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Development of an anti-CD2/CD3/CD28 bead-based T-cell proliferation assay

Development of an anti-CD2/CD3/CD28 bead-based T-cell proliferation assay BioscienceHorizons Volume 7 2014 10.1093/biohorizons/hzu012 Research article Development of an anti-CD2/CD3/CD28 bead-based T-cell proliferation assay 1,2, Louisa Green * Clinical Laboratory Immunology, Oxford University Hospitals NHS Trust, Churchill Hospital, Old Road, Headington, Oxford OX3 7LJ, England Oxford Brookes University, Gypsy Lane, Headington, Oxford OX3 0BP, England *Corresponding author: Tel: +44 07725748099. Email: lgreen2891@gmail.com Supervisor: Dr Lisa Ayers, Clinical Laboratory Immunology, Oxford University Hospitals NHS Trust, Churchill Hospital, Old Road, Headington, Oxford OX3 7LJ, England. Email: lisa.ayers@nhs.net Severe combined immunodeficiency (SCID) has many phenotypes , including those which result in the formation of T cells but render them incapable of an optimal level of proliferation following stimulation by an antigen. Phytohaemagglutinin (PHA) is a mitogen commonly used to assess T-cell proliferation, although anti-CD2/CD3/CD28-coated beads may provide a more physiological trigger. Some patients and healthy controls display poor T-cell proliferation (<45%) in response to PHA, but may be capable of an increased percentage proliferation when stimulated by another mitogen. This study aimed to develop an anti-CD2/CD3/CD28 bead-based T-cell proliferation assay and to compare T-cell proliferation generated by two mitogens both in healthy controls and in poor responders to PHA. A bead-based T-cell proliferation assay was optimized and validated. Peripheral blood mononuclear cells (PBMCs) were extracted from whole blood samples of 10 healthy controls and 3 poor PHA responders. PBMCs were incubated with carboxyfluorescin diacetate succinimidyl ester and three separate stimulants: PHA, Beads only and Beads plus IL-2. Flow cytometry was performed on Day 6, following fluorochrome staining with CD3 PerCP and CD4 APC, to identify CD4 T cells. No significant difference was found between the results generated by PHA- or Bead stimula - tion in 10 healthy controls. A strong positive correlation was observed between the use of the Beads alone and the addition of IL-2. A varied response to the Beads was produced among the poor PHA responders. It has been concluded that the bead- based assay will be run alongside every PHA test in the Oxford Immunology diagnostic laboratory. The step to add IL-2 on Day 3 will not be included as this did not significantly impact T-cell proliferation. The Bead assay provides a more physiological addition to the current PHA test and may be useful in assessing T-cell proliferation in those patients who respond poorly to PHA. However, some individuals who respond poorly to PHA may also respond poorly to the anti-CD2/CD3/CD28 Beads. Key words: PHA, anti-CD2/CD3/CD28 beads, T cell, proliferation, diagnosis, immunodeficiency Submitted on 14 June 2014; accepted on 17 November 2014 Introduction intrinsic genetic defects resulting in the complete absence or impaired function of a cell type or types (Geha et al., 2007). The immune system comprises a multitude of cells and path- In contrast, secondary immunodeficiencies are acquired in ways that work together to ultimately keep an organism safe later life and can arise secondary to infection, such as with from infectious disease and harm. However, a breakdown in human immunodeficiency virus (HIV), severe malnutrition or this networking can have a direct and compromising effect as a result of immunosuppressive drug use (Hall and Yates, on the successful functioning of the immune system, and the 2010). Whether the condition is congenital or acquired, both term used to cover and define such causative conditions is the innate and adaptive response types can be impaired (Geha immunodeficiency. Primary immunodeficiencies arise due to et al., 2007), and there is often an overlap with both T and B © The Author 2014. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Research article Bioscience Horizons • Volume 7 2014 lymphocytes being affected, even if only one of those cell laboratory currently performs an assay that assesses the ability of types is absent or does not function as it should. T cells to proliferate following stimulation with a mitogen called Phytohaemagglutinin (PHA). During this assay, peripheral blood An umbrella term used to cover many austere T-cell primary mononuclear cells (PBMCs) are first isolated from whole blood immunodeficiencies is severe combined immunodeficiency and incubated with carboxyfluorescin diacetate succinimidyl (SCID). The illness is termed severe because it is fatal and is ester (CFSE), a stable, membrane-permanent dye that covalently described as combined because although it is often directly couples to intracellular molecules. The T cells of healthy indi- T lymphocytes which are affected, B lymphocytes cannot ade- viduals would normally proliferate upon PHA stimulation, and quately function without the help of T cells and thus both cell- flow cytometric analysis should reveal a decline in CFSE staining mediated and humoural immunity are lost (Tasher and Dalal, within the PHA-stimulated cells as the dye is divided equally with 2012). SCID is considered a paediatric emergency with patients each cell division (Parish et al., 2009). However, this is not appearing well at birth but after a few months, following the observed in those patients whose T-cell proliferation is impaired subsequent loss of maternal antibody, often present with diar- (Fig. 1); thus, the PHA investigation is often a supportive test that rhoea, sepsis, otitis and an overall failure to thrive (Kelly et al., leads to a primary immunodeficiency diagnosis. 2013). Typically, children with SCID have a reduced spleen size Some patients and healthy controls show a reduced PHA and a small thymus weighing <1 g (Buckley, 2004), although response (a proliferation of <45%), but may be capable of these abnormalities are not usually noted until it becomes normal T-cell proliferation following stimulation by other apparent that the infant frequently falls ill and is not developing mitogens. The overall aim of this research was to establish at a normal rate. Opportunistic infections, caused by viral and develop a new test that uses anti-CD2/CD3/CD28-coated pathogens including cytomegalovirus and herpes simplex virus, Anti-Biotin MACSiBead™ particles to stimulate T-cell prolif- are another common medical concern for patients who have eration. More specifically, we wanted to conduct the PHA and T-cell deficiencies, and SCID sufferers often have such compro - Bead assays on at least 10 healthy control samples, compare mised immunity that even an attenuated vaccine can result in the results, identify any control samples that did not respond fatality (Kelly et al., 2013). well to PHA and evaluate the T-cell proliferation using the If SCID or another primary immunodeficiency is suspected, Beads. Furthermore, we aimed to investigate the impact of there are a set of standard differential tests undertaken to achieve IL-2 on T-cell proliferation. IL-2 plays a pivotal role in the a diagnosis. Crucially, it is important to rule out a secondary immune response and is secreted by T cells following the immunodeficiency, particularly HIV, and providing this result is binding of an antigen to the T-cell receptor (Cornish, Sinclair negative the next investigation should be the full blood count. and Cantrell, 2006). The production of IL-2 upon antigen The results of this test provide a breakdown of the amount of binding stimulates the up-regulation of the IL-2 receptor gene, each cell type comprising the blood; however, the main one of thus facilitating increased interaction between the two 9 −1 immunological interest is the lymphocyte count; 1–5 × 10 l for (Beadling and Smith, 2002). Moreover, the engagement of the young children is suggestive of an immunodeficiency. If this is the T-cell receptor CD3 complex on the surface of antigen- case, it should prompt a clinician to request analysis of specific presenting cells with the co-stimulatory molecule CD28 leads immunoglobulin levels to rule out a B-cell immunodeficiency , to the activation of numerous tyrosine kinase pathways, and then lymphocyte phenotyping to attain an absolute count which drive the transcription of the IL-2 gene. The interaction of lymphocyte subsets. In addition, the Oxford Immunology between IL-2 and its receptor stimulates the growth and Figure 1. Normal T-cell proliferation vs. impaired T-cell proliferation, and the effect of both on CFSE staining. 