Supplementary Data 1
Patient. The patient had no family history of ankylosing spondylitis. At the age of 20 (1984), he presented with polyarthritis and dactylitis. Later he complained of painful talocrural joint movement, pain and stiffness in the thoracic spine, and limited motion of the lumbar spine and was diagnosed with AS. During 2000-2005, he suffered from pain and stiffness of the cervical spine and recurrent uveitis symptoms. The patient had been treated with non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and celecoxib. Despite this, the Bath AS disease activity index (BASDAI) score and functional index (BASFI) score were >5.0 and >6.0, respectively. In 2005, radiographic examination of the pelvis and lumbar spine showed bilateral grade III-IV sacroiliitis and single syndesmophytes in the low thoracic spine. Bone scintigraphy showed increased uptake in the hip joints and spine. Due to high disease activity despite adequate treatment with NSAIDs, the patient started to receive monthly infusions of 400 mg infliximab (Remicade). Significant functional improvement was observed after initiating anti-TNF-alpha therapy in 2005; however, after 3 years of infliximab therapy, the remission periods shortened, and the patient showed clinical reactivation of AS. There was a complete absence of relief and acute relapse soon after the last infliximab injection in March, 2008. In autumn 2008, a single high dose of immunomodulator Licopid was administered, with subsequent blocking of induced inflammation by Humira that was further administered during the next six months. Licopid treatment did not result in any significant improvement (1) and Humira efficiency was moderate.
HSCT. Informed consent was obtained from the patient for these procedures. The study was approved by the local ethical committee. Stem cells were mobilized with G-CSF (10 mg/kg.b.wt.). Autologous stem cell aphaeresis was performed on a “Haemonetics MCS” instrument. No CD34+-positive selection was performed. The patient received 200 mg/kg of cyclophosphamide for 4 days with autologous blood stem cells for rescue and infusion of antithymocyte globulin (ATG) for in vivo T cell depletion. On day 0 (June 6, 2009), the patient received 2.4 x 106 kg.b.wt. of CD34+ stem cells. This number was determined by counting total number of cells and by FACS analysis of an aliquot of cells using a CD34-specific antibody. No life-threatening events occurred during transplantation. Post-transplant toxicity included neutropenia with a neutrophil count < 0.5×109/L (from day +2 to day + 9); thrombocytopenia, with a thrombocyte count < 50×109/L (from day +2 to day + 12); fever (from day +4 to day +5); and stomatitis.
Flow cytometry. PBMCs were isolated from the peripheral blood samples by Ficoll-Paque (Paneco, Russia) density-gradient centrifugation. Cells were washed twice with phosphate-buffered saline (PBS) and resuspended to a density of approximately 106 cells/ml. Cells were incubated with antibodies or MHC tetramer for 20 min at room temperature or at +8°C and washed twice with PBS. Phenotypic cell analysis was performed using a Cytomics FC 500 (Beckman Coulter) flow cytometer. Data were analyzed using the program Cytomics RXP Analysis (Beckman Coulter).
Antibodies. Cells were stained with the mouse anti-human antibodies from Invitrogen (USA): CD3-PC5 (clone UCHT1), and CD4-PC5 (clone S3.5), and from Beckman Coulter (USA): CD45RA-FITC (clone 2H4LDH11LDB9), CD27-PC5 (clone 1A4CD27), CD8-PC7 (clone SFCI21Thy2D3), and antibody mixtures: CD3-FITC/CD4-PE, CD3-FITC/CD16/56-PE, CD3-FITC/CD8-PE, CD3-FITC/CD19-PE.
Estimation of relative abundance of the NLV-specific T cells. PBMCs were incubated with a CD8-specific antibody and an HLA-A2 MHC tetramer loaded with the immunodominant cytomegalovirus (CMV) peptide NLVPMVATV for 20 min at room temperature in Ca2+- and Mg2+-free PBS /1 mM EDTA/1% BSA with added DNase I (Promega, USA) and analyzed by flow cytometry.
Reconstitution of the immune system after HSCT. Samples of the peripheral blood were collected before and at several time points after HSCT and were analyzed by blood count and flow cytometry. We examined the reconstitution of different lymphocyte populations in the peripheral blood, including B cells (defined as CD19+); NK cells (CD3-/CD16+ and/or CD56+); CD4 T cells (CD3+/CD4+); CD8 T cells (CD3+/CD8+); and naïve (CD27+/CD45RAhigh), activated effector-memory (CD45RA-CD27-) and effector (CD45RA+CD27-) CD4 and CD8 T cells. The results of the 2-year analysis are summarized in Supplementary Fig. 1B, C and Supplementary Table 1. The kinetics of the repopulation of the CD4 and CD8 T cells differed significantly. Relatively rapid expansion of the CD8 population and slow reconstitution of the CD4 pool were observed during the first 3-month post-graft period. The CD4/CD8 ratio dropped from 3:1 to 1:2 at 3 months after HSCT. At 24 months after HSCT, the CD4 and CD8 T cell populations equalized. Notably, the number of B cells declined approximately 5-fold at two weeks after treatment. The total numbers of T, B and NK cells reached the pre-graft levels at 8 months post-HSCT. The absolute counts of both CD4 and CD8 naïve T cells, defined as CD27+CD45RAhigh, dropped after HSCT 12-fold and 7-fold, respectively. In the next two years, both pools were gradually restored (Supplementary Fig. 1C). During the 2 years of observation after HSCT, the CD8 population consisted of >50% activated effector-memory (CD45RA-CD27-) and effector (CD45RA+CD27-) T cells (2). It was previously reported that the expansion of CD8+CD27- cells strongly correlates with CMV-positive status, especially the expansion of the CD45RA+CD27- subset (3, 4). In our study, flow cytometry analysis of T cells stained with an MHC tetramer loaded with the immunodominant CMV peptide NLVPMVATV (NLV) revealed pronounced expansion of NLV-specific T cells 4 months after HSCT. Similar changes were observed in the CD4 T cell population. The percentage of the CD4+CD27- T cells among all CD4 T cells increased 4-fold and the absolute counts increased 2-fold after HSCT. The absolute counts of CD4 T cells with the phenotype CD4+CD45RA+CD27-, representing terminally differentiated effector cells that have been reported to be mostly specific for CMV in CMV-positive donors (5), were increased by more than 20-fold (Supplementary Table 1).
References:
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