CD4 Molecules Research Articles

#1550 Incident hypertension: immune biomarkers and vascular phenotypes

Abstract Background and Aims Blood pressure (BP) regulation is mediated by cardiac, vascular, endocrine, renal, and immune system interactions. Hence, hypertension may exist as different phenotypic variants, with different clinical features and susceptibility to target organ damage. We aimed to analyse the circulating protein biomarker ‘signature’ of: 1) untreated hypertension; 2) hypertensive phenotypes identified by arterial and blood pressure parameters. Method 61 patients with hypertension, and 61 controls were assessed using 24hr ambulatory BP monitoring; endothelial function, arterial stiffness, carotid intima-media thickness (CIMT), and cardiovascular variability [1]. Circulating protein biomarkers analysed using Olink® Inflammation plasma biomarker panel, reported as NPX (Normalized Protein eXpression), in Log2 scale. Results 34 biomarkers dominated by cytokines and chemokines differed between normotensive and hypertensive subjects (Fig. 1), though failed to meet Bonferroni-adjusted threshold. Inflammatory biomarkers correlated with BP and arterial stiffness, BP variability, and CIMT, but not endothelial function. Associations were concordant across systolic and diastolic BP; TPP1, CCL7, CCL11, and CCL21 positively correlating; IL18R1, and KYNU negatively. These relationships were more pronounced in hypertensive subgroup, 85 biomarkers correlating including CD molecules (CD200R1, CD22, CD58, CD6, CD70) and cytokines (IL-5, IL-11, IL-15, IL-18, IL-32, IL-1RL2, IL-22RA1, IL-5RA). HGF, AGE, and CCL21 showed greatest between group differences and correlated with multiple BP or vascular parameters. Systolic nocturnal dipping demonstrated negative correlation with biomarkers relating to immune cell interactions and cellular adhesion (CTRC, EPHA1, LGALS4, SIT1, SMOC, IL-18 and TNFSF11). Machine learning techniques identified three phenotypes of hypertension, ‘arterially stiffened’, ‘vaso-protected’, and ‘non-dipper’ [1]. Sixteen of the 85 correlating biomarkers also differed between these phenotypic groups: Conclusion Hypertension is linked to alterations in circulating immune biomarkers. Multiple biomarkers correlated with both arterial stiffness and BP parameters. Some biomarkers are only correlated in the hypertensive group. Many also relate to nocturnal dipping, and differed across the hypertension endotypes. HGF is a promising biomarker for BP and arterial function; warranting further validation.

Immune response variation in mild and severe COVID-19 patients.

Sixty patients with COVID-19 infection were categorized into mild and severe groups, and their immune response was analyzed using flow cytometry and complete blood count. An observed increase in immune activation parameters, notably a higher percentage of CD4 lymphocytes co-expressing CD69 and CD25 molecules, and enhanced activity of the macrophage-monocyte cell line was noted in the mild group. Although Group 2 (severe COVID) had fewer CD4 cells, significant migration and proliferation were evident, with increased CD4CD69, CD8 HLA-DR+, and CD8CD69 lymphocytes. The CD4 to CD8 ratio in Group 1 suggested potential autoimmune reactions, while Group 2 indicated potential immunosuppression from severe infection and employing immunosuppressive drugs. Additionally, Group 2 exhibited an increased neutrophil count, hinting at possible bacterial co-infection. Group 1 showed differences in CD4RO and CD8RA lymphocyte populations, implying that cellular immunity plays a role in developing efficient postinfectious immunity. This intimation suggests that vaccination might mitigate the severity of the coronavirus infection and prevent complications, including long-term COVID-19.

Abstract PO3-25-06: Expression of CD28, Fas, PD1, and CTLA4 molecules on cellular immune response in the blood peripheral of patients with locally advanced triple-negative breast cancer

