Carboxyfluorescein succinimidyl ester-based proliferative assays for assessment of T cell function in the diagnostic laboratory

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1 Immunology and Cell Biology (1999) 77, Special Feature Carboxyfluorescein succinimidyl ester-based proliferative assays for assessment of T cell function in the diagnostic laboratory DA FULCHER and SWJ WONG Department of Immunopathology, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, New South Wales, Australia Abstract Immune deficiency diseases are often accompanied by abnormalities in one or both arms of the specific immune system. Impairment can often be detected as a decrease in the number of T or B lymphocytes or their products in the circulation, but questions are often asked as to the functional capabilities of T lymphocytes in patients with recurrent infections. Function of T cells has traditionally been measured by their uptake of [ 3 H]- thymidine following stimulation with antigen or mitogen in vitro. However, the ability of carboxyfluorescein succinimidyl ester (CFSE) to label lymphocytes intracellularly and track their mitotic activity by progressive twofold reduction in fluorescence intensity prompted an alternative methodology based on flow cytometry, an approach which has the advantage of allowing specific gating on particular T cell subsets and simultaneous assessment of activation markers. This method was therefore evaluated for T cell responses to mitogen and antigen. Phytohaemagglutinin-induced blast transformation of CFSE-labelled T cells was reflected by an increase in forward and orthogonal light scatter and a progressive two-fold decrease in CFSE fluorescence intensity. These changes allowed the derivation of various measures of mitotic activity, which correlated well with [ 3 H]-thymidine uptake. Patients with T cell functional deficiencies showed impairment in their responses by both assays, whereas the CFSE-based assay demonstrated that impaired blastogenesis was not simply due to depressed T cell numbers. Concomitant measurement of the activation markers CD69 and CD25 showed that CD69 was rapidly expressed on non-mitotic cells and that this expression was progressively diluted with subsequent rounds of cell division. In contrast, CD25 expression was unaffected by cell cycle, but was expressed in proportion to the PHA dose. Antigenspecific responsiveness to Candida was also assessed using a CFSE-based assay. Initial gating on the relatively minor population of T cells that underwent blast transformation demonstrated progressive twofold dilutions of CFSE intensity in responsive cells. These normal Candida responses, found in patients who had recovered from Candida infection, contrasted with those who had not been infected with Candida or who had chronic recurrent infection, in whom neither blast transformation nor significant mitosis could be detected. Again, there was good correlation with [ 3 H]-thymidine uptake. The CFSE-based assays are equivalent to traditional measures of mitogenand antigen-specific T cell responsiveness in the diagnostic laboratory and have significant advantages in terms of decreased labour intensiveness, avoidance of radioactivity, the ability to gate on a specific population of lymphocytes and the concomitant measurement of activation markers. Key words: [ 3 H]-thymidine, blastogenesis, carboxyfluorescein succinimidyl ester, CD25, CD69, T lymphocytes. Introduction Historically, the investigation of immunodeficiency diseases has centred on in vitro proliferation assays. It was noted early that various mitogenic substances would induce T or B lymphocytes to proliferate and that the diagnosis of certain immunodeficiency diseases could be supported by an absent or sluggish response. The usefulness of these assays has to a certain extent waned with the advent of flow cytometry to reliably and quickly quantify the relative and absolute number of the lymphocyte subsets in peripheral blood. On this basis, primary immunodeficiency diseases of the specific Correspondence: Dr DA Fulcher, Immunopathology, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW 2145, Australia. <davidf@westgate.wh.usyd.edu.au> Received 23 August 1999; accepted 23 August immune system can be divided into those manifest by predominantly B cell deficits, T cell deficits or a combined deficit. 1 Nevertheless these measures yield no useful information about the ability of cells to respond when exposed to their target antigen. Assessment of T cell function requires more complex assays measuring various aspects of T cell biology, typically their ability to divide or produce cytokines in response to mitogen or antigen. The classical means of measuring the T cell proliferative response to such stimuli has been via their uptake of [ 3 H]- thymidine during the final hours of a 3 to 5 day culture. This assay is rather cumbersome and labour-intensive, requiring handling of radioisotopes, scintillation fluid and expensive counting equipment. Interpretation of results is also problematic; a low level of [ 3 H]-thymidine uptake could be explained either by a depressed T cell count, impaired cell function or a poor batch of [ 3 H]-thymidine. The latter can be overcome by matching patient samples with appropriate

