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Accepted: 2016.09.26 Published: 2017.01.24

Increase in CD8+CD158a+ T Cells in Kidney Graft

Blood is Associated with Better Renal Function

ACDEF 1

Abelardo Caballero

ABDEF 2

Eulalia Palma

ABCDEF 2

Pedro Ruiz-Esteban

ABD 2

Eugenia Sola

ABD 2

Verónica López

ABD 2

Laura Fuentes

ABD 2

Edisson Rudas

ABD 2

Lara Perea

ACDEFG 2

Domingo Hernández

Corresponding Author: Domingo Hernández, e-mail: domingohernandez@gmail.com

Source of support: The present study was supported in part by grants from the Instituto de Salud Carlos III co-funded by the Fondo Europeo de Desarrollo Regional – FEDER (RD16/0009/0006; grant ICI14/00016) from the Spanish Ministry of Economy and Competitiveness

Background: Studies of liver and heart transplant patients have shown a gradual reconstruction of the CD8 KIR2D+ T cell subpopulations, measured in peripheral blood (PB), associated with better graft acceptance. The kinetics of these populations in kidney transplants, however, is still poorly understood, especially given the lack of stud-ies of blood samples from the kidney graft.

Material/Methods: Flow cytometry was used to measure CD8+CD158a/b/e T cells in 69 kidney transplant patients who had sta-ble renal function during follow-up. Measurements were made at 3, 6, and 12 months post-transplantation in graft capillary blood extracted by fine needle aspiration puncture (FNAP) and in PB.

Results: No progressive increase was found in the PB subpopulations. However, the CD8+CD158a+ subsets increased significantly at 12 months in the graft blood versus the PB samples (3.91±4.59 vs. 2.84±4.71; p=0.021). The ra-tio of the percentage of CD8+CD158a+ cells in graft blood compared to PB at 12 months was associated with better renal function in those patients with a ratio ³3 (66.6±14.53 vs. 55.7±21.6; p=0.032).

Conclusions: An increased ratio of CD8+CD158a+ cells, measured by flow cytometry, between graft blood and PB was asso-ciated with improved renal function.

MeSH Keywords: Biopsy, Fine-Needle • Peripheral Blood Stem Cell Transplantation • Receptors, KIR

Abbreviations: FNAP – fine needle aspiration puncture; PB – peripheral blood; KIR – killer-cell immunoglobulin-like receptor; GB – graft blood

Full-text PDF: http://www.annalsoftransplantation.com/abstract/index/idArt/900680

Authors’ Contribution: Study Design A Data Collection B Statistical Analysis C Data Interpretation D Manuscript Preparation E Literature Search F Funds Collection G

1 Department of Immunology, Carlos Haya Regional University Hospital, Institute of Biomedical Research in Malaga (IBIMA), University of Malaga, Malaga, Spain 2 Department of Nephrology, Carlos Haya Regional University Hospital, Institute of

Biomedical Research in Malaga (IBIMA), University of Malaga, Malaga, Spain

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Background

Killer-cell immunoglobulin-like receptor (KIR) molecules belong to a polygenic and polymorphic family of receptors [1], appear-ing on NK, Tgd, CD4, and CD8 cells [2–4]. Some of these mole-cules can have two or three extracellular domains (KIR2D/3D), the ligands of which are found in the HLA-C sequence of the molecules [5]. There exist activator KIR and inhibitory KIR, sig-nals which are generated via the immunoreceptor tyrosine-based activation motif (ITAM) or the immunoreceptor tyro-sine-based inhibition motif (ITIM) [6].

CD8+ T cell lymphocytes play an important role in both graft acceptance and graft rejection [7,8]. In this subpopulation the expression of KIRs starts to appear on the CD8 effector mem-ory cells, and an important fraction of terminally differentiat-ed effector CD8+ T cells are KIR+ [9]. The inhibitory KIRs can modulate the signalling of the T cell receptor and blunt the response of CD8+ T cells.