2 Bioscience Horizons • Volume 7 2014 Research article + + differentiation of antigen-specific CD4 T cells and CD8 chamber (Immune Systems Limited, UK), under a ×10 objec- T cells (Beadling and Smith, 2002). Our final aim was to tive lens, was conducted. establish a reference range of percentage proliferation values for the proposed Bead assay, with and without the addition of CFSE staining IL-2 on Day 3 of incubation. One microlitre of 2.25 µ M CFSE stock (Sigma-Aldrich, UK) was added to the PBMC resuspensions. Tubes were wrapped in Methods aluminium foil to reduce photosensitive CFSE reactions and placed into a water bath at 37°C for 15 min. Ten microlitres of Subjects ice-cold RPMI were added to prevent further proliferation and to ensure that all cells remained at the same point in the cell A cohort of subjects working in the Oxford Immunology cycle. The tubes were centrifuged at 483 g for 5 min. The super- laboratory was selected. All participants were over 18, and natant was discarded and the pellets were resuspended in 10 mL exclusion criterion was any known primary or secondary ice-cold RMPI. The tubes were again centrifuged at 483 g for a immunodeficiency. Demographics are shown in Table 1. further 5 min and the supernatant was again discarded. Ethics approval was obtained (05/Q1605/88), and all partici- pants gave informed consent prior to involvement. Current protocol: PHA PBMC separation The volume of culture medium AIM-V (Oxford Immunotec, 6 −1 All works performed were conducted under aseptic conditions. UK) required to achieve a cell concentration of 1 × 10 mL Blood from each control subject was drawn into three 3 mL was calculated for each control. A sterile 96-well, round- lithium heparin collection tubes (Becton Dickinson, UK) and bottomed plate (Sigma-Aldrich, UK) had wells marked for diluted 1 : 2 with RPMI (Sigma-Aldrich, UK) in a sterile Falcon unstimulated, PHA-stimulated and two Bead-stimulated tube. The blood–RMPI solution of each control was carefully PBMC populations. One hundred and ninety microlitres of the layered on the surface of an equal volume of Lymphoprep™ cell suspensions were added to the marked wells. Nothing fur- (Axis Shield, UK). The tubes were centrifuged (Sorvall, UK) at ther was added to the unstimulated wells. Ten microlitres of a 1000 g for 22 min. Care was taken not to disrupt the layers, and 1 : 2 PHA (Sigma-Aldrich, UK) dilution with RPMI were the small cloudy band of PBMCs was carefully removed using a added to each of the PHA-stimulated wells. sterile pastette (Sarstedt, UK) and added to a sterile 15 mL Falcon tube. The volume of PBMCs was made up to 10 mL with Proposed protocol: Anti-Biotin MACSiBead™ RPMI, and the tubes were centrifuged at 600 g for 7 min. The particles supernatant was discarded. The cell pellet of each control was resuspended in 10 mL RPMI, and the Falcon tubes were then The Anti-Biotin MACSiBead™ particles (Miltenyi Biotec, centrifuged at 350 g for a further 7 min. The supernatant was UK) were first coated with anti-CD2, -CD3 and -CD28. The again discarded and the pellets were resuspended in 1 mL RPMI. Miltenyi Biotec kit included the following: Cell counting • 2 mL Anti-Biotin MACSiBead™ particles, corresponding to 4 × 10 particles Ten microlitres of cell suspension were diluted with 40 µ L • 0.4 mL anti-CD2-biotin (100 µ g/mL) Trypan Blue (Sigma-Aldrich, UK) to give a 1:5 dilution. A cell • 0.4 mL anti-CD3-biotin (100 µ g/mL) count, using a 10-chamber Fast Read disposable counting • 0.4 mL anti-CD28-biotin (100 µ g/mL) A phosphate-buffered saline (PBS) solution was prepared Table 1. Participant demographics using 10 mL PBS, 0.05 g human serum albumin and 40 µ L 2 mM EDTA. The solution was filtered using a sterile 0.1 µ M filter (Millipore, UK) to remove any bacterial contamination. Healthy Poor PHA controls responders Five hundred microlitres of Beads and 100 µ L each of anti- CD2-, anti-CD3- and anti-CD28-biotin were added to a ster- Total number 10 3 ile cryovial tube, along with 200 µ L of the pre-prepared PBS. The cryovial was placed on a rotator in a 4°C cold room for Age median 29 27 2 h. One hundred microlitres of the solution were added to Age range 23–49 22–62 10 sterile tubes and all were stored at 4°C until use. The coated Beads may be stored for up to 3 months before expiry. Number of male subjects 4 1 The protocol provided with the Miltenyi Biotec kit is opti- Number of female subjects 6 2 6 −1 mized for use with a PBMC concentration of 5 × 10 mL . Poor responders were asymptomatic healthy control subjects within the To enable this assay to be compared with the current PHA laboratory without a known T-cell immunodeficiency, who gave a T-cell 6 −1 test, which requires a PBMC concentration of 1 × 10 mL , proliferation value of <45% (33.86, 18.42 and 29.45%) following stimulation the volumes were adjusted to allow for use with the lower by PHA. 3 Research article Bioscience Horizons • Volume 7 2014 PBMC concentration. Preliminary experiments were initially Results carried out on two control subjects using PBMC concentra- 6 6 −1 tions of both 1 × 10 and 5 × 10 mL . Rounds of proliferation 6 −1 For use with a PBMC concentration of 1 × 10 mL , 400 µ L A gating strategy was applied to select those cells within the AIM-V was added to a Falcon tube containing 20 µ L of the PBMC populations that were the least granular and which pre-prepared coated Beads and was vortexed thoroughly before also expressed CD3 and CD4, thus selecting CD4 T cells. An centrifugation at 300 g for 5 min. The supernatant was care- example of the gating strategy for both unstimulated and fully removed and a further 800 µ L AIM-V was added and vor- PHA-stimulated PBMC populations is shown in Fig. 2. A texed. Nineteen microlitres of the Bead suspension (to give a plot of side scatter vs. CD3 staining intensity was first applied bead-to-cell ratio of 1 : 2 as determined by Miltenyi Biotec) and cells that were strongly CD3 positive and low in granu- were added to the two Bead-stimulated PBMC populations of larity were gated. This was to ensure that the CD3 T lym- the round-bottomed plate. The plate was placed in the 5% CO phocytes were selected from the PBMC population (Fig. 2A). humidified 37°C incubator for 3 days. Thirty-eight microlitres This gate was applied to a second plot to gather a population −1 + + of IL-2 at a concentration of 200 IU mL were added to the of CD3 CD4 helper T cells, and a gate was drawn around second of the two Bead-stimulated PBMC populations. The this population (Fig. 2B). The same principle as in Fig. 2A plate was then returned to the incubator for a further 3 days. and B was applied to the PHA-stimulated population of The Miltenyi Biotec protocol is intended for T-cell expansion PBMCs, although stimulated T cells can be more granular so and recommends a 14-day assay, with fresh IL-2 advised to be a larger gate was drawn to include cells with an intermediate added every 2–3 days. However, to enable our Bead assay to be side scatter (Fig. 2C and D). The same principle was applied run alongside the current assay and to determine whether or for all controls (healthy and poor PHA responders) with not IL-2 addition significantly impacts T-cell proliferation, IL-2 unstimulated, PHA-, Beads only- and Bead plus IL-2- was added only on Day 3; halfway through the total 6-day stimulated PBMCs. incubation period. The data for each PBMC population (unstimulated, PHA- stimulated, Beads only and Beads plus IL-2) for each control Fluorochrome staining of PBMC were then displayed using histograms (Fig. 3). Numerical and flow cytometry analysis was obtained following the application of two gates, M1 and M2, enabling the percentage proliferation of each After a 6-day incubation, the contents of each well were resus- PBMC population to be calculated. Peaks, defined as a visible pended and like-wells were pooled together into sterile 5 mL apex or spike within the green line outside of the blue unstim- Falcon tubes (Becton Dickinson, UK). The cells were washed in ulated region, correspond to rounds of proliferation. Among 1 mL PBS, vortexed and then centrifuged at 483 g for 5 min. the 10 healthy controls tested, the mean number of peaks, The supernatant was decanted. Five microlitres of CD3 Percp and therefore rounds of proliferation, produced with PHA (Becton Dickinson, UK) and 5 µ L CD4 APC (Becton Dickinson, (Fig. 3A) was 1.70, increasing to 2.50 when the Beads were UK) were added and all tubes were vortexed and incubated at used alone (Fig. 3B) and further increasing to 3.10 following 4°C for 20 min. The cells were again washed in 1 mL PBS, vor- the addition of IL-2 on Day 3 of incubation (Fig. 3C). texed and centrifuged at 483 g for 5 min. The supernatant of each tube was discarded and 400 µ L one per cent formaldehyde Choosing the Anti-Biotin MACSiBead™ (to fix the cells) was added. All tubes were vortexed again and stored at 4°C before flow cytometry was performed using the particles concentration FACScalibur (Becton Dickinson, UK) following calibration Two healthy controls were set up using Bead and PBMCs at with Calibrite Beads (Becton Dickinson, UK). Fifty thousand 6 6 −1 + a concentration of both 1 × 10 and 5 × 10 mL . The per- CD3 events were acquired. centage proliferation yielded for both controls when the Beads were used alone and with the addition of IL-2 at Day Statistics 3 of incubation was not significantly different ( p > 0.05) All statistics were performed using the GraphPad Prism soft- at the lower concentration than the higher concentration ware, version 6.04. A non-parametric, paired ANOVA (Fried- (Fig. 4). However, there was a trend for the lower concentra- man) test was used for the comparison of the higher and lower tion to give greater proliferation. The decision was made at Bead and PBMC concentrations, and to analyse the data for this stage to only use PBMCs and Beads at the lower concen- the controls who responded poorly to the PHA assay. A non- tration, thus matching the PBMC concentration required for parametric, paired T-test (Wilcoxon) was used to compare the the current PHA assay, for the remaining control samples. percentage of T lymphocyte proliferation for the PHA and Bead assays. Correlations between the assays were determined Healthy controls: PHA vs. Beads vs. by a Spearman test. Values of p < 0.05 were considered signifi - Beads + IL-2 cant. The reference range of percentage proliferation for the Bead assay was determined by the 2.5th–97.5th percentiles as In order for conclusions on T-cell proliferation follow- the data gathered were not normally distributed. ing stimulation from the different mitogens to be made, 4 Bioscience Horizons • Volume 7 2014 Research article Figure 2. Representative gating strategy applied to unstimulated PBMCs (A and B) and PHA-stimulated PBMCs (C and