C-reactive protein induced T cell activation is an indirect monocyte-dependent mechanism involving the CD80/CD28 pathway

Abstract

Introduction: T cells are major components of the immune system. Their activation requires interaction between the T cell receptor and co-stimulatory molecules, crucial during infection, inflammation, and allogeneic rejection. Monomeric CRP (mCRP) is a known modulator of inflammation and particularly the innate immune response, however its interaction with T cells as part of the adaptive immune response remains unclear.

Methods: Peripheral blood mononuclear cells (PBMC) and T cells were isolated. Flow cytometric analysis was conducted to evaluate Fcγ receptor CD16 expression on T cells, the binding of CRP to T cells, and its impact on proliferation and apoptosis. T cell activation was assessed after 1, 2, 3, 5 and 7 days by assessing CD69 and CD25 expression, and under various conditions including coculture with monocytes and several inhibitory factors.

Results: T cells express CD16 that binds mCRP in a concentration-dependent manner, and particularly on activated T cells. While mCRP reduces apoptosis and accelerates proliferation in T cells, it does not independently activate them. However, activation of monocytes by mCRP leads to T cell activation, indicating a direct cell to cell interaction during CRP-induced activation. This effect could be alleviated by inhibition of the CD80/CD28 pathway.

Conclusion: CRP does not activate T Cells directly but via PI3-kinase-dependent activation of monocytes and subsequent CD80/CD28 cell to cell contact. The findings suggest the effects of CRP on T cells depend on their environment and the presence of other proinflammatory agents.

Keywords: Belatacept; C-reactive protein; CD80/CD28 pathway; T cells; innate immunity; monocytes.

Figure 1

(A) Expression of Fcγ receptor subtypes on T cells. Freshly taken whole blood was stained with antibodies against Fcγ receptor subtypes (CD16, CD32, CD64) and assessed in flow cytometry. CD16 is expressed by T cells, CD32 and CD64 not. n=3. (B) CD16 on activated vs. not activated T cell subtypes. PBMC were incubated with 12.5 µl/ml CD3/CD28 beads (1:2 bead number:cells) at 37°C and 5% CO₂ for 24 h and additionally stained with antibodies against CD4, CD8 and CD69 to assess subtype and activation state. CD16 is significantly more often expressed by activated (CD69+) than by non-activated T cells (CD69-) in CD4+ T cells. n=4. Paired t-test. (C) Concentration-dependent binding of CRP isoforms on CD4+ and CD8+ T cells. PBMC were incubated with 12.5 µl/ml CD3/CD28 (1:2 bead number:cells) beads at 37°C and 5% CO₂ for 24 h Binding of BSA, pCRP and mCRP at 25 µg/ml, 50 µg/ml and 100 µg/ml was assessed by conjugation with PE-Texas Red. Brakets indicate results of Friedman’s test in comparison to PBS. mCRP and pCRP bind significantly to CD4+ and CD8+ T cells in comparison to control. n=4. (D) Binding of mCRP after blocking of CD16. PBMC were incubated with 12.5 µl/ml CD3/CD28 beads (1:2 bead number:cells) at 37°C and 5% CO₂ for 24 h Before adding fluorophore-conjugated mCRP, cells were incubated with anti-CD16 for 15 min. Blocking of CD16 significantly reduced mCRP binding on CD4+ T cells. Results shown in mean and standard deviation. n=7. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant.

Figure 2

(A) Proliferation of CD4+ and CD8+ T cells. PBMC were incubated for 5 days at 37°C and 5% CO₂ with 12.5 µl/ml CD3/CD28 beads (1:2 bead number:cells). T cell proliferation was assessed with CFSE (CellTrace) to track following generations. 25 µg/ml pCRP and mCRP were added respectively. In the control group (PBS), no T cell proliferation was observed. Flow cytometry analysis revealed distinct peaks, each corresponding to a successive generation of dividing T cells and thereby reduced CFSE-fluorescence, with younger generations located towards the left of the x-axis. Stimulation with mCRP significantly enhanced the proliferation of both CD4+ and CD8+ T cells in comparison to CD3/CD28 beads, whereas pCRP did not have this effect. (B) Apoptosis of CD4+ and CD8+ T cells. PBMC were incubated for 5 days at 37°C and 5% CO₂. 25 µg/ml pCRP and mCRP were added. Apoptosis was assessed by Annexin V staining. mCRP decreases the percentage of apoptotic cells in comparison to PBS by 30% (CD4+) and by 17% (CD8+). n=4. (C) Influence of CRP isoforms on the activation of isolated T cells. T cells were isolated via magnetic sorting and incubated with PBS, 25 µg/ml pCRP, and 25 µg/ml mCRP for 3 and 7 days. There was no significant influence on the percentage of activated cells with early activation marker CD69+ nor late activation marker CD25+. Results are shown in mean and standard deviation. n=3. **p<0.01. ns = not significant.

Figure 3

Activation over time of CD4+ and CD8+ T cells, costimulation with CD3/CD28 beads. The curve displays a time course of activation markers CD69+ and CD25+ for CD4+ and CD8+ T cells when stimulated with CD3/CD28 beads only or additionally 50 µg/ml pCRP or mCRP. All results are displayed as means and standard deviation. n=5. *p<0.05, **p<0.01.

Figure 4

(A) Expression of CD69 on T cell subtypes in coculture with monocytes and with addition of mCRP. T cells were stimulated with 25 µg/ml mCRP either alone or in coculture with monocytes. T cells only served as an additional control group. Activation was assessed after 3 days via expression of surface marker CD69. The addition of mCRP to coculture with monocytes lead to a significant higher expression of CD69 on T cells in comparison to T cells only, monocytes or mCRP alone. n=7. (B) Expression of CD69 on T helper cell subtypes in coculture with monocytes and with addition of mCRP. As for CD4+ T helper cells, the addition of mCRP to coculture with monocytes lead to a significant higher expression of CD69 on T helper cell subtypes. Results are shown in mean and standard deviation. n=7. *p<0.05, **p<0.01, ***p<0.001. ns = not significant.

Figure 5

(A) Expression of CD80 on monocytes. Freshly taken whole blood was incubated with 50 µg/ml mCRP and 25 µg/ml Wortmannin (WM), a PI3-kinase inhibitor. Monocytes were identified as described previously (21) and stained with anti-CD80-APC. n=4. **p<0.01, ***p<0.001. ns = not significant. (B) Activation of T-cells in coculture with monocytes and mCRP, inhibition with Belatacept. A coculture of T-cells and monocytes was stimulated with 50 µg/ml mCRP. For inhibition, 10 µg/ml Belatacept was added and incubated for 3 days. T-cells were stained with anti-CD69 to assess activation. Data was normalized on T+M control group. n=9. *p<0.05. (C) Proposed mechanism of T cell activation via mCRP-stimulated monocytes and CD80/CD28 pathway. Monocytes are activated by mCRP via a PI3-kinase-dependent mechanism, which increases their expression of CD80. CD80 interacts with T-cells’ CD28, thereby increasing T cell activation in terms of CD69 expression. This interaction can be inhibited by Belatacept.