Role Of Cell-mediated Immunity Research Articles

Differential sensitivity of hemocyte subpopulations (Mytilus edulis) to aged polyethylene terephthalate micro- and nanoplastic particles

Bivalve hemocytes, particularly granulocytes and hyalinocytes, play a crucial role in cell-mediated immunity. However, their interactions with aged plastic particles, exhibiting altered properties that more closely resemble those in natural environments, remain largely underexplored. This study assesses the differential responses of hemocyte subpopulations (Mytilus edulis) to chemically aged polyethylene terephthalate (PET) microplastic (MPs) and nanoplastic (NPs) particles across multiple cellular effect endpoints. Particle characteristics were analyzed using Single Particle Extinction and Scattering, Raman Spectroscopy, Scanning Electron Microscopy, and Dynamic Light Scattering. In vitro experiments with aged PET MPs (1.9 µm) and NPs (0.68 µm) were conducted at three internally relevant concentrations: 10 (C1), 10³ (C2), and 10⁵ particles/mL (C3). Cellular responses were assessed by measuring lysosomal content stability, reactive oxygen species (ROS) production, cellular mortality, and morphological parameters using flow cytometry at 6, 12, 24, and 48 hours. Our findings provide mechanistic insights into the differential sensitivities of granulocytes and hyalinocytes to aged PET, influenced by particle size and concentration. Specifically, aged PET MPs and NPs induce distinct size and concentration-dependent patterns of lysosomal destabilization, coinciding with the loss of functional integrity. Elevated ROS levels were observed only in granulocytes and hyalinocytes exposed to high concentrations of aged PET NPs, underscoring the effects on oxidative stress. Both aged PET MPs and NPs induce significant increases in cellular mortality, particularly after 24 h of exposure at high concentrations. These findings reveal the complex cellular mechanisms underlying hemocyte functional impairment following exposure to aged PET particles under environmentally and biologically relevant conditions.

Peripheral Blood CD8+ T-Lymphocyte Immune Response in Benign and Subpopulations of Breast Cancer Patients.

Peripheral blood CD8+ T lymphocytes play a crucial role in cell-mediated immunity and tumor-related immune responses in breast cancer. In this study, label-free quantification analysis and gene set enrichment analysis (GSEA) of CD8+ T lymphocytes in the peripheral blood of benign patients and patients with different breast cancer (BC) subtypes, i.e., luminal A, luminal B, and triple-negative breast cancer (TNBC), were performed using nano-UHPLC and Orbitrap mass spectrometry. Differential protein expression in CD8+ T lymphocytes revealed significant downregulation (log2 FC ≥ 0.38 or ≤-0.38, adj. p < 0.05), particularly in proteins involved in cytotoxicity, cytolysis, and proteolysis, such as granzymes (GZMs) and perforin 1 (PRF1). This downregulation was observed in the benign group (GZMH, GZMM, and PRF1) and luminal B (GZMA, GZMH) subtypes, whereas granzyme K (GZMK) was upregulated in TNBC in comparison to healthy controls. The RNA degradation pathway was significantly downregulated (p < 0.05, normalized enrichment score (NES) from -1.47 to -1.80) across all BC subtypes, suggesting a potential mechanism for regulating gene expression during T cell activation. Also, the Sm-like proteins (LSM2, LSM3, and LSM5) were significantly downregulated in the RNA degradation pathway. Proteomic analysis of CD8+ T lymphocytes in peripheral blood across different breast cancer subtypes provides a comprehensive view of the molecular mechanisms of the systemic immune response that can significantly contribute to advancements in the diagnosis, treatment, and prognosis of this disease.

Specific T-cell subsets have a role in anti-viral immunity and pathogenesis but not viral dynamics or onwards vector transmission of an important livestock arbovirus.

