DOI: 10.1002/eji.201545455

Eur. J. Immunol. 2015. 45: 2017–2027

Immunomodulation

Allospecific CD4+ T cells retain effector function and are actively regulated by Treg cells in the context of transplantation tolerance Jian-Guo Chai1,2 , Kulachelvy Ratnasothy1 , R. Pat Bucy3 , Randolph J Noelle1,4 , Robert Lechler1 and Giovanna Lombardi1 1

MRC Centre for Transplantation, King’s College London, London, UK Therapeutic Immunology Group, Sir William Dunn School of Pathology, University of Oxford, Oxford, UK 3 Department of Pathology, University of Alabama, Birmingham, AL, USA 4 Department of Microbiology and Immunology, Dartmouth Medical School, Norris Cotton Cancer Center, Lebanon, NH, USA 2

Although donor-specific transfusion (DST) plus CD154 blockade represents a robust protocol for inducing transplantation tolerance, the underlying mechanisms are incompletely understood. In a murine T-cell adoptive transfer model, we have visualized alloantigenspecific, TCR-transgenic for H2-Ab /H2-Kd 54–68 epitope (TCR75) CD4+ T cells with indirect allospecificity during the course of tolerance induction. Three main observations were made. First, although the majority of TCR75 CD4+ T cells were deleted following DST plus CD154 blockade, the surviving TCR75 CD4+ T cells were capable of making IL-2, upregulating CD44, and undergoing cell division, suggesting that they were functionally active. Indeed, residual TCR75 CD4+ T cells reisolated from the primary recipients given DST plus CD154 blockade were fully capable of rejecting allografts upon secondary transfer. Second, in tolerant mice, TCR75 CD4+ T cells were not induced to express Foxp3 in the graft-draining lymph node. TCR75 CD4+ T cells were also absent in accepted graft tissues in which endogenous Treg cells were enriched. Finally, DST plus CD154 blockade resulted in an abortive expansion of TCR75 CD4+ T cells, a process that required the presence of endogenous Treg cells. Collectively, surviving TCR75 CD4+ T cells are immunocompetent but kept in check by an endogenous immunosuppressive network induced by DST plus CD154 blockade.

Keywords: CD40L blockade

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CD4 T cells

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Tolerance

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Transplantation r Treg

See accompanying Commentary by Luis Graca



Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction Donor-specific transfusion (DST) plus CD40L blockade represents one of the most powerful and best characterized regimens for the

Correspondence: Dr. Giovanna Lombardi e-mail: [email protected]  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

induction of tolerance of islet [1–3], skin [4, 5], and cardiac [6] allografts in murine models. Although the clinical application of intact FcR-binding anti-human CD40L mAb is challenging due to the development of thromboembolism [7, 8], alternative strategies to inactivate the CD40/CD154 pathway are under active investigation with some promising results [9–11]. Importantly, blockade of this particular costimulatory pathway has been recognized as one

