Baseline JAK phosphorylation profile of peripheral blood leukocytes, studied by whole blood phosphospecific flow cytometry, is associated with 1-year treatment response in early rheumatoid arthritis

The study included 35 patients diagnosed with early RA at the Division of Rheumatology, Helsinki University Central Hospital, Helsinki, Finland, between April 2012 and April 2013. The patients fulfilled the American College of Rheumatology/European League Against Rheumatism 2010 classification criteria [11]. At study entry, the patients had received no DMARDs or oral glucocorticoids. Twenty-one healthy volunteers (62% women; mean age 47 years [SD 13 years]) with no infectious symptoms or history of autoimmune disease and derived from hospital and laboratory staff served as reference subjects. All subjects were white Finnish persons residing in the Helsinki metropolitan area. The study protocol was approved by the ethical review board of the Joint Authority for the Hospital District of Helsinki and Uusimaa, and written informed consent was obtained from each patient.

A comprehensive clinical and laboratory evaluation was undertaken at entry concomitant to blood sampling. Joints were evaluated for swelling and pain (66 and 68 joints, respectively). Patient global assessment of disease activity was recorded on a 100-mm visual analogue scale. Laboratory measurements, including white blood cell count, erythrocyte sedimentation rate (ESR), and plasma C-reactive protein (CRP) level, were logged. The 28-joint Disease Activity Score (DAS28) was calculated [12]. After a mean 12-month follow-up, the patients were reexamined. Patients with no tender or swollen joints (68 and 66 joints, respectively) and normal ESR were considered to be in remission.

At study entry, an 8-ml blood sample was taken by venipuncture from the antecubital vein into a Falcon polypropylene tube (BD Biosciences, Bedford, MA, USA) supplemented with 800 μl of pyrogen-free acid citrate dextrose solution A (ACD-A; Baxter Healthcare Ltd, Thetford, UK). Cells were prepared for flow cytometry within 3 h of blood sampling.

The following antibodies were purchased from BD Biosciences (San Jose, CA, USA): fluorescein isothiocyanate (FITC)-conjugated anti-CD14 (MφP9), anti-CD4 (SK3), and anti-CD19 (SJ25C1); phycoerythrin (PE)-conjugated anti-CD3 (SP34-2); peridinin chlorophyll protein complex (PerCP)-conjugated anti-CD3 (SK7); Alexa Fluor 647-conjugated anti-CD8 (RPA-T8) mouse anti-human monoclonal antibodies; and PE-conjugated anti-rabbit secondary antibody. Anti-phosphorylated (anti-p)-JAK1 (PhosphoDetect Tyr1022/1023) rabbit anti-human antibody was purchased from EMD Millipore (Darmstadt, Germany). The following antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): anti-pJAK2 (Tyr1007/Tyr1008), anti-pJAK3 (Tyr980), anti-pTyk2 (Tyr1054) rabbit anti-human antibodies, and PerCP-conjugated anti-rabbit secondary antibody. The amounts of the antibodies were optimized, and their compatibility was assured in preliminary examinations.

Blood leukocytes were prepared for flow cytometry using a protocol and buffers by BD Biosciences [13]. Aliquots (50 μl) of blood were deposited into 12 polystyrene tubes (BD Biosciences). Four tubes were supplemented with anti-CD14-FITC, four with anti-CD4-FITC and anti-CD8-Alexa Fluor 647, and four with anti-CD19-FITC for surface marker staining of monocytes, CD4⁺/CD8⁺ T cells, and B cells, respectively. Following a 15-minute incubation at 37 °C, leukocytes were fixed and erythrocytes lysed by adding 1× Lyse/Fix Buffer (BD Biosciences). After being pelleted, leukocytes were permeabilized and washed by incubating them twice in 1× Perm/Wash Buffer (BD Biosciences) at room temperature protected from light for 15 minutes. Cells were pelleted and resuspended in Perm/Wash Buffer. One tube each containing anti-CD14, anti-CD4 and anti-CD8, and anti-CD19 were supplemented with either anti-pJAK1, anti-pJAK2, anti-pJAK3, or anti-pTyk2. The tubes were incubated at room temperature protected from light for 1 h, and then cells were pelleted and resuspended in Perm/Wash Buffer. Tubes containing the anti-pJAK1, anti-pJAK2, or anti-pTyk2 were supplemented with PE-conjugated secondary antibody, and tubes containing anti-pJAK3 were supplemented with PerCP-conjugated secondary antibody. The anti-CD4- and anti-CD8-containing tubes, as well as the anti-CD19-containing tubes, were also supplemented with the T-cell marker antibody anti-CD3-PerCP (for anti-pJAK1, anti-pJAK2, and anti-pTyk2 tubes) or anti-CD3-PE (for anti-pJAK3 tubes). Following incubation at room temperature protected from light for 40 minutes, cells were washed in Perm/Wash Buffer and suspended in Stain Buffer (BD Biosciences). The samples were kept on ice for a maximum of 3 h until flow cytometric acquisition.