Bluetongue virus (BTV) is an arthropod-borne Orbivirus that is almost solely transmitted by Culicoides biting midges and causes a globally important haemorrhagic disease, bluetongue (BT), in susceptible ruminants. Infection with BTV is characterised by immunosuppression and substantial lymphopenia at peak viraemia in the host. In this study, the role of cell-mediated immunity and specific T-cell subsets in BTV pathogenesis, clinical outcome, viral dynamics, immune protection, and onwards transmission to a susceptible Culicoides vector is defined in unprecedented detail for the first time, using an in vivo arboviral infection model system that closely mirrors natural infection and transmission of BTV. Individual circulating CD4+, CD8+, or WC1+ γδ T-cell subsets in sheep were depleted through the administration of specific monoclonal antibodies. The absence of cytotoxic CD8+ T cells was consistently associated with less severe clinical signs of BT, whilst the absence of CD4+ and WC1+ γδ T cells both resulted in an increased clinical severity. The absence of CD4+ T cells also impaired both a timely protective neutralising antibody response and the production of IgG antibodies targeting BTV non-structural protein, NS2, highlighting that the CD4+ T-cell subset is important for a timely protective immune response. T cells did not influence viral replication characteristics, including onset/dynamics of viraemia, shedding, or onwards transmission of BTV to Culicoides. We also highlight differences in T-cell dependency for the generation of immunoglobulin subclasses targeting BTV NS2 and the structural protein, VP7. This study identifies a diverse repertoire of T-cell functions during BTV infection in sheep, particularly in inducing specific anti-viral immune responses and disease manifestation, and will support more effective vaccination strategies.

TLR2, TLR3, TLR4, TLR9 and TLR11 expression on effector CD4+ T-cell subsets in Leishmania donovani infection

T-cells play a central role in cell-mediated immunity. While activation of T-cells is major histocompatibility-restricted, the Toll-like receptors (TLRs)- a family of proteins that recognize conserved molecular patterns present on the pathogens-are not well-studied for their expression and function in T-cells. As any association of TLR expression profiles with an effector T-cell subset is unknown, we analyze BALB/c mice-derived CD4+ and CD8+ T-cells’ TLR expression profiles. We report: CD4+t-bet+ T-cells are frequent in TLR2LowTLR3HighTLR4Low subpopulation, CD4+GATA3+ T-cells are frequent within the cells with intermediate expression of TLR2, TLR3, TLR4 and TLR11, CD4+FoxP3+ T-cells in TLR2HighTLR3High cells whereas CD4+RORγt + T-cells are frequent in TLR2LowTLR3LowTLR4LowTLR11Low cells. CD4+ effector T-cell subsets may therefore show association with TLRs- TLR3, in particular-expression. In Leishmania donovani infection in BALB/c mice, TLR3 expression on both CD4+ and CD8+ T-cells is reduced. Poly-I:C, a TLR3 ligand, do not have any distinctive effects on the CD4+ effector T-cell subsets. These data suggest that TLRs on T-cells may not function as a primary receptor that controls T-cell function but their distinctive expression profiles on different T-cell subsets suggest plausible immunomodulatory role.

Consensus Algorithm for Calculation of Protein Binding Affinity using Multiple Models

The major histocompatibility complex (MHC) molecules, which bind peptides for presentation on the cell surface, play an important role in cell-mediated immunity. In light of developing databases and technologies over the years, significant progress has been made in research on peptide binding affinity calculation. Several in techniques have been developed to predict peptide binding to MHC class I. Most of the research on MHC Class I due to its nature brings better performance and more. Considering the use of different methods and different technologies, and the approach of similar methods on different proteins, a classification was created according to the binding affinity of protein peptides. For this classification, MHC Class I was studied using the MHCflurry, NetMHCPan, NetMHC, NetMHCCons and ssmpmbec. In these simulations conducted within the scope of this thesis, no overall superiority was observed between the models. It has been determined that they are superior to each other in various points. Getting the best results may vary depending on the multiple uses of models. The important thing is to recognize the data and act with the appropriate model. But even that doesn’t make a huge difference. Since the consensus approach is directly related to the models, the better the models, the better. Xix

Advancements in Cancer Immunotherapies.