D). The same strategy was applied to all PBMC populations of 13 controls (healthy and poor PHA responders) for the unstimulated and each of the three stimulants: PHA, Beads only and Beads plus IL-2. proliferation was assessed by all three methods (PHA, the Poor responders to the current PHA assay Beads alone and the Beads plus IL-2) (Fig. 5) for 11 healthy The response to the Bead assay was varied among those con- controls. However, after analysing the percentage prolifera- trols which responded poorly to PHA. The first showed a tion values for all 11, it was found that 10 gave a PHA pro- decrease in proliferation with the Beads alone and then a liferation value above the normal range of 45% and one gave further slight decrease when IL-2 was added, the second a PHA proliferation below 45%. This 11th control was showed a slightly reduced proliferation when the Beads were therefore included in the statistical analysis for the poor PHA used alone but an increase in proliferation following the responders. addition of IL-2 and the third showed an increase in prolif- No significant difference ( p > 0.05) was observed between eration with the Beads alone and no change when IL-2 was the percentage proliferation when PHA and the Beads alone added (Fig. 6). were used (p-value obtained by a Wilcoxon test) (Fig. 5A). An association was observed, but this was not significant Establishing a percentage proliferation (r-value obtained by a Spearman correlation) (Fig. 5B). There reference range for the Anti-Biotin was also no significant difference ( p > 0.05) between the per- centage proliferation seen when PHA and the Beads plus IL-2 MACSiBead™ were used (p-value obtained by a Wilcoxon test) (Fig. 5C). For a normally distributed (parametric) set of data, a reference Again, an association was observed, but this was not signifi - range of values for diagnostic use is determined by calculating cant (r-value obtained by a Spearman correlation) (Fig. 5D). the mean value ±2 SD. However, as the data gathered for this Interestingly however, one healthy control showed a marked research were not normally distributed (non-parametric), the increase in proliferation with the Beads and following the 2.5th and 97.5th percentile values were used instead (Table 2). addition of IL-2 compared with PHA. No significant differ - ence (p > 0.05) was observed between the percentage prolif- eration seen when the Beads were used alone and when IL-2 Discussion was added on Day 3 across all healthy controls (p-value obtained by a Wilcoxon test) (Fig. 5E). A strong positive cor- A new assay has been developed within the Oxford Immu- relation was observed (Fig. 5F). nology diagnostic laboratory to assess the ability of T cells to 5 Research article Bioscience Horizons • Volume 7 2014 Figure 3. Three representative histogram plots observed following T-cell proliferation after incubation with CFSE. The blue population of each represents the unstimulated T lymphocytes and was used to set the two M gates. The green line shows the rounds of T-cell proliferation following stimulation when PBMCs were incubated with PHA (A), Beads only (B) and Beads plus the addition of IL-2 on Day 3 of incubation (C). proliferate following stimulation with anti-CD2/CD3/CD28- for PHA (<45%). A variable T-cell proliferation response was coated Anti-Biotin MACSiBead™ particles. seen among the three subjects when the Beads were used in place of PHA. A reference range of T-cell percentage prolif- No significant difference was seen between the percentage eration values has been established for the Bead assay. of T-cell proliferation when PHA and the Beads (with and without the addition of IL-2) were used as the stimulants for Justifications for performing the Anti-Biotin the healthy control samples. There was also no significant MACSiBead™ particles assay with the lower difference observed between the proliferation seen when IL-2 was added to the Bead assay on Day 3 of incubation com- bead and PBMC concentration pared with when the Beads were used alone. The decision to conduct the Bead assay with the lower PBMC −6 −1 Two known poor PHA responders and one healthy con- concentration of 1 × 10 mL over a concentration of −6 −1 trol gave a percentage proliferation below the normal range 5 × 10 mL was made given that there was no significant 6 Bioscience Horizons • Volume 7 2014 Research article to begin proliferating 40–50 h following incubation (Li and Kurlander, 2010). It was also noted that the cell populations incubated with the Beads took less time to acquire during flow cytometry; the more rounds of proliferation which occurred, the less time it took to reach the set number of 50 000 events. All T cells express CD3, and CD28 functions as a co-receptor and thus, one possible reason for the faster proliferation could be that, as the Beads are coated with both anti-CD3 and anti- CD28 simultaneously, a stronger proliferative signal is deliv- ered through co-stimulation by these two molecules. This double signal transduction causes an increase in the number of rounds of proliferation without inducing premature cell death (Li and Kurlander, 2010). This contrasts with PHA, which induces T-cell activation by bypassing the early TCR/ CD3 signal transduction (Pignata et al., 1996), and has previ- ously been shown to lead to functional impairment of PBMCs 6 −1 Figure 4. Higher Bead and PBMC concentration (5 × 10 mL ) vs. in vitro (Duarte et al., 2002 ). 6 −1 lower Bead and PBMC concentration (1 × 10 mL ) for two healthy controls (p-value obtained by a Friedman test). Anti-Biotin MACSiBead™ particles may increase the percentage of T-cell difference between the results for the Bead and PHA assays at proliferation in some healthy controls the two PBMC and Bead concentrations (Fig. 4). Ideally, the Bead assay would have been performed at the two PBMC compared with PHA concentrations on a larger number of healthy control sam- The difference between the percentage of T-cell proliferation ples. This may have strengthened the statistical analysis, and in the 10 healthy controls when the Beads were used in place perhaps a significant difference between the T-cell prolifera - of PHA was not significant (Fig. 5A). One control showed a tion values at the two different concentrations would have 37% increase in proliferation when the Beads were used com- been seen. However, conducting the assay at both concentra- pared with PHA, and three controls showed a slight decrease tions requires eight 3 mL lithium–heparin samples from each in proliferation. The same phenomenon was observed with participant. It was therefore not practical or realistic to con- the same controls following the addition of IL-2 on Day 3 duct the Bead assay at the two concentrations on all healthy compared with PHA. This shows that the Beads may increase controls. T-cell proliferation in some individuals, but also highlights the A second reason in favour of using the lower concentra- possibility that those subjects who show a normal prolifera- tion is that a smaller volume of blood would be required tive response to PHA may not produce a significantly different from the patient, which is ideal given that the Bead assay response when another mitogen is used. would be used on younger patients. Although there was no significant correlation between the Finally, the Bead assay would be used alongside the cur- use of the Beads alone and PHA with the healthy control rent PHA test that requires the lower PBMC concentration, subjects (Fig. 5B), there did appear to be a trend for a positive and thus, it is more practical for both assays to use the same association. It may be that this study was underpowered by cell concentration. the limited number of healthy controls available. Anti-Biotin MACSiBead™ particles induce Addition of IL-2 did not significantly more rounds of proliferation impact T-cell proliferation A trend noted throughout was that those PBMC populations No significant difference was found between the percentage incubated with the Beads typically underwent more rounds of proliferation yielded when the Beads were used alone and proliferation than when PHA was used (Fig. 3). All cell popu- when IL-2 was added on Day 3 (Fig. 5E). However, in all but lations were incubated for the same length of time, and thus, one of the 10 healthy controls, those cell populations incu- it can be concluded that, generally, the Beads may facilitate bated with IL-2 at Day 3 gave a slightly higher percentage more efficient T-cell proliferation. Moreover, a change in the proliferation. A strong positive correlation was observed colour of the culture medium from red to yellow, indicating between the use of the Beads only and the addition of IL-2 at nutrient utilization for proliferation, was seen typically Day 3 (Fig. 5F). This indicates that both assays give compa- 24–48 h sooner in those PBMC populations incubated with rable results and therefore suggests that the addition of IL-2 the Beads than with PHA. More specifically, in the presence is not necessary. Furthermore, the addition of IL-2 on Day 3 of anti-CD3/CD28-coated beads, T cells have been known limits the number of days that the assay can be performed so 7 Research article Bioscience Horizons • Volume 7 2014 Figure 5. Comparison of the percentage of T-cell proliferation and correlation statistics for 10 healthy controls using PHA vs. Beads alone (A and B), PHA vs. Beads plus IL-2 (C and D) and Beads alone vs. Beads plus IL-2 (E and F). as to allow for the work on Day 3 to be carried out during the to 14.79% when the Beads were used alone, and the value routine laboratory hours. fell by a further 0.72% in the PBMC population to which IL-2 was added on Day 3. Poor Responder 2 gave a percent- age proliferation of 18.42% with