Expression Of Granzyme Research Articles

Post-acute sequelae of COVID-19 is characterized by diminished peripheral CD8+β7 integrin+ T cells and anti-SARS-CoV-2 IgA response

Several millions of individuals are estimated to develop post-acute sequelae SARS-CoV-2 condition (PASC) that persists for months after infection. Here we evaluate the immune response in convalescent individuals with PASC compared to convalescent asymptomatic and uninfected participants, six months following their COVID-19 diagnosis. Both convalescent asymptomatic and PASC cases are characterised by higher CD8+ T cell percentages, however, the proportion of blood CD8+ T cells expressing the mucosal homing receptor β7 is low in PASC patients. CD8 T cells show increased expression of PD-1, perforin and granzyme B in PASC, and the plasma levels of type I and type III (mucosal) interferons are elevated. The humoral response is characterized by higher levels of IgA against the N and S viral proteins, particularly in those individuals who had severe acute disease. Our results also show that consistently elevated levels of IL-6, IL-8/CXCL8 and IP-10/CXCL10 during acute disease increase the risk to develop PASC. In summary, our study indicates that PASC is defined by persisting immunological dysfunction as late as six months following SARS-CoV-2 infection, including alterations in mucosal immune parameters, redistribution of mucosal CD8+β7Integrin+ T cells and IgA, indicative of potential viral persistence and mucosal involvement in the etiopathology of PASC.

Differential regulatory T cell signature after recovery from mild COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a range of symptoms in which host immune response have been associated with disease progression. However, the putative role of regulatory T cells (Tregs) in determining COVID-19 outcomes has not been thoroughly investigated. Here, we compared peripheral Tregs between volunteers not previously infected with SARS-CoV-2 (healthy control [HC]) and volunteers who recovered from mild (Mild Recovered) and severe (Severe Recovered) COVID-19. Peripheral blood mononuclear cells (PBMC) were stimulated with SARS-CoV-2 synthetic peptides (Pool Spike CoV-2 and Pool CoV-2) or staphylococcal enterotoxin B (SEB). Results of a multicolor flow cytometric assay showed higher Treg frequency and expression of IL-10, IL-17, perforin, granzyme B, PD-1, and CD39/CD73 co-expression in Treg among the PBMC from the Mild Recovered group than in the Severe Recovered or HC groups for certain SARS-CoV-2 related stimulus. Moreover, Mild Recovered unstimulated samples presented a higher Tregs frequency and expression of IL-10 and granzyme B than did that of HC. Compared with Pool CoV-2 stimuli, Pool Spike CoV-2 reduced IL-10 expression and improved PD-1 expression in Tregs from volunteers in the Mild Recovered group. Interestingly, Pool Spike CoV-2 elicited a decrease in Treg IL-17+ frequency in the Severe Recovered group. In HC, the expression of latency-associated peptide (LAP) and cytotoxic granule co-expression by Tregs was higher in Pool CoV-2 stimulated samples. While Pool Spike CoV-2 stimulation reduced the frequency of IL-10+ and CTLA-4+ Tregs in PBMC from volunteers in the Mild Recovered group who had not experienced certain symptoms, higher levels of perforin and perforin+granzyme B+ co-expression by Tregs were found in the Mild Recovered group in volunteers who had experienced dyspnea. Finally, we found differential expression of CD39 and CD73 among volunteers in the Mild Recovered group between those who had and had not experienced musculoskeletal pain. Collectively, our study suggests that changes in the immunosuppressive repertoire of Tregs can influence the development of a distinct COVID-19 clinical profile, revealing that a possible modulation of Tregs exists among volunteers of the Mild Recovered group between those who did and did not develop certain symptoms, leading to mild disease.