Recent work has suggested involvement of the immune system in biological therapies specifically targeting tumor microenvironment. Substantial advancement in the treatment of malignant tumors utilizing immune cells, most importantly T cells that play a key role in cell-mediated immunity, have led to success in clinical trials. Therefore, this article focuses on the therapeutic approaches and developmental strategies to treat cancer. This review emphasizes the immunomodulatory response, the involvement of key tumor-infiltrating cells, the mechanistic aspects, and prognostic biomarkers. We also cover recent advancements in therapeutic strategies.

Quantitative serum determination of CD3, CD4, CD8, CD16, and CD56 in women with primary infertility: The role of cell-mediated immunity.

Objective:Cellular adaptive immunity plays an essential role in the etiology of primary infertility. This study aimed to measure the T-lymphocyte subpopulations and natural killer (NK) cells in infertile women compared with healthy ones.Materials and Methods:From January to September 2021, we conducted this cross-sectional study among women with primary infertility, and healthy women were referred to Isfahan Fertility and Infertility Center affiliated with Najafabad University of medical sciences in Isfahan, Iran for immunological investigations. For each person, we determined quantitative serum measurements of CD3, CD4, CD8, CD4/CD8, CD16, CD56, and CD56+16.Results:This study included one hundred and fifty-one infertile women with a mean age of 31.4±4.7 years and 46 healthy women with a mean age of 31.5±3.4 years. Compared to the controls, immunophenotyping findings in infertile patients revealed a significant drop in CD8 T cells [p=0.01, 95% confidence interval (CI) 0.53 to 4.57] and the percentage of CD 56 NK cells (p=0.005, 95% CI 0.74 to 4.03) in infertile patients.Conclusion:Despite having a normal quantity of CD3 T cells, infertile women had lower CD8 T cells and CD56 NK cells than the controls. More studies are needed to confirm the role of cell-mediated assessments as a screening test in patients with primary infertility.

The role of cell-mediated immunity against influenza and its implications for vaccine evaluation

Influenza vaccines remain the most effective tools to prevent flu and its complications. Trivalent or quadrivalent inactivated influenza vaccines primarily elicit antibodies towards haemagglutinin and neuraminidase. These vaccines fail to induce high protective efficacy, in particular in older adults and immunocompromised individuals and require annual updates to keep up with evolving influenza strains (antigenic drift). Vaccine efficacy declines when there is a mismatch between its content and circulating strains. Current correlates of protection are merely based on serological parameters determined by haemagglutination inhibition or single radial haemolysis assays. However, there is ample evidence showing that these serological correlates of protection can both over- or underestimate the protective efficacy of influenza vaccines. Next-generation universal influenza vaccines that induce cross-reactive cellular immune responses (CD4+ and/or CD8+ T-cell responses) against conserved epitopes may overcome some of the shortcomings of the current inactivated vaccines by eliciting broader protection that lasts for several influenza seasons and potentially enhances pandemic preparedness. Assessment of cellular immune responses in clinical trials that evaluate the immunogenicity of these new generation vaccines is thus of utmost importance. Moreover, studies are needed to examine whether these cross-reactive cellular immune responses can be considered as new or complementary correlates of protection in the evaluation of traditional and next-generation influenza vaccines. An overview of the assays that can be applied to measure cell-mediated immune responses to influenza with their strengths and weaknesses is provided here.

