JC Virus-DNA Detection Is Associated with CD8 Effector Accumulation in Peripheral Blood of Patients with Multiple Sclerosis under Natalizumab Treatment, Independently from JC Virus Serostatus

Abstract

Although natalizumab (anti-α4 integrin) represents an effective therapy for relapsing remitting multiple sclerosis (RRMS), it is associated with an increased risk of developing progressive multifocal leukoencephalopathy (PML), caused by the polyomavirus JC (JCV). The aim of this study was to explore natalizumab-induced phenotypic changes in peripheral blood T-lymphocytes and their relationship with JCV reactivation. Forty-four patients affected by RRMS were enrolled. Blood and urine samples were classified according to natalizumab infusion number: 0 (N0), 1-12 (N12), 13-24 (N24), 25-36 (N36), and over 36 (N > 36) infusions. JCV-DNA was detected in plasma and urine. T-lymphocyte phenotype was evaluated with flow cytometry. JCV serostatus was assessed. Ten healthy donors (HD), whose ages and sexes matched with the RRMS patients of the N0 group, were enrolled. CD8 effector (CD8 E) percentages were increased in natalizumab treated patients with detectable JCV-DNA in plasma or urine compared to JCV-DNA negative patients (JCV-) (p < 0.01 and p < 0.001, resp.). Patients with CD8 E percentages above 10.4% tended to show detectable JCV-DNA in plasma and/or urine (ROC curve p = 0.001). The CD8 E was increased when JCV-DNA was detectable in plasma or urine, independently from JCV serology, for N12 and N24 groups (p < 0.01). As long as PML can affect RRMS patients under natalizumab treatment with a negative JCV serology, the assessment of CD8 E could help in the evaluation of JCV reactivation.

Figure 1

Discordance between the Stratify JCV assay and JCV-DNA detection in blood and urine samples. The pie charts represent the percentages of concordance and discordance between JCV antibody (Ab) and JCV-DNA detection in urine and plasma. Percentages of double positive JCV-DNA+ and JCV-Ab+ cases: 21% (4/19), 12% (6/50), 16.7% (7/42), 28.6% (4/14), and 33.3% (2/6) at N0, N12, N24, N36, and N > 36, respectively. Percentages of JCV-DNA− and JCV-Ab+ cases: 21% (4/19), 18% (9/50), 23,8% (10/42), 21.4% (3/14), and 50% (3/6) of cases at N0, N12, N24, N36, and N > 36, respectively. Percentages of cases with double negative JCV-DNA− and JCV-Ab−: 36,8% (7/19), 58% (29/50), 42,9% (18/42), 35.7% (5/14), and 16.7% (1/6) of cases at N0, N12, N24, N36, and N > 36, respectively. Percentages of discordant cases with JCV-DNA+ and JCV-Ab− (separated sector): 21.1% (4/19), 12% (6/50), 16.7% (7/42), 14.3% (2/14), and 0% (0/6) of cases at N0, N12, N24, N36, and N > 36, respectively. N0: 0 infusions, N12: from 1 to 12 infusions, N24: from 13 to 24 infusions, N36: from 25 to 36 infusions, and N > 36: over 36 infusions of natalizumab.

Figure 2

CD49d median fluorescence intensity (MFI) measured on CD4+ and CD8+ T-lymphocyte subsets. CD49d expression assessed on the overall CD4+ (a) and CD8+ (b) T-lymphocytes and their subpopulations in peripheral blood of RRMS patients under natalizumab treatment are represented after stratification according to natalizumab infusion number. Statistical analysis was performed using a one-way ANOVA test for nonparametrical data (Kruskal–Wallis): for CD4, p = 0.0004; CD4 N, p > 0,05; CD4 CM, p < 0,0001; CD4 EM, p = 0.0022; CD4 E, p < 0.0001; for CD8, p = 0.01; CD8 I, p < 0.0001; CD8 N, p > 0.05; CD8 CM, p < 0.0001; CD8 EM, p < 0.0001; CD8 E, p < 0.0001. Dunnett's posttest was performed comparing each group with N0, and asterisks represent the level of statistical significance. Dashed line represents median value of CD49d expression observed in healthy donors. CD49d expression on CD4+ and CD8+ T-lymphocytes decreased from N0 to N > 36 group with a linear trend (posttest for linear trend: p < 0,0001 and p = 0,0035, resp.). Lines and whiskers represent median values and interquartile ranges, respectively. N: naïve, CM: central memory, EM: effector memory, E: effectors, I: intermediate. N0: 0 infusions, N12: from 1 to 12 infusions, N24: from 13 to 24 infusions, N36: from 25 to 36 infusions, and N > 36: over 36 infusions of natalizumab. ^∗0.05 < p < 0.01; ^∗∗0.01 < p < 0.001; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 3

Evaluation of T-lymphocyte subset percentages in blood samples. Percentages of CD4+ (a) and CD8+ (b) T-lymphocyte subpopulations in peripheral blood of RRMS patients under natalizumab treatment are represented, after stratification according to natalizumab infusion number. Values are represented as percentage of the parental population (overall CD4 and CD8, resp.). Statistical analysis was performed using a one-way ANOVA test for nonparametrical data (Kruskal–Wallis, for CD4 N, p > 0.05; CD4 CM, p > 0.05; CD4 EM, p > 0.05; CD4 E, p > 0.05; CD8 N, p > 0.05; CD8 I, p > 0,05; CD8 CM, p = 0.02; CD8 EM, p = 0.0003; CD8 E, p < 0.0057). Dunnett's posttest was performed comparing each group with N0, and asterisks represent the level of statistical significance. Lines and whiskers represent median values and interquartile ranges, respectively. Dashed line represents median percentages observed in healthy donors. N: naïve, CM: central memory, EM: effector memory, E: effectors, and I: intermediate. N0: no infusions, N12: from 1 to 12 infusions, N24: from 13 to 24 infusions, N36: from 25 to 36 infusions, and N > 36: over 36 infusions of natalizumab. ^∗0.05 < p < 0.01; ^∗∗0.01 < p < 0.001; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 4

Immune activation and immune senescence levels of CD4+ and CD8+ T-lymphocytes. (a) CD4+ T-lymphocyte immune activation levels were assessed considering the percentages of HLA-DR and CD38 double positive CD4. N0: 1.6%  [0.9–1.8], N12: 1.6%  [0.9–2.3], N24: 1.7%  [1.3–2.2], N36: 2.7%  [1.6–3.2], and N > 36: 2.1  [1.8–2.4]. Statistical analysis was performed using a one-way ANOVA test for nonparametrical data (Kruskal–Wallis, p = 0.0099). Dunnett's posttest was performed comparing each group with N0, and asterisks represent the level of statistical significance. Dashed line represents median value of CD4+ T-lymphocyte immune activation levels observed in healthy donors. (b) CD8+ T-lymphocyte immune activation levels were assessed considering the percentages of HLA-DR and CD38 double positive CD8. N0: 1.5%  [1.0–1.9], N12: 2.3%  [1.4–4.7], N24: 2.7%  [1.7–4.8], N36: 3.6%  [2.3–4.3], and N > 36: 3.0%  [2.0–5.2]. Statistical analysis was performed using a one-way ANOVA test for nonparametrical data (Kruskal–Wallis, p = 0.0025). Dunnett's posttest was performed comparing each group with N0, and asterisks represent the level of statistical significance. Dashed line represents median value of CD8+ T-lymphocyte immune activation levels observed in healthy donors. Immune senescence levels were evaluated for CD4+ (c) and CD8+. (d) T-lymphocytes as the percentages of CD28 negative and CD57 positive cells. No statistical significant differences were found after performing a one-way ANOVA test for nonparametrical data (Kruskal–Wallis). Lines and whiskers represent median values and interquartile ranges, respectively. N0: no infusions, N12: from 1 to 12 infusions, N24: from 13 to 24 infusions, N36: from 25 to 36 infusions, and N > 36: over 36 infusions of natalizumab. ^∗0.05 < p < 0.01; ^∗∗0.01 < p < 0.001; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 5

CD8 E percentages according to JCV-DNA detection in biological samples and ROC analysis. (a) CD8 E percentages associated with JCV-DNA positive urine samples (JCVu+), JCV-DNA positive plasma samples (JCVp+), and JCV-DNA negative plasma and urine samples (JCV-DNA−) are shown: for JCVu+, 13.4%  [7.6–15.4], for JCVp+, 13.2%  [8.3–15.2], and for JCV-DNA−, 8.0%  [6.0–12.0]. Data are represented as median [interquartile range]. Statistical analysis was performed using a one-way ANOVA test for nonparametrical data (Kruskal–Wallis, p = 0.0001). Dunnett's posttest was performed comparing each group with JCV-DNA− group, and asterisks represent the level of statistical significance. (b) ROC analysis was performed using CD8 E percentages after stratification of samples according to JCV detection in plasma and/or urine in JCV-DNA+ and JCV-DNA−. The area under the curve is 0.73 with p < 0.0001. The cut-off of >10.6% shows a sensitivity of 67% (CI: 51% to 80%) and a specificity of 65% (CI: 54% to 75%). CI: confidence interval. Lines and whiskers represent median values and interquartile ranges, respectively. CD8 E: CD8 effectors. ^∗0.05 < p < 0.01; ^∗∗0.01 < p < 0.001; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 6