A162 RARE PRESENTATION OF EXTRANODAL NK/T-CELL LYMPHOMA INVOLVING STOMACH AND EYE: CASE REPORT AND LITERATURE REVIEW

Abstract Background Extranodal NK/T-cell lymphoma (ENKTL) is a rare and aggressive form of non-Hodgkin lymphoma. ENKTL are predominantly localized to nasal and upper aerodigestive sites, but extranasal involvement including gastrointestinal tract are rarely seen. Small and large intestines are primary sites of gastrointestinal ENKTL. Gastric involvements are exceedingly rare accounting for less than 5% of all gastrointestinal ENKTL. Purpose We present a literature review on gastrointestinal ENKTL and a case report of gastrointestinal bleed secondary to ENKTL involving stomach and left orbit. Method Case report and literature review. Result(s) 33-year-old female was admitted to a tertiary hospital with 3-week history of epigastric pain and left periorbital swelling. Abdominal CT showed edema and thickening of gastric folds. Head CT showed grossly enlarged left lateral rectus muscle and periorbital soft tissue swelling suggestive of left orbital pseudotumor. Esophagogastroduodenoscopy revealed multifocal Forrest classification II-C ulcerations throughout her stomach and duodenum. Biopsies from stomach showed gastric mucosa with extensive infiltration by an atypical lymphoid cell population. Immunohistochemistry demonstrated high grade lymphoid cells with uniform expression of CD2, CD3, CD30, CD56, TIA1, perforin and granzyme B. EBV-encoded small RNA in-situ hybridization (EBER ISH) was strongly positive. Findings were consistent with gastric ENKTL. Left orbital biopsy revealed similar morphology and phenotypic features consistent with concurrent ENKTL involvement of her orbit. Patient was initiated on intravenous corticosteroids, but unfortunately developed hemorrhagic shock secondary to gastrointestinal bleeding from gastric ulcer and passed away. Gastrointestinal ENKTL is a rare presentation of a rare disease. Gastric involvement is especially rare and described only in few case reports. Gastrointestinal ENKTL are often initially asymptomatic but can progress to abdominal pain, bleeding and even bowel perforation. Due its nonspecific clinical features and rarity, diagnosis can be difficult and requires careful examination by an experienced pathologist. This aggressive lymphoma is characterized by positive CD2, CD3, CD30, CD56, TIA, granzyme B, perforin and EBER ISH. Optimal treatment approach remains unclear due to lack of prospective clinical studies. Currently, treatment modalities used for other lymphomas including radiotherapy and non-anthracycline-based chemotherapy are used. Despite treatment, prognosis is grim with median overall survival period of 7-8 months. Image Conclusion(s) We present a case of gastric and orbital ENKTL with gastric ulcer bleeding. Gastric ENKTL disease is a rare presentation of a rare disease. Due to non-specific clinical features, diagnosis is often difficult and relies on careful pathology examination by experienced pathologist. Prognosis is poor without optimal treatment approach due to rarity of disease and lack of validated data. Please acknowledge all funding agencies by checking the applicable boxes below None Disclosure of Interest None Declared INFLAMMATORY BOWEL DISEASES MECHANISMS AND TREATMENTS

Experimental Study on the Characteristic Changes of the Immunological Microenvironment at the Maternal-Fetal Interface in IVF-ET Pregnancy

To investigate the characteristic functional changes of the decidual natural killer (NK) cells and γδ T cells, two immunocytes in the decidua, at the maternal-fetal interface in in vitro fertilization-embryo transfer (IVF-ET) pregnancy. Decidual samples were collected from 12 women of natural pregnancy (NP) and 32 women of IVF-ET pregnancy, who were enrolled in the NP group and the IVF-ET group, respectively. Then part of the decidual samples were paraffin-embedded for HE staining and immunofluorescence staining, while the rest of the samples were digested and Percoll was used for isolating decidual immunocytes (DICs) by gradient centrifugation. Flow cytometry was used to determine the cell counts of decidual NK cells and γδ T cells and the expression levels of their surface activation markers, CD69 and NKG2D in the NP and the IVF-ET groups. In addition, the expression levels of IFN-γ, TNF-α, IL-17A, and IL-10, the intracellular cytokines, and granzyme B, perforin, and granulysin, the cytolytic granules, were measured. The characteristic changes in the relevant immunological indicators were compared and analyzed. HE staining of the tissue specimens showed that the typical structure of decidua was observed, and that lymphocytes were enriched in the decidua. Immunofluorescence staining showed that the percentage of decidual NK (dNK) cells in nucleated cells of the IVF-ET group was significantly lower than that of the NP group ( P<0.05). Flow cytometry analysis of DICs showed that, compared with those of the NP group, the percentage of dNK cells of the IVF-ET group was decreased ( P<0.05) and the expression levels of IL-10 and perforin were significantly decreased in the IVF-ET group ( P<0.05). However, there was no significant difference in the decidual γδ T (dγδT) cell count between the two groups. The expression of IL-10, IL-17A, and perforin was downregulated in the IVF-ET group ( P<0.05). There was no significant difference in the expression of IFN-γ, TNF-α, granzyme B, and granulysin, the cellular function indicators ( P>0.05). The dNK cell count and the secretion of some intracellular cytokines of dNK and dγδT cells of women of IVF-ET pregnancy decreased to some degree, which suggests that certain changes may have taken place in the immunological microenvironment at the maternal-fetal interface. The specific effect of these changes on pregnancy outcomes needs further investigation.

