Thymic involution and proliferative T-cell responses in multiple sclerosis

Journal of Neuroimmunology 221 (2010) 73–80

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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

Thymic involution and proliferative T-cell responses in multiple sclerosis Danielle A. Duszczyszyn a,1, Julia L. Williams a,1, Helen Mason a, Yves Lapierre b, Jack Antel b, David G. Haegert a,⁎,1 a b

Department of Pathology, McGill University, Duff Medical Building, 3775 rue University, Montreal, Quebec, Canada H3A 2B4 Department of Neurology, McGill University, 3601 rue University, Montreal, Quebec, Canada H3A 2B4

a r t i c l e

i n f o

Article history: Received 27 September 2009 Received in revised form 8 January 2010 Accepted 8 February 2010 Keywords: T-cells MS Neuroimmunology T-cell receptors Homeostasis

a b s t r a c t We investigated naïve CD4 T-cell homeostasis in relapsing–remitting multiple sclerosis (RRMS). Quantification of signal joint T-cell receptor excision circles in FACS-isolated CD31hi cells, which correspond closely to CD4 recent thymic emigrants (RTEs), indicates that young patients have reduced generation of CD4 RTEs compared to age-matched controls. In RRMS, compared to controls, CXCR4 analyses indicate ageassociated thymic output of progressively immature CD4 RTEs, and Ki-67 data demonstrate altered T-cell proliferative responses that fail to maintain naïve CD4 T-cell numbers with age. Thus, RRMS patients have early thymic involution with compensatory homeostatic peripheral T-cell proliferative responses that may predispose patients to autoreactivity. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Current opinion invokes an autoimmune response targeting CNS antigens as the pathogenetic mechanism in MS (Hafler et al., 2005; Sospedra and Martin, 2005). Naïve CD4 T-cells have a central role in initiating autoimmune responses (Muraro et al., 2000; Sospedra and Martin, 2005). Naïve T-cell homeostasis balances thymic production, and peripheral T-cell proliferative responses, which compensate for age-associated thymic involution (Almeida et al., 2005; Jameson, 2002). In lymphopenic mice, homeostatic T-cell changes lead to serious autoimmune disease (King et al., 2004). Some investigators think that increased homeostatic T-cell proliferation leads to autoreactivity in the elderly (Kilpatrick et al., 2008; Prelog, 2006) and may trigger the onset of autoimmune disease (Datta and Sarvetnick, 2009; King et al., 2004). We hypothesized that RRMS patients have a thymic alteration that reduces generation of naïve CD4 T-cells, and in turn, leads to compensatory peripheral T-cell proliferative responses. Signal joint T-cell receptor excision circles (sjTRECs) form late during thymopoiesis and are often measured in T-cells or PBMC as an index of thymic output (Dion et al., 2004; Douek et al., 1998; Hazenberg et al., 2000; Kilpatrick et al., 2008; Kimmig et al., 2002; Kohler and Thiel, 2009; van

Abbreviations: MS, multiple sclerosis; RRMS, relapsing–remitting MS; TREC, TCR excision circle; sjTREC, signal joint TREC; RTE, recent thymic emigrant; MFI, mean fluorescence intensity. ⁎ Corresponding author. Tel.: +1 514 398 5599; fax: +1 514 398 3465. E-mail address: [email protected] (D.G. Haegert). 1 D. A. D., J. L. W., and D.G. H. contributed equally. 0165-5728/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2010.02.005

den Dool and de Boer, 2006). We used three strategies to investigate thymic output, based on sjTREC analyses; to avoid potential treatment influences on sjTREC levels, we recruited patients who had never been treated with cytotoxic agents, e.g. mitoxantrone, and who had not been treated with corticosteroids or immunomodulatory agents in the year preceding the study. Firstly, we quantified sjTRECs in constant naïve CD4 T-cell numbers, i.e. we determined sjTREC frequencies. We found that patients under age 40 have decreased sjTRECs compared to age-matched controls. Secondly, in an attempt to confirm that this decrease was caused by reduced thymic output, we determined the proportion of naïve CD4 T-cells expressing CD31, as some claim that CD31pos naïve CD4 T-cells are recent thymic emigrants (RTEs) (Kimmig et al., 2002; Kohler and Thiel, 2009). Our sjTREC frequency analysis indicated, however, that CD31pos cells proliferate yet retain CD31. Accordingly, in a third strategy, we isolated CD4 T-cells having highest CD31 expression (CD31hi) to see whether these cells alone would be a better marker of CD4 RTEs. These cells had significantly higher absolute sjTREC numbers than CD31lo or CD31neg naïve CD4 T-cells. We show that young patients have CD31hi absolute sjTREC numbers comparable to older controls, i.e. patients have early thymic involution. We used two strategies to investigate peripheral T-cell proliferative responses to thymic involution. Firstly, we quantified sjTREC frequencies in FACS-isolated naïve CD4 T-cells, CD31hi, CD31lo and CD31neg cells. Secondly, we measured the proliferation marker Ki-67 (Gerdes et al., 1984). In controls, the combined sjTREC frequency and Ki-67 data indicate naïve CD4 T-cell proliferation, particularly of CD31hi cells, maintains absolute naïve CD4 T-cell numbers as thymic involution occurs with age. In contrast, in patients, naïve CD4 T-cell