O-030 Disturbed homeostasis of regulatory T-cells and imbalanced signal transducer and activator of transcription 5 (STAT5) in women with ovarian endometriosis

Abstract Study question Do women with endometriosis have altered cytokine signalling and regulatory T-cell (Treg) homeostasis compared to controls? Summary answer Altered homeostasis of circulating Treg cells seems to be associated with STAT5 phosphorylation, which is significantly increased in women with ovarian endometriosis compared to controls. What is known already Cytokine signaling influences homeostasis of regulatory T-cells (Treg), which play a crucial role in cell-mediated immunity and are responsible for certain immune disorders. Endometriosis is a condition associated with altered immune response to endometrial cells. While some animal studies have shown that Treg deficiency facilitates ectopic implantation of endometriosis, a recent study performed in a small number of women with endometriosis has shown the decreased proportion of a specific-activated subset of Treg cells in the endometrioma compared to controls. There is no data characterising cytokine signalling in circulating Treg subsets, which could underlie their altered homeostasis in women with endometriosis. Study design, size, duration A prospective cohort study including 14 women with endometriosis and 10 controls at a university-based tertiary care centre for endometriosis. The data were collected from August 2020 to September 2021. Serum blood samples were collected in the early follicular phase of the menstrual cycle, in women not using any hormonal treatment. Early follicular steroid hormone levels were measured. A detailed expert ultrasound examination was performed to diagnose and accurately map endometriotic lesions. Participants/materials, setting, methods We investigated signal transducer and activator of transcription (STAT) signalling in peripheral blood Treg subsets, including CD45RA+FOXP3low+ naive Tregs (nTregs). Expression of proliferation marker Ki-67 and STAT activation also in FOXP3- conventional (Tcon) CD4+T-cells was monitored, hypothesising that defects in this pathway could be associated with altered Treg/Tcon homeostasis in endometriosis. Immunophenotyping and flow cytometry methods with phospho-specific antibodies that allow analysis of basal STAT activation/phosphorylation also in rare subsets of Treg cells were used. Main results and the role of chance Among women with endometriosis, 5 had evidence of ovarian and deep infiltrating endometriosis (DIE), 5 had only ovarian endometriosis and 4 had only evidence of DIE. Groups were comparable in their clinical characteristics, there was no difference in the median age of women with endometriosis and controls; 31 (range 23-45) vs. 34 (range 27-39). We demonstrated significantly higher levels of phosphorylated STAT5 (pSTAT5) in nTreg from women with ovarian endometriosis compared to controls (p = 0.026). To assess the homeostatic balance between the nTreg and Tcon, we compared pSTAT5 levels between these cell subsets in each patient. STAT5 phosphorylation was significantly higher in Tcon than in nTreg subset (p &amp;lt; 0.001). Considering STAT5 activation in nTreg subset, this was positively correlated with the proportion of nTreg among CD4+T-cells (rs=0.54, p = 0.027) in all women with endometriosis. We also found a significant positive correlation between pSTAT5 levels in Tcon from women with endometriosis and the percentage of Ki-67+ cells in the Tcon subset (rs=0.66, p = 0.005) as well a significant correlation between luteinizing hormone (LH) levels and pSTAT5 in Tcon subset of CD4 cells (rs=0.71, p = 0.02). This implies LH-dependent imbalanced STAT5 signalling that could be responsible for increased Tcon homeostatic proliferation. Limitations, reasons for caution Our sample size is relatively small and the findings should be validated in future prospective studies. Due to the sample size, there is a risk for Type I error, although the results are coherent in indicating disturbance of Treg homeostasis. Wider implications of the findings Our study suggests that altered STAT5 signalisation may be associated with disturbed nTreg/Tcon homeostasis, which seems to be mostly prominent in women with ovarian endometriosis. If confirmed by further studies in the future, targeted treatment options could be developed to supress progress of endometriosis in this group of women. Trial registration number not applicable

Toll-Like Receptor Signaling and Its Role in Cell-Mediated Immunity.

Innate immunity is the first defense system against invading pathogens. Toll-like receptors (TLRs) are well-defined pattern recognition receptors responsible for pathogen recognition and induction of innate immune responses. Since their discovery, TLRs have revolutionized the field of immunology by filling the gap between the initial recognition of pathogens by innate immune cells and the activation of the adaptive immune response. TLRs critically link innate immunity to adaptive immunity by regulating the activation of antigen-presenting cells and key cytokines. Furthermore, recent studies also have shown that TLR signaling can directly regulate the T cell activation, growth, differentiation, development, and function under diverse physiological conditions. This review provides an overview of TLR signaling pathways and their regulators and discusses how TLR signaling, directly and indirectly, regulates cell-mediated immunity. In addition, we also discuss how TLR signaling is critically important in the host’s defense against infectious diseases, autoimmune diseases, and cancer.