Abstract Background: Locally advanced triple-negative breast cancer (TNBC) represents a public health problem in Brazil; its standard treatment consists of neoadjuvant chemotherapy followed by surgical and radiotherapy, with highly variable success rates are depended on access to appropriate cancer treatment, and the type of pathological response. In the last decade, the immune system's role in breast cancer has gained space by demonstrating the favorable impact of lymphocyte tumor infiltrate (TILs) and gene expression of the immune response, especially in tumors with high proliferation rates and negative estrogen receptors. The cellular immune response is regulated by co-stimulatory and co-inhibitory proteins such as CD28, Fas, PD1, and CTLA4 molecules. Methods: This was a longitudinal study with follow-up performed between 2015 and 2017. The study was conducted at the Hospital de Cancer de Pernambuco and Translational Research Laboratory of the Instituto de Medicina Integral Prof. Fernando Figueira (IMIP). Thirty women (18 to 60 years) diagnosed with locally advanced TNBC submitted to neoadjuvant chemotherapy (NAC), and 30 healthy women as a control group were included. The co-stimulatory and co-inhibitory proteins analyses were performed in the peripheral blood of TNBC women before (Pre-NAC) and after NAC (Post-NAC) Results: High levels of CD28+CD3+ T, CD28+CD4+ T, PD1+CD3+ T, and CTLA4+CD3+ T cells in the pathological complete response (pCR) and non-pCR groups compared to the control group (p < 0.05). High levels of Fas+CD4+T cells and PD1+CD8+T cells in the non-pCR compared to the pCR group (p < 0.05). In the paired analysis, significant differences were observed in CD3+T and CD4+T levels with CD28, Fas, and PD1 expression between pre- and post-NAC. Conclusion: Fas+CD4+T and PD1+CD8+T levels in peripheral blood in TNBC are associated with pCR, suggesting potential predictive biomarkers of NAC response. Preliminary results allow us to infer that neoadjuvant chemotherapy modulates the cellular immune response in TNBC women. Citation Format: Marcelo Salgado, Fernando Soares, Denise Viana, Amanda Salgado, Carolina Vasconcelos, Carlos Eduardo Anunciação, Eduardo Salgado, Leuridan Torres. Expression of CD28, Fas, PD1, and CTLA4 molecules on cellular immune response in the blood peripheral of patients with locally advanced triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-06.

Advancing CD19 CAR T Cell Therapy for Treatment of Primary Biliary Cholangitis

Abstract CD19 Chimeric Antigen Receptor (CAR) T cell therapy is emerging as a revolutionary approach in autoimmune disease management, offering a promising alternative to conventional treatments like anti-CD20 antibody therapy. Unlike challenges associated with existing therapies, CD19 CAR T cells, engineered to recognize CD19 molecules on B cells, exhibit remarkable efficacy in achieving targeted B cell depletion. Upon target recognition, they become activated, efficiently eliminating the harmful B cells. Furthermore, the CAR T cells exhibit a propensity for proliferation upon target recognition, thereby ensuring their prolonged presence within the body. Our findings unequivocally establish the effectiveness of CD19 CAR T cells in specifically targeting and depleting B cells, both in vitro and in vivo. In a well-established PBC model involving NOD.c3 mice, the application of CD19 CAR T cell therapy led to a remarkable reduction in liver pathology scores, indicating a substantial decrease in tissue damage and inflammation compared to control groups. Beyond liver autoimmunity, our research holds promise for treating other autoimmune conditions where B cells play a detrimental role. These insights pave the way for innovative therapies that have the potential to revolutionize autoimmune disease management, offering a hope for affected individuals and improving their quality of life.

Enrichment using a selectable CD34t marker enhances TCR/CAR transduced T cell functionality and transgene expression in clinically relevant receptors

Abstract Gene-modified TCR transduced T cells and CAR T cells are used clinically and display a range of efficacy across multiple cancers. Many therapies have successfully controlled patient’s tumors; however, some have shown off-target adverse events which are harmful to patients’ overall outcomes. T cell-based therapies have high variation in patient outcome due to multiple factors, such as transduction efficiency and T cell phenotypes. To reduce product variability, our lab developed a selectable marker gene for use in cellular constructs. This selectable marker is a truncated, non-signaling CD34 molecule (CD34t), which can be used to remove the non-transduced T cells that function as bystanders. Our clinical trials have used two TCRs (TIL 1383I and HERV-E) and one CAR (CD19 CAR). Following transduction with these CD34t constructs, we determined transduction efficiency by expression of CD34t. CD34t expression correlates with transgene, allowing us to select high CD34t expressors, and thus high transgene expressors. The selected population produces higher levels of inflammatory cytokines and CD107a expression compared to the nonselected. This improved functionality is likely due to the selected population having higher levels of transgene across the population, whereas the nonselected has a broader range of transgene expressions with fewer expressors total. This selectable marker contributes to a more functional product, which may in turn improve patient response clinically.

T-Cell Engagers-The Structure and Functional Principle and Application in Hematological Malignancies.