The expression of KIR molecules on T lymphocytes is induced after chronic antigen stimulation [10], such that in the evolu-tion of the transplant, with its permanent presence of alloan-tigens, an increase in CD8+ KIR2D T cells can be expected to be associated with better graft acceptance [11].

Studies undertaken in these lymphocyte subpopulations in the transplant setting have used samples from peripheral blood (PB) [11,12]. The determination of these subpopulations has not been done using cells obtained from grafts using fine nee-dle aspiration puncture (FNAP) in patients with a stable kidney graft function. This may be because these lymphocyte popu-lations participate in the mechanisms of tolerance and FNAP is used in those cases having an important degree of lym-phocyte infiltrate, as happens in cases of acute rejection [13]. However, a sample obtained by FNAP is usually also accompa-nied by blood, which given the puncture site is of capillary or-igin, we refer to the sample obtained by FNAP as graft blood (GB). When the parenchymal infiltrate is reduced, as occurs in patients with stable graft function, the cells obtained by punc-ture are mostly from GB.

Studies comparing lymphocyte proportions in capillary blood and peripheral venous blood have found no significant dif-ferences between the two blood samples [14]. These studies, however, were undertaken with capillary blood from healthy tissue; no studies have yet used blood from inflamed tissues. The cell proportions in capillaries from inflamed tissue, may be influenced by the chemokines released in the tissue. This would result in the proportion of leukocytes differing when compared with peripheral blood, and this difference could pro-vide useful information about the state of the inflamed tissue, especially if it is significant.

Bearing in mind the aforementioned, the aim of this prelim-inary study was to determine, using flow cytometry, the per-centage of CD8+CD158a/b/e cells in capillary blood collected by FNAP of the renal allograft, with the idea of determining whether the percentages of these particular subpopulations differ between GB and PB (GB/PB ratio of CD8+CD158a/b/e cells) from transplant patients with stable renal function.

Material and Methods

Study design

We undertook a one-year, nonrandomized, controlled longitu-dinal study of all patients who received a kidney transplant at Carlos Haya Regional University Hospital in Malaga, Spain, from February 1, 2012 to June 30, 2013. The inclusion criteria were: a) age >18 years; b) deceased or living donor single kidney trans-plant, including retransplants; c) absence of any coagulation disorders being treated with oral anticoagulants or double anti-aggregation. The exclusion criteria were: a) dual transplants; b) multiple transplants (pancreas-kidney or kidney-liver); c) recipi-ent age <18 years; d) hemorrhagic coagulation disorder or treat-ment with anticoagulants or double anti-aggregation; e) bleeding during the immediate postoperative period that required reop-eration; f) obesity (body mass index >35 kg/m2). Patients were

also excluded if they had biopsy-diagnosed rejection.

Written informed consent was obtained from all the par-ticipants. Medical record review was performed according to Spanish law, and the study was approved by the Ethics Committee of Malaga Regional University Hospital and con-ducted according to the Declaration of Helsinki and Istanbul.

Flow cytometry analysis of lymphocyte subpopulations in peripheral blood

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antibodies: CD3, CD8, CD158a, and CD158b. In the second tube we added 10 μL of the following monoclonal antibodies: CD3, CD8, and CD158e. Labelled isotype control antibodies were used at equivalent concentrations. After incubation for 20 minutes the sample was lysed with BD FACS Lysing Solution. The lymphocyte population was gated in the FSC/SSC plot and then the CD3+ and CD8+ cell subpopulations were separated into their corresponding fractions. The expression of CD158a+, CD158b+, and CD158e+ was shown on the y-axis (Figure 1). The method used to consid-er the expression of CD158a+, CD158b+, and CD158e+ as posi-tive or negaposi-tive is shown and described in the legend to Figure 1. The gating and analyses were performed using BD FACSDiva™ 6.x software at www.bdbiosciences.com/facsdiva.

