Research ArticleRheumatoid Arthritis

Citrullinated peptide dendritic cell immunotherapy in HLA risk genotype–positive rheumatoid arthritis patients

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Science Translational Medicine  03 Jun 2015:
Vol. 7, Issue 290, pp. 290ra87
DOI: 10.1126/scitranslmed.aaa9301
  • Fig. 1. Concept and schema of immunomodulatory therapy with DCs and citrullinated peptides in HLA risk genotype–positive patients with RA.

    (A and B) HLA-DRB1 SE+ RA patients (A) are the target population for initial investigation of immunomodulatory therapy of modified autologous DCs (B) exposed to putative citrullinated autoantigenic epitopes (A). HLA-DR risk allotypes bind citrullinated self-peptides due to a critical lysine residue at position 71 of the peptide binding groove (A). DCs generated ex vivo from PB monocytes in the presence of the NF-κB inhibitor Bay11-7082 are exposed to citrullinated peptides and then injected intradermally (I.D.) (B). Immune response is expected in draining lymph nodes (LN) on Teff and Treg cells, with expected impact on joint symptoms through regulation of inflammation (A). (C) Schema for ex vivo production, injection, and follow-up. Each follow-up assessment included clinical and laboratory evaluation of toxicity (table S2); phone follow-up evaluated clinical evidence of toxicity only. Assessments included clinical and laboratory evaluation of RA disease activity, using DAS scores. At each assessment, PB mononuclear cells (PBMCs) and serum were collected for analysis of cell populations by flow cytometry (table S3), in vitro responses to antigens (table S4), serum analytes (table S5), anti-CCP titer, and peptide-specific ACPA (fig. S5 and table S4). Fasting insulin and glucose were measured at baseline and 1 month, and patients recorded fasting glucose measurements for the first 2 days after Rheumavax.

  • Fig. 2. Changes in Teff and Treg cells in treated and control individuals.

    (A to C) PB % CD4+CD25+CD127+ Teff (A), % CD25hiCD127CD4+ Treg cells (B), and Treg/Teff ratio (C) for individuals at baseline (month 0), 6 days, and 1 month after low- or high-dose Rheumavax, and controls at baseline and 1 month. (D) The maximum decrease in % CD4+CD25+CD127+ Teff and the maximum increase in % CD25hiCD127/CD25+CD127+CD4+ T cells (Treg/Teff) were calculated in each patient after Rheumavax relative to baseline (that is, Emax = peak value/baseline value × 100%; Emin = trough value/baseline value × 100%). Emin Teff and Emax Treg/Teff values are plotted with median and IQR. n = 9 each for low-dose (black symbols) and high-dose (green symbols) Rheumavax; n = 5 for controls.

  • Fig. 3. Changes in citrullinated antigen-specific IL-6 responses and tetanus toxoid–specific proliferative responses in treated and control individuals.

    PBMCs (2 × 105 per well) were incubated for 5 days with aggrecan84–103–Cit93, vimentin447–455–Cit450, fibrinogen α chain717–725–Cit720, and collagen type II1237–1249–Cit1240 (0 or 30 μg/ml) or with tetanus toxoid [4 Limes flocculation units (Lf)/ml]. IL-6 production and T cell proliferation were measured by cytokine bead array. The stimulation indices for peptide-stimulated/unstimulated IL-6 and T cell proliferation were calculated and are plotted for each individual at baseline (0) and 1 month. Black symbols, low dose; green symbols, high dose. (A) Citrullinated peptide–specific IL-6. (B) Tetanus toxoid–specific proliferation. n = 17 for Rheumavax; n = 10 for controls. Differences in Rheumavax-treated and control group responses over time were compared by LME for group ×time interaction. *P = 0.02.

  • Fig. 4. Changes in T cells, proinflammatory cytokines, and anti-CCP levels in patients treated with Rheumavax and controls.

    (A and B) The relationship between the change in DAS4v at 1 month after Rheumavax with the changes in Teff at day 6 (A) and Emax Treg/Teff (B) are plotted, showing regression lines. (C) Serum levels of anti-CCP antibodies were determined using the CCP3.1 enzyme-linked immunosorbent assay (ELISA) assay. The relationship between change in DAS4v at 1 month after Rheumavax with the change in anti-CCP level at day 6 after Rheumavax is plotted, showing regression line. P and R2 values are shown in Table 3. (D) Change in C-reactive protein (CRP) at 1 month plotted relative to DC dose/kg. The regression line is shown (r2 = 0.37). Black symbols, low dose; green symbols, high dose. (E) Serum levels of IL-15, CXCL11, CX3CL1, IL-29, and PYY were determined by luminex assay at baseline, day 6 (d6), and 1 month after Rheumavax (closed circles) and at a 1-month interval in controls (open circles). Log-transformed values are plotted as median and IQR. n = 9 for controls; n = 18 for Rheumavax. Differences at 1 month were estimated with the Mann-Whitney test (*P = 0.046, IL-15; P = 0.028, CXCL11; P = 0.024, CX3CR1; P = 0.045, IL-29; P = 0.027, PYY).