Ascites CD100 levels and immunomodulation effects in the peripheral blood of patients with liver cirrhosis combined with spontaneous bacterial peritonitis

Objective: To observe the level and detection of ascites CD100 on the activity of CD4(+) and CD8(+) T lymphocytes in vitro in the peripheral blood of patients with liver cirrhosis combined with spontaneous bacterial peritonitis. Methods: Peripheral blood and ascites were collected from 77 cases of liver cirrhosis (49 patients with liver cirrhosis combined with simple ascites and 28 patients with liver cirrhosis combined with SBP), and peripheral blood was collected from 22 controls. Soluble CD100 (sCD100) in peripheral blood and ascites was detected by an enzyme-linked immunosorbent assay. Flow cytometry was used to detect membrane-bound CD100 (mCD100) on the surface of CD4(+) and CD8(+)T lymphocytes. CD4(+) and CD8(+)T lymphocytes in ascites were sorted. CD4(+)T lymphocyte proliferation, key transcription factor mRNA, and secreted cytokine changes, as well as CD8(+)T lymphocyte proliferation, important toxic molecule mRNA, and secreted cytokine changes, were detected after CD100 stimulation. The killing activity of CD8(+)T cells was detected by direct contact and indirect contact culture systems. Data conforming to normality were compared using one-way ANOVA, a student's t-test, or a paired t-test. Data that did not conform to a normal distribution were compared using either the Krusal-Willis test or the Mann-Whitney test. Results: There was no statistically significant difference in plasma sCD100 level between patients with liver cirrhosis combined simple ascites (1 415 ± 434.1) pg/ml, patients with liver cirrhosis combined with SBP (1 465 ± 386.8) pg/ml, and controls (1 355 ± 428.0) pg/ml (P = 0.655). The ascites sCD100 level was lower in patients with liver cirrhosis combined with SBP than that of patients with simple ascites [(2 409 ± 743.0) pg/ml vs. (2825±664.2) pg/ml, P=0.014]. There was no statistically significant difference in the level of mCD100 in peripheral blood CD4(+) and CD8(+) T lymphocytes among the three groups (P > 0.05). The levels of mCD100 in ascites CD4(+) and CD8(+) T lymphocytes were higher in patients with liver cirrhosis combined with SBP than those in patients with simple ascites (P < 0.05). CD100 stimulation had no significant effect on the proliferation of CD4(+) and CD8(+)T lymphocytes in the ascites of patients with liver cirrhosis combined with SBP (P > 0.05). There were no significant effects on the expression of transcription factors in effector CD4(+)T lymphocytes (T-bet, retinoic acid associated solitary nuclear receptor γt, aromatic hydrocarbon receptor) or secretion of cytokines (interferon-γ, 17, and 22) (P > 0.05). CD100 stimulation had increased the relative expression of perforin, granzyme B, and granlysin mRNA and the levels of secreted interferon-γ and tumor necrosis factor-α, killing activity in ascites CD8+ T lymphocytes of patients with liver cirrhosis combined with SBP (P < 0.05). Conclusion: The active form of CD100 is sCD100 instead of mCD100. There is an imbalance between the expression of sCD100 and mCD100 in the ascites of patients with cirrhosis combined with SBP. sCD100 can enhance the function of CD8(+)T lymphocytes in the ascites of patients with cirrhosis combined with SBP and thus is one of the potential therapeutic targets.

Pan-HDAC inhibitors augment IL2-induced proliferation of NK cells via the JAK2-STAT5B signaling pathway

BackgroundNatural killer (NK) cells are a subtype of lymphocytes with the ability to quickly and efficiently identify and eliminate tumor cells. In the presence of IL2, NK cells can divide rapidly but in limited numbers. According to previous studies, in vivo treatment with histone deacetylase (HDAC) inhibitors did not impair NK-cell function. This study aimed to investigate the effect of HDAC inhibitors on NK-cell proliferation and the underlying regulatory mechanism. MethodsNK92 cells, primary NK (pNK) cells, and CD19-CAR-NK92 cells were treated with low concentrations of pan-HDACi Dacinostat (Dac) and Panobinostat (Pan) in the presence of IL2, and Cell Counting Kit-8 (CCK8), 5-ethynyl-2′-deoxyuridine (EdU), and flow cytometry assays were used to assess cell proliferation and apoptosis. The expression of granzyme B was detected by immunofluorescence, and the expression of CD107a and NKG2D was determined by flow cytometry. The downstream regulatory genes were identified by RNA-seq, and the “JAK-STAT signaling pathway”- and “Cell cycle signaling pathway”-related genes were detected by real-time quantitative polymerase chain reaction (RT–qPCR) and Western blot analysis. The JAK2V617F mouse model was constructed to simulate the upregulation of the JAK2 signaling pathway in vivo, and the NK proliferation was evaluated by flow cytometry. A tumor-bearing nude mouse model was constructed to determine the anti-tumor efficacy of NK92 cells following Dac treatment. ResultsIn the presence of IL2, the proliferation rate of NK92 cells, pNK cells, and CD19-CAR-NK92 cells treated with pan-HDACi Dac and Pan at low nanomolar doses was significantly increased, although cell function was unaffected. Low doses of Dac upregulated the JAK-STAT signaling pathway and enhance the cell cycle via that pathway. In addition, the in vivo experiment in nude mice showed that the capacity of Dac treated NK92 cells to eliminate tumor cells was unaffected. ConclusionLow nanomolar doses of Pan-HDACi enhanced IL2-induced NK cell proliferation without compromising the functioning of NK cells.

