Determination of Immune Cell Counts From Whole Blood

Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) is an immune check point to downregulate T-cell activity, and expressed highly on Treg cells. CTLA-4 mutations cause autoimmunity because T-cell activity and T-cell dependent B-cell activity cannot be controlled [56]. For this reason, adaptive immune response of CTLA-4 mutation is studied well while effect on innate immune system is not fully studied. In this part of the thesis, affects of CTLA-4 mutation on both innate and adaptive immune cells were investigated.

3.3.1.1 CD4/CD8 ratio was reversed only in one patient

Healthy immune system has a balance between CD4+ and CD8+ T-cells. If CD4+/CD8+ ratio is lower than 1 it implies viral infections such as HIV while increased ratio could be reasoned by autoimmunity. According to literature, ratio between 1.5-2.5 is normal [65]. Because CTLA-4 mutation cause autoimmunity in patients, CD4+/CD8+ ratios of 4 patients were assessed. The whole blood of healthy controls and CTLA-4 patients were stained with fluorochrome conjugated anti-human CD45, anti-human CD3, anti-human CD4 and anti-human CD8 antibodies, and analyzed by flow cytometer (Figure. 3.12). Ratios of the patients are 3, 1.8, 0.7 and 1.7 while one healthy donor has 1.6, and the other has 0.7. Therefore, one of the

54

healthy control probably have an infection as well as Patient 3, and Patient 1 experience autoimmunity.

Figure 3.12 CD4 and CD8 surface expression of CD45+ CD3+ cells. The 100 µl whole blood of healthy controls and CTLA-4 patients were stained with fluorochrome conjugated anti-human CD45, anti-human CD3, anti-human CD4 and anti-human CD8 antibodies. Gating strategy is performed as forward and side scattering of lymphocytes, further as singlets. A) Density plot of CD45+ CD3+ CD4+ CD8+ cells.

B) Representative bar graphs of CD4+ CD8+ cell percentages in CD3+ cells of healthy controls and patients.

A)

B) CD4/CD8 Ratio

%cells in CD3+ Cells

CD4+ CD8+

CD4+ CD8+

CD4+ CD8+

CD4+ CD8+

CD4+ CD8+

CD4+ CD8+ 0

2 0 4 0 6 0

8 0 H e a lt h y 1 H e a lt h y 2 P a t ie n t 1 P a t ie n t 2 P a t ie n t 3 P a t ie n t 4

55

3.3.1.2 Low density granulocytes were observed in two patients

Proinflammatory low density granulocytes (LDGs) play a role in systemic autoimmunity [66]. Therefore, LDG counts of PBMCs of healthy controls and patients were determined by flow cytometer which localize between lymphocytes and granulocytes (Figure 3.13). Average LDGs from a healthy person is 1.28% from PBMC count [67]. Patient 1,2 and 3 showed increased percentage of LDGs as 2.41%, 5.41%, 3.18% compared to healthy controls, 0.38% and 1.77%, which could be result of CTLA-4 mutation which make patients prone to autoimmunity.

A)

B) LDG %

% Low Density Granulocytes

Healthy 1 Healthy 2

Patient 1 Patient 2

Patient 3 Patient 4 0

2 4 6

56

Figure 3.13 Low density granulocyte (LDG) percentages in PBMCs. Gating strategy is performed as forward and side scattering of lymphocytes. A) Density plot of LDGs. B) Representative bar graphs LDG percentages in PBMCs of healthy controls and patients.

3.3.1.3 Monocyte percentage is higher in all patients

CTLA-4 is expressed by 3% monocytes on their surfaces [68]. The whole blood of healthy controls and CTLA-4 patients were stained with fluorochrome conjugated anti-human CD14 and anti-human CD16 antibodies to count monocyte percentage in PBMCs, and analyzed by the flow cytometer (Figure 3.14). Monocyte counts of healthy controls were 4.34% and 2.65% of PBMCs while patients had 5.89%, 6.83%, 11.68% and 3.50%, respectively. Patient 3 had elevated monocyte population in the PBMC.