2 560 DA Fulcher and S Wong controls, but this is often difficult, particularly when analysing hospital patients or children. Some investigators recommend analysing 10 or so controls and cryopreserving cells from the median three responders. 2 Newer, simpler methods may address some of these shortcomings. The use of carboxyfluorescein succinimidyl ester (CFSE) has generated widespread interest for tracking cells in vivo and also tracing their mitotic activity. 3 This fluorescent dye stains intracellular proteins and generates a fluorescent signal that progressively halves with each mitosis. Reduction in fluorescence intensity can be quantified by flow cytometry (FCM) and an algorithm used to evaluate the extent of blastogenesis. Furthermore, cultured cells can be stained for expression of other cell surface markers to define lineage or activation state. The present paper summarizes our experience with a CFSE-based assay for the assessment of mitogenic and antigenic T cell proliferation, using phytohaemagglutinin (PHA) and Candida antigen as candidate mitogenic and antigenic stimuli, respectively, in comparison to the traditional [ 3 H]-thymidine-based blastogenesis assay. Phytohaemagglutinin blastogenesis assay Carboxyfluorescein succinimidyl ester-based assessment of blastogenesis The traditional blastogenesis assay requires culture of peripheral blood mononuclear cells over a 3-day period with increasing doses of PHA, a [ 3 H]-thymidine pulse being added for the final 6 h. The CFSE-based assay is essentially identical to this, Figure 1 The 3 day blastogenic response of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled peripheral blood mononuclear cells to stimulation with PHA at a dose of 0 µg/ml (upper three panels), 1 µg/ml (middle three panels) and 5 µg/ml (lower three panels). The CFSE-based blastogenesis methodology has been described in detail. 4 Briefly, peripheral blood lymphocytes were labelled with CFSE by incubation at 37 C at a cell density of cells/ml with 7.5 µmol/l CFSE in Roswell Park Memorial Institute media (RPMI) without added protein. After 10 min, cells were washed in RPMI supplemented with 10% AB serum at 4 C and then cultured at /200 µl well. Cells were stimulated with phytohaemagglutinin (Murex Biotech Ltd, England, Cat HA16) at 0, 1 and 5 µg/ml then harvested after 3 days. Cells were then stained for CD3 (anti-cd3-pecy5, Immunotech, Marseille, France) and either CD69 (anti-cd69- PE, Becton Dickinson, San Jose, CA, USA) or CD25 (IL-2Rα) (anti-cd25-pe, Becton Dickinson). All plots were gated on CD3-positive cells (histograms not shown), while the histograms of (b) and (c) were also gated to include both resting lymphocytes (R1) and blasts (R2). (a) Alterations in light scatter characteristics; (b) expression of CD25 on CD3 + cells in culture; (c) progressive two-fold dilutions of CFSE that accompanied mitotic cell division. Application of an analysis algorithm (see text) results in a division index of 1.64 (1 µg/ml PHA) and 3.31 (5 µg/ml PHA) divisions per added T cell.

3 Proliferative assays of T cell function 561 with the addition of an initial labelling stage and the omission of the [ 3 H]-thymidine pulse. Blast transformation of T cells was represented by increased forward and orthogonal scatter of light, a response that was proportional to the dose of PHA (Fig. 1a). Their activated state was accompanied by upregulation of the IL-2Rα (Fig. 1b) and by cellular division represented by progressive halving of CFSE intensity (Fig. 1c). Even after 3 days, however, not all cells had entered cell cycle, particularly at the lower stimulation dose. Measuring mitotic activity with CFSE Figure 2 The PHA response, as determined by uptake of [ 3 H]- thymidine, in a control subject ( ) compared with the patient with systemic lupus erythematosus and CD4 lymphopenia ( ). Peripheral blood mononuclear cells were cultured with doses of PHA as indicated and their uptake of [ 3 H]-thymidine over the last 6 h of culture determined. Stimulation indices, representing mean c.p.m. in wells cultured with PHA divided by the mean of those cultured without PHA, are plotted against the PHA dose. The mitotic activity of the T cell population can be estimated based on the number of cell divisions, expressed as a proportion of the entire starting T cell population. This algorithm assumes that the presence of two cells in a lower CFSE fluorescence gate arose from the mitosis of a single cell of the next highest fluorescence intensity. A simple formula (see Angulo and Fulcher 4 ) yields the division index, a value of 1 representing