CD4<Superscript>+</Superscript>FOXP3<Superscript>+</Superscript> T regulatory cells decrease and CD3<Superscript>+</Superscript>CD8<Superscript>+</Superscript> T cells recruitment in TILs from melanoma metastases after electrochemotherapy

Electrochemotherapy (ECT) represents an effective local treatment for skin unresectable melanoma metastases with high overall objective response rate. ECT is based on the combination of anti-neoplastic drugs administration and cancer cells electroporation. Whether ECT can also activate the immune system is a matter of debate, however a significant recruitment of dendritic cells in melanoma treated metastases has been described. Herein we investigated immediate and late effects of ECT treatment on T cell subsets in ECT-treated lesions by fluorescent immunohistochemistry. Biopsies from melanoma patients (n = 10) were taken before ECT (t0), at d1 and d14 from treatment. At t0, CD3⁺CD4⁺ T cells were the most represented T cells, well detected in the perilesional dermis, particularly at tumour margin, while CD3⁺CD8⁺ T cells were less represented. CD4⁺FOXP3⁺ T regulatory (Treg) cells were present in the perilesional dermis and within the lesion. ECT induced a significant decrease of CD4⁺FOXP3⁺ Treg cells percentage in the perilesional dermis, observed at d1 and at d14 (p < 0.001). CD3⁺CD8⁺ T cells frequency significantly increased at d14 from treatment in the perilesional dermis (p < 0.001). Furthermore calreticulin translocation to the plasma membrane, a hallmark of immunogenic cell death, was observed in metastatic cells after ECT. The data reported here confirm that ECT induces a local response, with a lymphoid infiltrate characterized by CD4⁺FOXP3⁺ Treg cells decrease and CD3⁺CD8⁺ T cells recruitment in the treated lesions. These results might contribute to design novel combinational therapeutic approaches with ECT and immunotherapy in order to generate a systemic long-lasting anti-melanoma immunity.     

Reduced CD27 Expression on Antigen-Specific CD4<Superscript>+</Superscript> T Cells Correlates with Persistent Active Tuberculosis

Objective  

CD27, a member of the tumor necrosis factor receptor family, has important role in generation of T cell immunity. In this study, association of CD27 expression on mycobacterial antigen-specific CD4⁺ T cells with pulmonary tuberculosis (TB) was investigated.     

Greater frequency of CD5-negative CD8<Superscript>+</Superscript> T cells against human immunodeficiency virus type 1 than other viruses is consistent with adaptation to antigenic variation

Background

The CD5 protein antagonizes phosphorylation events downstream of T cell receptor (TCR) engagement to decrease T cell responsiveness. CD5-negative T cell clones respond preferentially over their CD5⁺ counterparts against cells with low human histocompatibility-linked leukocyte antigen (HLA) levels. In human immunodeficiency virus type 1 (HIV-1) infection, CD5^-CD8⁺ T cells increase in prevalence with disease progression.

Methods

To investigate potential causes of this expansion of CD5^-CD8⁺ T cells in HIV-1 infection, we compared CD5 expression on CD8⁺ T cells reactive against HIV-1 peptides, common viral peptides and a self peptide that together span a broad range of TCR avidities in the context of the common HLA-A2 class I restriction molecule. Following stimulation, CD5 expression on peptide-specific CD8⁺ T cells was assessed by flow cytometry.

Results

In healthy controls, there was no significant difference in the CD5⁺ percentage of CD8⁺ T cells specific for common viral peptides, but a lower percentage of those responding against a common self peptide expressed CD5. The same relationship occurred in HIV-infected individuals, however, a lower percentage of HIV peptide-specific CD8⁺ T cells than other viral peptide-specific CD8⁺ T cells expressed CD5. In terms of overall CD5 expression level at the peptide-specific responder population level, HIV-specific CD8⁺ T cells resembled those responsive against the self peptide, despite much higher avidity TCR/HLA/peptide interactions.

