Urinary T cells in active lupus nephritis show an effector memory phenotype

CHAPTER 6

Urinary T-cells in active lupus nephritis show an effector memory phenotype

Sebastian Dolff1,2, Wayel H. Abdulahad1, Marcory C.R.F. van Dijk3, Pieter C. Limburg1, Cees G.M. Kallenberg1, Marc Bijl1

1 Department of Rheumatology and Clinical Immunology, University Medical Center Groningen, University of Groningen, The Netherlands 2 Department of Nephrology, University Hospital Essen, University Duisburg-Essen, Germany 3 Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, The Netherlands

Ann Rheum Dis. 2010 Nov; 69 (11): 2034-41.

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Abstract Background: Systemic lupus erythematosus (SLE) is accompanied by

alterations in T-cell homeostasis including an increased effector response.

Migrated effector memory T-cells (CD45RO+CCR7-;TEM) appear to be involved

in tissue injury. The objective of this study was to investigate the distribution and

phenotype of effector memory T-cells in the peripheral blood (PB), and their

presence in renal biopsies and urine of SLE patients. We tested the hypothesis

that these TEM-cells migrate to the kidney during active disease.

Patients and Methods: Fourty-three SLE patients and twenty healthy controls

(HC) were enrolled. CD4+ TEM-cells and CD8+ TEM-cells were analysed in PB

and urine using flow cytometric analysis. In ten patients with active lupus

nephritis a parallel analysis was performed on the presence of TEM-cells in

kidney biopsies.

Results: The percentage of circulating CD8+ TEM cells in SLE patients was

significantly decreased versus HC (33.9 ±18.3 % vs. 42.9 ±11.0 %, p=0.008). In

patients with active renal involvement (n=12) this percentage was further

decreased to 30.4±15.9 %, p=0.01. Analysis of the urinary sediment in active

renal disease showed increased numbers of CD4+ T-cells (134 ±71 cells/ml)

and CD8+ T-cells (287 ±220 cells/ml), respectively, while in HC and patients

without active renal disease almost no T-cells were present. 73.6 ±8.3 % of

urinary CD4+T-cells and 69.3 ±26.0 % of urinary CD8+ T-cells expressed the TEM

phenotype. CD8+ cells were as well found in renal biopsies.

Conclusion: The data presented are compatible with the hypothesis that CD8+

effector memory cells migrate from the PB to the kidney and appear in the urine

during active renal disease in SLE patients. These cells could serve as an

additional marker of renal activity in patients with SLE.

Urinary effector memory T-cells in active lupus nephritis

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Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease characterized

by multiple organ manifestations. Inflammation of the kidney, in particular, is

associated with an unfavourable prognosis.1 Although the precise pathogenesis

of lupus nephritis (LN) has not been fully elucidated, disturbances in T-cell

homeostasis seem to contribute to the inflammatory pathology of LN.2;3

During maturation of CD45RO+ memory T-cells expression of the lymph

node homing chemokine receptor CCR7 distinguishes central memory T-cells

(TCM; CD45RO+CCR7+) from effector memory T-cells (TEM; CD45RO+CCR7-). In

several autoimmune diseases including SLE, disturbances have been described

in the distribution of these T-cell subsets in the peripheral blood.4-6 Remarkably,

CD8+ TEM have been thought to play a crucial role in T-cell homeostasis due to

their ability to produce cytokines and exert cytotoxic activity.7

Further evidence for the important role of T-cells in SLE is given by the

observation that mononuclear cells, predominantly T-lymphocytes, are a

frequent histological finding in proliferative forms (ISN/RPS class III and IV) of

LN. However, data regarding CD4+ and CD8+ cell counts and their ratio in renal

biopsies from lupus patients are conflicting.2;3 Especially, the presence of

periglomerular infiltrating CD8+T-cells has been shown to correlate with

histologic activity, clinical severity and bad prognosis of LN.2;3;8 Additionally,

several studies demonstrated the presence of mononuclear cells in urine of

patients with active IgA nephropathy, LN and Wegener’s granulomatosis.9;10

Thus, analysis of urinary cells reflecting renal inflammation in SLE could be a

useful tool in monitoring the course of renal disease.

In line with this, we recently reported an increase of urinary TEM (CD4+

CD45RO+CCR7-) cells in patients with ANCA-associated vascultis (AAV) with

active renal disease.11 Thus, analysis of urinary TEM-cells seems a promising

tool for detecting renal flares. Although T-cell infiltration in kidneys of SLE

patients has been reported, analysis of the state of activation of urinary CD4+ or

CD8+ T-cells has not been performed so far even though T-cells lacking CCR7

have a strong ability to migrate in vitro.12

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We hypothesize that T-cells with effector function migrate from peripheral blood

into the kidneys of SLE patients during active renal disease. This is reflected by

the presence of these T-cells in the urinary sediment. To test this hypothesis of

T-cell migration, we analyzed the peripheral blood and urine for the presence of

effector memory T-cells lacking CCR7 by 4-color flow-cytometry and evaluated

in parallel obtained renal biopsies of patients with active renal disease for the

presence of T-cells.


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Patients and methods Study population

Fourty-three SLE patients with a mean age of 41 ±12 years fulfilling at least four

of the American College of Rheumatology revised criteria for SLE and 20 sex

matched healthy controls (age 36 ±10 years) were enrolled in this study.13

Disease activity was assessed by SLEDAI (SLE Disease Activity Index).

Twenty-four patients had inactive disease (SLEDAI score ≤ 4) and nineteen

patients had active SLE (defined as SLEDAI score > 4). Mean disease activity

for all patients was 6.3 ±5.6 (Table 1). 25 patients had a current or former renal

biopsy consistent with LN while 18 had no history of renal involvement.

Currently active LN (n=12) was defined by a proliferative (class III or IV)

glomerulonephritis in a parallel obtained renal biopsy (n=11) or the presence of

an active urinary sediment with glomerular erythrocyturia (n=1) (Table 2). Seven

active LN patients presented with the first episode of LN. 12 patients did not

receive any immuno-modulating medication at the time of analysis, 6 of them

were newly diagnosed. 31 patients received immuno-modulating medication

(Table 1).

Materials

EDTA-blood and fresh urine samples were collected from patients and HC.

Urine samples from patients which were nitrite positive on a dip stick test or with

proof of bacterial contamination in the sediment were excluded. Percentages

and absolute counts of CD4+ and CD8+ T-cells were assessed immediately after

sampling by four-color flow cytometry in blood and urine samples.

Paraffin-embedded sections of renal biopsy specimens obtained from

eleven patients were included in the present study. Informed consent was

obtained from the patients after approval by the Local Ethics Committee. The

study was conducted according to the ethical guidelines of our institution and

the Declaration of Helsinki.

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Table 1: Baseline characteristics and medication of all patients included (n=43). SLEDAI: systemic lupus erythematosus disease activity index, MMF: mycophenolate mofetil, ¶ renal activity defined by a current biopsy proving active renal disease, or the presence of an active urine sediment in patients with previously known renal involvement.

Antibodies

The following antibodies were used in flow cytometry: phycoerythrin (PE)-

conjugated anti-CCR7 (clone 3D12), fluorescein (FITC)-conjugated anti-

CD45RO (clone UCHL-1), peridin-chlorophyll (PerCP)-conjugated anti-CD4

(clone SK3), allophycocyanin (APC)-conjugated anti-CD3 (clone UCHT1),

MultiTESTTM four-color antibodies (CD3-FITC, CD8-PE, CD45-PerCP and CD4-

APC), and isotype matched control antibodies of irrelevant specificity. All were

purchased from Becton-Dickinson ((BD), Amsterdam, The Netherlands).


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Sample preparation and flow cytometry

Immediately after voiding, 100 ml of urine was diluted 1:1 with cold phosphate-

buffered saline (PBS) and processed as decribed before.11 Briefly, isolated

mononuclear cells were resuspended in wash-buffer (1 % BSA in PBS) and

mixed with appropriate concentrations of anti-CD45RO-FITC, anti-CCR7-PE,

anti-CD4-PerCP, and anti-CD3-APC for 15 minutes at room temperature in the

dark. In parallel, blood samples were labeled with the aforementioned

monoclonal antibodies. Afterwards, cells were successively treated with 2 ml

diluted FACS lysing solution (BD, Amsterdam, The Netherlands) for 10 minutes

and samples were washed twice in wash-buffer and immediately analyzed by

flow cytometry. Four-color staining was analyzed on FACS-Calibur (BD,

Amsterdam, The Netherlands) and data were collected for 105 events for each

sample and plotted using Win-List software package (Verity Software House

Inc., ME, USA). Positively and negatively stained populations were calculated

by quadrant dot-plot analysis, as determined by the isotype controls.

