Renal allograft rejection: Examination of delayed differentiation of Treg and Th17 effector T cells

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Renal allograft rejection: Examination of delayed differentiation of Treg and Th17 effector T cells Marcin Pekalski a , Sarah E. Jenkinson a , Joseph D.P. Willet a , Elizabeth F.M. Poyner a , Abdulaziz H. Alhamidi b , Helen Robertson a , Simi Ali a , John A. Kirby a,∗ a b

Applied Immunobiology and Transplantation Research Group, Institute of Cellular Medicine, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK Clinical Laboratory Sciences Department, King Saud University, Riyadh 11421, Saudi Arabia

a r t i c l e

i n f o

Article history: Received 25 January 2012 Received in revised form 11 May 2012 Accepted 16 May 2012 Keywords: Kidney allograft Rejection TGF-␤ Th17 Treg FOXP3

a b s t r a c t Antigen presentation after kidney transplantation occurs in lymphoid tissues remote from the allograft, with activated T cells then migrating towards the graft. This study examined the possibility that these activated T cells can differentiate to acquire Th17 or Treg phenotypes after a time consistent with their arrival within renal allograft tissues. An immunocytochemical study was performed to demonstrate the response to intragraft TGF-␤ and the phenotype of lymphoid cells within rejecting human renal allograft tissue. A series of in vitro experiments was then performed to determine the potential to induce these phenotypes by addition of appropriate cytokines 3 days after initial T cell activation. During renal allograft rejection there was a strong response to TGF-␤, and both FOXP3 and IL-17A were expressed by separate lymphoid cells in the graft infiltrate. FOXP3 could be induced to high levels by the addition of TGF-␤1 3 days after the initiation of allogeneic mixed leukocyte culture. This Treg marker was enriched in the subpopulation of T cells expressing the cell-surface ␣E(CD103)␤7 integrin. The ROR␥t transcription factor and IL-17A were induced 3 days after T cell activation by the addition of TGF-␤1, IL-1␤, IL-6 and IL-23; many of these Th17 cells also co-expressed CD103. T cells can develop an effector phenotype following cytokine stimulation 3 days after initial activation. This suggests that the intragraft T cell phenotype may be indicative of the prevailing cytokine microenvironment. © 2012 Elsevier GmbH. All rights reserved.

Introduction Following activation, human CD4+ T cells can develop a number of specific phenotypes. These include Th17 cells which express the transcription factor ROR␥t and secrete IL-17A, and immunoregulatory T cells (Treg) which express the transcription factor FOXP3 and inhibit T cell activation (O’Garra et al. 2008). Recent studies suggest that the relative number of proinflammatory Th17 and immunoregulatory Treg cells within a transplanted kidney can regulate long-term allograft survival (Heidt et al. 2010). Attention has also been drawn to the spatial distribution of these cells within the kidney, with the description of specific features such as Treg tubulitis (Veronese et al. 2007). Study of the differentiation of Th17 and induced Treg cells typically involves the analysis of naive T cells after stimulation of the antigen and costimulatory receptors in the presence of a range of exogenous cytokines. A mixture of TGF-␤ with proinflammatory cytokines is generally used to induce Th17 cells (Veldhoen et al. 2009), whilst TGF-␤ alone increases the expression of FOXP3

∗ Corresponding author. Tel.: +44 0191 222 7057; fax: +44 0191 222 8514. E-mail address: [email protected] (J.A. Kirby).

