March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Generation of Anti-chemokine Monoclonal Antibodies with Migration Neutralizing Capacity: Their Potential as Therapeutic Agents in a Murine Model of High Risk Corneal Transplantation
Author Affiliations & Notes
  • Maitee Urbieta
    Ophthalmology, Bascom Palmer, Miami, Florida
  • Pirouz Daftarian
    Ophthalmology, Bascom Palmer, Miami, Florida
  • Victor L. Perez
    Ophthalmology, Bascom Palmer, Miami, Florida
  • Footnotes
    Commercial Relationships  Maitee Urbieta, None; Pirouz Daftarian, None; Victor L. Perez, None
  • Footnotes
    Support  (VLP) NIH R01 EY018624, NEI P30 EY014801, Research to Prevent Blindness
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 2373. doi:
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      Maitee Urbieta, Pirouz Daftarian, Victor L. Perez; Generation of Anti-chemokine Monoclonal Antibodies with Migration Neutralizing Capacity: Their Potential as Therapeutic Agents in a Murine Model of High Risk Corneal Transplantation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2373.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Recruitment of immune cell subsets to the site of transplantation is potentiated in part by primordial chemokines such as CXCL1/KC and later CXCL9/Mig and CXCL10/IP-10, which are markedly up-regulated in high risk (HR) allogeneic corneal transplant recipients but not in syngeneic or normal risk allogeneic recipients. The purpose of this study is to address the effect of newly generated anti-chemokine mAbs as potential therapy for diminishing the influx of inflammatory immune cells into HR corneal allografts, therefore leading to enhanced graft survival.

Methods: : Wild type (wt) C57BL/6 (B6), mice were immunized with CXCL1 peptide plus universal T cell epitope (PADRE) as adjuvant and boosted 14 days later with the same combination. Hybridoma lines were created by fusion of splenocytes from immunized mice with SP2/IL-6 myeloma cells. Anti-CXCL1/KC clones were selected based on their ability to bind whole CXCL1/KC by ELISA. Thioglycollate-elicited neutrophils or macrophages from peritoneal cavity (PerC) of wt B6 mice were used as input populations for testing mAb migration neutralizing activity in-vitro. In-vitro cell migration was quantified using various detection methods; colorimetric assays, fluorescence detection, and cell acquisition by flow cytometric equipment. High risk B6 recipients were systemically or locally treated with varying doses of anti-CXCL1/KC mAb (clones 1A4, M1G3, and mouse IgG Isotype control) at days 0, 1, 2, and 3 of transplantation with wt BALB/c corneas. Transplanted eyes were enucleated at days 1, 3, and 7 post-transplant and histologically examined for immune cell infiltration using H&E differential staining.

Results: : Two anti-CXCL1/KC lab-generated Ab clones, 1A4 and M1G3, were shown to bind whole,bioactive CXCL1/KC protein by ELISA. These clones were observed to have drastic differential effects on PerC neutrophil and macrophage migration in-vitro: 1A4 worked as an antagonist while M1G3 functioned as agonist. Flow cytometric cell acquisition proved to be the most consistent and reliable readout system for migration quantification in neutralization assays.

Conclusions: : Newly generated anti-chemokine monoclonal antibodies may represent useful tools for dissecting the kinetics and function of innate and adaptive immune cell infiltrates not only in HR corneal allotransplantation but in allogeneic tissue rejection responses in general. Future studies will continue to address the effect of single vs. combinatorial anti-chemokine antibody therapy on corneal allograft survival in HR corneal transplantation as well as in other types of solid organ transplants.

Keywords: cytokines/chemokines • transplantation 

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