Representative examples are shown in Figure 1.

Quantification of effector memory T-cells

T-cells were quantified in urine using TruCOUNTTM tubes (BD, Amsterdam, The

Netherlands). In brief, 20 µl of MultiTESTTM four-color antibodies (CD3-FITC,

CD8-PE, CD45-PerCP, and CD4-APC) and 50 µl of sample (urine or blood)

were added to bead-containing TruCOUNTTM tubes. The cell suspension was

processed and analysed as described elsewere.11 Afterwards, the absolute

counts for TEM cells in 1 ml urine were calculated as decribed before.11

Analysis and scoring of renal biopsies

Biopsies taken at the time of analysis of blood and urine samples were

processed. All biopsies were reviewed and classified by an experienced

nephropathologist (MvD) according to the revised criteria for LN. The activity

index (AI) and chronicity index (CI) were calculated for each specimen with

maximum scores of 24 for the AI and 12 for the CI.14 For this study

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methenaminesilver-stained slides (with HE-counterstaining), H&E and PAS

stained slides, were used.

The assessment was completed by determining the ISN/RPS2003

classification and activity and chronicity indices for LN. For these aspects of the

assessment, the definitions of the classification systems and the activity and

chronicity indices were used.

Table 2: Laboratory and histological data of 12 patients with active renal disease are shown. na: not assessed § m = male, f = female ¶ histological ISN/RPS classification, nc: not classified # serum creatinine expressed as µmol/l

Immunohistochemistry staining

All specimens were fixed in 10 % neutral buffered formalin and paraffin

embedded. Five-micrometer-thick sections were deparaffinized in xylene and

rehydrated in a series of different concentrations of ethanol. EDTA buffer,

pH 8.2, for heat-induced epitope retrieval was applied for 1 h, followed by

neutralization of endogenous peroxidase with 0.3 % H2O2. Incubation with a

monoclonal mouse anti-human CD8 (DAKO, Glostrup, Denmark) was

performed. Next, sections were washed and incubated with a HRP-conjugated

secondary antibody (EnvisonTM, DAKO, Glostrup, Denmark) for 30 min. at room


105

temperature. A DAB substrate was used for visualization. Washing with PBS

was performed after each incubation step. CD4 (Monosan, Uden, Netherlands)

was performed in the Benchmark Ultra (Ventana, Ventana Medical Systems

S.A. CEDEX France) with citrate buffer heat inducted antigen retrieval and

detected with the ultraView Universal Alkaline Phosphatase Red Detection Kit

(Ventana). Finally, the slides were counterstained with haematoxylin and

mounted with Kaiser’s glycerine gelatin (Merck, Darmstadt, Germany).

Cells were separately counted for the interstitium and glomeruli. Cells

with positive staining for CD8 and CD4 were counted per high powerfield (40 x

magnification). The average value was calculated for each biopsy.

Statistical analysis

Results are presented as mean ±SD and the nonparametric Mann-Whitney U-

test was used for comparison of values between groups. Correlation with

disease activity was assessed using Spearman’s rank correlation coefficient.

Two-tailed P-values less than 0.05 were regarded as statistically significant.

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Results SLE patients have decreased percentage of circulating CCR7-

CD45RO+CD8+ effector memory T-cells during active disease and even

less during active renal disease

We determined the percentages of naïve (CCR7+CD45RO- ;Tnaive), central

memory (CCR7+CD45RO+ ;TCM) and effector memory (CCR7-CD45RO+ ;TEM)

subsets of CD4+ and CD8+ T-cells in peripheral blood of HC and SLE patients.

No differences in the percentages of circulating Tnaive, TCM or TEM CD4+ T-cells

were found between HC and SLE patients. There was a significant difference in

the percentages of TCM and TEM CD8+ T-cells between HC and SLE patients

(TCM: 5.8 ±3.8 % vs. 10.9± 10.3 % p=0.02; TEM: 42.9 ±11.0 % vs. 33.9± 18. %,

p=0.008, Figure 2a and b). TCM were increased in SLE patients while TEM CD8+

T-cells were decreased as compared to HC. There was no difference between

the percentages of circulating naïve CD8+ T-cells in peripheral blood of HC and

SLE patients. Within the patient group there was no difference in the

percentages of circulating naïve CD8+ T-cells in peripheral blood between those

with or without immunomodulating medication.

