Immunophenotyping 101
Immunophenotyping of cells of the immune system is a staple in the workup of patients with immunologic disorders. According to the principle of "Form Follows Function", immunophenotyping is reflective of functional characteristics of the immune system (or lack thereof). Progress in immunophenotyping continues to be made, as demonstrated by the fact that new lymphocyte subsets, as well as important new characteristics of known subsets are discovered in a seemingly unrelenting pace. Ongoing developments in contemporary flow cytometry have been helpful in changing concepts of "multi" in multi-parameter flow cytometry, paralleled by continued refinement in analysis software, and a continued expansion of the repertoire of commercially available reagents. To illustrate the enduring power of immunophenotyping, examples of innovations will be featured on a regular basis in the Newsletter. Consistent with the mission of the DIL, these innovations will make their way into the menu of available tests.
One of the most significant discoveries in T-cell phenotyping has been the detection of alternatively spliced isoforms of the cell surface receptor CD45. The fact that these isoforms are differentially expressed on specific T-cell subsets is widely used in the phenotypic characterization of T-cells, despite the fact their receptors remain largely unknown. The basic premise is that the long isoform of CD45, CD45RA, is expressed on naïve T-cells, while the shorter isoform, CD45RO, is expressed on T-cells that have encountered (i.e. primed by) antigens.
In clinical immunology, this distinction between CD45RA+ versus CD45RO+ T-cell subsets is particular useful to determine the status of the naïve T-cell compartment relative to their thymic origin (i.e. the status of the thymus). Primary immunodeficiency disorders characterized by decreased/absent thymic output often show decreased CD45RA+ T-cells (see figure a). As can be seen in the upper dotplots, obtained from a healthy control (HC), CD4 and CD8 T-cells can be divided into CD45RA+/CD45RO- cells (naïve cells; R3/R7), CD45RA-/CD45RO+ T-cells (primed cells; R5/R9) and, typically, a small population of CD45RA+/CD45RO+ T-cells ("transitional" cells; R4/R8). In contrast, the lower dotplots, obtained from a patient (Pt) with a combined immunodeficiency disorder (CID) shows the almost complete lack of naïve CD4 and CD8 T-cells (R3 and R7, respectively).
The status of the thymus can similarly be followed using these markers in the context of immune reconstitution post stem cell transplantation. Large-scale immune activation in vivo can also induce shifts in the distribution between CD45RA+ and CD45RO+ T-cell subsets (e.g. EBV infection).
| Immuno-101: Markers Discussed |
|---|
| CD4 |
| CD8 |
| CD45RA |
| CD45RO |
| CCR7 |
| Immuno-101: Further Reading |
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| Merkenslager et al. Eur J Immunol. 1988; 18:1653. |
| Hamann et al. J Exp Med. 1997; 186:1407. |
| Sallusto et al. Nature. 1999; 401:708. |
| Amyes et al. J Immunol. 2006; 175-5765. |
An expansion of this basic model incorporates the expression of the chemokine receptor CCR7. Naïve cells express CD45RA and CCR7, while antigen-experienced/primed T-cells can be subdivided into central memory cells (CD45RO+ and CCR7+/bright; "TCM")) and effector memory cells (CD45RO+ and CCR7-/dim; "TEM"). In figure b, CCR7/CD45RO staining are shown from the patient and control in figure a. In this example one can see that the CD45RO positive cells are largely CCR7 negative, consistent with a TEM phenotype.
It has become clear that the distinction between naïve (CD45RA+) T-cells and antigenexperienced (CD45RO+) T cells is quite accurate for T-cells expressing CD4, but not for T-cells expressing CD8. Although an unidirectional differentiation pathway of CD45RA+ into CD45RO+ cells was originally proposed, it has become clear that certain CD8+ T-cells that express CD45RA, are not naïve. They are referred to as effector memory cells RA+ (TEMRA). In figure c, an example is shown from a patient with an immunodeficiency disorder, affecting T-cell generation and function. The upper left dotplot shows a lack of naïve (CD45RA+) CD4+ T-cells (R5).
The CD8+ T-cell compartment seems to show a preserved naïve population, based on the expression of CD45RA (R7), which would suggest that the basic defect in this patient affects CD4+ T-cells differently than CD8+ T-cells. Adding the CCR7 marker shows that the primed CD4+ T-cells indeed have a TEM phenotype (lower left dotplot). However, the presumed naïve CD8+ T-cells are not naïve, but are in fact TEMRA cells. Thus, this expanded immunophenotyping model more accurately identifies the relevant T-cell subsets, with respect to thymic origin, antigen experience and effector/memory classification.
There are many more layers of T-cell immunophenotyping that can be added to the model, for example by including markers that identify activation states (e.g. CD71, HLA-DR), (loss of) co-stimulatory receptors (CD27, CD28), cytokine receptors (e.g. CD127), markers of cytotoxicity (e.g. CD57, perforin) and homing/trafficking receptors (e.g. CD62L, CXCR5). This may be the subject of a future "Immuno-101" discussion. As with all immunologic studies, the power of the assays is determined by how it is used and by how the results are interpreted.
