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. 2014 Jul;78(3):168–175.

In-vitro immunosuppression of canine T-lymphocyte-specific proliferation with dexamethasone, cyclosporine, and the active metabolites of azathioprine and leflunomide in a flow-cytometric assay

Laura A Nafe 1, John R Dodam 1, Carol R Reinero 1,✉
PMCID: PMC4068407  PMID: 24982547

Abstract

A high rate of mortality, expense, and complications of immunosuppressive therapy in dogs underscores the need for optimization of drug dosing. The purpose of this study was to determine, using a flow-cytometric assay, the 50% T-cell inhibitory concentration (IC50) of dexamethasone, cyclosporine, and the active metabolites of azathioprine (6-mercaptopurine) and leflunomide (A77 1726) in canine lymphocytes stimulated with concanavalin A (Con A). Whole blood was collected from 5 privately owned, healthy dogs of various ages, genders, and breeds. Peripheral blood mononuclear cells, obtained by density-gradient separation, were cultured for 72 h with Con A, a fluorochrome-tagged cell proliferation dye, and various concentrations of dexamethasone (0.1, 1, 10, 100, 1000, and 10 000 μM), cyclosporine (0.2, 2, 10, 20, 30, 40, 80, and 200 ng/mL), 6-mercaptopurine (0.5, 2.5, 50, 100, 250, and 500 μM), and A77 1726 (1, 5, 10, 25, 50, and 200 μM). After incubation, the lymphocytes were labeled with propidium iodide and an antibody against canine CD5, a pan T-cell surface marker. Flow cytometry determined the percentage of live, proliferating T-lymphocytes incubated with or without immunosuppressants. The mean (± standard error) IC50 was 3460 ± 1900 μM for dexamethasone, 15.8 ± 2.3 ng/mL for cyclosporine, 1.3 ± 0.4 μM for 6-mercaptopurine, and 55.6 ± 22.0 μM for A77 1722. Inhibition of T-cell proliferation by the 4 immunosuppressants was demonstrated in a concentration-dependent manner, with variability between the dogs. These results represent the initial steps to tailor this assay for individual immunosuppressant protocols for dogs with immune-mediated disease.

Introduction

Immune-mediated hemolytic anemia (IMHA) and immune-mediated thrombocytopenia (ITP) are common and serious immunemediated diseases affecting dogs (1). Even with appropriate treatment, both of these diseases have a high mortality rate, 21% to 83% and 10% to 30%, respectively (2,3). In addition, the expense and complications of standard immunosuppressive therapy must be considered (4–6). Unfortunately, appropriate clinical trials evaluating the efficacy of different treatment protocols are lacking (5–9). As a result, decisions regarding immunosuppressive therapy for individual patients are often based on anecdotal experience and response to treatment. Pharmacokinetic monitoring helps to target therapeutic blood concentrations and reflects how the body handles the drug, whereas pharmacodynamic monitoring evaluates the effects of the drug on the body. In the case of immunosuppressants, pharmacodynamic monitoring allows assessment of specific effects on the components of the immune system (10). Pharmacodynamic assays to assess immune responses are expensive and time-consuming, and they require special expertise and equipment; thus, at this time they are only done in veterinary medicine in a research setting. For example, a recent study evaluated the in-vitro effects of cyclosporine on the expression of canine T-lymphocyte cytokines [interleukin (IL) 2 and 4 and interferon-γ], providing valuable information on the specific immune effects of that immunosuppressive agent (11,12). Given that canine IMHA and ITP remain challenging to treat, with high morbidity and mortality rates, further exploration of pharmacodynamic assays is warranted. Promising assays can subsequently undergo modification to make them more clinically applicable and available.