one mitosis for every cell added to culture. This analysis can be directed to a specific population of cells defined by both light scatter characteristics and cell surface antigen expression. In a typical analysis of PHA-stimulated cells, light scatter profiles of CD3-positive cells are used to calculate the percentage of T cells that have moved from the resting (Fig. 1, R1) to the blast (Fig. 1, R2) population ( percentage blast transformation, %BT). Histograms displaying CFSE and activation marker expression are gated by light scatter to include all viable T lymphocytes (i.e. both resting and blast cells), and the CFSE histogram is then used to determine the division index (DI). Both DI and %BT values are likely to underestimate overall mitotic activity, because cells that die in culture will not be included and cell divisions that follow dilution of CFSE intensity to autofluorescence levels cannot be evaluated. Correlation with [ 3 H]-thymidine incorporation To determine the validity of CFSE-based measures of blastogenic activity, DI and %BT values were compared with [ 3 H]-thymidine incorporation in 11 normal controls along with two patients with T cell deficiency. The latter included a patient with systemic lupus erythematosis, multiple opportunistic infections and marked CD4 lymphopenia, persistent after withdrawal of all immunosuppressive medication, and a 6-month old boy with di George syndrome. Given the greater cell numbers required for flow cytometry, CFSE analysis was restricted to three PHA concentrations (0, 1 and 5 µg/ml), while [ 3 H]-thymidine incorporation was measured at 0, 1, 2.5, 5, 10 and 25 µg/ml and the peak uptake determined. All 11 control subjects demonstrated normal blastogenic responses based on incorporation of [ 3 H]-thymidine, while the Figure 3 The PHA responses, as determined by carboxyfluorescein diacetate succinimidyl ester (CFSE), in a control subject and the patient with systemic lupus erythematosus and CD4 lymphopenia. Gating strategy is identical to the corresponding plots in Fig. 1.

4 562 DA Fulcher and S Wong Figure 4 Correlation between 1 µg/ml (a, c, e) and 5 µg/ml (b, d, f) PHA responses as determined by [ 3 H]-thymidine uptake (maximal c.p.m. obtained at any PHA dose) and the carboxyfluorescein diacetate succinimidyl ester (CFSE)-derived values division index, percentage blast transformation and increase in CD69- positive cells. The two patients with T cell immunodeficiency are depicted by filled circles. two patients with T cell deficiency had flat responses (see Fig. 2). The CFSE-based assay also reflected these differences. T cells from control subjects moved into the blast gate and divided whereas T cells from the patients with T cell deficiency did not respond in either fashion (Fig. 3). Furthermore, CFSE-derived values correlated well with the peak [ 3 H]-thymidine uptake, with good discrimination between responders and non-responders in this small cohort of subjects (Fig. 4). Quantitative analysis demonstrated that the two patients with T cell dysfunction showed reduced division indices (Fig. 4a,b) and blast transformation percentages (Fig. 4c,d), which corresponded with low uptake of radionucleotide. The inability of radionucleotide-based assays to discriminate between poor T cell function as opposed to decreased T cell numbers was overcome by the CFSE-based assay, which was able to determine the mitotic activity of T cells even when they constituted the minority of the cultured population. Activation markers and cell cycle T-cell responsiveness to mitogen can also be analysed according to the expression of a number of activation markers. CD69 is expressed soon after activation, peaking at 24 h, while expression of CD25 and CD71 is maximal after several days stimulation. 5 In the 13 subjects analysed above, the CD69 response did not differ between the two patients with T cell immunodeficiency and control subjects (Fig. 4e,f). This may have been due to harvesting the cells at a time well past that of maximal expression, a drawback that

5 Proliferative assays of T cell function 563 Figure 5 Relationship between cell cycle and expression of T cell activation markers CD69 (a, b) and CD25 (c, d) at two doses of PHA. Dot plots are gated by CD3- positivity and light scatter, the latter to include both resting lymphocytes and blasts (see legend to Fig. 1). may be overcome using other activation markers that peak at later timepoints. Staining for T-cell activation markers as well as with CFSE afforded a unique opportunity to examine the relationship between their expression and the cell cycle. After 3 days in culture, undivided cells demonstrated high expression of CD69, similar at both PHA doses examined, which then declined progressively with each cell division (Fig. 5). This demonstrated that the designation of CD69 as an early T-cell activation marker is an over-simplification, because expression was only early