EVs modulate induced immune responses

The immune modulating capacity of isolated EVs were accessed in pro-inflammatory immune responses that were induced by stimulation with anti-CD3. A comparative analysis and the determination of affected parameters, such as T cell proliferation, was enabled by treating these PBMC cultures simultaneously with either unstimulated EVs, cytokine stimulated EVs, PBS in equal volumes to EVs, or these stimulated PBMC cultures were left untreated. The frequencies of proliferated CD4⁺ as well as CD8⁺ T cells were significantly reduced in anti-CD3 stimulated PBMC cultures treated with both unstimulated or cytokine stimulated EVs from CardAP cells in comparison to the PBS control (Figure 31A). Interestingly, cytokine stimulated EVs diminished normalized CD4⁺ and CD8⁺ T cell proliferation even greater than their unstimulated counterpart, although both EV treatments exhibited significant diminishing effects in comparison to the PBS control (median normalized CD4⁺ T cell proliferation (range): PBS = 0.983 (0.764 – 1.23), EVs = 0.928 (0.524 – 1.00), EVs^(cyt) = 0.855 (0.548 – 0.983), ; median normalized CD8⁺ T cell proliferation (range): PBS = 1.00 (0.711 – 1.16); EVs = 0.903 (0.472 – 0.993); EVs^(cyt) = 0.929 (0.544 – 1.06); Figure 31B).

un PBS EVs EVs^(cyt)

0 2 4 6 8 10 12

**

CD25 CD62L CD62L

un PBS EVs EVs^(cyt)

70 75 80 85

90 *

*

un PBS EVs EVs^(cyt)

x = CD25; y = CD62L

Frequency [%]

A

B

CD4⁺ T cells in unstimulated PBMC cultures

65

Figure 31: EVs diminished anti-CD3 induced T cell pro-liferation in PBMC cultures.

CFSE labelled PBMCs (3x10⁵ cells/well) were stimulated with anti-CD3, and treated with 12 µg/mL unstimulated (EVs), cytokine stimulated EVs (EVs^(cyt)), PBS in equal volumes as EVs (PBS), or they were left untreated. After five days, harvested cells were analysed by flow cytometry. Obtained T cell proliferation frequencies were normalized to the untreated control. (A): Representative flow cytometry plots display the frequencies of proliferated CD4⁺ and CD8⁺ T cells in anti-CD3 stimulated PBMCs. (B): The normalized CD4⁺ or CD8⁺ T cell proliferation in anti-CD3 stimulated PBMC cultures is presented as median with data range (n = 9; four different CardAP donors; five different PBMC donors). Statistical analysis was performed by Friedman´s test with Dunn´s multiple comparison post hoc test (*** p < 0.001, * p < 0.05). Unstimulated and cytokine stimulated EVs reduce the T cell proliferation in anti-CD3 provoced immune responses of human isolated PBMCs.

CD4⁺ T cells of anti-CD3 stimulated PBMC cultures were analysed in more detail for some activation markers. As anticipated, the expression of CD25 as well as CD69 was enhanced on CD4⁺ T cell in anti-CD3 stimulated PBMC cultures (Figure 32A) in comparison to previous results in unstimulated PBMC cultures (Figure 29). Notably, treatments with unstimulated or cytokine stimulated EVs did not changed neither the expression level nor the frequency of these activation markers in comparison to PBS treated or untreated controls (median normalized MFI CD69: un = 2.61; PBS = 2.76; EVs = 2.74; EVs^(cyt) = 2.49; median normalized MFI CD25: un

= 14.78; PBS = 14.33; EVs = 16.82; EVs^(cyt) = 14.96; median normalized frequency CD69: un

= 18.25; PBS = 25.74; EVs = 18.59; EVs^(cyt) = 20.70; median normalized frequency CD25: un

= 91.40; PBS = 88.20; EVs = 90.70; EVs^(cyt) = 90.70; Figure 32A). Since the activation of CD4⁺ T cells was obtained at comparable levels between all treatments, it was of further interest whether the subset of regulatory T cells were affected by the treatment with isolated EVs. This is alo reasoned by the fact that this specific subset of T helper cells would be beneficial for the treatment of damaged cardiac tissue with prolonged or chronic inflammation. Indeed, PBS treated PBMC cultures exhibited lower frequencies of Tregs (viable, single CD3⁺ CD4⁺ CD127 -CD25⁺ Foxp3⁺) in comparison to both EV treatments (Figure 32B). Although both EVs significantely enhanced the frequency of Tregs, unstimulated EVs exhibited a greater trend to enhance frequency of regulatory T cells than cytokine stimulated EVs (median frequency of

***

66 Tregs (range): PBS = 1.68 (1.05 – 2.68) %; EVs = 2.55 (1.96 – 3.38) %; EVs(cyt) =2.40 (1.75 – 2.81) %; Figure 32B).

Figure 32: EVs increased the frequency of regulatory T cells, while CD4⁺ T cells showed comparable expression of activation markers in anti-CD3 induced PBMC cultures.

CFSElabelled PBMCs (6x10⁵ cells/well) were stimulated with anti-CD3 treated with 12 µg/mL unstimulated (EVs), cytokine stimulated EVs (EVs^(cyt)), PBS in equal volumes as EVs (PBS), or they were left untreated (un).

