Secretion of cytokines by T-cell populations cultured in the presence of the different combinations of sHSP70 and/or cytokines
| Analytes, pg/mL . | ||||||
|---|---|---|---|---|---|---|
| Experiment . | IFN-γ . | IL-1β . | IL-5 . | IL-6 . | IL-8 . | TNF-α . |
| Multiple cytokine analysis for CD3+ T cells | ||||||
| SEB | 1163.9 ± 875.7 | 21.4 ± 42.7 | 63.0 ± 51.3 | 54.4 ± 92.6 | 3655.8 ± 4308.5 | 200.0 ± 225.6 |
| sHSP70 | 462.9 ± 853.7 | 17.4 ± 34.8 | 37.8 ± 0 | 5.7 ± 14.3 | 2162.3 ± 2207.7 | 64.3 ± 128.6 |
| IL-2 | 24.7 ± 34.7 | 0 | 0 | 0 | 27.1 ± 135.2 | 42.8 ± 41.0 |
| sHSP70 + IL-2 | 985.8 ± 648.4* | 69.7 ± 0 | 844.9 ± 138.6** | 129.5 ± 147.6** | 4034.8 ± 4522.2* | 82.9 ± 88.2* |
| IL-7/IL-12/IL-15 | 0 | 0 | 0 | 0 | 376.8 ± 834.5 | 8.6 ± 17.1 |
| sHSP70 + IL-7/IL-12/IL-15 | 54.7 ± 91.4* | 0 | 0 | 68.0 ± 114.7* | 3895.9 ± 4195.4** | 29.5 ± 27.4* |
| Multiple cytokine analysis for CD4+ T cells | ||||||
| SEB | 418.7 ± 725.2 | 65.6 ± 69.7 | 92.2 ± 144.4 | 123.0 ± 177.7 | 7928.4 ± 3400.1 | 205.9 ± 211.7 |
| sHSP70 | 74.9 ± 79.3 | 0 | 0 | 99.7 ± 126.3 | 3403.4 ± 2671.0 | 0 |
| IL-2 | 74.0 ± 90.6 | 0 | 0 | 0 | 61.5 ± 300.8 | 0 |
| sHSP70 + IL-2 | 199.8 ± 92.4 | 0 | 256.5 ± 444.3* | 107.6 ± 109.8* | 6685.1 ± 0** | 21.7 ± 0 |
| IL-7/IL-12/IL-15 | 0 | 0 | 0 | 0 | 742.6 ± 1788.1 | 0 |
| sHSP70 + IL-7/IL-12/IL-15 | 0 | 0 | 0 | 28.7 ± 40.5* | 6051.7 ± 5533.6* | 0 |
| Multiple cytokine analysis for CD8+ T cells | ||||||
| SEB | 1428.0 ± 326.5 | 0 | 720.5 ± 647.2 | 0.0 | 1367.1 ± 1782.0 | 88.1 ± 80.8 |
| sHSP70 | 91.9 ± 4.1 | 0 | 0 | 5.2 ± 9.0 | 278.9 ± 355.5 | 0 |
| IL-2 | 44.3 ± 39.4 | 0 | 0 | 0 | 42.1 ± 4.1 | 0 |
| sHSP70 + IL-2 | 198.6 ± 24.1* | 0 | 0 | 0 | 1077.5 ± 0* | 0 |
| IL-7/IL-12/IL-15 | 0 | 0 | 0 | 5.2 ± 9.0 | 19.9 ± 0 | 0 |
| sHSP70 + IL-7/IL-12/IL-15 | 0 | 0 | 0 | 22.3 ± 19.6 | 621.3 ± 604.3* | 0 |
| Analytes, pg/mL . | ||||||
|---|---|---|---|---|---|---|
| Experiment . | IFN-γ . | IL-1β . | IL-5 . | IL-6 . | IL-8 . | TNF-α . |
| Multiple cytokine analysis for CD3+ T cells | ||||||
| SEB | 1163.9 ± 875.7 | 21.4 ± 42.7 | 63.0 ± 51.3 | 54.4 ± 92.6 | 3655.8 ± 4308.5 | 200.0 ± 225.6 |
| sHSP70 | 462.9 ± 853.7 | 17.4 ± 34.8 | 37.8 ± 0 | 5.7 ± 14.3 | 2162.3 ± 2207.7 | 64.3 ± 128.6 |
| IL-2 | 24.7 ± 34.7 | 0 | 0 | 0 | 27.1 ± 135.2 | 42.8 ± 41.0 |
