Ustekinumab Improves Psoriasis without Altering T Cell Cytokine Production, Differentiation, and T Cell Receptor Repertoire Diversity

<div><p>Ustekinumab is a fully human IgG1κ monoclonal antibody targeting interleukin (IL)-12/23 p40 subunit. The role of IL-12/23-mediated pathway in the mechanism of various inflammatory disorders especially psoriasis has been well recognized. Recently the long-term efficacy and safety of ustekinumab in patients with moderate-to-severe psoriasis has been evaluated in phase 2/3 clinical trials, and the results showed no significant risk for serious adverse effects, infections, or malignancies. Ustekinumab inhibits the function of the IL-12/23 p40 subunit, and therefore it is believed that inhibition of IL-12 p40 pathway decreases IFN-γ production. The major concern for the use of ustekinumab is the possibility of increased immunosuppression due to low IFN-γ production. However, the effects of ustekinumab on CD4⁺ T cell function have not been fully investigated so far. In this study, we explored changes in cytokine production by memory CD4⁺ T cells as well as in the differentiation of naïve T cells to helper T cell (Th) 1, Th2, or Th17 cells in psoriasis patients treated with ustekinumab. The effect of the treatment on T cell receptor repertoire diversity was also evaluated. The results showed that ustekinumab improves clinical manifestation in patients with psoriasis without affecting cytokine production in memory T cells, T cell maturation, or T cell receptor repertoire diversity. Although the number of patients is limited, the present study suggests that T cell immune response remains unaffected in psoriasis patients treated with ustekinumab.</p> </div

Background of five patients and five healthy controls.

<p>The PASI score of the patients was high before ustekinumab therapy, and improved dramatically after the treatment. However, the PASI score of case 5 was increased at one month after the third therapy. WBC counts and the ratio of lymphocytes in all patients and controls were preserved during all the course of the study.</p

Differentiation of naïve CD4⁺ T cells to cytokine-producing mature cells (Th1/Th2/Th17).

<p>Representative flow cytometry data are shown. (A) The percentage of CD45RA⁻CD45RO⁺IFN-γ⁺ cells in the CD4⁺ T cell population (B) The percentage of CD45RA⁻CD45RO⁺IL-4⁺ cells in the CD4⁺ T cell population (C) The percentage of CD45RA⁻CD45RO⁺IL-17⁺ cells in the CD4⁺ T cell population. T cell maturation was not influenced by ustekinumab treatment.</p

T cell receptor repertoire diversity.

<p>TCR BV subfamilies were preserved during treatment with ustekinumab as compared with normal volunteer.</p

Naturally occurring regulatory T cells.

<p>The percentage of nTreg (FoxP3⁺CD127^lowCD25^highCD4⁺ T cells/CD4⁺ T cells) was similar among the seven volunteers. Flow cytometry data from four patients and three healthy controls are shown.</p

Cytokine production by memory CD4⁺ T cells and differentiation of naïve CD4⁺ T cells to mature cytokine-producing T cells in psoriasis patients during treatment with ustekinumab and in healthy controls.

<p>(A) Memory CD4⁺ T cells were stimulated with PMA and ionomycin. Distinct cytokine production by mature cells was observed, and the production of all cytokines was not suppressed in patients with psoriasis treated with ustekinumab. (B) Naïve CD4⁺ T cells were differentiated to mature T cells (Th1/Th2/Th17). Ustekinumab treatment did not change the percentage of cytokine-producing mature T cells compared to the control group.</p

The effects of CsA and Tac on cytokine production from memory CD4⁺ T cells.

<p>Memory CD4⁺ T cells were stimulated with PMA and ionomycin in the absence or presence of CsA or Tac. Fluorescence profiles showed distinct cytokine productions from mature cells, and the production of all cytokines investigated here was strikingly suppressed with the addition of CsA or Tac, even at the lower concentration. Representative figures of ten independent experiments are shown (A). The percentages of IFN-γ⁺CD4⁺ T cells (B), IL-4⁺CD4⁺ T cells (C) or IL-17⁺CD4⁺ T cells (D) within the CD4⁺ T cell population were significantly suppressed with the addition of CsA or Tac. Data are expressed as the mean ± SEM.</p

The effects of CsA and Tac on the differentiation of naïve CD4⁺ T cells into cytokine-producing mature cells (Th1/Th2/Th17).

<p>Flow cytometric analysis showed abundant cytokine production from CD45RA⁻CD45RO⁺CD4⁺ T cells. Representative figures of ten independent experiments are shown (A). Addition of CsA or Tac lowered the percentage of CD45RA⁻CD45RO⁺IFN-γ⁺ cells significantly compared with the control group (B). The percentages of CD45RA⁻CD45RO⁺IL-4⁺ cells and CD45RA⁻CD45RO⁺IL-17⁺ cells within the CD4⁺ T cell population were also decreased compared with the control group (C,D). Data are expressed as the mean ± SEM.</p

Changes in cytokines mRNA expression levels in the ears of AD mice by vaccination of rhPIV2/Ag85B.

<p>Cytokines: IL-4 (panel A), IFN-γ (panel B), IL-10 (panel C), TGF-β (panel D), TNF-α (panel E), MIP2-α (panel F), IL-2 (panel G), IL-17 (panel H), mRNA expression in the ear lesions measured with Quantitative RT-PCR. Expressions of IL-4, TNF-α and MIP2-α mRNA were significantly decreased in the ear skin treated with intra-nasally rhPIV2/Ag85B treated group compared to those of control groups. Meanwhile, the expression levels of mRNA of IFN-γ, IL-10, TGF-β and IL-2 were significantly elevated in rhPIV2/Ag85B intra-nasally treated group compared to those of control groups. *P<0.05, **P<0.01, ***P<0.001.</p

Expression of EGFP from rhPIV2/EGFP.

<p><b>A.</b> HaCat cells were infected with rhPIV2/EGFP at an MOI of 0.5. Three days after, EGFP was clearly visualized using a fluorescence microscopy (x100). <b>B.</b> The rhPIV2/EGFP (5×10⁶ TCID₅₀) were administered to a wild type BALB/c mice intranasally EGFP was visualized clearly in the airway epithelial cells 4 days after administration (x200, upper right box, x400).</p