Flow cytometric data were acquired using a BD FACSCanto II flow cytometer and analyzed with BD FACSDiva software (BD Biosciences), as described previously [14]. CD3⁺CD4⁺ and CD3⁺CD8⁺ T lymphocytes, CD19⁺ B lymphocytes, and CD14⁺ monocytes were identified, and pJAK1-PE, pJAK2-PE, pJAK3-PerCP, and pTyk2-PE fluorescence intensities of the respective cell populations were determined as relative fluorescence units (RFU).

The data are presented as means with SDs and numbers of patients with percentages, and groups were compared using a permutation test or chi-square test. Exact logistic regression was used to investigate crude and adjusted relationships of the study entry variables to remission at 12 months. The 95% CIs of means were estimated by bias-corrected bootstrapping (5000 replications). pJAK1-PE, pJAK2-PE, pTyk2-PE, and pJAK3-PerCP fluorescence intensities are presented as standardized fluorescence intensities (mean 0, SD 1) of RFU. Correlation coefficients were calculated by the Spearman method using Šidák-adjusted probabilities. The Stata 14.1 statistical software package (StataCorp, College Station, TX, USA) was used for the analyses.

The study included 35 DMARD-naïve patients with recent-onset RA (Table 1). After blood sampling, 33 patients started sDMARD therapy (methotrexate, sulfasalazine, hydroxychloroquine) according to the Finnish national care guidelines [4] (Table 1). In addition, 16 patients (46%) started a course of low-dose (≤10 mg/day) oral prednisone. Intra-articular glucocorticoid injections were also applied to treat swollen joints according to the Finnish national guidelines [4].

During follow-up (mean duration 12 months, SD 3.6 months), the drug treatment was modified when appropriate, targeting remission. After follow-up, 33 patients (91%) were on sDMARDs, including 3 patients (9%) on oral prednisone. Twenty patients (57%) were in remission (no swollen or tender joints, and normal ESR). No patients were on biological DMARDs. The patients in remission started with a lower DAS28 score, lower number of tender joints, and lower patient global assessment at study entry (Table 1).

Baseline phosphorylation of levels of JAKs were compared between the patients who subsequently were in remission after 12-month follow-up and the patients who were not (Fig. 1). JAK3 phosphorylation in CD8⁺ and CD4⁺ T cells and CD19⁺ B cells was found to be higher in the patients who achieved remission than in those who did not (p values of 0.015, 0.024, and 0.038, respectively). No correlations were found between baseline JAK phosphorylation levels and plasma ESR or plasma CRP levels (data not shown).

In order to further elucidate the predictive value of JAK phosphorylation in each cell population, crude and adjusted exact logistic regression models of the baseline phosphorylation levels of JAK1, JAK2, JAK, and Tyk2 were constructed (Table 2). In the models adjusted for anti-cyclic citrullinated peptide, number of DMARDs started at entry, smoking, and white blood cell count, high phosphorylation level of JAK3 was associated with remission in all leukocyte types studied. In addition, low JAK2 phosphorylation level in CD14⁺ monocytes at baseline emerged to be associated with remission.

Tyk2 phosphorylation level was higher in patients than in healthy reference subjects in all leukocyte types studied (Table 3): CD4⁺ T cells (p = 0.0014), CD8⁺ T cells (p = 0.037), CD19⁺ B cells (p = 0.049), and CD14⁺ monocytes (p < 0.001). This indicated constitutive activation of Tyk2 in the patients’ circulating leukocytes. Also, JAK2 phosphorylation of CD14⁺ monocytes was higher in patients than in healthy reference subjects (p = 0.011) (Table 3).