Conclusions

This deficit in CD5 expression selective for HIV-specific CD8⁺ T cells is consistent with in vivo adaptation to low avidity HIV peptide variants and has potential consequences for CD8⁺ T cell expansion, cross-reactivity and autoreactivity.

     

The effects of leflunomide on CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>Foxp3<Superscript>+</Superscript> T regulatory cells in mice receiving allogeneic bone marrow transplantation

Objective and design  

Leflunomide (LEF) is effective not only in different animal models of autoimmune diseases and the therapy of patients with rheumatoid arthritisbut also in graft rejection. The effect of LEF on CD4⁺CD25⁺T regulatory cells (Treg) was determined in a mouse model of allogeneic bone marrow transplantation.     

Effects of simvastatin on the function of splenic CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cells in sepsis mice

Simvastatin may be beneficial for treating sepsis due to its immune-regulating properties, although the mechanisms remain elusive. Herein, we hypothesized simvastatin may attenuate T cell dysfunction induced by sepsis. To test this hypothesis, we used a model based on cecal ligation and puncture (CLP) to induce sepsis in mice. Male C57BL/6 mice were pre-treated with simvastatin (0.2 μg/g of body weight) before CLP. The expression of B and T lymphocyte attenuator (BTLA) on splenic CD4⁺ T cells and T cell apoptosis, CD4⁺ and CD8⁺ T cells were quantified by flow cytometry. Immunohistochemical staining was performed to evaluate the loss of immune effector cells. Formation of TNF-α and interleukin 10 (IL-10) in the spleen and plasma levels of presepsin, IL-1β, and IL-6 were determined using enzyme-linked immunosorbent assay. Simvastatin markedly inhibited the reduction in cytokine secretion from lipopolysaccharide (LPS)-stimulated splenocytes. Simvastatin-treated mice had significantly decreased the percentages of negative costimulatory receptor BTLA on CD4 T cell expression. Simvastatin markedly reduced T cell apoptosis through downregulating the Fas/FasL expression and decrease the percentage of caspase-3 activity in spleen tissue. There was significantly less depletion of splenic CD4⁺ and CD8⁺ T cells in simvastatin-treated mice. Simvastatin reduced plasma levels of presepsin, IL-1β, and IL-6. Simvastatin can be a powerful regulator of immune function under sepsis conditions by improving T cell function in sepsis.     

Flow Cytometric Identification of CD93 Expression on Naive T Lymphocytes (CD4<Superscript>+</Superscript>CD45RA<Superscript>+</Superscript> Cells) in Human Neonatal Umbilical Cord Blood

Human CD93 has a molecular weight of about 100 kDa and is selectively expressed by myeloid cell lineages in peripheral blood (PB) mononuclear cells. Although CD93 was initially identified as a receptor for complement component 1, subcomponent q phagocytosis (C1qRp) involved in the C1q-mediated enhancement of the phagocytosis of various antigens, several recent studies have reported that CD93 is not a receptor for the C1q-mediated enhancement of phagocytosis. The expression patterns of CD93 have been previously investigated in PB mononuclear cells (lymphocytes, monocytes, and granulocytes) from adult PB and neonatal umbilical cord blood (UCB), and the expression of CD93 was not found on lymphocytes from either normal adult PB or neonatal UCB. However, the detection of CD93 expression in neonatal UCB using CD93 monoclonal antibodies (mAbs) that recognize different antigenic epitopes remains poorly understood. In this study, we examined the expression of CD93 on lymphocytes, monocytes, and granulocytes from neonatal UCB using four different types of CD93 mAb detection probes, mNI-11, R139, R3, and X-2, using flow cytometric and western blot analyses. We found that CD93, as defined using all four mAbs, was expressed on monocytes and granulocytes in PB mononuclear cells from adult PB and neonatal UCB. On the other hand, we observed for the first time that the expression of CD93 on lymphocytes in neonatal UCB can only be detected using the mNI-11 mAb, established in our laboratory, and not with commercially available CD93 mAbs (R139, R3, and X-2). However, CD93 expression on lymphocytes from normal adults was not detected using any of the four CD93 mAbs. Two-color flow cytometric analyses showed that the CD93 recognized by mNI-11 mAb was expressed on CD3⁺ T lymphocytes (mainly CD4⁺ helper T lymphocytes), but not on CD19⁺ B lymphocytes or on CD8⁺ suppressor/cytotoxic T lymphocytes from neonatal UCB. In addition, CD93 was expressed on CD45RA⁺ (naive antigen) lymphocytes from neonatal UCB, but not on CD45RO⁺ (memory antigen) lymphocytes from neonatal UCB or on CD45RA⁺ and CD45RO⁺ lymphocytes from normal adult PB. Three-color flow cytometric analysis showed that CD93 was co-expressed on naive T lymphocytes (CD4⁺CD45RA⁺) from neonatal UCB. In a western blot analysis, the CD93 mAb (mNI-11) immunoprecipitated at a molecular weight of 98 kDa, identified as a CD93 molecule, in the CD4⁺CD45RA⁺ cells from neonatal UCB but not from adult PB, similar to the results in the human monocyte-like cell line U937 (human CD93-positive cells). Taken together, these results provide the first direct evidence of a novel/naive cell population (CD4⁺CD45RA⁺CD93⁺) in neonatal UCB that may have an important role in cell biology, transplantation, and immature/mature immune responses.     

