The Value of a Rapid Test of Human Regulatory T Cell Function Needs to be Revised

CD4⁺CD25⁺FoxP3⁺ human regulatory T_CELLS (T_REG) are promising candidates for reshaping undesired immunity/inflammation by adoptive cell transfer, yet their application is strongly dependent on robust assays testing their functionality. Several studies along with first clinical data indicate T_REG to be auspicious to use for future cell therapies, e.g., to induce tolerance after solid organ transplantation. To this end, T_REG suppressive capacity has to be thoroughly evaluated prior to any therapeutic application. A 7 h-protocol for the assessment of T_REG function by suppression of the early activation markers CD154 and CD69 on CD4⁺CD25⁻ responder T_CELLS (T_RESP) upon polyclonal stimulation via αCD3/28-coated activating microbeads has previously been published. Even though this assay has since been applied by various groups, the protocol comes with a critical pitfall, which is yet not corrected by the journal of its original publication. Our results demonstrate that the observed decrease in activation marker frequency on T_RESP is due to competition for αCD3/28-coated microbeads as opposed to a T_REG-attributable effect and therefore the protocol cannot further be used as a diagnostic test to assess suppressive T_REG function.

Introduction

Regulatory T_CELLS (T_REG) are key players in maintaining immune homeostasis, resolution of inflammation, and self (1). Exploiting those characteristics, T_REG have gained plenty of attention as promising candidates in immunotherapeutic applications for the prevention or reshaping of undesired immune responses such as in autoimmune diseases, chronic inflammation, and allograft rejections. Data from clinical trials identify T_REG as an encouraging cell type for use in cellular therapy (2). By the same token, a robust protocol to assess T_REG function is of utmost importance to ensure their suppressive function prior to adoptive cell-therapeutic clinical trials, as well as for application in basic research. So far, for assessing T_REG functionality, evaluating the suppressive capacity of T_REG to inhibit the proliferation of responder T_CELL (T_RESP) after a 4-day co-cultivation period has been the gold-standard protocol since a decade (3, 4). Recently, Canavan et al. (5) and Ruitenberg et al. (6) described a rapid 7 h assay for the evaluation of T_REG functionality by assessing their suppressive capacity using upregulation of the early T_CELL activation makers CD154 (CD40L) and CD69 on conventional CD4⁺CD25⁻ responder T_CELLS (T_RESP) upon CD3/28 engagement. CD3/28 stimulation is mediated by microbeads coupled with αCD3 and αCD28 antibodies. According to these studies, T_REG alleviate CD154 and CD69 expression on T_RESP in a dose-dependent manner. Even though this assay has since been frequently applied and cited more than 80 times (7, 8, 10), we observed that the protocol comes with a critical pitfall: T_RESP and T_REG both express the signaling molecule CD3 and T_CELL co-stimulatory receptor CD28 on the plasma membrane, potentially competing for binding αCD3/28 T_CELL activating microbeads applied in the rapid 7 h assay. We investigated whether the observed decreased frequencies of activated T_RESP can be claimed to be a T_REG-attributable effect or if it is rather a result of competition for αCD3/28-coated activating microbeads. We thus explored whether different ratios of αCD3/28 T_CELL activation microbeads-to-T_CELLS impact the outcome of this functional T_REG assay.

Materials and Methods

Study Design

The aim of this study was to investigate the influence of αCD3/CD28-coated activating microbeads on the expression of early activation markers CD69 and CD154, used for predicting T_REG functionality in basic and translational research. We compared the expression of CD69 and CD154 of T_RESP in T_REG co-cultures, which were either activated via αCD3/CD28-coated microbeads adjusted to T_RESP only or to the total cell number present in one well (T_RESP + T_REG). To verify the integrity of the T_REG used in this study, as well as to demonstrate the T_REG-mediated suppressive function in a bead-uncompetitive setting, T_RESP proliferation suppression experiments were performed.

Cell Isolation

Peripheral blood mononuclear cells from healthy donors were purified using Ficoll-Paque separation (Biochrom). CD4⁺ cells were enriched by magnetic-activated cell sorting (Miltenyi) according to manufacturer's instructions (purity>90%). For fluorescence-activated cell sorting (FACS Aria II, BD) of CD4⁺CD25^highCD127^low T_REG and CD4⁺CD25⁻ T_RESP, cells were stained with CD4 (SK3, Biolegend), CD25 (2A3, BD), and CD127 (R34.34, Beckman Coulter). Post-FACSort analysis by flow cytometry yielded CD25⁺FoxP3⁺ T_CELL purity of >95%.

