Circulating lymphocyte and T memory subsets in glucocorticosteroid versus IVIG treated patients with CIDP

Journal of Neuroimmunology 283 (2015) 17–22

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Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim

Circulating lymphocyte and T memory subsets in glucocorticosteroid versus IVIG treated patients with CIDP Juliane Klehmet a,⁎, Max Staudt a, Lena Ulm a, Nadine Unterwalder c, Andreas Meisel a, Christian Meisel b,c a b c

Department of Neurology, Charité Universitaetsmedizin, Charitéplatz 1, Berlin, Germany Department of Medical Immunology, Charité Universitaetsmedizin, Berlin, Germany Department of Immunology, Labor Berlin Charité Vivantes, Sylter Strasse 2, Berlin, Germany

a r t i c l e

i n f o

Article history: Received 2 January 2015 Received in revised form 10 March 2015 Accepted 12 March 2015 Available online xxxx Keywords: CIDP Glucocorticosteroids IVIG NK cells T memory subsets

a b s t r a c t The present study compared lymphocyte and T memory subsets in currently untreated patients with chronic inflammatory demyelinating polyneuropathy (CIDP) to glucocorticosteroid (GS) and intravenous immunoglobulin (IVIG) treated patients. Peripheral blood from 48 CIDP patients (21 untreated who were either treatment naïve or without treatment during the last 3 months, 17 IVIG and 10 GS treatment) and from 12 age-matched controls was evaluated using flow cytometric analysis. Our data demonstrate that long-term GS treatment is associated with reduced frequencies of total CD4+ T cells, CD4+ memory subsets and NK cells while long-term IVIG treatment is associated with alterations of the CD8+ memory compartment. Reduction of CD4+ naïve T cell counts may explain the observation that GS treatment induces prolonged clinical remission compared to IVIG treatment. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare chronic progressive or relapsing–remitting disease leading to temporary or persistent severe disability in about 50% (Lunn et al., 1999; Vallat et al., 2010). So far, the underlying pathophysiological mechanisms of CIDP are not fully understood. Both humoral immunity and cellular immunity are considered to be involved (Yuki et al., 1996; Dalakas, 2003). Activated T cells, mainly CD4+ T cells are increased in the circulation of CIDP patients (Adam et al., 1989; Van den Berg et al., 1995; Hughes et al., 2006). We (Klehmet et al., 2014) and others (Csurhes et al., 2005; Sanvito et al., 2009) demonstrated increased frequencies of PMP-22 and P2 specific T cells in peripheral blood of treatment naïve patients who subsequently responded to intravenous immunoglobulin (IVIG)

Abbreviations: CIDP, chronic inflammatory demyelinating polyneuropathy; EFNS, European Federation of Neurological Sciences; HC, healthy controls; INCAT, inflammatory neuropathy cause and treatment; IVIG, IV immunoglobulin; MRC, Medical Research Council Scale; PBMC, peripheral blood mononuclear cells; TCM, central memory T cells; TEM, effector memory T cells; TEMRA, terminally differentiated T cells; ON, other neuropathy. ⁎ Corresponding author at: Department of Neurology, Charitéplatz 1, 10117 Berlin, Germany. E-mail addresses: [email protected] (J. Klehmet), [email protected] (M. Staudt), [email protected] (L. Ulm), [email protected] (N. Unterwalder), [email protected] (A. Meisel), [email protected] (C. Meisel).

http://dx.doi.org/10.1016/j.jneuroim.2015.03.023 0165-5728/© 2015 Elsevier B.V. All rights reserved.

treatment, suggesting a pathogenic role for peripheral myelin antigenspecific T cells in CIDP (Klehmet et al., 2014). The immunological memory is an essential feature of the adaptive immune system leading to a fast and vigorous immune response to antigens that have been encountered previously. T cells can be divided into naïve and memory/effector cells according to the expression of CD45RA and the lymph node homing receptor CCR7 (Sallusto et al., 2000). There is evidence that the T cell memory compartment influences self-antigen responses in chronic autoimmune diseases (Sen et al., 2004; Abdulahad et al., 2006; Haegele et al., 2007). Based on their regulatory effects on T and B cells as well as changes in cellularity and functionality observed in different autoimmune disorders natural killer cells (NK cells) which are important modulators of innate and adaptive immune response (Degli-Esposti and Smyth, 2005; Perricone et al., 2008) have been implicated in the induction and/or persistence of inappropriate adaptive autoimmune responses (Fogel et al., 2013). The treatment with intravenous immunoglobulins (IVIGs) has been shown to be effective in CIDP (Hughes et al., 2008). Based on long clinical experience and randomized controlled trials (Hughes, 2001; Van Schaik et al., 2010) it is also widely accepted that glucocorticosteroid treatment (GS) is effective in CIDP. Both therapy regimens together with plasmapheresis are considered to be first-line treatments (EFNS guideline, 2010). Recently, in an Italian multicentric study IVIG treatment has been proven to be more tolerable and efficient in comparison to intravenous GS given over a 6 month period. However and most interestingly, patients receiving GS treatment had a better chance to