Accumulation of CD5<Superscript>+</Superscript>CD19<Superscript>+</Superscript> B lymphocytes expressing PD-1 and PD-1L in hypertrophied pharyngeal tonsils

Programmed death-1 (PD-1) is one of the most important inhibitory co-receptors expressed predominantly on activated T and B lymphocytes whose expression could be sustained by permanent antigenic stimulation accompanying chronic or recurrent tonsillitis. The expression of PD-1 and PD-1L was analyzed using flow cytometry on hypertrophied tonsils collected from 57 children. We observed high expression of PD-1 and PD-1L on certain lymphocytes subpopulations of hypertrophied tonsils; among T cells, the expression of PD-1 on protein level was higher on CD4⁺ cells (70.3 %) than on CD8⁺ cells (35 %). Interestingly, a limited expression of PD-1 was observed on CD19⁺ B lymphocytes (6.5 %), while CD5⁺CD19⁺ B cells overexpressed PD-1 (52.5 %). Moreover, the expression of PD-1L was also higher on CD5⁺CD19⁺ B cells (16.5 %) than on CD19⁺ B cells (3.5 %) and on CD4⁺ T cells (20 %) than on CD8⁺ T cells (10 %). PD-1 and PD-1L expressions correlated only on CD5⁺CD19⁺ cells. In conclusion, high expression of PD-1 and PD-1L on T and B cells could represent hallmark of immune system adaptation to chronic antigenic exposition in patients with tonsillitis.     

CD27<Superscript>+</Superscript>TIM-1<Superscript>+</Superscript> memory B cells promoted the development of Foxp3<Superscript>+</Superscript> Tregs and were associated with better survival in acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a rapid onset life-threatening condition involving uncontrolled propagation of inflammatory responses. Here, we observed that ARDS patients that survived presented significantly higher frequencies of TIM-1⁺ B cells, especially the CD27⁺TIM-1⁺ B cells, than the ARDS patients who succumbed to the condition. We then found that using BCR/CD40 antigen-dependent stimulation or Staphylococcus aureus Cowan (SAC) antigen-independent stimulation, TIM-1⁺ B cells presented significantly higher IL-10 secretion and/or TGF-β1 secretion, with SAC stimulation being more effective. CD4⁺ T cells that incubated with TIM-1⁺ B cells presented significantly elevated IL-10 secretion, TGF-β1 secretion, and Foxp3 expression, than CD4⁺ T cells that incubated with TIM-1^? B cells, suggesting TIM-1⁺ B cells promoted the in vitro development of Foxp3⁺ Treg cells. Interestingly, this TIM-1⁺ B cell-mediated promotion of Foxp3 expression was mostly dependent on TGF-β1 but not IL-10, since neutralization of TGF-β1, but not IL-10, resulted in the suppression of Foxp3 expression. We further showed that in TIM-1⁺ B cells, the CD27⁺ classical memory B cell subset demonstrated more regulatory potency than the CD27^? subset. Together, our results suggested that the TIM-1⁺ B cells, especially those that expressed CD27, could promote Foxp3 expression. Their clinical efficacy in treating ARDS should be examined in in vivo experiments.     

