Detection of the potentiality before the actuality: Measurement of T‐cell proliferation before cell division occurs

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 [3H]-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.1 Alternatively, intracellular detection of ki67,2-4 a nuclear antigen expressed by proliferating but not resting cells, or cell surface expression markers such as CD71 and CD78,5 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).6, 7 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.8 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 by a subsequent decline. The authors decided to analyze pSTAT5A 24 h after stimulation, when the levels are still high, to simplify the laboratory working routine. CMV-seropositive subjects showed an increase in pSTAT5A positive T cells after CMV stimulation, whereas CMV-seronegative subjects did not. As expected, CMV-seropositive but not seronegative subjects showed T-cell proliferation in response to CMV, observed after a 7-day culture. The percentage of pSTAT5A-positive T cells after CMV stimulation was able to discriminate between healthy immunocompetent subjects with or without memory T cells able to proliferate in response to CMV antigens, because of previous CMV infection. On the other hand, three patients with a congenital immunodeficiency due to impaired CD28 signaling (CARMIL-2 mutation) and suffering from chronic CMV infection had negligible levels of pSTAT5A-positive T cells and T-cell proliferation in response to CMV. The assay showed satisfactory reproducibility allowing it to be used in diagnostic application. Most of the rapid assays used thus far in the clinical settings are based on measurement of INFγ (and other cytokines), and include intracellular staining (ICS) by flow cytometry, enzyme-linked immunosorbent assay (ELISA), or enzyme-linked immunospot assay (ELISpot). ELISA-based assays are restricted to the detection of IFNγ producing CD8+ T cells and do not allow enumeration and single-cell analysis of activated T cells. Both ELISpot and ICS allow measurement of the percentage of antigen-specific T cells; in addition, ICS also allows a detailed analysis of the T-cell subsets involved, and the coupling of additional markers of T-cell activation, that is, cytokines or cell surface molecules.9 However, these assays cannot predict the complete functionality of the virus-specific T cells and their proliferation capacity, in particular, when only INFγ production is adopted as readout. In fact, dysfunctional or exhausted T cells can still produce INFγ, but are impaired in other cytokine production (such as IL-2) or in their proliferation capacity,10 since INFγ production is the last function to be lost during progressive T-cell impairment and it is retained until full T-cell exhaustion occurs. This is observed especially in the CD8+ T-cell compartment. Therefore, assays that measures only INFγ may not be able to distinguish between protective or dysfunctional T cells. After primary infection in the immunocompetent host, active CMV replication persists for months, while the CMV-specific T-cell pool is constituted mostly of effector-like T cells producing only IFNγ, and memory-like polyfunctional T cells producing IL-2 able to proliferate appear only months after infection.11 Some years ago, it was shown that patients infected by human immunodeficiency virus required the presence of T cells producing both IFNγ and IL-2 to control CMV infection.12 Similarly, in hematopoietic stem cell transplant recipients, patients protected from CMV infection showed the simultaneous production of IFNγ and IL-2, whereas T cells producing only IFNγ did not confer protection.13, 14 In recent years, several efforts have been directed to the analysis of a broader cytokine and activation markers profile, in order to provide a better definition of protective CMV-specific T cells. Polyfunctional CMV-specific CD8+ T cells, positive for additional markers (among IL2, TNFα, CD107A CD40L, MIP1β, granzyme-B, and perforin) besides IFNγ, are detected at higher levels in patients able to spontaneously control CMV infection than in patients at risk of contracting the disease. The novel assay described by Bitar and colleagues (in this issue page 774–783) provides an additional tool for the rapid detection of CMV-specific protective T cells. Measurement of pSTAT5A can be coupled with ICS for cytokine production in order to better identify functional subsets of CMV-specific T cells with different protective potential. Cells retaining proliferative capacity (pSTAT5A-positive after antigenic stimulation) would represent a more efficient subset than T cells producing only IFNγ (or other activation markers) but lacking proliferative potential, thus representing an impaired T-cell subset (Figure 1). The pSTAT5A assay was performed in immunocompetent subjects and in three patients with congenital immunodeficiency. Larger studies in immunocompromised patients are now required to verify the reliability of the novel assay (alone or in combination with the classical ICS for cytokine production) in detecting patients able to spontaneously control CMV infection, who can avoid antiviral interventions, from patients lacking immune protection and thus requiring prophylactic or pre-emptive interventions. If confirming the predictive potential for protective T-cell immunity against CMV, the pSTAT5A assay could also be adapted to analyze the immune response to other pathogens. Chiara Fornara: Writing-review and editing.