title: Characterisation of the clinical and activated T cell response to repeat delayed-type hypersensitivity skin challenges in human subjects, with KLH and PPD, as a potential model to test T cell-targeted therapies.
abstract: OBJECTIVE
  To characterise the delayed-type hypersensitivity (DTH) skin reaction to repeated challenges of keyhole limpet hemocyanin (KLH) and tuberculin purified protein derivative (PPD) in healthy volunteers, as a potential model to test T cell-targeted investigational agents.
  Forty-nine subjects received either KLH, PPD, or PBS repeat skin challenges, and clinical assessments including induration, erythema and Laser Doppler Imaging. Skin biopsies or suction blisters were taken after challenge to investigate the cellular infiltrate of the challenge site, the T cell activation status, as determined by LAG-3 expression, and, specifically for the blister, the concentrations of inflammatory cytokines. Point estimates, estimates of variation and corresponding 95% confidence intervals were constructed for each type of challenge and timepoint.
  
  
  RESULTS
  The DTH response could be measured at 48 and 120 h post-KLH and PPD challenge with induration, erythema and Laser Doppler Imaging, with 48 h post-challenge demonstrating the peak of the response. PPD was well tolerated in subjects after multiple challenges, however, a significant number of KLH-treated subjects demonstrated an injection site reaction 6-7 days following the SC injection. PPD demonstrated a boost effect on the second challenge as measured by increased induration, where as this was not noted consistently for KLH. Compared to unchallenged and PBS control-injected skin, increased T cell numbers were detected in the challenge site by both the skin suction blister and biopsy technique, at either time point following KLH or PPD challenge. Use of the T cell activation marker LAG-3 demonstrated the activated phenotype of these cells. In skin blisters, higher numbers of LAG-3+ T cells were detected at 48 h post-challenge, whereas in the biopsies, similar numbers of LAG-3+ cells were observed at both 48 and 120 h. Analysis of blister T cell subpopulations revealed some differences in phenotypes between the time points and between the CD4 and CD8 T cells. Blister cytokine analysis revealed a pro-inflammatory dominated signature in PPD-challenged skin.
  
  
  CONCLUSIONS
  In summary, our data support the use of a repeat KLH and PPD DTH challenge in clinical trials and that the clinical measures of induration and to a lesser extent erythema are appropriate to monitor the clinical DTH response. Both the blister and biopsy can be utilised to assess and quantify activated T cells and at the dose used, PPD was better tolerated than KLH and hence may be optimal for future studies.
authors.affiliation: GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK. alex.s.belson@gsk.com.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Belson
authors.affiliation: GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK.
authors.forename: Tim
authors.initials: T
authors.lastname: Schmidt
authors.affiliation: Clinical Unit Cambridge, Addenbrooke's Centre for Clinical Investigation, GlaxoSmithKline, Hills Road, Cambridge, CB2 0QQ, UK.
authors.forename: Disala
authors.initials: D
authors.lastname: Fernando
authors.affiliation: GlaxoSmithKline, Stockley Park West, 1-3 Iron Bridge Road, Uxbridge, Middlesex, UB11 1BT, UK.
authors.forename: Kelly
authors.initials: K
authors.lastname: Hardes
authors.affiliation: GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK.
authors.forename: Nicola
authors.initials: N
authors.lastname: Scott
authors.affiliation: GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK.
authors.forename: Sara
authors.initials: S
authors.lastname: Brett
authors.affiliation: Clinical Unit Cambridge, Addenbrooke's Centre for Clinical Investigation, GlaxoSmithKline, Hills Road, Cambridge, CB2 0QQ, UK.
authors.forename: Deborah
authors.initials: D
authors.lastname: Clark
authors.affiliation: Clinical Unit Cambridge, Addenbrooke's Centre for Clinical Investigation, GlaxoSmithKline, Hills Road, Cambridge, CB2 0QQ, UK.
authors.forename: João Joaquim
authors.initials: JJ
authors.lastname: Oliveira
authors.affiliation: Clinical Unit Cambridge, Addenbrooke's Centre for Clinical Investigation, GlaxoSmithKline, Hills Road, Cambridge, CB2 0QQ, UK.
authors.forename: Bill
authors.initials: B
authors.lastname: Davis
authors.affiliation: Clinical Unit Cambridge, Addenbrooke's Centre for Clinical Investigation, GlaxoSmithKline, Hills Road, Cambridge, CB2 0QQ, UK.
authors.forename: Simon
authors.initials: S
authors.lastname: McHugh
authors.affiliation: GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK. john.d.stone@gsk.com.
authors.forename: John
authors.initials: J
authors.lastname: Stone
mesh_terms: D000328:Adult; D016207:Cytokines / Q000276:immunology; D005260:Female; D006433:Hemocyanins / Q000276:immunology*; D006801:Humans; D006968:Hypersensitivity, Delayed / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D012867:Skin / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*; D014373:Tuberculin / Q000276:immunology*; D014611:Vaccination
chemical_list: D016207:Cytokines; D014373:Tuberculin; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin
keywords: DTH; KLH; PPD; Skin blister; Skin challenge; T cell