to RE pathogenesis is unclear.MethodsHHVs infection and relevant immune response were compared among brain tissues from RE, temporal lobe epilepsy (TLE) and traumatic brain injury (TBI) patients. Viral antigen or genome, CD8+ T cells, microglia and innate immunity molecules were analyzed by immunohistochemical staining, DNA dot blot assay or immunofluorescence double staining. Cytokines were measured by multiplex flow cytometry. Cell apoptosis was visualized by TUNEL staining. Viral infection, immune response and the severity of unihemispheric atrophy were subjected to correlation analysis.ResultsAntigens of various HHVs were prevalent in RE and TLE brains, and the cumulative viral score of HHVs positively correlated with the unihemispheric atrophy in RE patients. CD8+ T cells infiltration were observed in both RE and TLE brains and showed co-localization with HHV antigens, but their activation, as revealed by Granzyme B (GZMB) release and apoptosis, was found only in RE. In comparison to TLE, RE brain tissues contained higher level of inflammatory cytokines, but the interferon-β level, which was negatively correlated with cumulative viral score, was relatively lower. In line with this, the DNA sensor STING and IFI16, rather than other innate immunity signaling molecules, were insufficiently activated in RE.ConclusionsCompared with TBI, both RE and TLE had prevalently HHV infection and immune response in brain tissues. However, in comparison to TLE, RE showed insufficient activation of antiviral innate immunity but overactivation of cytotoxic T cells. Our results show the relatively lower level of antiviral innate immunity and overactivation of cytotoxic T cells in RE cases upon HHV infection, the overactivated T cells might be a compensate to the innate immunity but the causative evidence is lack in our study and need more investigation in the future.

Granzyme B+ CD4 T cells accumulate in the colon during chronic HIV-1 infection

ABSTRACT Chronic HIV-1 infection results in the sustained disruption of gut homeostasis culminating in alterations in microbial communities (dysbiosis) and increased microbial translocation. Major questions remain on how interactions between translocating microbes and gut immune cells impact HIV-1-associated gut pathogenesis. We previously reported that in vitro exposure of human gut cells to enteric commensal bacteria upregulated the serine protease and cytotoxic marker Granzyme B (GZB) in CD4 T cells, and GZB expression was further increased in HIV-1-infected CD4 T cells. To determine if these in vitro findings extend in vivo, we evaluated the frequencies of GZB+ CD4 T cells in colon biopsies and peripheral blood of untreated, chronically infected people with HIV-1 (PWH). Colon and blood GZB+ CD4 T cells were found at significantly higher frequencies in PWH. Colon, but not blood, GZB+ CD4 T cell frequencies were associated with gut and systemic T cell activation and Prevotella species abundance. In vitro, commensal bacteria upregulated GZB more readily in gut versus blood or tonsil-derived CD4 T cells, particularly in inflammatory T helper 17 cells. Bacteria-induced GZB expression in gut CD4 T cells required the presence of accessory cells, the IL-2 pathway and in part, MHC Class II. Overall, we demonstrate that GZB+ CD4 T cells are prevalent in the colon during chronic HIV-1 infection and may emerge following interactions with translocated bacteria in an IL-2 and MHC Class II-dependent manner. Associations between GZB+ CD4 T cells, dysbiosis and T cell activation suggest that GZB+ CD4 T cells may contribute to gut HIV-1 pathogenesis.

Targeting CD96 overcomes PD-1 blockade resistance by enhancing CD8+ TIL function in cervical cancer

BackgroundNovel therapies are needed to treat recurrent and advanced cervical cancer (CC), as their prognosis remains very poor. Although therapies targeting the programmed cell death protein 1 (PD-1) pathway have...