PHA, and this value Poor responders to PHA may also respond dropped to 17.08% when the Beads were used alone. poorly to Anti-Biotin MACSiBead™ particles However, the addition of IL-2 on Day 3 increased the prolif - The response of the three poor PHA responders to the Bead eration to 30.08%. Poor Responder 3 (the 11th healthy assay was varied (Fig. 6). Poor Responder 1 gave a percent- control that gave an unexpected poor PHA response) yielded age proliferation value of 33.86% with PHA. This dropped a T-cell proliferation of 29.45% with PHA, 40.55% when 8 Bioscience Horizons • Volume 7 2014 Research article 95% of the results are covered by the range, and therefore there is a 1 in 20 chance that a result for a healthy individual will fall outside this interval. This can be illustrated by the three poor PHA responders analysed who gave a T-cell per- centage proliferation of <45% and therefore fell outside the normal range set by the clinical Immunology laboratory. None of these subjects have a known primary or secondary immunodeficiency and so would appear to simply be healthy individuals who happen to not fall within the 95% included in the PHA reference range. Following the calculation of reference range values for the Bead assay both with and without the addition of IL-2 on Day Figure 6. Proliferation values for three poor PHA responders (p-value obtained by a Friedman test). Poor Responders 1 and 2 were known to 3 (Table 2), it can be concluded that only the proliferation give a percentage proliferation value of <45%, and the third was yielded for Poor Responder 3 when the Beads were used alone initially tested as a healthy control but unexpectedly gave a fell into the established reference range. None of the poor proliferation of <45% in response to PHA. responders fell within the reference interval for the Beads plus IL-2, and Poor Responders 1 and 2 did not fall into either Table 2. Median, 2.5th and 97.5th reference range values for the reference interval. proposed Anti-Biotin MACSiBead™ particles assays Limitations and further work PHA (% Beads only  Beads + IL-2 (% proliferation) (% proliferation) proliferation) Perhaps the most significant limitation of the practical work carried out was that all subjects used were adults. Primary Median 79 87 82 immunodeficiencies are typically diagnosed at a young age, and therefore, the reference ranges calculated for the Bead 2.5th 46 38 43 assay may not be reflective of a younger cohort. Unfortunately, percentile the ethical implications of recruiting healthy children for a 97.5th continuation of this research are great. 92 95 96 percentile Secondly, only 10 healthy controls were available. To Reference strengthen the results and to validate the reference range, 100 range 45–92 38–95 43–96 healthy controls would ideally be recruited but this was simply established not feasible. PHA reference range determined previously and is currently used in the Oxford laboratory. To enhance the likelihood of attaining a significant differ - ence in the T-cell percentage proliferation between poor PHA responders and the proposed Bead assay, a larger number of poor PHA responders should be recruited. However, this the Beads were used alone and 40.72% when IL-2 was added would be challenging in practical terms as most healthy indi- on Day 3. viduals respond well to PHA. Given that no significant difference between the results for To further investigate the capability of T-cell proliferation the poor PHA responders across the two assays was found, a in healthy individuals who show a poor response to PHA, definitive conclusion as to whether the Bead assay yields a other stimuli could be used and compared with the Beads. different T-cell proliferation percentage in those who respond Phytolacca Americana, more commonly known as Pokeweed, poorly to PHA cannot be made. is a perennial plant found in North and South America, East Asia and New Zealand. When extracted from the roots with Significance of the Anti-Biotin MACSiBead™ saline, pokeweed has been shown to have mitogenic proper- particles reference range in a diagnostic ties such that it can stimulate the proliferation of both T and setting B lymphocytes (Bodger et al., 1979). Concanavalin A is a sec- ond mitogen capable of inducing T-cell proliferation (McCole A reference range is a range of values where 95% of the results et al., 1998). These mitogens may show a significant differ - in healthy individuals fall within, thus excluding the upper ence in T-cell proliferation compared with PHA. and lower 2.5% (Ahmed, 2011). A reference range of values for any given diagnostic test can help a clinician to decide whether or not a result is abnormal and therefore a cause for Conclusions concern; however, the whole clinical picture should always be taken into account. If a test result falls outside the reference An anti-CD2/CD3/CD28-coated Anti-Biotin MACSiBead™ range, it does not necessarily mean that the patient is unwell; particles assay has been established. The Bead and PBMC 9 Research article Bioscience Horizons • Volume 7 2014 concentration has been optimized for diagnostic use. The Bead References assay was successfully performed, both with and without the addition of IL-2, on 10 healthy control subjects. The assay was Ahmed, N. (2011) Clinical Biochemistry, Oxford University Press, Oxford. conducted on three controls who respond poorly to PHA and Beadling, C. and Smith, K. A. (2002) DNA array analysis of interleukin- comparisons between the two assays were made. Following 2-regulated immediate/early genes, Medical Immunology, 1 (2), 1–14. the calculation of a reference range for the new assay, it was found that two out of the three individuals who responded Bodger, M. P., McGiven, A. R. and Fitzgerald, P. H. (1979) Mitogenic pro- poorly to PHA also responded poorly to the Beads, and one teins of pokeweed, Immunology, 37, 785–792. poor responder to PHA fell into the reference range for the Buckley, R. H. (2004) Molecular defects in human severe combined Bead assay. immunodeficiency and approaches to immune reconstitution, After taking the results into account, the proposed Bead Annual Review of Immunology, 22, 625–655. assay will now be offered as a diagnostic test, alongside the Cornish, G. H., Sinclair, L. V. and Cantrell, D. A. (2006) Differential regula - current PHA assay, within the routine Oxford Immunology tion of T-cell growth by IL-2 and IL-15, Blood, 108 (2), 600–608. laboratory. The newly developed assay will not include the addition of IL-2 on Day 3. Duarte, R. F., Chen, F. E., Lowdell, M. W. et  al. (2002) Functional impair- ment of human T-lymphocytes following PHA-induced expansion The Bead assay provides a more physiological addition to and retroviral transduction: implications for gene therapy, Gene the current PHA test and may be useful in determining the Therapy, 9 (20), 1359–1368. ability of T cells to proliferate in some patients who respond poorly to PHA. Geha, R. S., Notarangelo, L. D., Casanova, J–L. et al. (2007) Primary immuno- deficiency diseases: an update from the International Union of Immunological Societies Primary Immunodeficiency Diseases Classi- Acknowledgements fication Committee, The Journal of Allergy and Clinical Immunology, 120 I thank all staff working at the Oxford Immunology labora- (4), 776–794. tory, and a special thank you goes to Dr Lisa Ayers for all her Hall, A. and Yates, C. (2010) Immunology, Oxford University Press, timeless help, guidance and support throughout. Oxford. Kelly, B. T., Tam, J. S., Verbsky, J. W. et  al. (2013) Screening for severe Author Biography combined immunodeficiency in neonates, Clinical Epidemiology, 5, 363–369. Louisa Green studied for her BSc (Hons) Biomedical Science degree at Oxford Brookes University over 4 years Li, Y. and Kurlander, R. J. (2010) Comparison of anti-CD3 and anti-CD28- whilst working as a Trainee Biomedical Scientist at the coated beads with soluble anti-CD3 for expanding human T cells: Oxford University Hospitals NHS Trust. After spending differing impact on CD8T cell phenotype and responsiveness to the first 2 and a half years of her course rotating through - restimulation, Journal of Translational Medicine, 8 (104), 1–15. out each laboratory discipline for 6 months at a time, she McCole, D. F., Dohertv, M. L., Baird, A. W. et  al. (1998) Concanavalin chose to spend the final 18 months of her employment spe - A-stimulated proliferation of T cell subset-depleted lymphocyte cializing in Immunology. Here, Louisa completed both her populations isolated from Fasciola hepatica-infected cattle, undergraduate research project and generic registration Veterinary Immunology and Immunopathology, 66 (3–4), 289–300. Portfolio for the Institute of Biomedical Science, and she is now a fully qualified Biomedical Scientist. She graduated Parish, C. R., Glidden, M. H., Quah, B. J. C. et  al. (2009) Use of the from Oxford Brookes University in June 2014 winning the intracellular fluorescent dye CFSE to monitor lymphocyte migration Best in Year project prize, and within the next year plans to and proliferation, Current Protocols in Immunology, 84, 4.9.1–4.9.10. apply for a 4-year integrated MRes/PhD in Infection and Pignata, C., Sanghera, J. S., Soiffer, R. J. et al. (1996) Defective activa- Immunity. tion of mitogen-activated protein kinase after allogeneic bone marrow transplantation. Blood, 88 (6), 2334–2341. Funding Tasher, D. and Dalal, I. (2012) The genetic basis of severe combined Funding was kindly provided by Oxford University Hospitals immunodeficiency and its variants, Journal of the Application of NHS Trust. Clinical Genetics, 5, 67–80. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Development of an anti-CD2/CD3/CD28 bead-based T-cell proliferation assay

