Inhibition of T Cell Proliferation By Granulocyte Membrane-Associated Proteinase 3 Is Reversed By Blocking the Interaction of Calreticulin with LRP on T Cells

Proteinase 3 (P3), a serine protease constitutively expressed on the membrane on a fraction of resting granulocytes and commonly existed in primary granules, is the target of autoimmunity in Wegener’s granulomatosis (WG) and of anti-leukemia immunity mediated by P3-derived PR1-specific cytotoxic T lymphocytes (CTL). We showed that soluble P3 and membrane expressed (mP3) peripheral blood polymorphonuclear neutrophils (PMNs) significantly inhibited healthy donor T-cell proliferation stimulated with anti-CD3/CD28 antibodies in a reversible, does-dependent, and enzyme-independent manner. Furthermore, there is an inverse correlation between percent proliferation of T cells and the amount of mP3 on AML (R=0.45, P<0.001). This inhibition was blocked by anti-P3 antibody. In addition, mP3-expressing AML blasts similarly inhibited T cell proliferation whereas AML blasts expressing no mP3 showed little effect. Interestingly, mP3 expression is significantly higher in bone marrow myeloid derived suppressor cells (MDSC) from leukemia patients compared to MDSC from healthy donors, (79.4±5.23% (n=7) versus (22.4±11.55% (n=3), respectively (P<0.001).

MP3-mediated T cell inhibition was independent of arginase I but suppression of reactive oxygen species (ROS) formation partially reversed mP3-mediated inhibition of CD4⁺ and CD8⁺ T cell proliferation by 15% and 23%, respectively, implicating the ROS pathway in mP3-mediated T cell inhibition.

Because mP3 inhibited proliferation of T cells stimulated via the T cell receptor (TCR), i.e. anti-CD3/CD28, we evaluated the effects of mP3 on ZAP70 signaling by phospho-flow cytometry. ZAP70 phosphorylation in CD8⁺ and CD4⁺ T cells co-incubated with mP3⁺ PMNs plus anti-CD3/CD28 mAbs for 5 minutes, was reduced by 51% and 70%, respectively, compared with T cells stimulated with anti-CD3/CD28 mAbs alone (n=4, P<0.001 for CD8 cells and P<0.05 for CD4 cells), suggesting that P3-mediated inhibition of T cell proliferation involves downstream TCR signaling pathways. Calreticulin (CRT), which binds to low-density lipoprotein receptor-related protein (LRP), is associated with mP3 expression on apoptotic PMNs. We showed that LRP expression increased significantly on CD4⁺ and CD8⁺ T cells (P<0.05) after stimulation with anti-CD3/CD28. Because LRP binds to CRT, which is co-associated with mP3 on apoptotic PMNs, we studied role of LRP+CRT in mP3 inhibition. To test this, CFSE-labeled PBMC stimulated with anti-CD3/CD28 were co-cultured with PMNs treated with anti-CRT or untreated at a ratio of 5:1 or in the presence or absence of anti-LRP for 5 days. Anti-LRP reversed mP3-expressing PMNs-mediated inhibition of CD4⁺ and CD8⁺ T cells by 86% and 53%, respectively. Similarly, the capacity of PMNs to suppress T cell proliferation was reduced in the presence of anti-CRT mAb. In conclusion, our data supports a role for mP3, which is co-associated with CRT on PMNs, on blocking the interaction of CRT with LRP on activated T cells. Taken together, these data implicate a novel role for mP3 in regulating T cell proliferation via CRT-LRP interactions.