The pathogenesis of IMHA is believed to center on autoreactive T-lymphocyte activation of B-lymphocytes, resulting in antierythrocyte antibody production against certain red blood cell antigens (13). A similar pathogenesis is suspected for ITP (14,15). In most cases of canine IMHA and ITP an underlying cause is not identified, and treatment is aimed at nonspecific immunosuppression and various ancillary forms of therapy (e.g., thromboembolic prophylaxis and red blood cell transfusions). The mainstay of immunosuppressive therapy is administration of glucocorticoids, to which it is common to add a 2nd immunosuppressive agent (16). Glucocorticoids exert immunosuppressive effects by inhibiting the expression of various genes necessary to the activity of multiple inflammatory mediators (e.g., IL-2) and to the expression of adhesion molecules (e.g., intercellular adhesion molecule 1 and vascular cell adhesion molecule 1) (17). In addition, Fc (fragment, crystallizable)-receptor expression and antigen processing and presentation, which help amplify the immune response, are down-regulated by glucocorticoids (17). Although glucocorticoids are effective at suppressing the immune response in many patients, long-term use at high dosages predisposes to complications (e.g., infections and thromboembolic disease). Moreover, a subset of patients may be glucocorticoid-resistant, have intolerable side effects, or have concurrent disease that precludes long-term, high-dose steroid treatment (18,19). For these reasons, an effective 2nd immunosuppressive agent for the treatment of IMHA and ITP is frequently sought. Cytotoxic and immunomodulatory agents, such as azathioprine, cyclosporine, and leflunomide, have been used in veterinary medicine for the treatment of various immune-mediated diseases, including IMHA and ITP (6,11,16,20–23). Although through different mechanisms, all of these agents aim to suppress lymphocyte production and/or function (24).

Further investigation of lymphocyte-suppression profiles for specific immunosuppressive agents is warranted when deciding on the most efficacious therapeutic agent(s) for an individual patient. Understanding the specifics of T-cell suppression will aid in targeting immunosuppressive agents toward individual patients and specific diseases. Previous studies have evaluated the ex-vivo lymphocyte suppression of various immunosuppressive agents in multiple species, including dogs (25,26). However, to the authors’ knowledge, no study has assessed, using a flow-cytometric assay, the effects of a panel of immunosuppressants on live T-lymphocytes undergoing proliferation. The objective of the present study was to determine the concentration of dexamethasone, cyclosporine, and the active metabolites of azathioprine (6-mercaptopurine) and leflunomide (A77 1726) that would result in 50% inhibition of canine T-lymphocyte proliferation (IC50). We hypothesized that the panel of immunosuppressant drugs would suppress T-cell proliferation at a specific titrated concentration when evaluated in vitro in healthy dogs.

Materials and methods

Animals

Five privately owned, healthy dogs of various ages, breeds, and genders that were not receiving any medications other than parasite preventatives were included in the study with informed consent of the owners. The study was conducted with the approval of the Animal Care and Use Committee, University of Missouri, Columbia, Missouri, USA (protocol 7370). Health screening done before the study included a physical examination and complete blood count.

Cell proliferation and its suppression

Whole blood was collected by jugular venipuncture into lithium heparinized tubes and processed within 1 h after collection. Density-gradient centrifugation with Histopaque 1077 (density 1.077; Sigma-Aldrich, St. Louis, Missouri, USA) was done to harvest peripheral blood mononuclear cells (PBMCs). A commercially available cell-proliferation dye (CPD), eFluor 670 (eBioscience, San Diego, California, USA), was used to assess lymphocyte proliferation. This fluorochrome-tagged dye binds to cellular proteins containing primary amines and is distributed equally to daughter cells upon division; thus, as cells divide, fluorescent staining becomes less bright. To optimize the assay the following conditions were tried. First, CPD eFluor 670 was added at concentrations of 2.5, 5.0, and 7.5 μM to washed PBMCs (105 cells, 5 × 105 cells, and 106 cells). The cells were incubated for 10 min at 37°C in the dark, and labeling was stopped by the addition of 5 volumes giving (500 mL of cold complete RPMI (cRPMI) medium (RPMI 1640; Sigma-Aldrich), with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, Massachusetts, USA), 5 mL of 1-M Hepes (Life Technologies, Grand Island, New York, USA), 0.35 L of diluted β-mercaptoethanol (Sigma-Aldrich), 5 mL of penicillin–streptomycin–glutamine (Life Technologies), and 1.1 g of sodium bicarbonate (Thermo Fisher Scientific). Optimal conditions occurred with 5.0-μM CPD eFluor 670 and 5 × 105 cells (data not shown). The cells were washed 3 times with cRPMI and transferred to 96-well plates (Costar #3595; Corning Inc., Corning, New York, USA) in the presence of the T-cell mitogen concanavalin A (Con A; Sigma-Aldrich), 5 μg/mL (previously tested concentrations ranged between 2.5 and 20 μg/mL), and increasing concentrations of dexamethasone (0.1, 1, 10, 100, 1000, and 10 000 μM; Sigma-Aldrich), cyclosporine (0.2, 2, 10, 20, 30, 40, 80, and 200 ng/mL; Sigma-Aldrich), 6-mercaptopurine (0.5, 2.5, 50, 100, 250, and 500 μM; Sigma-Aldrich), and A77 1726 (1, 5, 10, 25, 50, and 200 μM; EMD Chemicals, Gibbstown, New Jersey, USA). The plates were incubated at 37°C in humidified 5% CO2/95% air (Hera Cell 150; Kendro Laboratory Products, Langenselbold, Germany) for 3 d before the cells were labeled for flow-cytometric analysis.