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reach clinical remission compared to IVIG treatment (Eftimov et al., 2012; Nobile-Orazio et al., 2012). The aim of the present study was to examine possible differences in the lymphocyte compartment as well as in the T memory repertoire in patients with CIDP who were either treated with GS or with IVIGs in comparison to untreated CIDP patients and healthy controls (HC). 2. Methods

2.5. Statistical analysis All statistical tests were performed using GraphPadPrism 5.0 software. GS- or IVIG-treated patient measurements were compared with untreated group and HC using Kruskal–Wallis Test followed by posthoc unpaired t-test or Mann–Whitney-test, level of significance was defined as p b 0.05. For sex and CIDP diagnosis, Fisher's exact test was used. Level of significance was defined as p b 0.05 for all comparative tests.

2.1. Patients 3. Results A total of 48 patients with CIDP were recruited in the outpatient clinic of the Charité Department of Neurology. Patients fulfilled diagnostic criteria according to the European Federation of Neurological Sciences (EFNS, 2010) of possible, probable or definite CIDP. The group of untreated patients consisted of patients who were either treatment naïve or had not received prior immunosuppressive/immunomodulatory treatments for at least 3 months. All patients of the untreated group were clinically progressive (n = 21). In contrast, patients of the maintenance treatment group (IVIG or GS) were all clinically stable with regard to the MRC sum score, walking distance and to their subjective evaluation. Patients in the IVIG (n = 17) and GS (n = 10) group had received treatment for at least 6 months on an individual dosage tailored regimen (IVIG: 0.4–1 g/kg body weight, GS: range from orally 4–20 mg prednisolone or methylprednisolone daily or intravenous pulse therapy of 3 × 500 mg methylprednisolone monthly). All CIDP patients were clinically evaluated using Medical Research Council (MRC) sum score and walking distance. The MRC sum score ranges from 0–70 (including shoulder abduction, elbow flexion, wrist extension, hip flexion, knee extension, foot dorsiflexion, extension of big toe). Data from part of the IVIG treatment group (n = 13) and part of the untreated patients (n = 16) were published previously (Klehmet et al., 2014). Twelve age-matched healthy individuals were used as controls (mean age 49, range from 44–63 years). 2.2. Standard protocol approvals, registration, and patients' consents The present study has been approved by the local ethics committee (Ethikkommission, Charité Universitätsmedizin Berlin) and clinical investigations were conducted according to the Declaration of Helsinki. All patients gave written informed consent to the study. 2.3. Peripheral blood monocytes' (PBMC) preparation Peripheral blood was obtained at the day of first IVIG or pulsed steroids treatment immediately before starting the infusion by venipuncture and sampled into CPT tubes or at any day from patients taking daily oral glucocorticosteroids (prednisolone or methylprednisolone). PBMC were isolated within 5 h after venipuncture by density gradient centrifugation at 1500 g for 20 min. 2.4. Flow cytometry Flow cytometric analysis of human lymphocyte subsets in EDTA whole blood was performed as described recently (Klehmet et al., 2014). Briefly, the following mouse anti-human fluorescently-labelled monoclonal antibodies were used for quantification of lymphocytes and T cell subsets: cluster of differentiation (CD)3 AllophycocyanineAlexa Fluor 750 (APC-A750), CD4 energy coupled dye (ECD), CD8 APC, CD14 Fluorescein isothiocyanate (FITC), CD16 Phycoerythrine (PE), CD19 PE-Cy5.5, CD56 PE, CD45RA Pacific-Blue (PB), CD45 KromeOrange (KrO), (all from Beckman Coulter) and CCR7 Phycoerythrine (PE) (R&D Systems). Stained samples were acquired on a ten-colour Navios flow cytometer and analyzed using Navios Software (Beckman Coulter). Immunophenotyping analyses were performed according to the lymphocyte subset gating strategy shown in Fig. 1.