in terms of progression through the cell cycle rather than time in culture. One explanation for these observations was that synthesis and expression of CD69 occurred soon after activation, at a stage preceding cell division, and that the subsequent decline occurred as a result of a mitotic dilutional effect. By contrast, expression of CD25 was directly proportional to the mitogenic stimulus and bore no apparent relationship to cell cycle (Fig. 5), suggesting active synthesis throughout the culture period irrespective of mitosis. Antigen-specific assays Phytohaemagglutinin induces almost all T cells to enter cell cycle, hence the kinetic activity of stimulated cells proved relatively easy to measure. In the case of antigenic T-cell stimulation, it was expected that only a small minority of the cultured T cells would respond to any given antigen and therefore that mitotic activity may be difficult to detect by flow cytometry above the background of unstimulated cells. Indeed, this proved to be the case when the entire population of CFSE-labelled T cells from Candida-exposed women were cultured with Candida antigen (data not shown). However, by directing analysis to cells which migrated into the blast light scatter gate, the typical CFSE dilutional pattern was found. Thus, subjects who had not been infected with Candida, or had chronic candidiasis, showed no blast transformation and minimal cell division, whereas T cells from Candida-responsive controls divided normally (Fig. 6). Similar to the PHA analysis, there was good correlation between CFSE-derived indices (DI, %BT and the derived product of these two values) and [ 3 H]-thymidine incorporation in the 5-day culture. 4 It should be noted that in occasional non-responders, a significant number of CD3 + cells were sometimes found in the blast scatter gate but these T cells were non-mitotic, emphasizing the need to measure mitotic activity in conjunction with alterations in cell size. This prompted the mutliplication of the two values (DI and %BT) to generate the weighted division index, which combined these parameters into a single derived value. Expression of CD69 was also analysed in this antigenspecific assay by gating on the entire T cell population in culture. In contrast to the PHA studies, there did appear to be differences in the two patient populations, with Candida responders demonstrating an increased proportion of CD69 + T cells in comparison to non-responders. 4 However, there was

6 564 DA Fulcher and S Wong Figure 6 Candida-specific T-cell blastogenesis in a typical responder (left panels) and a non-responder (right panels). Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled peripheral blood mononuclear cells were cultured with Candida antigen (Bayer) at 0, 0.78 and 12.5 mg/ml and harvested after 5 days. Dot-plots are gated on CD3-positive cells, while CFSE histograms are gated on CD3- positive blasts (R2). significant overlap between the two groups and differences were less useful than mitotic values. Our interest has focused on Candida blastogenesis, due to a clinical interest in patients with recurrent Candida infections, although the same principles should be generally applicable to the measurement of T-cell responsiveness to other antigens. Conclusion The close relationship between traditional measures of T-cell responsiveness and CFSE-derived values has supported a change in our diagnostic laboratory away from cumbersome radioisotope-based assays towards newer CFSE-based assays employing flow cytometry. The latter are: (i) less labour intensive; (ii) cheaper to perform; (iii) able to focus analysis on particular lymphocyte subsets; and (iv) able to measure expression of activation markers directly and in terms of cell cycle. Their utility in the assessment of patients with suspected immunodeficiency disorders is the subject of ongoing evaluation. Acknowledgements We would like to thank Rudy Angulo for his help in performing the Candida proliferation assays, and the laboratory volunteers who donated blood for the normal controls. References 1 Report of a WHO Scientific Group. Primary immunodeficiency diseases. Clin. Exp. Immunol. 1997; 109 (Suppl. 1): S Maluish AE, Strong DM. Lymphocyte proliferation. In: Rose NR, Friedman HF, Fahey JL (eds). Manual of Clinical Laboratory Immunology. Washington, DC: American Society for Microbiology, 1986; Lyons AB, Parish CR. Determination of lymphocyte division by flow cytometry. J. Immunol. Meth. 1994; 171: Angulo R, Fulcher DA. Measurement of Candida-specific blastogenesis Comparison of carboxyfluorescein succinimidyl ester-labelling of T-cells, thymidine incorporation and CD69 expression. Comm. Clin. Cytometry 1998; 34: Biselli R, Matricardi PM, D Amelio R, Fattorossi A. Multiparametric flow cytometric analysis of the kinetics of surface molecule expression after polyclonal activation of human peripheral blood T lymphocytes. Scand. J. Immunol. 1992; 35: