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.     

Quantitative differences in lipid raft components between murine CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cells

Background  

Lipid rafts have been shown to play a role in T cell maturation, activation as well as in the formation of immunological synapses in CD4⁺ helper and CD8⁺ cytotoxic T cells. However, the differential expression of lipid raft components between CD4⁺ and CD8⁺ T cells is still poorly defined. To examine this question, we analyzed the expression of GM1 in T cells from young and aged mice as well as the expression of the glycosylphosphatidylinositol (GPI)-linked protein Thy-1 and cholesterol in murine CD4⁺ and CD8⁺ T cell subpopulations.     

Adoptive immunotherapy of cancer with polyclonal, 10<Superscript>8</Superscript>-fold hyperexpanded,CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cells

T cell-mediated cancer immunotherapy is dose dependent and optimally requires participation of antigen-specific CD4+ and CD8+ T cells. Here, we isolated tumor-sensitized T cells and activated them in vitro using conditions that led to greater than 108-fold numerical hyperexpansion of either the CD4+ or CD8+ subset while retaining their capacity for in vivo therapeutic efficacy. Murine tumor-draining lymph node (TDLN) cells were segregated to purify the CD62Llow subset, or the CD4+ subset thereof. Cells were then propagated through multiple cycles of anti-CD3 activation with IL-2 + IL-7 for the CD8+ subset, or IL-7 + IL-23 for the CD4+ subset. A broad repertoire of TCR Vbeta families was maintained throughout hyperexpansion, which was similar to the starting population. Adoptive transfer of hyper-expanded CD8+ T cells eliminated established pulmonary metastases, in an immunologically specific fashion without the requirement for adjunct IL-2. Hyper-expanded CD4+ T cells cured established tumors in intracranial or subcutaneous sites that were not susceptible to CD8+ T cells alone. Because accessibility and antigen presentation within metastases varies according to anatomic site, maintenance of a broad repertoire of both CD4+ and CD8+ T effector cells will augment the overall systemic efficacy of adoptive immunotherapy.     

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.     

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.     

Intravital imaging of CD8<Superscript>+</Superscript> T cell function in cancer

Recent technological advances in photonics are making intravital microscopy (IVM) an increasingly powerful approach for the mechanistic exploration of biological processes in the physiological context of complex native tissue environments. Direct, dynamic and multiparametric visualization of immune cell behavior in living animals at cellular and subcellular resolution has already proved its utility in auditing basic immunological concepts established through conventional approaches and has also generated new hypotheses that can conversely be complemented and refined by traditional experimental methods. The insight that outgrowing tumors must not necessarily have evaded recognition by the adaptive immune system, but can escape rejection by actively inducing a state of immunological tolerance calls for a detailed investigation of the cellular and molecular mechanisms by which the anti-cancer response is subverted. Along with molecular imaging techniques that provide dynamic information at the population level, IVM can be expected to make a critical contribution to this effort by allowing the observation of immune cell behavior in vivo at single cell-resolution. We review here how IVM-based investigation can help to clarify the role of cytotoxic T lymphocytes (CTL) in the immune response against cancer and identify the ways by which their function might be impaired through tolerogenic mechanisms.     

Tumor resistance to CD8<Superscript>+</Superscript> T cell-based therapeutic vaccination

CD8⁺ cytotoxic T lymphocytes (CTLs) play an important role in antitumor immunity. Induction of tumor-specific CTLs is one major strategy for tumor immunotherapy. However, therapeutic vaccinations used to treat firmly established tumors are generally ineffective. A thorough understanding of the mechanisms underlying tumor resistance to CTL-based therapeutic vaccination is very important in the tumor immunology field. There are two main mechanisms by which tumors develop resistance to CTL-based therapeutic vaccinations. One is that tumors induce peripheral tolerance of tumor-specific CD8⁺ T cells. The other is that tumor cells themselves develop immune evasion mechanisms to prevent recognition and killing by CTLs. This review focuses on recently reported cellular and molecular mechanisms of CD8⁺ T cell tolerance and immune evasion in tumors and discusses about the possibilities to improve tumor immunotherapy.     

CD4<Superscript>+</Superscript> T Cells Downregulate Bcl-2 in Germinal Centers

Germinal centers (GCs) are the main site of T cell-dependent antibody responses. Upon antigen challenge, GCs comprise mostly B cells undergoing proliferation, somatic hypermutation and antigen-affinity selection. GC B cells down-modulate the expression of Bcl-2 protein and are highly sensitive to apoptosis to eliminate autoreactive or low-affinity cells. Bcl-2 is still expressed in a few GC cells, whose identity remains unclear. To address this issue, we examined by confocal microscopy the expression of Bcl-2 by different GC lymphocyte subsets in hyperplastic tonsils. We found that the vast majority of Bcl-2⁺ GC cells are T lymphocytes. Conversely, while in the mantle zone and in the interfollicular areas T cells are almost exclusively Bcl-2⁺, in the GC, most T lymphocytes are Bcl-2⁻. In addition, most of the CD4⁺ GC T cells are Bcl-2⁻, while nearly 100% of the CD8⁺ GC T cells are Bcl-2⁺. The Bcl-2 downregulation by both B and CD4⁺ T GC cells supports the concept that these two subsets may undergo a selection process in this microenvironment.     

