Circulating CD3+HLA-DR+ Extracellular Vesicles as a Marker for Th1/Tc1-Type Immune Responses

Abstract

Extracellular vesicles (EVs) are known to contain unique proteins that reflect the cells of origins. Activated T cells are reported to secrete various EVs. To establish T cell subset-specific biomarkers, we performed proteomic analysis with Th1- and Th2-derived EVs and identified HLA-DR as a Th1-dominated EV membrane protein. We designed a measurement system for CD3⁺CD4⁺, CD3⁺CD8⁺, and CD3⁺HLA-DR⁺ EVs to specifically determine EV subpopulations derived from CD4⁺, CD8⁺, and Th1-type T cells, respectively. In vitro analysis showed that CD3⁺CD4⁺ EVs and CD3⁺CD8⁺ EVs were selectively secreted from activated CD4⁺ and CD8⁺ T cells, respectively, and that CD3⁺HLA-DR⁺ EVs were actively secreted from not only Th1 but also activated CD8⁺ T (probably mostly Tc1) cells. To evaluate the clinical usefulness of these EVs, we measured the serum levels in patients with inflammatory diseases, including Epstein-Barr virus (EBV, n = 13) infection, atopic dermatitis (AD, n = 10), rheumatoid arthritis (RA, n = 20), and osteoarthritis (OA, n = 20) and compared the levels with those of healthy adults (n = 20). CD3 ⁺ CD4 ⁺ EVs were significantly higher in all of EBV infection, AD, RA, and OA while CD3⁺CD8⁺ EVs were higher in EBV infection, lower in RA, and not different in AD and OA relative to the control. The levels of CD3⁺HLA-DR⁺ EVs were markedly higher in EBV infection and significantly lower in AD. The results suggest that these EV subpopulations reflect in vivo activation status of total CD4⁺, total CD8⁺, and Th1/Tc1-type T cells, respectively, and may be helpful in T cell-related clinical settings, such as cancer immunotherapy and treatment of chronic infection, autoimmune diseases, and graft-versus-host disease.

Figure 1

Identification of Th1-derived EV-dominant proteins. (a) Chemiluminescence images. Th1- and Th2-derived EVs were subjected to western blot analysis with anti-CD3 and anti-CD4 mAbs. (b) Fluorescent nanoparticle tracking image. Th1-derived EVs were stained with Qdot655-conjugated anti-CD3 antibody and analyzed on the NanoSight. Results in light scatter (black line) and fluorescence (grey line) modes. (c, d) Two parts of 2D-DIGE images of Th1-derived EV proteins. (e, f) 2D-DIGE images of Th2-derived EV proteins in the same areas as c and d, respectively. Protein spots that showed differences in the fluorescence intensities were picked up for MS analysis. The protein spots indicated by circles (panels c and e, mw = 37067 and pI = 4.8) and dotted circles (panels d and f, mw = 31322 and pI = 7.8) were identified as alpha- and beta-chains of HLA-DR, respectively. Data from one out of six pairs of EVs are shown. The results were similar for the five out of six pairs.

Figure 2

Comparison of various marker proteins between Th1 and Th2 cells. Five pairs of established Th1 and Th2 cells were restimulated with mAbs to CD3 and CD28 under Th1- and Th2-inducing conditions, respectively. After three-day culture, the expression levels of CD3 (a), CD4 (b), CD63 (c), and HLA-DR (d) on the cell surface were measured by FCM. ^∗∗P < 0.01, by the Mann–Whitney U test between Th1 and Th2.

Figure 3

Correlation between concentrations of EVs and AlphaLISA signals. Purified EVs derived from CD4⁺ T cells (a), CD8⁺ T cells (b), and Th1 (c) were subjected to AlphaLISA assay for CD3⁺CD4⁺ EVs, CD3⁺CD8⁺ EVs, and CD3⁺HLA-DR⁺ EVs, respectively. Protein concentrations of EVs were determined with the bicinchoninic acid assay. The correlation coefficient (r) between data sets of CD3⁺CD4⁺ EVs, CD3⁺CD8⁺ EVs, and CD3⁺HLA-DR⁺ EVs were 1.000, 0.999, and 0.999, respectively.