Flow cytometry analysis of lymphocyte subpopulations in capillary graft blood

Fine-needle aspiration puncture (FNAP) was done according to the technique of Hayry [15] at months 3, 6, and 12 post-transplantation at the same time as the venous blood punc-ture. FNAP was done by ultrasound-guided puncture (Sonsite Fujifilm, Sitelink Image Manager 2.2), without anaesthesia, at the external cortical pole of the graft (upper or lower), thus avoiding the large vessels, using a 0.8 mm/25G needle mount-ed in a Cameco-Gun device. This material was placmount-ed in 10 mL of Hepes culture medium buffered with heparin and human

serum albumin (HAM solution). The sample was centrifuged at 1,600 rpm and the pellet resuspended in 100 µL of RPMI and 50 µL was added to each tube. The subpopulations were mea-sured as described for the PB samples. Only when the plot of the sample obtained by FSC/SSC analysis was similar to that of the peripheral blood, was the study considered valid (Figure 1).

Statistical analysis

A descriptive analysis was made of the results, expressing the quantitative variables as mean ± standard deviation (SD) and the qualitative variables as relative percentages. Normality was tested (Kolmogorov-Smirnov and Shapiro-Wilk) to determine the distribution of the data. The Spearman correlation coeffi-cient was used to analyse unadjusted relationships between continuous data and hypothesis contrasts using parametric or nonparametric tests (Student t or Mann-Whitney U and chi square or Fisher test). An analysis of repeated measures was used to detect changes during the follow-up of the quantitative variables. The ratio of the percentage of CD8+CD158a+ cells in GB compared to that in the PB (GB/PB ratio of CD8+CD158a+ cells) was calculated at 12 months.

The statistical analyses were performed with SPSS Statistics 15.0 for Windows (SPSS Inc., Chicago, IL, USA) and significance was set at p<0.05.

PB SSC-A SSC-A (×1.000) 250 200 150 100 50 (×1.000) 250 200 150 100 50 SSC-A (×1.000) 250 200 150 100 50 SSC-A (×1.000) 250 200 150 100 50 GB

50 100 150 200 250 (×1.000) FSC-A

102 103 104 105

CD3PerCP-Cy5-5-A 102 CD8APC-Cy7-A103 104 105 10

2 103 104 105

CD8ACP-Cy7-A CD158aPE-A CD158bFIT C-A CD158ePE-A

102 103 104 105

CD8ACP-Cy7-A 102 103 104 105

CD8ACP-Cy7-A 50 100 150 200 250

(×1.000) FSC-A

Lymphocites fraction

CD3+

fraction fractionCD8+

Lymphocites fraction CD3+ CD8+ CD158a+ CD158a– CD158b+ CD158b– CD158e+ CD158e– 105 104 103 102 105 104 103 102 105 104 103 102

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Results

Patients

Of the patients who received a kidney transplant at our cen-ter during the study inclusion period, 69 had stable renal func-tion during the follow-up. The main cause of chronic kidney

disease in these patients was glomerulonephritis (24.2%) fol-lowed by polycystosis (16.7%). All the patients were receiving standard triple immunosuppression therapy during the study period (calcineurin inhibitors, mycophenolate mofetil, and ste-roids) and 78.2% received induction therapy. Delayed kidney function occurred in 31.9% of patients and 13% had received a previous kidney transplant. Renal function (MDRD) in the Donor

Age, yr, mean ±SD 52.61±12.36

Gender (male) (%) 66.7

Cause of death (%) Stroke

Traumatic brain injury Living donors Others

69.1 19.1 8.8 2.9

Cold ischaemia time, h, mean ±SD 14.16±4.84

Recipient

Age, yr, mean ±SD 55.15±15.61

Gender (male) (%) 68.7

Time on dialysis in months, mean ±SD 35.68±38.32

Cause of CKD (%) Diabetes

Interstitial nephropathy Familial nephropathy Glomerulonephritis Polycystic kidney disease Nephroangiosclerosis Unknown aetiology Others

6.1 7.6 1.5 24.2 16.7 6.1 21.2 16.7 HLA incompatibility (%)

0/1/2/3/4/5/6 1.5/2.9/8.8/25.0/32.4/22.1/7.4

Retransplant (%) 13.0

Delayed graft function (%) 31.9

Immunosuppression treatment (%) TaC+MMF+P

CsA+MMF+P

97.1 2.9 Induction treatment (%)

Basiliximab Thymoglobulin None

50.7 27.5 21.7

MDRD at three months (mL/min/1.73 m2) 48.14±19.54

MDRD at six months (mL/min/1.73 m2) 52.90±21.34

MDRD at twelve months (mL/min/1.73 m2) 59.40±19.39

Table 1. Clinical and demographic characteristics of the study population (n=69).