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proliferation, particularly of CD31neg and CD31hi cells, was insufficient to compensate for early thymic involution in patients, as indicated by decreasing absolute naïve CD4 T-cell numbers with age. We also considered the possibility that patients differ from controls in thymic responses to age-associated thymic involution. To that end, we compared CD31hi cells from patients and age-matched controls for expression of the chemokine receptor CXCR4, which plays a critical role in thymic output of CD4 RTEs (Poznansky et al., 2002). Our CXCR4 findings show that early thymic involution in patients is associated with thymic output of progressively immature CD4 RTEs with age. 2. Materials and methods 2.1. Participants Initially, we recruited a total of 68 RRMS patients (mean age 39.5), diagnosed according to accepted criteria (McDonald et al., 2001) and in clinical remission, and a total of 68 healthy controls (mean age 41). Table 1A provides further information about the total patient and control groups; we did not match for sex, as sjTRECs did not differ between male and female controls (see Suppl Fig. 1). For detailed analyses of naïve CD4 T-cell subsets by FACS/flow cytometry (see Fig. 1), we studied a total of 12 patients and 12 age-matched controls (see Table 1B); we recruited 6 patients from the patient group in Table 1A and 6 additional patients. All patients were recruited from the Montreal Neurological Hospital MS clinics. For all studies, patient exclusion criteria included a history of treatment with cytotoxic agents at any time and treatment with immunomodulatory agents or corticosteroids in the previous year. We bled patients and controls on a single occasion for analysis of isolated naïve CD4 T-cells and on a single occasion for analysis of naïve CD4 T-subsets. Ethical approval for the study was obtained from The Institutional Review Board, McGill University.

version 7.2 (Tree Star, Inc., Ashland OR, USA) to analyze gated cells triple-stained with antibodies to CD4, CD45RA, CD31 plus either Bcl-2, CD127, CXCR4 or Ki-67. For absolute cell counts, e.g. for CD31hi cells, the Royal Victoria Hospital Immunohematology Laboratory (Montreal) determined CD3+CD4+CD45RA+counts/ml in whole blood, which we multiplied by the % CD4+CD45RA+CD31hi cells among CD3+CD4+ CD45RA+ cells from FACS analysis. 2.3. TRECs We quantitated sjTRECs in triplicate using real-time quantitative TaqMan PCR as reported (Duszczyszyn et al., 2006). For βTRECs (Dion et al., 2004; van den Dool and de Boer, 2006) (see Results section), we used Bluescript plasmids (from M.L. Dion, University of Montreal, Montreal, Canada), containing CD3γ plus one of 6 βTRECS (β1.3–2.2) and nested quantitative real-time TaqMan PCR, including primers, probes and second-round quantitation reported by Dion et al. (2004). To determine absolute sjTRECs (sjTRECs/ml), we determined T-cell numbers (# T-cells) for each T-subset, using CD3γ probes and CD3γ standard curves, in each PCR reaction as described (Dion et al., 2004; Duszczyszyn et al., 2006). Thus, for CD31hi cells: sjTRECs/ml=sjTRECs÷#T-cells/ CD31hi cells/ml. 2.4. Statistics Statistical analyses were performed using MINITAB, version 13.1. Probability distributions were determined by maximum likelihood and least squares analysis. For TREC and CD31 analyses we used Mann–Whitney U and Spearman's correlation (ρ and p). We used Student's t test and Pearson correlations, r and p, for data having a normal distribution. A p ≤ 0.05 was considered significant. We used box–whisker-plots to identify data more than 1.5 times the interquartile range, i.e. statistical outliers; these data were removed from statistical analyses.