G9a/GLP inhibition during ex vivo lymphocyte expansion increases in vivo cytotoxicity of engineered T cells against hepatocellular carcinoma

Engineered T cells transiently expressing tumor-targeting receptors are an attractive form of engineered T cell therapy as they carry no risk of insertional mutagenesis or long-term adverse side-effects. However, multiple rounds of treatment are often required, increasing patient discomfort and cost. To mitigate this, we sought to improve the antitumor activity of transient engineered T cells by screening a panel of small molecules targeting epigenetic regulators for their effect on T cell cytotoxicity. Using a model for engineered T cells targetting hepatocellular carcinoma, we find that short-term inhibition of G9a/GLP increases T cell antitumor activity in in vitro models and an orthotopic mouse model. G9a/GLP inhibition increases granzyme expression without terminal T cell differentiation or exhaustion and results in specific changes in expression of genes and proteins involved in pro-inflammatory pathways, T cell activation and cytotoxicity.

Lenvatinib enhances antitumor immunity by promoting the infiltration of TCF1+ CD8+ T cells in HCC via blocking VEGFR2.

Lenvatinib is the favorable treatment for advanced hepatocellular carcinoma (HCC), and it is currently undergoing phase III clinical trials. However, the specific effects of lenvatinib on PD1+ CD8+ T cells in HCC microenvironment have not been systematically studied. Here, we established an orthotopic hepa1-6 mouse model treated with lenvatinib to investigate CD8+ T cells' role in the tumor and spleen. We found an increasing proportion of TCF-1+ in PD1+ CD8+ T cells and proliferation of PD1+ CD8+ T cells after lenvatinib treatment. Meanwhile, lenvatinib treatment upregulated the expression of granzyme B on PD1+ CD8+ T cells both in vitro and in vivo. Lenvatinib activated the endogenous mTOR pathway of exhausted CD8+ T cells, and mTOR pathway blockade eliminated the antitumor effect of lenvatinib and function of PD1+ CD8+ T cells. The effects of the mTOR pathway on PD1+ CD8+ T cells after lenvatinib treatment were mediated by VEGFR2 inhibition. Overall, our work provides insight into the mechanism of lenvatinib's antitumor efficacy through exhausted CD8+ T cells in HCC treatment.

Human pegivirus-1 replication influences NK cell reconstitution after allogeneic haematopoietic stem cell transplantation.

Human pegivirus-1 (HPgV-1) is a so-called commensal virus for which no known associated organ disease has been found to date. Yet, it affects immune-reconstitution as previously studied in the HIV population, in whom active co-infection with HPgV-1 can modulate T and NK cell activation and differentiation leading to a protective effect against the evolution of the disease. Little is known on the effect of HPgV-1 on immune-reconstitution in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients, a patient population in which we and others have previously reported high prevalence of HPgV-1 replication. The aim of this study was to compare the immune reconstitution after allo-HSCT among HPgV-1-viremic and HPgV-1-non-viremic patients. Within a cohort study of 40 allo-HSCT patients, 20 allo-HSCT recipients positive in plasma sample for HPgV-1 by rRT-PCR during the first year (1, 3, 6, 12 months) after transplantation were matched with 20 allo-HSCT recipients negative for HPgV-1. T and NK cell reconstitution was monitored by flow cytometry in peripheral blood samples from allo-HSCT recipients at the same time points. We observed no significant difference in the absolute number and subsets proportions of CD4 and CD8 T cells between patient groups at any analysed timepoint. We observed a significantly higher absolute number of NK cells at 3 months among HPgV-1-viremic patients. Immunophenotypic analysis showed a significantly higher proportion of CD56bright NK cells mirrored by a reduced percentage of CD56dim NK cells in HPgV-1-positive patients during the first 6 months after allo-HSCT. At 6 months post-allo-HSCT, NK cell phenotype significantly differed depending on HPgV-1, HPgV-1-viremic patients displaying NK cells with lower CD16 and CD57 expression compared with HPgV-1-negative patients. In accordance with their less differentiated phenotype, we detected a significantly reduced expression of granzyme B in NK cells in HPgV-1-viremic patients at 6 months. Our study shows that HPgV-1-viremic allo-HSCT recipients displayed an impaired NK cell, but not T cell, immune-reconstitution compared with HPgV-1-non-viremic patients, revealing for the first time a potential association between replication of the non-pathogenic HPgV-1 virus and immunomodulation after allo-HSCT.

