Immunophenotyping 101
Neither CD4 nor CD8: Double-Negative T-Cells
In the late 1970s, the dichotomy between Tlymphocyte subsets, based on CD4 and CD8 expression was firmly established. It seems that no other immunophenotypic marker that is exclusively expressed on CD4+ or CD8+ T cells has been found since (the Reader is kindly invited to submit references indicating otherwise). In the traditional dual-parameter dotplot; CD4 and CD8 are shown on alternate (X or Y) axes, corresponding to two different fluorescence (FL) channels (see left dotplot in figure 4a). As can be seen in this example, T cells are predominantly CD4+ (lower right [LR] quadrant), or CD8+ (upper left [UL] quadrant). In peripheral blood, few T cells express both CD4 and CD8 (UR quadrant).
In this edition of Immuno-101, the focus will be on the LL quadrant: T cells that express neither CD4 nor CD8. These cells are referred to as double-negative T cells (DNTCs). These cells can be anything from a curiosity to a diagnostic finding.
After the discovery of the CD4/CD8 dichotomy it became apparent that CD4 and CD8 are not the only two choices: a small population of DNTCs can be found in most humans. This is shown in a different way in the right dotplot in figure 4a. CD4 and CD8 are represented by the same FL channel on the Y-axis, while the Xaxis corresponds to T cells expressing the alpha/beta T-cell receptor (α/β-TcR). Thus, DNTCs are found in the LL and LR quadrants, while single-positive T cells (SPTCs; since one cannot determine which cell population corresponds to CD4 or CD8) are in the upper quadrants. Based on the Xaxis, T cells can be classified as being α/β-TcR-positive or α/β-TcR-negative (and thus gamma/delta TcR-positive, since T cells express either the α/β-TcR or the γ/δ-TcR).
DNTCs in humans are typically of the γ/δ-TcR variety. This is illustrated in figure 4b. The majority of cells are α/β-TcR+/SPTCs (either CD4+ or CD8+ T cells; 70.9% in the UR quadrant). The large population below the horizontal quadrant marker is defined as a γ/δ-TcR+/DNTC population (LL quadrant; 24.9% of T cells). This is also shown in a different format on the right (gated on all lymphocytes). The populations of cells in the left dotplot that remain to be defined are in the UL quadrant (γ/δ-TcR+/SPTCs; 7.6% of T cells) and in the LR quadrant (α/β−TcR+/DNTCs; ~1.1% of T cells).
Thus, upon finding DNTCs, the first question to ask is whether they are gamma/delta T cells (more common) or alpha/beta T cells (less common).
The rare population of α/β-TcR+/DNTCs received notoriety when it was discovered that this T-cell subset is expanded in patients with autoimmune lymphoproliferative syndrome (ALPS). An example, obtained from an ALPS panel, is shown in figure 4c. There are ALPS patients who show only small DNTC expansions (especially if immunosuppressive drugs are used). In addition, similarly expanded populations of α/β-TcR+/DNTCs can sometimes be found in healthy young children, as well as in patients with other immunological disorders. Distinguishing between these different scenarios can be difficult, but not impossible if additional immunophenotypic markers are included.
B220 (an isoform of CD45) is a particularly useful marker, as it appears to signify defective Fas-mediated apoptosis (the principle defect in ALPS). In figure 4c, several examples are shown of expanded α/β-TcR+/DNTC populations (left dotplots) in combination with B220 expression (right dotplots; gated on the cells in LR). The dotplots on top are from a patient with ALPS; the second set are derived from a patient with autoimmune manifestations, not on the basis of ALPS, while the third and fourth sets represent two patients with immunodeficiency disorders (a child with an undetermined T-cell defect and a child with DiGeorge syndrome, respectively). All dotplots were derived from the ALPS panel.
Taken in combination with the presence or absence of other immunophenotypic markers (e.g. HLA-DR, CD45RO), it appears that the origin (and perhaps disorder-specific relevance) of these DNTCs is different than those found in ALPS (e.g. extra-thymic differentiation versus end-stage T-cell differentiation and lack of apoptotic clearance).
There are other – equally rare – T-cell populations that can be found within the DNTC compartment (they may also have a counterpart within a SPTC compartment). For example, natural killer T cells (NKT cells) are defined by the expression of an α/β-TcR, defined in humans as Valpha24 and Vbeta11, in combination with NK-cell and other markers and sharing both "T-cell" and "NKcell" functional characteristics. Other rare populations of DNTCs have been reported in mice. It remains unclear if these DNTCs exist in humans.
Lastly, it should be considered that DNTCs are in fact thymocytes inappropriately "caught" in peripheral blood at the doublenegative state. Immunophenotypic markers that are specific for thymocytes, such as belonging to the CD1 family, in combination with markers that thymocytes typically do not express, can assist in distinguishing thymocytes from mature T cells.
Using a palette of immunophenotyping markers that focus both on absence and presence of cell surface and intracellular markers, including markers that may reflect pathogenesis (e.g. B220), as well as a more in-depth analysis of the immune system in general, one should be able to determine the identity of most (clinically relevant) DNTC populations and tie those populations to immunologic scenarios. As with all immunologic data, it is important to take age of the individual into consideration.