Bioscience Horizons , Volume 7 – Nov 6, 2014

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BioscienceHorizons Volume 7 2014 10.1093/biohorizons/hzu012 Research article Development of an anti-CD2/CD3/CD28 bead-based T-cell proliferation assay 1,2, Louisa Green * Clinical Laboratory Immunology, Oxford University Hospitals NHS Trust, Churchill Hospital, Old Road, Headington, Oxford OX3 7LJ, England Oxford Brookes University, Gypsy Lane, Headington, Oxford OX3 0BP, England *Corresponding author: Tel: +44 07725748099. Email: lgreen2891@gmail.com Supervisor: Dr Lisa Ayers, Clinical Laboratory Immunology, Oxford University Hospitals NHS Trust, Churchill Hospital, Old Road, Headington, Oxford OX3 7LJ, England. Email: lisa.ayers@nhs.net Severe combined immunodeficiency (SCID) has many phenotypes , including those which result in the formation of T cells but render them incapable of an optimal level of proliferation following stimulation by an antigen. Phytohaemagglutinin (PHA) is a mitogen commonly used to assess T-cell proliferation, although anti-CD2/CD3/CD28-coated beads may provide a more physiological trigger. Some patients and healthy controls display poor T-cell proliferation (<45%) in response to PHA, but may be capable of an increased percentage proliferation when stimulated by another mitogen. This study aimed to develop an anti-CD2/CD3/CD28 bead-based T-cell proliferation assay and to compare T-cell proliferation generated by two mitogens both in healthy controls and in poor responders to PHA. A bead-based T-cell proliferation assay was optimized and validated. Peripheral blood mononuclear cells (PBMCs) were extracted from whole blood samples of 10 healthy controls and 3 poor PHA responders. PBMCs were incubated with carboxyfluorescin diacetate succinimidyl ester and three separate stimulants: PHA, Beads only and Beads plus IL-2. Flow cytometry was performed on Day 6, following fluorochrome staining with CD3 PerCP and CD4 APC, to identify CD4 T cells. No significant difference was found between the results generated by PHA- or Bead stimula - tion in 10 healthy controls. A strong positive correlation was observed between the use of the Beads alone and the addition of IL-2. A varied response to the Beads was produced among the poor PHA responders. It has been concluded that the bead- based assay will be run alongside every PHA test in the Oxford Immunology diagnostic laboratory. The step to add IL-2 on Day 3 will not be included as this did not significantly impact T-cell proliferation. The Bead assay provides a more physiological addition to the current PHA test and may be useful in assessing T-cell proliferation in those patients who respond poorly to PHA. However, some individuals who respond poorly to PHA may also respond poorly to the anti-CD2/CD3/CD28 Beads. Key words: PHA, anti-CD2/CD3/CD28 beads, T cell, proliferation, diagnosis, immunodeficiency Submitted on 14 June 2014; accepted on 17 November 2014 Introduction intrinsic genetic defects resulting in the complete absence or impaired function of a cell type or types (Geha et al., 2007). The immune system comprises a multitude of cells and path- In contrast, secondary immunodeficiencies are acquired in ways that work together to ultimately keep an organism safe later life and can arise secondary to infection, such as with from infectious disease and harm. However, a breakdown in human immunodeficiency virus (HIV), severe malnutrition or this networking can have a direct and compromising effect as a result of immunosuppressive drug use (Hall and Yates, on the successful functioning of the immune system, and the 2010). Whether the condition is congenital or acquired, both term used to cover and define such causative conditions is the innate and adaptive response types can be impaired (Geha immunodeficiency. Primary immunodeficiencies arise due to et al., 2007), and there is often an overlap with both T and B © The Author 2014. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Research article Bioscience Horizons • Volume 7 2014 lymphocytes being affected, even if only one of those cell laboratory currently performs an assay that assesses the ability of types is absent or does not function as it should. T cells to proliferate following stimulation with a mitogen called Phytohaemagglutinin (PHA). During this assay, peripheral blood An umbrella term used to cover many austere T-cell primary mononuclear cells (PBMCs) are first isolated from whole blood immunodeficiencies is severe combined immunodeficiency and incubated with carboxyfluorescin diacetate succinimidyl (SCID). The illness is termed severe because it is fatal and is ester (CFSE), a stable, membrane-permanent dye that covalently described as combined because although it is often directly couples to intracellular molecules. The T cells of healthy indi- T lymphocytes which are affected, B lymphocytes cannot ade- viduals would normally proliferate upon PHA stimulation, and quately function without the help of T cells and thus both cell- flow cytometric analysis should reveal a decline in CFSE staining mediated and humoural immunity are lost (Tasher and Dalal, within the PHA-stimulated cells as the dye is divided equally with 2012). SCID is considered a paediatric emergency with patients each cell division (Parish et al., 2009). However, this is not appearing well at birth but after a few months, following the observed in those patients whose T-cell proliferation is impaired subsequent loss of maternal antibody, often present with diar- (Fig. 1); thus, the PHA investigation is often a supportive test that rhoea, sepsis, otitis and an overall failure to thrive (Kelly et al., leads to a primary immunodeficiency diagnosis. 2013). Typically, children with SCID have a reduced spleen size Some patients and healthy controls show a reduced PHA and a small thymus weighing <1 g (Buckley, 2004), although response (a proliferation of <45%), but may be capable of these abnormalities are not usually noted until it becomes normal T-cell proliferation following stimulation by other apparent that the infant frequently falls ill and is not developing mitogens. The overall aim of this research was to establish at a normal rate. Opportunistic infections, caused by viral and develop a new test that uses anti-CD2/CD3/CD28-coated pathogens including cytomegalovirus and herpes simplex virus, Anti-Biotin MACSiBead™ particles to stimulate T-cell prolif- are another common medical concern for patients who have eration. More specifically, we wanted to conduct the PHA and T-cell deficiencies, and SCID sufferers often have such compro - Bead assays on at least 10 healthy control samples, compare mised immunity that even an attenuated vaccine can result in the results, identify any control samples that did not respond fatality (Kelly et al., 2013). well to PHA and evaluate the T-cell proliferation using the If SCID or another primary immunodeficiency is suspected, Beads. Furthermore, we aimed to investigate the impact of there are a set of standard differential tests undertaken to achieve IL-2 on T-cell proliferation. IL-2 plays a pivotal role in the a diagnosis. Crucially, it is important to rule out a secondary immune response and is secreted by T cells following the immunodeficiency, particularly HIV, and providing this result is binding of an antigen to the T-cell receptor (Cornish, Sinclair negative the next investigation should be the full blood count. and Cantrell, 2006). The production of IL-2 upon antigen The results of this test provide a breakdown of the amount of binding stimulates the up-regulation of the IL-2 receptor gene, each cell type comprising the blood; however, the main one of thus facilitating increased interaction between the two 9 −1 immunological interest is the lymphocyte count; 1–5 × 10 l for (Beadling and Smith, 2002). Moreover, the engagement of the young children is suggestive of an immunodeficiency. If this is the T-cell receptor CD3 complex on the surface of antigen- case, it should prompt a clinician to request analysis of specific presenting cells with the co-stimulatory molecule CD28 leads immunoglobulin levels to rule out a B-cell immunodeficiency , to the activation of numerous tyrosine kinase pathways, and then lymphocyte phenotyping to attain an absolute count which drive the transcription of the IL-2 gene. The interaction of lymphocyte subsets. In addition, the Oxford Immunology between IL-2 and its receptor stimulates the growth and Figure 1. Normal T-cell proliferation vs. impaired T-cell proliferation, and the effect of both on CFSE staining. 