Silencing of circCRIM1 Drives IGF2BP1-Mediated NSCLC Immune Evasion.

Circular RNAs (circRNAs) have been found to have significant impacts on non-small cell lung cancer (NSCLC) progression through various mechanisms. However, the mechanism of circRNAs modulating tumor immune evasion in NSCLC has yet to be well-revealed. Through analyzing the expression profiles of circRNAs in NSCLC tissues, RNA FISH, pull-down assay, mass spectrometry analysis, and RIP, circCRIM1 was identified, and its interaction with IGF2BP1 was confirmed. The effects of circCRIM1 on modulating tumor immune evasion were explored via co-culture in vitro and in tumor xenograft models. Subsequently, we evaluated the regulatory effects of circCRIM1 on IGF2BP1 and screened its target genes through RNA sequencing. Finally, we explored the underlying molecular mechanisms that circCRIM1 could regulate the stability of target mRNA. circCRIM1 was downregulated in NSCLC, and its expression was positively correlated with favorable prognoses. Furthermore, circCRIM1 was more stable than its linear transcript and was mainly localized in the cytoplasm. Mechanistically, circCRIM1 destabilized HLA-F mRNA via competitive binding to IGF2BP1. Importantly, the overexpression of circCRIM1 suppressed the immune evasion of NSCLC and promoted the expressions of Granzyme B, IFN-γ, and TNF-α of CD8+ T and NK cell in vitro co-culture assays and tumor xenograft models. This study identifies circCRIM1 as a new tumor suppressor that inhibits tumor immune evasion through a competitive combination with IGF2BP1 to destabilize HLA-F mRNA.

Primary central nervous system T-cell lymphoma in children and adolescents: a clinicopathological analysis of five cases

Objective: To study the clinicopathological characteristics, and further understand primary central nervous system T-cell lymphoma (PCNSTCL) in children and adolescents. Methods: Five cases of PCNSTCL in children and adolescents were collected from December 2016 to December 2021 at the First Affiliated Hospital of Zhengzhou University. The clinicopathological characteristics, immunophenotypic, and molecular pathologic features were analyzed, and relevant literatures reviewed. Results: There were two male and three female patients with a median age of 14 years (range 11 to 18 years). There were two peripheral T-cell lymphomas, not otherwise specified, two anaplastic large cell lymphoma, ALK-positive and one NK/T cell lymphoma. Pathologically, the tumor cells showed a variable histomorphologic spectrum, including small, medium and large cells with diffuse growth pattern and perivascular accentuation. Immunohistochemistry and in situ hybridization showed CD3 expression in four cases, and CD3 was lost in one case. CD5 expression was lost in four cases and retained in one case. ALK and CD30 were expressed in two cases. One tumor expressed CD56 and Epstein-Barr virus-encoded RNA. All cases showed a cytotoxic phenotype with expression of TIA1 and granzyme B. Three cases had a high Ki-67 index (>50%). T-cell receptor (TCR) gene rearrangement was clonal in two cases. Conclusions: PCNSTCL is rare, especially in children and adolescents. The morphology of PCNSTCL is diverse. Immunohistochemistry and TCR gene rearrangement play important roles in the diagnosis.

NK- and T-cell granzyme B and K expression correlates with age, CMV infection and influenza vaccine-induced antibody titres in older adults.

