April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Dendritic cell derived thrombospondin-1 modulation of primary versus secondary T cell responses in a mouse model of ocular allergy
Author Affiliations & Notes
  • Rachel Smith
    Immunology, Duke University, School of Medicine, Durham, NC
  • Nancy Reyes
    Ophthalmology, Duke University, School of Medicine, Durham, NC
  • Daniel R Saban
    Immunology, Duke University, School of Medicine, Durham, NC
    Ophthalmology, Duke University, School of Medicine, Durham, NC
  • Footnotes
    Commercial Relationships Rachel Smith, None; Nancy Reyes, None; Daniel Saban, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 1871. doi:
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      Rachel Smith, Nancy Reyes, Daniel R Saban; Dendritic cell derived thrombospondin-1 modulation of primary versus secondary T cell responses in a mouse model of ocular allergy. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1871.

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

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Abstract

Purpose: Thrombospondin-1 (TSP-1) is a matricellular protein whose expression by dendritic cells (DCs) contributes to regulation of the immune system. Consistent with this, our lab has previously shown that TSP-1 null (TSP-1-/-) mice exhibit exacerbated clinical disease in an ocular allergy model. We therefore tested the hypothesis that DCs aberrant in TSP-1 expression increase allergen reactive T cell responses, which in turn contribute to the exacerbated clinical phenotype seen in TSP-1-/- mice.

Methods: To generate CD11b+ DCs, the immunopathogenic DC subset in ocular allergy, marrow was collected from bones of C57BL/6 (B6) CD45.2 WT and TSP-1-/- mice and cultured with GM-CSF for 6 days. On the 7th day, WT and TSP-1-/- DCs were pulsed overnight with 1 mg/ml OVA or 1 mg/ml OVA + 10-5 M histamine. DCs were thoroughly washed and co-cultured with purified and CFSE stained splenic T cells from B6 CD45.1 naïve or immunized mice (14 days post i.p. immunization with OVA in pertussis toxin and aluminum hydroxide). After 3 days, cultures were restimulated with a PMA, ionomycin, golgi plug cocktail for 4 hours. CFSE T cells were collected and stained for viability, CD45.1, CD45.2, CD4, IL-4, IL-13, IL-17, and IFN-γ to run 9-color flow cytometry analysis.

Results: Overall proliferation of primed CD4+ T cells (percent of CD45.1 CFSE daughter cells) was markedly increased (2.6-fold) to stimulation with TSP-1-/- DCs, as compared with WT counterparts. This was mostly due to increased proliferation of IL-13 (4.5-fold), IL-17 (3.8-fold), IFN-γ (3.1-fold), and IL-4 (1.5-fold) producing CD4+ T cells. Interestingly, such increases in proliferation were not observed with naïve T cells. These same trends were observed with stimulation of histamine pre-treated DCs as well.

Conclusions: We conclude that DC-derived TSP-1 indeed regulates allergen reactive T cell responses, and this may explain the exacerbated clinical phenotype seen in TSP-1-/- mice. Interestingly, however, increased responses due to absence of TSP-1 in DCs plays a more prominent role in primed T cells as compared to naïve counterparts, suggesting a mechanism most relevant in secondary allergen responses as opposed to primary responses (i.e. sensitization).

Keywords: 423 antigen presentation/processing • 480 cornea: basic science • 555 immunomodulation/immunoregulation  
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