2.2. Cell isolation and immunophenotyping

3. Results

We isolated peripheral blood CD4+CD45RA+ (naive CD4) T-cells by MACS technology as described previously (Duszczyszyn et al., 2006). We used the FACSAria™ (Becton Dickinson, San Diego) in one experiment to isolate naïve CD4, CD31pos and CD31neg T-cells and, in a second experiment, to isolate naïve CD4, CD31hi, CD31lo and CD31neg T-cells (see Fig. 1). Naïve CD4 T-cell purity was always above 98%, indicated by co-expression of CD4 and CD45RA and negative staining for CD45RO. Naïve CD4 T-subset purity was always above 95%, indicated by flow cytometry analysis of aliquots of the FACSisolated subsets (CD31pos, CD31hi, CD31lo and CD31neg cells) stained with antibodies to CD4, CD45RA and CD31. We used FlowJo,

3.1. Naïve CD4 T-cells and sjTRECs

Table 1 RRMS patients and controls. Total age-matched patients (59 women, 9 men) and controls (40 women, 28 men) included in the study, showing mean ± SD, with the range of disease duration and EDSS shown in brackets. Age-matched patients (7 women, 5 men) and controls (7 women, 5 men) included in the detailed FACS/flow cytometry analyses of naïve CD4 T-subsets.

A. Patients (n = 68) Controls (n = 68)

B. Patients (n = 12) Controls (n = 12)

Age

Disease duration

EDSS

41 ± 8.6 (21–57) 39.5 ± 10.1 (22–57)

11.4 ± 7.6 (1–32)

1.7 ± 1.5 (0–6)

39.8 ± 10.2 (23–55) 39.9 ± 10.2 (23–54)

11.03 ± 6.8 (1–21)

1.04 ± 1.03 (0–1.5)

Initially, to investigate thymic output, we compared sjTREC frequencies in isolated CD4+CD45RA+ (naïve CD4) T-cells from all patients and controls. These frequencies decreased exponentially with age, corresponding in controls to an ∼1 log decrease between ages 22 and 57 (slope of −0.0696 sjTRECs/year), and in patients to an ∼0.5 log decrease between ages 21 and 57 (slope of − 0.048 TRECs/year). Patients had lower sjTRECs than controls (Fig. 2). Since sjTRECs decreased exponentially with age, we anticipated that total patient–control differences in sjTREC frequencies would be most pronounced in young individuals. As reported in several other sjTREC studies (Geenen et al., 2003; Dion et al., 2004), we divided patients and controls into under-40 and over-40 age groups. Controls under age 40 had higher sjTRECs than age-matched patients or controls over 40. sjTRECs did not differ between controls and patients over 40 (Fig. 3). Thus, total patient–control differences in naïve CD4 T-cell sjTRECs were due essentially to reduced sjTRECs in under-40 patients. In total patients, sjTRECs did not correlate with disease duration (ρ = −0.187, p = 0.175). 3.2. Naïve CD4 T-cells: CD31 and sjTRECs In an attempt to see whether initial sjTREC findings in the total patient group (Fig. 2) were due to reduced thymic output, we measured the proportion of naïve CD4 T-cells expressing CD31; some claim that this proportion correlates with thymic generation of CD4 RTEs (Kimmig et al., 2002; Kohler and Thiel, 2009). The similar age-associated decreases in this proportion in patients (ρ = −0.34; p = 0.001) and

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Fig. 1. Flow cytometry plot from a representative control. Flow cytometry gates are shown for naïve CD4 (CD4+CD45RA+) T-cells and four naïve CD4 T-subsets defined by CD31 expression (CD31pos, CD31neg, CD31hi, and CD31lo cells).

controls (ρ = −0.41; p = 0.005) (see Suppl Fig. 2) might suggest that the two groups have comparable decreases in thymic output with age. Comparison of sjTREC frequencies in FACS-isolated naïve CD4, CD31pos

and CD31neg T-cells from controls (see Suppl Fig. 3), confirms reports (Kimmig et al., 2002; Kohler and Thiel, 2009) that CD31pos cells contain the majority of naïve CD4 T-cell sjTRECs. sjTREC frequencies decreased with age in CD31pos (ρ = −0.843; p = 0.03) but not CD31neg cells. Thus, proliferation of CD31pos but not CD31neg cells, contributes to the age-associated reduction in naïve CD4 T-cell sjTREC frequencies (see Fig. 2). The retention of CD31 on proliferating cells led us to conclude

