Immune Escape

Tumor induced immune suppression and immune escape Mechanisms and Possible Solutions Theresa L. Whiteside, Michael Campoli & Soldano Ferrone University of Pittsburgh Cancer Institute and Departments of Pathology, Immunology and Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA (T.L.W.) Roswell Park Cancer Institute and Department of Immunology, School of Medicine, State University of New York at Buffalo, Buffalo, NY, USA (M.C. & S.F.) Abstract It has been a...

Production And Validation Of Tetramers

The production of tetramers is a time-consuming, labor-intensive process which requires skill and experience that limited the widespread use of tetramers initially. However, tetramers are now widely available through www.emory.edu WHSC TETRAMER ) and commercial (Beckman Coulter Immunomics, www.immunomics.com ) sources. The quality of tetramer reagents has also improved steadily. Excellent reagents can now be obtained, especially from commerical sources as they undergo rigorous quality control...

Autologous Ifny Elispot Assay 31 Material and Methods

Blood samples were obtained from patients with Stage III or IV follicular center cell lymphoma grade 1 or grade 2 at various time points before and after vaccination with the patient-specific tumor-derived Id protein incorporated into liposomes along with recombinant human IL-2 as described previously (27). Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation with Ficoll Isopaque (ICN Biomedicals Inc., Aurora, OH) and cryopreserved for immunological assays....

Data Interpretation

3.1 Emergence of TCR clonality and preferential usage of TCR chains In general, healthy, 'antigen-experienced' individuals may show clonally altered, or even monoclonal TCRs. These 'private' alterations in the TCR repertoire have been reported in a number of apparently healthy individuals and identified in the CD8+ T-cell population, preferentially in the CD57+ T- cell subset (18). Monoclonal or oligoclonal TCR VB families may occur in EBV or CMV-specific T-cell responses and reflect the...

Essential Components Of An Effective Taspecific T Cellbased Immune Response

Although mechanisms of tumor-specific immunity and its role in the development and progression of cancer in man have been much debated, newer evidence suggest that the components necessary for mounting the anti-tumor immune response are present in cancer patients. Tumor-antigens. Both the SEREX, T-cell-based and reverse immunology approaches have identified a variety of tumor antigens (TA), which can be broadly classified as tumor antigens that are capable of being recognized by the...

Dynamic Monitoring Of Anticancer Immune Responses

Although genetic background may be responsible for immune responsiveness, it is also possible that the unstable nature of cancer cell phenotypes can strongly influence the susceptibility of cancerous lesions to immune attack. In fact very little is known about the algorithm that may determine the occurrence of immune-induced cancer regression in humans (56). The introduction of gene profiling arrays is particularly suited to circumstances when little is known about a biological event to...

NK and NKT cells in cancer

The innate arm of the immune response can discriminate between tumor and normal cells. The ability to detect and cause the elimination of tumor cells in vivo without prior sensitization was the founding definition of natural killer (NK) cells. The evidence that they do so is overwhelming, but until recently only indirect (27). Tumors sensitive to NK killing in vitro grew more slowly, or were rejected, in vivo (28, 29). Because many of the features of NK cells are shared with other lymphocyte...

Mechanisms Underlying The Lack Of Correlation Between Clinical And Immune Responses

To date, a large number of TA-specific immunotherapy trials have been conducted in patients with malignant disease. It is clear from these studies that the various types of tumor vaccines i) have limited or no toxicity and ii) are able to induce TA-specific immune responses and or augment already established TA-specific immune responses in immunized patients. Nevertheless, the results of these studies have highlighted two challenges facing tumor immunologists and clinical oncologists. The first...

Tracking cell divisions

Immediately after labeling, the intensity of CFSE within cells is extremely bright. The high fluorescence of freshly labeled cells diminishes rapidly over the first 24 hours, presumably due to the excretion of activated but unconjugated CFSE, as well as the catabolism of many labeled proteins. The initial strong intensity of staining often complicates flow cytometer measurement. Early time points in a time series may be off scale, and cannot be used in conjunction with other fluorochromes such...

Methodology Of Intracellular Cytokine Staining

Intracellular Cytokine Staining

There are a several methodological variations of the ICC assay described 2, 8-11 . T cells are typically stimulated with peptide epitopes for 6 to 18 hours. We usually stimulate cells with 10 g ml of the peptide and use an irrelevant peptide as negative control, e.g., HIV in HIV-seronegative subjects, binding to the corresponding HLA-A allele. PBMC stimulated with 1 g ml pokeweed mitogen can be used as positive control. After two hours a secretion inhibitor, mostly, brefeldin A Sigma,...

Creating Tcell receptor diversity

Defining the TCR binding region T-cell receptor fingerprinting measures the length of the complementarity determining region 3 CDR3 Figure 1 . In most cases, rearrangement of the TCR loci results from deletional joining. The V genes are localized upstream from the J or D and C genes. Two genes, the recombination activating gene 1 and 2 RAG1 and RAG2 were identified in 1989 and 1990. RAG1 and RAG2 are transcribed in lymphocytes that show recombinase activity i.e. B or T-cells . These genes are -...

Quantitative Analysis Of Cfse Data

Fluorescent division tracking data can be analyzed in a semi-quantitative or fully quantitative manner. Visual inspection of histograms such as that shown in Figure 2 can reveal whether division has occurred, and whether cells in one culture have divided further than another. Simply inspecting division peaks can be misleading when comparing cultures as CFSE profiles provide no indication of cell numbers. For example, two different culture conditions might exhibit similar CFSE profiles however,...

Elispot Assay Formats

The ELISPOT assay can be used for analysis of various types of specimens 6, 7, 8 . The responder cells may be whole PBMC, isolated CD4 and or CD8 T cells, cultured PBMC activated by in vitro sensitization IVS with a stimulating antigen or cultured T-cell lines. Antigen-presenting cells APC may consist of autologous PBMC, dendritic cells DC or a specific T-cell line such as T2 cells. Broadly, the two widely-used formats of ELISPOT can be distinguished as direct or indirect assays. The former...

Measuring cell number

The most direct method to establish proliferation is to sample cells from culture and counting live cells under a microscope using a haemocytometer. Increases in cell number over time establish that cell division has occurred and can give an indication of the rate and approximate time between divisions. It should be remembered, however, that the cell number increase can be offset by the death of cells and that often both processes will occur in a T cell culture. It is possible and common for...