Suppression of Primary Allogenic Response by CD8<Superscript>+</Superscript> Memory Cells

Immune response to allogenic tumor cells is associated with the appearance of long-living CD8⁺ memory cells capable of rapid restimulation and lysis of tumor cells in case of repeated injection of these cells. In order to acquire the effector function, allorestricted memory cells need antigen restimulation for 2 days, which is a specific feature of central memory cell population. These cells can suppress proliferation of naive splenocytes in vitro. In mixed lymphocyte cultures containing memory cells, antigen stimulation induces more intensive IL-10 production and deeper suppression of IL-2 production in comparison with cultures containing naive cells. The conditions for activation of naive cells during secondary immune response are not optimal. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 11, pp. 556–560, November, 2005     

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.     

Diversity in CD8<Superscript>+</Superscript> T Cell Function and Epitope Breadth Among Persons with Genital Herpes

CD8⁺ T cells are known to be important in clearing herpes simplex virus (HSV) infections. However, investigating the specific antiviral mechanisms employed by HSV-2-specific T cell populations is limited by a lack of reagents such as CD8⁺ T cell epitopes and specific tetramers. Using a combination of intracellular cytokine staining flow cytometry and ELISpot methods, we functionally characterized peripheral HSV-2-specific CD8⁺ T cells from peripheral blood mononuclear cell (PBMC) that recognize 14 selected HSV-2 open-reading frames (ORFs) from 55 HSV-2 seropositive persons; within these ORFs, we subsequently identified more than 20 unique CD8⁺ T cell epitopes. CD8⁺ T cells to HSV-2 exhibited significant heterogeneity in their functional characteristics, proliferation, production of inflammatory cytokines, and potential to degranulate ex vivo. The diversity in T cell response in these ex vivo assessments offers the potential of defining immune correlates of HSV-2 reactivation in humans.     

Cytokine-facilitated priming of CD8<Superscript>+</Superscript> T cell responses by DNA vaccination

Immune responses elicited by plasmid DNA vaccination can be enhanced and modulated by codelivery of cytokine-encoding plasmids. We studied whether priming of cytotoxic T lymphocyte (CTL) responses against hepatitis B surface antigen (HBsAg) by DNA vaccines injected either intramusculary or intradermally with the gene gun is enhanced by codelivery of cytokine-encoding plasmids. From a panel of tested cytokine plasmids only mouse IFNbeta, IL-15, and GM-CSF encoding plasmids showed an effect. Intradermal gene gun vaccination with 1 micro g plasmid DNA encoding intracellular HBsAg (large LS) showed enhanced CTL priming when IFNbeta, IL15, or GM-CSF encoding plasmids were codelivered; this was not observed when a DNA vaccine encoding secreted HBsAg (small S) was injected. Intramuscular injection of low (5 micro g) doses of a DNA vaccine encoding large HBsAg did not prime CTL when delivered without cytokines, with IFNbeta or IL15-encoding plasmids. However, codelivery with GM-CSF encoding plasmid DNA primed potent, specific CTL immunity detected either in a cytotoxic assay or by determining the frequency of L(d)-restricted CD8(+) T cells specifically inducible to IFNgamma production. The codelivery of GM-CSF encoding plasmids with the DNA vaccine furthermore enhanced CTL priming to a subdominant, D(d)-restricted epitope of HBsAg. The adjuvant effect of cytokine-encoding plasmids on CTL priming by DNA vaccines is thus complex and depends on: (a) the type of cytokine (or combination of cytokines) codelivered, (b) the type (intracellular vs. secreted) and dose (1-50 micro g) of the DNA vaccine, (c) the method of DNA vaccine delivery ("naked" vs. particle-coated DNA), and (d) the (intramuscular vs. intradermal) route of delivery of the DNA vaccine.     

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.     

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).     

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.     

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.     

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.     

Expression of P2X Receptor Subtypes on CD34<Superscript>+</Superscript> Cells and c-kit<Superscript>+</Superscript> Cells of Human Umbilical Blood

The presence of several subtypes of P2X receptors on early hemopoietic precursors (CD34⁺) from human umbilical blood was detected by flow cytometry. The expression of P2X receptors on umbilical blood lymphocytes was an order of magnitude higher than that on adult human blood cells. Our results attest to early involvement of P2X receptors in differentiation of human hemopoietic cells.