2 Bioscience Horizons • Volume 7 2014 Research article + + differentiation of antigen-specific CD4 T cells and CD8 chamber (Immune Systems Limited, UK), under a ×10 objec- T cells (Beadling and Smith, 2002). Our final aim was to tive lens, was conducted. establish a reference range of percentage proliferation values for the proposed Bead assay, with and without the addition of CFSE staining IL-2 on Day 3 of incubation. One microlitre of 2.25 µ M CFSE stock (Sigma-Aldrich, UK) was added to the PBMC resuspensions. Tubes were wrapped in Methods aluminium foil to reduce photosensitive CFSE reactions and placed into a water bath at 37°C for 15 min. Ten microlitres of Subjects ice-cold RPMI were added to prevent further proliferation and to ensure that all cells remained at the same point in the cell A cohort of subjects working in the Oxford Immunology cycle. The tubes were centrifuged at 483 g for 5 min. The super- laboratory was selected. All participants were over 18, and natant was discarded and the pellets were resuspended in 10 mL exclusion criterion was any known primary or secondary ice-cold RMPI. The tubes were again centrifuged at 483 g for a immunodeficiency. Demographics are shown in Table 1. further 5 min and the supernatant was again discarded. Ethics approval was obtained (05/Q1605/88), and all partici- pants gave informed consent prior to involvement. Current protocol: PHA PBMC separation The volume of culture medium AIM-V (Oxford Immunotec, 6 −1 All works performed were conducted under aseptic conditions. UK) required to achieve a cell concentration of 1 × 10 mL Blood from each control subject was drawn into three 3 mL was calculated for each control. A sterile 96-well, round- lithium heparin collection tubes (Becton Dickinson, UK) and bottomed plate (Sigma-Aldrich, UK) had wells marked for diluted 1 : 2 with RPMI (Sigma-Aldrich, UK) in a sterile Falcon unstimulated, PHA-stimulated and two Bead-stimulated tube. The blood–RMPI solution of each control was carefully PBMC populations. One hundred and ninety microlitres of the layered on the surface of an equal volume of Lymphoprep™ cell suspensions were added to the marked wells. Nothing fur- (Axis Shield, UK). The tubes were centrifuged (Sorvall, UK) at ther was added to the unstimulated wells. Ten microlitres of a 1000 g for 22 min. Care was taken not to disrupt the layers, and 1 : 2 PHA (Sigma-Aldrich, UK) dilution with RPMI were the small cloudy band of PBMCs was carefully removed using a added to each of the PHA-stimulated wells. sterile pastette (Sarstedt, UK) and added to a sterile 15 mL Falcon tube. The volume of PBMCs was made up to 10 mL with Proposed protocol: Anti-Biotin MACSiBead™ RPMI, and the tubes were centrifuged at 600 g for 7 min. The particles supernatant was discarded. The cell pellet of each control was resuspended in 10 mL RPMI, and the Falcon tubes were then The Anti-Biotin MACSiBead™ particles (Miltenyi Biotec, centrifuged at 350 g for a further 7 min. The supernatant was UK) were first coated with anti-CD2, -CD3 and -CD28. The again discarded and the pellets were resuspended in 1 mL RPMI. Miltenyi Biotec kit included the following: Cell counting • 2 mL Anti-Biotin MACSiBead™ particles, corresponding to 4 × 10 particles Ten microlitres of cell suspension were diluted with 40 µ L • 0.4 mL anti-CD2-biotin (100 µ g/mL) Trypan Blue (Sigma-Aldrich, UK) to give a 1:5 dilution. A cell • 0.4 mL anti-CD3-biotin (100 µ g/mL) count, using a 10-chamber Fast Read disposable counting • 0.4 mL anti-CD28-biotin (100 µ g/mL) A phosphate-buffered saline (PBS) solution was prepared Table 1. Participant demographics using 10 mL PBS, 0.05 g human serum albumin and 40 µ L 2 mM EDTA. The solution was filtered using a sterile 0.1 µ M filter (Millipore, UK) to remove any bacterial contamination. Healthy Poor PHA controls responders Five hundred microlitres of Beads and 100 µ L each of anti- CD2-, anti-CD3- and anti-CD28-biotin were added to a ster- Total number 10 3 ile cryovial tube, along with 200 µ L of the pre-prepared PBS. The cryovial was placed on a rotator in a 4°C cold room for Age median 29 27 2 h. One hundred microlitres of the solution were added to Age range 23–49 22–62 10 sterile tubes and all were stored at 4°C until use. The coated Beads may be stored for up to 3 months before expiry. Number of male subjects 4 1 The protocol provided with the Miltenyi Biotec kit is opti- Number of female subjects 6 2 6 −1 mized for use with a PBMC concentration of 5 × 10 mL . Poor responders were asymptomatic healthy control subjects within the To enable this assay to be compared with the current PHA laboratory without a known T-cell immunodeficiency, who gave a T-cell 6 −1 test, which requires a PBMC concentration of 1 × 10 mL , proliferation value of <45% (33.86, 18.42 and 29.45%) following stimulation the volumes were adjusted to allow for use with the lower by PHA. 3 Research article Bioscience Horizons • Volume 7 2014 PBMC concentration. Preliminary experiments were initially Results carried out on two control subjects using PBMC concentra- 6 6 −1 tions of both 1 × 10 and 5 × 10 mL . Rounds of proliferation 6 −1 For use with a PBMC concentration of 1 × 10 mL , 400 µ L A gating strategy was applied to select those cells within the AIM-V was added to a Falcon tube containing 20 µ L of the PBMC populations that were the least granular and which pre-prepared coated Beads and was vortexed thoroughly before also expressed CD3 and CD4, thus selecting CD4 T cells. An centrifugation at 300 g for 5 min. The supernatant was care- example of the gating strategy for both unstimulated and fully removed and a further 800 µ L AIM-V was added and vor- PHA-stimulated PBMC populations is shown in Fig. 2. A texed. Nineteen microlitres of the Bead suspension (to give a plot of side scatter vs. CD3 staining intensity was first applied bead-to-cell ratio of 1 : 2 as determined by Miltenyi Biotec) and cells that were strongly CD3 positive and low in granu- were added to the two Bead-stimulated PBMC populations of larity were gated. This was to ensure that the CD3 T lym- the round-bottomed plate. The plate was placed in the 5% CO phocytes were selected from the PBMC population (Fig. 2A). humidified 37°C incubator for 3 days. Thirty-eight microlitres This gate was applied to a second plot to gather a population −1 + + of IL-2 at a concentration of 200 IU mL were added to the of CD3 CD4 helper T cells, and a gate was drawn around second of the two Bead-stimulated PBMC populations. The this population (Fig. 2B). The same principle as in Fig. 2A plate was then returned to the incubator for a further 3 days. and B was applied to the PHA-stimulated population of The Miltenyi Biotec protocol is intended for T-cell expansion PBMCs, although stimulated T cells can be more granular so and recommends a 14-day assay, with fresh IL-2 advised to be a larger gate was drawn to include cells with an intermediate added every 2–3 days. However, to enable our Bead assay to be side scatter (Fig. 2C and D). The same principle was applied run alongside the current assay and to determine whether or for all controls (healthy and poor PHA responders) with not IL-2 addition significantly impacts T-cell proliferation, IL-2 unstimulated, PHA-, Beads only- and Bead plus IL-2- was added only on Day 3; halfway through the total 6-day stimulated PBMCs. incubation period. The data for each PBMC population (unstimulated, PHA- stimulated, Beads only and Beads plus IL-2) for each control Fluorochrome staining of PBMC were then displayed using histograms (Fig. 3). Numerical and flow cytometry analysis was obtained following the application of two gates, M1 and M2, enabling the percentage proliferation of each After a 6-day incubation, the contents of each well were resus- PBMC population to be calculated. Peaks, defined as a visible pended and like-wells were pooled together into sterile 5 mL apex or spike within the green line outside of the blue unstim- Falcon tubes (Becton Dickinson, UK). The cells were washed in ulated region, correspond to rounds of proliferation. Among 1 mL PBS, vortexed and then centrifuged at 483 g for 5 min. the 10 healthy controls tested, the mean number of peaks, The supernatant was decanted. Five microlitres of CD3 Percp and therefore rounds of proliferation, produced with PHA (Becton Dickinson, UK) and 5 µ L CD4 APC (Becton Dickinson, (Fig. 3A) was 1.70, increasing to 2.50 when the Beads were UK) were added and all tubes were vortexed and incubated at used alone (Fig. 3B) and further increasing to 3.10 following 4°C for 20 min. The cells were again washed in 1 mL PBS, vor- the addition of IL-2 on Day 3 of incubation (Fig. 3C). texed and centrifuged at 483 g for 5 min. The supernatant of each tube was discarded and 400 µ L one per cent formaldehyde Choosing the Anti-Biotin MACSiBead™ (to fix the cells) was added. All tubes were vortexed again and stored at 4°C before flow cytometry was performed using the particles concentration FACScalibur (Becton Dickinson, UK) following calibration Two healthy controls were set up using Bead and PBMCs at with Calibrite Beads (Becton Dickinson, UK). Fifty thousand 6 6 −1 + a concentration of both 1 × 10 and 5 × 10 mL . The per- CD3 events were acquired. centage proliferation yielded for both controls when the Beads were used alone and with the addition of IL-2 at Day Statistics 3 of incubation was not significantly different ( p > 0.05) All statistics were performed using the GraphPad Prism soft- at the lower concentration than the higher concentration ware, version 6.04. A non-parametric, paired ANOVA (Fried- (Fig. 4). However, there was a trend for the lower concentra- man) test was used for the comparison of the higher and lower tion to give greater proliferation. The decision was made at Bead and PBMC concentrations, and to analyse the data for this stage to only use PBMCs and Beads at the lower concen- the controls who responded poorly to the PHA assay. A non- tration, thus matching the PBMC concentration required for parametric, paired T-test (Wilcoxon) was used to compare the the current PHA assay, for the remaining control samples. percentage of T lymphocyte proliferation for the PHA and Bead assays. Correlations between the assays were determined Healthy controls: PHA vs. Beads vs. by a Spearman test. Values of p < 0.05 were considered signifi - Beads + IL-2 cant. The reference range of percentage proliferation for the Bead assay was determined by the 2.5th–97.5th percentiles as In order for conclusions on T-cell proliferation follow- the data gathered were not normally distributed. ing stimulation from the different mitogens to be made, 4 Bioscience Horizons • Volume 7 2014 Research article Figure 2. Representative gating strategy applied to unstimulated PBMCs (A and B) and PHA-stimulated PBMCs (C and D). The same strategy was applied to all PBMC populations of 13 controls (healthy and poor PHA