Chemokine receptors CXCR3 and CCR4 mediate corneal CD4⁺ T cell homing during herpes simplex virus-1 keratitis

Abstract

Purpose : Herpes simplex virus (HSV)-1 keratitis is a leading cause of corneal blindness. The profound role of effector CD4⁺ T cells in the pathogenesis of this disease has been confirmed. The chemokine receptors CCR4, CCR5 & CXCR3, are expressed in primed Th1 & Th17 effector T cells. This study aimed to examine the chemokine receptors involved corneal CD4⁺ T cell homing during HSV-1 keratitis.

Methods : Corneas from wild-type (WT) C57BL/6 mice were scarified with a 25-gauge needle and inoculated with 1X10⁴ PFU of McKrae HSV-1. CD4⁺ T cells were harvested from spleens and draining lymph nodes, stained for PEcy7 and sorting by flow cytometry at 8 days post infection (dpi). Competitive homing of CD4⁺ T cells to the HSV-infected cornea was evaluated in 8dpi 8-week old C57BL/6 hosts (n=6/group). Calcein green- and red-stained CD4⁺ T cells homing to the infected cornea were first examined at 8, 16, 24 & 30 hours post-adoptive transfer, to assess the timepoint yielding the greatest T cell density. Infected host mice received 1x10⁶ calcein red-stained donor CD4⁺ T cells that were blocked by antibodies/antagonist to CXCR3, CCR4 or CCR5, and 1x10⁶ calcein green-stained CD4⁺ T cells incubated with respective IgG controls or treated with diluent of antagonists. Stained CD4⁺ T cells were systemically delivered via intravenous injection. Mice were anesthetized and excised corneas were imaged with multi-photon microscopy, and the number of labelled CD4⁺ T cells homed to the cornea were compared.

Results : The greatest density of calcein-green (13.27±4.08cells/mm²) and red CD4⁺ T cells (11.22±4.08) in the cornea was observed at 16 h post-adoptive transfer (P=0.01). The blockade of CXCR3 (1.92±3.70 cells/mm²) & CCR4 (0.60±1.54 cells/mm²) in CD4⁺ T-cells significantly decreased their homing to HSV-1 infected corneas as compared to isotype control-treated CD4⁺ T cells (11.06±7.71 and 6.81±4.33 cells/mm², respectively) (both P<0.001). In contrast CCR5 blockade resulted in decreased the CD4⁺ T cell homing, but did not reach significance (2.56±4.73 vs 6.57±7.25 cells/mm², P=0.06).

Conclusions : The chemokine receptors CXCR3 and CCR4 mediate CD4⁺ T cell homing durung HSV-1 keratitis. Blockade of cornea specific chemokine receptors may inhibit CD4⁺ T cell homing and allow for decreased severity of HSV-1 keratitis.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.