Fig. 2. Naïve CD4 T-cells from patients have reduced sjTREC frequencies. Best-fit exponential lines show that naïve CD4 T-cell sjTRECs decrease with age in controls (Spearman ρ = − 0.546, p b 0.001) and patients (ρ = − 0.285, p = 0.04) but patients have significantly lower sjTRECs (Mann Whitney U, p = 0.004).

Fig. 3. sjTREC frequencies differ between controls and patients under age 40. Controls (n = 23) had higher sjTREC frequencies than age-matched patients under age 40 (median copies 35,881 vs. 16,152; p = 0.01). Above age 40, controls (n = 26) did not differ from age-matched patients (median copies, 12,330 vs. 8910; p = 0.41).

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that CD31 expression is an unreliable measure of thymic output. Thus, definite conclusions about thymic output were not possible from these analyses.

3.3. sjTRECs in T-subsets We FACS-isolated naïve CD4, CD31hi, CD31lo and CD31neg cells (see Fig. 1) from 10 patients and 10 age-matched controls to see whether CD31hi cells alone would serve as a better marker of CD4 RTEs. CD31 mean fluorescence intensities (MFI) were: CD31pos cells (controls, 6424 vs. patients, 5714; p = NS) CD31hi cells (controls, 8607 vs. patients, 8449; p = NS), and CD31lo cells (controls, 1630 vs. patients 1532; p = 0.04). CD31hi cells had higher absolute sjTREC numbers than CD31lo cells, which had higher sjTREC numbers than CD31neg cells (Fig. 4). Thus, these subsets are progressively distant in origin from the thymus. Absolute sjTREC numbers in CD31hi cells from controls showed a statistically significant decrease with age (Fig. 5), corresponding to an ∼ 0.5 log decrease from ages 23 to 54 (slope − 0.07 sjTRECs/year) whereas corresponding patient sjTRECs showed a minimal decrease (slope −0.019 sjTRECs/year). The best-fit exponential line for CD31hi sjTRECs was shifted downwards ∼ 0.5 log compared with the corresponding exponential line from controls and the two lines intercept around ages 50–55. The findings indicate that young patients have CD31hi sjTREC numbers comparable to controls 25–30 years older (see Fig. 5), i.e. RRMS patients have early thymic involution. The findings suggest further that some mechanism maintains the relative constancy of sjTREC numbers in older patients (see CXCR4 findings below). Since proliferation dilutes T-cell sjTREC content, we quantified sjTREC frequencies (sjTRECs/106 cells), as a measure of proliferative T-cell responses to thymic involution (data not shown). In controls, sjTREC frequencies decreased with age in CD31hi (slope −0.071 sjTRECs/year; ρ=−0.843, p=0.004) and CD31lo cells (slope −0.078 sjTRECs/year; ρ=−0.396), indicating age-associated proliferation of both subsets. In patients, sjTREC frequencies increased slightly in CD31hi cells (slope +0.0013 sjTRECs/year; p=NS) and decreased minimally in CD31lo cells (slope −0.00898 sjTRECs/year; p=NS). These findings might suggest reduced proliferation of CD31hi and CD31lo cells in patients but our Ki-67 and CXCR4 data below point towards a different interpretation (see Discussion section). We could not determine whether sjTREC frequencies decreased with age in CD31neg cells, as sjTRECs were undetectable in four patients and three controls.

Fig. 4. Differences in absolute sjTREC numbers between isolated T-subsets. CD31hi cells had higher sjTREC numbers than CD31lo cells (controls, p = 0.004; patients, p = 0.02), which had higher sjTREC numbers than CD31-cells (controls, p = 0.006; patients, p = 0.002). sjTREC numbers did not differ between controls and patients for any T-subset (p = NS).

Fig. 5. In patients, absolute sjTREC numbers do not decrease with age in CD31hi and CD31lo cells. In controls, absolute sjTREC numbers decreased with age in CD31hi (Spearman ρ = − 0.693, p = 0.039) and CD31lo cells (ρ = − 0.76, p = 0.017) but did not decrease in patients (p = NS).