Recent advancements in cancer immunotherapy have made directing the cellular immune response onto cancer cells a promising strategy for the treatment of hematological malignancies. The introduction of monoclonal antibody-based (mAbs) targeted therapy has significantly improved the prognosis for hematological patients. Facing the issues of mAb-based therapies, a novel bispecific antibody (BsAb) format was developed. T-cell engagers (TCEs) are BsAbs, which simultaneously target tumor-associated antigens on tumor cells and CD3 molecules present on T-cells. This mechanism allows for the direct activation of T-cells and their anti-tumor features, ultimately resulting in the lysis of tumor cells. In 2014, the FDA approved blinatumomab, a TCE directed to CD3 and CD19 for treatment of acute lymphoblastic leukemia. Since then, numerous TCEs have been developed, allowing for treating different hematological malignancies such as acute myeloid leukemia, multiple myeloma, and non-Hodgkin lymphoma and Hodgkin lymphoma. As of November 2023, seven clinically approved TCE therapies are on the market. TCE-based therapies still have their limitations; however, improving the properties of TCEs, as well as combining TCE-based therapies with other forms of treatment, give hope to find the cures for currently terminal diseases. In this paper, we summarized the technical basis of the TCE technology, its application in hematology, and its current issues and prospects.

Fcγ-Receptor-Independent Controlled Activation of CD40 Canonical Signaling by Novel Therapeutic Antibodies for Cancer Therapy.

The activation of CD40-mediated signaling in antigen-presenting cells is a promising therapeutic strategy to promote immune responses against tumors. Most agonistic anti-CD40 antibodies currently in development require the Fcγ-receptor (FcγR)-mediated crosslinking of CD40 molecules for a meaningful activation of CD40 signaling but have limitations due to dose-limiting toxicities. Here we describe the identification of CD40 antibodies which strongly stimulate antigen-presenting cells in an entirely FcγR-independent manner. These Fc-silenced anti-CD40 antibodies induce an efficient upregulation of costimulatory receptors and cytokine release by dendritic cells. Finally, the most active identified anti-CD40 antibody shows activity in humanized mice. More importantly, there are no signs of obvious toxicities. These studies thus demonstrate the potent activation of antigen-presenting cells with anti-CD40 antibodies lacking FcγR-binding activity and open the possibility for an efficacious and safe combination therapy for cancer patients.

Runx3 Regulates CD8+ T Cell Local Expansion and CD43 Glycosylation in Mice by H1N1 Influenza A Virus Infection.

We have recently reported that transcription factor Runx3 is required for pulmonary generation of CD8+ cytotoxic T lymphocytes (CTLs) that play a crucial role in the clearance of influenza A virus (IAV). To understand the underlying mechanisms, we determined the effects of Runx3 knockout (KO) on CD8+ T cell local expansion and phenotypes using an inducible general Runx3 KO mouse model. We found that in contrast to the lungs, Runx3 general KO promoted enlargement of lung-draining mediastinal lymph node (mLN) and enhanced CD8+ and CD4+ T cell expansion during H1N1 IAV infection. We further found that Runx3 deficiency greatly inhibited core 2 O-glycosylation of selectin ligand CD43 on activated CD8+ T cells but minimally affected the cell surface expression of CD43, activation markers (CD44 and CD69) and cell adhesion molecules (CD11a and CD54). Runx3 KO had a minor effect on lung effector CD8+ T cell death by IAV infection. Our findings indicate that Runx3 differently regulates CD8+ T cell expansion in mLNs and lungs by H1N1 IAV infection. Runx3 is required for CD43 core 2 O-glycosylation on activated CD8+ T cells, and the involved Runx3 signal pathway may mediate CD8+ T cell phenotype for pulmonary generation of CTLs.

Effect of LAG3 deficiency on natural killer cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis

To investigate the effect of LAG-3 deficiency (LAG3-/-) on natural killer (NK) cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis. C57BL/6 mice, each weighing (20 ± 2) g, were divided into the LAG3-/- and wild type (WT) groups, and each mouse in both groups was inoculated with 3 000 E. multilocularis protoscoleces via the hepatic portal vein. Mouse liver and spleen specimens were collected 12 weeks post-infection, sectioned and stained with sirius red, and the hepatic lesions and fibrosis were observed. Mouse hepatic and splenic lymphocytes were isolated, and flow cytometry was performed to detect the proportions of hepatic and splenic NK cells, the expression of CD44, CD25 and CD69 molecules on NK cell surface, and the secretion of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-4, IL-10 and IL-17A. Sirius red staining showed widening of inflammatory cell bands and hyperplasia of fibrotic connective tissues around mouse hepatic lesions, as well as increased deposition of collagen fibers in the LAG3-/-group relative to the WT group. Flow cytometry revealed lower proportions of mouse hepatic (6.29% ± 1.06% vs. 11.91% ± 1.85%, P < 0.000 1) and splenic NK cells (4.44% ± 1.22% vs. 5.85% ± 1.10%, P > 0.05) in the LAG3-/- group than in the WT group, and the mean fluorescence intensity of CD44 was higher on the surface of mouse hepatic NK cells in the LAG3-/- group than in the WT group (t = -3.234, P < 0.01), while no significant differences were found in the mean fluorescence intensity of CD25 or CD69 on the surface of mouse hepaticNK cells between the LAG3-/- and WT groups (both P values > 0.05). There were significant differences between the LAG3-/- and WT groups in terms of the percentages of IFN-γ (t = -0.723, P > 0.05), TNF-α (t = -0.659, P > 0.05), IL-4 (t = -0.263, P > 0.05), IL-10 (t = -0.455, P > 0.05) or IL-17A secreted by mouse hepatic NK cells (t = 0.091, P > 0.05), and the percentage of IFN-γ secreted by mouse splenic NK cells was higher in the LAG3-/- group than in the WT group (58.40% ± 1.64% vs. 50.40% ± 4.13%; t = -4.042, P < 0.01); however, there were no significant differences between the two groups in terms of the proportions of TNF-α (t = -1.902, P > 0.05), IL-4 (t = -1.333, P > 0.05), IL-10 (t = -1.356, P > 0.05) or IL-17A secreted by mouse splenic NK cells (t = 0.529, P > 0.05). During the course of E. multilocularis infections, LAG3-/- promotes high-level secretion of IFN-γ by splenic NK cells, which may participate in the reversal the immune function of NK cells, resulting in aggravation of hepatic fibrosis.

Abstract LB441: An intravenous compatible oncolytic vaccinia virus treated hepatic metastasis of colon cancer through extensive cancer cell killing and activation of cytotoxic effector differentiation

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide and nearly 50% of CRC patients develop liver metastasis during the course of their disease. Treatment of colorectal cancer liver metastasis (CRLM) has been particularly challenging due to tolerogenic nature of liver immune microenvironment as well as an eventual development of therapy resistance to standard chemotherapy regimen. One promising area of cancer immunotherapy is oncolytic virotherapy that can selectively destroy tumor cells and stimulate anti-tumor immune response. In this study, we systemically administered mSJ-650, an intravenous compatible oncolytic Wyeth strain vaccinia virus that expresses human complement regulatory protein CD55 and murine GM-CSF in place of viral thymidine kinase gene and has a deletion of the viral K2L gene, and evaluated its anti-tumor efficacy in CRLM. Mouse CRLM was induced by intrasplenic injection of 5×105 MC38-OVA cancer cells. Following tumor nodules formation in the liver, low dose (1×106 pfu) or high dose (3×106 pfu) of mSJ-650 was administered multiple times via tail vein and liver was harvested for evaluation of anti-tumor efficacy. Flow cytometry analysis was performed to investigate changes in hepatic and tumor immune microenvironment upon mSJ-650 treatment. Liver and tumor tissue were stained for immunofluorescence imaging of viral replication within the tumor cells and subsequent oncolysis by the virus. Systemic administration of mSJ-650 showed potent anti-tumor efficacy and led to significant reduction in metastatic nodule formation in liver even at low dose. The anti-tumor efficacy was mediated by tumor cell-specific viral distribution and replication, followed by lytic egress of mSJ-650 from infected tumor cells. Anti-tumor efficacy of mSJ-650 also accompanied a dramatic shift in immunologically tolerant hepatic microenvironment to immune-stimulatory one by decreasing proportion of myeloid-derived suppressor cells, Kupffer cells, and monocyte-derived macrophages (MDMs) in the liver while increasing CD8+ T cell infiltration by 2~3 folds. Moreover, mSJ-650 treatment was associated with phenotypic change in hepatic immune cells, as evidenced by Kupffer cell and MDM polarization to M1 phenotype and elevated expression of activation markers and cytotoxic molecules in CD8+ T cells. Changes in hepatic immune microenvironment were also observed in tumor nodules, especially in those completely surrounded by adjacent liver tissue, suggesting that anti-tumor immune response in tumor bed is potentiated by reprogramming of tumor-adjacent hepatic immune microenvironment. Systemic administration of mSJ-650 demonstrated superior anti-tumor efficacy in CRLM which was mediated by direct tumor cell killing and tumor-adjacent hepatic immune microenvironment reprogramming by the virus. Citation Format: Eunhye Kim, Yeonsoo Yang, Solchan Won, Namhee Lee, Songyi Lee, Mi-Ju Park, Byung-Jin Jung, Yun-Kyoung Hong, Hang-Rae Kim, Dong-Sup Lee, Keunhee Oh. An intravenous compatible oncolytic vaccinia virus treated hepatic metastasis of colon cancer through extensive cancer cell killing and activation of cytotoxic effector differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB441.