3.1. Clinical characterization of the untreated, IVIg and steroid treatment group Patients in untreated (n = 21), GS (n = 10) and IVIG (n = 17) groups did not differ in regard to sex, age, time since first clinical onset of symptoms, diagnostic categories, treatment duration or clinical severity (MRC). For detailed information see Table 1. 3.2. Quantitative analysis of peripheral blood leukocyte subsets Peripheral blood leukocyte subsets in untreated patients with CIDP compared to GS and IVIG treated patients were quantified using flow cytometry. Monocytes were more frequent in untreated CIDP-patients compared to healthy controls (p b 0.01, Fig. 2A). Furthermore, we found significantly reduced frequencies of NK cells in GS-treated patients compared to untreated patients and IVIG-treated patients (p b 0.001, Fig. 2B). In addition, B cell counts were reduced in the GS treatment group compared to untreated patients (p b 0.05, Fig. 2C). 3.3. Quantitative analysis of T memory subsets We next studied the distribution of naïve (CD45RA+ CCR7+), central memory (TCM, CD45RA − CCR7 +), effector memory (TEM, CD45RA− CCR7−) and TEMRA (CD45RA+ CCR7−) populations within CD4+ and CD8+ T cells of group of untreated patients and patients who received maintenance therapy with either GS or IVIG. Representative flow cytometric dot plots are shown in Fig. 1. We did not observe differences in absolute numbers of total T cells and regulatory T cells (Tregs) (data not shown). However, CD4 + T cells were increased in the group of untreated patients compared to HC (Fig. 3A; p b 0.01) and to GS-treated (p b 0.001; Fig. 3A). There were no differences in absolute CD8+ T cells counts (Fig. 3B). Analysing CD4+ T cell subsets we found elevated effector/memory CD4 + T cells in untreated patients compared to GS-treated patients (p b 0.05, Fig. 3C) as well as to HC (p b 0.001, Fig. 3C). In contrast, central/memory CD4 + cells were increased in untreated patients compared to both IVIG- (p b 0.01, Fig. 3C) and GS-treated patients (p b 0.05, Fig. 3C) as well as to HC (p b 0.05, Fig. 3C). In addition, we found reduced numbers of naïve CD4 + T cells in the GS group compared to untreated patients (p b 0.05; Fig. 3C). Looking at CD4 + TEMRA cells, GS treated patients had significantly lower counts compared to untreated patients (p b 0.05; Fig. 3C). For CD8+ memory subsets, CD8 + TCM were increased in the untreated group compared to IVIG and HC (untreated versus IVIG p b 0.05; untreated versus HC p b 0.01; Fig. 3D). Interestingly, patients treated with IVIGs had lower counts of CD8+ TEMRA compared to untreated patients (IVIG vs. untreated p b 0.05, Fig. 3D). 4. Discussion In the present study we investigated changes in lymphocyte subsets as well as T memory repertoire in currently untreated patients with chronic inflammatory demyelinating polyneuropathy (CIDP) compared to GS and IVIG treated patients with CIDP.

J. Klehmet et al. / Journal of Neuroimmunology 283 (2015) 17–22

B

C

SSC

SSC

CD3

SSC

F

SSC

CD8

CD4

CD3

I CD45RA

H CD45RA

G

SSC

CD16/CD56

E CD19

D

CD14

CD45

CD16/CD56

A

19

CCR7

CCR7

Fig. 1. Gating strategy for leukocyte subsets in human peripheral blood. Leukocyte subsets were analysed by flow cytometry as followed. Briefly, the gate was set on CD45+ mononuclear leukocytes using the CD45 and SSC parameters (A). Neutrophils were identified as CD16brightSSCbright cells in a CD16 vs. SSC dot plot (B). Monocytes were identified as CD14+ cells in a CD14 vs. SSC dot plot (C). T and B cells were identified as CD3+ and CD19+ lymphocytes in a CD3 vs. SSC and CD19 vs. SSC dot plot, respectively (D, E). NK cells were identified as CD3− CD16/ CD56+ lymphocytes using the CD3 and CD16/CD56 parameters (F). Naïve and memory CD4+ and CD8+ T cell subsets (H, I) were identified according to their differential expression of the CD45RA and CCR7 (naïve: CD45RA+ CCR7+; central memory: CD45RA− CCR7+; effector memory: CD45RA− CCR7−; CD45RA+ effector memory (TEMRA): CD45RA+ CCR7−).

