High-risk oncogenic HPV genotype infection associates with increased immune activation and T cell exhaustion in ART-suppressed HIV-1-infected women

Figures & data

Table 1. Patients characteristics.

Variable	Negative for high-risk HPV, Negative histopathology (group 1, n = 16)	Positive for high-risk HPV, Negative histopathology (group 2, n = 15)	Positive for high-risk HPV, CIN1/2/3 (group 3, n = 24)
Age (y), median (25th, 75th percentile)	35 (31.5, 39.75)	37 (29, 41)	34.5 (28.25, 39.75)
CD4^+ T cell count at recruitment (cells/mm^3), median (25th, 75th percentile)	568.5 (395.7, 754)	517 (399.5, 602)	435.5 (308, 673.75)
Nadir CD4^+ T cell count (cells/mm^3), median (25th, 75th percentile)	158 (93.2, 226.7)	98 (58.5, 142)	150 (88, 206)
Absolute number of subjects using the following drug combinations:
Two NRTI with either one NNRTI or CCR5 inhibitor	14	12	19
One PI with either two (or three) NRTI or one NNRTI	2	3	5
Low-risk HPV type frequency (%)	6 (12.5%); 61, 62, 72 (6.25%)	61, 72 (20%); 62, 81 (13.3%); 11, 53, 70, 71, 83, CP6108, (6.67%)	61 (20.8%); 55 (16.6%); 62, 81, 83 (12.5%); 26, 53, 72, (8.3%); 6, 11, 42, 69, 84, CP6108 (4.16%)
High-risk HPV-type frequency (%)	0	16 (53.3%); 56 (20%); 18, 45, 68 (13.3%); 35, 51, 52, 59, 66 (6.67%)	52 (37.5%); 16 (33.3%); 35, 45, 51 (20.8%); 58 (16.6%); 18 (12.5%); 31, 33, 68 (8.3%); 56 (4.16%)
Age for first sexual encounter (y), median (25th, 75th percentile)	17.5 (16, 18.75)	18 (16, 20)	17.5 (16, 19.75)
Number of sexual partners, median (25th, 75th percentile)	4.5 (3, 6)	5 (3, 9)	5.5 (4, 98)
Age for first pregnancy (y), median (25th, 75th percentile)	20 (19, 25)	20.5 (18, 22)	20 (18, 23.75)
Number of pregnancies, median (25th, 75th percentile)	2 (2, 4)	3 (1, 3)	2 (1, 3)
Absolute number of women using contraception	9	12	20
Absolute number of women smoking cigarettes or chewing tobacco	2	1	5
Absolute number of women using marijuana/intravenous drugs	0	0	0
Absolute number of women drinking alcohol	4	0	2

Figure 1. Association between cervical histopathology status and increased cervical cell density per square millimeter in ART-treated HIV⁺/HPV⁺ women. Immunohistochemical staining for CD4⁺, CD8⁺, CD68 and CD11c is shown in epithelium and stroma from cervical biopsies of representative women in each group: HR (-) HPV with negative cervical histopathology (group 1, study subject TJA1019081, top panel), HR (+) HPV with negative cervical histopathology (group 2, study subject TJA1040434, middle panel), and HR (+) HPV with CIN1 or CIN2/3 (group 3, study subject TJA1104317, bottom panel).

Table 2. Differences in cervical cell density per square millimeter amongst study groups.