Highly enriched CD133<Superscript>+</Superscript>CD44<Superscript>+</Superscript> stem-like cells with CD133<Superscript>+</Superscript>CD44<Superscript>high</Superscript> metastatic subset in HCT116 colon cancer cells

Stem-like cancer cells (SLCCs) are distinct cellular subpopulation in colon cancer that is essential for tumor maintenance. Previous studies indicated that SLCCs accounted for only a minor subset in a given cancer model. However, we found that SLCCs frequency varied among a panel of colon cancer cell lines, with HCT116 cells composed mainly of SLCCs, as demonstrated by colonosphere forming capability and CD133 expression. Indeed, flow cytometric analysis revealed more than 60% HCT116 cells co-expressed the putative SLCCs markers CD133 and CD44. Compared with non-CD133⁺CD44⁺ cells, FACS sorted CD133⁺CD44⁺ cells were undifferentiated, endowed with extensive self-renewal and epithelial lineage differentiation capacity in vitro. CD133⁺CD44⁺ exhibited enhanced tumorigeneicity in NOD/SCID mice. One thousand CD133⁺CD44⁺ cells initiated xenograft tumors efficiently (3/6) while 1 × 10⁵ non-CD133⁺CD44⁺ cells could only form palpable nodule with much slower growth rate (1/6). More interestingly, long-term cultured self-renewing CD133⁺CD44⁺ cells enriched CD133⁺CD44^high subset, which expressed epithelial to mesenchymal transition marker, were more invasive in vitro and responsible solely for liver metastasis in vivo. In conclusion, these data demonstrated for the first time that CD133⁺CD44⁺ SLCCs were highly enriched in HCT116 cells and that metastatic SLCCs resided exclusively in a CD133⁺CD44^high subpopulation.     

Monitoring of CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T-Cell Responses After Dendritic Cell-Based Immunotherapy Using CFSE Dye Dilution Analysis

CFSE dye dilution analysis and [³H] thymidine incorporation were used side by side to assess proliferative responses of peripheral blood mononuclear cells (PBMCs) after vaccination of renal cell carcinoma patients (n=6) with antigen-loaded dendritic cells. Immune responses against the control antigen keyhole limpet hemocyanin (KLH) were induced in all patients. While [³H] thymidine incorporation revealed a 4 to 977-fold increase in KLH-induced proliferation (mean: 209-fold), CFSE-labeling experiments demonstrated that the KLH-responsive population of postvaccination PBMCs represented 7–53% (mean: 23%). Combining CFSE-labeling with T-cell subset analysis confirmed the presence of CD4⁺ KLH-reactive T cells but also revealed a substantial population of CD8⁺ KLH-reactive T cells in one patient as well as minor populations of CD8⁺ KLH-reactive T cells in three other patients. Our data indicate that CFSE dye dilution analysis is a valuable tool for immune monitoring after dendritic cell vaccination.     

Role of immune activation in CD4<Superscript>+</Superscript> T-cell depletion in HIV-1 infected Indian patients

Objectives

The correlation of immune activation with CD4⁺ depletion and HIV-1 disease progression has been evidenced by several studies involving mainly clade B virus. However, this needs to be investigated in developing countries such as India predominately infected with clade C virus.

Materials and methods

In a cross-sectional study of 68 antiretroviral treatment naïve, HIV-1 infected Indian patients, we studied the association between CD4⁺ T cells, plasma HIV-1 RNA levels, and immune activation markers using unadjusted and adjusted correlative analyses.

Results

Significant negative correlations of higher magnitude were observed between the CD4⁺ T cell percentages and plasma HIV-1 RNA levels in the study population when adjusted for the effects of immune activation markers. However, the negative association of CD4⁺ T cells with immune activation markers remained unaffected when controlled for the effects of plasma HIV-1 RNA levels.