CD1 molecules: Beyond antigen presentation

CD1 molecules are well known for their role in binding and presenting lipid antigens to mediate the activation of CD1-restricted T cells. However, much less appreciated is the fact that CD1 molecules can have additional “unconventional” roles which impact the activation and functions of CD1-expressing cells, ultimately controlling tissue homeostasis as well as the progression of inflammatory and infectious diseases. Some of these roles are mediated by so-called reverse signalling, by which crosslinking of CD1 molecules at the cell surface initiates intracellular signalling. On the other hand, CD1 molecules can also control metabolic and inflammatory pathways in CD1-expressing cells through cell-intrinsic mechanisms independent of CD1 ligation. Here, we review the evidence for “unconventional” functions of CD1 molecules and the outcomes of such roles for health and disease.

The Influence of Microglia on Neuroplasticity and Long-Term Cognitive Sequelae in Long COVID: Impacts on Brain Development and Beyond.

Microglial cells, the immune cells of the central nervous system, are key elements regulating brain development and brain health. These cells are fully responsive to stressors, microenvironmental alterations and are actively involved in the construction of neural circuits in children and the ability to undergo full experience-dependent plasticity in adults. Since neuroinflammation is a known key element in the pathogenesis of COVID-19, one might expect the dysregulation of microglial function to severely impact both functional and structural plasticity, leading to the cognitive sequelae that appear in the pathogenesis of Long COVID. Therefore, understanding this complex scenario is mandatory for establishing the possible molecular mechanisms related to these symptoms. In the present review, we will discuss Long COVID and its association with reduced levels of BDNF, altered crosstalk between circulating immune cells and microglia, increased levels of inflammasomes, cytokines and chemokines, as well as the alterations in signaling pathways that impact neural synaptic remodeling and plasticity, such as fractalkines, the complement system, the expression of SIRPα and CD47 molecules and altered matrix remodeling. Together, these complex mechanisms may help us understand consequences of Long COVID for brain development and its association with altered brain plasticity, impacting learning disabilities, neurodevelopmental disorders, as well as cognitive decline in adults.

Abstract 3595: A retrospective analysis of the clinical characteristics of metastastic solid cancer patients with cytokine-induced killer cells combined immune checkpoint inhibitor immunotherapy

Abstract Background: Cytokine-induced killer cells (CIKs), obtained through ex vivo expasion from peripheral blood mononuclear cells, are phenotypically and functionally shared with both T and NK cells. Within the T cell group, it can be classified into a group that simultaneously expresses CD3 and CD56 molecules (range: 30% to 70%) and a group representing CD3+ CD56- phenotype (range: 20% to 60%). It also contains a small population (&amp;lt; 30%) of CD3 CD56+ NK cells. They can exhibit potent MHC-unrestricted cytotoxicity for both hematologic and solid tumors, but they are not harmful in hematopoietic precursors and normal tissues. Anti-programmed death-1 (PD-1) antibodies reinvigorate T cell-mediated antitumor immunity. PD-1 blockade antibody might significantly increase the cytotoxic potency of CIK cells via increasing proliferative capacity and IFN-r secretion. This retrospective study is the first to combine CIK cells with PD-1/PD-L1/CTLA-4 blockade(Immune checkpoint inhibitors, ICIs) in patients with metastatic solid cancers. Methods: We analyzed 225 heavily treated metastastic solid cancer patients receiving CIK cell (Immunecell-LC, GC cell inc provide.) and with ICIs. Blood Sampling for CIK cells production was performed after 3 days after ICIs administration. Patients received a single intravenous infusion of activated autologous CIK cells every 2 weeks. ICIs was administered according to the protocol of each drug regardless of the CIK cells administration date. The primary end points were safety and adverse event (AE) profiles. Objective responses, overall survival (OS), progression-free survival (PFS), and diseae control rate were secondary end points. Results: Treatment-related AEs occurred in 154/225 patients. Grade 3 or 4 toxicities, including fever and chills, were observed in 28 patients. All treatment-related AEs were tolerable and no death due to autoimmune disease. When administration ICIs, CIK cells exhibited excellent antitumor properties and increased IFN-γ secretion. Objective responses (complete or partial responses) were observed in 67 of the 225 patients. One patients with hepatocellular carcionma had complete response. In most patients who showed an objective response, the effectiveness was maintained for more than 6 months. The overall disease control rate in the patients was 58.9%. At the time of this report, the median OS and PFS were 18.6 and 7.1 months in hepatocellular carcinoma patients and were 13.1 and 6.5 months in triple negative breast cancer patients, respectively. Conclusion: Treatment with activated autologous CIK cells and ICIs was safe and exerted encouraging antitumor activity in metastastic solid cancers. Phase 2 clinical studies for each tumor type will be conducted. Keywords: Cytokine-induced killer cells, Immune check inhibitors, Safety, Objective response Citation Format: Jonggwon Choi, Jong Yeup Kim, Dong Won Yang, Gun He Nam. A retrospective analysis of the clinical characteristics of metastastic solid cancer patients with cytokine-induced killer cells combined immune checkpoint inhibitor immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3595.