(Shevach et al. 2008). Although this T cell differentiation may occur in response to TGF-␤ generated directly by antigen-presenting dendritic cells (Travis et al. 2007), the activated parenchymal (van Kooten and Daha 2001) and inflammatory cells (Cornell et al. 2008) within a renal allograft also produce cytokines relevant to T cell differentiation. Donor dendritic cells are rapidly recruited to the spleen after transplantation (Larsen et al. 1990) and it is likely that both direct (Baratin et al. 2004) and indirect (Brennan et al. 2009) alloantigen presentation initially occurs at this site. After T cell activation and early clonal expansion, these cells lose CD69 expression (Matloubian et al. 2004; Cyster et al. 2010) and leave this lymphoid tissue. They can then be recruited by passage across chemokine-expressing, activated endothelium within the allograft (Lo et al. 2011). The trafficking of activated T cells into a kidney allograft reaches a peak some 3–5 days after transplantation (Nemlander et al. 1983). As these T cells penetrate allograft tissues they encounter a complex and variable cytokine milieu produced by activated cells which can secrete IL-1␤ (Tesch et al. 1997), IL-6 (Fukatsu et al. 1993; de Haij et al. 2005), IL-23 (Langrish et al. 2004; Kastelein et al. 2007) and TGF-␤ (Robertson et al. 2001). This suggests that primary differentiation of T cells might occur within the graft at a site remote from initial alloantigen presentation and T

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Please cite this article in press as: Pekalski, M., et al., Renal allograft rejection: Examination of delayed differentiation of Treg and Th17 effector T cells. Immunobiology (2012), http://dx.doi.org/10.1016/j.imbio.2012.05.014

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cell activation. However, this possibility of intragraft T cell differentiation is only relevant if T cells can be induced to differentiate by contacting cytokines some days after initial T cell activation. Our group and others have shown that some activated T cells which penetrate renal tubules during acute rejection acquire the cell-surface ␣E(CD103)␤7 integrin, allowing chemokine-enhanced binding to the epithelial adhesion molecule E-cadherin (Al-Hamidi et al. 2008). The distribution of CD103 expressing T cells becomes more widespread during chronic rejection (Robertson and Kirby 2003). Previous studies have shown that T cells activated in the absence of TGF-␤ in vitro show little CD103 expression. However this integrin is induced by TGF-␤1, which activates Smad transcription factors that bind CAGA boxes within the proximal promoter of the ␣E integrin gene (Kilshaw 1999). Significantly, this integrin can be induced equally efficiently if TGF-␤1 is added at the time of T cell activation or some days later (Wong et al. 2003). The lack of expression of CD103 by T cells within the interstitial infiltrate during early acute renal rejection suggests that this differentiation is only induced when T cells are exposed to TGF-␤ within the kidney tubules rather than during initial antigen presentation within the spleen. The current study was designed to demonstrate the relative distribution of cells which express IL-17A and FOXP3 within rejecting renal allograft biopsy sections and to explore the potential for induction of these important phenotypes within the allograft by stimulation with appropriate cytokine mixtures 3 days after initial T cell activation. A further series of experiments was performed to assess the potential for overlap between CD103 expression and either the Th17 or Treg phenotypes. Materials and methods Rejection tissue and immunohistochemistry Normal human kidney tissue and diagnostic renal allograft biopsy samples (n = 10) were obtained as formalin-fixed and paraffin-embedded blocks from the archives of the Newcastle upon Tyne Teaching Hospitals NHS Trust. Frozen sections were also prepared from diagnostic biopsies from transplanted kidneys. All biopsy sections were used in accordance with Newcastle and North Tyneside Local Research Ethics Committee approval. Immunohistochemical procedures were performed according to previously published protocols (Robertson et al. 2001). Formalinfixed kidney sections were treated for heat-induced epitope retrieval then labelled with primary mouse monoclonal antibodies specific for FOXP3 (clone 221D/D3; a gift from Dr Alison Banham, Oxford, UK) and IL-17A (biotin-conjugated; R&D Systems, UK); a rabbit polyclonal antibody was used to label phosphoSmad 2/3 (pSmad2/3: Santa Cruz). Frozen sections were treated with mouse monoclonal anti-CD103 (BerAct-8; DAKO). Bound primary antibodies were detected with biotin-conjugated rabbit anti-mouse or swine anti-rabbit antibodies (both DAKO) followed by addition of a streptavidin–biotin–peroxidase complex (Vector, UK) with diaminobenzidine (DAB) or nickel-enhanced DAB (NiDAB: Sigma, UK) chromogenic substrate. Biotinylated anti-IL-17A was detected directly by application of streptavidin–biotin–peroxidase complex with NiDAB development. Sections were counterstained with Mayer’s haematoxylin except for those labelled to detect pSmad 2/3, which were counterstained with alcoholic fast green. T cell isolation and activation Human T cells were isolated from heparinised blood taken from normal donors by negative selection using RosetteSep human T cell enrichment cocktail (Stemcell Technologies, Grenoble, France) in