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of the most robust procedures to induce transplantation tolerance in nonhuman primate models [12–15]. Thus, there is considerable interest in elucidating the detailed cellular and molecular events underlying DST plus anti-CD154-induced graft tolerance. Two well-defined pathways are involved in the recognition of allogeneic MHC molecules [16]. The first, the direct pathway, is the mechanism by which recipient T cells recognize determinants on intact donor MHC:peptide complexes displayed on the surface of donor cells. The second, known as the indirect pathway, refers to recognition of processed peptides of allogeneic MHC antigens presented by recipient MHC in a self-restricted manner. The indirect alloresponse is the major immune driver of chronic rejection and it is thought to be the major threat to long-term survival of allografts [17, 18]. The indirect pathway is critical for the induction of transplantation tolerance, because regulatory CD4+ T cells that are capable of transferring tolerance have indirect allospecificity [19, 20]. In addition, it is becoming increasingly clear that indirect recognition of alloantigen presented by host MHC class II molecules is required for long-term allograft survival by DST plus CD154 blockade [21–23]. In other words, DST serves only as an alloantigen depot, providing the source of allopeptides to be presented by host APCs [24]. The use of TCR-transgenic CD4+ T cells with indirect allospecificity to study the mechanisms of tolerance induced by DST plus CD154 blockade has yielded inconsistent, and sometimes contradictory conclusions, varying from T-cell anergy [22–24] to T-cell regulation [25–27]. One of the reasons for the lack of concordance in the conclusions reached by these studies is the breadth of differences inherent in the approaches used. Differences in TCR affinity and avidity, the dose/timing of T-cell adoptive transfer, type of allografts (skin versus heart), nature of alloantigen (surrogate OVA versus natural MHC), and status of host’s immune system (lymphocyte deficient versus lymphocyte sufficient) have all contributed to difficulties in direct comparisons across these studies. Second, the definition of T-cell anergy appears to be very limited, because most studies have restricted their measurements to IL-2 production or in vitro secondary responsiveness. We would propose that the most reliable readout for evaluating graft-reactive T cell functionality is to assess the capacity of T cells from the primary recipients to mediate rejection of a fresh allograft in secondary recipients. Unfortunately, few studies have taken this second donor T-cell adoptive transfer approach. Third, for tracking allospecific T cells, most studies have exclusively focused on activities of T cells in the graft-draining LN, and little information is available regarding frequency of graft-infiltrating T cells in longterm tolerant mice. Finally, for the measurement of Foxp3 expression, the majority of reports have concentrated on donor-specific T cells in graft-draining LN during a short time period of antigen exposure, rather than those in the graft tissue that have survived indefinitely. To track the fate of CD4+ T cells with indirect allospecificity in mice rendered tolerant, we have utilized an adoptive transfer model with na¨ıve TCR-transgenic CD4+ T cells from TCR-transgenic for H2-Ab /H2-Kd 54–68 epitope (TCR75) mice [28]. These T cells recognize an allopeptide (Kd 54–68 ) derived from a  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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major alloantigen, H2-Kd , presented by self-H2-Ab [28]. TCR75 mice are one of the few available TCR-Tg mouse lines with indirect allospecificity, and have been proven useful in the study of in vitro differentiation of adaptive Treg and their impact on linked suppression of CD8 T cells [29], priming of the indirect pathway [30], the generation of Treg cells with defined indirect allospecificity by TCRαβ gene transfer [31, 32], and the induction of transplantation tolerance by targeting dendritic cells [33]. To our knowledge, this is the first study to exploit the special assets provided by the TCR75 model to dissect the cellular events, which underlie the transplantation tolerance induced by DST and anti-CD154.

Results DST+αCD40L induces long-term transplantation tolerance in B6 recipients of TCR75 CD4+ T cells DST+αCD40L treatment is one of the most powerful toleranceinducing regimens [34]. Although DST+αCD40L failed to achieve long-term acceptance of fully mismatched BALB/c skin grafts on B6 mice [35], we consistently observed that indefinite survival of B6. Kd skin grafts in B6 mice could be readily induced following a standard DST+αCD40L treatment (Fig. 1A). As reported previously [[4, 5, 22–24, [34–36]], untreated hosts or hosts treated with irrelevant mAb or self-DST rejected alloskin grafts with similar kinetics (approximately with 14 days). Importantly, tolerance to Kd skin could still be achieved in B6 mice previously injected with a small number of alloantigen-specific na¨ıve TCR Tg CD4+ T cells (Fig. 1B), purified from TCR75 mice [28]. This finding represents a platform and starting point for further investigations of the cellular events that underlie the tolerance induction by DST+αCD40L.

Costimulation blockade induces abortive expansion to alloantigen in TCR75 CD4+ T cells To evaluate the cellular events during the early stage of T cell activation, we employed an adoptive T-cell transfer model (Fig. 1C). The adoptively transferred TCR75 CD4+ T cells were readily identified by the coexpression of Thy1.1 and Vβ8.3 (Fig. 1D). The proportion of residual TCR75 CD4+ T cells detected in the axillary LNs of the DST group was significantly higher than in the DST+αCD40L group (Fig. 1E). The same results were obtained when the absolute numbers of TCR75 CD4+ T cells between the two groups were compared (Fig. 1F). The reduced frequency of residual TCR75 CD4+ T cells by DST+αCD40L treatment may be due to weak activation and/or impaired proliferation. However, a subsequent analysis of carboxyfluorescein succinimidyl ester (CFSE) dye dilution and the upregulation of CD44, (Fig. 2A and B), revealed that neither the increase of CD44 expression nor the progression of cell division was significantly affected by αCD40L treatment. However, αCD40L treatment significantly reduced the absolute number of residual CD44high TCR75 CD4 T cells detected in axillary LNs (Fig. 2C), www.eji-journal.eu