responders) for the unstimulated and each of the three stimulants: PHA, Beads only and Beads plus IL-2. proliferation was assessed by all three methods (PHA, the Poor responders to the current PHA assay Beads alone and the Beads plus IL-2) (Fig. 5) for 11 healthy The response to the Bead assay was varied among those con- controls. However, after analysing the percentage prolifera- trols which responded poorly to PHA. The first showed a tion values for all 11, it was found that 10 gave a PHA pro- decrease in proliferation with the Beads alone and then a liferation value above the normal range of 45% and one gave further slight decrease when IL-2 was added, the second a PHA proliferation below 45%. This 11th control was showed a slightly reduced proliferation when the Beads were therefore included in the statistical analysis for the poor PHA used alone but an increase in proliferation following the responders. addition of IL-2 and the third showed an increase in prolif- No significant difference ( p > 0.05) was observed between eration with the Beads alone and no change when IL-2 was the percentage proliferation when PHA and the Beads alone added (Fig. 6). were used (p-value obtained by a Wilcoxon test) (Fig. 5A). An association was observed, but this was not significant Establishing a percentage proliferation (r-value obtained by a Spearman correlation) (Fig. 5B). There reference range for the Anti-Biotin was also no significant difference ( p > 0.05) between the per- centage proliferation seen when PHA and the Beads plus IL-2 MACSiBead™ were used (p-value obtained by a Wilcoxon test) (Fig. 5C). For a normally distributed (parametric) set of data, a reference Again, an association was observed, but this was not signifi - range of values for diagnostic use is determined by calculating cant (r-value obtained by a Spearman correlation) (Fig. 5D). the mean value ±2 SD. However, as the data gathered for this Interestingly however, one healthy control showed a marked research were not normally distributed (non-parametric), the increase in proliferation with the Beads and following the 2.5th and 97.5th percentile values were used instead (Table 2). addition of IL-2 compared with PHA. No significant differ - ence (p > 0.05) was observed between the percentage prolif- eration seen when the Beads were used alone and when IL-2 Discussion was added on Day 3 across all healthy controls (p-value obtained by a Wilcoxon test) (Fig. 5E). A strong positive cor- A new assay has been developed within the Oxford Immu- relation was observed (Fig. 5F). nology diagnostic laboratory to assess the ability of T cells to 5 Research article Bioscience Horizons • Volume 7 2014 Figure 3. Three representative histogram plots observed following T-cell proliferation after incubation with CFSE. The blue population of each represents the unstimulated T lymphocytes and was used to set the two M gates. The green line shows the rounds of T-cell proliferation following stimulation when PBMCs were incubated with PHA (A), Beads only (B) and Beads plus the addition of IL-2 on Day 3 of incubation (C). proliferate following stimulation with anti-CD2/CD3/CD28- for PHA (<45%). A variable T-cell proliferation response was coated Anti-Biotin MACSiBead™ particles. seen among the three subjects when the Beads were used in place of PHA. A reference range of T-cell percentage prolif- No significant difference was seen between the percentage eration values has been established for the Bead assay. of T-cell proliferation when PHA and the Beads (with and without the addition of IL-2) were used as the stimulants for Justifications for performing the Anti-Biotin the healthy control samples. There was also no significant MACSiBead™ particles assay with the lower difference observed between the proliferation seen when IL-2 was added to the Bead assay on Day 3 of incubation com- bead and PBMC concentration pared with when the Beads were used alone. The decision to conduct the Bead assay with the lower PBMC −6 −1 Two known poor PHA responders and one healthy con- concentration of 1 × 10 mL over a concentration of −6 −1 trol gave a percentage proliferation below the normal range 5 × 10 mL was made given that there was no significant 6 Bioscience Horizons • Volume 7 2014 Research article to begin proliferating 40–50 h following incubation (Li and Kurlander, 2010). It was also noted that the cell populations incubated with the Beads took less time to acquire during flow cytometry; the more rounds of proliferation which occurred, the less time it took to reach the set number of 50 000 events. All T cells express CD3, and CD28 functions as a co-receptor and thus, one possible reason for the faster proliferation could be that, as the Beads are coated with both anti-CD3 and anti- CD28 simultaneously, a stronger proliferative signal is deliv- ered through co-stimulation by these two molecules. This double signal transduction causes an increase in the number of rounds of proliferation without inducing premature cell death (Li and Kurlander, 2010). This contrasts with PHA, which induces T-cell activation by bypassing the early TCR/ CD3 signal transduction (Pignata et al., 1996), and has previ- ously been shown to lead to functional impairment of PBMCs 6 −1 Figure 4. Higher Bead and PBMC concentration (5 × 10 mL ) vs. in vitro (Duarte et al., 2002 ). 6 −1 lower Bead and PBMC concentration (1 × 10 mL ) for two healthy controls (p-value obtained by a Friedman test). Anti-Biotin MACSiBead™ particles may increase the percentage of T-cell difference between the results for the Bead and PHA assays at proliferation in some healthy controls the two PBMC and Bead concentrations (Fig. 4). Ideally, the Bead assay would have been performed at the two PBMC compared with PHA concentrations on a larger number of healthy control sam- The difference between the percentage of T-cell proliferation ples. This may have strengthened the statistical analysis, and in the 10 healthy controls when the Beads were used in place perhaps a significant difference between the T-cell prolifera - of PHA was not significant (Fig. 5A). One control showed a tion values at the two different concentrations would have 37% increase in proliferation when the Beads were used com- been seen. However, conducting the assay at both concentra- pared with PHA, and three controls showed a slight decrease tions requires eight 3 mL lithium–heparin samples from each in proliferation. The same phenomenon was observed with participant. It was therefore not practical or realistic to con- the same controls following the addition of IL-2 on Day 3 duct the Bead assay at the two concentrations on all healthy compared with PHA. This shows that the Beads may increase controls. T-cell proliferation in some individuals, but also highlights the A second reason in favour of using the lower concentra- possibility that those subjects who show a normal prolifera- tion is that a smaller volume of blood would be required tive response to PHA may not produce a significantly different from the patient, which is ideal given that the Bead assay response when another mitogen is used. would be used on younger patients. Although there was no significant correlation between the Finally, the Bead assay would be used alongside the cur- use of the Beads alone and PHA with the healthy control rent PHA test that requires the lower PBMC concentration, subjects (Fig. 5B), there did appear to be a trend for a positive and thus, it is more practical for both assays to use the same association. It may be that this study was underpowered by cell concentration. the limited number of healthy controls available. Anti-Biotin MACSiBead™ particles induce Addition of IL-2 did not significantly more rounds of proliferation impact T-cell proliferation A trend noted throughout was that those PBMC populations No significant difference was found between the percentage incubated with the Beads typically underwent more rounds of proliferation yielded when the Beads were used alone and proliferation than when PHA was used (Fig. 3). All cell popu- when IL-2 was added on Day 3 (Fig. 5E). However, in all but lations were incubated for the same length of time, and thus, one of the 10 healthy controls, those cell populations incu- it can be concluded that, generally, the Beads may facilitate bated with IL-2 at Day 3 gave a slightly higher percentage more efficient T-cell proliferation. Moreover, a change in the proliferation. A strong positive correlation was observed colour of the culture medium from red to yellow, indicating between the use of the Beads only and the addition of IL-2 at nutrient utilization for proliferation, was seen typically Day 3 (Fig. 5F). This indicates that both assays give compa- 24–48 h sooner in those PBMC populations incubated with rable results and therefore suggests that the addition of IL-2 the Beads than with PHA. More specifically, in the presence is not necessary. Furthermore, the addition of IL-2 on Day 3 of anti-CD3/CD28-coated beads, T cells have been known limits the number of days that the assay can be performed so 7 Research article Bioscience Horizons • Volume 7 2014 Figure 5. Comparison of the percentage of T-cell proliferation and correlation statistics for 10 healthy controls using PHA vs. Beads alone (A and B), PHA vs. Beads plus IL-2 (C and D) and Beads alone vs. Beads plus IL-2 (E and F). as to allow for the work on Day 3 to be carried out during the to 14.79% when the Beads were used alone, and the value routine laboratory hours. fell by a further 0.72% in the PBMC population to which IL-2 was added on Day 3. Poor Responder 2 gave a percent- age proliferation of 18.42% with PHA, and this value Poor responders to PHA may also respond dropped to 17.08% when the Beads