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patients improved significantly (p<0.001) during the follow-up period (Table 1).

Percentage of CD8+CD158a/b/e subpopulations in graft blood and peripheral blood samples

Table 2 shows the mean percentage and evolution of CD8+CD158a/b/e cells obtained at the different times for the two sample types studied. Only the CD8+CD158a+ subpopu-lation at 12 months differed significantly between the GB and PB samples (2.84±4.31 vs. 3.93±4.59; p=0.021). No significant differences were seen for the other cell populations between the two sample types. Repeated measures analysis showed no significant changes during the study.

Relationship between GB/PB ratio of CD8+CD158a+ cells and renal function

Considering that the CD8+CD158a+ subpopulation has an inhib-itory effect on the immune system, we examined whether there was a relation between the GB/PB of CD8+CD158a+ cells ratio and renal function. The median GB/PB ratio of CD8+CD158a+ cells was 2 (interquartile range 1.00–5.37). No significant dif-ferences were found between patients who were above or below the average ratio (data not shown). However, patients who had a ratio ³3 had better kidney function than patients with a ratio below 3 (66.6±14.53 vs. 55.7±21.6; p=0.032). A value of 3 was chosen because this was the minimum medi-an GB/PB ratio of CD8+CD158a+ cells that gave a significmedi-ant value for renal function.

PB (%) GB (%) p

CD3+ CD8+ CD158a+ cells

Three months 2.13±3.72 3.37±5.86 0.145

Six months 1.96±3.65 2.96±4.36 0.080

Twelve months 2.84±4.71 3.93±4.59 0.021

CD3+ CD8+ CD158b+ cells

Three months 5.44±6.84 6.68±7.27 0.306

Six months 5.73±5.87 6.73±6.35 0.271

Twelve months 5.99±6.51 6.99±7.32 0.385

CD3+ CD8+ CD158e+ cells

Three months 1.16±1.88 1.01±1.68 0.546

Six months 1.59±3.26 1.56±3.16 0.744

Twelve months 1.44±1.86 1.09±1.42 0.516

Table 2. Statistical comparison of CD8+CD158a/b/e subpopulations between peripheral blood and graft blood at three, six and twelve months post-transplantation.

Mean ± standard deviation. PB – peripheral blood; GB – graft blood.

Spearman’s rank correlation coefficient

between GB vs. PB P value

CD3+ CD8+ CD158a+ cells

Three months 0.447 0.001

Six months 0.573 <0.001

Twelve months 0.654 <0.001

CD3+ CD8+ CD158b+ cells

Three months 0.744 <0.001

Six months 0.868 <0.001

Twelve months 0.781 <0.001

CD3+ CD8+ CD158e+ cells

Three months 0.783 <0.001

Six months 0.657 <0.001

Twelve months 0.555 <0.001

Table 3. Spearman’s correlation between graft blood and peripheral blood at three, six and twelve months.

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Internal validation

Internal validation using the Spearman correlation coefficient showed a good positive correlation between GB and PB for each of the subpopulations in the various study periods (Table 3).

Discussion

The main finding of this study was a significant increase 12 months post-transplantation in the CD8+CD158a+ subpopu-lation in GB compared with PB. In addition, this increased ra-tio was associated with improved renal funcra-tion, which could indicate a better graft acceptance. Although measurement of lymphocyte subpopulations using flow cytometry in samples from aspiration cytology has been used before as a diagnostic tool for acute rejection [16], this is the first time it has been employed to assess grafts which have a stable function. The most important difficulty when using FNAP in a graft that is minimally inflamed concerns obtaining sufficient cells from the parenchyma. However, this technique usually also obtains capillary blood, and our study used the CD8+CD158a/b/e T cell populations from this blood, which were then compared with the results found in PB samples. The origin of the blood in our graft samples were confirmed by the plot obtained by FSC/SSC analysis (Figure 1).