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Immunomodulation

Figure 1. Induction of transplantation tolerance in B6 or B6 recipients of TCR75 CD4+ T cells by the DST+αCD40L treatment. (A) B6 mice received a single DST (25 × 106 B6 Kd SP cells) on day −6 together with four i.p. injections of MR1 mAb (0.25 mg per injection) on day −6, −3, 0 and day 3 (DST+MR1, n = 12). B6 mice given DST alone (n = 10) or MR1 alone (n = 7) were used as controls. On day 0, all mice were transplanted with B6.Kd skin grafts. Data of graft survival were pooled from three independent experiments and analyzed by longrank test. (B) On day −7, B6 mice were adoptively transferred with 1 × 105 TCR75 CD4+ T cells and were subsequently treated with DST alone (n = 10), MR1 alone (n = 4) or DST+MR1 (n = 10) as described in Fig. 1A. Data were pooled from two independent experiments and analyzed by long-rank test. (C) Experiment design. (D) Representative FACS dot plots show the identification of TCR75 CD4+ T cells by coexpression of Vβ8.3 and Thy1.1. E and F. Numbers show percentages of donor TCR75 T cells in the axillary LN of total number of leukocytes. (E) The quantitation of TCR75 CD4+ T cells in axillary LN is represented as both percentage and (F) absolute cell number, which means the absolute donor TCR75 T cells recovered from the same LN. Data were presented as mean ± SEM pooled from two independent experiments (n = 4), and analyzed by two-tailed Student’s t-test.

suggesting that αCD40L administration dramatically impairs the accumulation of activated TCR75 CD4+ T cells, presumably by diminishing cell survival and/or enhancing cell death. Similar conclusions were apparent when the analysis was extended to CD62L and CD69 (Supporting Information Fig. 1). The function of residual TCR75 CD4+ T cells from mice treated with DST and DST+αCD40L was compared based on their capacity to produce cytokines. In terms of the frequency of IL-2-producing TCR75 CD4+ T cells, there was not a significant difference between the two groups (Fig. 2D and E). However, costimulation blockade significantly limited the absolute numbers of IL-2-producing TCR75 CD4+ T cells (Fig. 2F). Analysis of IFN-γ and TNF-α production yielded similar results (Supporting Information Fig. 2). Therefore, in response to DST in the presence of CD40L blockade, TCR75 CD4+ T cells underwent an abortive expansion that resulted in a profound reduction in T cell numbers yet the residual cells retained the capacity to make IL-2.

iments (Fig. 3A). We found that, like those from the DST only group, TCR75 CD4+ T cells from the DST+αCD40L group rapidly rejected skin allografts with similar kinetics (Fig. 3B) to nontolerized TCR75 CD4+ T cells, indicating that residual TCR75 CD4+ T cells, which survived after DST+αCD40L treatment are functionally active, despite a much lower frequency in the hosts. We also employed an alternative second adoptive transfer model to evaluate the functionality of residual TCR75 CD4+ T cells (Fig. 3C). As expected, without TCR75 CD4+ T-cell transfer, anti-Thy1.2-treated hosts could not reject B6. Kd grafts (Fig. 3D). In addition, we found that TCR75 CD4+ T cells reisolated from the DST+αCD40L group were as efficient as those from the DST alone group in rejecting B6. Kd skin grafts in T cell-depleted hosts (Fig. 3D). Collectively, data from these secondary transfer experiments using two independent models provided compelling evidence that residual TCR75 CD4+ T cells in DST+αCD40L-treated mice were functionally competent.

Residual TCR75 CD4+ T cells in DST+αCD40L-treated mice still reject grafts after secondary transfer

Presence and Foxp3 expression of residual TCR75 CD4+ T cells in long-term tolerant mice

Above analysis suggested that the residual TCR75 CD4+ T cells in the DST+αCD40L hosts appear to be functionally intact. To provide direct evidence, we conducted the secondary transfer exper-

In the adoptive transfer model using OT-II T cells, transplantation tolerance induced by DST+αCD40L correlated with the induction of Foxp3-expressing Tregs [27]. Having established that B6

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Figure 2. Residual TCR75 CD4+ T cells in DST+αCD40L-treated mice are able to upregulate CD44 and produce IL-2. T-cell adoptive transfer, DST and MR1 treatment were conducted as described in Figure 1C. (A) Gated TCR75 CD4+ T cells were analyzed for CFSE dilution and CD44 expression by flow cytometry. Numbers show percentages of donor TCR75 T cells in the axillary LN of total number of leukocytes. (B, C) The quantitation of CD44high TCR75 CD4+ T cells is represented as both percentage (B) and absolute cell number (C). Data were presented as mean ± SEM pooled from two independent experiments (n = 4), and analyzed by two-tailed Student’s t-test. (D) Gated TCR75 CD4+ T cells were also analyzed for CFSE dilution and intracellular IL-2 production by flow cytometry. Numbers show percentages of donor TCR75 T cells in the axillary LN of total number of leukocytes. (E, F) The quantification of IL-2-producing TCR75 CD4+ T cells is represented as both percentage (E) and absolute cell number (F). Data were presented as mean ± SEM pooled from two independent experiments (n = 4), and analyzed by two-tailed Student’s t-test.

recipients of TCR75 CD4+ T cells can be successfully rendered tolerant to B6. Kd skin grafts following DST+αCD40L treatment (Fig 1.B), we next evaluated Foxp3 expression by TCR75 CD4+ T cells in graft-draining axillary LN (dLN) in long-term tolerant mice, which had accepted grafts for approximately 100 days. DSTtreated mice, which had rejected B6. Kd skin grafts approximately 90 days previously were used as controls (Fig. 4A). We found that the frequency of Foxp3-expressing TCR75 CD4+ T cells in the dLN of tolerant mice was not significantly greater than those in control mice (Fig. 4B and C). Therefore, tolerance did not seem to be associated with peripheral Treg conversion of TCR75 CD4+ T cells. Residual TCR75 CD4+ T cells in the spleen (SP) of tolerant mice were present at a very low percentage (Supporting Information Fig. 3A), they were able to produce IFNγ although the frequency of IFNγ-producing TCR75 CD4+ T cells appeared to be modestly reduced compared with those in control mice (Supporting Information Fig. 3B). On the other hand, IL10-producing TCR75 CD4+ T cells were not detectable in either of the groups (Supporting Information Fig. 3C), indicating that TCR75 CD4+ T cells were not converted to IL10-producing regulatory T cells type 1 (Tr1) under costimulation blockade. In tolerant mice, TCR75 CD4+ T cells were present at a very low frequency in graft-draining LNs, but were barely detectable  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

within skin grafts (Supporting Information Fig. 4), indicating that residual TCR75 CD4+ T cells either did not migrate to grafts or failed to expand/survive within grafts. In contrast, within the same dLN and graft tissues tested, endogenous CD4+ T cells (i.e., of host origin) including both conventional and Foxp3-expressing Tregs were readily detectable (Fig. 4D). More importantly, the proportion of endogenous CD4+ Foxp3+ T cells in the tolerated graft tissues was higher than that in graft-draining LNs (Fig. 4E). This established local immunoregulatory network could prevent residual TCR75 CD4+ T cells from migrating into graft tissues or/and impair their accumulation.

Foxp3 expression in graft-infiltrated TCR75 CD4+ T cells during the early stage of tolerance induction The absence of TCR75 CD4+ T cells in tolerated skin grafts could be due to the inability to migrate to the grafted tissues. To test this possibility, we next analyzed the presence of TCR75 CD4+ T cells and their Foxp3 expression within the skin grafts during the early stages of tolerance induction, as illustrated in Figure 5A. Ten days after transplantation, we were able to detect the graftinfiltrated TCR75 CD4+ T cells in the DST+αCD40L-treated mice, however their frequency (3%) was approximately threefold lower www.eji-journal.eu

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Immunomodulation

Figure 3. Residual TCR75 CD4+ T cells recovered from the DST+αCD40L-treated mice were functional competent. (A) Secondary transfer. TCR75 CD4+ T cells reisolated from the DST or DST+MR1 groups on day 15 or day 20 were adoptively transferred into Rag1−/− B6 mice, which had been transplanted with B6. Kd skin grafts previously. (B) Graft survival in the B6 Rag−/− recipients of TCR75 CD4+ T cells reisolated from DST-treated mice (n = 6, black circles), from DST+MR1-treated mice (n = 6, black squares) and no T-cell transfer (n = 6, black triangles). Data were pooled from two independent experiments and analyzed by long-rank test (NS between DST and DST+MR1, and p < 0.001 between no T-cell transfer and other two groups). (C) Alternative secondary transfer that involved the transfer of TCR75 CD4+ T cells (Thy1.1+ve Thy1.2−ve ) into WT Thy1.2 B6 mice, which were previously treated with depleting anti-Thy1.2 mAb and subsequently transplanted with a B6. Kd skin graft. (D) Graft survival in recipients of TCR75 CD4+ T cells reisolated from DST-treated mice (n = 4, black circles), from the DST+MR1-treated mice (n = 5, black squares) and no T-cell transfer (n = 6, black triangles). Data were pooled from two independent experiments and analyzed by long-rank test (NS between DST and DST+MR1, and p < 0.001 between no T-cell transfer and other two groups).

than in the DST-treated group (9%) (Fig. 5B and C). These differences were recaptured when a similar analysis was conducted in the graft-draining LNs (not shown). Thus TCR75 CD4+ T cells were able to migrate into skin grafts in the early phase of tolerance induction. However, neither graft-infiltrated TCR75 CD4+ T cells from the DST+αCD40L nor those from the DST group were positive for Foxp3 (Fig. 5D and E), suggesting that TCR75 CD4+ T cells were not induced to express Foxp3 after migrating to the graft tissues.