3.4. Ki-67 expression We also analyzed Ki-67 expression in gated T-subsets from 12 patients and 12 age-matched controls in order to assess peripheral T-cell proliferative responses; the study group included the 10 patients and 10 controls shown in Fig. 5 plus two additional age-matched patients and controls (ages 51 and 55) (Fig. 6). In controls, CD31hi cells had a significantly higher proliferation fraction (% Ki-67 positivity) than CD31lo cells whereas in patients both CD31hi and CD31neg cells had higher proliferation fractions than CD31lo cells. CD31neg cells from patients also showed a trend towards higher proliferation than CD31neg cells from controls. Thus, all three subsets proliferate in patients and controls but only in patients do CD31neg cells proliferate at relatively high levels.

Fig. 6. Increased Ki-67 expression in different T-subsets from patients and controls. CD31hi cells had consistently higher % Ki-67 expression than CD31lo cells (controls; mean 1.20% vs. 0.67%, p = 0.001 and patients; mean 1.32% vs. 0.77%; p = 0.02). In patients, CD31neg cells had higher % Ki-67 than CD31lo cells (median 1.26 vs. 0.77, p = 0.01) and showed a trend towards higher % Ki-67 than control CD31neg cells (p = 0.094). In controls, % Ki-67 did not differ between CD31neg and CD31lo cells (p = NS).

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3.5. Absolute T-cell numbers We wanted to determine whether proliferative T-cell responses compensate for thymic involution (see Fig. 7 for CD31hi and naïve CD4 T-cells). Accordingly, we measured absolute numbers of T-cells in various naïve CD4 T-subsets from the 12 patients and 12 age-matched controls described in the Ki-67 analyses. T-cell numbers from these patients and controls had a lognormal distribution. CD31neg numbers did not change with age in either group (data not shown). In controls, CD31pos (ρ = −0.708, p = 0.01) and CD31hi numbers decreased with age, whereas CD31lo cell numbers remained constant (ρ = −0.156), indicating that thymic involution normally affects only CD31hi cells. In controls, the relatively constant naïve CD4 T-cell numbers indicate that proliferative T-cell responses compensate adequately for thymic involution. In patients, naïve CD4, CD31pos (ρ = −0.635; p = 0.02), CD31hi and CD31lo (ρ = −0.712; p = 0.006) numbers decreased with age, indicating that proliferative responses cannot maintain adequate cell numbers as the thymus involutes. In an analysis of 4 patients, b1% of CD31pos, CD31hi and CD31lo cells from each patient co-expressed CD45RO; 0.63–1.46% of naïve CD4 T-cells co-expressed CD45RO; and 0.61–3.1% of CD31neg cells coexpressed CD45RO. That is, T-subset contamination by CD45RO+ memory cells was minimal. 3.6. βTRECs βTRECs form earlier than sjTRECs during thymopoiesis (Dion et al., 2004; van den Dool and de Boer, 2006). Thymocyte and T-cell proliferation affect T-cell βTREC frequencies. Some claim that the sj/βTREC ratio is unaffected by peripheral proliferation and so provides a measure of early thymocyte proliferation (Dion et al., 2007; Dion et al., 2004; van den Dool and de Boer, 2006). This ratio is calculated after quantification of 6 or 10 βTRECs, then extrapolation to 13 βTRECs (Dion et al., 2007; Dion et al., 2004). We hoped to use this ratio to compare thymocyte proliferation in patients vs. controls. Since patient–control sjTREC differences occurred only in the under-40 groups (Fig. 3), we quantified six βTRECs in 15 under-40 patients and age-matched controls selected from the total patient and control groups. We found that individual βTREC frequencies differed significantly (Suppl Fig. 4A), indicating that extrapolation to un-quantified βTRECs and calculation

Fig. 7. Patients fail to maintain absolute naïve CD4 T-cell numbers with age. CD31hi cell numbers decreased with age in controls (ρ = − 0.59, p = 0.03) and patients (ρ = − 0.735, p = 0.004), whereas naïve CD4 T-cell numbers decreased with age in patients (ρ = − 0.738, p = 0.004) but not in controls (ρ = − 0.34, p = 0.165).