Conclusions

Our results support the important role of immune activation in CD4⁺ T cell depletion and disease progression during untreated HIV-1 infection.

     

Lenalidomide potentiates CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>Treg-related suppression of lymphoma B-cell proliferation

We have previously found that ex vivo expanded human CD4⁺CD25⁺Treg cells suppress proliferation of lymphoma B-cell lines. Here we demonstrate that the immunomodulatory drug lenalidomide potentiates suppression of lymphoma B-cell proliferation by freshly isolated CD4⁺CD25⁺Tregs, as well as suppression by Tregs expanded polyclonally in the presence of rapamycin from CD4⁺CD25⁺T cells or CD4⁺CD25⁺CD127^loT cells. The regulation of lymphoma cell proliferation by Tregs pre-expanded with “third-party” allogeneic MoDCs in the presence of rapamycin was also potentiated by lenalidomide. Lenalidomide contributed to the suppression exerted by Tregs despite concomitant downregulation of Treg proliferation. Lenalidomide did not reduce the suppression of conventional T cells by expanded Tregs. The exposure of polyclonally expanded Tregs to lenalidomide did not significantly alter their phenotype. There was no uniform pattern of lenalidomide effect on Treg-mediated regulation of lymphoma B cells freshly isolated from patients. Freshly isolated lymphoma cells activated with multimeric CD40L and IL-4 to support their survival in vitro varied in their sensitivity to lenalidomide, and the regulatory effect of Tregs on such lymphoma cells ranged from suppression to help in individual patients. Lenalidomide potentiated or attenuated Treg effects on the survival of freshly isolated lymphoma cells. A combination of lenalidomide treatment with adoptive transfer of CD4⁺CD25⁺Tregs or CD4⁺CD25⁺CD127^loTregs expanded ex vivo could be used to suppress proliferation of residual lymphoma in select patients with lymphoma responsive to the regulation by Tregs and sensitive to lenalidomide.     

Expression of Markers of Regulatory CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>foxp3<Superscript>+</Superscript> Cells in Atherosclerotic Plaques of Human Coronary Arteries

The content of marker foxp3 of regulatory T cells and chemokines in atherosclerotic plaques of human coronary arteries was measured by the polymerase chain reaction. In vitro migration of regulatory CD4⁺CD25⁺foxp3⁺ cells in the CD4⁺ lymphocyte population from healthy donors was studied after treatment with chemokines I-309, IP-10, and SDF-1. mRNA for the factor foxp3 and chemokines SDF-1, I-309, and MIP-1β were found in the majority of samples from atherosclerotic plaques. SDF-1 induced maximum migratory response of CD4⁺CD25⁺foxp3⁺ cells.     

Expansion of CD11b<Superscript>+</Superscript>Ly6G<Superscript>high</Superscript> and CD11b<Superscript>+</Superscript>CD49d<Superscript>+</Superscript> myeloid cells with suppressive potential in mice with chronic inflammation and light-at-night-induced circadian disruption

Objective

Myeloid-derived suppressor cells (MDSCs) are important negative regulators of immune processes in cancer and other pathological conditions. We suggested that MDSCs play a key role in pathogenesis of chronic inflammation, which precedes and, to a certain extent, induces carcinogenesis. The present study aimed at investigation of MDSCs arising during chronic inflammation and light-at-night (LN)-induced stress, which is shown to accelerate chronic diseases.

Subjects

67 CD-1 mice and in vitro MDSC cultures.

Treatment

Adjuvant arthritis was induced by a subdermal injection of complete Freund’s adjuvant. LN was induced by illumination of 750 lx at night.

Methods

Flow cytometry for evaluation of cell phenotypes and MTT standard test for cell proliferation were used.

Results

Increased levels of splenic CD11b⁺Ly6G^high and CD11b⁺CD49d⁺ myeloid cells possessing suppressive potential in mice with adjuvant arthritis are shown. LN amplifies the process of CD11b⁺Ly6G^high expansion in mice with adjuvant arthritis. Expression of CD62L and CD195 is elevated on the myeloid cells during exposure to LN.