Staining for viability and CD5

The PBMCs were stained with 0.0625 μg of perCP-eFluor 710 antibody against canine CD5, a pan T-cell surface marker (clone YKIX322.3; eBioscience), for 30 min at 4°C in the dark, according to the manufacturer’s instructions, in a final volume of 50 μL. After being washed twice with fluorescence-activated cell-sorting (FACS) buffer (phosphate-buffered saline/3% FBS) the cells were stained with 2.5 μL of propidium iodide (PI, eBioscience; previously optimized with 1, 2.5, and 5 μL/well; data not shown), according to the manufacturer’s recommendations, for 20 min at 4°C in the dark in a final volume of 50 μL. The cells were then washed and resuspended in FACS buffer for analysis.

Flow-cytometric analysis

Analysis was done with a CyAn ADP Flow Cytometer (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA), a minimum of 20 000 gated events being collected. Lymphocytes were identified on a plot of forward scatter (FSC) versus side scatter (SSC), and live cells were identified as PI-negative on a plot of SSC versus fluorescence intensity (FL) 2. The remaining analyses were done on live lymphocytes applied to a dot plot of proliferating daughter cells (CPD eFluor 670 in the FL8 channel) versus T-cells (CD5 perCP-eFluor 710 in the FL4 channel). The daughter population was defined as cells to the left of the gate set from cells incubated in CPD eFluor 670 before culture (Figures 1A and 1B). Data were represented by the percentage of cells double-positive for CD5 and CPDdim (daughter cells).

Figure 1A.

Figure 1A

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Plot of side scatter (SSC) versus forward scatter (FSC) from flow-cytometric analysis of canine peripheral blood mononuclear cells harvested by means of density-gradient centrifugation. R1 indicates lymphocytes.

Figure 1B.

Figure 1B

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After culture for 3 d in the presence of the T-cell mitogen concanavalin A (Con A), the population of stimulated lymphocytes was identifiable (R1) as larger and more complex cells.

Calculating the IC50

The percentage of live, proliferating T-lymphocytes incubated with or without immunosuppressants was determined by means of flow cytometry. The IC50 for each immunosuppressant was determined with the use of a 4- or 5-parameter logistic-regression analysis (SigmaPlot; Systat Software, San Jose, California, USA). Results were reported in a descriptive fashion and in the form of scatter plots.

Results

Compared with unstimulated lymphocytes harvested immediately after density-gradient centrifugation (Figure 1A), lymphocytes stimulated with Con A for 3 d were a distinct population on a scatter plot: as a whole they were larger and more complex (Figure 1B). Once stimulated lymphocytes were identified, a 2nd gate was applied around PI-negative cells (Figure 1C), which allowed dead cells to be excluded from further analysis. In this assay, viability exceeded 90% (usually 97%). Live unstimulated lymphocytes were used to establish the parent cells staining with CPD (Figure 2A), which constituted more than 97% of this population. After 3 d of incubation with Con A, proliferating daughter cells were identified to the left of the parent population (Figure 2B). This population was more variable between the dogs, ranging from 26% to 70%. Addition of the CD5 pan T-cell marker allowed identification of proliferating T-cells in the whole proliferating lymphocyte population; this population also varied between dogs, ranging from 49% to 70%.

Figure 1C.

Figure 1C

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Staining with propidium iodide allows the exclusion of dead lymphocytes (R3) from the subsequent analyses. R2 — viable lymphocytes.

Figure 2A.

Figure 2A

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A commercially available cell-proliferation dye (CPD), eFluor 670, was used to track lymphocyte division. A gate was placed around CPDhi cells in the live cell population incubated without Con A to identify the parent cells that had not undergone proliferation (R5).