7 h Diagnostic Test for T_REG Function and αCD3/28 Microbead Titration

Assays were performed as described by Canavan et al. (5). Briefly, CFSE-labeled T_RESP were co-cultured with autologous T_REG at T_RESP/T_REG ratios ranging from 1:1 to 32:1. In two parallel setups, cells were either stimulated with αCD3/28-coated microbeads (Dynabeads^® Human T-Activator CD3/CD28, Thermo Fisher Scientific) at a bead/cell ratio of 0.2 adjusted to the T_RESP cell number per well (5, 6) or adapting the ratio of 0.2 to the total cell number per well including T_REG. Stimulated and unstimulated T_RESP without T_REG were included as controls. For the microbead titration, T_RESP were cultured alone at bead/T_RESP ratios ranging from 0.1 to 0.4 (mimicking the presence of T_REG). αCD154 (24–31) was added at start of incubation. Cells were incubated at 37°C for 7 h. All cell cultures were performed in X-Vivo-15 medium supplemented with 10% FCS (Lonza & Biochrom) and 100 IU/ml Penicillin/Streptomycin. After harvesting, cells were stained with CD3 (OKT3), CD4 (SK3), CD137 (4B4-4), and CD69 (FN50), all Biolegend. Dead cells were excluded (LIFE/DEAD^TM Fixable Blue Dead Cell Stain Kit, Thermo Fisher Scientific).

Proliferation Suppression Assay

CFSE-labeled T_RESP were cultured alone or with autologous T_REGS at T_RESP/T_REG ratios ranging from 1:1 to 16:1. The cells were stimulated with αCD3/28-coated microbeads (T_REG Suppression Inspector, Miltenyi) at a cell/bead ratio of 1:1 and 1:2 adjusted to the total cell number per well and incubated at 37°C for 96 h. Thereafter, cells were stained with CD3 (OKT3), CD4 (SK3), all Biolegend. Dead cells were excluded (Thermo Fisher Scientific). Proliferation was assessed by CFSE dilution and percentage suppression of proliferation was calculated by relating the percentage of proliferating T_RESP in the presence and absence of T_REG, respectively.

Flow Cytometry Analysis

Data were acquired on a LSR-II Fortessa flow cytometer (BD) and analyzed using FlowJo V10 (TreeStar).

Statistics

Analysis was performed with GraphPad Prism software (version 6, GraphPad, La Jolla, CA) and R (version 3.4.1) (9). We have tested for significant interaction, i.e., non-parallel response profiles of the two bead adjustment methods to the different T_RESP:T_REG ratios, using a non-parametric rank-based ANOVA-type statistic [as implemented in the nparLD package (11)] in a two-way factorial repeated measures design. For bead titration experiments, non-parametric two-tailed Wilcoxon matched-pairs signed rank tests were used to determine significance in pairwise comparison. Data indicate means ± SEMs in all bar graphs. P < 0.05 was considered significant.

Results

T_CELL Early Activation Marker Expression Is Dependent of TCR Engagement

We first examined T_REG functionality according to the protocols published by Canavan et al. (5) and Ruitenberg et al. (6), whereby ex vivo FACSorted and CFSE-labeled T_RESP were co-cultured in the presence and absence of autologous T_REG and stimulated with αCD3/28-coated activating microbeads at a ratio of 0.2 microbeads per T_RESP (Figure 1A). After 7 h, the mean frequency of CD154⁺ and CD69⁺ T_CELLS of unstimulated T_RESP was 0.14 and 0.45%, respectively and 57.25 and 78.26% on CD3/28-stimulated T_RESP, respectively (Figure 1B). When T_RESP were stimulated in the presence of T_REG at ratio 1:1, the mean frequency of CD154⁺ and CD69⁺ T_CELLS decreased to 47.77 and 69.86%, respectively. With increasing T_RESP/T_REG ratios both, CD154 and CD69 expression, increased in a linear fashion (Figure 1C, quantified in E, F, red columns). We next determined whether the total T_CELL/bead ratio influences T_REG-induced activation marker suppression. Accordingly, we adjusted the bead numbers to the total cell numbers, including T_REG, thereby eluding the bead competition in contrast to Canavan et al. (5) and Ruitenberg et al. (6). In that case, T_RESP activation in the presence of T_REG equaled control T_RESP cultures without T_REG (Figure 1D, quantified in E, F, blue bars), indicating that indeed T_RESP and T_REG compete for CD3/28-binding microbeads. Serving as a negative control, we co-cultured T_RESP with CD4⁺CD25⁻ non-T_REG/effector T_CELLS in place of T_REG. When the bead number was adjusted to T_RESP only we observed similar reductions of CD154 and CD69 expression (Figures 1G,H, red bars) as when T_RESP were co-cultured with T_REG (Figures 1E,F, red bars). Correspondingly, when adjusting the bead number to the total cell number (Figures 1E,H, blue bars), the expression of CD154 and CD69 is similar to the conditions with T_RESP only (Figures 1E–H, gray bars). To mimic the competition for the activating microbead stimuli, we stimulated T_RESP with different amounts of αCD3/28-coated microbeads in the absence of T_REG. We set the actual bead/T_CELL ratio according to the published T_RESP/T_REG co-culture approach, in which the activation bead/T_RESP ratio is adjusted to T_RESP only, i.e., calculated the actual bead/T_CELL ratio in each setting. CD154 and CD69 expression decreased in a dose-dependent manner with highest expression levels at a bead/T_RESP ratio of 0.4 (69.83 and 89.47%, respectively) and lowest at a ratio of 0.1 (37.80 and 53.33%, respectively). The T_RESP activation pattern with the different bead ratios ranging from 0.1 to 0.194 indicate a strong bead/T_RESP ratio dependency (Figures 1I,J).