In addition, subsets were compared between SLE patients with active

and inactive disease. No differences were present between the percentages of

circulating naïve (Tnaive), central memory (TCM) or effector memory (TEM) CD4+ T-

cells of HC as compared to active and inactive SLE patients, respectively

(Figure 2c). Within the CD8+ T-cell populations, a significant increase of

circulating TCM cells was observed in SLE patients with active disease as

compared to HC (TCM: 14.4 ±13.3 % vs. 5.8 ±3.8 % p=0.009). The TEM

population was significantly decreased in active disease as compared to HC

(TEM: 31.6 ±14.9 % vs. 42.9 ±11.0 % p=0.004, Figure 2d).

Next, we assessed the percentages of CD4+ and CD8+ T-cell subsets in

SLE patients in relation to the presence of active renal disease. The percentage

of circulating TCM cells in SLE patients without active LN was significantly

increased compared to HC (TCM: 10.1 ±9.0 % vs. 5.8 ±3.8 % p=0.02) whereas

circulating peripheral TEM cells were decreased in patients with inactive renal


107

disease as compared to healthy controls (TEM: 35.2 ±19.2 % vs. 42.9 ±11.0 %

p=0.03). They were even more decreased in patients with active renal disease

(TEM: 30.4 ±15.9 % vs. 45.1 ±9.4 % p=0.01, Figure 2f) but not statistically

significant compared to inactive disease. There was no difference between

patients with a first episode of LN and those with relapsing renal disease

regarding circulating memory T-cell subsets (data not shown).

Urinary T-cells with effector memory phenotype are associated with active

renal disease in SLE

In parallel to the analysis of peripheral blood we collected urine to quantify the

absolute numbers of CD4+ and CD8+ cells (Figure 3a). The absolute count of

CD4+ T-cells was significantly increased in SLE patients with active lupus

nephritis (LN) as compared to SLE patients without active LN and healthy

controls, respectively (134 ±71 cells/ml vs. 15 ±30 cells/ml, p=0.0001 and vs.

2 ±4 cells/ml, p=0.002). Furthermore, the absolute count of CD8+ T-cells was

significantly increased in SLE patients with active LN as compared to SLE

patients without active LN and HC, respectively (287 ±220 cells/ml vs.

22 ±28 cells/ml, p<0.0001 and vs. 1 ±1 cells/ml, p=0.002). Both increased CD4+

T-cell and CD8+ T-cell counts/ml in urine were associated with active renal

disease and correlated with disease activity as assessed by SLEDAI (CD4+:

r=0.62, p<0.001; CD8+: r=0.68, p<0.001). There was no correlation between

these cell counts and other renal parameters in the subgroup of patients with

active renal disease, such as serum levels of creatinine or 24 h-proteinuria.

Also, histological scores as the activity index (AI) or chronicity index (CI) did no

correlate with the absolute cell count (Table 2).

Additionally, urinary T-cells were assessed for the expression of

CD45RO and CCR7 in order to differentiate between naive T-cells, central

memory T-cells and effector memory T-cells. The majority of urinary CD4+T-

cells as well as CD8+T-cells were CD45RO+CCR7- effector memory cells

(59 ±23 % and 62 ±26 %, respectively). As shown in figure 4d almost no Tnaive

and TCM were present in the urine of these SLE patients.

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Figure 1: Decreased percentages of circulating CD8+ effector memory (TEM) cells in active SLE patients. Representative dot plots of circulating naïve (Tnaive), central memory (TCM) or effector memory (TEM) cells in the peripheral blood of healthy controls, inactive and active patients with SLE are illustrated.