Abstract 3979: PD-1 signals regulate NKG7 that is required for CD8+ T cells to sustain cytotoxicity and durable antitumor memory responses

Abstract Cytotoxic CD8+ T cells play a key role in response to anti-PD-1/PD-L1 immune checkpoint inhibitors (ICI) therapy. However, the exact effector molecules that are regulated by PD-1 signals in cytotoxic CD8+ T cells have not been completely defined. Lack of this knowledge is a crucial problem in addressing how to overcome the low clinical response rate to ICI therapy. When exploring why certain patients not responding to ICI therapy, we found non-responders did not have an elevated NKG7 expression in CD8+ T cells as responders to ICI therapy. The difference in NKG7 expression in CD8+ T cells between non-responders and responders to anti-PD-1 therapy prompted us to investigate the potential role of PD-1 signaling in regulating NKG7 expression. To that end, we generated a CD8+ T cell-specific Pdcd1 conditional knockout mice (CD8 E8ICre-Pdcd1fl/fl, also known as CD8-PD-1 cKO) where Pdcd1 is deleted after thymic selection of single positive CD8+ T cells. We observed a delayed tumor growth and prolonged survival in CD8-PD-1 cKO mice compared to control mice after B16-OVA melanoma tumor challenge. On Day 12, when the CD8-PD-1 cKO mice demonstrated their ability to control tumor growth, we conducted single cell RNA sequencing on isolated tumor infiltrating CD8+ T cells, revealing a significant increase of Nkg7 expression in effector CD8+ T cells in the Pdcd1 deficient CD8+ T cells compared to the control wild type T cells. Pdcd1-deficient effector CD8+ T cells also had increased expression of genes linked to NFAT/Calcineurin signaling pathway including Nfatc1, S100a4, Tbet and Tox, suggesting Nkg7 could be regulated by Pdcd1 via the NFAT pathway. To determine the impact of increased NKG7 expression in CD8+ T cells, we used OVA protein as a surrogate tumor antigen in vaccination. We found a complete B16-OVA tumor rejection in CD8-PD-1 cKO mice compared to control mice when tumor cells were injected on day 7 after vaccination with OVA protein and poly (I:C) (as an adjuvant), but only a partial rejection on day 21. The CD8-PD-1 cKO mice that were tumor-free for 30-40 days rejected 2nd tumor challenge with B16-OVA tumors. This long-term protection is tumor antigen (OVA) specific because these mice did not reject B16F10 tumor cells that do not express OVA antigen. We also found effector memory CD8+ T cells with high cytotoxicity increased in the spleen of CD8-PD-1 cKO mice after OVA antigen immunization compared to control. In conclusion, we found NKG7 is one of the effector molecules in cytotoxic CD8+ T cells, which can be regulated by PD-1 signals through NFAT signaling. Targeting the PD-1-NFAT pathway may be a new option to overcome low responses of T cells to ICI therapy in treatment of refractory cancer. Citation Format: Zhiming Mao, Joanina Gicobi, Jacob Hirdler, Xin Liu, Michelle Hsu, Emilia Dellacecca, Wenjing Zhang, Fabrice Lucien-Matteoni, Hai dong. PD-1 signals regulate NKG7 that is required for CD8+ T cells to sustain cytotoxicity and durable antitumor memory responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3979.

Humoral and cellular immune responses induced by serogroup W135 meningococcal conjugate and polysaccharide vaccines

Investigating the mechanisms by which W135 meningococcal conjugate (PSW135-TT) activates adaptive immune responses in mice can provide a comprehensive understanding of the immune mechanisms of bacterial polysaccharide conjugate vaccines. We compared B-cell and T-cell immune responses immunized with W135 meningococcal capsular polysaccharides (PSW135), tetanus toxoid (TT) and PSW135-TT in mice. The results showed that PSW135-TT could induce higher PSW135-specific and TT-specific IgG antibodies with a significant enhancement after two doses. All serum antibodies immunized with PSW135- TT had strong bactericidal activity, whereas none of the serum antibodies immunized with PSW135 had bactericidal activity. Besides, IgM and IgG antibodies immunized with PSW135-TT after two doses were positively correlated with the titer of bactericidal antibodies. We also found Th cells favored Th2 humoral immune responses in PSW135-TT, PSW135, and TT-immunized mice, especially peripheral blood lymphocytes. Furthermore, PSW135-TT and TT could effectively activate bone marrow derived dendritic cells (BMDCs) and promote BMDCs to highly express major histocompatibility complex Ⅱ (MHCⅡ), CD86 and CD40 molecules in mice, whereas PSW135 couldn’t. These data verified the typical characteristics of PSW135-TT and TT as T cell dependent antigen (TD-Ag) and PSW135 as T cell independent antigen (TI-Ag), which will be very helpful for further exploration of the immune mechanism of polysaccharide-protein conjugate vaccines and improvement of the quality of bacterial polysaccharide conjugate vaccines in future.