Endogenous Treg cells cause abortive expansion of TCR75 CD4+ T cells induced by DST+αCD40L The in vivo depletion (by anti-CD25) or inactivation (by antiGITR) of endogenous Tregs abrogates the tolerogenic effects of DST+αCD40L on adoptively transferred T cells [24]. However, the interpretation of the above data is complicated by the fact that neither anti-CD25 nor anti-GITR treatment is Treg specific. To overcome these limitations, we have taken advantage of Foxp3DTR mice in which Foxp3-expressing Tregs can be selectively and efficiently removed in vivo following diphtheria toxin (DT) administration [37]. To this end, Rag1−/− B6 mice, were reconstituted with Foxp3DTR SP cells. T-cell adoptive transfer, DST, αCD40L treatment, and DT administration were executed as indicated in Figure 6A. When intact endogenous Tregs were present, TCR75 CD4+ T cells failed to expand in the DST+αCD40L-treated mice (Fig. 6B), as shown earlier (Fig. 1F). However, when endogenous Tregs were absent, TCR75 CD4+ T cells expanded equally in the DST and the DST+αCD40L groups (Fig. 6C). Thus, we conclude that endogenous Tregs play a critical role in the regulation  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

of expansion of TCR75 CD4+ T cells in the presence of CD154 blockade.

Residual TCR75 CD4+ T cells in DST+αCD40L-treated mice are not anergic to TCR stimulation in vitro PMA and ionomyin stimulation had been so far used to assess the function of residual TCR75 CD4+ T cells, raising the question whether these T cells would be responsive to TCR-mediated activation signals. We have taken two different approaches to address this point. The first approach was to test whether residual TCR75 CD4+ T cells would be able to undergo proliferation in response to TCR engagement. To this end, we compared the expansion of TCR75 CD4+ T cells derived from either DST or DST+αCD40L treated groups, after in vitro stimulation with splenic CD11c+ (DC) cells generated from B6.Kd mice. First, we demonstrated that the frequency of TCR75 CD4+ T cells in PBL of the DST group was significantly higher than in PBL of DST+αCD40L group, thus confirming the previous analysis with peripheral LN (Fig. 1D and E). After 5 days restimulation in vitro with B6.Kd DC cells, the percentages of TCR75 CD4+ T cells from the DST as well as the DST+αCD40L group were increased significantly, compared with those prior to the culture, indicating that they underwent extensive expansion. When the fold of expansion as a measure of the degree of proliferation was compared between these two groups, we found no significant differences (Fig. 7D). These results demonstrated that the residual TCR75 CD4+ T cells in the DST+αCD40L group, like those in the DST group, were www.eji-journal.eu

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competent in recognizing the cognate peptide/MHC complexes on the surface of B6.Kd DC. In the second approach, we used Kd 54–68 peptide and B6 BMDC (bone marrow-derived DC) instead of PMA and ionomycin to stimulate TCR75 CD4 T cells to produce cytokines. The T-cell adoptive transfer, DST and clone for anti-CD40L (MR1) treatment were performed as illustrated in Figure 1C. In terms of the frequency of TNFα-producing TCR75 CD4+ T cells, there was not a significant difference between the two groups (left panel of Supporting Information Fig. 5). However, MR1 treatment significantly reduced the absolute numbers of TNFα-producing TCR75 CD4+ T cells (right panel of Supporting Information Fig. 5). Thus the same conclusion was made the use of peptide and APC stimulation, although the use of PMA and ionomycin was able to trigger greater cytokine production.

Discussion The first observation was that residual TCR75 CD4+ T cells, which survived in the DST+αCD40L-treated mice, appeared to be functionally competent. This conclusion is supported by the secondary adoptive transfer experiments (Fig. 3). In addition, residual TCR75 CD4+ T cells in the DST+αCD40L-treated mice, like those in the DST-treated mice, were able to produce cytokines, upregulate activation markers, and undergo cell division (Fig. 2, Supporting Information Figs. 1 and 2). Therefore, residual TCR75 CD4+ T cells, which have survived after DST+αCD40L administration, cannot be regarded as anergic [22–24]. As reported recently [38], we showed that MR1 alone cannot induce tolerance to Kd skin grafts in B6 recipients of TCR75 T cells. These observations led us to focus on the comparison between DST versus DST+MR1 group exclusively. There are indeed other combinational therapies with MR1 that can result in allospecific skin tolerance without DST. For example, thymectomy coupled with CD8 depletion, or by combining MR1 with a nonlytic antiCD8 mAb in the euthymic CBA mice results in long-term alloskin survival [39]. Although MR1 alone cannot induce tolerance to Kd skin grafts in B6 recipients of TCR75 cells, it remains to be determined whether MR1 without DST could result in an abortive expansion of TCR75 cells. It is still controversial over whether MR1 treatment