Here, we found reduced NK cell counts in GS treated compared to untreated patients. It has been demonstrated that high dose GS treatment leads to reduction of monocytes and NK cells in patients suffering from multiple sclerosis (Mirowska-Guzel et al., 2006). NK cells have high immunoregulatory abilities and are capable of killing their target cells through receptor–ligand interactions (natural cytotoxicity) and antibody-dependent cell-mediated cytotoxicity (Colucci et al., 2003; Ahmad et al., 2005). Recently it has been shown in 10 CIDP patients that IVIG treatment leads to the reduction of NK cell numbers by 36% (Bohn et al., 2011). In contrast, primarily reduced frequencies of NK cells in the peripheral blood of untreated CIDP patients have been found (Sanvito et al., 2009). Here, we found in the GS but not in the IVIG treatment group reduced NK cell counts compared to the untreated patient group. However, in the present study we investigated the long-term effect at the end of an infusion interval whereas others (Bohn et al., 2011) investigated NK frequencies immediately after IVIG infusion which may explain the different results between both studies. We therefore cannot exclude short-lasting diminishing effect to NK cells by IVIG treatment.

We found primarily increased monocyte counts in the group of untreated patients. This is in line with a recent study published by Sanvito and colleagues (Sanvito et al., 2009). However, unlike others (MirowskaGuzel et al., 2006), we could not observe changes in monocyte numbers in the GS treatment group which may be due to the fact that our patients did not receive high dose GS treatment but rather personalized dose regimen on a long-term basis according to their individual stable clinical status. In accordance with a previous study we did not find primarily increased frequencies of B cells (Sanvito et al., 2009) but decreased B cell counts in the GS but not in the IVIG treatment group. In accordance with previous studies (Trebst et al., 2006; Sanvito et al., 2009), we did not find any changes in total numbers of T cells in the groups of steroid or IVIG treatment. However, we detected significantly reduced numbers of CD4 + T cells in GS treated patients compared to untreated patients. This is in accordance with a number of previous studies demonstrating the ability of GS therapy to induce apoptosis preferentially in CD4+ T cells (Migita et al., 1997; Leussink et al., 2001). Our results of increased CD4+ T cell counts in untreated patients with CIDP are also in line with several studies demonstrating increased

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Table 1 Clinical information for untreated patients and maintenance treatment group.

Sex

Male Female Mean Range Mean Range Definite Probable Possible Baseline MRC sum score (SD) None Steroids Plasmapheresis IVIG other

Age (years) Time since first onset (years) CIDP

Clinical severity Previous treatment

Untreated

GS

IVIG

p-Values

13/21 (64%) 8/21 (37%) 65.43 33–84 7.053 1–27 12/21 (57%) 7/21 (33%) 2/21 (9%) 65.9 (4.460) 7/21 (33%) 10/21 (48%) 1/21 (5%) 7/21 (33%) 3/21 (14%)

8/10 (80%) 2/10 (20%) 58.4 31–78 5.400 1–23 3/10 (33%) 4/10 (40%) 3/10 (33%) 65.0 (4.6) 0/10 (0%) 10/10 (100%) 4/10 (40%) 4/10 (40%) 4/10 (40%) 20.5 6–70

10/17 (59%) 7/17 (41%) 62.82 29–82 7.8 1–23 12/17 (70%) 4/17 (23%) 1/17 (5%) 64.1 (5.4) 3/17 (18%) 14/17 (82%) 3/17 (18%) 17/17 (100%) 2/17 (12%) 34 6–74

0.4059⁎

Duration of current treatment (months) Time since previous treatment Mean (range in months)

Steroids Plasmapheresis IVIG other

0.3959 0.1265 0.1819⁎⁎ 0.0709⁎⁎ 0.8590

0.1467

12.3 (3–30) 3 25.4 (3–132) 73.0 (3–204)

⁎ Fisher´s exact test of male vs. female in IVIG vs. steroid maintenance group. ⁎⁎ Fisher´s exact test of definite vs. probable or definite vs. possible diagnosis in IVIG vs. steroid maintenance group.