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of the sj/βTREC ratio are questionable. The βTREC sum was lower in patients than controls (Suppl Fig. 4B), raising the possibility of increased naïve CD4 proliferation in patients; increased thymocyte proliferation seems unlikely, since thymocyte proliferation declines with age (Dion et al., 2004) and causes the decreasing thymic output associated with thymic involution (Almeida et al., 2001). 3.7. Bcl-2 and CD127 expression (MFI data not shown) We performed subsequent phenotypic analyses on constant T-cell numbers, as age would clearly affect numbers of cells expressing various markers. We questioned whether survival signals Bcl-2 and CD127 (IL-7 receptor α) (Jiang et al., 2005; Marrack and Kappler, 2004) differ between the 12 patients and 12 age-matched controls described in the Ki-67 analyses. In both groups, CD31neg cells had highest % Bcl-2 expression (Fig. 8A), higher Bcl-2 MFI than CD31lo cells (controls, 598.6 vs. 549.7, p= 0.01; patients 623.5 vs. 571.82, p= 0.04) and showed a trend towards higher MFI than CD31hi cells (controls, 598.6 vs. 485.9, p= 0.067; patients 623.5 vs. 566.7, p =0.057). In patients, CD31neg cells had higher % Bcl-2 expression than in controls. Thus, CD31neg cells consistently up-regulate Bcl-2, but more CD31neg cells up-regulate Bcl-2 in patients. In controls, CD31hi cells had higher CD127 MFI (767.1) than CD31lo (686.0, p b 0.001) or CD31neg cells (682.8, p b 0.001). Similarly, patient CD31hi cells had higher CD127 MFI (747.5) than CD31lo (665.2, p b 0.001) or CD31neg cells (659.4, p b 0.001). Thus, CD127 MFI seemingly decreases according to distance in origin from the thymus. CD127 MFI did not differ between patients and controls for any T-subset. In both groups, CD31hi cells had highest % CD127 expression levels but

Fig. 8. % Bcl-2 and CD127 expression levels differ between patients and controls. (A) CD31neg cells had higher mean % Bcl-2 expression than either CD31lo cells (controls; 92.67 vs. 90.61, p = 0.02 and patients; 95.17 vs. 92.50, p = 0.005) or CD31pos cells (controls; 92.67 vs. 90.48, p = 0.047 and patients; 95.17 vs. 92.26, p = 0.02). Mean % Bcl-2 expression was higher in CD31neg cells from patients than controls (p = 0.01). (B) CD31hi cells had higher mean % CD127 positivity than CD31 lo (controls; 91.19 vs. 82.87, p = 0.001 and patients; 93.83 vs. 91.38; p = 0.005) or CD31neg cells (controls; 91.19 vs. 81.15, p = 0.001 and patients, 93.83 vs. 90.28, p = 0.001). In patients, every T-subset had higher mean % CD127 expression than controls (all comparisons; p b 0.01).

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all five T-subsets from patients had higher % CD127 expression than controls (Fig. 8B). Clearly, more cells from every T-subset express this receptor in patients than controls. 3.8. CXCR4 expression Thymocytes express CXCR4 (Poznansky et al., 2002). In the 12 patients and 12 age-matched controls described in the Ki-67 analyses, CD31hi cells had highest % CXCR4 expression but all T-subsets had higher % CXCR4 expression in patients than controls (Fig. 9). Further, each T-subset had higher CXCR4 MFI in patients than controls (Fig. 10 shows CD31hi and CD31lo data). In controls, CXCR4 MFI did not vary with age in any T-subset. In patients, however, CXCR4 MFI increased with age in CD31hi and CD31lo cells (Fig. 10), and in CD31neg (r = 0.758, p = 0.004), CD31pos (r = 0.662, p = 0.02) and naïve CD4 T-cells (r = 0.693, p = 0.012). Notably, the increasing CXCR4 MFI in CD31hi cells from patients suggests that CD4 RTEs exit the thymus progressively earlier during their maturation process with age. 4. Discussion Our objective was to clarify differences in naïve CD4 T-cell homeostasis between RRMS patients and controls. Whereas most RRMS patients are treated with disease-modifying agents (Freedman et al., 2008), we deliberately selected patients in our study to exclude possible effects of these treatments on sjTREC levels. In the initial assessment of thymic output, we quantified sjTRECs in isolated CD4+CD45RA+ cells (naïve CD4 T-cells), as these cells do not respond to recall antigens (Muraro et al., 2000). sjTREC frequencies decreased exponentially with age in both patients and controls (Fig. 2), as expected (Douek et al., 1998) and patients under 40 had significantly reduced sjTRECs (Fig. 3). This finding of reduced sjTRECs in RRMS is compatible with other reports on sjTREC levels in RRMS (Duszczyszyn et al., 2006; Haas et al., 2007; Hug et al., 2003; Puissant-Lubrano et al., 2008; Thewissen et al., 2005; Thewissen et al., 2007) and suggests that our data apply to the general MS population even though our patients had relatively benign MS with a mean EDSS of 1.7. sjTRECs are influenced not only by thymic output but also by peripheral T-cell proliferation, which reduces sjTREC levels (Dion et al., 2004; Douek et al., 1998; Hazenberg et al., 2000; Kilpatrick et al., 2008; Kimmig et al., 2002; Kohler and Thiel, 2009; van den Dool and de Boer, 2006).Consequently, some claim reduced thymic output in RRMS whereas others report increased T-cell proliferation (Duszczyszyn et al., 2006; Haas et al., 2007; Hug et al., 2003; Puissant-Lubrano et al., 2008; Thewissen et al., 2005; Thewissen et al., 2007). In order to determine whether the decreased sjTRECs in RRMS (Fig. 2) reflects reduced thymic output, we analyzed the proportion of naïve CD4 T-cells

