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. 2019 Apr 5;14(4):e0214938.
doi: 10.1371/journal.pone.0214938. eCollection 2019.

Antibody-mediated targeting of cleavage-specific OPN-T cell interactions

Bettina Wanko  1   2   3 , Matteo Tardelli  4 , Alexander Jürets  1 , Angelika Neuhofer  1 , Gerhard Prager  5 , John Morser  2   3 , Lawrence L Leung  2   3 , Günther Staffler  6 , Maximilian Zeyda  1 , Thomas M Stulnig  1
Affiliations

Antibody-mediated targeting of cleavage-specific OPN-T cell interactions

Bettina Wanko et al. PLoS One. .

Abstract

T cells are crucial players in obesity-mediated adipose tissue inflammation. We hypothesized that osteopontin (OPN), an inflammatory protein with enhanced activity when proteolytically cleaved, affects the number of viable T cells in adipose tissue and assessed inhibition of the interaction between T cells and thrombin and matrix metalloproteinases-cleaved OPN using antibodies and postimmune sera. Gene expression of T cell markers in adipose tissue from wild-type (wt) and Spp1-/- (OPN deficient) mice was analyzed after 16 weeks of high fat diet (HFD) or low fat diet (LFD) feeding. CD3, CD8 and OPN gene expression in omental adipose tissue from individuals with obesity was measured. OPN-T cell interactions were assessed with a fluorescence-based adhesion assay and blocked with antibodies targeting OPN. Comparison of T cell gene expression in adipose tissue from wt and Spp1-/- mice showed that OPN affected the number of T cells while in humans, levels of OPN correlated with T cell markers in omental adipose tissue. The interaction between T cells and cleaved OPN was blocked by postimmune sera following OPN peptide vaccinations and with monoclonal antibodies. In conclusion, levels of OPN affected the number of T cells in obesity and antibodies against cleaved OPN antagonize OPN-T cell interactions.

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Conflict of interest statement

The author G. Staffler is employee of AFFiRiS AG. GS was involved in selecting the peptide antigens for the vaccines described in this manuscript and in vaccine design as well as in vaccine preparation. A patent protecting the antibodies mAb 9-3 and mAb 21-5 was filed (official patent number: PCT/EP2015/064701). AFFiRiS AG is a supporting member of the Christian Doppler Society. MZ and TMS received consultant honoraria from AFFiRiS AG. The authors confirm that the commercial affiliation of GS from AFFiRiS AG does not alter the authors' adherence to all PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. OPN affects T cell numbers in mouse.
wt (n = 26) and Spp1-/- mice (n = 19) were fed HFD or wt were fed LFD (n = 24) for 16 weeks. Gonadal fat was isolated and analyzed by RT-PCR for expression of CD3, CD4 and CD8; mean ± SEM depicted.
Fig 2
Fig 2. Relative amount of T cells in adipose tissue.
The CD3, CD4 and CD8 immunofluorescence intensity in gonadal fat was measured and divided by the number of nuclei (stained with DAPI) resulting in a relative quantification. 2–5 pictures were taken per mouse, 4–5 mice were analyzed. Comparison of groups with wt HFD group; **** p < 0.0001.
Fig 3
Fig 3. CD3, CD8 and GATA3 correlate significantly with OPN gene expression in human adipose tissue.
Omental adipose tissue from individuals with obesity was isolated and (A) CD3 and OPN gene expression were analyzed by RT-PCR (n = 17) Spearman coefficient: r = 0.723, p = 0.0005 (B) CD8 and OPN gene expression (n = 17): r = 0.5637, p = 0.0203; (C) GATA3 and OPN gene expression (n = 16): r = 0.5206, p = 0.04; (D) Tbet and OPN (n = 12): r = 0.4825, p = 0.1154; (E) Foxp3 and OPN (n = 16): r = 0.3971, p = 0.1289; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Best fit line as well as the 95% confidence interval are indicated by lines in the graphs.
Fig 4
Fig 4. FL-OPN, Thr-cOPN and MMP-cOPN increase the number of viable T cells.
Peripheral T cells (n = 3, 3 independent experiments) were incubated with FL-OPN, Thr-cOPN or MMP-cOPN under serum free conditions for 72 h. Release of ATP was assessed and ascribed to viable cells. Data was calculated by normalizing to the values without OPN (negative control). Complete medium included 10% FCS; mean ± SEM depicted; Comparison of OPN treated groups with the respective BSA control; * p < 0.05, ** p < 0.01.
Fig 5
Fig 5. T cells bind to OPN.
(A) Jurkat cells (2 experiments, each run in duplicate) and (B) primary T cells (4 experiments utilizing 4 donors, each run in duplicate) were analyzed for their adhesion to FL-OPN, Thr-cOPN and MMP-cOPN.
Fig 6
Fig 6. The interaction between T cells and cleaved OPN can be blocked with monoclonal antibodies.
(A-C) Blockade of the interaction of primary human T cells with OPN with monoclonal antibodies 21–5 and 9–3. 1 experiment, 2 donors, each run in duplicate (A) Blockade of T cell binding to FL-OPN. (B) Blockade of T cell binding to Thr-cOPN. (C) Blockade of T cell binding to MMP-cOPN; mean ± SEM depicted; Comparison with the isotype control antibody; * p < 0.05, ** p < 0.01 and *** p < 0.001; **** p < 0.0001.
Fig 7
Fig 7. The OPN-T cell interaction can be antagonized by serum from active vaccination with OPN peptides.
(A) 3 peptides derived from the OPN sequence plus a scrambled peptide were injected into mice. The MMP and thrombin cleavage sites are delineated by the vertical lines. The RGD binding site for αvβ3 integrins is marked with a red box and the SVVYGLR binding site for α4β1 and α4β9 integrins is marked with a mauve box. (B) Interference of postimmune serum with binding of Jurkat T cells to FL-OPN. (C) Interference of postimmune serum with binding of Jurkat T cells to Thr-cOPN. (D) Interference of postimmune serum with Jurkat T cells binding to MMP-cOPN; The different sera for the experiments were pooled from 5 mice with the OPN-T cell interaction determined once with each point detected in duplicate; mean ± SD depicted.
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