Decrease In Activation Markers Research Articles

Reduced monocyte proportions and responsiveness in convalescent COVID-19 patients.

The clinical manifestations of acute severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) suggest a dysregulation of the host immune response that leads to inflammation, thrombosis, and organ dysfunction. It is less clear whether these dysregulated processes persist during the convalescent phase of disease or during long COVID. We sought to examine the effects of SARS-CoV-2 infection on the proportions of classical, intermediate, and nonclassical monocytes, their activation status, and their functional properties in convalescent COVID-19 patients. Peripheral blood mononuclear cells (PBMCs) from convalescent COVID-19 patients and uninfected controls were analyzed by multiparameter flow cytometry to determine relative percentages of total monocytes and monocyte subsets. The expression of activation markers and proinflammatory cytokines in response to LPS treatment were measured by flow cytometry and ELISA, respectively. We found that the percentage of total monocytes was decreased in convalescent COVID-19 patients compared to uninfected controls. This was due to decreased intermediate and non-classical monocytes. Classical monocytes from convalescent COVID-19 patients demonstrated a decrease in activation markers, such as CD56, in response to stimulation with bacterial lipopolysaccharide (LPS). In addition, classical monocytes from convalescent COVID-19 patients showed decreased expression of CD142 (tissue factor), which can initiate the extrinsic coagulation cascade, in response to LPS stimulation. Finally, we found that monocytes from convalescent COVID-19 patients produced less TNF-α and IL-6 in response to LPS stimulation, than those from uninfected controls. SARS-CoV-2 infection exhibits a clear effect on the relative proportions of monocyte subsets, the activation status of classical monocytes, and proinflammatory cytokine production that persists during the convalescent phase of disease.

PI3K-δ Inhibition Influences T-Cell Populations and Anti-Tumoral Immune Function in Preclinical Models

Chronic lymphocytic leukemia (CLL) is characterized by continuously activated B-cell receptor (BCR) signaling. Phosphoinositide 3-kinase delta (PI3K-δ) is an important mediator of BCR signaling, which controls cell survival and proliferation through activation of AKT. The PI3K-δ inhibitor idelalisib (IDELA) is a highly effective treatment option for relapsed/refractory CLL patients. In the frontline setting, IDELA in treatment-naïve patients was associated with frequent adverse effects (AEs), such as an increased infection rate, colitis, and pneumonitis. Therefore, understanding the impact of PI3K-δ inhibition on the immune cells in the CLL microenvironment is of importance to identify novel treatment strategies to overcome these AEs.Methods and Results: In this study, we specifically focused on the effect of PI3K-δ inhibition on T-cell function in CLL using the Eµ-TCL1 adoptive transfer (TCL1 AT) model. In brief, Eµ-TCL1 tumor cells were transferred intravenously into syngeneic littermates, followed by continuous treatment after reaching a tumor load of 50% in peripheral blood. GS-649443, a tool compound with more favorable murine pharmacokinetic properties than IDELA, was used for PI3K-δ inhibition in vivo . Tumor load, as well as the immune microenvironment, was analyzed after 2 weeks of continuous treatment of the drug.Since PI3K-δ signaling has been previously demonstrated to be important for CD4+ regulatory T-cell (Treg)-mediated suppression of antitumor immunity, we studied the impact of PI3K-δ inhibition on differentiation and activation of Tregs. GS-649443 treatment resulted in a dramatic reduction in numbers of Foxp3+ CD25+ CD4+ Tregs, as well as a decrease in Treg maturation and activation markers (CD103, GITR, KLRG1, and CD69) compared to vehicle treatment.We further assessed the impact of PI3K-δ inhibition on TCL1 AT-induced changes in CD8+ T-cell differentiation and subset distribution. Notably, treatment with GS-649443 resulted in a dose-dependent decrease in effector T-cell numbers, accompanied by a decrease in activation markers such as CD69 and CD137. In line with these findings, effector cell proliferation, as measured by Ki-67, significantly decreased in GS-649443-treated mice. In accordance with that, degranulation capacity, granzyme B, and IFNγ production by CD8+ T cells was substantially reduced by GS-649443 treatment. These results were further confirmed in a treatment study with PI-3065, another PI3K-δ-specific inhibitor.To investigate a direct impact of PI3K-δ inhibition on CD8+ T cells, we performed in vitro studies with GS-649443. For that purpose, we used splenocytes from Nur77-GFP mice, a reporter mouse model for T-cell receptor (TCR) activity, and stimulated these with aCD3 antibody after pre-treatment with GS-649443 or vehicle. PI3K-δ inhibition led to a profound reduction of TCR signaling, which was accompanied by decreased expression of activation markers like CD69, CD44, CD25, and CD137 on CD8+ T-cells. Furthermore, GS-649443 pre-treatment resulted in significant dose-dependent decrease in CD8+ T-cell proliferation rates, as measured by CFSE dilution. These findings were confirmed using aCD3-stimulated human peripheral blood mononuclear cells that were pretreated with IDELA in vitro, which resulted in an impairment of CD8+ T-cell activation measured by CD69 and CD25 expression, as well as proliferation measured by CFSE dilution.Conclusion: This study clearly shows that PI3K-δ inhibition abrogates the immunosuppressive activity of Tregs in an in vivo model of CLL. Additionally, PI3K-δ inhibition of CD8+ T cells leads to decreased activation and proliferation of these cells, which might contribute to the immune-mediated AEs observed with IDELA when used as frontline treatment. DisclosuresStilgenbauer:Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding.