Fig. 9. Patients have increased % CXCR4 expression levels on all T-subsets. CD31hi cells had higher mean % CXCR4 expression than either CD31lo (controls; 79.22 vs. 74.57, p = 0.002 and patients; 86.89 vs. 82.61, p = 0.009) or CD31neg cells (controls 79.22 vs. 71.66, p = 0.01 and patients; 86.89 vs. 81.01, p = 0.009). The % CXCR4 was higher in patients for all T-subsets (all comparisons; p b 0.001).

Fig. 10. CXCR4 MFI increases with age in T-subsets from patients. CXCR4 MFI did not correlate significantly with age in controls, whereas, in patients, CXCR4 MFI increased with age in CD31hi (r = 0.683, p = 0.014) and CD31lo cells (r = 0.77, p = 0.003).

expressing CD31, as a putative marker of CD4 RTEs (Haas et al., 2007; Kimmig et al., 2002; Kohler and Thiel, 2009). In contrast to findings in an earlier study of 28 RRMS patients and age-matched controls (Haas et al., 2007), this proportion (see Suppl Fig. 2) did not differ between total patients (n = 68) and total controls (n = 68). However, our sjTREC frequency analysis indicated that some CD31pos cells proliferate yet retain CD31 expression. Also, in vitro, cytokines induce proliferation of CD31pos cells without loss of CD31 expression (Kohler et al., 2005). This retention of CD31 on proliferating naïve CD4 T-cells led us to conclude that CD31 expression is an unreliable measure of thymic output. We asked, therefore, whether CD31hi cells alone might be a better marker of CD4 RTEs. Among isolated naïve CD4 T-subsets, CD31hi had highest absolute sjTREC numbers (Fig. 4), i.e. include CD4 RTEs, but Ki-67 data (Fig. 6) show that these cells also proliferate at low levels, suggesting that some CD31hi cells are the progeny of RTEs. The downward shift of the best-fit exponential line for CD31hi sjTRECs from patients, compared with the corresponding exponential line from controls (Fig. 5), provides first direct evidence that patients have reduced thymic output of CD4 RTEs. That is, early thymic involution occurs in RRMS. In patients, the CXCR4 findings clarify the apparent discrepancies between Ki-67 data, suggesting significant CD31hi proliferation, and the sjTREC frequency data, suggesting minimal CD31hi proliferation. CXCR4 MFI results (Fig. 10) indicate that patients have progressive, age-associated immaturity of CD4 RTEs. By definition, immature RTEs replicate fewer times than mature RTEs after thymocyte generation of sjTRECs. Consequently, although absolute CD31h and CD31lo numbers decreased with age at a higher rate in patients than controls, absolute sjTREC numbers in CD31hi and CD31lo cells decreased much more slowly in patients (Fig. 5). That is, thymic release of CD4 RTEs having progressively increasing sjTRECs with age maintains CD31hi sjTREC content in spite of their ongoing proliferation. The absolute sjTREC numbers (Fig. 4) suggest that CD31hi, CD31lo and CD31neg cells are part of a continuum of naïve CD4 T-cell proliferation and differentiation. The decreasing CD127 and CXCR4 MFI between these T-subsets are consistent with that interpretation. Phenotypic reversion of memory cells with re-expression of CD31 cannot explain the different sjTREC numbers in these T-subsets, as more than 95% of CD4+CD45RA+ cells in patients and controls coexpress CD27 (Duszczyszyn et al., 2006), a marker lost permanently