accordance with the manufacturer’s instruction. These cells were resuspended at 1 × 106 cells/ml in RPMI 1640 (Sigma, Dorset, UK) for studies of FOXP3 expression, or Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen, Paisley, UK) for studies of IL-17A production (Veldhoen et al. 2009); both media were supplemented with 10% FCS (Sigma, Poole, UK). Naïve (CD45RA+) CD4+ T cells were isolated from peripheral blood mononuclear cells by negative immunomagnetic selection (EasySep; StemCell Technologies) in accordance with the manufacturer’s instructions. In all cases, cell purity was greater than 98%. The T cell populations were activated by stimulation with immobilized CD3 (clone OKT3; Janssen-Cilag, High Wycombe, UK) and CD28 (clone 28.2; Becton Dickinson, Oxford, UK) antibodies, or by allogeneic mixed leukocyte culture (MLC) with a ␥-irradiated, EBV-transformed B cell line at an optimal ratio of 1:1. At time zero or after 72 h some cultures were variably supplemented with optimal concentrations of TGF-␤1 (5 ng/ml), IL-6 (25 ng/ml), IL-1␤ (10 ng/ml) and IL-23 (25 ng/ml); all cytokines were from R&D Systems (Abingdon, UK). Some cultures were supplemented with the TGF-␤ type I receptor (ALK5) inhibitor, SB-505124 (Sigma) at 1 ␮M; this non-toxic concentration was optimised for complete receptor inhibition using a sensitive, TGF-␤ responsive reporter cell assay (Tesseur et al. 2006). The T cells were recovered up to 10 days later for phenotypic or functional analysis; culture medium was collected for IL-17 ELISA (R&D Systems).

Addition of CD103+ or CD103− T cells to MLC Purified T cells were activated by allogeneic MLC for 72 h before addition of TGF-␤1. After culture for a further 5 days the cells were labelled with PE-conjugated mouse monoclonal antibody specific for CD103 (DAKO, Ely, UK). The CD103+ and CD103− subpopulations were then separated to 98% purity by cell sorting (FACSVantage; Becton Dickinson). Naïve syngeneic T cells were labelled with 5 ␮M carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen), mixed with either CD103+ or CD103− T cells at a ratio of 1:1 and stimulated with the original EBV-transformed B cell line for 4 days before flow cytometric measurement of CFSE dilution.

Immunofluorescence and immunocytochemistry Activated T cells were fixed, permeabilised and labelled with fluorochrome-conjugated monoclonal antibodies specific for CD103 (eBioscience, Hatfield, UK), IL-17A (Biolegend, Cambridge, UK) and FOXP3 (Biolegend). Appropriate fluorochrome-conjugated, isotype-matched control antibodies were used in all cases. After washing, the cells were analysed by flow cytometry (BD LSRII; Becton Dickinson). In some cases, cytosmears (Cytopspin; Shandon) were prepared from T cell suspensions after activation by MLC. These were fixed and incubated with monoclonal anti-FOXP3 antibodies (clone 221D/D3 kindly provided by Dr. A. Banham, Oxford, UK); labelled cells were identified after development with a biotinylated rabbit anti-mouse secondary antibody (DAKO) for immunoperoxidase development.

Quantitative realtime PCR RNA was isolated from cultured cells using Trizol reagent (Sigma) and transcribed to cDNA using superscript II (Invitrogen). Changes in expression of mRNA encoding FOXP3, the ␣E (CD103) integrin, TGF-␤1 and ROR␥t were measured by quantitative realtime PCR (ABI Prism 7700 using Assays-onDemand; Applied Biosystems, Warrington, UK); 18S RNA was also

Please cite this article in press as: Pekalski, M., et al., Renal allograft rejection: Examination of delayed differentiation of Treg and Th17 effector T cells. Immunobiology (2012), http://dx.doi.org/10.1016/j.imbio.2012.05.014