Figure 4. TCR75 CD4+ T cells in tolerance mice were not converted to Foxp3-expressing Treg cells. (A) Experiment design. (B) Representative FACS dot plots show Foxp3 expression by gated TCR75 CD4+ T cells in the graft-draining axillary LN (dLN) cells from the DST (rejected) and the DST+MR1 (tolerant) groups. (C) Quantification of proportion of Foxp3-expressing TCR75 CD4+ T cells in dLN between the DST (n = 8, black circles) and the DST+MR1 group (n = 6, black squares). Data were presented as mean ± SEM pooled from three independent experiments, and analyzed by two-tailed Student’s t-test. (D, E) Representative FACS dot plots and quantitation of Foxp3 expression by CD4+ T cells of host origin (Thy1.1−ve CD4+ve ) in the graft (open circles) and draining axillary LN (dLN) (open squares) from the DST+MR1 (tolerant) groups. Data were presented as mean ± SEM pooled from three independent experiments (n = 6), and analyzed by two-tailed Student’s t-test.  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 6. The role of endogenous Treg cells in regulation of abortive expansion of TCR75 CD4+ T cells induced by DST+αCD40L. (A) Experiment design. The reconstitution and DT treatment were detailed in the Materials and methods. (B, C) On day 6, both untreated (B) and DTtreated mice (C) were analyzed for the representation of TCR75 CD4+ T cells in axillary LN in DST (n = 5) or DST+MR1 groups (n = 8–10). Data were presented as mean ± SEM pooled from two independent experiments and analyzed by two-tailed Student’s t-test.

Figure 5. Graft-infiltrated TCR75 CD4+ T cells were not induced to express Foxp3 in DST+αCD40L-tretaed mice 10 days posttransplantation. (A) Experiment design. (B, C) Representative FACS dot plots and the quantification of graft-infiltrated TCR75 CD4+ T cells in the DST (n = 3) and the DST+MR1 groups (n = 4). Data were presented as mean ± SEM and analyzed by two-tailed Student’s t-test. One representative experiment of two is shown. (D, E) Representative FACS dot plots and the quantification of Foxp3 expression by graft-infiltrated TCR75 CD4+ T cells in the DST (n = 3) and the DST+MR1 groups (n = 4). Data were presented as mean ± SEM and analyzed by two-tailed Student’s t-test. One representative experiment of two is shown.

alone would impact the activation of graft-reactive CD4 T cells in vivo [22, 40]. The second observation is that na¨ıve TCR75 CD4+ T cells are not converted to Foxp3-expressing Tregs in either graft-draining LNs or grafts following DST+αCD40L treatment (Fig. 4 and 5). Although this conclusion is in line with one report [40], it seems to be in conflict with others [27, 36]. We offer the following inter C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

pretations of the disparities. First, there is a huge gap in timing of the analysis. We conducted Foxp3 analysis at both 10 and 100 days posttransplantation whereas others performed this at 7 and 14 days only [27, 36]. It has been indicated that expression of Foxp3 could be transient and simply represent a phase of T-cell activation, and it does not necessarily indicate a regulatory property [41]. Second, there are model-dependent differences including skin versus heart allografts, and low versus high doses of allospecific T cells infused [36]. In addition, Kd , as an MHC class I molecule, significantly differs from membrane-bound OVA [27]. Finally, a difference in TCR affinity and avidity between TCR75, Tea, and OT-II CD4+ T cells may also contribute to the apparent differences in the findings. In vitro na¨ıve TCR75 CD4+ T cells are readily converted to Foxp3-expressing Tregs following stimulation with cognate peptide plus TGFβ [29], suggesting that they have no intrinsic defect in the induction of Foxp3. It remains to be determined if other www.eji-journal.eu

