Long-term immune reconstitution and T cell repertoire analysis after autologous hematopoietic stem cell transplantation in systemic sclerosis patients

We selected 10 SSc patients treated with aHSCT at Saint-Louis Hospital (Paris, France) and for whom repeated simultaneous clinical and immunological monitoring had been obtained until long-term, at least 6 years, after HSCT. All patients gave a written informed consent. They had received CD34+-selected aHSCT, without or with rabbit antithymocyte globulin (rATG, Genzyme) as part of the previously published ISAMAIR [ 3] or ASTIS [ 6] protocols approved by the ethics committee. As a control group, 18 healthy donors were tested to determine the reference values of TCR diversity.

Transplant procedure was previously described [ 3, 6]. In brief, mobilization and collection of peripheral blood hematopoietic stem cells (PBSC) using cyclophosphamide at 2 g/m ²/day on two consecutive days followed 4 days later by rHu G-CSF (Lenograstim®, Aventis and Chugai Pharma France) at 5 μg/kg/day subcutaneously until the last apheresis. PBSC were collected when CD34+ cells were above 20/μL in peripheral venous blood and CD34+ cells were selected using immunomagnetic bead technique (Nexel Isolex®300i Stem Cell Collection System). Conditioning was performed at least 4 weeks later, using cyclophosphamide at 50 mg/kg/day from day 5 to day 2 prior to CD34+-selected HSC reinjection without or with rATG. All patients received rHu G-CSF after the graft until neutrophil recovery.

Clinical follow-up was performed as previously described during the first year after aHSCT [ 8] and thereafter at yearly intervals plus or minus 6 months. Clinical response after aHSCT was assessed by the same observer using repeated functional and physical examination of organ involvement. Relapse was defined by any of the following criteria [ 15]: an increase of skin score by 25% from best improvement, as assessed by modified Rodnan skin score (mRSS), or a decline in forced vital capacity (FVC, %) by 10%, renal crisis, start of total parenteral nutrition, or restarting of immune suppressive or modulating medication. According to the clinical response at long-term, SSc patients were retrospectively classified as long-term responders (group A) and non-responders or relapse or necessitating immunosuppression (group B) within the first 6 years post-aHSCT. We analyzed their IR profiles and evolution of TCR repertoire according to the clinical outcome after aHSCT.

At time of each clinical evaluation before and after AHSCT, blood and serum samples were drawn for IR analysis. Prospective lymphocyte immunophenotyping was performed on freshly collected whole blood samples, using a FACS Canto II flow cytometer and FACS DIVA software (BD Biosciences, le Pont de Claix, France). Absolute counts were determined using the TruCount system (BD Biosciences) with BD Multitest (BD Biosciences). Eight color labeling was performed with the following mAbs: CD3, CD4, CD8, CD16, CD56, CD45RA, CD45RO, and CD19. Data were analyzed using FACS Diva (BD Biosciences). Anti-Scl-70 antibody level detection was performed by enzyme-linked immunosorbent assay as previously described [ 8] and after the year 2010 by BioPlex ANA Screen (Bio-Rad, Hercules, CA).

Determination of TCR-Vβ usage was made from total RNA using quantitative “Immunoscope” [ 8]. Briefly, RNA was purified using TriReagent (Molecular Research Center, Cincinnati, OH). Synthesis of complementary DNA (cDNA), complementarity determining region 3 (CDR3) gene amplifications, run-off using an internal β-chain gene constant fluorescent primer, gel running, and Immunoscope software analysis were performed [ 8]. The definition of Immunoscope profiles as polyclonal, skewed, or negative was based on the identification of peaks that deviate from the normal distribution curve [ 8].

All results are shown as mean ± standard deviation (SD). Significant differences ( P ≤ 0.05) between patient groups related to individual conditions were assessed with Mann-Whitney test and ANCOVA for general linear regression analysis, using SPSS software. To reduce intra-group variability induced by individual differences at entry, we used both mean absolutes values and percentages of the total number of cells at inclusion that remained at each time follow-up post-aHSCT for IR analysis, and global trends were expressed using linear regression slope (LRS) [ 8].