Variable	Group 1 Median (25th, 75th percentile)	n	Group 2 Median (25th, 75th percentile)	n	Group 3 Median (25th, 75th percentile)	n	P (1 vs. 2)	P (1 vs. 3)	P (2 vs. 3)
CD4^+ epithelium	22.31 (9.8, 34.64)	16	31.4 (11.3, 59.3)	15	55.7 (35.75, 91.6)	24	ns	0.003	ns
CD4^+ stroma	44.8 (32.1, 56.8)	16	47.5 (29.2, 157)	15	131.5 (88.4, 195.22)	24	ns	<0.001	ns
CD4^+ Total	66.64 (47.8, 87.9)	16	97.3 (44.74, 213.82)	15	211.5 (123.83, 320.22)	24	ns	<0.001	ns
CD8^+ epithelium	22.02 (9.01, 39.2)	16	17.3 (6.9, 71.5)	15	34.15 (15.9, 47.52)	24	ns	ns	ns
CD8^+ stroma	39.95 (28.04, 56.8)	16	67.34 (34.52, 147.95)	15	183.85 (78.28, 257.35)	24	ns	<0.001	ns
CD8^+ Total	68.46 (45.74, 81.31)	16	93.11 (47.7, 208.16)	15	224 (117.1, 286.02)	24	ns	<0.001	ns
CD68^+ epithelium	4.77 (2.57, 10.82)	16	7.81 (3.95, 19.8)	15	34.95 (14.7, 43.54)	24	ns	<0.001	0.012
CD68^+ stroma	20.9 (13.75, 35.05)	16	28 (22.33, 104.5)	15	67.33 (43.24, 90.25)	24	ns	<0.001	ns
CD68^+ Total	28.65 (17.19, 45.43)	16	35.4 (27.33, 134.63)	15	98.9 (76.1, 137.7)	24	ns	<0.001	ns
CD11c^+ epithelium	7.1 (1.56, 12.1)	16	8.06 (3.25, 13.7)	15	24.15 (10.03, 61.8)	24	ns	0.006	ns
CD11c^+ stroma	9.92 (6.61, 13.4)	16	13.9 (4.6, 38.7)	15	36.3 (19.05, 70.8)	24	ns	<0.001	ns
CD11c^+ Total	15.1 (10.5, 27.15)	16	25.8 (8.22, 54.5)	15	68.7 (35.8, 125.9)	24	ns	<0.001	ns

ns: non-significant, p >0.05

Figure 2. Association between HR HPV and systemic T cell activation and Tregs cell subsets. CD3⁺CD8⁺CD38⁺ percentages (%) of lymphocytes (top panel) and CD4⁺CD25⁺FoxP3⁺ (Tregs) % of lymphocytes (bottom panel) are shown for women in groups 1, 2 and 3. Data are shown as interquartile box plots with median and outliers for each group, and significant (<0.05) p values.

Figure 3. Association between HR HPV and systemic immune exhaustion. Left panel shows CD3⁺CD8⁺PD1⁺ (top), CD3⁺CD8⁺PD1^-CTLA-4⁺ (middle), and CD3⁺CD8⁺BTLA⁺CD160⁺ (bottom) % of lymphocytes, while right panel shows Lin2^-HLA-DR⁺CD11c⁺PDL1⁺PDL2⁺ (top), Lin2^-HLA-DR⁺CD11c⁺CD86⁺CD40⁺ (middle), and Lin2^-HLA-DR⁺CD11c⁺HVEM⁺ (bottom) % of live cells for women in groups 1, 2 and 3. Data are shown as interquartile box plots with median and outliers for each group, and significant (<0.05) p values.

Table 3. Significant differences in immune variables amongst study groups.