Peritubular capillaries that contain mononuclear cells suggest cell-mediated acute rejection [17]. The severity of the rejection is associated with the number of cells contained, both in the capillaries and in the interstitium. Accordingly, subclinical rejec-tion may be apparent only on biopsy of the organ and, in the absence of renal dysfunction, can damage the allograft [18]. In our study, the significant increase in the CD8+CD158a+ sub-population in GB compared to PB could be the result of a T cell-mediated reaction. Bearing in mind that these patients had a stable renal function they might therefore be experiencing a subclinical inflammation, but unfortunately we did not per-form protocol biopsies to confirm this possibility.

The presence of Treg cells in asymptomatic cell infiltrates de-tected on protocol biopsies may be a reliable biomarker to dis-tinguish between an infiltrate with features of rejection and one with protective characteristics. In addition, the FoxP3+ Treg/CD3+ T cell ratio has been found to correlate positively with graft function two years post-transplantation, suggest-ing that an increassuggest-ing proportion of Treg cells in the infiltrate above the total number of T cells may facilitate kidney graft function [19].

Evidence suggests that the CD8+CD158a+ subpopulation also has a down-regulatory function in the immune response. Renal tumors are known to contain infiltrating CD8+ T cells that

express the CD158a molecule. In vitro, these CD8+ T lympho-cytes correspond to highly lytic effectors, but their functional activity against tumor cells is strongly inhibited by this recep-tor, thus provoking the escape of the immune response [20].

The results suggest that the difference, which was significant, between the CD8+CD158a+ population in GB and PB may be due to the presence in most of these patients of a certain de-gree of subclinical inflammation whose cytokines probably influence the increase in these cells in graft capillary blood, especially 12 months post-transplant. This would again sug-gest that a stable renal function does not imply absence of inflammatory activity. The type of cells present in the inflam-matory infiltrate could determine future graft status. The pres-ence of cells with immunosuppressive characteristics, such as CD8+CD158a+, would be associated with better survival. In addition, the relatively harmless use of FNAP compared with a biopsy would enable more frequent sampling to determine the progress of the transplanted organ, with application of any adjustments of treatment on a personalized basis.

We also examined whether a greater GB/PB ratio of CD8+CD158a+ cells was associated with the different induc-tion therapy regimens. The results showed no significant dif-ferences in this respect (data not shown), indicating that the increase in CD8+CD158a+ cells in GB 12 months post-trans-plant in these patients could be due to other as yet unknown factors. Nevertheless, this requires confirmation with a larger study that includes biopsy samples as well.

As suggested in our study of kidney transplant recipients, in liver and heart transplants a better graft acceptance has been associated with low modulation of CD28 in T cells soon after transplantation and a gradual reconstitution of CD28–

CD8P+KIR2D+. Control of the expression of the CD28 and KIR2D

receptors on T cells is therefore considered to act as a sensor of the immune status in these patients [12]. In our study, how-ever, we failed to find a significant progressive increase in the CD8+KIR2D subpopulation assayed (CD158a/b) in the PB

sam-ples, even though the patients had a stable renal function and presented a progressive increase in the glomerular filtration rate values (MDRD). Thus, at least in kidney transplants, the measurement of these subpopulations just in PB may be insuf-ficient to evaluate the immunological status of the transplant.

Conclusions

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evaluate the potential for this technique to be used as a tool to monitor kidney transplant recipients.

Acknowledgments

The authors thank the renal transplantation team of Carlos Haya University Regional Hospital for their collaboration. We also thank Ian Johnstone for linguistic assistance in the prep-aration of the text.