Conclusions

Our study raises the possibility that CD11b⁺Ly6G^high and CD11b⁺CD49d⁺ MDSCs play an important role in the induction of immunosuppressive environment typical for chronic inflammation. Also, LN can affect immune responses during chronic inflammation through recruitment of MDSCs from the bone marrow.

     

Human thymic stromal lymphopoietin enhances expression of CD80 in human CD14<Superscript>+</Superscript> monocytes/macrophages

Objective  

Human thymic stromal lymphopoietin (TSLP), an epithelial cell-derived cytokine, promotes inflammatory T helper type 2 cell (Th2) differentiation of naive CD4⁺ T cells. TSLP is highly produced in keratinocytes of patients with atopic dermatitis and bronchial epithelia of patients with asthma and was thought to be a master switch for allergic inflammation. We sought to examine the effect of TSLP in human monocytes/macrophages.     

Age-dependent hantavirus-specific CD8<Superscript>+</Superscript> T-cell responses in mice infected with Hantaan virus

Summary. To investigate age-dependent differences in hantavirus-specific CD8⁺ T-cell responses, mice were inoculated with 0.1 50% newborn mouse lethal dose of Hantaan virus (HTNV) at 0, 3, 7, 14, or 35 days after birth. HTNV-specific CD8⁺ T cells producing gamma interferon (IFN-) were measured on day 30 after HTNV inoculation. Although no IFN--producing HTNV-specific CD8⁺ T cells were detected in most of the mice inoculated with HTNV on day 0 after birth, most mice inoculated at 3, 7, 14, or 35 days had HTNV-specific CD8⁺ T cells. The production of tumor necrosis factor alpha (TNF-) by IFN--producing CD8⁺ T cells and the cytotoxic activity against HTNV-infected target cells were similar in immature and adult mice. However, the number of IFN--producing HTNV-specific CD8⁺ T cells was significantly less in mice inoculated with HTNV at 3 days than in older mice. In addition, a strong correlation between HTNV persistence and a lack of HTNV-specific CD8⁺ T cells was observed. These results suggest that mice over 7 days old have the ability to induce functional HTNV-specific CD8⁺ T-cell responses that are indistinguishable from the responses of adult mice, and that HTNV-specific CD8⁺ T cells are important for clearance of HTNV.     

Specific Immunotherapy to Birch Allergen Does not Enhance Suppression of Th2 Cells by CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript> Regulatory T Cells During Pollen Season

Introduction  

The aim of this study was to investigate the suppressive capacity of CD25⁺ regulatory T cells on birch allergen-induced T-cell responses during the first birch pollen season after initiation of specific immunotherapy (SIT).     

Not All Tetramer Binding CD8<Superscript>+</Superscript> T Cells Can Produce Cytokines and Chemokines Involved in the Effector Functions of Virus-Specific CD8<Superscript>+</Superscript> T Lymphocytes in HIV-1 Infected Children

In the pediatric human immunodeficiency virus type-1 (HIV-1) infection, the presence of cytotoxic T lymphocytes (CTL) is associated with a slow progression to AIDS. The secretion of cytokines by CTLs may be critical in the control of viral infection. We used the combination of cell surface and intracellular staining to study the functionality of tetramer binding CD8⁺ T cells recognizing two HIV-1 immunodominant epitopes, in peripheral blood mononuclear cells from HIV-1-infected children. A fraction of tetramer positive CD8⁺ T cells produce cytokines (IFN-, TNF-) or chemokines (CCL4, CCL5) after ex vivo stimulation with the cognate peptide. There was a negative correlation between the plasma viral load and the percentage of CD8⁺ Tetramer Gag⁺ T cells secreting IFN-. This is the first report in the context of pediatric HIV-1 infection showing that only a fraction of HIV-1-specific CD8⁺ T cells have the capacity to produce cytokines and chemokines implicated in their antiviral functions.