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Figure 7. Residual TCR75 CD4+ T cells from DST+αCD40L-treated mice are not anergic in response to antigenic restimulation in vitro. (A) Experimental protocol. (B, C) Comparison of the proportion of TCR75 CD4+ T cells in the PBL before and after culture with B6.Kd DC. The PBL from DST (n = 4) or DST+MR1 group (n = 9) and cultured cells were stained with antiVβ8.3FITC , anti-CD4PE , and anti-Thy1.1PerCP and the percentage of TCR75 CD4+ T cells was determined by CD4+ Thy1.1+ Vβ8.3+ cells by flow cytometry. (D) Comparison of the fold of expansion of TCR75 CD4+ T cells between DST and DST+MR1 group. The fold of expansion was determined by the frequency after culture divided by the frequency before culture. (B–D) Data were presented as mean ± SEM pooled from two independent experiments and analyzed by two-tailed Student’s t-test.

powerful tolerance-inducing agents could induce Foxp3 in TCR75 CD4+ T cells in vivo. Analysis of graft-infiltrated CD4+ T cells and their Foxp3 expression (both host and adoptively transferred) (Fig. 4 and 5) is another special feature of this study. Although residual TCR75 CD4+ T cells were still present in the draining LN of long-term tolerant mice, they were completely absent in skin grafts, which had been accepted for 100 days following the DST+αCD40L treatment (Fig. 4). Given TCR75 CD4+ T cells are present in recently transplanted skin grafts (Fig. 5), we conclude that although TCR75 CD4+ T cells can migrate to antigen-expressing skin grafts, they are unable to survive for a long term. If residual TCR75 CD4+ T cells are functionally active and not converted to Tregs by DST+αCD40L, why do they fail to reject grafts in the initial B6 recipients? We speculate that their activity is under the control of antigen-specific Tregs of host origin induced by DST+αCD40L. Peripheral generation of antigenspecific Foxp3+ Tregs is the principal mechanism for transplantation tolerance induced by the DST+αCD40L treatment [26, 34]. We noticed that within tolerant grafts the ratio of Treg:Teff of host origin (20:80) (Fig. 4D) was greater than that in graft-draining LNs (10:90), indicating that a tolerance-biased microenvironment is established within the graft. The lack of TCR75 CD4+ T cells within accepted grafts could be due to the presence of graftinfiltrated host Tregs, which could impair their persistence. In this regard, a significant reduction of graft-infiltrating TEα CD4+ T cells after the DST+αCD40L therapy was also observed by others [24]. The discrepancies regarding induction of Treg cells in different experiments models involving CD154 blockade is an important

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issue. With nondepleting anti-CD4, it was shown that a circulating source of antigen the resulting tolerance relies on Foxp3independent mechanisms [42], something that is different with a solid transplant [43]. These observations raise a possibility that the nature of antigen influence the selection of tolerance mechanisms. The final observation was that the DST+αCD40L treatment resulted in an abortive expansion of TCR75 CD4+ T cells, a process that appears to be required the presence of endogenous Foxp3+ Tregs (Fig. 6). This was because diminished accumulation of TCR75 CD4+ T cells in the DST+αCD40L group was completely prevented following DT-mediated removal of Foxp3DTR expressing Tregs. However, data interpretation is complicated by the fact that DT-treatment affects the number of TCR75 cells in both DST-treated and DST+MR1-treated mice (Fig. 6B). It is possible that the difference in cell numbers between the groups is lost when both T-cell populations increase after Treg depletion and that this result simply mean that a plateau was reached.

Materials and methods Mice, culture media, reagents, and mAbs Thy1.2 B6, Rag−/− B6, TCR75 (Rag1−/− TCR-transgenic specific for I-Ab /H2-Kd 54–68 epitope) [28], and B6.Kd mice (H2-Kd transgenic on B6 background) [44] were bred and maintained under the SPF condition in the Biological Services Unit of the KCL. The SP cells of Foxp3DTR mice [37] were provided by Dr. R Boyton

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(the ICL). The H2-Kd 54–68 peptide was purchased from Proimmune (Oxford, UK). RPMI 1640 supplemented with FCS (10%), HEPES (10 mM), penicillin/streptomycin (100 μg/mL), 2-ME (5 × 10−5 M), and L-glutamine (2 mM) was used as culture medium. PMA, ionomycin, brefeldin A, and DT were purchased from SigmaAldrich. CFSE was provided by Molecular Probes. All FACS mAbs were purchased from BD Biosciences or eBiosciences. Anti-CD40L (MR1)[45] and anti-Thy1.2 mAb (30H12) were purchased from Bio X Cell (West Lebanon, NH).

Immunomodulation

CD4+ T cells were sorted on a BD FACS Aria II and then adoptive transferred to Rag1−/− B6 mice (25 × 103 /mouse), which had been transplanted with B6. Kd skin grafts 28 days previously. Alternatively, Thy1.2 B6 mice were i.p. injected with 30H12 mAb (0.5 mg/mouse, on day −1, 0, 1, 7, and 14) before receiving reisolated TCR75 CD4+ T cells on day 0 and B6.Kd skin grafts on day 1.