Category	Variable	Group 1 Median (25th, 75th percentile)	n	Group 2 Median (25th, 75th percentile)	n	Group 3Median (25th, 75th percentile)	n	P (1 vs. 2)	P (1 vs. 3)	P (2 vs. 3)
Activation (T cells)	CD3^+CD8^+CD38^+ %	15.05 (11.2, 18.9)	16	20.25 (14.73, 23.15)	14	25.15 (17.85, 35.7)	24	ns	0.002	ns
	MFI of CD38 on CD3^+CD8^+	241 (171.75, 359.25)	16	351.5 (299.75, 397)	14	460 (289, 550.5)	24	ns	0.047	ns
	MFI of CD38 on CD3^+CD8^-	397 (250.5, 464.75)	16	595 (432.5, 789.25)	14	643.5 (386.5, 751)	24	0.02	0.02	ns
Exhaustion (T cells)	CD4^+CD25^hiFoxP3^+ %	0.92 (0.34, 3.51)	16	6.5 (5.51, 9.1)	14	5.51 (2.43, 9.3)	24	0.003	0.02	ns
	CD3^+CD8^+PD1^+ %	0.7 (0.43, 1.4)	16	3.21 (1.6, 4.52)	14	4.05 (2.02, 6.22)	23	0.003	<0.001	ns
	CD3^+CD8^+CTLA-4^+ %	0.33 (0.3, 0.4)	16	0.6 (0.44, 0.82)	14	0.61 (0.42, 0.8)	23	0.01	ns	ns
	MFI of CTLA-4 on CD3^+CD8^+	31 (26.42, 34.52)	16	24.95 (23.6, 26.3)	14	23.7 (21.25, 27.8)	24	0.045	0.01	ns
	CD3^+CD8^+BTLA^+ %	28.83 (23.5, 33.7)	16	32.7 (25.81, 42.3)	14	36.7 (33.4, 44.9)	24	ns	<0.001	ns
	CD3^+CD8^+CD160^+ %	6.32 (3.23, 8.62)	15	9.2 (6.43, 14.5)	14	12.65 (9.25, 15.55)	24	ns	0.001	ns
	MFI of CD160 on CD3^+CD8^+	176 (122, 185)	15	241 (157.75, 289.5)	14	245 (219.5, 294)	24	ns	0.005	ns
Exhaustion (DC)	Lin2^-HLADR^+CD11c^+PDL1^+PDL2^+%	0.02 (0.02, 0.05)	16	0.06 (0.04, 0.09)	14	0.07 (0.04, 0.14)	22	ns	0.01	ns
	Lin2^-HLADR^+CD11c^+HVEM^+ %	0.44 (0.07, 0.8)	16	0.81 (0.6, 1.2)	14	1.05 (0.43, 1.8)	22	ns	0.03	ns
	MFI of HVEM on Lin2^-HLADR^+CD11c^+	386.5 (129.75, 444.75)	16	581 (467.25, 657.25)	14	513 (414.75, 621)	22	0.03	ns	ns
	MFI of HVEM on Lin2^-HLADR^+CD123^hi	417 (215.75, 486.75)	16	588.5 (521.5, 688.5)	14	519.5 (452.5, 593.5)	22	0.002	0.04	ns
	Lin2^-HLADR^+CD11c^+CD86^+CD40^+ %	0.1 (0.03, 0.35)	16	0.5 (0.25, 0.72)	14	0.51 (0.3, 0.93)	24	ns	0.02	ns
NK cell subsets	CD3^-CD56^bright %	0.4 (0.31, 0.5)	15	0.6 (0.4, 1.02)	14	0.3 (0.13, 0.41)	24	ns	ns	0.009
	CD3^-CD56^brightCD94^+ %	0.4 (0.22, 0.43)	15	0.6 (0.4, 1.02)	14	0.25 (0.13, 0.4)	24	0.04	ns	0.006
	CD3^-CD56^brightCD16^- %	0.25 (0.1, 0.34)	15	0.33 (0.24, 0.71)	14	0.2 (0.1, 0.4)	24	ns	ns	0.01

ns: non-significant, P>0.05

Table 4. Significant correlations with CD4⁺ T cell count.