Figure 2B.

Figure 2B

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After 3 d of incubation with Con A, a population of dividing daughter cells (R4) was easily identified to the left of the parent population (R5).

The addition of increasing concentrations of immunosuppressants in vitro resulted in a concentration-dependent reduction in lymphocyte proliferation. The mean IC50 (± standard error), as determined by flow cytometry for the 5 dogs, was 3460 ± 1900 μM for dexamethasone, 15.8 ± 2.3 ng/mL for cyclosporine, 1.3 ± 0.4 μM for 6-mercaptopurine, and 55.6 ± 22.0 μM for A77 1722. Marked variability between the dogs was noted, especially for dexamethasone and A77 1722. Despite the variability between dogs and between immunosuppressant drugs, all the immunosuppressant agents resulted in a concentration-dependent decrease in T-lymphocyte proliferation, as illustrated for 1 dog in Figure 3 and for each drug in Figures 4 to 7.

Figure 3A.

Figure 3A

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Representative data for the lymphocytes from 1 of the 5 dogs evaluated. Plot of lymphocytes staining with perCP-eFluor 710 antibody against canine CD5, a pan T-cell surface marker, and CPD eFluor 670 after incubation with Con A. R6 — live, proliferating T-lymphocytes.

Figure 4.

Figure 4

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Percentage inhibition of T-lymphocyte proliferation by various concentrations of dexamethasone for the 5 individual dogs.

Figure 7.

Figure 7

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Percentage inhibition of T-lymphocyte proliferation by various concentrations of A77 1726, the active metabolite of leflunomide, for the 5 individual dogs.

Discussion

This article describes the development and validation of a canine-specific flow-cytometric assay for assessing live, proliferating T-lymphocytes in the presence of mitogen and a panel of immunosuppressant compounds at various concentrations. Compared with previous canine and feline studies that have evaluated the effects of immunosuppressants on lymphocyte proliferation using radioactive thymidine (27–29), our assay has the advantage of assessing viable populations of proliferating T-lymphocytes. In addition, the use of flow cytometry allows for flexibility in that it can be adapted to look at multiple cell populations (e.g., B-lymphocytes and monocytes) if this is indicated. The IC50 was the set point in this study, as it is a well-established parameter used in pharmacodynamic studies when the effects of drugs on the immune system are being assessed (30). This pharmacodynamic assay has great potential for detecting lymphocyte proliferation in dogs with immune-mediated diseases (e.g., IMHA and ITP) in need of immunosuppressant therapy. In addition, it can be used for either in-vitro evaluation (taking blood and adding drug outside the body) or ex-vivo evaluation (taking blood from dogs treated with the drug in vivo) to study the effects of immunosuppressive drugs on lymphocyte proliferation in individual patients and tailor therapy accordingly.

Current methods of assessing response to therapy in patients with immune-mediated disease are centered on monitoring clinical response (by such means as measuring the hematocrit in the patient with IMHA) and conducting a pharmacokinetic evaluation if a commercial assay is available (e.g., as with cyclosporine and leflunomide). Although pharmacokinetic analysis is the primary method of determining the absorption, distribution, metabolism, and excretion of a particular drug, it does not evaluate the pharmacologic effects of that agent on the immune cells of the patient, a concept known as pharmacodynamics (10). A patient may have the therapeutic concentration of a particular medication in the blood stream, but this may not correlate with pharmacodynamics and clinical response. The concentration of a drug that a cell is exposed to in vivo depends on many pharmacokinetic factors, including speed of distribution, volume of distribution, clearance, degradation, and protein binding. There are complex factors relevant to pharmacodynamics that could influence immunologic responses in vivo. For example, lymphocytes in vivo may travel to draining lymph nodes and receive additional signals that could affect survival, activation, and phenotype. In vitro, factors aside from lymphocyte response to a mitogen and an immunosuppressant cannot be assessed; therefore, a direct correlation between drug concentrations in vivo and in vitro is not really appropriate. It must be understood that our proposed in-vitro studies are intended as a surrogate for invasive in-vivo therapeutic trials. The assay described would allow clinicians to evaluate the pharmacodynamics of a panel of immunosuppressants in individual patients to select the ones that are likely to achieve the optimal inhibition of lymphocyte proliferation that would correspond to an effective reduction in the harmful immune response.