Foxp3 expression by residual TCR75 CD4+ T cells Tolerance induction and skin transplantation To induce tolerance to B6.Kd skin grafts, B6 mice received a single DST (by i.v. injection of 25 × 106 B6.Kd SP cells) and they were also given three i. p. MR1 mAb injections. Skin grafting was conducted as previously described [31–33].

Both graft tissues and graft-draining LN cells of long-term tolerant and rejected mice were stained with anti-Vβ8.3PE , antiThy1.1PerCP , and anti-CD4APC , fixed and permeabilized, and then stained with anti-Foxp3FITC . Skin graft-infiltrated TCR75 CD4+ T cells and host’s endogenous CD4+ T cells were identified as CD4+ Thy1.1+ Vβ8.3+ or CD4+ Thy1.1− , respectively, were further analyzed for the expression of Foxp3.

Adoptive transfer of TCR75 CD4+ T cells After RBC lysis and FcR blockade, TCR75 SP and LN cells were staining with anti-CD62LFITC , anti-Vβ8.3PE , anti-CD4PerCP , and anti-CD44APC . FACS sorting for na¨ıve TCR75 CD4+ T cells was conducted on BD-FACS-Aria II. A total of 1 × 105 na¨ıve TCR75 CD4+ T cells were adoptively transferred to Thy1.2 B6 mice. In some experiments, CFSE-labeled TCR75CD4+ T cells were used.

Tracking of TCR75 CD4+ T cells in vivo Thy1.2 B6 recipients of TCR75 CD4+CFSE+ T cells were given a single DST without or with αCD40L treatment. Axillary LN cells of DST or DST+αCD40L group were stained with anti-Vβ8.3PE , antiThy1.1PerCP , and anti-CD44APC . The absolute number of TCR75 CD4+ T cells was determined by multiplying the percentage of Thy1.1+ Vβ8.3+ T cells by the total number of cells.

Intracellular cytokine staining This was performed as described previously [31–33]. In brief, LN cells (1 × 106 /well) were stimulated with 50 ng/mL PMA and 500 ng/mL ionomycin in the presence of Brefeldin A (5 μg/mL) for 4 h. The cells were stained with anti-Vβ8.3PE and anti-Thy1.1PerCP , fixed and permeabilized and then stained with anti-IL-2APC . The absolute number of IL-2-producing TCR75 CD4+ T cells was determined by multiplying the percentage of IL-2+ Thy1.1+ Vβ8.3+ T cells by the total number of LN cells.

The secondary adoptive transfer of TCR75 CD4+ T cells Thy1.2 B6 recipients of TCR75 CD4+ T cells were given DST without or with MR1 mAbs. On day 15–20, pooled LN and SP cells were stained with anti-CD4FITC , anti-Vβ8.3PE , anti-Thy1.1PerCP . TCR75  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

In vivo depletion of Foxp3+ Treg cells before adoptive transfer Rag1−/− B6 mice reconstituted with Foxp3DTR SP cells were untreated or treated with a standard dose of DT. After Treg depletion, TCR75 CD4+ T cells were adoptively transferred to recipients, which were then given a single DST without or with MR1 injections. The mice were euthanized for the analysis of the representation of TCR75 CD4+ T cells in axially LN.

Statistical analysis Graft survival data were analyzed using the Kaplan–Meier method, with the Wilcoxon rank test and the log-rank test used to verify the significance of difference between the groups (GraphPad Prism). Statistical analysis of other data was performed using the twotailed Student’s t-test for unpaired samples with unequal variance.

Acknowledgments: This work was supported by the British Heart Foundation.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

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Abbreviations: DST: donor-specific transfusion · TCR75: TCR-transgenic for H2-Ab/H2-Kd54–68 epitope · MR1: clone for anti-CD40L · SP: spleen · DT: diphtheria toxin

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Full correspondence: Dr. Giovanna Lombardi, Department of Nephrology and Transplantation, King’s College London, 5th Floor Thomas Guy House, Guy’s Hospital Campus, London SE1 9RT, UK e-mail: [email protected] Additional correspondence: Dr. Jian-Guo Chai, Therapeutic Immunology Group, The Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford Ox1 3RE, UK e-mail: [email protected]

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See accompanying Commentary: http://dx.doi.org/10.1002/eji.201545762 Received: 7/1/2015 Revised: 12/3/2015 Accepted: 30/4/2015 Accepted article online: 6/5/2015

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