Group	Category	Variable	n	p	rho
High-risk HPV (group 2 + group 3)	Activation (T cells)	CD3^+CD8^-CD38^+ %	38	0.01	0.40
	Exhaustion (T cells)	CD3^+CD8^+CD160^+ %	38	0.02	−0.37
	Exhaustion (DC)	Lin2^-HLA-DR^+CD11c^+HVEM^+ %	36	0.02	−0.37
		Lin2^-HLA-DR^+CD11c^+PDL1^+PDL2^+HVEM^+ %	36	0.04	−0.35
		MFI of PDL1PDL2 on Lin2^-HLA-DR^+CD11c^+	36	0.02	−0.38
	NK cell subsets	CD3^-CD56^dimCD16^- %	38	0.009	−0.42
No high-risk HPV (group 1)	Activation (T cells)	CD3^+CD8^+CD38^+ %	16	0.03	0.55
	Exhaustion (T cells)	CD3^+CD8^+PD1^+ %	16	0.04	0.53
	Exhaustion (DC)	Lin2^-HLA-DR^+CD11c^+PDL1^+PDL2^+HVEM^- %	16	0.006	−0.66
	NK cell subsets	CD3^-CD56^brightCD16^-CD94^+ %	15	0.03	−0.57

Figure 4. Association between systemic CD8⁺ T cell activation and systemic immune activation/exhaustion. (A) Correlation between MFI of CD38 on CD3⁺CD8⁺ T cells and systemic immune activation/exhaustion [MFI HVEM on Lin2^-HLA-DR⁺CD11c⁺ (top left), and MFI HVEM on Lin2^-HLA-DR⁺CD123^hi (top right)] in women with HR HPV (group 2 + 3); (B) Correlation between CD3⁺CD8⁺CD38⁺ % of lymphocytes and systemic immune activation/exhaustion [CD3⁺CD8⁺PD1⁺ (bottom left), and CD3⁺CD8⁺BTLA⁺ (bottom right) % of lymphocytes] in women with HR HPV (group 2+3). Data are shown as regression lines, with number of subjects (n), correlation and p values.

Table 5. Significant correlations with CD8⁺ T cell activation.

Group	Category	Variable	Correlation with		n	p	rho
High-risk HPV (group 2+ group 3)	Exhaustion (T cells)	CD3^+CD8^+PD1^+ %	CD8^+CD38^+ %		37	0.001	0.52
		CD3^+CD8^+CTLA-4^+ %			37	0.01	0.42
		MFI of PD1 on CD3^+CD8^-			38	<0.001	0.55
		CD3^+CD8^+BTLA^+ %			38	<0.001	0.64
		CD3^+CD8^+CD160^+ %			38	0.01	0.41
	Exhaustion (DC)	MFI of HVEM on Lin2^-HLA-DR^+CD123^hi	MFI of CD38 on CD3^+CD8^+		36	0.01	0.41
		MFI of HVEM on Lin2^-HLA-DR^+CD11c^+			36	<0.001	0.58
		MFI of PDL1PDL2 on Lin2^-HLA-DR^+CD123^hi			36	0.007	0.45
		MFI of PDL1PDL2 on Lin2^-HLA-DR^+CD11c^+			36	0.002	0.51
	NK cell subsets	CD3^-CD56^bright %	CD8^+CD38^+ %		38	0.02	−0.37
		CD3^-CD56^brightCD94^+ %			38	0.02	−0.38
		CD3^-CD56^brightCD16^+ %			38	<0.001	−0.57
		CD3^-CD56^dim %			38	0.04	−0.34
		CD3^-CD56^dimCD94^+ %			38	0.03	−0.35
No high-risk HPV (group 1)	Exhaustion (T cells)	MFI of CD160 on CD3^+CD8^+	MFI of CD38 on CD3^+CD8^+		15	0.006	0.68
	Exhaustion (DC)	Lin2^-HLA-DR^+CD11c^+CD86^-CD40^+ %	CD8^+CD38^+ %		16	0.03	0.56
		Lin2^-HLA-DR^+CD11c^+HVEM^+ %	MFI of CD38 on CD3^+CD8^+		16	0.04	0.52
	NK cell subsets	CD3^-CD56^bright %	CD8^+CD38^+ %		15	0.03	−0.57
		CD3^-CD56^brightCD94^+ %			15	0.048	−0.52