FIGURE 1

Figure 1. T_CELL early activation marker expression is dependent of TCR engagement and cannot be used for T_REG functional evaluation. FACSorted CD4⁺CD25⁻ T_RESP with and without autologous T_REG co-culture were stimulated with anti-CD3/CD28-coated microbeads and analyzed for early activation marker expression. (A) For precise T_RESP/T_REG discrimination, T_RESP were labeled with CFDA-SE (CFSE). (B) Representative plots of CD154 and CD69 expression on unstimulated and stimulated T_RESP cultured without T_REG. (C) Representative plots of CD154 and CD69 expression of T_RESP co-cultured with T_REG at different T_RESP:T_REG ratios and stimulated with anti-CD3/CD28-coated microbeads adjusted to T_RESP. (D) Representative plots of CD154 and CD69 expression of T_RESP co-cultured with T_REG at different T_RESP:T_REG ratios and stimulated with anti-CD3/CD28-coated microbeads adjusted to total cell number. (E,F) Quantified data from (C,D), respectively. CD154 and CD69 of CFSE⁺T_RESP co-cultured with FACSorted T_REG at different T_RESP:T_REG ratios and stimulated with anti-CD3/CD28-coated microbeads adjusted to T_RESP (red columns) and to total cell numbers (blue columns). For clarification, the table summarizes the experimental setups. n = 7. Non-parametric rank-based ANOVA-type statistic **p < 0.001 (CD154: p = 1.90E-06, CD69: p = 5.527256E-16). (G) Expression of CD154 and (H) expression of CD69 of CFSE⁺T_RESP co-cultured with FACSorted T_NON−TREG at different T_RESP:T_REG ratios and stimulated with anti-CD3/CD28-coated microbeads adjusted to T_RESP (red columns) and to total cell numbers (blue columns). n = 3. (I) Expression of CD154 and (J) expression of CD69 of CFSE⁺T_RESP after different anti-CD3/CD28-coated microbead:T_RESP ratio stimulation. For clarification, the table summarizes the experimental setups. n = 7. *p < 0.05, Wilcoxon matched-pairs signed rank test. T_RESP:T_REG/T_NON−TREG co-cultures (E,F) and corresponding bead titration (I,J) experiments were performed simultaneously using the same donor cells. Median data of independent experiments are shown and error bars represent SEM.

T_REG Demonstrate a Dose-Dependent T_RESP Proliferation Suppression in a Bead-Uncompetitive Setting

To confirm T_REG functionality in an environment where the number of αCD3/28-activation microbeads is adjusted to the total cell number, the gold-standard T_RESP proliferation suppression assay was performed. The proliferation assay was conducted with T_CELLS of the same donors in parallel to the experiments shown in Figure 1. Following activation, T_RESP proliferation alone yielded 52.03% and dose-dependently decreased in the presence of T_REG to 15.51% at a T_RESP/T_REG ratio of 1:1 (Figures 2A–C, quantified in Figure 2D, green bars). Thus, we conclude that the T_REG employed in this study are able to suppress T_RESP proliferation in a standardized bead-competitive setting. To ascertain the reduction of proliferation to be T_REG-mediated, we have added non-T_REG/effector T_CELLS instead of T_REG to T_RESP and observed no decrease in T_RESP proliferation, indicating the suppression of T_RESP proliferation to be a T_REG-attributable effect (Figure 2D, blue bars). Even when T_CELLS are stimulated with twice the number of activating αCD3/CD28 microbeads, the T_REG-specific impact in suppressing T_RESP proliferation can be seen (Figure 2E).