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upon memory/effector-cell differentiation (Messele et al., 1999). Phenotypic reversion of CD31neg cells is also unlikely, as in vitrostimulated CD31neg cells proliferate but do not re-express CD31 (Kohler et al., 2005). In controls, sjTREC frequency and Ki-67 data show that peripheral proliferative T-cell responses, particularly of CD31hi cells, maintain relatively constant absolute naïve CD4 T-cell numbers with age. In patients, proliferation was most pronounced in CD31neg and CD31hi cells, as analyzed by Ki-67 expression (Fig. 6), but failed to maintain absolute naïve CD4 T-cell numbers with age (Fig. 7). Naïve CD4 T-cell survival requires IL-7 and self-MHC/TCR interaction (Jiang et al., 2005; Marrack and Kappler, 2004). Plasma IL-7 did not differ between patients and controls (data not shown). IL-7-CD127 binding modulates Bcl-2 expression (Marrack and Kappler, 2004). Bcl-2 up-regulation may favor survival of proliferating CD31neg cells, which had higher % Bcl-2 expression in patients than controls. Differences in CD127 expression cannot explain the reduced naïve CD4 T-cell numbers in patients vs. controls, as all patients' T-subsets had higher % CD127 expression than controls. We conclude that age-associated decreasing thymic output of CD4 RTEs in controls is adequately compensated by peripheral T-cell proliferative responses, and replacement of CD31pos by CD31neg cells. In contrast, early thymic involution in patients reduces thymic output of CD4 RTEs, which leads to thymic output of progressively immature CD4 RTEs and to compensatory proliferative T-cell responses, particularly of CD31hi and CD31neg cells, which fail to maintain naïve CD4 numbers. We reported previously that healthy and affected members of identical twin pairs discordant for RRMS have TCR repertoire shifts (Haegert et al., 2003), suggesting a genetic basis for thymic alterations in RRMS, particularly since genetic background determines thymic output (Dulude et al., 2008). We found no correlation between HLA-DR2 (15) and sjTRECs in RRMS (Duszczyszyn et al., 2006), i.e. the MS-associated DR15 haplotype (Haegert et al., 1996) and linked promoters (Ramagopalan et al., 2009) cannot explain our findings. We did not address other potential genetic influences on the thymus, e.g. IL-7 receptor polymorphisms (Gregory et al., 2007; Lundmark et al., 2007). Also, we did not investigate whether early thymic involution impacts regulatory T-cell generation in patients. CD31pos cells proliferate in response to cytokines, whereas CD31neg cells seemingly proliferate after TCR engagement (Kohler et al., 2005). Assuming that CD31neg cells proliferate in response to self-peptides, the increased CD31neg cell proliferation in patients may select and expand autoreactive T-cells analogous to what is observed in an autoimmune diabetic mouse model (Calzascia et al., 2008) and in human recipients of islet cell transplants for type I diabetes (Monti et al., 2008), i.e. favor the development of autoimmunity. Thus, thymic alterations and compensatory homeostatic T-cell proliferation may contribute to RRMS pathogenesis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jneuroim.2010.02.005. References Almeida, A.R., Borghans, J.A., Freitas, A.A., 2001. T cell homeostasis: thymus regeneration and peripheral T cell restoration in mice with a reduced fraction of competent precursors. J. Exp. Med. 194, 591–599. Almeida, A.R., Rocha, B., Freitas, A.A., Tanchot, C., 2005. Homeostasis of T cell numbers: from thymus production to peripheral compartmentalization and the indexation of regulatory T cells. Semin. Immunol. 17, 239–249. Calzascia, T., Pellegrini, M., Lin, A., Garza, K.M., Elford, A.R., Shahinian, A., Ohashi, P.S., Mak, T.W., 2008. CD4 T cells, lymphopenia, and IL-7 in a multistep pathway to autoimmunity. Proc. Natl. Acad. Sci. U. S. A. 105, 2999–3004. Datta, S., Sarvetnick, N., 2009. Lymphocyte proliferation in immune-mediated diseases. Trends Immunol. 30, 430–438.

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