A)

Patient 4

57

Figure 3.14 CD14 and CD16 surface expression of lymphocytes. The 100 µl whole blood of healthy controls and CTLA-4 patients were stained with fluorochrome conjugated anti-human CD14 and anti-human CD16 antibodies. Gating strategy is performed as forward and side scattering of lymphocytes, further as singlets. A) Density plot of CD14+ CD16- cells. B) Representative bar graphs of CD14+ CD16- cell percentages in PBMCs of healthy controls and patients.

3.3.1.4 Three patients have low pDC count

Plasmacytoid dendritic cells (pDC) express CD86 which is a CTLA-4 ligand. By the CD86 and CTLA-4 ligation, Treg cells are stimulated, and T-cell priming is inhibited [69]. The whole blood of healthy controls and CTLA-4 patients were stained with fluorochrome conjugated anti-human CD123 and anti-human CD303 antibodies to examine pDC count from PBMCs by flow cytometric analysis (Figure 3.15). Patient 2, 3 and 4 had reduced count of pDCs while Patient 1 had normal pDC count compared to healthy controls.

B) Monocyte %

%CD14+CD16- Cells

Healthy 1 Healthy 2

Patient 1 Patient 2

Patient 3 Patient 4 0

5 10 15

58

Figure 3.15 CD123 and CD303 surface expression of lymphocytes. The 100 µl whole blood of healthy controls and CTLA-4 patients were stained with fluorochrome conjugated anti-human CD123 and anti-human CD303 antibodies. Gating strategy is performed as forward and side scattering of lymphocytes, further as singlets. A) Density plot of CD123+ CD303+ cells. B) Representative bar graphs of CD123+

CD303+ cell percentages in PBMCs of healthy controls and patients.

A)

B) pDC %

%CD123+ CD303+ Cells

Healthy 1 Healthy 2

Patient 1 Patient 2

Patient 3 Patient 4 0.0

0.2 0.4 0.6 0.8

59

3.3.1.5 B-cell percentage is normal in patients

CTLA-4 prevent T-cell mediated autoimmunity by downregulating T-cell response, and T-cell dependent B-cell response is also controlled by CTLA-4 indirectly. The whole blood of healthy controls and CTLA-4 patients were stained with fluorochrome conjugated anti-human CD45, anti-human CD3 and anti-human CD19 antibodies to determine B-cell percentage in PBMCs, and analyzed by flow cytometer (Figure 3.16). All patients had normal B-cell percentage as compared to healthy controls.

Figure 3.16 CD19 surface expression of CD45+ CD3- cells. The 100 µl whole blood of healthy controls and CTLA-4 patients were stained with fluorochrome conjugated anti-human CD45, anti-human CD3 and anti-human CD19 antibodies. Gating strategy is performed as forward and side scattering of lymphocytes, further as singlets. A) A)

B) B-cell %

CD3- CD19+ Cells

Healthy 1 Healthy 2

Patient 1 Patient 2

Patient 3 Patient 4 0

2 4 6

60

Density plot of CD3- CD19+ cells. B) Representative bar graphs of CD19+ cell percentages in CD45+ CD3- cells of healthy controls and patients.

3.3.1.6 Treg count is decreased in three patients

Treg cells contain CTLA-4 protein to regulate T-cell activity by downregulation. The whole blood of controls and patients were stained with fluorochrome conjugated anti-human CD45, anti-anti-human CD3, anti-anti-human CD4, anti-anti-human CD25 and anti-anti-human CD127 antibodies to stain Treg cell population inside the PBMCs (Figure 3.17).

Patient 2,3 and 4 had lower Treg count as 1.81%, 1.43% and 2.12%, respectively, while Patient 1 and controls had 2 fold more Treg percentage as 3.29% and 3.68%.

A)

61

Figure 3.17 CD25 and CD127 surface expression of CD45+ CD3+ CD4+ cells.

The 100 µl whole blood of controls and CTLA-4 patients were stained with fluorochrome conjugated human CD45, human CD3, human CD4, anti-human CD25 and anti-anti-human CD127 antibodies. Gating strategy is performed as forward and side scattering of lymphocytes, further as singlets. A) Density plot of CD45+ CD3+ CD4+ CD25+ CD127dim cells. B) Representative bar graphs of CD4+

CD25+ CD127dim cell percentage of healthy controls and patients.