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Fig. 1. Immunohistochemical analysis of rejecting human renal allograft tissue. (A) Expression of (p)hosphorylated Smad 2/3 in the nuclei of renal tubular epithelial cells and infiltrating leukocytes in a representative biopsy showing infiltration, tubular damage and interstitial expansion. The inset panel shows pSmad 2/3 in the nuclei of the tubular epithelial cells in normal kidney (NK). Graft infiltrating cells expressed cell surface ␣E(CD103)␤7 integrin (B), nuclear FOXP3 (C) and cytoplasmic IL-17A (D). (E) Two colour immunohistochemistry showing cells within the graft infiltrate expressing IL-17A (black) and FOXP3 (brown). (F) High magnification showing cells expressing IL-17A or FOXP3 in close proximity to each other in a rejection biopsy. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

quantified for normalization. The results were analysed using Rest 8000 software (CorbettLifescience.com). Statistical analysis All data were analysed and presented using Prism 3 software (Graphpad.com). Statistical tests included paired and un-paired Student’s t-tests or ANOVA with Bonferroni’s post-test as appropriate; P < 0.05 was considered significant. Results Immunohistochemical analysis of renal allograft biopsy tissue showing features of rejection, including tubulitis, tubular atrophy and interstitial fibrosis (Solez et al. 2008), showed widespread accumulation of the activate, phosphorylated form of Smad 2/3 (pSmad 2/3) in the nuclei of damaged epithelial cells and leukocytes within the tubular epithelium and expanded interstitium (Fig. 1A). The epithelial cells in normal kidney (inset panel; Fig. 1A) showed less intense, constitutive activation of pSmad 2/3 and no

leukocytes were observed. A proportion of the graft infiltrating mononuclear cells expressed cell surface CD103 (Fig. 1B), nuclear FOXP3 (Fig. 1C) and cytoplasmic IL-17A (Fig. 1D). Two colour development (Fig. 1E) showed separate cells expressing nuclear FOXP3 (brown) and cytoplasmic IL-17A (black) in the same rejection sections. Higher magnification (Fig. 1F) showed cells expressing FOXP3 and IL-17A in close proximity. Immunocytochemical analysis of FOXP3 expression by T cells activated in MLC for 8 days showed only a low level of expression of this antigen in the absence of exogenous TGF-␤1 (Fig. 2A), but a high level of nuclear expression was apparent in a proportion of the alloreactive T cells following culture with TGF-␤1 (Fig. 2B); analysis of results using responder T cells from 4 different donors (Fig. 2C) showed an increase in expression of FOXP3 after stimulation with TGF-␤1 (P < 0.05), with a mean 19.7 ± 4.8% of the treated cells expressing this transcription factor. The increase in FOXP3 expression was produced by addition of TGF-␤1 on either day 0 or day 3 of the 8 day culture. The low level expression of FOXP3 observed in the absence of exogenous TGF-␤1 could be further reduced but not eliminated by inhibition of the TGF-␤1 receptor

Please cite this article in press as: Pekalski, M., et al., Renal allograft rejection: Examination of delayed differentiation of Treg and Th17 effector T cells. Immunobiology (2012), http://dx.doi.org/10.1016/j.imbio.2012.05.014

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Fig. 2. Analysis of FOXP3 expression following T cell activation. Representative immunocytochemical detection of FOXP3 expression by activated T cells isolated after 8 days in MLC; in the absence of TGF-␤1 (A) the cells show a low levels of FOXP3, but 19.7 ± 4.8% of the cells expressed FOXP3 in the presence of TGF-␤1 from the start of the culture (B). (C) Significant induction of FOXP3 in TGF-␤1 treated MLC using responder T cells isolated from 4 different donors. (D) FOXP3 expression by T cells from 6 separate donors after 8 days of polyclonal expansion; the expression of FOXP3 was increased by the addition of TGF-␤1 on either day 0 or day 3 (P = 0.002). (E) Results from quantitative realtime PCR analysis showed little expression of the FOXP3 gene in resting T cells but this gene was expressed at an increased level after stimulation in MLC for 8 days in the absence of TGF-␤1; addition of TGF-␤1 at the start of the MLC produced a mean 9-fold increase in FOXP3 gene expression by day 8. (F) The ␣E integrin gene was expressed at a very low level in resting T cells or cells which had been stimulated in MLC for 8 days in the absence of TGF-␤1; addition of TGF-␤1 at the start of the MLC produced a mean 280-fold increase in expression of this gene by day 8. Gene expression data are representative of 3 similar experiments, error bars show SEM.

with SB-505124 (Fig. 2D). Quantification of mRNA encoding FOXP3 showed a 3-fold increase in MLC without exogenous TGF-␤1 but this was increased to 9-fold by addition of TGF-␤1 at the start of the culture period (Fig. 2E). The level of IL-17A produced by these cells was not increased by TGF-␤1 stimulation. After culture for 8 days the expression of mRNA encoding the ␣E(CD103) integrin showed a similar trend (Fig. 2F), with cells activated in the absence of TGF-␤ showing little expression of the ␣E integrin gene, whilst a strong induction of this gene was observed in the presence of TGF-␤1. A representative dot plot (Fig. 3A) showing CD103 and FOXP3 expression by T cells activated for 8 days in MLC with TGF-␤1 added

on day 3 demonstrates a higher proportion of FOXP3 expressing cells in the CD103+ve (44%) than the CD103−ve fraction (29%). Summary data from 3 similar experiments using responder T cells from different donors demonstrates the reproducibility of this result (P = 0.02; Fig. 3B). Flow cytometric analysis of the dilution of CFSE in the responder T cells after MLC for 4 days shows that 12% of the labelled T cell population is dividing (top panel; Fig. 3C). Mixture of an equal number of CFSE labelled responder cells at a 1:1 ratio with syngeneic CD103− T cells sorted from a TGF-␤1 supplemented MLC reduced the proportion of proliferating cells to 6% (middle panel; Fig. 3C); the proportion of proliferating cells in

Please cite this article in press as: Pekalski, M., et al., Renal allograft rejection: Examination of delayed differentiation of Treg and Th17 effector T cells. Immunobiology (2012), http://dx.doi.org/10.1016/j.imbio.2012.05.014

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Fig. 3. Examination of the immunoregulatory function of CD103+ T cells. (A) Representative flow cytometric dot-plot showing the expression of cell surface CD103 and intracellular FOXP3 by T cells stimulated in MLC for 8 days with TGF-␤1 added on day 3. (B) Summary results from experiments using T cells from 3 separate donors showing a higher proportion of CD103+ than CD103− T cells co-express FOXP3 after MLC for 8 days with TGF-␤1 added on day 3. (C) Alloreactive CD103+ve and CD103−ve T cells were separated by FACS from TGF-␤1 stimulated MLCs. These cells were then added at a 1:1 ratio to CFSE-labelled resting syngeneic T cells; the mixed population of T cells was then activated using the same antigen presenting cells as the initial MLC; CFSE dilution was analysed by flow cytometry 4 days later. The upper histogram shows CFSE dilution by T cells which have not been mixed with either sorted CD103−ve or CD103+ve T cells and indicates that 12% of the CFSE-labelled T cells have divided. The shaded middle and lower histograms show CFSE dilution in cultures supplemented with sorted CD103−ve and CD103+ve T cells respectively; the open histogram is from the upper panel (for reference). (D) Summary results from experiments using T cells from 3 different donors showing that the addition of sorted CD103−ve allospecific T cells reduced T cell proliferation in MLC by a mean 39% (P < 0.05); addition of CD103+ve T cells inhibited proliferation by 82% (P < 0.01).