Inhibitor of glutamine metabolism V9302 promotes ROS-induced autophagic degradation of B7H3 to enhance antitumor immunity

Despite the enormous successes of anti-PD-1/PD-L1 immunotherapy in multiple other cancer types, the overall response rates of breast cancer remain suboptimal. Therefore, exploring additional immune checkpoint molecules for potential cancer treatment is crucial. B7H3, a T-cell coinhibitory molecule, is specifically overexpressed in breast cancer compared with normal breast tissue and benign lesions, making it an attractive therapeutic target. However, the mechanism by which B7H3 contributes to the cancer phenotype is unclear. Here we show that the expression of B7H3 is negatively related to the number of CD8+ T cells in breast tumor sites. In addition, analysis of the differentially expressed B7H3 reveals that it is inversely correlated to autophagic flux both in breast cancer cell lines and clinical tumor tissues. Furthermore, block of autophagy by bafilomycin A1 (Baf A1) increases B7H3 levels and attenuates CD8+ T cell activation, while promotion of autophagy by V9302, a small-molecule inhibitor of glutamine metabolism, decreases B7H3 expression and enhances granzyme B (GzB) production of CD8+ T cells via regulation of reactive oxygen species (ROS) accumulation. We demonstrate that combined treatment with V9302 and anti-PD-1 monoclonal antibody (mAb) enhances antitumor immunity in syngeneic mouse models. Collectively, our findings unveil the beneficial effect of V9302 in boosting antitumor immune response in breast cancer and illustrate that anti-PD-1 together with V9302 treatment may provide synergistic effects in the treatment of patients insensitive to anti-PD-1 therapy.

Mesenchymal stromal cells equipped by IFN\u03b1 empower T cells with potent anti-tumor immunity

Cancer treatments have been revolutionized by the emergence of immune checkpoint blockade therapies. However, only a minority of patients with various tumor types have benefited from such treatments. New strategies focusing on the immune contexture of the tumor tissue microenvironment hold great promises. Here, we created IFNα-overexpressing mesenchymal stromal cells (IFNα-MSCs). Upon direct injection into tumors, we found that these cells are powerful in eliminating several types of tumors. Interestingly, the intra-tumoral injection of IFNα-MSCs could also induce specific anti-tumor effects on distant tumors. These IFNα-MSCs promoted tumor cells to produce CXCL10, which in turn potentiates the infiltration of CD8+ T cells in the tumor site. Furthermore, IFNα-MSCs enhanced the expression of granzyme B (GZMB) in CD8+ T cells and invigorated their cytotoxicity in a Stat3-dependent manner. Genetic ablation of Stat3 in CD8+ T cells impaired the effect of IFNα-MSCs on GZMB expression. Importantly, the combination of IFNα-MSCs and PD-L1 blockade induced an even stronger anti-tumor immunity. Therefore, IFNα-MSCs represent a novel tumor immunotherapy strategy, especially when combined with PD-L1 blockade.

Chloroquine Suppresses Effector B-Cell Functions and Has Differential Impact on Regulatory B-Cell Subsets.

ObjectivesChloroquine (CQ) is approved for treatment of B-cell mediated diseases such as rheumatoid arthritis and systemic lupus erythematosus. However, the exact mode of action in these diseases has not been studied and it remains unclear which effect CQ has on B-cells. Thus, it was the aim of this study to investigate to which extent CQ affects functionality of effector and regulatory B-cell.MethodsFor this purpose, B-cells were isolated from peripheral blood of healthy controls and renal transplant patients. B-cells were stimulated in presence or absence of CQ and Interleukin-10 (IL-10) and Granzyme B (GrB) secretion were assessed. In addition, effector functions such as plasma cell formation, and Immunoglobulin G (IgG) secretion were studied.ResultsCQ suppressed Toll-Like-Receptor (TLR)-9 induced B-cell proliferation in a dose-dependent manner. IL-10pos regulatory B-cells were suppressed by CQ already at low concentrations whereas anti-IgG/IgM-induced GrB secreting regulatory B-cells were less susceptible. Plasma blast formation and IgG secretion was potently suppressed by CQ. Moreover, purified B-cells from renal transplant patients were also susceptible to CQ-induced suppression of effector B-cell functions as observed by diminished IgG secretion.ConclusionIn conclusion, CQ had a suppressive effect on IL-10 regulatory B-cells whereas GrB secreting regulatory B-cells were less affected. Effector functions of B-cells such as plasma blast formation and IgG secretion were also inhibited by CQ. Effector B-cells derived from renal transplant patients already under immunosuppression could be suppressed by CQ. These findings may partly explain the clinical efficacy of CQ in B-cell mediated autoimmune diseases. The application of CQ in other disease contexts where suppression of effector B-cells could offer a benefit, such as renal transplantation, may hypothetically be advantageous.

The immune impact of PI3K-AKT pathway inhibition in colorectal cancer.