FIGURE 2

Figure 2. Ex vivo isolated T_REG demonstrate a dose-dependent T_RESP proliferation suppression in a bead-uncompetitive setting. FACSorted CFSE⁺CD4⁺CD25⁻ T_RESP were co-cultured with autologous FACSorted T_REG and stimulated with anti-CD3/CD28-coated microbeads for 96 h. T_RESP proliferation was analyzed by CFSE dilution. Representative plots depicting (A) CFSE-labeling strategy to accurately analyze T_RESP proliferation; (B) proliferation of unstimulated and stimulated CFSE⁺T_RESP cultured without T_REG and (C) CFSE⁺T_RESP proliferation after co-culture with different T_REG ratios. (D) Percentage of T_RESP proliferation after co-culture with decreasing T_RESP:T_REG ratios (green bars) and T_RESP:T_NON-_TREG ratios (blue bars) stimulated with a total cell number:bead ratio of 1:1. n = 7 T_RESP:T_REG co-cultures, n = 3 T_RESP:T_NON−TREG co-cultures. (E) Percentage of T_RESP proliferation after co-culture with decreasing T_RESP:T_REG ratios (green bars) and T_RESP:T_NON-_TREG ratios (blue bars) stimulated with a total cell number:bead ratio of 1:2. n = 3. Median data of independent experiments are shown and error bars represent SEM.

Discussion

In conclusion, when adjusting the αCD3/28-bead numbers to only T_RESP in co-cultures of T_RESP and T_REG, activation marker expression was comparable to approaches where T_RESP were cultured alone at same bead/total cell ratio present in the T_RESP/T_REG co-culture. When normalizing αCD3/28-bead competition by adjusting the bead number to total cell numbers, T_REG-mediated suppression of activation marker upregulation is nullified. Even more strikingly, when titrating non-T_REG/effector T_CELLS to T_RESP and adjusting the αCD3/28-bead numbers to T_RESP only, we observe the same decrease in activation marker expression as in T_RESP:T_REG co-cultures. We thereby demonstrate that the suppression of activation marker expression on T_RESP observed in co-cultures with T_REG are due to competitive T_CELL receptor and CD28 engagement limited by αCD3/28 microbead availability rather than by suppressive activity of T_REG (Supplementary Figure 1). There is a pressing demand for a fast assay to evaluate T_REG functionality, especially in the light of upcoming clinical trials needing a robust diagnostic test to assess the suppressive function as a release criterion for their T_REG cell products. Nonetheless, the T_RESP proliferation suppression analysis should still be considered as the gold-standard T_REG functional assay as it is performed by adjusting the activation bead to T_CELL ratios in experimental setups with decreasing T_REG cell numbers (to assess T_REG dose-dependent suppression). Since we firmly believe that activation bead to T_CELL receptor competition should be kept constant throughout all conditions within a T_REG functional assay, we claim that the rapid assessment for human T_REG function proposed by Canavan et al. (5) and Ruitenberg et al. (6) does not result in reliable evidence of functional suppression since the putative T_REG-mediated suppression of T_RESP activation is to be ascribed to competitive T_CELL receptor and CD28 engagement. Hence, we suggest that the previously published protocol is unsuitable as a diagnostic test to assess suppressive T_REG function.

Ethics Statement

The Charité Ethics Committee (IRB) approved the study protocol and all blood donors provided written informed consent.

Author Contributions

DW designed the research, performed experiments, analyzed and interpreted the data, and wrote the manuscript. LA performed experiments and revised the manuscript. SS performed statistical analyses. PR revised the manuscript. H-DV interpreted the data and revised the manuscript. MS-H led the project, designed the research, analyzed and interpreted the data, and wrote the manuscript.

Funding

The study was generously supported in parts by the Deutsche Forschungsgemeinschaft (DFG-SFB-TR36-project A3–H-DV, PR, and MS-H) and the German Federal Ministry of Education and Research (Berlin-Brandenburg Center for Regenerative Therapies grant—all authors). We acknowledge support from the German Research Foundation (DFG) and the Open Access Publication Fund of Charité—Universitätsmedizin Berlin.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We acknowledge the assistance of the BCRT Flow Cytometry Core Lab, Dr. D. Kunkel and J. Hartwig. Mathias Streitz for flow cytometry assistance. Karolina Grzeschik and Anke Jurisch for technical assistance. Dr. Toralf Roch for critical discussions. We likewise acknowledge support from the German Research Foundation (DFG) and the Open Access Publication Fund of Charité — Universitätsmedizin Berlin.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2019.00150/full#supplementary-material

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