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Chapter 28 : Lymphocyte Activation

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Lymphocyte Activation, Page 1 of 2

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Abstract:

Measurement of lymphocyte activation is a key diagnostic component in the work-up of several immunological diseases: either primary in nature or altered as a result of immune-modifying therapies or other diseases that are not genetic immune deficiencies. There are many ways to measure lymphocyte activation, but the two main approaches in the diagnostic immunology laboratory include immunophenotyping (measurement of cell surface or intracellular activation markers after lymphocyte stimulation) and functional assays (where there is direct assessment of a certain function, e.g., proliferation after lymphocyte activation or cytotoxicity). Among the lymphocyte subsets that are typically assessed for activation status as a measure of immune competence, T cells are the most common, though in some contexts it is also appropriate to evaluate B cell and NK cell activation or function. This chapter focuses primarily on discussing assays and methods related to measurement of T cell activation and function.

Citation: Abraham R. 2016. Lymphocyte Activation, p 269-279. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch28
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Figures

Image of FIGURE 1
FIGURE 1

(A) Flow cytometric measurement of cellular proliferation with Edu. The flow cytometry results show a detailed assessment of proliferating lymphocytes after stimulation with PHA. Proliferating cells are apparent in comparing the unstimulated (upper panels) to stimulated (lower panels) samples, and the use of specific antibodies (CD45, total lymphocytes; and CD3, total T cells) allows further characterization. (B) Assessment of cell viability for lymphocyte proliferation assays. The CD45 marker is used to identify lymphocytes. Viable, apoptotic, and dead cells are identified in the original sample used for cell proliferation, prior to initiation of cell culture, by using a combination of two fluorescent dyes: annexin V and 7-AAD. Viable cells are negative for both markers, while dead cells are positive for both annexin V and 7-AAD. Only apoptotic cells are positive for annexin V alone.

Citation: Abraham R. 2016. Lymphocyte Activation, p 269-279. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch28
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Image of FIGURE 2
FIGURE 2

Ki-67-based cell proliferation. There are several markers available for flow cytometric measurement of cellular proliferation, including CFSE, Edu, and Ki-67. Ki-67 is a nuclear marker that assesses cell proliferation, and the use of specific antibodies in conjunction with this marker permits identification of proliferating subsets of T cells.

Citation: Abraham R. 2016. Lymphocyte Activation, p 269-279. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch28
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Image of FIGURE 3
FIGURE 3

Activated T cells produce various cytokines in a temporally regulated manner. The important cytokines produced during the temporal course of T cell activation and differentiation include IL-2, IFN-γ, and TNF-α. (A) Polyfunctional T cell analysis of CD4 T cells. The cytokines are measured by intracellular flow cytometry analysis of CD4 T cells. The top panels show normal production of these cytokines in activated CD4 T cells from a healthy donor after cell stimulation with PHA. The bottom panels show the absence of cytokine production after stimulation of CD4 T cells from a patient with compromised cellular function. (B) Polyfunctional T cell analysis of CD8 T cells. The cytokines are measured by intracellular flow cytometry analysis of CD8 T cells. The top panels show normal production of these cytokines in activated CD8 T cells from a healthy donor after cell stimulation with PHA. The bottom panels show the absence of cytokine production after stimulation of CD8 T cells from a patient with compromised cellular function.

Citation: Abraham R. 2016. Lymphocyte Activation, p 269-279. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch28
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Image of FIGURE 4
FIGURE 4

(A) Flow cytometry analysis of T cell proliferation in response to PHA. PHA is a commonly used mitogen for global assessment of T cell function. The top panels show normal proliferative responses to PHA for a healthy individual, and the bottom panels show decreased T cell proliferation for a patient with T cell dysfunction. (B) Flow cytometry analysis of cell proliferation in response to PWM. PWM is a relatively weak T cell mitogen compared to PHA. However, PWM also induces B cell proliferation; hence, it is still used in the clinical laboratory. The top panels show normal proliferative responses to PWM for a healthy individual, and the bottom panels show decreased proliferation in both T and B cells for a patient with immunodeficiency.

Citation: Abraham R. 2016. Lymphocyte Activation, p 269-279. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch28
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Image of FIGURE 5
FIGURE 5

(A) Flow cytometry analysis of T cell proliferation in response to anti-CD3 stimulants for a healthy donor. Anti-CD3 antibody is often used to assess T cell functional competence, particularly in contexts where a detailed evaluation of T cell proliferative responses is essential. Soluble anti-CD3 alone delivers a weak mitogenic signal, although the addition of anti-CD28 or exogenous IL-2 to soluble anti-CD3 improves the proliferative response, as shown here. (B) Flow cytometry analysis of T cell proliferation in response to anti-CD3 stimulants in a patient with immunodeficiency. Soluble anti-CD3 alone delivers a weak signal, and addition of anti-CD28 does not show incremental proliferation. However, addition of exogenous IL-2 to soluble anti-CD3 modestly improves the proliferative response, though it remains outside the normal values for age.

Citation: Abraham R. 2016. Lymphocyte Activation, p 269-279. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch28
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