  • Fig. 5. Clinical effect of Rheumavax.

    (A) Disease activity score (DAS4v) at baseline and 1 month after treatment in RA patients treated with low and high doses of DCs and in controls. Patients with active disease (SJC ≥ 1) and inactive disease (SJC = 0) are denoted by the dashed and solid lines, respectively. (B) SJC at baseline and 1 month after treatment in RA patients in low- and high-dose DC groups and in controls. (C) Change in DAS relative to baseline for 6 months plotted in active and inactive groups and controls. Grouped data are displayed as median and IQR. n = 9 for Rheumavax groups; n = 11 for control group. Groups were compared at each time point using the Kruskal-Wallis test with Dunn’s multiple comparison test: **P = 0.005, Rheumavax active versus inactive; P = 0.033, Rheumavax active versus control; *P = 0.013, active versus inactive; P = 0.040, inactive versus control. Control data not available at 2 months.

  • Table 1. Demographic details of treated and control patients.

    DAS, disease activity score; RF, rheumatoid factor; MTX, methotrexate; SSZ, sulfasalazine; HCQ, hydroxychloroquine; LEF, leflunomide; TNFi, tumor necrosis factor inhibitor; N/A, not applicable.

    Rheumavax Low
    dose (n = 9)
    Rheumavax High
    dose (n = 9)
    Control
    Group 1 (n = 11)
    Control
    Group 2 (n = 5)
    Age, mean (SD)56.8 (9)55.1 (10.1)57.6 (9.8)49.8 (10.8)
    Females, n (%)5 (55)8 (89)6 (54)4 (88)
    Disease duration (years),
    median (IQR)
    3 (1.5–4)2 (1–4)1 (1–2)2 (1–11)
    Baseline DAS,
    median (IQR)
    2.43 (1.54–3.81)2.2 (1.56–3.3)2.81 (2.1–3.63)3.29 (2.75–3.83)
    RF+, n (%)7 (78)9 (100)10 (91)4 (88)
    Anti-CCP+, n (%)9 (100)9 (100)11 (100)5 (100)
    HLA-DR SE+, n (%)9 (100)9 (100)11 (100)5 (100)
    Other treatment, n (%)
      MTX7 (78)9 (100)7 (64)5 (100)
      SSZ2 (22)6 (67)5 (45)5 (100)
      HCQ5 (56)6 (67)8 (73)4 (88)
      LEF0 (0)0 (0)2 (18)1 (20)
    TNFi2 (22)0 (0)0 (0)0 (0)
    Rheumavax
      Dose/kg (range)7.2 × 103 to 1.7 × 1042.7 × 104 to 6.2 × 104N/AN/A
      Adverse events, n (%)4 (44)5 (56)N/AN/A
  • Table 2. Clinically relevant adverse events (treatment and possibly treatment-related).

    ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase.

    Systemic class
    of adverse event
    Adverse eventNumber of
    patients affected
    Dose group (1 = low dose,
    2 = high dose) and time
    of adverse events
    after Rheumavax
    Blood/bone marrowLymphopenia (0.5 × 109 to
    0.9 × 109/liter)
    31 (day 30), 2 (day 30),
    2 (day 90)
    Leukopenia (3.9 × 109 to 3.0 × 109/liter)31 (day 30), 2 (day 60), 2 (day 60)
    Neutropenia (1.9 × 109 to 1.5 × 109/liter)22 (day 30), 2 (day 60)
    Hemoglobin (100–134 g/liter)32 (days 30, 60, 90), 2 (day 90),
    2 (days 30, 60, 90)
    Metabolic/laboratoryGlucose (2–2.9 mM)12 (day1)
    HepaticALP (120–300 U/liter)11 (day 180)
    AST (36–86 U/liter)21 (day 90), 1 (day 90)
    HepaticBilirubin (19–35 mM)11 (day 180)
    ALT (46–112 U/liter)21 (day 180)
    MusculoskeletalHeadache12 (day 90)
  • Table 3. Linear regression analysis of change in DAS at day 30 on laboratory features.