Strategies to reduce dendritic cell activation through functional biomaterial design

Dendritic cells play a key role in determining adaptive immunity, and there is growing interest in characterizing and manipulating the interactions between dendritic cells and biomaterial surfaces. Contact with several common biomaterials can induce the maturation of immature dendritic cells, but substrates that reduce dendritic cell maturation are of particular interest within the field of cell-based therapeutics where the goal is to reduce the immune response to cell-laden material carriers. In this study, we use a materials-based strategy to functionalize poly(ethylene glycol) hydrogels with immobilized immunosuppressive factors (TGF-β1 and IL-10) to reduce the maturation of immature dendritic cells. TGF-β1 and IL-10 are commonly employed as soluble factors to program dendritic cells in vitro, and we demonstrate that these proteins retain bioactivity towards dendritic cells when immobilized on hydrogel surfaces. Following stimulation with lipopolysaccharide (LPS) and/or cytokines, a dendritic cell line interacting with the surfaces of immunosuppressive hydrogels expressed reduced markers of maturation, including IL-12 and MHCII. The bioactivity of these immunomodulatory hydrogels was further confirmed with primary bone marrow-derived dendritic cells (BMDCs) isolated from non-obese diabetic (NOD) mice, as quantified by a decrease in activation markers and a significantly reduced capacity to activate T cells. Furthermore, by introducing a second signal to promote BMDC-material interactions combined with the presentation of tolerizing signals, the multifunctional PEG hydrogels were found to further increase signaling towards BMDCs, as evidenced by greater reductions in maturation markers.