the primary MLC was decreased to 2% by 1:1 mixture of the CFSE labelled cells with CD103+ T cells (bottom panel; Fig. 3C). Summary data from 3 similar experiments using responder T cells from different donors shows the reproducibility of these differences in responder cell proliferation (Fig. 3D). Purified CD4+CD45RA+ (naive) T cells were activated in the presence of TGF-␤1 and a range of proinflammatory cytokines and the expression of mRNA encoding ROR␥t (Fig. 4A) and the secretion of IL-17A (Fig. 4B) were measured after 72 h. In both cases, TGF-␤1 alone had little effect. However, further addition of IL-1␤, or IL-1␤ and IL-6, or IL-1␤, IL-6 and IL-23 all increased the expression of ROR␥t (P < 0.01); the greatest secretion of IL-17A was produced in

the presence of TGF-␤1 and the three proinflammatory cytokines (P < 0.05; Fig. 4B). Flow cytometric dot plots (Fig. 5A) demonstrate that IL-17A expression was expressed at day 8 following stimulation of activated CD4+ T cells with TGF-␤1, IL-1␤, IL-6 and IL-23 added on either day 0 or day 3 of culture; FOXP3 expression was not elevated in these cultures. In both cases, CD103 expression was also increased with some cells expressing both IL-17A and CD103. Summary results using CD4+ T cells purified from different donors show the proportion of IL-17A expressing T cells (Fig. 5B) was increased 7-fold (P < 0.05) by the presence of exogenous cytokines from day 0 but by a greater 9.5-fold (P < 0.01) when these cytokines were added

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Fig. 4. Analysis of the cytokine requirement for Th17 differentiation after T cell activation. Quantitative realtime PCR analysis showing changes in expression of the ROR␥t gene (A) and ELISA quantification of IL-17A (B) in culture supernatant 72 h after activation of purified CD4+CD45RA+ T cells in culture containing a range of cytokine supplements. All data are representative of three similar experiments; error bars show SEM.

on day 3. The proportion of CD103+ T cells which also expressed IL-17A (Fig. 5C) was increased a mean 4-fold (P < 0.05) by addition of the cytokine mixture on day 0 but by a greater 5.1-fold (P < 0.001) when the start of cytokine stimulation was delayed until day 3. Discussion Previous studies have shown that renal tissue contains a reservoir of TGF-␤ which is greatly expanded during episodes of acute rejection (Robertson et al. 2001). This growth factor induces gene transcription following receptor activation and specific phosphorylation of Smad-2 and -3 (Moustakas et al. 2001). Normal kidney expresses low levels of pSmad 2/3 in the tubular epithelial cells but this is increased in the nuclei of both epithelial cells and infiltrating leukocytes during chronic rejection (Tyler et al. 2006), demonstrating a general response to TGF-␤. Previous studies have also shown the production of IL-1␤ and IL-6 by stressed tubular epithelial cells (Fukatsu et al. 1993; Tesch et al. 1997) whilst IL-23 can be produced by activated macrophages (Kastelein et al. 2007), which are present within the graft infiltrate (Ricardo et al. 2008). Immunohistochemical analysis of biopsy sections with features of active inflammation and chronic rejection (Solez et al.