154 Background: Our prior work has shown that PI3K-altered colorectal cancer (CRC), with PIK3CA mutation or PTEN loss, has increased expression of key immune checkpoints (including PD-L1) resulting in immune evasion, despite increased immune engagement. Here, we investigated the impact of PI3K-AKT inhibition on the immune repertoire of CRC. Methods: Multiplex immunofluorescence was performed using two Vectra panels [1: AE1/AE3, CD3, CD8, PD-1, PD-L1, CD68; and 2: AE1/AE3, CD3, CD8, Granzyme B (GzB), CD45RO, FoxP3] on paired biopsies (baseline and cycle 1 day 15) from 6 patients with PI3K-altered metastatic CRC (mCRC) treated with AKT inhibitor, MK2206 (200 mg oral weekly), on a phase 2 clinical trial. Separately, one million CT26 CRC cells were implanted in BALB/C-e mice. After 48 hours, 10 mice/group were randomized for treatment with pan-PI3K inhibitor copanlisib (C, 10 mg/Kg IV 2x/week), anti-PD-1 (P, 200 µg IP 2x/week), copanlisib + anti-PD-1 (C+P), or control (Ct), for 21 days. Mouse tumors were stained with 6-plex immunohistochemistry (CD3, CD8, PD-L1, Ki67, GzB, AE1/AE3). Data were analyzed using related-samples Wilcoxon Signed-Rank test, Mann-Whitney U test, Kruskal-Wallis test, and Student’s t-test, as appropriate. Results: In PI3K-altered mCRC patients, AKT inhibition resulted in a trend towards increased median densities of intratumoral CD8+ T cells (0.8 vs 4.8 density/mm2, P = 0.14) and memory T cells (0 vs 10.3, P = 0.07), and decreased density of macrophages (12.4 vs 0, P = 0.07). No antigen experienced T cells were seen and activated CD8+ T cells were present in 1 patient only. In CT26 mice, PI3K and PD-1 co-inhibition resulted in the smallest mean tumor volumes (C+P 12% of Ct vs C 40% and P 42% of Ct, P < 0.05 for both), and the highest median % of intratumoral CD8+Ki67+ T cells as compared to all other treatment arms (C+P 1.6% vs C 0.5%, P 0.4%, Ct 0.6%, P < 0.05 for each pairwise comparison). C+P also increased the % of total CD3+ and CD8+ cells as compared to Ct and C (P < 0.05 for all). C alone did not increase immune infiltration in this non-PI3K activated model. Conclusions: PI3K-AKT pathway inhibition has the potential to improve effector T cell infiltration in PI3K-altered CRC. PI3K inhibitor synergizes with anti-PD-1 to improve treatment efficacy and CD8+ T cell proliferation. The mechanisms behind this immune repertoire shift are yet to be elucidated, such as via cytokine modulation. Therapeutic approaches to activate the proliferating CD8+ cells would be useful, and may require PI3Kα/β specific inhibitors to allow early T cell activation through PI3Kδ/γ isoforms.

Cytofluorimetric assay to investigate variability in blinatumomab in vitro response.

