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.     

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.     

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.     

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.     

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.     

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.     

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.     

The specific NK cell response in concert with perforin prevents CD8<Superscript>+</Superscript> T cell-mediated immunopathology after mouse cytomegalovirus infection

Natural killer (NK) and CD8⁺ T cells play a crucial role in the control of mouse cytomegalovirus (MCMV) infection. These effector cells exert their functions by releasing antiviral cytokines and by cytolytic mechanisms including perforin activation. In addition to their role in virus control, NK cells play an immunoregulatory role since they shape the CD8⁺ T cell response to MCMV. To investigate the role of perforin-dependent cytolytic mechanism in NK cell modulation of CD8⁺ T cell response during acute MCMV infection, we have used perforin-deficient C57BL/6 mice (Prf1^?/?) and have shown that virus control by CD8⁺ T cells in Prf1^?/? mice is more efficient if NK cells are activated by the engagement of the Ly49H receptor with the m157 MCMV protein. A lack of perforin results in severe liver inflammation after MCMV infection, which is characterized by immunopathological lesions that are more pronounced in Prf1^?/? mice infected with virus unable to activate NK cells. This immunopathology is caused by an abundant infiltration of activated CD8⁺ T cells. The depletion of CD8⁺ T cells has markedly reduced pathohistological lesions in the liver and improved the survival of Prf1^?/? mice in spite of an increased viral load. Altogether, the results of our study suggest that a lack of perforin and absence of the specific activation of NK cells during acute MCMV infection lead to an unleashed CD8⁺ T cell response that is detrimental for the host.     

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.     

Programmed cell death-1 (PD-1) at the heart of heterologous prime-boost vaccines and regulation of CD8<Superscript>+</Superscript> T cell immunity

Developing new vaccination strategies and optimizing current vaccines through heterologous prime-boost carries the promise of integrating the benefits of different yet synergistic vectors. It has been widely thought that the increased immunity afforded by heterologous prime-boost vaccination is mainly due to the minimization of immune responses to the carrier vectors, which allows a progressive build up of immunity against defined epitopes and the subsequent induction of broader immune responses against pathogens. Focusing on CD8⁺ T cells, we put forward a different yet complementary hypothesis based primarily on the systematic analysis of DNA vaccines as priming agents. This hypothesis relies on the finding that during the initiation of immune response, acquisition of co-inhibitory receptors such as programmed cell death-1 (PD-1) is determined by the pattern of antigen exposure in conjunction with Toll-like receptor (TLR)-dependent stimulation, critically affecting the magnitude and profile of secondary immunity. This hypothesis, based upon the acquisition and co-regulation of pivotal inhibitory receptors by CD8⁺ T cells, offers a rationale for gene-based immunization as an effective priming strategy and, in addition, outlines a new dimension to immune homeostasis during immune reaction to pathogens. Finally, this model implies that new and optimized immunization approaches for cancer and certain viral infections must induce highly efficacious T cells, refractory to a broad range of immune-inhibiting mechanisms, rather than solely or primarily focusing on the generation of large pools of vaccine-specific lymphocytes.     

A Distinct Role of CD4<Superscript>+</Superscript> Th17- and Th17-Stimulated CD8<Superscript>+</Superscript> CTL in the Pathogenesis of Type 1 Diabetes and Experimental Autoimmune Encephalomyelitis

Both CD4(+) Th17-cells and CD8(+) cytotoxic T lymphocytes (CTLs) are involved in type 1 diabetes and experimental autoimmune encephalomyelitis (EAE). However, their relationship in pathogenesis of these autoimmune diseases is still elusive. We generated ovalbumin (OVA)- or myelin oligodendrocyte glycoprotein (MOG)-specific Th17 cells expressing RORγt and IL-17 by in vitro co-culturing OVA-pulsed and MOG(35-55) peptide-pulsed dendritic cells (DC(OVA) and DC(MOG)) with CD4(+) T cells derived from transgenic OTII and MOG-T cell receptor mice, respectively. We found that these Th17 cells when transferred into C57BL/6 mice stimulated OVA- and MOG-specific CTL responses, respectively. To assess the above question, we adoptively transferred OVA-specific Th17 cells into transgenic rat insulin promoter (RIP)-mOVA mice or RIP-mOVA mice treated with anti-CD8 antibody to deplete Th17-stimulated CD8(+) T cells. We demonstrated that OVA-specific Th17-stimulated CTLs, but not Th17 cells themselves, induced diabetes in RIP-mOVA. We also transferred MOG-specific Th17 cells into C57BL/6 mice and H-2K(b-/-) mice lacking of the ability to generate Th17-stimulated CTLs. We further found that MOG-specific Th17 cells, but not Th17-activated CTLs induced EAE in C57BL/6 mice. Taken together, our data indicate a distinct role of Th17 cells and Th17-stimulated CTLs in the pathogenesis of TID and EAE, which may have great impact on the overall understanding of Th17 cells in the pathogenesis of autoimmune diseases.     