Granzymes are a family of serine-proteases that act as critical mediators in the cytolytic and immunomodulatory activities of immune cells such as CD8+ T-cells and natural killer (NK) cells. Previous work indicates that both granzyme B (GZB) and K (GZK) are increased with age in CD8+ T-cells, and in the case of GZB, contribute to dysfunctional immune processes observed in older adults. Here, we sought to determine how GZB and GZK expression in NK-cells, and CD4+, CD8+, and gamma-delta T-cells, quantified in terms of positive cell frequency and mean fluorescence intensity (MFI), differed with age, age-related health-traits and the antibody response to high-dose influenza vaccine. We found that the frequency and MFI of GZB-expressing NK-cells, and CD8+ and Vδ1+ T-cells, and GZK-expressing CD8+ T-cells was significantly higher in older (66-97years old; n = 75) vs. younger (24-37years old; n = 10) adults by up to 5-fold. There were no significant associations of GZB/GZK expression with sex, frailty or plasma levels of TNF or IL-6 in older adults, but those who were seropositive for cytomegalovirus (CMV) exhibited significantly higher frequencies of GZB+ NK-cells, and CD4+, CD8+ and Vδ1+ T-cells, and GZK+ CD8+ T-cells (Cohen's d = .5-1.5). Pre-vaccination frequencies of GZB+ NK-cells were positively correlated with vaccine antibody responses against A/H3N2 (d = .17), while the frequencies of GZK+ NK and CD8+ T-cells were inversely associated with A/H1N1 (d = -0.18 to -0.20). Interestingly, GZK+ NK-cell frequency was inversely correlated with pre-vaccination A/H1N1 antibody titres, as well as those measured over the previous 4years, further supporting a role for this subset in influencing vaccine antibody-responses. These findings further our understanding of how granzyme expression in different lymphoid cell-types may change with age, while suggesting that they influence vaccine responsiveness in older adults.

PHD2 Constrains Antitumor CD8+ T-cell Activity.

The prolyl hydroxylase domain/hypoxia-inducible factor (PHD/HIF) pathway has been implicated in a wide range of immune and inflammatory processes, including in the oxygen-deprived tumor microenvironment. To examine the effect of HIF stabilization in antitumor immunity, we deleted Phd2 selectively in T lymphocytes using the cre/lox system. We show that the deletion of PHD2 in lymphocytes resulted in enhanced regression of EG7-OVA tumors, in a HIF-1α-dependent manner. The enhanced control of neoplastic growth correlated with increased polyfunctionality of CD8+ tumor-infiltrating lymphocytes, as indicated by enhanced expression of IFNγ, TNFα, and granzyme B. Phenotypic and transcriptomic analyses pointed to a key role of glycolysis in sustaining CTL activity in the tumor bed and identified the PHD2/HIF-1 pathway as a potential target for cancer immunotherapy.

Effects of Newcastle Disease/Infectious Bronchitis Vaccine and Feeding Yeast Products on the Innate Immune System in the Proventriculus and Ileum of Broiler Chicks.

The aim of this study was to determine whether Newcastle disease/infectious bronchitis (ND/IB) vaccination and yeast product diet supplementation modulate the expression of innate immune molecules in the proventriculus and ileum of broiler chicks. One-day-old male broiler chicks were divided into four groups (V-Y- (control), V-Y+, V+Y-, and V+Y+ groups, where V and Y represent vaccination and yeast product supplementation, respectively). Chicks in the V+Y- and V+Y+ groups were immunized with the live ND/IB vaccine, whereas chicks in the V-Y- and V-Y+ groups were not. Chicks in the V-Y+ and V+Y+ groups received feed containing yeast products from day 4, whereas chicks in the V-Y- and V+Y- groups did not. The proventriculus and ileum were collected on day 7 to analyze the expression of seven Toll-like receptors (TLRs) and Dectin-1. In the proventriculus, compared with those of the V-Y- control group, the TLR7 and TLR21 expression levels were higher in the V+Y- group; however, there were no differences in the expression levels of any TLR or Dectin-1 in the ileum. There were also no differences in the expression of avian β-defensins and cathelicidin-1 in the proventriculus and ileum between the control and treatment groups. The expression of granzyme in cytotoxic cells and interleukin (IL)-1B was upregulated by ND/IB vaccination in the proventriculus. Supplementation with yeast products upregulated only granzyme expression in the ileum and downregulated IL-6 expression in the proventriculus in chicks immunized with the ND/IB vaccine. Thus, we concluded that ND/IB vaccination is effective at enhancing the innate immune system in the proventriculus of chicks, at least until day 7 post-hatching, whereas the effects of diet supplementation with yeast products may be limited, at least under the present study conditions.

