February 25, 2011  |  Education, Flow Cytometry  |  By  |  0 Comments

Immunophenotyping Tumor-Infiltrating T cells

Dataset #4659: Testing Set – T Cell Immunophenotype (trimmed)

Quantifying the percentage of cells expressing a protein of interest is a frequent goal in both basic research and clinical studies. Paired with per-cell comparisons of the level of protein expression, this approach provides a powerful way to track and immunophenotype populations of cells present in a particular sample.

One widely recognized application of flow cytometric immunophenotyping is determining the percentage of CD4+ cells in a gated lymphocyte population in order to determine prognosis for an HIV patient. Other applications include measuring a series of markers in order to distinguish between different forms of leukemia.

In Cytobank, you can use the “percent in gate” statistic to measure and display the percentage of cells in a selected gate as compared to each active population in your figure. To illustrate with a simple example, let’s examine a sample dataset looking at the percentage of CD25+ cells in a CD3+ T cell population.

CD25 is a cell surface protein and the alpha chain of the IL-2 receptor. CD25 expression is often observed on activated T cells and FOXP3+ regulatory T (TReg) cells [1]. There is an interest in phenotyping the T cells infiltrating tumors, because these cells may regulate the immune response to the tumor and thus play a role in the course of the patient’s disease.

The figure above quantifies the CD25+ fraction of CD3+ cells in healthy human blood (“Healthy PBMC”) and follicular lymphoma tumors from five individual patients using “percent in gate” on Cytobank. The y-axis shows CD25 expression and the x-axis shows CD4 expression. The percentage of CD25+ T cells varied significantly from patient to patient. In some follicular lymphoma patients, more than 25% of the tumor-infiltrating T cells were CD25+. These data are from a larger immunophenotyping study performed in parallel with analysis of B and T cell signaling networks in the same follicular lymphoma tumor samples [2].

Visit our support article to learn how to do percent positive in gate in Cytobank.

Questions about immunophenotyping or “percent in gate”? Let us know.


[1] Campbell DJ and Koch MA. Phenotypical and functional specialization of FOXP3 regulatory T cells. Nat Rev Immunol. 2011 Feb;11(2):119-30. PMID 21267013.

[2] Irish JM, et al. B-cell signaling networks reveal a negative prognostic human lymphoma cell subset that emerges during tumor progression. Proc Natl Acad Sci U S A. 2010 Jul 20;107(29):12747-54. Epub 2010 Jun 11. PMID 20543139.