    Features with P ≤ 0.05 (t test) are shown in the table. The intercepts are not shown. % Foxp3+ Treg is the % of the CD4+CD25hiCD127lo Treg cells expressing Foxp3. Emax = peak value/baseline value × 100%; Emin = trough value/baseline value × 100%. FDR, false discovery rate (calculated using Benjamini-Hochberg correction).

    FeaturesEstimatePFDRR2
    % Teff, day 60.780.0040.0560.41
    % Foxp3+ Treg, day 6−0.650.0090.140.36
    % CD16hiCD14+ monocytes, day 30−0.660.020.420.35
    Emin Teff0.730.0050.0640.37
    Emax Treg/Teff−0.710.0010.0090.57
    Anti-CCP IgA/IgG titer, day 67.420.020.0460.42
    Anti-CCP IgA/IgG titer, day 301.730.0050.0460.39

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/290/290ra87/DC1

    Fig. S1. Flowchart of the study.

    Fig. S2. Gating strategy for CD4+ Teff and Treg cells.

    Fig. S3. Gating strategy for B cell/T cell/Tfh and NKT/DC/monocyte/NK panels shown in table S3.

    Fig. S4. Effects of Rheumavax on disease activity, Teff and Treg cells, tetanus toxoid immunity, and anti-CCP titer in each treated patient.

    Fig. S5. Proportion of each group with citrullinated peptide–specific ACPA for the duration of the study.

    Fig. S6. Ratio of HLA-DR and CD40 expression by modified DC relative to DC before administration.

    Fig. S7. Frequency of anchor residues from naturally eluted peptides from HLA-DRB1*01:01, DRB1*04:01, and DRB1*04:04.

    Fig. S8. Generation of monocyte-derived DCs from RA PB in the presence of increasing concentrations of Bay11-7082 reduces HLA-DR expression and the capacity to stimulate allogeneic T cells.

    Table S1. Characteristics, clinical outcomes, and treatment side effects for the 18 study subjects.

    Table S2. Rheumavax safety toxicity criteria.

    Table S3. Flow cytometry panels used in the analysis of PBMCs.

    Table S4. Peptides used in this study.

    Table S5. Serum analytes measured in this study.

    Table S6. LME model of serum analytes.

    Source data (excel file)

  • Supplementary Material for:

    Citrullinated peptide dendritic cell immunotherapy in HLA risk genotype–positive rheumatoid arthritis patients

    Helen Benham, Hendrik J. Nel, Soi Cheng Law, Ahmed M. Mehdi, Shayna Street, Nishta Ramnoruth, Helen Pahau, Bernett T. Lee, Jennifer Ng, Marion E. G. Brunck, Claire Hyde, Leendert A. Trouw, Nadine L. Dudek, Anthony W. Purcell, Brendan J. O'Sullivan, John E. Connolly, Sanjoy K. Paul, Kim-Anh Lê Cao, Ranjeny Thomas*

    *Corresponding author. E-mail: ranjeny.thomas{at}uq.edu.au

    Published 3 June 2015, Sci. Transl. Med. 7, 290ra87 (2015)
    DOI: 10.1126/scitranslmed.aaa9301

    This PDF file includes:

    • Fig. S1. Flowchart of the study.
    • Fig. S2. Gating strategy for CD4+ Teff and Treg cells.
    • Fig. S3. Gating strategy for B cell/T cell/Tfh and NKT/DC/monocyte/NK panels shown in table S3.
    • Fig. S4. Effects of Rheumavax on disease activity, Teff and Treg cells, tetanus toxoid immunity, and anti-CCP titer in each treated patient.
    • Fig. S5. Proportion of each group with citrullinated peptide–specific ACPA for the duration of the study.
    • Fig. S6. Ratio of HLA-DR and CD40 expression by modified DC relative to DC before administration.
    • Fig. S7. Frequency of anchor residues from naturally eluted peptides from HLADRB1* 01:01, DRB1*04:01, and DRB1*04:04.
    • Fig. S8. Generation of monocyte-derived DCs from RA PB in the presence of increasing concentrations of Bay11-7082 reduces HLA-DR expression and the capacity to stimulate allogeneic T cells.
    • Table S1. Characteristics, clinical outcomes, and treatment side effects for the 18 study subjects.
    • Table S2. Rheumavax safety toxicity criteria.
    • Table S3. Flow cytometry panels used in the analysis of PBMCs.
    • Table S4. Peptides used in this study.
    • Table S5. Serum analytes measured in this study.
    • Table S6. LME model of serum analytes.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Source data (excel file)

    [Download Source Data File]

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