1.0 0.8

*

0.6 0.4 0.2

C H

er oi ds st

G

0.0

IV I

0.0

B cells

at ed

abs. number of B cells/nl

0.2

C

C H

st er oi ds

IV IG

0.0

0.4

H

0.2

0.6

st er oi ds

0.4

0.8

IV IG

0.6

***

at ed

0.8

C

1.0

un tr e

abs. number of NK cells/nl

** 1.0

at ed

NK cells

B

Monocytes

un tr e

abs. number of monocytes/nl

A

of the key auto-antigens in multiple sclerosis has been shown to emerge mainly from CD45RA+ naïve T cells (Muraro et al., 2000). Considering the results from recent clinical trials with GS and IVIG treatment in CIDP, in which a prolonged remission phase in GS versus IVIG treated patients could be demonstrated (Eftimov et al., 2012; Nobile-Orazio et al., 2012), this is an interesting finding. Given that naïve CD4 + T cells embody the source of antigen-specific T cells, downregulation of CD4 + naïve T cells in GS treated patients may explain the longerlasting immune modulating effect even after withdrawal of the treatment. Most of the patients tested in this study received a low maintenance steroid dose which was adapted individually according to their individual clinical course. This was similar to patients of the IVIG treatment group who received individually tailored IVIG treatment regimen with individually determined infusion intervals. However, in contrast to the GS group, IVIG treated patients had no clear alterations of CD4 + subsets but revealed more pronounced changes on the CD8+ T memory repertoire with lower counts of CD8 + TCM and TEMRA cells compared to untreated patients. TEMRA cells are known to have an enhanced capacity to respond very quickly to antigens (Sallusto et al., 1999; Willinger et al., 2005).

un tr e

CD4 + T cell counts in the circulation of CIDP patients (Van den Berg et al., 1995; Hughes et al., 2006). Looking at the naïve/memory T cell compartment, untreated patients showed increased TEM as well as TCM CD4 + T cell counts compared to HC. Elevated TEM counts have been found in other autoimmune diseases such as type 1 diabetes and multiple sclerosis (Matteucci et al., 2011; Mikulkova et al., 2011). Persistent antigen presentation increases the number of TEM (Jabbari et al., 2006; Masopust et al., 2006) whereas vaccination or infection with temporary antigen exposure leads to upregulation of TCM (Marzo et al., 2005; Van Faassen et al., 2005; Badovinac and Harty, 2007). Here, we found increased numbers of CD4 + TEM in untreated CIDP patients which were reduced in the GS treatment group. Interestingly, we also observed lower counts of CD4 + naïve T cells in the GS treatment group. In a previous study, cyclophosphamide has been shown to target preferentially naïve (CD4RA +) T cells in patients suffering from multiple sclerosis and CIDP (Gladstone et al., 2007). Naïve T cells downregulate the CD45RA+ isoform and upregulate CD45RO isoform after antigen priming and differentiation towards effector/memory T cells (Yamane and Paul, 2013). The T cell response to myelin basic protein which is one

Fig. 2. Quantitative analysis of peripheral blood leukocyte subsets in CIDP patients. Absolute counts of monocytes (A), NK cells (B), and B cells in untreated CIDP patients (n = 22) compared to IVIG-treated (n = 17), GS treated patients (n = 10) and HC (n = 12). Increased frequencies of monocytes in untreated CIDP patients compared to HC (A) as well as for NK cells and B cells in untreated patients compared to HC (B and C). Data are shown as box plot Tukey with whiskers of max. 1.5 interquartile range (IQR). (*p b 0.05, **p b 0.01, ***p b 0.001).