Examining the Effect of Kindlin-3 Binding Site Mutation on LFA-1-ICAM-1 Bonds by Force Measuring Optical Tweezers.

Integrins in effector T cells are crucial for cell adhesion and play a central role in cell-mediated immunity. Leukocyte adhesion deficiency (LAD) type III, a genetic condition that can cause death in early childhood, highlights the importance of integrin/kindlin interactions for immune system function. A TTT/AAA mutation in the cytoplasmic domain of the β2 integrin significantly reduces kindlin-3 binding to the β2 tail, abolishes leukocyte adhesion to intercellular adhesion molecule 1 (ICAM-1), and decreases T cell trafficking in vivo. However, how kindlin-3 affects integrin function in T cells remains incompletely understood. We present an examination of LFA-1/ICAM-1 bonds in both wild-type effector T cells and those with a kindlin-3 binding site mutation. Adhesion assays show that effector T cells carrying the kindlin-3 binding site mutation display significantly reduced adhesion to the integrin ligand ICAM-1. Using optical trapping, combined with back focal plane interferometry, we measured a bond rupture force of 17.85 ±0.63 pN at a force loading rate of 30.21 ± 4.35 pN/s, for single integrins expressed on wild-type cells. Interestingly, a significant drop in rupture force of bonds was found for TTT/AAA-mutant cells, with a measured rupture force of 10.08 ± 0.88pN at the same pulling rate. Therefore, kindlin-3 binding to the cytoplasmic tail of the β2-tail directly affects catch bond formation and bond strength of integrin–ligand bonds. As a consequence of this reduced binding, CD8+ T cell activation in vitro is also significantly reduced.

Pasteurella multocida PlpE Protein Polytope as a Potential Subunit Vaccine Candidate.

Pasteurella multocida is the causative agent of a range of animal, and occasionally human, diseases. Problems with antimicrobial treatment of P. multocida highlight the need to find other possible ways, such as prophylaxis, to manage infections. Current vaccines against P. multocida include inactivated bacteria, live attenuated and nonpathogenic bacteria; they have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence. Using bioinformatics approaches, potentially immunogenic and protective epitopes were identified and merged to design the most optimally immunogenic triple epitope PlpE fusion protein of P. multocida as a vaccine candidate. This triple epitope (PlpE1 + 2 + 3) was cloned into the pBAD/gIII A plasmid (pBR322-derived expression vectors designed for regulated, secreted recombinant protein expression and purification in Escherichia coli), expressed in Top 10 E. coli and purified in denatured form using Ni-NTA chromatography and 8 M urea. The immunogenicity of the purified proteins in BALB/c mice was assayed by measuring immunoglobulin G (IgG) responses. The protection potential was evaluated by challenging with 10 LD50 of serotype A:1, X-73 strain of P. multocida and compared with commercially available inactivated fowl cholera vaccine and PlpE protein. IgG levels elicited by the polytope fusion protein of P. multocida PlpE were higher than both commercially available inactivated fowl cholera vaccine and PlpE protein. Surprisingly, protection was independent of IgG level; commercially available inactivated fowl cholera vaccine (100% protection) was more protective than the polytope fusion protein (69% protection) and PlpE protein (69% protection). These results also confirm that IgG level is not a reliable indicator of protection. Further studies to evaluate the other antibody classes, such as immunoglobulin A or M, are required. The role of cell-mediated immunity should also be considered as a potential protection pathway.