Chemical Complementarity of Breast Cancer Resident, T-Cell Receptor CDR3 Domains and the Cancer Antigen, ARMC3, is Associated With Higher Levels of Survival and Granzyme Expression

Introduction: One of the most pressing goals for cancer immunotherapy at this time is the identification of actionable antigens. Methods: This study relies on the following considerations and approaches to identify potential breast cancer antigens: (i) the significant role of the adaptive immune receptor, complementarity determining region-3 (CDR3) in antigen binding, and the existence cancer testis antigens (CTAs); (ii) chemical attractiveness; and (iii) informing the relevance of the integration of items (i) and (ii) with patient outcome and tumor gene expression data. Results: We have assessed CTAs for associations with survival, based on their chemical complementarity with tumor resident T-cell receptor (TCR), CDR3s. Also, we have established gene expression correlations with the high TCR CDR3-CTA chemical complementarities, for Granzyme B, and other immune biomarkers. Conclusions: Overall, for several independent TCR CDR3 breast cancer datasets, the CTA, ARMC3, stood out as a completely novel, candidate antigen based on multiple algorithms with highly consistent approaches. This conclusion was facilitated by use of the recently constructed Adaptive Match web tool.

Clinicopathological and predictive value of MAIT cells in non-small cell lung cancer for immunotherapy

BackgroundImmune-checkpoint inhibitors (ICIs) remain ineffective in a large group of non-small cell lung cancer (NSCLC) patients. Mucosal-associated invariant T (MAIT) cells, a population of unconventional innate-like T lymphocytes abundant in...

Small intestine and colon tissue-resident memory CD8+ T cells exhibit molecular heterogeneity and differential dependence on Eomes

Tissue-resident memory CD8+ T (TRM) cells are a subset of memory Tcells that play a critical role in limiting early pathogen spread and controlling infection. TRM cells exhibit differences across tissues, but their potential heterogeneity among distinct anatomic compartments within the small intestine and colon has not been well recognized. Here, by analyzing TRM cells from the lamina propria and epithelial compartments of the small intestine and colon, we showed that intestinal TRM cells exhibited distinctive patterns of cytokine and granzyme expression along with substantial transcriptional, epigenetic, and functional heterogeneity. The T-box transcription factor Eomes, which represses TRM cell formation in some tissues, exhibited unexpected context-specific regulatory roles in supporting the maintenance of established TRM cells in the small intestine, but not in the colon. Taken together, these data provide previously unappreciated insights into the heterogeneity and differential requirements for the formation vs. maintenance of intestinal TRM cells.

Analysis of scRNA-seq and bulk RNA-seq demonstrates the effects of EVI2B or CD361 on CD8+ T cells in osteosarcoma.

Osteosarcoma (OS) is a common primary malignant tumor of the bone in children and adolescents. The five-year survival rate is estimated to be ~70% based on the currently available treatment modalities. It is well known that tumor-infiltrating immune cells (TIICs) that are the most important components in the tumor microenvironment can exert a killing effect on tumor cells. Therefore, in the present study, 85 RNA-sequencing OS samples were categorized into high- and low-immune score groups with ESTIAMATE. Based on the immune score groups, 474 differentially expressed genes (DEGs) were acquired using the LIMMA package of R language. Subsequently, 86 DEGs were taken through univariate COX regression analysis, of which 14 were screened out by least absolute shrinkage and selection operator regression analysis. Furthermore, multivariate COX regression analysis was performed to obtain 4 DEGs. Finally, ecotropic virus integration site 2B (EVI2B) or CD361 gene was screened out via Kaplan-Meier analysis. In addition, CIBERSORT algorithm was used to evaluate the proportion of 22 kinds of TIICs in OS. Correlation analysis revealed that the high expression level of EVI2B can elevate the infiltrated proportion of CD8+ T cells. Moreover, analysis of single cell RNA-sequencing transcriptome datasets and immunohistochemical staining uncovered that EVI2B was mainly expressed on CD8+ T cells and that EVI2B could promote the expression of granzyme A and K of CD8+ T cells to exhibit a potent killing effect on tumor cells. Therefore, EVI2B was identified as a protective immune-related gene and contributed to good prognosis in OS patients.

Indolent T-cell lymphoproliferative disorder of gastrointestinal tract with unusual clinical courses: report of 6 cases and literature review.