After three days, cells were harvested and analysed by flow cytometry. (A): The frequency and expression as geometrical mean fluorescence (MFI) were determined for CD25 and CD69 in the CD4⁺ T cell population in these anti-CD3 stimulated PBMC cultures. The obtained MFIs were additionally normalized to the respective unstained control. Individual data point of the normalized MFI (upper graphs) or frequencies (lower graphs) are shown as median with data range for CD69 (left), CD25 (middle) and HLA-DR Obtained individual geometrical mean fluorescence intensities (MFI) are displayed as median with data range for an early activation (CD69), mediate activation (CD25). (B): Representative flow cytometry plots (left) show the frequencies of regulatory T cells (CD25⁺ Foxp3⁺ T cells with previous gating on viable single CD3⁺ CD4⁺ CD127^-) in anti-CD3 stimulated PBMCs.

The individual frequencies of regulatory T cells (viable single CD3⁺ CD4⁺ CD127^- CD25⁺ Foxp3⁺ T cells) as median with data range (n = 8; five different CardAP donors; five different PBMC donors). Statistical analysis was performed by Friedman´s test with Dunn´s multiple comparison post hoc test (* p < 0.05). Unstimulated and cytocine stimulated EVs enhanced the frequency of regulatory T cells without affecting the general activation level of total CD4⁺ T cells in anti-CD3 stimulated PBMC cultures.

Moreover, the treatment with isolated EVs contributed towards a reduced pro-inflammatory cytokine profile in anti-CD3 stimulated PBMC cultures (Figure 33). Two pro-inflammatory

unPBS

CD4⁺T cells in stimulated PBMC cultures

un PBS

67 cytokines, namely IFNγ and TNFα, were significantly lowered up to 30% in its concentration in anti-CD3 stimulated PBMCs treated with unstimulated EVs or cytokine stimulated EVs in comparison to the PBS treated control (median IFNγ concentration (range): PBS = 28.82 (9.17 - 77.93) µg/mL; EVs = 18.49 (5.78 - 80.24) µg/mL; EVs^(cyt) = 21.86 (5.08 - 44.56) µg/mL;

median TNFα concentration (range): PBS = 1.36 (0.32 - 2.92) µg/mL; EVs = 0.76 (0.20 - 1.66) µg/mL; EVs^(cyt) = 0.49 (0.12 - 1.98) µg/mL). Not only concentrations of pro-inflammatory but also anti-inflammatory cytokines were affected in these stimulated immune responses. Anti-CD3 stimulated PBMC cultures exhibited significantly more active TGFβ in cultures treated with unstimulated EVs or cytokine stimulated EVs in comparison to the PBS control (median TGFβ concentrations (range): PBS = 15.06 (2.2 - 20.57) pg/mL; EVs = 22.57 (3.56 - 26.29) pg/mL; EVs^(cyt) = 22.84 (7.38 - 26.47) pg/mL). Interestingly, significantly more IL-10 was measured upon treatment with cytokine stimulated EVs in anti-CD3 stimulated PBMC cultures in comparison to the respective PBS control, while unstimulated EVs enhanced solely by trend the IL-10 concentration in the respective samples (median IL-10 concentrations (range): PBS = 159.10 (23.76 - 369.10) pg/mL; EVs = 204.70 (23.66 - 369.10) pg/mL; EVs^(cyt) = 232.40 (37.90 - 379.90) pg/mL; Figure 33). In addition IL-17a and IL-1ß were also investigated for the respective concentrations. While comparable results were determined for IL-17a, a reduced tendency of IL-1ß concentrations were detected in anti-CD3 stimulated PBMC cultures treated with unstimulated or cytokine stimulated EVs (median IL-1ß concentrations: PBS = 660.80 pg/mL; EVs = 496.40 pg/mL; EVs^(cyt) = 469.30 pg/mL; median IL-1ß concentrations: PBS = 164.10 pg/mL; EVs = 188.10 pg/mL; EVs^(cyt) = 197.4 pg/mL; Figure 33).

Figure 33: EVs attenuated the in-flammatory milieu in anti-CD3 stimulated PBMC cultures.

PBMCs (3x10⁵/well) were stimulated with anti-CD3, treated with 12 µg/mL unstimulated (EVs), cytokine stimula-ted EVs (EVs^(cyt)), PBS in equal volumes as EVs (PBS), or they were left untreated. After three days, the conditioned medium was collected and the concentrations of released cytokines investigated by ELISAs (IFNγ, active TGFβ) or bead-based Multiplex assay (IL-10, TNFα, IL-17a, IL-1β). The obtained individual cytokine concentrations are displayed as median with data range (n = 8, four different CardAP donor, four different PBMC donors). Statistical analysis was performed by Friedman´s test with Dunn´s multiple comparison post hoc test (** p < 0.01, * p < 0.05).

PBS EVs EVs^(cyt)

68

Treatment with unstimulated and cytokine stimulated EVs resulted in a reduced pro-inflammatory cytokine milieu of anti-CD3 stimulated PBMC cultures.

4.6.3 EVs modulate induced immune responses in a CD14

cell