Microglia/Macrophages and CD4+CD25+ T Cells Enhance the Ability of Injury-Activated Lymphocytes to Reduce Traumatic Optic Neuropathy In Vitro

Inflammation after acute CNS injury plays a dual role. The interplay between immune cells and inflammatory mediators is critical to the outcome of injured neurons. Microglia/macrophages are the first sensors and regulators of the immune response. We previously found that the enhancement of macrophages on neuron survival does not persist in thymectomized rats. How T lymphocytes and macrophages interact and benefit neuron survival is not fully elucidated. To this point, we introduce and characterize a cell-retina co-culture model that mimics the recruitment of peripheral lymphocytes at the injury site. Three-day post-optic nerve transection (ONT) in Fischer 344 rats, transected retinas were co-cultured with either peripheral lymph node-derived lymphocytes (injury-activated) or from intact rats as the control. The injury-activated lymphocytes preserved retinal ganglion cells (RGCs) and caused extensive retina microglial/macrophage infiltration. CD4+CD25+ T cells were upregulated in the injury-activated lymphocytes and increased RGC survival, suggesting that CD4+CD25+ T cells suppressed the cytotoxicity of control lymphocytes. When microglia/macrophages were depleted by clodronate, neuron loss was more extensive, the cytotoxicity of control lymphocytes on RGCs was alleviated, and the neuroprotective effect of injury-activated lymphocytes remain unchanged Cytokine detection showed an increase in IL-6 and TNF-α levels that were reduced with microglia/macrophage depletion. Our results suggest that microglial/macrophage infiltration into axotomized retinas promotes RGC survival by secreting cytokines to induce CD4+CD25+ T cells and suppress T cell-mediated RGC toxicity. These findings reveal a specific role for microglia/macrophage and CD4+CD25+ T cells in inflammation after CNS injury, thereby adding to the mechanistic basis for the development of microglial/macrophage modulation therapy for traumatic CNS injury.

CD30-Positive Extracellular Vesicles Enable the Targeting of CD30-Negative DLBCL Cells by the CD30 Antibody-Drug Conjugate Brentuximab Vedotin

CD30, a member of the TNF receptor superfamily, is selectively expressed on a subset of activated lymphocytes and on malignant cells of certain lymphomas, such as classical Hodgkin Lymphoma (cHL), where it activates critical bystander cells in the tumor microenvironment. Therefore, it is not surprising that the CD30 antibody-drug conjugate Brentuximab Vedotin (BV) represents a powerful, FDA-approved treatment option for CD30+ hematological malignancies. However, BV also exerts a strong anti-cancer efficacy in many cases of diffuse large B cell lymphoma (DLBCL) with poor CD30 expression, even when lacking detectable CD30+ tumor cells. The mechanism remains enigmatic. Because CD30 is released on extracellular vesicles (EVs) from both, malignant and activated lymphocytes, we studied whether EV-associated CD30 might end up in CD30– tumor cells to provide binding sites for BV. Notably, CD30+ EVs bind to various DLBCL cell lines as well as to the FITC-labeled variant of the antibody-drug conjugate BV, thus potentially conferring the BV binding also to CD30– cells. Confocal microscopy and imaging cytometry studies revealed that BV binding and uptake depend on CD30+ EVs. Since BV is only toxic toward CD30– DLBCL cells when CD30+ EVs support its uptake, we conclude that EVs not only communicate within the tumor microenvironment but also influence cancer treatment. Ultimately, the CD30-based BV not only targets CD30+ tumor cell but also CD30– DLBCL cells in the presence of CD30+ EVs. Our study thus provides a feasible explanation for the clinical impact of BV in CD30– DLBCL and warrants confirming studies in animal models.

Immune Alveolitis in Interstitial Lung Disease: an Attractive Cytological Profile in Immunocompromised Patients.

