Surface markers of lymphocyte activation and markers of cell proliferation

Clinica Chimica Acta 413 (2012) 1338–1349

Contents lists available at SciVerse ScienceDirect

Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim

Invited critical review

Surface markers of lymphocyte activation and markers of cell proliferation Maria Shipkova, Eberhard Wieland ⁎ Zentralinstitut für Klinische Chemie und Laboratorioumsmedizin, Klinikum Stuttgart, Stuttgart, Germany

a r t i c l e

i n f o

Article history: Received 29 May 2011 Received in revised form 1 November 2011 Accepted 4 November 2011 Available online 19 November 2011 Keywords: T cell activation Cell proliferation Immunosuppressants Pharmacodynamics Mitogen stimulation Transplantation

a b s t r a c t The individualization of immunosuppression is an approach for preventing rejection in the early phase after transplantation and for avoiding the long-term side effects of over immunosuppression. Pharmacodynamic markers, either specific or nonspecific, have been proposed as complementary tools to drug monitoring of immunosuppressive drugs. A key event in graft rejection is the activation and proliferation of the recipient's lymphocytes, particularly T cells. Activated T cells express surface receptors, such as CD25 (the IL-2 receptor) and CD71 (the transferrin receptor), or co-stimulatory molecules (CD26, CD27, CD28, CD30, CD154 or CD40L, and CD134). Both surface marker expression and cell proliferation are predominately assessed by flow cytometry. Protocols have been established and utilized for both in vitro and ex vivo investigations with either isolated lymphocytes or whole blood. This article reviews the current body of research regarding the use of lymphocyte proliferation and surface activation markers with an emphasis on T cells. Experimental and clinical results related to these markers, as well as methodological issues and open questions, are addressed. © 2011 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Why markers of lymphocyte proliferation and activation? . . . . . . . . . . . . . . . . Transplant rejection, lymphocyte activation and proliferation . . . . . . . . . . . . . . Markers of lymphocyte proliferation and activation . . . . . . . . . . . . . . . . . . . 3.1. Proliferation markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Markers of cell activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Methods for assessing markers of lymphocyte proliferation and activation . . . . . . . . 5. Analytical performance of methods for measuring T cell activation and proliferation . . . 6. The effect of immunosuppressants on lymphocyte proliferation and activation in vitro. . . 7. The effect of immunosuppressants on lymphocyte proliferation and activation ex vivo . . . 8. Association between markers of lymphocyte proliferation, as well as activation and clinical 9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disclosure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . events . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

1339 1339 1339 1339 1339 1340 1344 1344 1345 1347 1347 1347 1347 1347

Abbreviations: APC, Antigen Presenting Cell; BrdU, Bromodeoxyuridine; CD, Cluster of Differentiation; CNI, Calcineurin Inhibitor; CON, Concanvalin A; CFSE, Carboxyfluorescein Succinimidyl Ester; CTLA, Cytotoxic T Lymphocyte Antigen; CYA, Cyclopsorine A; EVER, Everolimus; GITR, Glucocorticoid-induced TNFR-Related Protein; HVEM, Herpes Virus Entry Mediator; ICAM-1, Intercellular Adhesion Molecule 1; ICOS, Inducible T Cell Co-stimulator; IL, Interleukin; IL-2R, Interleukin 2 Receptor; IFN-γ, Interferon γ; ION, Inonomycin A; KIR, Killer-Cell Immunoglobulin-like Receptors; LFA, Lymphocyte Function Associated Antigen; MPA, Mycophenolic Acid; MHC, Major Histocompatibility Complex; NK, Natural Killer Cells; PD, Pharmacodynamic; PCNA, Proliferating-Cell-Nuclear-Antigen; PMA, Phorbol Myristate Acetate; PWM, Pokeweed Mitogen; TAC, Tacrolimus; TCR, T Cell Receptor; TIM, Type 1 Trans-membrane Glycoprotein; TGF-β, Tissue Growth Factor β; TNF α, Tumor Necrosis Factor α; SRL, Sirolimus; VLA-4, Very Late Antigen 4. ⁎ Corresponding author at: Klinikum Stuttgart, Zentralinstitut für Klinische Chemie und Laboratoriumsmedizin, Kriegsbergstrasse 60, D-70174 Stuttgart, Germany. Tel.: +49 711 27834800; fax: +49 711 27834809. E-mail address: [email protected] (E. Wieland). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.11.006

M. Shipkova, E. Wieland / Clinica Chimica Acta 413 (2012) 1338–1349

1. Why markers of lymphocyte proliferation and activation? Modern immunosuppressive regimens have paved the way to achieving low acute rejection rates in the early phases after the transplantation of solid organs. Rejection rates are generally less than 20% within the first year after the transplantation of liver and kidney grafts [1,2]. However, the long-term toxic effects of immunosuppressants include malignancy, infection, and metabolic disorders, such as diabetes, hypertension, and dyslipidemia [3]. In addition, in kidney transplantation, chronic allograft rejection is not effectively treated with current immunosuppressive drugs and is responsible for long-term allograft failure [4]. An ongoing challenge of transplantation medicine, therefore, is to tailor immunosuppression to maintain efficacy while minimizing toxicity. At present, maintenance immunosuppressive therapy is “fine-tuned” according to drug concentrations in the blood, biomarkers of organ function, clinical symptoms, and biopsy results. However, noninvasive, reliable, and reproducible indicators, which would enable individualization of the immunosuppressive treatment protocol, are needed. Drug specific-pharmacodynamic (PD) effects have been monitored successfully through such methods as measuring target enzyme activities or monitoring drug-specific gene expression profiles [5]. However, immunosuppressive protocols generally consist of more than one drug and involve induction therapy with antibodies directed against T cells or the interleukin 2 receptor (IL-2R). Current combination therapies generally rely on a combination of T cell activation and proliferation inhibitors, such as cyclosporine A (CYA) or tacrolimus (TAC), and/or an antiproliferative drug, such as mycophenolic acid (MPA), sirolimus (SRL), or everolimus (EVER). Therefore, a non-specific, more general approach reflecting T cell activation and proliferation might be promising. 2. Transplant rejection, lymphocyte activation and proliferation During organ transplantation, the immune systems of the graft and of the recipient are activated by surgical stress, as well as by ischemia/reperfusion injury of the graft [6]. This activation leads to the production and release of proinflammatory cytokines, which mediate the infiltration of the graft by lymphocytes of the recipient [7]. The immune response of a host to an allogeneic graft is characterized by a cellular, lymphocyte-dependent reaction and a humoral, antibody-mediated reaction [8]. T cells, which are characterized by the expression of CD3 (CD = cluster of differentiation), play the central role in the cellular lymphocyte-mediated process of graft rejection. T cell activation can be divided into three phases: the induction phase, the expansion phase, and the effector phase [9]. In the induction phase, T helper (Th) cells (CD4 +) and cytotoxic T cells (CD8 +) cells interact with the alloantigens present on the cells of the donor graft or on the antigen presenting cells (APC) of the transplant recipient through their T cell receptors (TCRs). Antigens presented by APCs through major histocompatibility complex (MHC) class II molecules primarily react with CD4 + cells, whereas MHC class I molecules mainly interact with CD8 + T cells [8], although the interactions are not exclusive [10]. There is accumulating evidence that the innate immune system enhances the activation of the adaptive alloimmune response. Tolllike receptors (TLR) are germline encoded pathogen recognition receptors expressed on APCs. Activation of TLRs results in the release of proinflammatory cytokines priming the adaptive immune response [11]. In addition, TLRs are also expressed on T cells complementing T cell receptor induced signals to enhance T cell proliferation and cytokine production [12]. Emerging data also show that immune cell derived complement is an important regulator of T cell immune responses [13]. Furthermore, natural killer (NK) cells also get activated in response to stress ligands expressed on allografts. They have cytolytic effector functions and also release proinflammatory cytokines