The T-cell engager antibody blinatumomab (BlincytoT⁢M) represents a promising rescue therapy for relapsed/refractory CD19+ acute lymphoblastic leukemia (B-ALL), although ~20-30% of patients still do not respond to treatment. Blinatumomab creates a tight synapsis between CD3+ T-lymphocytes and leukemic CD19+ B-cells, resulting in a granzyme B (GzB)-mediated specific lysis of leukemic cells. Aim of the study was to provide evidence that variability in blinatumomab response could have a genetic basis in PAX5, one of the most often mutated genes in B-ALL, affecting the CD19 surface expression on lymphoblasts, and could be explored in vitro by means of a cytofluorimetric assay, staining both surface antigens (CD45, CD19 and CD3) and intracytoplasmic markers (7AAD, Syto16). Two human immortalized B-ALL cell lines (NALM6 and REH) were chosen for their different PAX5 and CD19 protein levels, as verified by western blot and flow cytometry, respectively. In contrast to NALM6, REH cells do not express the full-length PAX5 protein and show less CD19 on the cell surface (fluorescence peak median intensity: 9155 versus 28895). Co-cultures of CD3+ T-lymphocytes from healthy donors and B-ALL cell lines were seeded at an effector-to-target cell ratio of 1:10 for simulating the condition existing in the bone marrow due to the malignant invasion of blast cells. Co-cultures were exposed in vitro to blinatumomab and the simultaneous increase in blast mortality and T-lymphocytes activation induced by the drug was observed at day +7 (both effects: p < 0.0001 versus untreated, two-way ANOVA, Bonferroni post-test), and was particularly pronounced in REH compared to NALM6 co-cultures (p < 0.05). Surprisingly, daily release of GzB in supernatants, measured by an ELISA assay, was significantly lower in drug-exposed REH co-cultures compared to NALM6 at early time-points (days +3 and +4, p-value < 0.0001, three-way ANOVA), reaching a comparable plateau only towards the end of the incubation period (at day +5). Only 2 out of 5 primary co-cultures of leukemic and mononuclear cells isolated from bone marrow aspirates of B-ALL patients (age: median 10.7 years, interquartile range (IQR) 3.4; males: 60%) responded to the drug in vitro (simultaneous blast mortality and T-lymphocyte activation: both effects: p < 0.0001 versus untreated) and at different drug concentrations. Results were unrelated to the percentages of immature CD19+ B-cells in the diagnostic samples. In conclusion, cytofluorimetric analysis can highlight the different response induced by blinatumomab among co-cultures. Whether and how this difference is affected by PAX5-regulated CD19 expression is unclear and whether it is predictive of in vivo response to therapy remains to be established. Further dedicated studies are required to investigate these issues in detail.

P009 Integrin-b7+ cytotoxic MAIT cells are expanded in the colon of Ulcerative Colitis patients, correlate with disease severity and are targeted by vedolizumab therapy

Abstract Background Mucosal-associated invariant T (MAIT) cells are major histocompatibility complex (MHC) class Ib-restricted innate-like lymphocytes with anti-bacterial functions that are enriched at mucosal sites. Their role in ulcerative colitis (UC) is unknown. Methods Colonic biopsies and peripheral blood were collected from patients with active UC (n=34) and healthy control subjects (n=14). Peripheral blood mononuclear cells (PBMC) and Lamina propria mononuclear cells (LPMC) were isolated and activated in vitro with PMA/ionomycin followed by surface and intracellular staining and multiparametric flow cytometry analysis. T cell populations, including gamma delta T cells (TCR gamma delta+), iNKT cells (TCR Vα24-Jα18+), MAIT cells (TCR Vα7.2+ CD161+), CD4, CD8 T cells and their intracellular expression of IL17A, TNF, IFN-g, IL22, IL13 and granzyme B (GZMB) were determined. MAIT cells were further characterised as CD8+, CD4+, CD8 CD4 double positive (DP), CD8 CD4 double negative (DN) cells and expression of the gut homing integrin-b7. Results DN cells were the most common MAIT subset in the colon and were significantly increased in both UC patients and HC subjects compared to peripheral blood. In UC, DN MAIT cells exhibited a cytotoxic phenotype, with significantly higher production of Granzyme B (median 45.0% of cells) compared to DN MAIT cells in HC (median 17%, P&amp;lt;0.005). DN MAIT cells in UC gut also co-produced significantly higher proportion of IL17A (median 22% in UC vs 7% in HC, P&amp;lt;0.006) and TNF (median 61% in UC vs 10% in HC, P&amp;lt;0.02) compared to DN MAIT cells from HC. In UC patients, the proportional abundance of cytotoxic DN MAIT cells accumulating in the colon significantly correlated with disease activity (UCEIS) score (r2=0.34, P&amp;lt;0.0007). The number of DN MAIT cells expressing integrin-b7 was also significantly higher in UC patients compared to HC (median 66% in UC vs 32% in HC, p&amp;lt;0.001). In UC patients initiated on vedolizumab therapy, the proportional abundance of cytotoxic, DN MAIT cells accumulating in the colon significantly fell at week 14 in comparison with their pre-treatment abundance (median 25% vs 51%, P&amp;lt;0.001). Following vedolizumab induction, the reduction in the abundance of colonic cytotoxic DN MAIT cells correlated with endoscopic improvement (r2=0.45, p=0.0085). Conclusion DN MAIT cells with cytolytic activity and augmented expression of multiple cytokines are enriched in the colon of UC patients in numbers that correlate with the magnitude of mucosal injury. Vedolizumab treatment reduces DN MAIT cell accumulation in the colon and the magnitude of reduction correlates with treatment response.