The ability to evaluate the pharmacodynamic profile of a panel of immunosuppressive drugs in individual patients is useful for 2 reasons. First, recognition of resistance to front-line immunosuppressants in vitro may lead to earlier use of 2nd-line drugs, which are frequently used only after primary drug failure. Second, the selection of another immunosuppressant has been based predominantly on anecdotal evidence or small retrospective studies, which have recognized limitations (4–9). An assay tailored to recognize the immune response to a panel of immunosuppressive drugs in vitro will provide scientific evidence that may guide a logical choice for therapy. The current assay focused on evaluation of T-lymphocyte proliferation as a method of assessing response to immunosuppressive drugs. Although T-lymphocytes play a crucial role in immunemediated diseases through cytokine production and activation of B-lymphocytes, the mechanisms of action of the drugs studied are different, and it is possible that the immune response of interest is different for each drug. Additionally, the immune response to these agents may vary between individual patients and between diseases. Studies need to be done to determine if lymphocyte-proliferation assays guiding drug therapy such as the one described here will correlate with clinical response.

Most protocols for the treatment of immune-mediated diseases in dogs use glucocorticoids in sole or front-line therapy. However, in clinical practice it is not uncommon for monotherapy with glucocorticoids to fail to control disease in dogs. Glucocorticoid resistance has been well-documented in humans (19,31,32). Glucocorticoid treatment failure has been correlated with continued ex-vivo lymphocyte proliferation in humans with ulcerative colitis, further demonstrating glucocorticoid resistance in a population of human subjects with inflammatory disease (32). Up to 30% of healthy humans are considered steroid-resistant on the basis of decreased ex-vivo lymphocyte sensitivity (18). Glucocorticoid resistance has also been described in dogs with cancer (33,34). An in-vitro assay that assesses the effects of glucocorticoids on lymphocyte proliferation in dogs may help predict in which dogs treatment with this drug is likely to fail, which would allow earlier intervention with a different immunosuppressant.

Although this type of flow-cytometric assay appears to have promise for clinical application in pet dogs requiring immunosuppressive therapy, it has some limitations. The current assay required approximately 30 mL of whole blood, which is not a feasible volume to collect from anemic patients. However, this large volume was required for the validation studies to determine the IC50 (i.e., with 4 drugs and up to 8 concentrations for each drug), whereas a smaller volume would be needed in clinical situations. The minimum volume of blood required for a clinical assay that would just test drug concentrations at or around the IC50 has yet to be established. Another limitation was that samples were processed and put into culture within hours to facilitate proliferation and reduce cell death. Studies are needed to determine how long samples could be stored before processing, but it is possible that this assay will work optimally only for patients on site. A 3rd limitation is that the assay is time-consuming and requires 3 to 4 d of cell culture before results are obtained. Finally, the variation in IC50 between healthy dogs, as seen in this study, is a limitation when one considers using this assay in clinical practice and emphasizes the importance of optimization of the assay. Although the collected data are valuable, this is still an in-vitro assay, and in-vivo correlates are needed to determine clinical utility. Future studies should focus on pairing pharmacodynamic results with pharmacokinetic data and clinical response.

Figure 3B.

Figure 3B

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to 3E. With the in-vitro addition of increasing concentrations of 6-mercaptopurine (2.5, 100, 250, and 500 μM), the percentage of proliferating lymphocytes in the same doge decreased, in a concentration-dependent manner.

Figure 5.

Figure 5

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Percentage inhibition of T-lymphocyte proliferation by various concentrations of cyclosporine for the 5 individual dogs.

Figure 6.

Figure 6

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Percentage inhibition of T-lymphocyte proliferation by various concentrations of 6-mercaptopurine, the active metabolite of azathioprine, for the 5 individual dogs.

Acknowledgments

The authors thank the Cisco Fund for Immunologic Research, University of Missouri, Columbia, Missouri, for funding a portion of the study and Dr. Chee-Hoon Chang and Hong Liu for technical assistance.

Footnotes

This manuscript represents a portion of a thesis submitted by Dr. Nafe to the University of Missouri Department of Veterinary Biomedical Sciences as partial fulfillment of the requirements for a Master of Science degree.

Presented in abstract form at the 2012 American College of Veterinary Internal Medicine Forum, New Orleans, Louisiana, USA, May 30 – June 2, 2012.

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