Indolent T-cell lymphoproliferative disorder of the gastrointestinal tract (iTLPD-GI) is a rare neoplasm usually having an indolent clinical course and easily misdiagnosed as inflammatory bowel disease or other T-cell lymphomas. A subset of the disorders that progressed to overt peripheral T-cell lymphoma have been reported, and the etiology and pathogenesis are poorly understood. The current study retrospectively examined the pathological, molecular, and clinical features of 6 cases of iTLPD-GI. Hematoxylin and eosin staining, immunohistochemistry, in situ hybridization, T-cell receptor gene rearrangement, and next-generation sequencing (NGS) were performed with the diseased tissues. All the 6 patients were immunocompetent Chinese men, who presented with recurrent abdominal pain and diarrhea for 4 to 13years. Histologically, the intestinal tissue was expanded by lymphoid infiltration, composed of small-to-medium-sized lymphocytes with gland intact. The neoplastic cells were CD4 - /CD8 + with expression of TIA1 and variable granzyme B in five cases, and the other one was CD4 + /CD8 - . Two of the 5 patients progressed to more aggressive T-cell lymphoma and died of disease with complications. NGS identified TET2 and DDX3X mutations in patient 1, and BIRC6 and REV3L mutations in patient 2. Literature review indicated that iTLPD-GI with CD4 - /CD8 + immunophenotype was more commonly reported in Chinese cases. Our limited data indicated CD4-/CD8 + iTLPD-GI have similar potential to progress to more aggressive T-cell lymphoma as that of CD4 + /CD8 - , and gradually increased expression of granzyme B and Ki-67 may be early signs of the disease progression. Gain of novel gene mutations may be indicators of the pathogenesis.

Abstract A50: IFNλ1 is a marker of DNA damage-triggered STING-signaling in lung cancer that induces immune activation through macrophage stimulation

Abstract Immunotherapy is a fundamental pillar in the treatment of lung cancer but remains inefficient in half of the treated patients. Recent clinical trials showed synergistic effects of combining chemotherapy and immunotherapy for lung cancer, suggesting that increased DNA damage imposed by chemotherapy modulates the tumor microenvironment to aid the efficacy of immunotherapy. But the immunological mechanisms related to this remains to be fully elucidated. The cGAS-STING pathway senses cytosolic DNA introduced by DNA-damaging agents such as chemotherapy. Activation of this pathway and induction of inflammatory cytokines may contribute to better immunotherapy efficacy. In particular, the release of type I IFN is a hallmark for STING pathway activation. However, we also know that IFNλ – a type III IFN – can be induced by STING-signaling. Despite the relevance of IFNλ in controlling inflammatory responses, its contribution in cancer remains unclear. Here, we investigated cGAS-STING expression and signaling in a panel of lung cancer cells. We identified IFNλ1 as an alternative hallmark for cGAS-STING DNA-sensing with higher expression after DNA-stimulation than IFNb in 6 out of 8 cell lines with one cell line having no IFNb expression at all. We then tested IFN induction after chemotherapeutic treatment in three different NSCLC cell lines and saw significant increase in both IFNb and IFNλ1 expression across four different chemotherapeutics, but with a higher fold change for IFNλ1. The induction of both IFNb and IFNλ1 was abolished when STING KO cell lines were treated with doxorubicin pointing to a STING-dependent response. To access the effect of IFNλ1 on macrophages, a prevalent cell type in the tumor microenvironment expressing IFNLR1, we performed RNA-sequencing on four primary human macrophage donors stimulated with IFNλ1. This stimulation led to a significant increase in well-known interferon-stimulated genes including the chemokines CXCL10 and CXCL11. We also explored whether IFNλ1 stimulation led to increased macrophage-dependent activation of autologous CD8+ T-cells. Interestingly, we found increased expression of both IFNg (p = 0.0215, n = 6) and Granzyme B (p = 0.0033, n = 6) measured by flow cytometry and ELISA (p &amp;gt; 0.0001, n = 6 for both). The addition of IFNλ1 during direct co-culture of macrophages and lung tumor organoids furthermore dampened the induction of the signal regulatory protein-a (SIRPa), a phagocytosis-inhibiting receptor, (p = 0.0041, n = 4) otherwise induced by the tumor organoids, but not matched healthy organoids. In support of this, there was an increased activation of CD8+ T-cells measured by IFNg production in an organoid-macrophage-CD8+ T-cell co-culture model. To summarize, we demonstrate that IFNλ1 may be a strong and broad marker of STING activation induced by chemotherapy in NSCLC and that IFNλ1 has the ability to prime a wider immune response by targeting macrophages. Hence, we propose that an IFNλ1 induced immune response has the potential to support and boost the response induced by existing immunotherapies. Citation Format: Kristine R Gammelgaard, Stine H Godsk, Albert G Olivier, Maria Riedel, Silke D Nielsen, Allard van Renterghem, Trine V Larsen, Anders Etzerodt, Emile Voest, Martin R Jakobsen. IFNλ1 is a marker of DNA damage-triggered STING-signaling in lung cancer that induces immune activation through macrophage stimulation [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A50.