1339

enhancing adaptive T cell responses after ligation of activating receptors such as NKG2D a C-type lectin receptor [14]. On the other hand NK cells can promote tolerance by killing donor APCs and secreting anti-inflammatory cytokines such as IL-10 and TGF-β thus preventing allospecific T cell activation [15]. Other players for T cell activation are B cells, which can be critical for optimal T cell activation trough antigen presentation and cytokine production [16]. Once activated, the specific T cells proliferate in the lymph nodes and become competent to react to subsequent signals presented by the APCs. During this expansion phase, proliferating T cells divide rapidly and secrete interleukin 2 (IL-2) and a variety of other cytokines. CD4+ cells further differentiate into subtypes, such as Th1 and Th2. In general, Th1 cells are mainly involved in cell-mediated immunity, whereas Th2 cells are involved in humoral immunity, supporting B cell activation and maturation, although the Th1 and Th2 cells may exhibit some synergism in their activities [17]. IL-2 and interferon γ (IFN-γ) are crucial for T cell proliferation [18]. In the effector phase, alloactivated T cells interact with their cognate antigens on target cells, causing cytotoxicity, cell destruction, and apoptosis. An allograft carries a number of APCs, also called “passenger cells,” in the form of interstitial dendritic cells or B cells, which can directly stimulate the recipient's T cells. In addition, T cells can be activated by indirect mechanisms if the alloantigens are presented as peptides by self-APCs [6]. T cell-derived cytokines, such as tumor necrosis factor α (TNF α), and chemokines (e.g., CCL2, CCL5, and CX3CL1) are involved in the effector phase, promoting intense macrophage infiltration into the allograft. T cell activation and proliferation can be achieved in vitro in a nonspecific manner by lectins such as concanavalin A (CON A), pokeweed mitogen (PWM), and phytohemagglutinin (PHA). Phorbol myristate acetate (PMA) activates protein kinase C, and ionomycin (ION) artificially increases intracellular calcium [19]. T cell activation can also be mimicked by ligation of the TCR and co-stimulatory receptors through specific antibodies and third party cells or, more specifically, by allogeneic stimulation with donor-specific APCs in a mixed-lymphocyte reaction [20,21]. While the mitogens ION and PMA are more suitable for T cell activation, CON A induces proliferation more effectively [22]. 3. Markers of lymphocyte proliferation and activation 3.1. Proliferation markers The proliferation and division of eukaryotic cells is separated into four phases. The first phase [G1] prepares for DNA replication, while the second phase [S] results in the production of two identical sets of chromosomes. In the third phase [G2], significant protein synthesis occurs. The M phase consists of nuclear and cytoplasmic division. Most assays to follow cell proliferation are based on either the incorporation of a labeled nucleoside DNA precursor into newly synthesized DNA in the S phase, the expression of nuclear antigens, such as proliferating cell nuclear antigen (PCNA) an auxiliary protein of DNA polymerase and Ki-67 [23,24], or the assessment of the mitochondrial activity. PCNA is induced in the late G1 and S phases and is necessary for DNA synthesis and cell cycle progression. Alternatively, a dye that is equally distributed between the daughter cells can also be employed. 3.2. Markers of cell activation Although activation markers can be observed on most lymphocytes, including B cells, NK cells, dendritic cells, and cells of the myeloid lineage (neutrophils, monocytes, macrophages, and eosinophils) most data concerning pharmacodynamic monitoring of immunosuppressive therapy have been generated with T cells. Activated T cells

1340

M. Shipkova, E. Wieland / Clinica Chimica Acta 413 (2012) 1338–1349

express molecules on their cell surfaces, which clearly distinguishes them from naïve T cells. These molecules include receptor proteins, co-stimulatory molecules, adhesion molecules, chemokine receptors, and MHC class II molecules (Fig. 1). Examples of receptor proteins observed to be upregulated on proliferating lymphocytes include CD25 (the interleukin-2 receptor), CD69 (the early activation antigen), CD70 (the ki24 antigen), CD71 (the transferrin receptor), and CD95 (the Fas receptor) [25–29]. Co-stimulatory molecules can be classified based on their molecular structures. They either belong to the immunoglobulin G (IgG) superfamily, the tumor necrosis factor–tumor necrosis factor receptor (TNF–TNFR) family, or the TIM (type 1 trans-membrane glycoprotein) family. Members of the IgG superfamily include CD28, CD152 or CTLA-4 (cytotoxic T lymphocyte antigen 4), ICOS (inducible T cell co-stimulator), and PD-1 (programmed cell death 1; CD279). Whereas CD28 is constitutively expressed on naïve and activated T cells, the other co-stimulatory molecules are expressed or upregulated only upon T cell activation. Members of the TNF–TNF-receptor superfamily include CD27, CD30, CD154 or CD40L, CD134 or OX40, CD137 or 41BB (CD137), GITR (glucocorticoid-induced TNFR-related protein), and HVEM (herpes virus entry mediator). The TIM family has been only recently been studied, and TIM1 and TIM2 are observed on activated T cells in humans [30]. Another co-stimulatory molecule that is upregulated on activated T cells is CD26. CD26 is a multifunctional protein, which possesses adenosine binding properties and dipeptidyl peptidase activity [31]. Dipeptidyl peptidase IV is a serine peptidase member of the S9B protein family. CD26 is preferentially expressed on activated CD4 + T cells. Recently, a high level of CD26 expression on CD8 + was detected and has been suggested to be a marker of T memory formation [32]. T cell surface molecules, such as soluble CD30 or CD26 (sCD30 or sCD26), can be released into the bloodstream. Discussing the promising results obtained with these biomarkers, which can be easily measured by ELISA techniques in serum samples from transplant patients, is beyond the scope of this review. Examples of adhesion molecules on activated T cells include members of the integrin family, such as LFA-1 (lymphocyte function associated antigen 1) or CD11a, and very late antigen 4 (VLA-4), which is a

dimer of CD49d and CD29. Other adhesion molecules, such as ICAM-1 (intercellular adhesion molecule 1, also known as CD54) and CD2 (LFA-2) belong to the immunoglobulin superfamily [33]. Adhesion molecules permit the extraversion of alloreactive T cells from the lymph nodes into the inflamed graft and, therefore, contribute to graft rejection. The chemokine superfamily constitutes a group of structurally related cytokines that play pivotal roles in inflammatory and immunological responses by recruiting selective types of leukocytes. Currently, the family is divided into the CXC, CC, C, and CX3C subfamilies based on the motif formed by the first two N-terminal cysteine residues [32]. Chemokine receptors, such as CXCR3 and CCR5, are strongly upregulated in activated T cells and are involved in transplant rejection [34,35]. MHC class II molecules expressed on activated T cells encompass all isotypes (HLA-DR, HLA-DQ, and HLA-DP). MHC molecules are observed to be upregulated only after 3–5 days [36]. Whereas the constitutively expressed CD28 co-stimulatory molecule can be directly assessed within 2 h on T cells [37] other co-stimulatory molecules are usually detectable on the cell surface within 1–3 days after stimulation [38]. A summary of activation markers assessed in transplant patients is presented in Table 1. In the following paragraphs, we will mainly focus on cell proliferation and the expression of CD markers from the group of receptor proteins and co-stimulatory molecules. 4. Methods for assessing markers of lymphocyte proliferation and activation T cell activation markers can be easily detected on the cell surface using fluorescence labeled monoclonal antibodies and modern multicolor benchtop flow cytometers. For cell proliferation, the intracellular upregulation of PCNA can also be assessed by flow cytometry after lysis of the lymphocytes [39]. An alternative approach is to determine PCNA mRNA expression by real-time PCR [40]. Whereas PCNA protein expression requires approximately 72 h to become reliably measurable, PCNA mRNA expression can be assessed 24 h after the onset of stimulation. In addition, a number of other methods

T Cell

APC

CD71 CD95 CXCR3 CCR5 CD26 CD27 CD28* CD30 Signal 1 PCNA; Ki67 OX40

CD70 CD69 CD25

OPCNA

CD40L CD134 Co-stimulation CD137 BrdU CD154 LFA-2 4-1BB LFA-1 GITR Mitogens: VLA-4 HVEM PWM ICAM-1 TIM2 TIM1 Signal 2

3H-Thymidine;

Co-stimulatory molecules

MHC TCR

ION PMA CON A

Fig. 1. T cell surface activation and proliferation markers. * = constitutively expressed. For details see text.APC = Antigen Presenting Cell; BrdU = Bromodeoxyuridine; CON = Concanvalin A; GITR = Glucocorticoid-induced TNFR-Related Protein; HVEM = Herpes Virus Entry Mediator; ICAM-1 = Intercellular Adhesion Molecule 1; ICOS = Inducible T Cell Co-stimulator; ION = Inonomycin A; LFA = Lymphocyte Function Associated Antigen; MHC = Major Histocompatibility Complex; PCNA = Proliferating-Cell-Nuclear-Antigen; PMA = Phorbol Myristate Acetate; PWM = Pokeweed Mitogen; TCR = T Cell Receptor; TIM = Type 1 Trans-membrane Glycoprotein; and VLA-4 = Very Late Antigen 4.

M. Shipkova, E. Wieland / Clinica Chimica Acta 413 (2012) 1338–1349

1341

Table 1 Cell surface activation markers under investigation to assess immunosuppression. CD number

Alternate name

Expression

CD 11a

Integrin alpha-L

All leukocytes

CD 25

Interleukin 2 receptor, alpha chain, IL2RA

CD 26

Dipeptidyl peptidase IV, EC 3.4.14.5

CD 27

CD27 antigen, tumor necrosis factor receptor superfamily member

CD 28

T cell-specific surface glycoprotein, constitutively expressed

CD 54

Intercellular adhesion molecule-1, ICAM-1

CD 69

Early activation antigen CD69

CD 70

CD27-ligand; Ki-24 antigen

CD 71

Transferrin receptor protein 1

CD 86

T lymphocyte activation antigen CD86; B7-2; B70

CD 95

Tumor necrosis factor receptor superfamily member 6, apoptosismediating surface antigen FAS, APO-1

CD 134

Tumor necrosis factor receptor superfamily member 4; OX40

CD 137

Tumor necrosis factor receptor superfamily, member 94-1BB; ILA

CD 154

CD40 ligand

NKG2D

Natural Killer Group 2, member D

Function

Forms together with the beta 2 chain the integrin lymphocyte function-associated antigen-1 (LFA-1), which plays a central role in leukocyte intercellular adhesion through interactions with ICAMs 1–3 and co-stimulatory signaling. Activated T cells, B cells, monocytes/macrophages The interleukin 2 (IL2) receptor alpha (IL2RA) and beta (IL2RB) chains, together with the common gamma chain (IL2RG), constitute the high-affinity IL2 receptor. – Dipeptidyl peptidase IV activity. Regulates various Mature thymocytes, activated T cells, B cells NK physiological processes by cleaving peptides (chemokines, cells, macrophages, renal and small intestinal mitogenic growth factors, neuropeptides, peptide hormones) epithelium, biliary canaliculae, and splenic sinus in the circulation. lining cells etc. – Co-stimulatory molecule in T cell activation. Involved in induction of T cell proliferation and NF-kappa-B activation in a T cell receptor/CD3-dependent manner. – Binds adenosine deaminase (ADA) thus regulating lymphocyte-epithelial cell adhesion. – Involved in the pericellular proteolysis of the extracellular matrix (ECM), the migration and invasion of endothelial cells into the ECM. Most T lymphocytes, memory-type B cells, NK cells Receptor for CD70/CD27L. May play a role in survival of activated T cells and apoptosis. Involved in regulation of B cell activation and immunoglobulin synthesis. Mature CD3 + thymocytes, most peripheral T Co-stimulation of T cell proliferation and cytokine production, by lymphocytes, plasma cells binding to its ligands CD80 or CD86, co-stimulates T cell effector function and T cell-dependent antibody production. Binds to integrins of type CD11a/CD18, or CD11b/CD18 and acts Activated endothelial cells, T and B lymphocytes, monocytes, inducible on epithelial and fibroblastic as a cellular receptor for rhinovirus. Ligand for LFA-1 (see above). cells Appears to be the earliest inducible cell surface glycoprotein Activated T cells, B-cells, NK cells, neutrophils, acquired during lymphoid activation. Involved in lymphocyte eosinophils, epidermal Langerhans cells and proliferation and functions as a signal-transmitting receptor in platelets lymphocytes, natural killer (NK) cells, and platelets. Activated T and B lymphocytes Cytokine, member of the tumor necrosis factor (TNF) ligand family. Involved in T cell activation, proliferation and generation of cytolytic T cells; regulation of B-cell activation, cytotoxic function of NK cells, and immunoglobulin synthesis. All proliferating cells Involved in iron homeostasis and necessary for development of erythrocytes and the nervous system. Receptor in the co-stimulatory signaling. Essential role for T DC in T zones of secondary lymphoid organs, Langerhans cells and peripheral blood DC; T and B- lymphocyte proliferation and interleukin-2 production, by binding CD28 or CTLA-4 as well as in the early events of T cell lymphocytes, monocytes activation, such as deciding between immunity and anergy. Isoform 2 interferes with the formation of CD86 clusters, and thus acts as a negative regulator of T cell activation. Activated T and B cells Mediation of apoptosis-inducing signals. May be related to the induction of peripheral tolerance as well as to the antigenstimulated suicide of mature T cells. Role in transduction of proliferating signals in normal diploid fibroblast and T cells. Activated T and B Cells, cultured endothelial cells as Involved in activation of NFkB, potential role in suppression of well as DC apoptosis as well as in CD4 + T cell response, T cell-dependent B cell proliferation and differentiation. Co-stimulatory molecule. Contributes to the clonal expansion, Predominantly on activated CD8+, but also on survival, and development of T cells, can induce proliferation in CD4 + T cells, on DC, NK cells, granulocytes, and peripheral monocytes, enhance T cell apoptosis, and regulate endothelial cells at sites of inflammation. CD28 co-stimulation to promote Th1 cell responses. Predominantly on mature, activated CD4 + T cells, Co-stimulatory molecule involved in antigen-presenting cell but also in a small population of activated CD8 − T (APC) activation, CD4 + and CD8 + T cell priming, and effector T cells, platelets, mast cells, macrophages, basophils, cell maturation. Regulates B cell function by engaging CD40 on the B cell surface. NK cells, B lymphocytes, smooth muscle cells, endothelial cells, and epithelial cells. NK cells, T cells Activation receptor that allows natural killer cells to detect diseased host cells

DC = Dendritic Cells; NK = Natural Killer. Data sources: http://prow.nci.nih.gov/; http://www.ncbi.nlm.nih.gov/; http://www.ncbi.nlm.nih.gov/omim; http://expasy.org/sprot/.

have been developed to study cell proliferation in various cell populations. The most important parameters to be employed are metabolic activity and DNA synthesis. Cell division and growth require energy, and metabolic activity assays are based on this premise. The cells are incubated with a colorimetric substrate, such as MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide), which is reduced by mitochondrial succinate dehydrogenase [41]. Another approach to follow the cell's metabolic activity and

proliferation is to measure the intracellular ATP-concentration (iATP) [42]. For this purpose a FDA cleared assay is already commercially available (Immuknow® assay, Cylex, Inc., Columbia, USA). As outlined above, during the S phase the cell undergoes DNA synthesis and replicates its genome. If labeled DNA precursors, such as radioactive thymidine (3H-thymidine) or the thymidine analog BrdU (bromoedoxyuridine), are added to the cells, then they are incorporated into the newly synthesized DNA. Levels of 3H-thymidine can be detected

1342

M. Shipkova, E. Wieland / Clinica Chimica Acta 413 (2012) 1338–1349

Table 2a In vitro effects of immunosuppressive agents on surface marker expression. CD number

Immunosuppressive Cell population drug investigated

CD 11a

MPA + TAC, CYA or SRL SRL + TAC or CYA

CD 25

TAC, CYA, SRL, MPA, CD3+CD25 + PRD

CD3+CD11a +

MMF + SRL

CD19+CD25 +

MPA + TAC, CYA or SRL SRL + TAC or CYA

CD3+CD25 +

CD 26

MPA, CYA, TAC, SRL, CD3+CD26 + EVER

CD 27

MPA

CD4+CD27 +

CD 28

MPA

CD4+CD28 +

CD 54

CYA + SRL

CD19+CD54 +

MMF + SRL

CD19+CD54 +

CD 69

MMF + SRL

CD19+CD69 +

CD 70

MPA

CD4+CD70 +

CYA, TAC, EVER, MPA

CD3+CD70 +

CD 71

TAC, CYA, SRL, MPA, CD3+CD71 + PRD

MMF + SRL

CD19+CD71 +

CD3+CD71 +

Experimental details

In vitro effect described

Incubation for 3 days. Stimulation with CON A Whole blood cultures from HV. Flow cytometry.

Concentration-dependent inhibition by all drugs. Stronger with CYA and TAC than with SRL and MPA. Higher IC50 values for all drugs compared to the other markers investigated. Synergistic to antagonistic effects depending on drug combination. Concentration-dependent inhibition by all drugs (only slightly by SRL).

Incubation for 3 days. Stimulation with CON A Whole blood cultures from HV. Flow cytometry. PBMC cultures from HV blood suspended in autologous serum. Incubation for 24 h. PWM stimulation. Flow cytometry. Incubation for 3 days. Stimulation with CON A Whole blood cultures from HV. Flow cytometry.

PBMC cultures from HV blood. Incubation for 1–3 days. PHA stimulation. Flow cytometry. Stimulation with anti-CD3+/antiCD28 mAb-coated beads. Cultures of isolated and purified CD4 + cells (HV blood) for 6 days. Flow cytometry. Stimulation with anti-CD3+/antiCD28 mAb-coated beads. Cultures of isolated and purified CD4 + cells (HV blood) for 6 days. Flow cytometry. PBMC cultures from HV blood suspended in autologous serum. Incubation for 24 h. PWM stimulation. Flow cytometry. (as described above)

Concentration-dependent inhibition by MMF and SRL, whether used alone or in combination (synergistic effects).

Comment

Reference [57]

[47]

The experiments were performed with MMF and not with the active moiety MPA that may influence the results.

[55]

Concentration-dependent inhibition by all drugs. Stronger with CYA and TAC than with SRL and MPA. Synergistic to antagonistic effects depending on drug combination and concentration. Concentration- and time-dependent inhibition by all drugs.

[57]

Down regulation.

[59]

Down regulation.

[59]

Synergistic inhibition.

[60]

[58]

[55] The experiments were done with MMF and not with the active moiety MPA that may influence the results. [55] Concentration-dependent inhibition The experiments were PBMC cultures from HV blood by MMF and SRL, whether used alone done with MMF and not suspended in autologous serum. with the active moiety or in combination (synergistic Incubation for 24 h. PWM MPA that may influence effects). stimulation. Flow cytometry. the results. Concentration-dependent up Stimulation with anti-CD3+/anti[59] Controversial results to CD28 mAb-coated beads. Cultures of regulation, directly linked to target van Rijen et al. [58] maybe isolated and purified CD4 + cells (HV enzyme inhibition. According to the due to different cell blood) over 6 days. Flow cytometry. authors comment this result presents population investigated a unique feature of MPA among other and methodological differences. immunosuppressive drugs tested (A77 1726, SRL, CYA, dexamethasone). However, data related to these experiments are not shown. [61] Only 2 concentrations tested. Nearly PMA/ION do not trigger PBMC cultures from HV blood. complete inhibition of expression at the mTOR signal Stimulation with PMA/ION (60 h transduction pathway, incubation) as well as allogenic with MPA concentration of 4 mg/L with irradiated mature monocyte-derived both stimulations. Inhibition by CYA which precludes and TAC only with PMA/ION. Partial investigation of EVER DCs (5 days incubation). Flow effects. inhibition by EVER with allogenic cytometry. stimulation. [47] Concentration-dependent inhibition The experiments were Incubation for 3 days. Stimulation by all drugs. done with MMF and not with CON A Whole blood cultures with the active moiety from HV. Flow cytometry. MPA that may influence the results. [55] Concentration-dependent inhibition PBMC cultures from HV blood by MMF and SRL, whether used alone suspended in autologous serum. or in combination (synergistic Incubation for 24 h. PWM effects). stimulation. Flow cytometry. Concentration-dependent inhibition [57] by all drugs. Stronger with CYA and Concentration-dependent inhibition by MMF and SRL, whether used alone or in combination (synergistic effects).

M. Shipkova, E. Wieland / Clinica Chimica Acta 413 (2012) 1338–1349

1343

Table 2a (continued) CD number

CD 86

Immunosuppressive Cell population drug investigated

Experimental details

In vitro effect described

MPA + TAC, CYA or SRL SRL + TAC or CYA

Incubation for 3 days. Stimulation with Con A Whole blood cultures from HV. Flow cytometry.

TAC than with SRL and MPA. Synergistic to antagonistic effects depending on drug and concentrations. No inhibition

CYA + SRL

CD19+CD86 +

MMF + SRL

CD19+CD86 +

CYA + SRL

CD19+CD95 +

MMF + SRL

CD19+CD86 +

MPA + TAC, CYA or SRL SRL + TAC or CYA

PBMC cultures from HV blood suspended in autologous serum. Incubation for 24 h. PWM stimulation. Flow cytometry. (as given above).

Concentration-dependent inhibition by MMF and SRL, whether used alone or in combination (synergistic effects).

PBMC cultures from HV blood suspended in autologous serum. Incubation for 24 h. PWM stimulation. Flow cytometry. (as described above).

Synergistic inhibition

CD3+CD95 +

Incubation for 3 days. Stimulation with CON A Whole blood cultures from HV Flow cytometry.

CYA, TAC, EVER, MPA

CD3+CD134 +

MPA + TAC, CYA or SRL SRL + TAC or CYA

CD3+CD134 +

PBMC cultures from HV blood. Stimulation with PMA/ION (60 h incubation) as well as allogenic with irradiated mature monocyte-derived DCs (5 days incubation). Flow cytometry. Incubation for 3 days. Stimulation with CON A Whole blood cultures from HV Flow cytometry.

Concentration-dependent inhibition by all drugs. Stronger with CYA and TAC than with SRL and MPA. Synergistic to antagonistic effects depending drug combination and concentrations. Only 2 concentrations for each drug tested. No inhibition by TAC and CYA, inhibition by MPA regardless of stimulation and partial inhibition by EVER only with allogenic stimulation.

CD 137

CYA, TAC, EVER, MPA

CD3+CD137 +

PBMC cultures from HV blood. Stimulation with PMA/ION (60 h incubation) as well as allogenic with irradiated mature monocyte-derived DCs (5 days incubation). Flow cytometry.

CD 154

CYA, TAC, EVER, MPA

CD3+CD154 +

PBMC cultures from HV blood. Stimulation with PMA/ION (60 h incubation) as well as allogenic with irradiated mature monocyte-derived DCs (5 days incubation). Flow cytometry.

MPA + TAC, CYA or SRL SRL + TAC or CYA

CD3+CD154 +

Incubation for 3 days. Stimulation with CON A Whole blood cultures from HV Flow cytometry.

CYA

CD56 (NK cells) Incubation for 7 days. Stimulation and NK cell with IL-2 and IL-15. Enriched NK cells subsets isolated from PMNCs. (CD56brightCD16− and CD56dimCD16+)

CD 95

CD 134

NKG2D

by a scintillation counter, while BrdU can be detected by a quantitative cellular enzyme immunoassay using monoclonal antibodies [43,44]. The succinimidyl ester of carboxyfluorescein diacetate (CFSE) can also be utilized to assess cell proliferation. CFSE irreversibly binds to intracellular and cell-surface proteins by reacting with lysine and other amine groups. During cell division, CFSE labeling is distributed equally between the daughter cells, which, therefore, lose

Concentration-dependent inhibition by MMF and SRL, whether used alone or in combination (synergistic effects).

Concentration-dependent inhibition by all drugs. Stronger with CYA and TAC than with SRL and MPA. Synergistic to antagonistic effects depending on drug combination and concentrations. Only 2 concentrations for each drug tested. Significant but incomplete inhibition by CYA and TAC and nearly complete inhibition by MPA with both stimulations. Partial inhibition by EVER with allogenic stimulation and no inhibition with PMA/ION. Only 2 concentrations for each drug tested. Significant but incomplete inhibition by CYA and TAC and nearly complete inhibition by MPA with both stimulations. Partial inhibition by EVER with allogenic stimulation and no inhibition with PMA/ION. Concentration-dependent inhibition by all drugs. Stronger with CYA and TAC than with SRL and MPA. Synergistic to antagonistic effects depending on drug and concentrations. Decrease in NKG2D expression.

Comment

Reference

[60]

The experiments were done with MMF and not with the active moiety MPA that may influence the results.

[55]

[60]

[55] The experiments were done with MMF and not with the active moiety MPA that nay influence the results. [57]

PMA/ION do not trigger the mTOR signal transduction pathway, which precludes investigation of EVER effects.

[61]

[57]

PMA/ION do not trigger the mTOR signal transduction pathway, which precludes investigation of EVER effects.

[61]

[61]

[57]

CYA had different effects on proliferation of NK cell subsets.

[56]

fluorescence compared with the parent cells in a process that can be observed by flow cytometry [45]. To date, most approaches for assessing an individual's immune status under immunosuppressive therapy have employed ex vivo incubation systems in which either whole blood or isolated mononuclear cells are stimulated by mitogens. To mimic better the situation in vivo, stimulation of the recipient lymphocytes in a mixed

1344

M. Shipkova, E. Wieland / Clinica Chimica Acta 413 (2012) 1338–1349

leukocyte reaction (MLR) with donor APCs or antibodies and thirdparty cells has been proposed. Antigen-specific assays have the advantage that patients exhibiting an increased immune response directed against a certain antigen can be identified, as well as patients with donor-specific hyporesponsiveness, which is an indicator of graft tolerance [46]. However, as pointed out by Böhler et al., allogeneic stimulation of only one T cell clone hampers the assessment of the inter-individual sensitivity of various subjects to various immunosuppressants and precludes investigations into the pre-transplant conditions [47]. Whole blood assays have the advantage over incubations of isolated cells, as the immunosuppressant taken by the graft recipients are not washed out, thereby preserving the equilibrium between drugs bound to the plasma proteins and their distribution into blood cells. In addition, whole blood assays require less blood and prevent the selective loss of immune cells, thereby better preserving physiological conditions [48]. Cell proliferation in response to PMA was more rapid in whole blood cultures compared with isolated mononuclear cells, which exhibited a delay of 4–6 h when followed by flow cytometric determination of BrdU incorporation into dividing cells [49]. Incubation conditions and times vary between various protocols and hinder comparisons of the results. To date, the standardization of these assays has not been achieved, although each assay has been validated individually. A further disadvantage of this approach to monitoring lymphocyte activation is the prolonged assay time of up to 72 h, which currently limits its introduction into routine clinical monitoring of immunosuppression. In addition, whole blood stability, cell isolation, and incubation render multi-center trials problematic. 5. Analytical performance of methods for measuring T cell activation and proliferation To date, scarce findings have been published on the performance of assays for assessing T cell activation and proliferation. Böhler et al. have studied intracellular PCNA expression, as well the surface expression of CD25 and CD71, on CD3 + cells stimulated by CON A. Heparinized whole blood was incubated for up to 120 h ex vivo before flow cytometry was performed [45]. An incubation time of 72 h and a CON A concentration of 7.5 μg/mL were determined to be optimal. Whole blood samples can be stored for 24 h at room temperature without any significant effect on PCNA, CD25, and CD71 expression. After surface marker staining, samples must be analyzed immediately for assessing CD71 expression [47]. Overnight storage at 4 °C exhibited an effect only on PCNA. After staining, cells can be stored overnight at 4 °C except for CD71 expression, which was observed to be significantly increased. Intra-assay and inter-individual variability were determined after 72 h of incubation of whole blood from volunteers (n = 7) in the presence and absence of various concentrations of tacrolimus (1–1000 nmol/l). Intra-assay variability ranged between 10.5% and 33.1% for PCNA, 2.8% and 22.4% for CD25, and 7.6% and 20.1% for CD71. Intra-individual median values of variability were 31.3% for PCNA, 20.7% for CD25, and 18.4% for CD71. Inter-individual variability was also observed to be in this range. In tacrolimus-treated blood from one healthy donor, the IC50 value determined in three experiments indicated a large variation. The maximal difference between the values was 30% for PCNA, 45% for CD25, and 41% for CD71. These results demonstrated that the assessment of biomarkers in cell-based assays is less precise and reproducible than current methods for the drug monitoring of immunosuppressants, which are required to achieve an intra- and inter-assay imprecision of between 10 and 15% [50] and some nowadays achieve even less than 10%. Taken together, flow cytometry to assess the PD effects of immunosuppressants should be performed as quickly as possible. Whole blood samples should be stored for no more than 24 h, preferably at

room temperature. Measurements should be conducted immediately after staining. Recently, efforts have been undertaken to stabilize whole blood to enable extended storage at room temperature before cytometry or to freeze samples after staining. Commercial products, such as Cyto-Chex® blood collection tubes (Streck, Omaha, USA), provide reliable storage of whole blood for up to 7 days for markers, such as chemokine receptors. However, the T cell activation markers CD25 and HLA-DR were only stable for 72 h and 48 h, respectively [51]. For testing cell proliferation, whole blood samples should not be stored for more than 24 h at room temperature [52]. When studying mRNA expression, pre-analytical factors must also be considered. The degradation of mRNA can occur, or cell–cell interactions can stimulate the mRNA expression of activation markers. Stabilization can be achieved through the PAXgene Blood RNA Kit (Qiagen, Hilden, Germany), although a systematic evaluation concerning the mRNA expression of lymphocyte activation markers or PCNA is lacking [53]. The ideal assay would allow the direct assessment of cell proliferation and/or cell activation in whole blood without any cell isolation and pre-incubation steps. We have recently observed that CD26 was already strongly stimulated on the T cells (CD3+) of kidney transplant patients who were prone to rejection, which supports the feasibility of monitoring in whole blood without stimulation [54]. This approach is currently under investigation in a further trial with patients who are evaluated both soon after transplantation and in the maintenance phase.

6. The effect of immunosuppressants on lymphocyte proliferation and activation in vitro The effect of various immunosuppressants on cell proliferation and activation has been investigated in various in vitro cell culture models. Activation markers have been predominately studied on T cells (CD3 +) and T cell subsets, which were stimulated either by mitogens or alloantigens. The mitogens employed were CON A and PWM, as well as PMA in combination with ION. For allogeneic stimulation, either antibody-coated beads or dendritic cells were predominately utilized. One group studied activation markers on B cells in PWM-stimulated whole blood and observed synergistic effects between mycophenolate mofetil and SRL in inhibiting CD25, CD54, CD69, CD71, CD86, and CD95 [55]. In general, all of the immunosuppressants studied inhibited both T cell activation and proliferation. Synergistic effects became apparent when the drug combinations were studied. Although there are very limited data available on the effect of immunosupressants on other cells than T cells there are some preliminary reports on the effect of CYA on NK cell surface receptor expression. CYA was shown to increase cytotoxicity of NK cells which was accompanied by an altered expression of their activating and inhibitory receptors such as KIR (killer-cell immunoglobulin-like receptors) or NKG2D [56]. Tables 2a and 2b present an overview of the literature investigating the in vitro effects of immunosuppressants induced by the incubation of either stimulated whole blood or isolated cells. The activation markers are summarized in Table 2a, while the effects on cell proliferation are depicted in Table 2b. The nonspecific assessment of lymphocyte function, as described above, offers the advantage that the synergistic effects of drug combinations can be studied, which may better reflect the in vivo situation. However, the effect of the utilization of either whole blood or isolated cells, mitogen- or allogeneicstimulated cells, and the employment of autologous serum or plasma has not been fully elucidated, which currently precludes a clear recommendation of which technique may become the most promising tool for applications in clinical routines, as systematic investigations comparing the assays under standardized conditions have not been extensively undertaken.

M. Shipkova, E. Wieland / Clinica Chimica Acta 413 (2012) 1338–1349

1345

Table 2b In vitro effects of immunosuppressive agents on proliferation markers. Marker

Immunosuppressive drug

Cell population investigated

PCNA/DNA expression TAC, CYA, SRL, MPA, PRD PCNA + PIhigh in lymphocytes lymphocytes MPA + TAC, CYA or SRL SRL + TAC or CYA

PCNA + PIhigh lymphocytes

(3H)-TdR incorporation

MPA

CD4 + cells

PCNA mRNA

TAC, CYA, MPA, PRD

Leukocytes

BrdU incorporation

MPA and metabolites

PBMC

Experimental details

In vitro effect described

Incubation for 3 days. Stimulation with CON A Whole blood cultures from HV. Flow cytometry. Incubation for 3 days. Stimulation with CON A Whole blood cultures from HV. Flow cytometry.

Concentration-dependent inhibition by all drugs.

[47]

Concentration-dependent inhibition by all drugs. Stronger with CYA and TAC than with SRL and MPA. Synergistic to antagonistic effects depending on drug combination and concentrations. Concentration-dependent inhibition of cell proliferation

[57]

Concentration-dependent inhibition by all drugs

[40]

Concentration-dependent inhibition by MPA and its acyl glucuronide.

[44]

Stimulation with anti-CD3+/anti-CD28 mAb-coated beads. Cultures of isolated and purified CD4 + cells (HV blood) for 4 days. Analysis by scintillation counter. Pre-incubation for 3 h with the single drugs followed by overnight stimulation with PMA and calcium ionophore. Whole blood cultures from HV. RT-PCR. PBMC cultures from HV blood. Stimulation with PMA/ION and incubation over 48 h. ELISA.

Comment Reference

[59]

BrdU = Bromodeoxyuridine; CON A = Concanvalin A; CYA = Cyclosporine A; DC = Dendritic Cells; ELISA = Enzyme Linked Immunsosorbent Assay; EVER = everolimus; FACS = Fluorescence-Activated-Cell-Sorter; (3H)-TdR = 3H-Thymidine; HV = Human Volunteers; ION = Ionomycin; mAB = Monoclonal Antibody; MMF = Mycophenolate Mofetil; MPA = Mycophenolic Acid; NK = Natural Killer; PBMC = Peripheral Blood Mononuclear Cells; PCNA = Proliferating-Cell-Nuclear-Antigen; PIhigh = propidium iodide high; PMA = Phorbol Myristate Acetate; PRD = Prednisolone; PWM = Pokeweed Mitogen; RT-PCR = Real Time Polymerase Chain Reaction; TAC = Tacrolimus; and SRL = Sirolimus.

7. The effect of immunosuppressants on lymphocyte proliferation and activation ex vivo In the preceding paragraph, we described that immunosuppressants, alone or in combination, inhibit lymphocyte activation and proliferation in vitro. However, determining whether activation markers behave differently in whole blood or lymphocyte cultures obtained from transplant patients treated with immunosuppressive drug combinations in vivo is important. Most of the published data are derived from models in which diluted whole blood was stimulated by mitogens, such as PMA, PHA, CON A, and ionomycin. Stadler et al. have investigated activation markers on CD3+ cells (CD25, CD71, CD11a, CD95, and CD154) and cell proliferation (PCNA and 3H-thymidine) in whole blood collected from stable kidney transplant patients. Patients were treated with prednisone, MMF, and CYA, and lymphocyte stimulation was achieved with CON A for 72 h. T cell activation and lymphocyte proliferation was significantly suppressed when compared with healthy controls [62]. Based on their results, the authors conclude that immune-function assays may possess the potential to tailor immunosuppression. A transient MPA concentrationdependent decrease in T cell expression of CD25 and CD71 and a strong inhibition of T cell proliferation was observed after a single dose of MPA in liver transplant candidates on the waiting list [63]. An inhibition level of 50% was observed at MPA concentrations ≤ 2 mg/l. Similar observations were described by Kamar et al., who detected a transient decrease of CD25 and CD71 within 1 h in response to the first dose of MMF prior to kidney transplantation in a whole blood stimulation assay [64]. In addition, PCNA expression was observed to decrease along with MPA concentrations and the inhibition of inosine monophosphate dehydrogenase activity. In keeping with Premaud et al. [61], cell proliferation was inhibited at low MPA concentrations, demonstrating an IC50 value of 1.55 mg/l [64]. In a comparative study with renal transplant patients under various immunosuppressive regimens, Hutchinson et al. indicated that combination therapies containing MMF were more effective in suppressing cell proliferation in whole blood stimulated with PHA than therapies including azathioprine [65]. In stable kidney allograft recipients, cell proliferation (PCNA) and CD25, as well as CD71 expression, tended to increase when patients were switched from

MMF to enteric-coated mycophenolate sodium (EC-MPS), possibly indicating a trend toward lower immunosuppression [66]. Direct assessment of activation markers on T and B cells from heart transplant recipients indicated a significant inhibition of CD25 expression on T cell subsets in heart transplant recipients receiving triple therapy with CYA, azathioprine, and prednisone [67]. Switching from azathioprine to MMF resulted in a further decrease of CD25, particularly on CD16 + cells (NK cells). A dual therapy with MMF and steroids demonstrated surprising efficacy in suppression of cell activation, matching that of the standard triple therapy, which included CYA [68]. Conversion of CYA to TAC in heart and lung transplant recipients undergoing combination therapy with MMF demonstrated no effect on T cell activation (CD25) and cell proliferation (PCNA) [69]. In a second investigation, the same group compared activation markers and cell proliferation in two subgroups, which were switched from a CYA-based immunosuppressive therapy to either TAC or SRL. In addition, drug concentrations were measured in whole blood and correlated to pharmacodynamics. The study observed that 2 h after CYA dosing, PCNA, CD25, CD95, and CD134 were significantly downregulated on T cells and that this effect correlated with both the CYA dose and the concentration. In keeping with previous observations mentioned above, TAC indicated no significant additional effect on either CD25 or CD95 expression [70]. In contrast, PCNA and CD134 levels were decreased. The expression of PCNA was not altered after conversion from CYA to SRL, whereas CD25 and CD95 levels were elevated [70]. PCNA mRNA expression was observed in 55 renal transplant patients in isolated whole blood stimulated with PMA + ION. The patients' blood was collected before transplantation and at various time points after transplantation for up to 3 months. Patients were receiving a CNI-based immunosuppressive triple therapy and received induction therapy with basiliximab. Compared with healthy volunteers and samples collected pre-transplantation, PCNA mRNA expression declined during the next 2 weeks, but returned to baseline levels after 4 weeks [40]. Although different assay conditions, stimuli, patient groups, and incubation conditions were employed, we can conclude from these data, generated ex vivo, that MPA exerts a particularly strong effect on lymphocyte activation and cell proliferation. Whereas in vitro data indicate distinct differences among immunosuppressive drugs

1346

M. Shipkova, E. Wieland / Clinica Chimica Acta 413 (2012) 1338–1349

and their combinations, this “fine tuning” of drug treatments is less clearly defined if the lymphocytes of immunosuppressed transplant patients on combination therapies are investigated. Cell activation and proliferation may be particularly suitable as pharmacodynamic markers for patients treated with either MMF or EC-MPS.

NK cell function as assessed by IFN-γ production and degranulation (CD170a expression) was decreased early after kidney transplantation in patients under CNI therapy. In late transplant patients CYA treatment was associated with a significant reduction of NK cell degranulation and IFN-γ production compared to tacrolimus therapy [71].

Table 3 Association of cell surface activation markers and cell proliferation markers with clinical events in human solid organ transplantation. Biomarker

Transplant population

Immunosuppressive therapy

CD 25

Cardiac, n = 22

CD3+/CD25 + monocytes: ↓in patients with allograft dysfunction early after transplantation, probably due to over immunosuppression. CYA (n = 27) or TAC (n = 1), AZA, CD4+CD25 + among others steroids. helpful to distinguish patients with CYA nephrotoxicity from patients with rejection or infection. Anti-lymphocyte globulin, AZA, CD4+CD25+: ↑in patients with CYA and steroids. grade 2 rejection. Basiliximab, EC-MPS, TAC, CD26+CD3 + non stimulated steroids. cells: ↑risk of rejection. Rabbit anti-human thymocyte Donor-specific CD8+CD28 − globulin, steroid-free TAC/SRL. cells: more often absent with rejection.

Renal, n = 28

Cardiac, n = 15 CD 26

Renal, n = 35

CD 28

Liver (n = 7), liver–kidney (n = 1) and small bowel (n = 4), pediatric Liver, n = 52

Association with clinical events

Comments

Combination therapy of TAC or CYA and steroids.

References [72]

Rabbit anti-human thymocyte globulin, CYA, AZA and steroids.

[73]

[74] At day 7 ± 1 post transplantation

CD3+CD28 + early after CD38 expression on CD3 + cells transplantation: ↑risk of rejection. was also related to rejection, whereas CD25 was not ↓CD28+CD8+ (b40%): ↑risk of de Drawback of the study is the Liver, n = 134 Various combinations of TAC or retrospective design that limits CYA with steroids and/or MMF or novo malignancies in long-term implications. survivors. AZA. CD 54 Liver, pediatric, n = 25 Rabbit anti-human thymocyte ↑EC50 of tacrolimus CD54+ Activation using PWM, CD86 + globulin, steroid-free TAC. CD19+: ↑risk of rejection. and CD25 + on CD19 + cells have been investigated, but did also not allow discrimination. Urine flow cytometry with serial Renal with biopsy confirmed CYA/steroids or CYA/AZA/steroids. CD54 + cells in urine sediment: ↑in patients with steroid resistant monitoring of sediments over acute rejection, n = 17 rejection and correlated with the 1 month after rejection. (3 pediatric, 24 rejections). need for antilymphocytic therapy and irreversible graft damage. CD 71 Cardiac, n = 22 Rabbit anti-human thymocyte CD71+/CD14 + monocytes: ↑in globulin, CYA, AZA and steroids. patients with allograft dysfunction early after transplantation probably due to over immunosuppression. Renal, n = 35 Basiliximab, EC-MPS, TAC, steroids ↓CD71 + CD3+: ↑risk of At day 7 ± 1 post transplantation. infections. CD 154 Intestinal, pediatric, n = 32 Rabbit anti-human thymocyte IR ≥ 1,351 of CD154+CD19 + IR = ratio of donor- and globulin. cells: ↑risk of rejection. third-party-induced T cells. Investigated cell populations: allospecific CD19 + B cells and allospecific T cytotoxic memory cells. Investigated cell populations: Liver, pediatric, n = 58 Rabbit anti-human thymocyte IR> 1.13 of memory CD154+ allospecific T helper and globulin, steroid-free TAC. CD4− cells: ↑risk of rejection. T helper cells more strongly inhibited cytotoxic T memory cells. Donorand third-party induction. by TAC than cytotoxic T cells. Patients were included in an PCNA/DNA expression in PCNA/DNA expression Liver, long-term surviving, Monotherapy with CYA, TAC or CD8 + cells: ↑ in patients with in lymphocytes n = 24 l MMF or biotherapy with CYA/ rejection (p = 0.06). immunosuppression weaning MMF or TAC/MMF. protocol with dose reduction over 6–9 months until complete withdrawal. Stimulation index b2 early post PCNA mRNA Renal, n = 55 Basiliximab, CNI (CYA or TAC), antimetabolite (MMF, mizoribine transplant: ↑risk of virus infections/reactivation; of 4 or cyclophosphamide) and patients with acute rejection 3 steroids. had a stimulation index ≥2. BrdU incorporation Renal, n = 35 Basiliximab, EC-MPS, TAC, Reduced values : ↑risk of At day 7 ± 1 post transplantation. steroids. leucopenia. CFSE dilution assay Small bowel, pediatric, Rabbit anti-human thymocyte IR > 1: ↑risk of rejection. IR = ratio of donor- and thirdn = 28 globulin. party-induced T cell proliferation.

[54] [78]

[75]

[37]

[77]

[76]

[72]

[54] [79]

[80]

[81]

[40]

[54] [79]

AZA = Azathioprine; BrdU = Bromodeoxyuridine; CFSE = carboxyfluorescein succinimidyl; CYA = Cyclosporine A; CNI = Calcineurine Inhibitor; MMF = Mycophenolate Mofetil; EC-MPS = Enteric Coated Mycophenolate sodium; PCNA = Proliferating-Cell-Nuclear-Antigen; TAC = Tacrolimus; and SRL = Sirolimus.

M. Shipkova, E. Wieland / Clinica Chimica Acta 413 (2012) 1338–1349

8. Association between markers of lymphocyte proliferation, as well as activation and clinical events As indicated in the preceding paragraph, the mechanisms of lymphocyte activation and proliferation are important in the development of immunosuppressive therapies. However, to serve as a biomarker to guide immunosuppressive therapy requires relating laboratory data to clinical events, such as rejection or infection, as indications of over or under immunosuppression. In this regard, evidence has been obtained with the activation markers CD25, CD26, CD28, CD54, CD71, and CD154. Clinical data linked to cell proliferation have been published using such assays as PCNA protein and mRNA expression, BrdU incorporation, and CFSE dilution. CD25 expression on CD3+ and CD4+ cells distinguished allograft dysfunction from rejection in heart and kidney graft recipients receiving a CNI-based therapy [72–74]. The results from our group suggest that kidney transplant patients with a spontaneously low level of CD26 expression on CD3+ cells within the first week after transplantation are less prone to rejection, whereas a low level of CD71 expression was associated with more infections, probably indicating over immunosuppression [54]. The data obtained regarding CD28 are conflicting. However, Boleslawski et al. reported an association between a decreased frequency of CD8 + cells expressing CD28 and malignancies in long-term liver transplant patients, as well as an association between increased expression and rejection [37,75]. This finding is of particular interest, as an important goal of an individualized immunosuppression is to avoid the long-term side effects of over immunosuppression. In this respect, evidence from the activation and proliferation markers is scarce. One group has observed CD54 + cells in the urine of kidney transplant patients and determined a relationship with steroid-resistant rejection [76]. Sindhi et al. reported in liver transplant patients that the EC50 of TAC for CD54 expression in ex vivo mitogen-stimulated lymphocytes was twofold greater in rejectors compared with non-rejectors [77]. A link between cell proliferation and clinical events was observed for both rejection and infections in renal and small bowel transplant recipients [40,54,78]. Table 3 summarizes a number of the studies published in recent years. 9. Conclusions Recognition of the importance of personalized medicine in drug treatment has increased in recent years. Accordingly, the management of immunosuppression is moving toward individualization based on the immunological risk and immune status of the patient receiving the transplant. In this non-comprehensive review, we have focused on the most commonly employed analytical tools and models to assess T cell proliferation and activation in solid organ transplantation. Most data are available from kidney transplant patients. Lymphocyte proliferation and activation can be observed by various techniques available in many laboratories around the world. Particularly, flow cytometry, which can be utilized to monitor proteins both expressed on the cell surface and expressed intracellularly, has become the main method for assessing the pharmacodynamic effects of immunosuppressants on the graft recipient's immune cells. The inhibition of lymphocyte proliferation and activation induces nonspecific markers, which allow monitoring of the synergistic effects of drug combinations on the immune system. This ability is a clear strength of these approaches compared with markers measuring the effects of a single drug only. Promising fields for applying such markers could be the evaluation of inter-individual sensitivities to various drugs or drug combinations, risk stratification in patients, and proof-of-concept/proof-of-effect studies conducted during preclinical and clinical trials. In addition, these markers may be helpful in improving understanding the mechanisms of drug actions and interactions. However, the number of activation markers that can be

1347

considered for such purposes is large and the evidence available with single markers, conversely, is limited. In fact, the clinical data available were generated only in studies with a small power and represent single-center experience. Important limitations in this regard include the poor standardization of the methods (e.g., matrix employed, stimulation protocols, antibodies/labeling, cell populations investigated) and the sparse data on method validation. Such standardization of well-evaluated protocols is an ultimate prerequisite for performing large prospective interventional studies, as well as in comparing results generated in different analytical units. This step is needed to estimate the diagnostic value of these markers and to approach open questions: which are the clinically meaningful target ranges; when is the most appropriate time to perform the assay; should specifics of different population be taken into account for interpretation of results; and when should a change be considered significant. Another notable problem with markers that require a stimulation step is the incubation time. Whereas early activation molecules (CD69 and CD154), the proliferation PCNA-mRNA assay, and the measurement of CD26 expression can be performed within less than 24 h, investigations of the expression of all of the other markers, e.g., CD71, CD25, and PCNA/DNA require more than 3 days until obtaining a reportable result. It is questionable whether such an extended analytical procedure is suitable in supporting the clinical decision, particularly in regard to acute rejection in the early phase after transplantation. However, the introduction of new immunosuppressant drugs with pharmacological activities based on the costimulation blockade offer new perspectives in the clinical utilization of cell surface markers. For example, data regarding the extent to which belatacept binds to its targets (CD80 and CD86) in transplantation patients, thus influencing the CD28-mediated activation of T-cells, may enable the correlation of its exposure with receptor saturation as a pharmacodynamic measure of co-stimulation blockade [82]. Furthermore B cells, B cell subsets, plasma cells, and NK cells as well as B and NK cell activation have been recently found to be associated with transplant tolerance [16,83,84] a fact that should be given more attention when looking for biomarkers to personalize immunosuppressive treatment regimens particularly immunosuppressant sparing protocols. However, data related to pharmacodynamic biomarkers based on activation or proliferation of cells of the innate or humoral immune system are missing and so far there is also no proof that adjustment of dose or changes in immunosuppressive drugs based on T cell surface marker expression or proliferation can influence the clinical outcome. A promising approach to identify transplant patients who require less or no immunosuppression may be the assessment of gene expression signatures as suggested by the recent findings of the RISET (Reprogramming the Immune System for Reestablishment of Tolerance) EU consortium or the US Immune Tolerance network (ITN). We conclude that the evaluation of biomarkers of cell activation and proliferation is still in its early stages, is based on single center experience, and many open questions remain to be answered before these methods can be introduced into clinical practice. Disclosure Nothing to disclose. Funding None. References [1] Waki K. UNOS Liver Registry: ten year survivals. Clin Transpl 2006:29–39. [2] Kaneku HK, Terasaki PI. Thirty year trend in kidney transplants: UCLA and UNOS Renal Transplant Registry. Clin Transpl 2006:1–27.

1348

M. Shipkova, E. Wieland / Clinica Chimica Acta 413 (2012) 1338–1349

[3] Srinivas TR, Meier-Kriesche HU. Minimizing immunosuppression, an alternative approach to reducing side effects: objectives and interim result. Clin J Am Soc Nephrol 2008(Suppl. 2):S101–16. [4] Meier-Kriesche HU, Schold JD, Srinivas TR, Kaplan B. Lack of improvement in renal allograft survival despite a marked decrease in acute rejection rates over the most recent era. Am J Transplant 2004;4:378–83. [5] Wieland E, Olbricht CJ, Süsal C, et al. Biomarkers as a tool for management of immunosuppression in transplant patients. Ther Drug Monit 2010;32:560–72. [6] Bharat A, Mohanakumar T. Allopeptides and the alloimmune response. Cell Immunol 2007;248:31–43. [7] El-Sawy T, Fahmy NM, Fairchild RL. Chemokines: directing leukocyte infiltration into allografts. Curr Opin Immunol 2002;14:562–8. [8] Nankivell BJ, Alexander SI. Rejection of the kidney allograft. N Engl J Med 2010;363:1451–62. [9] Barrett AJ, Rezvani K, Solomon S, et al. New developments in allotransplant immunology. Hematology Am Soc Hematol Educ Program 2003;102:350–71. [10] Wucherpfennig KW, Allen PM, Celada F, et al. Polyspecificity of T cell and B cell receptor recognition. Semin Immunol 2007;19:216–24. [11] Pasare C, Medzhitov R. Toll-like receptors: linking innate and adaptive immunity. Adv Exp Med Biol 2005;560:11–8. [12] Kabelitz D. Expression and function of Toll-like receptors in T lymphocytes. Curr Opin Immunol 2007;19:39–45. [13] Raedler H, Heeger PS. Complement regulation of T-cell alloimmunity. Curr Opin Organ Transplant 2010;16:54–60. [14] Suárez-Alvarez B, López-Vázquez A, Baltar JM, Ortega F, López-Larrea C. Potential role of NKG2D and its ligands in organ transplantation: new target for immunointervention. Am J Transplant 2009;9:251–7. [15] Yu G, Xu X, Vu MD, Kilpatrick ED, Li XC. NK cells promote transplant tolerance by killing donor antigen-presenting cells. J Exp Med 2006;203:1851–8. [16] Clathworthy MR. Targeting B cells and antibody in transplantation. Am J Transplant 2011;11:1359–67. [17] Csencsits KL, Bishop DK. Contrasting alloreactive CD4 + and CD8 + T cells: there's more to it than MHC restriction. Am J Transplant 2003;3:107–15. [18] Häyry P. Molecular pathology of acute and chronic rejection. Transplant Proc 1994;26:3280–4. [19] Chatila T, Silverman L, Miller R, Geha R. Mechanisms of T cell activation by the calcium ionophore ionomycin. J Immunol 1989;143:1283–9. [20] Zarling JM, McKeough M, Bach FH. A sensitive micromethod for generating and assaying allogeneically induced cytotoxic human lymphocytes. Transplantation 1976;21:468–76. [21] Trickett A, Kwan YL. T cell stimulation and expansion using anti-CD3/CD28 beads. J Immunol Methods 2003;275:251–5. [22] Barten MJ, Gummert JF, van Gelder T, Shorthouse R, Morris RE. Flow cytometric quantitation of calcium-dependent and -independent mitogen-stimulation of T cell functions in whole blood: inhibition by immunosuppressive drugs in vitro. J Immunol Methods 2001;253:95–112. [23] Leonardi E, Girlando S, Serio G, et al. PCNA and Ki67 expression in breast carcinoma: correlations with clinical and biological variables. J Clin Pathol 1992;45:416–9. [24] Palutke M, Tabaczka PM, Kukuruga DL, Kantor NL. A method for measuring lymphocyte proliferation in mixed lymphocyte cultures using a nuclear proliferation antigen, Ki-67, and flow cytometry. Am J Clin Pathol 1989;91:417–21. [25] Schlegel PG. The role of adhesion and costimulation molecules in graft-versus-host disease. Acta Haematol 1997;97:105–17. [26] Testi R, Phillips JH, Lanier LL. T cell activation via Leu-23 (CD69). J Immunol 1989;143:1123–8. [27] Hintzen RQ, Lens SM, Lammers K, Kuiper H, Beckmann MP, van Lier RA. Engagement of CD27 with its ligand CD70 provides a second signal for T cell activation. J Immunol 1995;154:2612–23. [28] Schuurman HJ, van Wichen D, de Weger RA. Expression of activation antigens on thymocytes in the ‘common thymocyte’ stage of differentiation. Thymus 1989;14: 43–53. [29] Miyawaki T, Uehara T, Nibu R, et al. Differential expression of apoptosis-related Fas antigen on lymphocyte subpopulations in human peripheral blood. J Immunol 1992;149:3753–8. [30] Li XC, Rothstein DM, Sayegh MH. Costimulatory pathways in transplantation: challenges and new developments. Immunol Rev 2009;229:271–93. [31] Ohnuma K, Takahashi N, Yamochi T, Hosono O, Dang NH, Morimoto C. Role of CD26/dipeptidyl peptidase IV in human T cell activation and function. Front Biosci 2008;13:2299–310. [32] Ibegbu CC, Xu YX, Fillos D, Radziewicz H, Grakoui A, Kourtis AP. Differential expression of CD26 on virus-specific CD8+ T cells during active, latent and resolved infection. Imunology 2009;126:346–53. [33] Heemann UW, Tullius SG, Azuma H, Kupiec-Weglinsky J, Tilney NL. Adhesion molecules and transplantation. Ann Surg 1994;219:4–12. [34] Tan J, Zhou G. Chemokine receptors and transplantation. Cell Mol Immunol 2005;2:343–9. [35] Qin S, Rottman JB, Myers P, et al. The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J Clin Invest 1998;101:746–54. [36] Holling TM, Schooten E, van Den Elsen PJ. Function and regulation of MHC class II molecules in T-lymphocytes: of mice and men. Hum Immunol 2004;65: 282–90. [37] Boleslawski E, Othman SB, Aoudjehane L, et al. CD28 expression by peripheral blood lymphocytes as a potential predictor of the development of de novo malignancies in long-term survivors after liver transplantation. Liver Transpl 2011;17: 299–305.

[38] Biselli R, Matricardi PM, D'Amelio R, Fattorossi A. Multiparametric flow cytometric analysis of the kinetics of surface molecule expression after polyclonal activation of human peripheral blood T lymphocytes. Scand J Immunol 1992;35: 439–47. [39] Barten MJ, Streit F, Boeger M, et al. Synergistic effects of sirolimus with cyclosporine and tacrolimus: analysis of immunosuppression on lymphocyte proliferation and activation in rat whole blood. Transplantation 2004;77:1154–62. [40] Niwa M, Miwa Y, Kuzuya T, et al. Stimulation index for PCNA mRNA in peripheral blood as immune function monitoring after renal transplantation. Transplantation 2009;87:1411–4. [41] Böhler T, Waiser J, Budde K, et al. The in vivo effect of rapamycin derivative SDZ RAD on lymphocyte proliferation. Transplant Proc 1998;30:2195–7. [42] Augustine NH, Pasi BM, Hill HR. Comparison of ATP production in whole blood and lymphocyte proliferation in response to phytohemagglutinin. J Clin Lab Anal 2007;21:265–70. [43] Wu J, Palladino MA, Figari IS, Morris RE. Comparative immunoregulatory effects of rapamycin, FK 506 and cyclosporine on mitogen-induced cytokine production and lymphoproliferation. Transplant Proc 1991;23:238–40. [44] Shipkova M, Wieland E, Schütz E, et al. The acyl glucuronide metabolite of mycophenolic acid inhibits the proliferation of human mononuclear leukocytes. Transplant Proc 2001;33:1080–1. [45] Lyons AB, Parish CR. Determination of lymphocyte division by flow cytometry. J Immunol Methods 1994;71:131–7. [46] Babel N, Reinke P, Volk HD. Lymphocyte markers and predicition of long-term renal allograft acceptance. Curr Opin Nephrol Hypertens 2009;18:489–94. [47] Böhler T, Nolting J, Kamar N, et al. Validation of immunological biomarkers for the pharmacodynamic monitoring of immunosuppressive drugs in humans. Ther Drug Monit 2007;29:77–86. [48] Dambrin C, Klupp J, Morris RE. Pharmacodynamics of immunosuppressive drugs. Curr Opin Immunol 2000;12:557–62. [49] Kubbies M, Schindler D, Hoehn H, Rabinovitch PS. Cell cycle kinetics by BrdUHoechst flow cytometry: an alternative to the differential metaphase labeling. Cell Tissue Kinet 1985;18:551–62. [50] Shaw LM, Annesley TM, Kaplan B, Brayman KL. Analytic requirements for immunosuppressive drugs in clinical trials. Ther Drug Monit 1995;17:577–83. [51] Davis C, Wu X, Li W, Fan H, Reddy M. Stability of immunophenotypic markers in fixed peripheral blood for extended analysis using flow cytometry. J Immunol Methods 2011;363:158–65. [52] Fletcher MA, Baron GC, Ashman MR, Fischl MA, Klimas NG. Use of whole blood methods in assessment of immune parameters in immunodeficiency states. Diagn Clin Immunol 1987;5:69–81. [53] Rainen L, Oelmueller U, Jurgensen S, et al. Stabilization of mRNA expression in whole blood samples. Clin Chem 2002;48:1883–90. [54] Wieland E, Shipkova M, Martius Y, et al. Association between pharmacodynamic biomarkers and clinical events in the early phase after kidney transplantation: a single-center pilot study. Ther Drug Monit 2011;33:341–9. [55] Berry V, Magill A, Yost M, Janosky J, Sindhi R. Individualizing combination of two antiproliferative immunosuppressants with pharmacodynamic modeling of stimulated lymphocyte responses. Cytometry A 2006;69:95–103. [56] Wang H, Grzywacz B, Sukovich D, et al. The unexpected effect of cyclosporin A on CD56+CD16 − and CD56+CD16 + natural killer cell subpopulations. Blood 2007;110:1530–9. [57] Barten MJ, Dhein S, Chang H, et al. Assessment of immunosuppressive drug interactions: inhibition of lymphocyte function in peripheral human blood. J Immunol Methods 2003;283:99–114. [58] Shipkova M, Krizan E, Klett C, Leicht S, Wieland E. Effect of immunosuppressants on CD26/dipetidyl peptidase IV expression on CD3 in vitro. Ther Drug Monit 2009;31:660. [59] He X, Smeets RL, Koenen HJ, et al. Mycophenolic acid-mediated suppression of human CD4 + T cells: more than mere guanine nucleotide deprivation. Am J Transplant 2011;11:439–49. [60] Sindhi R, Allaert J, Gladding D, et al. Modeling individual variation in biomarker response to combination immunosuppression with stimulated lymphocyte responses-potential clinical implications. J Immunol Methods 2003;272:257–72. [61] Van Rijen MM, Metselaar HJ, Hommes M, Ijzermans JN, Tilanus HW, Kwekkeboom J. Mycophenolic acid is a potent inhibitor of the expression of tumour necrosis factor- and tumour necrosis factor-receptor superfamily costimulatory molecules. Immunology 2003;109:109–16. [62] Stalder M, Bîrsan T, Holm B, Haririfar M, Scandling J, Morris RE. Quantification of immunosuppression by flow cytometry in stable renal transplant recipients. Ther Drug Monit 2003;25:22–7. [63] Prémaud A, Rousseau A, Johnson G, et al. Inibition of T-cell activation and proliferation by mycophenolic acid in patients awaiting liver transplantation: PK/PD relationships. Pharmacol Res 2011;63:432–8. [64] Kamar N, Glander P, Nolting J, et al. Pharmacodynamic evaluation of the first dose of mycophenolate mofetil before kidney transplantation. Clin J Am Soc Nephrol 2009;4:936–42. [65] Hutchinson P, Chadban SJ, Atkins RC, Holdsworth SR. Laboratory assessment of immune function in renal transplant patients. Nephrol Dial Transplant 2003;18: 983–9. [66] Böhler T, Canivet C, Galvani S, et al. Pharmacodynamic monitoring of the conversion from mycophenolate mofetil to enteric-coated mycophenolate sodium in stable kidney-allograft recipients. Int Immunopharmacol 2008;8:769–73. [67] Weigel G, Griesmacher A, Karimi A, Zuckermann AO, Grimm M, Mueller MM. Effect of mycophenolate mofetil therapy on lymphocyte activation in heart transplant recipients. J Heart Lung Transplant 2001;21:1074–9.

M. Shipkova, E. Wieland / Clinica Chimica Acta 413 (2012) 1338–1349 [68] Canivet C, Böhler T, Galvani S, et al. T-cell function in maintenance renal transplant patients receiving mycophenolate mofetil and steroids with or without tacrolimus. Transpl Proc 2008;40:3422–3. [69] Barten MJ, Rahmel A, Garbade J, et al. Pharmacodynamic monitoring of the conversion of cyclosporine to tacrolimus in heart and lung transplant recipients. Transplant Proc 2005;37:4532–4. [70] Barten MJ, Tarnok A, Garbade J, et al. Pharmacodynamics of T-cell function for monitoring immunosuppression. Cell Prolif 2007;40:50–63. [71] Morteau O, Blundell S, Chakera A, et al. Renal transplant immunosuppression impairs natural killer cell function in vitro and in vivo. PLoS One 2010;12(5):e13294. [72] Deng MC, Erren M, Roeder N, et al. T-cell and monocyte subsets, inflammatory molecules, rejection, and hemodynamics early after cardiac transplantation. Transplantation 1998;65:1255–61. [73] Beik AI, Morris AG, Higgins RM, Lam FT. Serial flow cytometric analysis of T-cell surface markers can be useful in differential diagnosis of renal allograft dysfunction. Clin Transplant 1998;12:24–9. [74] Chang DM, Ding YA, Kuo SY, Chang ML, Wei J. Cytokines and cell surface markers in prediction of cardiac allograft rejection. Immunol Invest 1996;25:13–21. [75] Boleslawski E, Ben Othman S, Grabar S, et al. CD25, CD28 and CD38 expression in peripheral blood lymphocytes as a tool to predict acute rejection after liver transplantation. Clin Transplant 2008;22:494–501. [76] Roberti I, Reisman L. Serial evaluation of cell surface markers for immune activation after acute renal allograft rejection by urine flow cytometry—correlation with clinical outcome. Transplantation 2001;71:1317–20. [77] Sindhi R, Magill A, Bentlejewski C, et al. Enhanced donor-specific alloreactivity occurs independently of immunosuppression in children with early liver rejection. Am J Transplant 2005;5:96–102.

1349

[78] Sindhi R, Manavalan JS, Magill A, Suciu-Foca N, Zeevi A. Reduced immunosuppression in pediatric liver-intestine transplant recipients with CD8+CD28 − Tsuppressor cells. Hum Immunol 2005;66:252–7. [79] Ashokkumar C, Bentlejewski C, Sun Q, et al. Allospecific CD154 + B cells associate with intestine allograft rejection in children. Transplantation 2010;90:1226–31. [80] Ashokkumar C, Talukdar A, Sun Q, et al. Allospecific CD154+ T cells associate with rejection risk after pediatric liver transplantation. Am J Transplant 2009;9:179–91. [81] Millán O, Benitez C, Guillén D, et al. Biomarkers of immunoregulatory status in stable liver transplant recipients undergoing weaning of immunosuppressive therapy. Clin Immunol 2010;137:337–46. [82] Latek R, Fleener C, Lamian V, et al. Assessment of belatacept-mediated costimulation blockade through evaluation of CD80/86-receptor saturation. Transplantation 2009;87:926–33. [83] Newell KA, Asare A, Kirk AD, et al. Identification of a B cell signature associated with renal transplant tolerance in humans. J Clin Invest 2010;120:1836–47. [84] Sagoo P, Perucha E, Sawitzki B, et al. Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans. J Clin Invest 2010;120: 1848–61.

WEB REFERENCES http://prow.nci.nih.gov/ http://www.ncbi.nlm.nih.gov/ http://www.ncbi.nlm.nih.gov/omim http://expasy.org/sprot/