Ten severe SSc patients, five males, mean age of 37.4 ± 13.9 years, were included in the study. According to the observed clinical response during an overall mean follow-up of 7.8 ± 0.8 years, five patients were long-term responders (group A) and the five others (group B) had either no response or clinical relapse or necessitated immunosuppression post-aHSCT. All were severe dcSSc patients with similar functional status and organ involvement before aHSCT. There was no clinical difference between groups A ( n = 5) and B ( n = 5) before transplant, including for mRSS (28 ± 5.7 vs 31 ± 15.2, ns) and FVC% (80 ± 22.2 vs 61 ± 8.8, ns) (Table  1). During the month prior to transplant (baseline), patients received no treatment ( n = 6) or low dose oral steroids below or equal to 10 mg/day ( n = 4). The individual patients’ graft characteristics and engraftment duration after CD34+ aHSCT without ( n = 4) or with rATG ( n = 6) are in Table  1. The number of reinjected CD34+ cells was higher in group B versus A (4.1 ± 1.3 vs 6.9 ± 2.0, P = 0.02), while time to hematopoietic platelets and neutrophils reconstitution were similar in both groups. After transplant, patients received either no treatment up to long-term follow-up ( n = 5, group A) or mycophenolate mofetil 1–2 g/day ( n = 5, group B). Low-dose oral steroids below or equal to 10 mg/day were given in three out of five group A and B patients. Compared to pretransplant mRSS values (29 ± 11.0, n = 10), all SSc patients had a significant decrease in skin score at 1 year (19 ± 11.1, P = 0.029) which was sustained until 6 years (8 ± 9.3, P = 0.006, Fig.  1a). The significant regression of skin score, with a fall in mRSS greater than 25% compared to pretransplant values, was present in both groups throughout all follow-up (Fig.  1b) and was significantly more pronounced at long-term in group A than in group B patients ( P = 0.004, Fig.  1b). Compared to pretransplant FVC values (in % of normal, Fig.  1c), there was no overall change in the lung function post-aHSCT when considering all 10 SSc patients. However, the relative FVC% changes compare to pre-transplant values differed significantly between the two groups during follow-up ( P = 0.040, ANCOVA), and at long-term, significant improvement of FVC above 10% compared to pretransplant values was observed in group A ( P = 0.026, Fig.  1d).

At inclusion, the absolute values of T cell subsets, CD4+, CD4+CD45RA+, CD8+ T cells, and number of NK-cells were in normal ranges for all patients and remained stable after aHSCT with no difference between groups A and B (Table  2). The absolute number of B cells was lower in group A than in healthy donors ( P < 0.05) and was comparable to controls in group B (Table  2). Due to lymphocyte counts variations between patients, we expressed data as percentage of cells compared to numbers before transplant. After aHSCT, there was an overall trend toward increased CD3+ (LRS = 16.8) and CD3+CD4+ (LRS = 12.5) T cell counts in group B compared to group A (LRS = 8.7 and 9.7, respectively) (Fig.  2a, b). A rapid increase in CD3+CD8+ T cell counts starting 2–3 years post-transplant was observed in group B (LRS = 21.8) patients although under immunosuppressive drugs, while they remained almost stable in group A (LRS = 5.0, Fig.  2c). The relative increase in the number of CD4+CD45RA+ was higher in group B (LRS = 12.9) than in group A (LRS = 2.6) patients (Fig.  2d). Memory CD4+CD45RO+ T cell IR profiles were similar in groups A (LRS = 7.2) and B (LRS = 10.5, Fig.  2e). The percentage of B cell change differed with a sustained positive slope in group A (LRS = 11.1), while the slope remained negative in group B (LRS = −11.6, Fig.  2f). Four out of five group A and three out of four group B patients seropositive for anti-Scl-70 before aHSCT became negative after (Table 3). There was no correlation between B cell counts and the presence of high levels of Scl-70 autoantibody (data not shown).

TCR-Vβ family quantifications showed the same relative usage of each family in all the 10 SSc patients before and at long-term after transplantation as observed in healthy subjects (Fig.  3a). As we [ 8] and others [ 16] previously reported in SSc patients, the TCR-Vβ family usage did not differ from age-matched controls. CDR3 size distribution was very disturbed with few polyclonal TCR-Vβ families and overexpression of skewed and/or negative families (data not shown). At long-term, distinct changes in repertoire compared to pretransplant TCR-Vβ profiles were observed (Fig.  3b). Two profiles were noted, with either the recovery of a polyclonal profile—similar to healthy individuals—as opposed to a skewed and disturbed repertoire before transplant or the persistence of disturbed profile with still oligoclonaly expanded TCR-Vβ families (Fig.  3b). Overall, T cell diversity improved in almost all long-term patients compared to baseline, and the percentage of polyclonal TCR-Vβ families increased significantly ( P = 0.046) with no significant difference between groups A and B (Fig.  3c).