Immunohistochemical Evaluation of NKP46 Receptor Expression and the Number of NK Cells in the Endometrium of Patients with Endometriosis.

Endometriosis is a medical condition that can cause infertility in women. Women with endometriosis experience a decrease in NK cell cytotoxic activity against endometrial cells, ultimately contributing to the spread of these cells. To assess the frequency of NK cells and the expression of the NKP46 receptor in endometrial tissue from patients with endometriosis using immunohistochemistry. 30 endometrial tissue specimens were collected from three groups of cases with mild (n=11), moderate (n=10), and severe endometriosis (n=9), respectively. Additionally, 20 normal endometrial tissue specimens were collected as the control group. Immunohistochemical staining was carried out using specific human monoclonal antibodies against CD56 and NKP46 molecules. Cases with severe endometriosis had a significantly higher number of CD56+ uterine NK cells (26.19±2.50) compared to fertile women (15.02±0.622) and women with mild to moderate endometriosis (p<0.001). However, there was no significant difference between the mild to moderate patients compared with the healthy women (p>0.05). Endometrial NKp46 expression was lower in women with severe endometriosis (0.447±0.0829) compared to fertile women (0.987±0.115, p=0.03). The NKp46+/CD56+ cell ratio was also lower in women with severe endometriosis (0.019±0.003) compared to fertile women (0.072±0.011, p=0.01). Women with severe endometriosis demonstrated an increased rate of infiltrated uterine NK cells and a significant decrease in NKP46 expression compared to fertile women. Therefore, NK cells and the NKp46 receptor may be involved in the development of endometriosis.

Tissue-plasminogen activator effects on the phenotype of splenic myeloid cells in acute inflammation

Tissue-plasminogen activator (tPA) is a serine protease well known for its fibrinolytic function. Recent studies indicate that tPA could also modulate inflammation via plasmin generation and/or by receptor mediated signalling in vitro. However, the contribution of tPA in inflammatory processes in vivo has not been fully addressed. Therefore, using tPA-deficient mice, we have analysed the effect of lipopolysaccharide (LPS) challenge on the phenotype of myeloid cells including neutrophils, macrophages and dendritic cells (DCs) in spleen. We found that LPS treatment upregulated the frequency of major histocompatibility class two (MHCII+) macrophages but also, paradoxically, induced a deep downregulation of MHCII molecule level on macrophages and on conventional dendritic cells 2 (cDC2). Expression level of the CD11b integrin, known as a tPA receptor, was upregulated by LPS on MHCII+ macrophages and cDC2, suggesting that tPA effects could be amplified during inflammation. In tPA−/− mice under inflammatory conditions, expression of costimulatory CD86 molecules on MHCII+ macrophages was decreased compared to WT mice, while in steady state the expression of MHCII molecules was higher on macrophages. Finally, we reported that tPA deficiency slightly modified the phenotype of DCs and T cells in acute inflammatory conditions. Overall, our findings indicate that in vivo, LPS injection had an unexpectedly bimodal effect on MHCII expression on macrophages and DCs that consequently might affect adaptive immunity. tPA could also participate in the regulation of the T cell response by modulating the levels of CD86 and MHCII molecules on macrophages.

The Role of the CD28 Family Receptors in T-Cell Immunomodulation.

The CD28 family receptors include the CD28, ICOS (inducible co-stimulator), CTLA-4 (cytotoxic T-lymphocyte antigen-4), PD-1 (programmed cell death protein 1), and BTLA (B- and T-lymphocyte attenuator) molecules. They characterize a group of molecules similar to immunoglobulins that control the immune response through modulating T-cell activity. Among the family members, CD28 and ICOS act as enhancers of T-cell activity, while three others-BTLA, CTLA-4, and PD-1-function as suppressors. The receptors of the CD28 family interact with the B7 family of ligands. The cooperation between these molecules is essential for controlling the course of the adaptive response, but it also significantly impacts the development of immune-related diseases. This review introduces the reader to the molecular basis of the functioning of CD28 family receptors and their impact on T-cell activity.