In vitro and in vivo anti-Cryptosporidium and anti-inflammatory effects of Aloe vera gel in dexamethasone immunosuppressed mice

Cryptosporidiosis has been considered as a serious diarrheal disease, especially in immunodeficient patients, where they failed to clear the infection leading to several consequences of infection (i.e death). The role of cell mediated immunity in clearing the infection was demonstrated by the increased susceptibility of HIV/AIDS patients to infection. To date, no specific treatment has been proven for cryptosporidiosis in immunodeficient patients. The study aimed to evaluate the efficacy of Aloe vera gel for the treatment of cryptosporidiosis in immunocompetent and dexamethasone immunosuppressed mice in comparison to that of nitazoxanide. Mice were orally administrated with Aloe vera gel, in a daily dose of 250 mg/L in drinking water, for 14 consecutive days post infection. Parasitological, molecular and immunological measurements were recorded on the 7th, 14th, 21st and 32nd days post infection. Our in vitro results showed that 250 mg/L of prepared gel achieved the highest parasitic reduction. The body weights of Aloe vera treated mice on the 21st and 32nd day post infection, either in immunocompetent or immunosuppressed groups, were nearly the same as those of their corresponding control groups. Aloe vera gel succeeded in clearing cryptosporidiosis with a percent reduction of 100% in immunocompetent mice and 99.67% in immunosuppressed mice. The anti-inflammatory effect of Aloe vera reduced the levels of IFN-γ, IL-4, -6 and -17. The success of Aloe vera gel, in clearing cryptosporidiosis in immunosuppressed mice, was obvious either from the reduction of Cryptosporidium DNA or the oocysts in stool samples; and from the improvement of histopathological sections.

25052 Ustekinumab-induced disseminated verrucosis

Ustekinumab is a biologic agent with FDA approval for the treatment of moderate-to-severe plaque psoriasis, psoriatic arthritis, ulcerative colitis, and Crohn disease. It functions to inhibit interleukin (IL)-12 and IL-23, proinflammatory cytokines that not only play an important role in autoinflammatory and autoimmune disorders but also play a role in cell-mediated immunity against viral, bacterial, and fungal pathogens. Due to its immunosuppressive effect, ustekinumab may increase the risk of infection and reactivation of latent infections, including human papillomavirus.

Characterisation and analysis of IFN-gamma producing cells in rainbow trout Oncorhynchus mykiss

IFN-γ is one of the key cytokines involved in Th1 immune responses. It is produced mainly by T cells and NK cells, which drive both innate and adaptive responses to promote protection against infections. IFN-γ orthologues have been discovered to be functionally conserved in fish, suggesting that type I immunity is present in early vertebrates. However, few studies have looked at IFN-γ protein expression in fish and its role in cell mediated immunity due to a lack of relevant tools. In this study, four monoclonal antibodies (mAbs) V27, N2, VAB3 and V91 raised against short salmonid IFN-γ peptides were developed and characterised to monitor IFN-γ expression. The results show that the IFN-γ mAbs specifically react to their peptide immunogens, recognise E. coli produced recombinant IFN-γ protein and rainbow trout IFN-γ produced in transfected HEK 293 cells. The mAb VAB3 was used further, to detect IFN-γ at the cellular level after in vitro and in vivo stimulation. In flow cytometry, a basal level of 3–5% IFN-γ secreting cells were detected in peripheral blood leucocytes (PBL), which increased significantly when stimulated in vitro with PAMPs (Aeromonas salmonicida bacterin), a mitogen (PHA) and recombinant cytokine (IL-2). Similarly, after injection of live bacteria (Aeromonas salmonicida) or poly I:C the number of IFN-γ+ cells increased in the lymphoid population of PBL, as well as in the myeloid population after infection, with the myeloid cells increasing substantially after both treatments. Immunohistochemistry was used to visualise the IFN-γ+ cells in spleen and head kidney following vaccination, which increased in intensity of staining and number relative to tissue from saline-injected control fish. These results show that several types of cells can produce IFN-γ in trout, and that they increase following infection or vaccination, and likely contribute to immune protection. Hence monitoring IFN-γ producing cells/protein secretion may be an important means to assess the effectiveness of Th1 responses and cell mediated immunity in fish.

