Supplementary Information

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1 Supplementary Information Methods Lymphocyte subsets analysis was performed on samples of 7 subjects by flow cytometry on blood samples collected in ethylenediaminetetraacetic acid (EDTA)-containing tubes and processed immediately (subjects 4, 5, 6, 8, 10, 11, and 13). 100 L of whole blood was incubated for 30 min at 4 C with pre-titrated saturating dilutions of the following fluorochromelabeled monoclonal antibodies (BD Biosciences, San Jose, CA; clone designated in parenthesis): CD3 (SK7), CD4 (RPA-T4), CD8 (RPA-T8), CD45 (Hi30), CD45RA (HI100) and CCR7 (150503; R&D systems, Minneapolis, MN). After lysis of red blood cells with ACK (Ammonium-Chloride-Potassium) lysing buffer samples were washed twice in staining buffer (PBS supplemented with 0.5% bovine serum albumin and 2 mm EDTA). Intracellular staining for FOXP3 (PCH101) was performed using the FOXP3 kit (FOXP3-phycoerythrin, ebioscience, San Diego, CA) according to the manufacturer s instructions. Dead cells were excluded from these assays by staining with 2 g/ml 7-amino-actinomycin D (Molecular Probes, Eugene, OR) for 5 min prior to analysis. Appropriate isotype-matched negative controls were used to assess background fluorescence intensity. Absolute cell counts were performed using a lyse/no-wash protocol with SPHERO AccuCount fluorospheres (Spherotech Inc) as previously described. 1 Samples were analyzed with a BD Biosciences FACScan flow cytometer modified with a second, 633-nm laser and data were analyzed using FlowJo software (TreeStar, Ashland, OR). Statistical comparisons of flow cytometry data from subjects with cgvhd and normal controls were performed using an unpaired Student t-test (GraphPad Prism). P-values 0.05 were considered significant. Results Samples were collected from 7 subjects at various time-points (0, 3, 6, 9, 12 mo) after starting pomalidomide. Although there were no consistent alterations in total CD45+ cells and Tregs, numbers of CD4+ and CD8+ T-cells decreased in 5 of 6 subjects (Figure S1A). We also observed an increased relative percent of Tregs within the CD3+CD4+ T-cell subset in 5 of 7 subjects studied (Figure S1B). To better characterize T-cells, we assessed relative percents of naïve, memory and effector cell subsets by evaluating CD45RA expression combined with CCR7. Within the CD4+ T-cell subset, 5 of 7 subjects had a decrease in the relative percent of naïve cells with a concomitant increase in the frequencies of central and effector memory cells during pomalidomide therapy (Figure S1B). Four of seven subjects had a relative decrease in the naïve CD8+ T-cell population with an increase in the effector memory and/or effector cell subsets (Figure S1C).

2 Discussion The immune-modulatory effects of pomalidomide could improve or worsen cgvhd. Although we cannot make definitive conclusions based on our few subjects, our data suggest that pomalidomide decreases the number of CD4+ and CD8+ T-cells in cgvhd patients. This T-cell depletion was associated with a trend toward increased relative percents (but not absolute numbers) of Tregs and memory CD4+ T-cells with a concomitant decrease in naïve CD4+ T cells during pomalidomide therapy. Similar alterations in the relative percent of CD4+ T-cells are reported following lenalidomide therapy. 2, 3 Although studies of the effects of pomalidomide on CD8+ T-cells showed no consistent trends, lenalidomide was shown to significantly increase the relative percent of CD8+ memory T-cells. 2, 3 Recent studies report pomalidomide exerts at least some of its immune-modulatory effects by binding cereblon and promoting degradation of the transcription factors Ikaros and Aiolos. 4, 5 Because the Ikaros family of zinc-finger transcription factors plays a central role in T-cell fate decisions, 3 it will be interesting to determine if a similar mechanism is responsible for pomalidomide-induced alterations in mature naïve, memory and effector T-cell subsets. In this small study, and with limited samples collected, it is impossible to make associations between observed laboratory changes and response to pomalidomide. References 1. Uy GL, Rettig MP, Motabi IH, McFarland K, Trinkaus KM, Hladnik LM et al. A phase 1/2 study of chemosensitization with the CXCR4 antagonist plerixafor in relapsed or refractory acute myeloid leukemia. Blood 2012; 119(17): doi: /blood Gandhi AK, Shi T, Li M, Jungnelius U, Romano A, Tabernero J et al. Immunomodulatory effects in a phase II study of lenalidomide combined with cetuximab in refractory KRAS-mutant metastatic colorectal cancer patients. PLoS One 2013; 8(11): e doi: /journal.pone Thomas RM, Chunder N, Chen C, Umetsu SE, Winandy S, Wells AD. Ikaros enforces the costimulatory requirement for IL2 gene expression and is required for anergy induction in CD4+ T lymphocytes. J Immunol 2007; 179(11): Gandhi AK, Kang J, Havens CG, Conklin T, Ning Y, Wu L et al. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.). Br J Haematol 2014; 164(6): doi: /bjh.12708

3 5. Lopez-Girona A, Mendy D, Ito T, Miller K, Gandhi AK, Kang J et al. Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide. Leukemia 2012; 26(11): e-pub ahead of print 2012/05/04; doi: /leu

4 Tables Supplementary Table 1. N subjects with a NIH organ-specific scores at study-entry Organ Systems Score 0 Score 1 Score 2 Score 3 Skin Mouth Eyes GI Tract Liver Lungs Joint and Fascia GYN (7 women) GYN, gynecological; GI, gastrointestinal. Supplementary Table 2. Response to pomalidomide Organ-system CR PR Skin erythema 2 3 Skin movable sclerosis 0 1 Mouth 2 0 Eyes 1 1 GI 2 0 Complete overall response Partial overall response Stable disease Progression CR, complete response; PR, partial response; GI, gastrointestinal.

5 Figures Supplemental Figure 1. Peripheral blood T cell subsets before and after pomalidomide treatment. (A) Effect of pomalidomide on peripheral blood cell subsets. Serial measurements of peripheral blood cell subset numbers were determined for 7 patients who were treated with at least 3 cycles of pomalidomide by flow cytometry. Aliquots of peripheral blood were stained with monoclonal antibodies specific for total CD45+ leukocytes, CD3+ T cells, CD3+CD4+ T cells, CD3+CD8+ T cells and CD3+CD4+FOXP3+ Tregs. (B-C) Relative frequencies of CD4+ and CD8+ T cell subsets before and after pomalidomide treatment. Serial measurements of the relative frequencies of peripheral blood CD4+ (B) or CD8+ (C) T cells that were naïve, central memory, effector memory, terminal effector, or FoxP3+ Treg cells in 7 cgvhd patients who were treated with at least 3 cycles of pomalidomide. The frequency of each subset is represented as a percentage of the total CD4+ or CD8+ T cells.