were used alone. poorly to Anti-Biotin MACSiBead™ particles However, the addition of IL-2 on Day 3 increased the prolif - The response of the three poor PHA responders to the Bead eration to 30.08%. Poor Responder 3 (the 11th healthy assay was varied (Fig. 6). Poor Responder 1 gave a percent- control that gave an unexpected poor PHA response) yielded age proliferation value of 33.86% with PHA. This dropped a T-cell proliferation of 29.45% with PHA, 40.55% when 8 Bioscience Horizons • Volume 7 2014 Research article 95% of the results are covered by the range, and therefore there is a 1 in 20 chance that a result for a healthy individual will fall outside this interval. This can be illustrated by the three poor PHA responders analysed who gave a T-cell per- centage proliferation of <45% and therefore fell outside the normal range set by the clinical Immunology laboratory. None of these subjects have a known primary or secondary immunodeficiency and so would appear to simply be healthy individuals who happen to not fall within the 95% included in the PHA reference range. Following the calculation of reference range values for the Bead assay both with and without the addition of IL-2 on Day Figure 6. Proliferation values for three poor PHA responders (p-value obtained by a Friedman test). Poor Responders 1 and 2 were known to 3 (Table 2), it can be concluded that only the proliferation give a percentage proliferation value of <45%, and the third was yielded for Poor Responder 3 when the Beads were used alone initially tested as a healthy control but unexpectedly gave a fell into the established reference range. None of the poor proliferation of <45% in response to PHA. responders fell within the reference interval for the Beads plus IL-2, and Poor Responders 1 and 2 did not fall into either Table 2. Median, 2.5th and 97.5th reference range values for the reference interval. proposed Anti-Biotin MACSiBead™ particles assays Limitations and further work PHA (% Beads only  Beads + IL-2 (% proliferation) (% proliferation) proliferation) Perhaps the most significant limitation of the practical work carried out was that all subjects used were adults. Primary Median 79 87 82 immunodeficiencies are typically diagnosed at a young age, and therefore, the reference ranges calculated for the Bead 2.5th 46 38 43 assay may not be reflective of a younger cohort. Unfortunately, percentile the ethical implications of recruiting healthy children for a 97.5th continuation of this research are great. 92 95 96 percentile Secondly, only 10 healthy controls were available. To Reference strengthen the results and to validate the reference range, 100 range 45–92 38–95 43–96 healthy controls would ideally be recruited but this was simply established not feasible. PHA reference range determined previously and is currently used in the Oxford laboratory. To enhance the likelihood of attaining a significant differ - ence in the T-cell percentage proliferation between poor PHA responders and the proposed Bead assay, a larger number of poor PHA responders should be recruited. However, this the Beads were used alone and 40.72% when IL-2 was added would be challenging in practical terms as most healthy indi- on Day 3. viduals respond well to PHA. Given that no significant difference between the results for To further investigate the capability of T-cell proliferation the poor PHA responders across the two assays was found, a in healthy individuals who show a poor response to PHA, definitive conclusion as to whether the Bead assay yields a other stimuli could be used and compared with the Beads. different T-cell proliferation percentage in those who respond Phytolacca Americana, more commonly known as Pokeweed, poorly to PHA cannot be made. is a perennial plant found in North and South America, East Asia and New Zealand. When extracted from the roots with Significance of the Anti-Biotin MACSiBead™ saline, pokeweed has been shown to have mitogenic proper- particles reference range in a diagnostic ties such that it can stimulate the proliferation of both T and setting B lymphocytes (Bodger et al., 1979). Concanavalin A is a sec- ond mitogen capable of inducing T-cell proliferation (McCole A reference range is a range of values where 95% of the results et al., 1998). These mitogens may show a significant differ - in healthy individuals fall within, thus excluding the upper ence in T-cell proliferation compared with PHA. and lower 2.5% (Ahmed, 2011). A reference range of values for any given diagnostic test can help a clinician to decide whether or not a result is abnormal and therefore a cause for Conclusions concern; however, the whole clinical picture should always be taken into account. If a test result falls outside the reference An anti-CD2/CD3/CD28-coated Anti-Biotin MACSiBead™ range, it does not necessarily mean that the patient is unwell; particles assay has been established. The Bead and PBMC 9 Research article Bioscience Horizons • Volume 7 2014 concentration has been optimized for diagnostic use. The Bead References assay was successfully performed, both with and without the addition of IL-2, on 10 healthy control subjects. The assay was Ahmed, N. (2011) Clinical Biochemistry, Oxford University Press, Oxford. conducted on three controls who respond poorly to PHA and Beadling, C. and Smith, K. A. (2002) DNA array analysis of interleukin- comparisons between the two assays were made. Following 2-regulated immediate/early genes, Medical Immunology, 1 (2), 1–14. the calculation of a reference range for the new assay, it was found that two out of the three individuals who responded Bodger, M. P., McGiven, A. R. and Fitzgerald, P. H. (1979) Mitogenic pro- poorly to PHA also responded poorly to the Beads, and one teins of pokeweed, Immunology, 37, 785–792. poor responder to PHA fell into the reference range for the Buckley, R. H. (2004) Molecular defects in human severe combined Bead assay. immunodeficiency and approaches to immune reconstitution, After taking the results into account, the proposed Bead Annual Review of Immunology, 22, 625–655. assay will now be offered as a diagnostic test, alongside the Cornish, G. H., Sinclair, L. V. and Cantrell, D. A. (2006) Differential regula - current PHA assay, within the routine Oxford Immunology tion of T-cell growth by IL-2 and IL-15, Blood, 108 (2), 600–608. laboratory. The newly developed assay will not include the addition of IL-2 on Day 3. Duarte, R. F., Chen, F. E., Lowdell, M. W. et  al. (2002) Functional impair- ment of human T-lymphocytes following PHA-induced expansion The Bead assay provides a more physiological addition to and retroviral transduction: implications for gene therapy, Gene the current PHA test and may be useful in determining the Therapy, 9 (20), 1359–1368. ability of T cells to proliferate in some patients who respond poorly to PHA. Geha, R. S., Notarangelo, L. D., Casanova, J–L. et al. (2007) Primary immuno- deficiency diseases: an update from the International Union of Immunological Societies Primary Immunodeficiency Diseases Classi- Acknowledgements fication Committee, The Journal of Allergy and Clinical Immunology, 120 I thank all staff working at the Oxford Immunology labora- (4), 776–794. tory, and a special thank you goes to Dr Lisa Ayers for all her Hall, A. and Yates, C. (2010) Immunology, Oxford University Press, timeless help, guidance and support throughout. Oxford. Kelly, B. T., Tam, J. S., Verbsky, J. W. et  al. (2013) Screening for severe Author Biography combined immunodeficiency in neonates, Clinical Epidemiology, 5, 363–369. Louisa Green studied for her BSc (Hons) Biomedical Science degree at Oxford Brookes University over 4 years Li, Y. and Kurlander, R. J. (2010) Comparison of anti-CD3 and anti-CD28- whilst working as a Trainee Biomedical Scientist at the coated beads with soluble anti-CD3 for expanding human T cells: Oxford University Hospitals NHS Trust. After spending differing impact on CD8T cell phenotype and responsiveness to the first 2 and a half years of her course rotating through - restimulation, Journal of Translational Medicine, 8 (104), 1–15. out each laboratory discipline for 6 months at a time, she McCole, D. F., Dohertv, M. L., Baird, A. W. et  al. (1998) Concanavalin chose to spend the final 18 months of her employment spe - A-stimulated proliferation of T cell subset-depleted lymphocyte cializing in Immunology. Here, Louisa completed both her populations isolated from Fasciola hepatica-infected cattle, undergraduate research project and generic registration Veterinary Immunology and Immunopathology, 66 (3–4), 289–300. Portfolio for the Institute of Biomedical Science, and she is now a fully qualified Biomedical Scientist. She graduated Parish, C. R., Glidden, M. H., Quah, B. J. C. et  al. (2009) Use of the from Oxford Brookes University in June 2014 winning the intracellular fluorescent dye CFSE to monitor lymphocyte migration Best in Year project prize, and within the next year plans to and proliferation, Current Protocols in Immunology, 84, 4.9.1–4.9.10. apply for a 4-year integrated MRes/PhD in Infection and Pignata, C., Sanghera, J. S., Soiffer, R. J. et al. (1996) Defective activa- Immunity. tion of mitogen-activated protein kinase after allogeneic bone marrow transplantation. Blood, 88 (6), 2334–2341. Funding Tasher, D. and Dalal, I. (2012) The genetic basis of severe combined Funding was kindly provided by Oxford University Hospitals immunodeficiency and its variants, Journal of the Application of NHS Trust. Clinical Genetics, 5, 67–80.

Journal

Bioscience HorizonsOxford University Press

Published: Nov 6, 2014

Keywords: PHA anti-CD2/CD3/CD28 beads T cell proliferation diagnosis immunodeficiency

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