Reciprocal alterations in circulating and hepatic gamma-delta T cells in patients with primary biliary cholangitis.

Gamma-delta (γδ) T cells are involved in the development of diverse liver and autoimmune diseases, whereas the role of γδ T cells in primary biliary cholangitis (PBC) remains unclear. We analyzed the number, phenotypes, and functional molecules of both circulating and hepatic γδT cells in PBC patients and healthy controls (HCs) by flow cytometric analysis and immunohistochemistry. We identified two distinct functional subsets of circulating γδT cells according to the CD3/TCRγδ complex: the TCRγδhigh and TCRγδlow subsets. Approximately, three-quarters of cells in the TCRγδhigh subset were Vδ1 T cells, while Vδ2 T cells were enriched in the TCRγδlow subset in HCs. The frequency and absolute number of circulating TCRγδlow cells were significantly decreased in PBC patients compared with HCs (p < 0.001). Furthermore, the frequency of TCRγδlow cells was correlated with disease severity and ursodeoxycholic acid (UDCA) response. TCRγδlow cells exhibited a similar apoptotic and proliferative phenotype, but enhanced liver-homing chemokine receptor (CXCR6) expression in PBC patients compared with HCs. In addition, circulating TCRγδlow cells were more activated and produced higher granzyme B (GZMB) in PBC patients compared with HCs. Finally, compared with heathy liver controls, hepatic γδT cells were increased and infiltrated in the inflamed portal tracts in PBC liver. Furthermore, the number of hepatic γδ T cells was correlated with cholestatic markers and UDCA response. The circulating TCRγδlow subset may migrate to the liver via the CXCR6-CXCL16 axis and be involved in the pathogenesis of PBC by increasing GZMB production.

The Critical and Diverse Roles of CD4-CD8- Double Negative T Cells in Nonalcoholic Fatty Liver Disease.

Background & AimsHepatic inflammation is a hallmark of nonalcoholic fatty liver disease (NAFLD). Double negative T (DNT) cells are a unique subset of T lymphocytes that do not express CD4, CD8, or natural killer cell markers, and studies have suggested that DNT cells play critical and diverse roles in the immune system. However, the role of intrahepatic DNT cells in NAFLD is largely unknown.MethodsThe proportions and RNA transcription profiling of intrahepatic DNT cells were compared between C57BL/6 mice fed with control diet or methionine-choline–deficient diet for 5 weeks. The functions of DNT cells were tested in vitro and in vivo.ResultsThe proportion of intrahepatic DNT cells was significantly increased in mice with diet-induced NAFLD. In NAFLD mice, the proportion of intrahepatic TCRγδ+ DNT cells was increased along with elevated interleukin (IL) 17A; in contrast, the percentage of TCRαβ+ DNT cells was decreased, accompanied by reduced granzyme B (GZMB). TCRγδ+ DNT cell depletion resulted in lowered liver IL17A levels and significantly alleviated NAFLD. Adoptive transfer of intrahepatic TCRαβ+ DNT cells from control mice increased intrahepatic CD4 and CD8 T cell apoptosis and inhibited NAFLD progression. Furthermore, we revealed that adrenic acid and arachidonic acid, harmful fatty acids that were enriched in the liver of the mice with NAFLD, could induce apoptosis of TCRαβ+ DNT cells and inhibit their immunosuppressive function and nuclear factor kappa B (NF-κB) or AKT signaling pathway activity. However, arachidonic acid facilitated IL17A secretion by TCRγδ+ DNT cells, and the NF-κB signaling pathway was involved. Finally, we also confirmed the variation of intrahepatic TCRαβ+ DNT cells and TCRγδ+ DNT cells in humans.ConclusionsDuring NAFLD progression, TCRγδ+ DNT cells enhance IL17A secretion and aggravate liver inflammation, whereas TCRαβ+ DNT cells decrease GZMB production and lead to weakened immunoregulatory function. Shifting of balance from TCRγδ+ DNT cell response to one that favors TCRαβ+ DNT regulation would be beneficial for the prevention and treatment of NAFLD.