Changes in Cell-Mediated Immunity (IFN-γ and Granzyme B) Following Influenza Vaccination.

Interferon gamma (IFN-γ) is considered a key moderator of cell-mediated immunity. However, little is known about its association with granzyme B, which plays an important role in the effector function of cytotoxic T lymphocytes (CTLs). In the present study, we collected blood samples from 32 healthy adults before and after vaccination with inactivated influenza vaccine in 2017/18 to measure the levels of IFN-γ and granzyme B, which play roles in cell-mediated immunity, and hemagglutination inhibition (HAI) antibody, which plays a role in humoral immunity. The levels of IFN-γ and granzyme B were significantly correlated both before and after vaccination. Furthermore, the post-vaccine fold increases in the IFN-γ and granzyme B levels were significantly correlated. The levels of IFN-γ and granzyme B decreased five months after vaccination in more than half of the subjects who exhibited an increase in IFN-γ and granzyme B at two weeks post-vaccination. This is the first study to investigate the correlation between IFN-γ and granzyme B levels following influenza vaccination. Our study suggests that both IFN-γ and granzyme B can be used as markers of cell-mediated immunity.

Cytokine saga in visceral leishmaniasis

In humans, infection with Leishmania manifests into a spectrum of diseases. The manifestation of the diseases depend on the resultant evasion of the parasite to immune responses namely by macrophages, which is an exclusive host of Leishmania. The B cells valiantly mount antibody responses, however, to no avail as the Leishmania parasites occupy the intracellular niches of the macrophages and subvert the immune response. Extensive studies have been documented on the role of cell-mediated immunity (CMI) in protection and counter survival strategies of the parasites leading to downregulation of CMI. The present review attempts to discuss the cytokines in progression or resolution of visceral form of leishmaniasis or kala-azar, predominantly affecting the Indian subcontinent. The components/cytokine(s) responsible for the regulation of the critical balance of T helper cells and their subsets have been discussed in the perspective.Therefore, any strategy involving the treatment of visceral leishmania (VL) needs to consider the balance and regulation of T cell function.

Cellular Uptake of siRNA-Loaded Nanocarriers to Knockdown PD-L1: Strategies to Improve T-cell Functions.

T-cells are a type of lymphocyte (a subtype of white blood cells) that play a central role in cell-mediated immunity. Currently, adoptive T-cell immunotherapy is being developed to destroy cancer cells. In this therapy, T-cells are harvested from a patient’s blood. After several weeks of growth in culture, tumor-specific T-cells can be reinfused into the same cancer patient. This technique has proved highly efficient in cancer treatment. However, there are several biological processes that can suppress the anti-cancer responses of T-cells, leading to a loss of their functionality and a reduction of their viability. Therefore, strategies are needed to improve T-cell survival and their functions. Here, a small interfering RNA (siRNA)-loaded nanocarrier was used to knockdown PD-L1, one of the most important proteins causing a loss in the functionality of T-cells. The biocompatibility and the cellular uptake of siRNA-loaded silica nanocapsules (SiNCs) were investigated in CD8+ T-cells. Then, the PD-L1 expression at protein and at mRNA levels of the treated cells were evaluated. Furthermore, the effect of the PD-L1 knockdown was observed in terms of cell proliferation and the expression of specific biomarkers CD25, CD69 and CD71, which are indicators of T-cell functions. The results suggest that this siRNA-loaded nanocarrier showed a significant potential in the delivery of siRNA into T-cells. This in turn resulted in enhanced T-cell survival by decreasing the expression of the inhibitory protein PD-L1. Such nanocarriers could, therefore, be applied in adoptive T-cell immunotherapy for the treatment of cancer.

Modeling and simulation of spatial-temporal calcium distribution in T lymphocyte cell by using a reaction-diffusion equation.