CD8 E percentages in JCV-DNA+ and JCV-DNA− samples, after stratification according to natalizumab infusion number. Comparison of CD8 E percentages between JCV-DNA+ (plasma and/or urine) and JCV-DNA− samples for N0: 6.6%  [3.8–11.9] versus 5.1%  [1.9–9.5], N12: 14.7%  [12.4–16.9] versus 11.1%  [6.8–13.6], N24: 13.6%  [11.8–16.2] versus 8.0%  [4.6–10.8], N36: 9.5%  [7.0–14.8] versus 8.1%  [6.5–9.7], and N > 36: 12.5%  [11.3–13.7] versus 6.7%  [6.4–7.4]. Data are shown as median [interquartile range]. Statistical analysis was performed using the 2-tailed Mann–Whitney test (p = 0.3, p = 0.002, p < 0.0001, p = 0.2, and p = 0.1, for N0, N12, N24, N36, and N > 36, resp.). Lines and whiskers represent median values and interquartile ranges, respectively. CD8 E: CD8 effectors. N0: no infusions, N12: from 1 to 12 infusions, N24: from 13 to 24 infusions, N36: from 25 to 36 infusions, and N > 36: over 36 infusions of natalizumab. ^∗0.05 < p < 0.01; ^∗∗0.01 < p < 0.001; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 7

CD8 E percentages in JCV-DNA+ and JCV-DNA− samples, after stratification according to anti-JCV specific serology and natalizumab infusion number. (a) Comparison of CD8 E percentages between JCV-DNA+ (plasma and/or urine) and JCV-DNA− samples in patients with either positive or negative JCV serology (JCV-Ab+ and JCV-Ab−, resp.). For JCV-Ab+, 13.4%  [8.2–14.8] versus 7.8%  [6.2–9.9]; for JCV-Ab−, 13.2%  [7.1–16.2] versus 8.2%  [5.8–12.7], respectively. Data are shown as median [interquartile range]. Statistical analysis was performed using the 2-tailed Mann–Whitney test (p = 0.0007 and p = 0.004 for JCV-Ab+ and JCV-Ab− groups, resp.). (b) Comparison of CD8 E percentages between JCV-DNA+ (plasma and/or urine) and JCV-DNA− samples in patients with positive JCV serology (JCV-Ab+), after stratification according to natalizumab infusion number: for N0: 6.4%  [2.8–12.1] versus 7.0%  [2.5–12.3], N12: 14.1%  [8.4–15.7] versus 7.8%  [6.5–11.8], N24: 13.4%  [12.6–16.1] versus 7.3%  [2.8–8.8], N36: 11.3%  [7.2–15.8] versus 10.2%  [8.1–23.2], and N > 36: 12.5%  [11.3–13.7] versus 6.5%  [6.4–6.8]. Data are shown as median [interquartile range]. Statistical analysis was performed using the 2-tailed Mann–Whitney test (p = 0.8, p = 0.049, p < 0.0012, p = 0.86, and p = 0.2, for N0, N12, N24, N36, and N > 36, resp.). (c) Comparison of CD8 E percentages between JCV-DNA+ (plasma and/or urine) and JCV-DNA− samples in patients with negative JCV serology (JCV-Ab−), after stratification according to natalizumab infusion number: for N0: 6.6%  [4.2–11.6] versus 5.1%  [1.4–6.2], N12: 15.4%  [13.8–18.8] versus 12.0%  [6.7–13.7], N24: 13.8%  [9.2–16.8] versus 8.4%  [4.9–11.3], N36: 8.9%  [7.1–10.8] versus 6.6%  [6.1–8.2], and N > 36: not applicable. Data are shown as median [interquartile range]. Statistical analysis was performed using the 2-tailed Mann–Whitney test (p = 0.2, p = 0.004, p < 0.008, p = 0.38, and p = not applicable, for N0, N12, N24, N36, and N > 36, resp.). Lines and whiskers represent median values and interquartile ranges, respectively. CD8 E: CD8 effectors. N0: no infusions, N12: from 1 to 12 infusions, N24: from 13 to 24 infusions, N36: from 25 to 36 infusions, and N > 36: over 36 infusions of natalizumab. ^∗0.05 < p < 0.01; ^∗∗0.01 < p < 0.001; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.