2008) showed the presence of CD103+ T cells within both the tubules and the interstitial infiltrate. Cells expressing nuclear FOXP3 or cytoplasmic IL-17A showed a similar distribution, with a proportion of both cell-types associated with the tubules. The presence of FOXP3 expressing T cells within renal tubules has been observed previously during rejection and is termed Treg tubulitis (Veronese et al. 2007); the potential for co-expression of FOXP3 and CD103 provides a basis for the retention of Treg within the tubules. Sometimes FOXP3 and IL-17A expressing cells were found in close proximity to each other. This observation is consistent with several models. Firstly, T cell differentiation may occur in response to different cytokine populations with a very restricted tissue distribution. Secondly, Treg and Th17 cells can express a similar repertoire of chemokine receptors, including CXCR3, CCR2, CCR5 and CCR6 (Sato et al. 2007; Turner et al. 2010; Campbell and Koch 2011) which will respond to rejection-associated chemokines such as CXCL10, CCL2, CCL5 and CCL20 (Robertson et al. 2000; Woltman et al. 2005; Cornell et al. 2008) driving convergent migration of these 2 cell types within the allograft. Finally, it has been suggested that Treg and Th17 cells retain some functional plasticity, allowing cells to switch between these phenotypes following appropriate cytokine stimulation (Murphy and Stockinger 2010). There was no evidence of single cells expressing both FOXP3 and IL-17A. This is consistent with the potential of FOXP3 to block Th17 differentiation by direct inhibition of the ROR␥t transcription factor (Zhou et al. 2008). In the absence of exogenous TGF-␤ a small proportion of responding T cells in MLC showed low levels of nuclear FOXP3 expression. The failure to reduce this to baseline levels by blockade of the TGF-␤ receptor suggests that Smad signalling is not necessary to support this low level expression of FOXP3 by human T cells. A previous study has shown that the activation of NF-AT is sufficient to increase FOXP3 expression, although this was increased synergistically by Smad activation (Tone et al. 2008). The addition of TGF-␤ 3 days after initiation of the MLC was also sufficient to induce a high level of FOXP3 expression, with a greater proportion of FOXP3+ cells developing within the CD103+ fraction than the CD103−ve fraction in these cultures. The greater immunoregulatory function of the CD103+ fraction was demonstrated by addition of sorted CD103+ and CD103− T cells to CFSE labelled responder cells in a primary MLC. Analysis of naïve CD4+ T cells after activation in the presence of TGF-␤1 and a mixture of proinflammatory cytokines demonstrated the requirement for IL-1␤, IL-6 and IL-23 for optimal induction of ROR␥t expression and IL-17A secretion. This is consistent with previous studies (Veldhoen et al. 2009). The proportion of IL-17A expressing T cells was increased by delaying the addition of exogenous cytokines for 3 days after T cell activation. The proportion of CD103+ T cells which co-expressed IL-17A in these cultures was also increased by delaying addition of the cytokines for 3 days after T cell activation. The presence of Th17 cells is associated with renal injury (Kitching and Holdsworth 2011) whilst stimulation of renal tubular epithelial cells with IL-17 enhances the production of CXCL8 and CCL2 (Woltman et al. 2000), potentially increasing allograft inflammation. This study demonstrates that activated allospecific T cells can acquire a functionally relevant, differentiated phenotype when stimulated with appropriate cytokines 3 days after initial activation. This phenotype may develop in response to microenvironmental cytokines within the allograft tissues, with TGF-␤ alone favouring the development of immunoregulatory T cells, whilst contact with TGF-␤ in the presence of additional, stressrelated innate inflammatory cytokines will favour development of pro-inflammatory Th17 cells. The study also shows that FOXP3

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Fig. 5. Demonstration that delayed cytokine addition does not alter Th17 differentiation. (A) Representative flow cytometric dot-plots showing expression of cell surface CD103 and intracellular IL-17A by CD4+CD45RA+ T cells after activation for 8 days in the presence of either no exogenous cytokine or a mixture of TGF-␤1, IL-1␤, IL-6 and IL-23 added on day 0 or day 3; quadrant markers were placed using isotype control results. (B) Summary results from replicate experiments using CD4+CD45RA+ T cells from different donors showing that the proportion of activated T cells expressing IL-17A on day 8 was increased by the addition of cytokines on day 0 (P < 0.05) or day 3 (P < 0.01) after T cell activation. (C) The proportion of CD103+ T cells co-expressing IL-17A was also increased by addition of cytokines on day 0 (P = 0.05) or day 3 (P < 0.001).

and Th17 cells can exist in close proximity to each other within a rejecting allograft. Finally, CD103 expression was shown to overlap immunoregulatory and Th17 markers as well as the previously reported cytotoxic function (Yuan et al. 2005), conferring the potential for adhesive retention of all these T cell phenotypes within the renal tubules. Acknowledgements This work is supported by grants from Kidney Research UK and the Genzyme Renal Innovations Programme (GRIP); MP is supported by an EU FP6 Marie Curie EST Fellowship (ROSAT project). References Al-Hamidi, A., Pekalski, M., Robertson, H., Ali, S., Kirby, J.A., 2008. Renal allograft rejection: the contribution of chemokines to the adhesion and retention of alphaE(CD103)beta7 integrin-expressing intratubular T cells. Mol. Immunol. 45, 4000–4007.

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Please cite this article in press as: Pekalski, M., et al., Renal allograft rejection: Examination of delayed differentiation of Treg and Th17 effector T cells. Immunobiology (2012), http://dx.doi.org/10.1016/j.imbio.2012.05.014