Background: Bronchoalveolar lavage (BAL) is a major diagnostic tool in interstitial lung disease (ILD). Its use remains largely quantitative, usually focused on cell differential ratio. However, cellular morphological features provide additional valuable information. The significance of the "immune alveolitis" cytological profile, characterized by lymphocytic alveolitis with activated lymphocytes and macrophages in epithelioid transformation or foamy macrophages desquamating in cohesive clusters with lymphocytes, remains unknown in ILD. Our objective was to describe patients’ characteristics and diagnoses associated with an immune alveolitis profile in undiagnosed ILD.Methods: We performed a monocentric retrospective observational study. Eligible patients were adults undergoing diagnostic exploration for ILD and whose BAL fluid displayed an immune alveolitis profile. For each patient, we collected clinical, radiological and biological findings as well as the final etiology of ILD. Results: Between January 2012 and December 2018, 249 patients were included. Mean age was 57±16 years, 140 patients (56%) were men, and 65% of patients were immunocompromised. The main etiological diagnosis was Pneumocystis pneumonia (PCP) (24%), followed by drug-induced lung disease (DILD) (20%), viral pneumonia (14%) and hypersensitivity pneumonitis (HP) (10%). All PCP were diagnosed in immunocompromised patients while HP was found in only 8% of this subgroup. DILD and viral pneumonia were also commonly diagnosed in immunocompromised patients (94% and 80%, respectively).Conclusions: Our study highlights the additional value of BAL qualitative description in ILD. We suggest incorporating the immune alveolitis profile for the diagnosis and management of ILD, especially in immunocompromised patients, since it guides towards specific diagnoses.

Analysis of Same Selected Immunomodulatory Properties of Chorionic Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) represent a population of adherent cells that can be isolated from multiple adult tissues. MSCs have immunomodulatory capacity and the ability to differentiate into many cell lines. Research study examines the immunomodulatory properties of MSCs isolated from chorion (CMSCs). Following the stimulation process, it was found that MSCs are capable of immunomodulatory action via the release of bioactive molecules as well as through direct contact with the immune cells. Immunomodulatory potential of the CMSCs was analyzed by modifying proliferative capacity of mitogen-activated lymphocytes. CMSCs and lymphocytes were tested in cell-to-cell contact. Lymphocytes were stained with carboxyfluorescein diacetate succinimidyl ester. Inhibition of the proliferation of activated lymphocytes was observed. Following the co-cultivation, the expression of markers involved in the immune response modulation was assessed. Afterwards, an increase in CMSCs expression of IL-10 was detected. Following the co-cultivation with activated lymphocyte, adhesion molecules CD54 and CD44 in the CMSCs increased. An increase of CD54 expression was observed. The properties of CMSCs, adherence and differentiation ability, were confirmed. The phenotype of CMSCs CD105+, CD90+, CD73+, CD44+, CD29+, CD45−, CD34−, CD54+ was characterized. It was demonstrated that chorion-derived MSCs have important immunomodulatory effects.

Thiopental Elevates Steady-State Levels of Intracellular Ca2+ and Zn2+ in Rat Thymic Lymphocytes

Thiopental is an ultra-short-acting barbiturate and has been used commonly in the induction phase of general anesthesia. However, the toxic effect of thiopental is not completely clear. The effect of thiopental on intracellular Ca2+ ([Ca2+]i) levels was investigated in non-excitable cells. Experiments were carried out using a flow-cytometric technique, rat thymic lymphocytes (as non-excitable cells), and appropriate fluorescent probes. Treatment of cells with 300 µM thiopental increased Fluo-3 fluorescence intensity, indicating elevation of [Ca2+]i. This increase was partially attenuated by a chelator of intracellular Zn2+. Thus, thiopental elevated both [Ca2+]i and intracellular Zn2+ ([Zn2+]i) levels. Under intracellular Zn2+-free conditions, 100–300 µM thiopental was still able to induce a statistically significant increase in [Ca2+]i, whereas removal of extracellular Ca2+ greatly reduced the increase in [Ca2+]i induced by this dose of thiopental. Therefore, the thiopental-induced increase in [Ca2+]i was mainly due to an increased influx of Ca2+. Treatment of cells with 300 µM thiopental increased FluoZin-3 fluorescence intensity, indicating the presence of [Zn2+]i, both in the presence and absence of extracellular Zn2+. The thiopental-induced elevation of [Zn2+]i was due to an increase in both influx of Zn2+ and intracellular Zn2+ release. Concanavalin A (10 µg/mL) augmented Fluo-3 fluorescence in the presence of an intracellular Zn2+ chelator. The combination of concanavalin A and 100–300 µM thiopental synergistically increased [Ca2+]i. Results suggest that thiopental increases [Ca2+]i in both quiescent and activated lymphocytes, possibly resulting in modulation of immune system function.

PROSPECTS OF COMBINING INTERLEUKIN-2 WITH IMMUNE CHECKPOINT INHIBITORS FOR CANCER THERAPY

The review summarizes results of pre-clinical and clinical investigations of combination therapies with interleukin-2 and immune checkpoint inhibitors. It presents data of the current registered clinical trials of immune checkpoint combinations either with interleukin-2 or the cytokine-activated lymphocytes. The up-to-date experimental and preliminary clinical data evidence that such an immune therapy strategy is highly promising for cancer treatment.