Cytometry Part A | 2021
Detection of the potentiality before the actuality: Measurement of T‐cell proliferation before cell division occurs
Abstract
After an encounter with an infectious agent, naïve T lymphocytes that recognize the antigens of the pathogen expand and acquire effector functions (i.e., cytokine production and cytotoxic activity), enabling control of the infection and clearance of the infected cells. After antigen clearance, while the majority of expanded effectors disappear, a subset of the expanded antigen-specific T cells persists, constituting a pool of memory T cells, which promptly proliferate and differentiate into effector cells upon re-exposure to the antigen. The rapid and strong recall response is the hallmark of adaptive immunity that allows control of the infection and makes disease prevention possible. For years, measurement of T-cell proliferation in vitro in response to recall antigens has been used to determine the efficacy of the cellular immunity in different clinical settings, and especially in immunocompromised patients. Initially, assays for lymphocyte proliferation were based on the measurement of [H]-thymidine uptake by proliferating cells. Subsequently, flow cytometry assays have been developed. Currently, the most widely used assays are based on T-cell staining with vital dyes before stimulation, and they measure dye dilution and serial fluorescence intensity halving that occur in dividing cells as a consequence of cell proliferation. Alternatively, intracellular detection of ki67, a nuclear antigen expressed by proliferating but not resting cells, or cell surface expression markers such as CD71 and CD78, has been proposed as readout of T-cell proliferation. However, these assays require cell culture for 3 days (to measure proliferation to nonspecific polyclonal stimuli, such as anti-CD3/CD28 or PHA) or for 5–7 days (to measure antigen-specific proliferation). Therefore, rapid and simple methods enabling the detection of lymphocytes entering into proliferation cycles are sought, especially with the aim of implementing immune monitoring assays in the clinical practice. The measurement of antigen-specific immune response is entering into the range of diagnostic tools, guiding the management of human cytomegalovirus (CMV) infection in immunocompromised patients, namely, solid organ, or hematopoietic stem cell transplant recipients. CMV belongs to the family of the herpesviruses and infects the great majority of the population worldwide. After primary infection, CMV establishes a lifelong relationship with the human host, entering into a latency status with intermittent reactivation episodes, under the control of T-cell immunity. The infection is usually asymptomatic or mildly symptomatic in the immunocompetent host, but becomes dangerous when CMV infects women during pregnancy and is transmitted to the fetus, or when it is in the immunocompromised patient. CMV infection represents a major complication in transplant recipients, leading to life-threatening disease in patients lacking CMV immunity if antiviral interventions are not promptly implemented. Measurement of CMV-specific T-cell response would provide fundamental information on the patient s immune protection against CMV, therefore, allowing decisions on the necessity to implement preventive antiviral interventions (in the absence of protective immunity) or stop them (when in the presence of immunity). In this issue of Cytometry Part A (page 774–783), Bitar and colleagues present a novel and rapid method for evaluating CMV-specific T-cell entry into a proliferation program. The assay measures, by flow cytometry, the phosphorylation of signal transducer and activator of transcription 5 A (STAT5A) after 24 h stimulation with CMV antigens. After binding of the cognate ligand to the T-cell receptor (TCR), the nuclear factor of activated T cells (NFAT) is activated and migrates into the nucleus, where it activates, in turn, its target genes, among which IL-2. Subsequently, binding of IL-2 to the IL-2 receptor (IL-2R)βγ activates Janus kinase 3 (Jak3), which in turn phosphorylates (STAT5). Phosphorylated STAT5 (pSTAT5) translocate to the nucleus, leading to the transcription of target genes including the IL-2Rα and the subsequent formation of the high-affinity IL-2Rαβγ. The activation of the IL-2 and high-affinity IL-2Rαβγ system is mandatory for T-cell entry into the proliferation program. The same authors previously showed that detection of pSTAT5A can reliably assess T-cell proliferation to polyclonal stimuli. T cells stimulated with peptides of immunodominant CMV antigens show detectable levels of pSTAT5A 6 h after stimulation, with peak levels of pSTAT5A positive cells 12 h after stimulation, followed Received: 13 January 2021 Accepted: 19 January 2021