Rituximab Directly Decreases T Cell Activation, Both In Vivo and In Vitro

AbstractAbstract 3935Rituximab, a chimeric anti-C20 monoclonal antibody, has been extensively used over the last decade for the therapy of B cell malignancies. Recent clinical data suggest that rituximab may affect T cell function, increasing the risk of T cell dependent infections in heavily-treated patients. The current study was designed to investigate the effect of rituximab on T cell activation and assess T cell function following the addition of rituximab to purified T cells. The T cell activation profile, dependent on rituximab administration, was evaluated in vivo and in vitro. Peripheral blood mononuclear cells (PBMCs) generated from B-cell non-Hodgkin lymphoma (NHL) patients prior and immediately after the administration of 375 mg/m2 rituximab, were examined for the expression of inflammatory cytokines. The in vitro studies were performed by using CD25 depleted PBMCs or B cell depleted T cells (CD3+CD25-CD19-). The obtained cells were stimulated with allogeneic dendritic cells (DCs), in the absence or presence or 2 mg/ml rituximab. T cell activation was evaluated using immunophenotypic markers, cytokine profile and T cell proliferation assay. Eight NHL patients participated in the study. The level of T cells expressing inflammatory cytokines was significantly decreased following the administration of a single dose of rituximab. T cells expressing IL-2 declined from a mean level of 26.5% to 11.5% and the level of IFN- γ decreased from 22% to 4.2%. Further administration of rituximab, up to 4 weekly doses, resulted in an additional decline in the amount of inflammatory cytokine producing T cells to a level of 1.4% for IL-2 and 3.5% for IFN-g. However, repeated evaluation, performed at 4 months after completing rituximab, showed restoration of the inflammatory population. In accord with this inhibitory effect, in vitro stimulation of T cells with allogeneic DCs, in the presence of rituximab, resulted in a significant decrease in activation markers (CD25, GITR and CTLA-4) (Table 1). These changes were accompanied by a marked reduction in inflammatory cytokine production and proliferative capacity. Of interest, these inhibitory effects were also obtained whilst using B cell depleted T cells (CD3+CD25-CD19-). In conclusion, rituximab administration results in a transient T cell inactivation, demonstrated through the reduction in inflammatory cytokine production and T cell proliferation capacity. This effect appears to be non-B cell dependent, being obtained in the absence of B cell in the culture, and may account for clinical observations in ameliorating T-cell dependent disorders, such as graft-versus-host disease. Disclosures:No relevant conflicts of interest to declare.

Activated human renal tubular cells inhibit autologous immune responses

Renal proximal tubule epithelial cells (PTEC) respond and contribute to the pathological process in a range of kidney diseases. Within this disease setting, PTEC up-regulate surface antigens which may enable them to act as non-professional antigen-presenting cells and become targets for infiltrating T cells in the context of disease and allograft rejection. In order to define, for the first time, whether PTEC modulate immune responses within the autologous human system, we monitored their interaction with T and B cells in the presence of stimuli which mimic immunological signalling. The expression of PTEC surface antigen in response to inflammatory mediators was monitored by flow cytometry. Purified T and B lymphocyte subsets and peripheral blood mononuclear cells were cultured in the presence or absence of autologous activated PTEC, and their responses to specific activators were monitored by proliferation, cytokine secretion and surface antigen expression. Some experiments were performed in the presence of blocking antibodies to PD-L1. The presence of activated primary autologous PTEC resulted in significantly decreased T- and B-cell proliferative responses, which were only partly mediated by programmed death ligand 1. This modulation was not induced by a decrease in activation markers or an increase in T regulatory cells but was accompanied by strong significant skewing of cytokine profiles. Significant decreases in gamma-interferon, interleukin-2 and tumour necrosis factor and increases in interleukin-4 were detected in the presence of PTEC, indicating that these cells induce a shift away from an inflammatory Th1 effector profile to a Th2 type profile. Human PTEC do modulate autologous immune responses. We hypothesize that such mechanisms may have developed to help dampen inflammatory responses and macrophage activation seen within kidney interstitium in many immune-mediated kidney diseases.

Innate Immune Function in Placenta and Cord Blood of Hepatitis C – Seropositive Mother-Infant Dyads