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Figure 2: CD4+ and CD8+ naïve and memory T-cell subsets in the peripheral blood of SLE patients (n=43) and healthy controls (n=20). The percentages of naïve (Tnaive), central memory (TCM) or effector memory (TEM) T-cells within the CD4+ (a) and CD8+ T-cell population (b) in 43 SLE patients (SLE) and 20 healthy controls (HC) are illustrated. The open symbols within the patient group represent patients without immuno-modulating medication (n=12), solid symbols represent patients with immuno-modulating medication. A subanalysis was performed for active (SLE active, n=19) and inactive SLE patients (SLE inactive, n=24) according to the SLEDAI score (c/d). Figure (e) and (f) show data of patient groups analysed according to renal disease activity. Patients with active renal lupus nephritis (SLE with aLN, n=12) were compared to patients without active lupus nephritis (SLE without aLN, n=31) and healthy controls. Bars represent mean percentages. P-valus were calculated using the nonparametric Mann-Whitney U-test.

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Figure 3: Absolute numbers of urinary T-cells. Absolute counts of urinary CD4+ and CD8+ of SLE patients with active lupus nephritis (LN) (n=11), SLE patients without active LN (n=24) and healthy controls (n=5) are shown. Bars represent mean values ±SD. Significant differences are indicated (p< 0.005 = **, p< 0.001 = ***) (a). Urinary CD8+ cells (b and c) show an effector memory phenotype (d).


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CD8+ cells in renal biopsies

To determine the presence and localization of CD8+ T-cells and CD4+ T-cells in

renal biopsies, immunohistochemistry staining with anti-CD8 and anti-CD4 was

performed.

CD8+ T-cells were present in all renal biopsies investigated. The

average amount of CD8+ cells was 6.8 ±6.2 cells/high powerfield. This was

significantly higher than the average amount of CD4+T-cells (2.6 ±4.9 cells/high

powerfield; p=0.02). In ~90 % of specimens CD8+ T-cells were distributed as

peritubular infiltrates, in 50 % of renal biopsies CD8+ T-cells were localized

periglomerularly as well (Figure 4). Intraglomerularly almost no CD8+ T-cells

could be found. The amount of infiltrated CD8+ T-cells did not correlate with the

number of urinary CD8+ T-cells.

Figure 4: Immunhistochemical staining of CD8+ cells in a renal biopsy of a lupus nephritis patient. Periglomerular (a) and interstitial (b) T-cell infiltrates are positive for CD8 (brown).

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Discussion This study demonstrates disturbed frequencies of CD8+ TEM-cells and TCM-cells

in the peripheral blood of SLE patients with active disease, especially a

decrease of CD8+ TEM-cells in active renal disease, consistent with increased

numbers of urinary CD8+ TEM-cells during renal flares. Moreover, infiltrating

CD8+ T-cells could be observed in renal biopsies of patients with active renal

disease. These data support the hypothesis that effector memory T-cells

migrate during active renal disease from the peripheral blood to the kidney and

appear in the urinary sediment.

Previous studies have also reported aberrant CD8+ memory T-cell

populations in the peripheral blood of SLE patients.6;15 Sen et al. described

increased percentages of CD8+ TCM-cells producing Th2 cytokines (IL-4 and IL-

5) in active SLE patients as compared to inactive patients and healthy

controls.15 A decrease of CD8+ TEM-cells was not reported and a subanalysis of

SLE patients with renal involvement was not performed. In accordance with our

results these alterations were restricted to the CD8+ T-cells. In contrast to the

latter and our findings, a recent study by Fritsch et al. reported a shift in the

CD4+ memory T-cell balance towards an increased CCR7-CD4+ memory

population in SLE independent of disease activity.6 However, in that study no

information was given on the characteristics of disease activity and SLEDAI was

relatively low (3.9 ±1.6). As we included 12 patients with active LN, differences

in lymphocyte subsets between both studies might be explained by differnces in

disease activity and/or organ involvement.

Beside significantly reduced numbers of CCR7-CD45RO+ TEM-cells in

the peripheral blood of patients with active SLE, in particular during active renal

disease, which have not been reported so far, this is the first study investigating

the effector phenotype of memory T-cells with special regard to renal disease.

There is increasing evidence from animal and human studies that T-cells, in

particular CD8+ T-cells, contribute to the pathogenesis of LN.3;16 Analysis of 26

renal biopsies of SLE patients with the aim to identify renal infiltrating

leukocytes showed a CD4/CD8 ratio of 0.71 due to a relative increase of the


113

mean percentage of CD8+ cells in LN biopsies which was found to correlate with

histologic activity.3 This finding was confirmed in a recent study where

predominantly infiltrating CD8+ cells were found in kidneys of SLE patients.