Evaluation of the expression of early activation marker CD69 by CD8<Superscript>+</Superscript> lymphocytes in HIV-infected patients

We studied the expression of CD69 antigen on CD8⁺ lymphocytes in response to nonspecific activator phytohemagglutinin in 202 patients with HIV infection. It was found that the number of CD8⁺CD69⁺ lymphocytes in HIV-infected patients increased; this parameter negatively correlated with relative and absolute content of CD4⁺ cells and positively correlated with IgG concentration. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 12, pp. 661–663, December, 2007     

CD34<Superscript>+</Superscript> fibrocytes in tubular carcinomas and radial scars of the breast

The present study was undertaken to analyze whether and to what extent CD34⁺ fibrocytes and -smooth muscle actin (-SMA)-positive myofibroblasts occur in the stroma of radial scars and tubular carcinoma of the breast. We investigated a total of 24 radial scars obtained from 23 females and a total of 43 tubular carcinomas. All tubular carcinomas showed a virtually complete loss of CD34⁺ fibrocytes paralleled by a gain of -SMA-positive myofibroblasts. The peripheral parts of radial scars harbored CD34⁺ fibrocytes comparable to normal breast tissue. In 23 of 24 radial scars, the central core was characterized by a loss of CD34⁺ fibrocytes accompanied by the presence of -SMA-positive myofibroblasts in six cases, a pattern that up to now has only been described in malignant breast lesions. This finding underlines the close relationship between tubular carcinomas and radial scars. We conclude CD34- and -SMA immunohistochemistry to be valuable adjunctive tools in distinguishing radial scars from tubular carcinomas. In the present study, the presence of CD34⁺ fibrocytes excluded malignancy. The absence of CD34⁺ fibrocytes paralleled by the presence of -SMA myofibroblasts indicated malignancy in most cases, although it has to be carefully considered that in a minority of cases (6 of 24) the central core of radial scars may disclose this stromal composition.This revised version was published online September 2003 with corrections to size and format of Fig. 1     

CD34<Superscript>+</Superscript> fibrocytes in melanocytic nevi and malignant melanomas of the skin

CD34(+) fibrocytes are constitutive elements of the human connective tissue. The stroma associated with invasive carcinomas is characterized by a stereotypic loss of CD34(+) fibrocytes and a phenotype change towards CD34(-) alpha-Smooth muscle actin (SMA)(+) myofibroblasts. Secreted protein acidic and rich in cysteine (SPARC) is an important mediator of tumor-associated stromal remodeling. Melanocytic lesions of the skin have not been investigated as to this aspect up to now. Thus, we investigated a total of 20 malignant melanomas and 29 melanocytic nevi. The normal dermis and benign melanocytic nevi showed numerous CD34(+) fibrocytes, whereas malignant melanomas were devoid of this cell type. alpha-SMA-positive myofibroblasts were absent from the normal dermis, melanocytic nevi, and malignant melanomas. SPARC was positive in malignant melanoma cells and negative in their associated stroma, while all melanocytic nevi were completely negative. The stromal phenotype of malignant melanomas (CD34(-) alpha-SMA(-)) differs from that of invasive carcinomas (CD34(-) alpha-SMA(+)) suggesting different pathogenic mechanisms involved in tumor-associated stromal remodeling. SPARC expression appears to be closely related to malignancy in melanocytic lesions.     

CD28<Superscript>neg.</Superscript> T lymphocytes of a melanoma patient harbor tumor immunity and a high frequency of germline-encoded and public TCRs

Increased numbers of CD8⁺CD28^neg. T cells have been detected in the peripheral blood of patients with several types of malignancies. However, the role of these cells in anticancer immunity are not yet clear and CD8⁺CD28^neg. T cells are a controversially discussed subpopulation reported both as immunosuppressive and cytotoxic. In this study, we examined the T cell receptor (TCR) repertoire and complementarity-determining region 3 sequences of CD28^neg. T cells in a melanoma patient with recurrent disease who achieved long-term disease-free status. As a result, the patient’s oligoclonal CD8⁺CD28^neg. T cell compartment holds TCRs that are public and specific for Melan-A as well as several public TCRs reported for common viral antigens. While over 80% of his CD8⁺CD28^neg. T cells expressed a cytotoxicity marker, CD57, only 0.01% of CD8⁺ CD28^neg. T cells were positive for Foxp3. In conclusion, our results demonstrate that besides virus-specific also tumor-associated self-antigen targeting T cells accumulate in the CD28^neg. compartment of the immunological memory. Since the patient is in ongoing complete remission for more than 9 years, CD8⁺CD28^neg. T cells with the Melan-A-specific TCR might contribute to antitumor immunity in this patient.     

CD34<Superscript>+</Superscript> fibrocytes in chronic cystitis and noninvasive and invasive urothelial carcinomas of the urinary bladder

CD34⁺ fibrocytes are constitutive elements of the connective tissue where they play a role in matrix synthesis and tumor-associated stromal remodeling. Secreted protein, acidic, and rich in cysteine (SPARC) is a pivotal mediator of stromal remodeling precipitated by invasive carcinomas. The present study was undertaken to investigate CD34⁺ fibrocytes in the stroma of the tumor-free urinary bladder, chronic cystitis, and urothelial carcinomas together with stromal expression of α-smooth muscle actin (α-SMA), CD117, and SPARC. In tumor-free urinary bladder and chronic cystitis, CD34⁺ fibrocytes were found in the deep lamina propria and tunica muscularis, whereas the superficial lamina propria disclosed a CD34-negative and α-SMA-positive fibrocyte-like cell. Invasive urothelial carcinomas revealed a complete loss of CD34⁺ fibrocytes and concomitant appearance of α-SMA-reactive myofibroblasts which showed strong expression of SPARC. CD117 expression of tumor-free and tumor-associated stroma revealed no differences. We in this study for the first time describe CD34⁺ fibrocytes in the urinary bladder and an up-to-now unknown population of α-SMA-positive fibrocytes exclusively occurring in the superficial lamina propria. Stromal remodeling associated with invasive carcinomas in the urinary bladder is characterized by a loss of CD34⁺ fibrocytes paralleled by a gain of α-SMA-positive myofibroblasts and increased expression of SPARC.