Vertical transmission accounts for the majority of pediatric cases of hepatitis C viral (HCV) infection. In contrast to the adult population who develop persistent viremia in ∼80% of cases following exposure, the rate of mother-to-child transmission (2–6%) is strikingly low. Protection from vertical transmission likely requires the coordination of multiple components of the immune system. Placenta and decidua provide a direct connection between mother and infant. We hypothesized that innate immune responses would differ across the three compartments (decidua, placenta and cord blood) and that hepatitis C exposure would modify innate immunity in these tissues. The study was comprised of HCV-infected and healthy control mother and infant pairs from whom cord blood, placenta and decidua were collected with isolation of mononuclear cells. Multiparameter flow cytometry was performed to assess the phenotype, intracellular cytokine production and cytotoxicity of the cells. In keeping with a model where the maternal-fetal interface provides antiviral protection, we found a gradient in proportional frequencies of NKT and γδ-T cells being higher in placenta than cord blood. Cytotoxicity of NK and NKT cells was enhanced in placenta and placental NKT cytotoxicity was further increased by HCV infection. HCV exposure had multiple effects on innate cells including a decrease in activation markers (CD69, TRAIL and NKp44) on NK cells and a decrease in plasmacytoid dendritic cells in both placenta and cord blood of exposed infants. In summary, the placenta represents an active innate immunological organ that provides antiviral protection against HCV transmission in the majority of cases; the increased incidence in preterm labor previously described in HCV-seropositive mothers may be related to enhanced cytotoxicity of NKT cells.

Clinical and immunological benefits from highly active antiretroviral therapy in spite of limited viral load reduction in HIV type 1 infection.

Both naive and memory T lymphocyte responses are lost during advanced HIV infection. Treatment with highly active antiretroviral therapy (HAART) is associated with an increase in T lymphocytes and a reduction in viral load. However, the viral response to HAART in patients with low levels of helper T lymphocytes and a high viral load is often not satisfactory. We investigated the capacity of long-term HAART to reconstitute the immune system in severely ill patients. A nonselected longitudinal patient population with high baseline viral levels and CD4(+) cells below 100 x 10(6)/liter were monitored for 2 years during HAART. Markers to estimate the therapeutic effects included viral levels and cell surface markers representing naive and memory T lymphocytes as well as activation markers, B cells, NK cells, and clinical events. After 2 years of treatment, viral load was reduced to undetectable levels in 55% (viral responders, vRs) and less than 1 log (median value) from baseline in 45% (viral low responders, vLRs). Elevated numbers of memory and naive CD4(+) and CD8(+) cells as well as a decrease in activation markers were seen in both vRs and vLRs. However, the magnitude was greater in vRs. No differences in the clinical outcome were observed between vRs and vLRs. We conclude that most patients, even in advanced stages of HIV disease, benefited from HAART. The magnitude of the response was related to good viral reduction, but even patients with poor viral reduction had a recovery of naive and memory CD4(+) and CD8(+) cells. Even a small reduction in viral load is thus of importance for health and potentially also for years of survival.

T cell changes after combined nucleoside analogue therapy in HIV primary infection.

To characterize the immune changes after treatment of acute HIV-1 infection with triple nucleoside analogue therapy. Immunological and virological parameters were monitored from day 0 to weeks 36-44 in eight patients [median CD4 cells = 451 cells/microl (range: 149-624), viral load = 4.8 log10 copies/ml (range: 6.5-3.3)] who started at time of primary HIV infection (PHI) a therapy including zidovudine (ZDV), didanosine (ddl), and lamivudine (3TC). Lymphoid subsets were evaluated on peripheral blood lymphocytes by four-colour flow cytometry using a panel of mAbs directed against differentiation and activation markers. We observed a median -2.1 (range: -1; -3.3) log10 copies/ml viral load decrease and a median +158 cells/microl (range: +7 to +316) CD4 cell count increase at week 4 reaching normal CD4 cell count values of 761 CD4 cells/microl (range: 389-1153) at weeks 36-44. Virus undetectability was obtained at week 24 for all subjects. A rapid CD4 T cell amplification involved both memory and naive CD4 T cells. This was associated with a very rapid and significant decrease in activation markers [human leukocyte antigen-DR (HLA-DR), CD38] on both CD4 and CD8 T cell subsets together with a CD8+CD28+ cell increase as early as week 4. These results show that early therapy with nucleoside analogues can correct the immunological abnormalities observed in CD4 and CD8 T cell subsets at the time of PHI. This early kinetics in T cell recovery appears to be faster than in established disease.