Evaluation of the TLR3 involvement during Schistosoma japonicum-induced pathology

BackgroundDespite the functions of TLRs in the parasitic infections have been extensively reported, few studies have addressed the role of TLR3 in the immune response to Schistosoma japonicum infections. The aim of this study was to investigate the properties of TLR3 in the liver of C57BL/6 mice infected by S. japonicum.MethodsThe production of TLR3+ cells in CD4+T cells (CD4+CD3+), CD8+T cells (CD8+CD3+), γδT cells (γδTCR+CD3+), NKT cells (NK1.1+CD3+), B cells (CD19+CD3−), NK (NK1.1−CD3+) cells, MDSC (CD11b+Gr1+), macrophages (CD11b+F4/80+), DCs (CD11c+CD11b+) and neutrophils (CD11b+ Ly6g+) were assessed by flow cytometry. Sections of the liver were examined by haematoxylin and eosin staining in order to measure the area of granulomas. Hematological parameters including white blood cell (WBC), red blood cell (RBC), platelet (PLT) and hemoglobin (HGB) were analyzed. The levels of ALT and AST in the serum were measured using biochemical kits. The relative titers of anti-SEA IgG and anti-SEA IgM in the serum were measured by enzyme-linked immunosorbent assay (ELISA). CD25, CD69, CD314 and CD94 molecules were detected by flow cytometry.ResultsFlow cytometry results showed that the expression of TLR3 increased significantly after S. japonicum infection (P < 0.05). Hepatic myeloid and lymphoid cells could express TLR3, and the percentages of TLR3-expressing MDSC, macrophages and neutrophils were increased after infection. Knocking out TLR3 ameliorated the damage and decreased infiltration of inflammatory cells in infected C57BL/6 mouse livers.,The number of WBC was significantly reduced in TLR3 KO-infected mice compared to WT-infected mice (P < 0.01), but the levels of RBC, platelet and HGB were significantly increased in KO infected mice. Moreover, the relative titers of anti-SEA IgG and anti-SEA IgM in the serum of infected KO mice were statistically decreased compared with the infected WT mice. We also compared the activation-associated molecules expression between S.japonicum-infected WT and TLR3 KO mice.ConclusionsTaken together, our data indicated that TLR3 played potential roles in the context of S. japonicum infection and it may accelerate the progression of S. japonicum-associated liver pathology.

MiR-940 modulates CD47 to suppress biological functions of lung adenocarcinoma cells.

mir-940 and CD47 play regulatory and immunoregulatory roles in lung cancer. While previous study found that the expression of mir-940 decreased, associated with the increasing of CD47 in lung adenocarcinoma. However, their inherent correlations remain elusive. Herein, this experiment intends to search for the relevant molecular mechanisms regulating the biological function of non-small cell lung cancer. The cancer and adjacent tissue samples were collected from 20 pairs of newly diagnosed non-small cell lung cancer patients without applying radiotherapy and chemotherapy. We performed immunohistochemistry containing 45 lung adenocarcinoma tissues to investigate the relationship between the clinicopathological features and CD47 expression. The expressions of mir-940 and CD47 were detected by real-time quantitative polymerase chain reaction (qRT-PCR). Lung epithelial and lung adenocarcinoma (A549, H1299, GLC-82, PC-9) cell lines were cultured to detect the expression of mir-940 and CD47 molecules in each cell line. According to the expression situation, 2 cell lines were selected for mimic and siRNA transfection, and the transfection efficiency was also verified by qRT-PCR and western blot. CCK-8, transwell migration, transwell invasion, and colony formation assays were used to detect the changes in biological functions of lung adenocarcinoma cells after transfection, such as enhanced proliferation, migration, invasion, and cloning. The changes of related protein molecules after transfection were detected by western blot. The dual-luciferase experiment verified the targeting regulation relationship between mir-940 and CD47. Finally, flow cytometry analysis of apoptosis and cell cycle were carried out to detect apoptosis cells and change phase of cell cycle distribution. CD47 expression was not associated with clinicopathologic factors in lung adenocarcinoma. The proliferation, migration, invasion, and cloning abilities of lung adenocarcinoma cells were weakened after transfection with mir-940 mimic and siRNA-CD47. Overexpression of CD47 could promote proliferation, migration, invasion, cloning abilities, reduce apoptosis rate and attenuate the antitumor effect of mir-940 on lung adenocarcinoma. Dual luciferase experiments confirmed that mir-940 can target CD47 molecules. mir-940 can inhibit the biological function of lung adenocarcinoma cells by targeting CD47.