T lymphocytes are white blood cells that play a central role in cell-mediated immunity. Ca has its major signaling function when it is elevated in the cytosolic compartment. The free cytosolic Ca dynamics plays a very important role in the activation, and fate decision process in the T lymphocytes. Here, we develop a quantitative spatio-temporal Ca dynamic model which includes, the Ca releasing channels ER leak and voltage-gated Ca channel, buffering and re-uptaking mechanism in the T lymphocytes. In this model, the cell is represented as a circular-shaped geometrical domain. This representation introduces modeling flexibility needed for detailed representation of the properties of Ca dynamics in the cell including important parameters. The proposed mathematical model is solved using a finite difference method and the finite element method. Appropriate initial and boundary conditions are incorporated in the model based on biophysical conditions of the problem. Computer simulations in MATLAB R2010a are employed to investigate mathematical models of reaction-diffusion equation. The estimation is based on reaction-diffusion equation associated with biophysical and biochemical reactions taking place in the cell. From our results, it is observed that, the coordinated combination of the incorporated parameters plays a significant role in Ca regulation in T lymphocytes. ER leak and voltage-gated Ca channel provides the necessary Ca to the cell when required for its proper functioning, while on the other side buffers and Na/Ca exchanger makes balance in the Ca concentration, so as to prevent the cell from death as higher concentration for longer time is harmful for the cell and can cause cell death. These results have been used to study the relationship of Ca concentration with parameters like VGCC, Na/Ca exchanger, ER leak and buffers. The significance of the study reveals that there is a significant variation in Ca profiles due to the effect of VGCC, Na/Ca exchanger, ER leak, and buffers. The results give us better insights of coordinated effect of VGCC, Na/Ca exchanger, ER leak, and buffers on Ca distribution in T lymphocytes. T lymphocytes are the primary host cells to receive the viral infections which transmits the signal then to other cell types. The proper quantity of Ca concentration makes T lymphocytes more active and healthier to fight the infection properly and can protect the immune system from various fatal viral infections. Thus, the application of the study lies in the field of immunology to protect a susceptible from various viral infectious diseases like HIV, HBV, HINI, etc. by strengthening the immune system. The outcomes of the study reveal that the applied finite element method is computationally very strong and effective to analyze differential equations that arise in Ca dynamics.

Abstract A55: Uncovering the mechanism of Trib1 in cancer immunotherapy

Abstract T cells provide a fundamental role in cell-mediated immunity, but during chronic antigen exposure such as in the case of cancer, T cells begin to lose effector function and undergo exhaustion. Our goal is to find a target within T cells that could be manipulated to prolong T-cell function and reduce tumor growth. Initial experiments focused on the pseudokinase Trib1, as data suggest that Trib1 acts as a negative regulator of T-cell activation. T cells isolated from mice in which Trib1 was specifically ablated within the T-cell compartment produced more IL-2 compared to wild-type T cells. T cells lacking Trib1 also proliferated more than wild-type T cells. In mice lacking Trib1 in grafted tumor models, tumor growth was reduced. These observations suggest that Trib1 restricts T-cell activation and function. Trib1 is a pseudokinase that is thought to act as a scaffolding molecule to facilitate protein interactions. To understand the mechanism by which Trib1 regulates T-cell function, we performed a proteomic screen to identify interacting partners. The screen was performed in Jurkat cells, an immortalized human T-cell line, expressing a FLAG/strep tagged Trib1. A FLAG pulldown was performed to isolate Trib1, and interacting partners were identified by mass spectrometry. The screen revealed two interacting partners: RFWD2, an E3 ubiquitin ligase also known as COP1, and DET1, a binding partner of COP1. These results provide evidence that Trib1 interacts with COP1 in T cells and suggest that this interaction might be important in the regulation of T-cell function and the regression of tumor growth. Note:This abstract was not presented at the conference. Citation Format: Franklin Iheanacho, Sarah J. Stein, Kelly S. Rome, Martha S. Jordan, Warren S. Pear. Uncovering the mechanism of Trib1 in cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A55.