References:

1. Vilches C, Parham P: KIR: Diverse, rapidly evolving receptors of innate and adaptive immunity. Annu Rev Immunol, 2002; 20: 217–51

2. Battistini L, Borsellino G, Sawicki G et al: Phenotypic and cytokine analysis of human peripheral blood gamma delta T cells expressing NK cell recep-tors. J Immunol, 1997; 159: 3723–30

3. van Bergen J, Kooy-Winkelaar EMC, van Dongen H et al: Functional killer Ig-like receptors on human memory CD4+ T cells specific for cytomegalo-virus. J Immunol, 2009; 182: 4175–82

4. Anfossi N, Doisne J-M, Peyrat M-A et al: Coordinated expression of Ig-like inhibitory MHC class I receptors and acquisition of cytotoxic function in human CD8+ T cells. J Immunol, 2004; 173: 7223–29

5. Parham P: MHC class I molecules and KIRs in human history, health and survival. Nat Rev Immunol, 2005; 5: 201–14

6. Long EO: Regulation of immune responses through inhibitory receptors. Annu Rev Immunol, 1999; 17: 875–904

7. Su CA, Iida S, Abe T, Fairchild RL: Endogenous memory CD8 T cells directly mediate cardiac allograft rejection. Am J Transplant, 2014; 14(3): 568–79 8. Krupnick AS, Lin X, Li W et al: Central memory CD8+ T lymphocytes

medi-ate lung allograft acceptance. J Clin Invest, 2014; 124(3): 1130–43 9. Young NT, Uhrberg M, Phillips JH et al: Differential expression of leukocyte

receptor complex-encoded Ig-like receptors correlates with the transition from effector to memory CTL. J Immunol, 2001; 166: 3933–41

10. Vivier E, Anfossi N: Inhibitory NK-cell receptors on T cells: Witness of the past, actors of the future. Nat Rev Immunol, 2004; 4: 190–98

11. López-Alvarez MR, Moya-Quiles MR, Minguela A et al: HLA-C matching and liver transplants: Donor-recipient genotypes influence early outcome and CD8+KIR2D+ T-cells recuperation. Transplantation, 2009; 88: S54–61

Statement

The authors declare they have no conflicts of interest with this study and confirm that the manuscript was not prepared or funded by a commercial organization. The only funding re-ceived was from government grants.

12. Blanco-García RM, López-Álvarez MR, Garrido IP et al: CD28 and KIR2D re-ceptors as sensors of the immune status in heart and liver transplantation. Hum Immunol, 2011; 72: 841–48

13. Olteanu H, Schur BC, Bredeson C et al: Expression of natural killer recep-tors in T- and NK-cells: comparison of healthy individuals, patients with prior stem cell transplant, and patients undergoing chemotherapy. Leuk Lymphoma, 2010; 51: 481–87

14. Hollis VS, Holloway JA, Harris S et al: Comparison of venous and capillary differential leukocyte counts using a standard hematology analyzer and a novel microfluidic impedance cytometer. PLoS One, 2012; 7: e43702 15. Häyry PJ: Fine-needle aspiration biopsy in renal transplantation. Kidney Int,

1989; 36: 130–41

16. Xavier PDP, Lema GL, Magalhães MC et al: Flow cytometry assessment of graft-infiltrating lymphocytes can accurately identify acute rejection in kid-ney transplants. Clin Transplant, 2014; 28: 177–83

17. Nankivell BJ, Alexander SI: Rejection of the kidney allograft. N Engl J Med, 2010; 363: 1451–62

18. Nankivell BJ, Borrows RJ, Fung CL-S et al: The natural history of chronic al-lograft nephropathy. N Engl J Med, 2003; 349: 2326–33

19. Bestard O, Cruzado JM, Rama I et al: Presence of FoxP3+ regulatory T Cells predicts outcome of subclinical rejection of renal allografts. J Am Soc Nephrol, 2008; 19: 2020–26

Figure

Figure 1.   Flow scheme used to establish the areas to calculate the percentages of CD3+ CD8+ CD158a/b/e positive cells in both  peripheral blood and graft blood
Table 1.  Clinical and demographic characteristics of the study population (n=69).
Table 2.   Statistical comparison of CD8+CD158a/b/e subpopulations between peripheral blood and graft blood at three, six and twelve  months post-transplantation.

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