| sHSP70 + IL-2 | 985.8 ± 648.4* | 69.7 ± 0 | 844.9 ± 138.6** | 129.5 ± 147.6** | 4034.8 ± 4522.2* | 82.9 ± 88.2* |
| IL-7/IL-12/IL-15 | 0 | 0 | 0 | 0 | 376.8 ± 834.5 | 8.6 ± 17.1 |
| sHSP70 + IL-7/IL-12/IL-15 | 54.7 ± 91.4* | 0 | 0 | 68.0 ± 114.7* | 3895.9 ± 4195.4** | 29.5 ± 27.4* |
| Multiple cytokine analysis for CD4+ T cells | ||||||
| SEB | 418.7 ± 725.2 | 65.6 ± 69.7 | 92.2 ± 144.4 | 123.0 ± 177.7 | 7928.4 ± 3400.1 | 205.9 ± 211.7 |
| sHSP70 | 74.9 ± 79.3 | 0 | 0 | 99.7 ± 126.3 | 3403.4 ± 2671.0 | 0 |
| IL-2 | 74.0 ± 90.6 | 0 | 0 | 0 | 61.5 ± 300.8 | 0 |
| sHSP70 + IL-2 | 199.8 ± 92.4 | 0 | 256.5 ± 444.3* | 107.6 ± 109.8* | 6685.1 ± 0** | 21.7 ± 0 |
| IL-7/IL-12/IL-15 | 0 | 0 | 0 | 0 | 742.6 ± 1788.1 | 0 |
| sHSP70 + IL-7/IL-12/IL-15 | 0 | 0 | 0 | 28.7 ± 40.5* | 6051.7 ± 5533.6* | 0 |
| Multiple cytokine analysis for CD8+ T cells | ||||||
| SEB | 1428.0 ± 326.5 | 0 | 720.5 ± 647.2 | 0.0 | 1367.1 ± 1782.0 | 88.1 ± 80.8 |
| sHSP70 | 91.9 ± 4.1 | 0 | 0 | 5.2 ± 9.0 | 278.9 ± 355.5 | 0 |
| IL-2 | 44.3 ± 39.4 | 0 | 0 | 0 | 42.1 ± 4.1 | 0 |
| sHSP70 + IL-2 | 198.6 ± 24.1* | 0 | 0 | 0 | 1077.5 ± 0* | 0 |
| IL-7/IL-12/IL-15 | 0 | 0 | 0 | 5.2 ± 9.0 | 19.9 ± 0 | 0 |
| sHSP70 + IL-7/IL-12/IL-15 | 0 | 0 | 0 | 22.3 ± 19.6 | 621.3 ± 604.3* | 0 |
The capacity of T-cell subpopulations (CD3+ T cells, CD4+ T cells, CD8+ T cells) from 4 healthy donors to proliferate in the presence of sHSP70 and different cytokines was assessed by determining the secretion levels of Th-type 1 (Th1) cytokine IFN-γ and proinflammatory cytokines using a bead-based flow cytometric assay (picograms per milliliter). The basal cytokine expression by unstimulated cells was subtracted from the values measured after stimulation. There were no secretion of IL-5, IL-6, and IL-8 in both CD3+ and CD4+ T cells by unstimulated T cells detectable. Therefore stimulation with sHSP70 resulted in the de novo production of these inflammatory mediators. Cells stimulated with staphylococcal enterotoxin B (SEB) served as positive secretion controls. Statistically significant differences between the stimulation with sHSP70 plus IL-2 versus IL-2 and sHSP70 plus IL-7/IL-12/IL-15 versus IL-7/IL-12/IL-15 are indicated with asterisks (*P < 0.05 or **P < 0.01).