J. Klehmet et al. / Journal of Neuroimmunology 283 (2015) 17–22

B

C

C

0.0

un tr e *

0.4 0.2

IVIG

0.8

GS

** *

0.6

HC

0.4 0.2 0.0

TE M R A

TE M

TC M

na iv e

0.0

untreated

TE M R A

* **

0.6

*** *

M

*

*

TE

0.8

1.0

TC M

*

na iv e

1.0

abs. number of CD8+ subsets/nl

D

C abs. number of CD4+ subsets/nl

0.5

H

st er oi ds

IV IG

at ed

0.0

1.0

H

0.5

1.5

st er oi ds

1.0

CD8+ T cells

IV IG

1.5

abs. number of CD8+T cells/nl

** ***

at ed

CD4+ T cells

un tr e

abs. number of CD4+T cells/nl

A

21

Fig. 3. Quantitative analysis of T memory subsets in CIDP patients. Absolute counts of CD4+ T cells (A), and CD8+ T cells (B) in currently untreated CIDP patients (n = 22) compared to maintenance IVIG treated patients (n = 17), GS maintenance treatment patients (n = 10) and HC (n = 12). Increased frequencies of CD4+ T cells in currently untreated patients compared to GS treated patients and HC (A). No significant differences in CD8+ T cells between CIDP patient groups and HC (B). C Absolute counts of CD4+ subsets (naïve, central memory, effector memory, and TEMRA) in untreated patients (n = 22) compared to IVIG treated (n = 17), and GS treated (n = 10) patients as well as HC (n = 12). D Absolute counts of CD8+ subsets (naïve, central memory, effector memory, and TEMRA) in untreated patients (n = 22) compared to IVIG treated (n = 17), and GS treated (n = 10) patients as well as HC (n = 12). Increased numbers of CD4 naïve T cells were observed in untreated patients compared to GS treatment group, higher frequencies of CD4+ TCM in the untreated group compared to all other groups (IVIG, GS and HC). CD4+ TEM were elevated in the group of untreated patients compared to GS treatment group and HC, higher CD4+ TEMRA counts in untreated patients compared to GS treatment group (C). For CD8+ subsets, there were higher frequencies of TCM in untreated patients compared to HC and IVIG treatment group as well as elevated CD8+ TEMRA counts in untreated patients compared to IVIG treatment group. Data are shown as box plot tukey with whiskers of max. 1.5 interquartile range (IQR). (*p b 0.05, **p b 0.01, ***p b 0.001).

Only a few studies have addressed the possible role of the adaptive immune system in the pathogenesis of CIDP so far. SchneiderHohendorf et al. have shown that the T-cell receptor repertoire of cells infiltrating the peripheral nerve has a strong monoclonal or oligoclonal restriction (Schneider-Hohendorf et al., 2012). In addition, an oligoclonal restriction pattern of CD8 + T cells has been shown in CIDP patients which could be normalized by IVIG treatment. We showed recently, that treatment naïve CIDP patients who were responsive to IVIG treatment had increased frequencies of T cells reactive for peripheral myelin antigens PMP-22 and P2, which decreased by longterm IVIG treatment (Klehmet et al., 2014). Reduction of specific autoreactive T-cell responses was paralleled by reduced CD8 + TEM frequencies after long-term IVIG treatment (Klehmet et al., 2014). Here, we show that GS treatment is associated with reduced counts of NK cells and both naïve and memory CD4 + T cells whereas IVIG treatment was associated with the reduction of the CD8 + memory compartment compared to untreated CIDP patients. The reduced counts of CD4+ naïve T cells which were observed only in GS-treated patients may explain the longer-lasting remission phase after treatment withdrawal compared to IVIG treatment. However, our study size population is relatively small. In addition, we have not performed longitudinal assessments in single patients in order to prove causal relation between treatment and changes in the T memory compartments. Finally, we cannot exclude that the difference in the immunological phenotype observed between GS and IVIg treated patients might result from other factors, e.g. different disease activities at the time of leukocyte subset analysis. Therefore, further research and long-term

immunological monitoring of CIDP patients with specific therapy in a greater (prospective) cohort study will be needed for a better understanding of the relationship between efficacy of different first line therapies in CIDP and T cell subsets. Conflicts of interest and funding The study was funded by a research grant from Octapharma and supported by the Deutsche Forschungsgemeinschaft = German Research Foundation (NeuroCure Cluster of Excellence, Exc 257). J. Klehmet has received personal compensation for activities with Grifols, CSL Behring. M. Staudt, L. Ulm, A. Meisel, and C. Meisel report no relevant financial activities outside the submitted work from any organisation for the submitted work; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years, no other relationships or activities that could appear to have influenced the submitted work disclosures. References Abdulahad, W.H., van der Geld, Y.M., Stegeman, C.A., Kallenberg, C.G., 2006. Persistent expansion of CD4+ effector memory T cells in Wegener's granulomatosis. Kidney Int. 70, 938–947. Adam, A.M., Atkinson, P.F., Hall, S.M., Hughes, R.A., Taylor, W.A., 1989. Chronic experimental allergic neuritis in Lewis rats. Neuropathol. Appl. Neurobiol. 15, 249–264. Ahmad, A., Ahmad, R., Iannello, A., Toma, E., Morisset, R., Sindhu, S.T., 2005. IL-15 and HIV infection: lessons for immunotherapy and vaccination. Curr. HIV Res. 3, 261–270. Badovinac, V.P., Harty, J.T., 2007. Manipulating the rate of memory CD8+ T cell generation after acute infection. J. Immunol. 179, 53–63.

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