Moreover, the authors showed that a high number of periglomerular CD8+ cells

were a characteristic feature of severe and active forms of LN.8

Based on the assumption that these histopathological changes are

reflected by the appearance of effector cells in urine, analysis of urine became

an attractive goal over the last years. Microscopic examination of urine showed

a significantly increased amount of CD3+ cells in SLE patients with active renal

disease.10 A deeper insight in urinary T-cells was provided by an elegant and

cohesive study of CD4+ T-cells in the urine and in renal biopsies of active LN

patients.17 The authors concluded from the presence of these cells in biopsies

and the urinary sediment that CD4+ T-cells were potentially recruited to the

kidney via CXCR3 and finally appeared in the urine. A selective accumulation of

CD4+ cells expressing CCR4 was described as well in renal biopsies of LN-

patients suggesting that this chemokine receptor plays a pivotal role in

recruiting T-cells to the kidney.18 These studies are confirmed by the presence

of CD4+ T-cells in kidney biopsies as well as urinary CD4+ T-cells in our cohort.

However, in contrast to the aforementioned investigations, we observed a

predominant appearance of CD8+ T-cells in biopsies and the urine of patients

with active renal disease. This is a new finding in SLE and corresponds with a

previous study in which mainly CD8+ T-cells were detected in the urine of

patients with IgA nephropathy, Henoch-Schönlein purpura nephritis and anti-

neutrophil cytoplasmatic antibody associated GN.9 Additionally, urinary CD8+ T-

cells, but also urinary CD4+ T-cells were correlated with disease activity. The

phenotypic analysis of these urinary CD8+ T-cells and the observation of renal

infiltrating CD8+ T-cells provide additional evidence that TEM-cells might enforce

inflammatory processes in lupus nephritis. Remarkably, an overwhelming

amount of T-cells in the urine displayed an effector memory cell type, in contrast

to the peripheral blood, supporting the hypothesis of selective migration of TEM-

cells into the inflamed tissue, in particular the kidney.

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The migratory abilities as well as the harmful cytotoxic properties of TEM-cells

are not completely understood so far, but recent investigations may reveal

underlying mechanisms. Evidence for the high migrating capacity of TEM-cells

comes from in vitro experiments demonstrating selective accumulation of TEM-

cells at the site of inflammation.12;19;20 Roberts et al. demonstrated an

enrichment of TEM-cells in the peritoneal cavity of patients on peritoneal

dialysis.19 Gattorno et al. observed an enrichment of TEM-cells in the synovial

fluid of patients with juvenile arthritis as compare to the peripheral blood.21 The

authors conclude that these TEM-cells migrate selectively from peripheral blood

into the inflamed tissue.

Urinary and infiltrating TEM-cells are presumed to have cytotoxic

capacities. A characteristic feature of CD8+ TEM-cells is the expression of

perforin and granzyme B which mediate tissue injury.22;23 In renal transplant

tissue the amount of infiltrated CD8+ TEM-cells, being strongly positive for

granzyme B, has been found to be associated with the severity of graft

damage.24 Apart from perforin and granzyme B, cytotoxic effector T-cells are

able to release IFN-γ upon stimulation.25 Further investigations are necessary to

reveal the mechanisms underlying migration and cell mediated cytoxicity of

CD8+ TEM-cells in the pathogenesis of LN.

The present study is the first investigating effector memory T-cells in the

peripheral blood, renal biopsy and urine of SLE patients. The data provide

strong evidence that CD8+ TEM-cells migrate from the peripheral blood to the

kidney and appear in the urine during active LN. Therefore, CD8+ TEM-cells

could be a useful monitoring tool in SLE patients with renal involvement.

Additionally, based on the potential pathological role of these CD8+ TEM-cells,

they could represent a new therapeutic target.


115

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characterisation of CCR7+ and. Arthritis Res. Ther. 7:R256-R267.


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23. Blanco,P., V.Pitard, J.F.Viallard, J.L.Taupin, J.L.Pellegrin, and J.F.Moreau. 2005.

Increase in activated CD8+ T lymphocytes expressing perforin and granzyme B

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25. Ahn,J.K., H.Chung, D.S.Lee, Y.S.Yu, and H.G.Yu. 2005. CD8brightCD56+ T cells

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175:6133-6142.

Chapter 6

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Urinary T cells in active lupus nephritis show an effector memory phenotype

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