Figure 4

Comparison of T cell-derived EVs between Th1 and Th2 cultures. Three pairs of established Th1 and Th2 cells were restimulated with CD3 and CD28 antibodies under Th1- and Th2-inducing conditions with a starting cell density of 1.0 × 10⁶ cells/ml. After 5-day culture, the cell density was determined by the trypan blue dye-exclusion test (a) and levels of CD3⁺CD4⁺ EVs (b), CD3⁺CD63⁺ EVs (c), and CD3⁺HLA-DR⁺ EVs (d) in the culture supernatants were measured by AlphaLISA. AlphaLISA counts were normalized by dividing the value by the cell number. Data are presented as the mean ± SD. ^∗∗P < 0.01, by the Mann–Whitney U test between Th1 and Th2.

Figure 5

Levels of T cell-derived EVs in culture supernatants of CD4⁺ and CD8⁺ T cells. CD4⁺ and CD8⁺ T cells were purified from PBMCs obtained from three healthy donors and cultured with (sti) or without (unsti) stimulation by CD3 and CD28 antibodies with a starting cell density of 1.0 × 10⁶ cells/mL. After 6-day culture, the cell numbers (a, e) and supernatant levels of CD3⁺CD4⁺ EVs (b, f), CD3⁺CD8⁺ EVs (c, g), and CD3⁺HLA-DR⁺ EVs (d, h) were examined for CD4⁺ T cells (a-d) and CD8⁺ T cells (e-h). AlphaLISA counts were normalized by dividing the value by the cell number. Data are presented as the mean±SD. ^∗P < 0.05 and ^∗∗P < 0.01, by the Mann–Whitney U test between sti and unsti cell groups.

Figure 6

Correlation between the signal and EVs. Three sera from patients with EBV infection (◆◇, ●○, ▲△)were diluted with EV-free NHS (solid lines) or reaction buffer (dotted lines) and subjected to AlphaLISA assay for CD3⁺CD4⁺ EVs (a), CD3⁺CD8⁺ EVs (b), and CD3⁺HLA-DR⁺ EVs (c). The correlation coefficients (r) between alpha signal and serum volume in CD3⁺CD4⁺ EV, CD3⁺CD8⁺ EV, and CD3⁺HLA-DR⁺ EV assay ranged from 0.997 to 1.000 when diluted with EV-free NHS, while they ranged from 0.976 to 0.999 when diluted with reaction buffer. EVs were isolated from nine human sera using a commercial isolation kit, suspended to their original volume with EV-free NHS, and their CD3⁺CD4⁺ EVs (d), CD3⁺CD8⁺ EVs (e), and CD3⁺HLA-DR+ EVs (f) were measured together with original sera. The correlation coefficients (r) of nine sera between before and after isolation were 0.997, 0.996, and 0.998 for CD3⁺CD4⁺ EVs, CD3⁺CD8⁺ EVs, and CD3⁺HLA-DR⁺ EVs, respectively. Data are presented as the mean ± SD (n = 2).

Figure 7

Levels of T cell-derived EVs in sera from patients with various inflammatory diseases. Levels of CD3⁺HLA-DR⁺ EVs (a), CD3⁺CD4⁺ EVs (b), and CD3⁺CD8⁺ EVs (c) in sera from patients with EBV infection (EBV, n = 12), atopic dermatitis (AD, n = 10), rheumatoid arthritis (RA, n = 20), and osteoarthritis (OA, n = 20) were measured by AlphaLISA and compared with those of healthy subjects (H, n = 20). The bars represent the mean ± SD^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗∗P < 0.0001, by the Mann–Whitney U test.