Transplant Immunology 24 (2011) 119–126

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Transplant Immunology

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Adoptive transfer of DNT cells induces long-term cardiac allograft survival andaugments recipient CD4+Foxp3+ Treg cell accumulation

Zhu-Xu Zhang a,b,c,⁎, Dameng Lian a,c, Xuyan Huang a,c, Shuang Wang a,c, Hongtao Sun a,c, Weihua Liu a,b,c,Bertha Garcia a,b,c, Wei-Ping Min a,b,c, Anthony M. Jevnikar a,b,c,⁎a The Multi-Organ Transplant Program, London Health Sciences Centre, London, Ontario, Canadab Departments of Medicine, Microbiology & Immunology, and Pathology, University of Western Ontario, London, Ontario, Canadac Lawson Health Research Institute, London, Ontario, Canada

⁎ Corresponding authors. Department of Medicine, ULondon Health Sciences Centre, C9-116, 339 WinderCanada, N6A 5A5. Tel.: +1 519 685 8500x32945; fax: +

E-mail addresses: zhuxu.zhang@lhsc.on.ca (Z.-X. Zha(A.M. Jevnikar).

0966-3274/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.trim.2010.11.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 August 2010Received in revised form 3 November 2010Accepted 4 November 2010

Keywords:DNT cellTreg cellsTransplantationHeartRapamycin

Regulatory T (Treg) cells play an important role in the regulation of immune responses but whether Treg willinduce tolerance in transplant recipients in the clinic remains unknown. Our previous studies have shownthat TCRαβ+CD3+CD4−CD8−NK1.1− (double negative, DN) T cells suppress T cell responses and prolongallograft survival in a single locus MHC-mismatched mouse model. In this study, we investigated the role ofDNT cells in a more robust, fully MHC-mismatched BALB/c to C57BL/6 transplantation model, which may bemore clinically relevant. Adoptive transfer of DNT cells in combination with short-term rapamycin treatment(days 1–9) induced long-term heart allograft survival (101±31 vs. 39±13 days rapamycin alone, pb0.01).Furthermore adoptive transfer DNT cells augmented CD4+Foxp3+ Treg cells accumulation in transplantrecipients while depletion of CD4+ Treg cells by anti-CD25 inhibited the effect of DNT cells on long-term graftsurvival (48±12 days vs. 101±31 days, pb0.001). In conclusion, DNT cells combined with short-termimmunosuppression can prolong allograft survival, which may be through the accumulation of CD4+Foxp3+

Treg cells in the recipient. Our result suggests that allograft tolerancemay require the co-existence of differenttype Treg cell phenotypes which are affected by current immunosuppression.

niversity of Western Ontario,mere Road, London, Ontario,1 519 663 3295.ng), jevnikar@uwo.ca

l rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Donor organ tolerance refers ideally to a state of permanent graftacceptance in the absence of long-term immune suppression. Variousapproaches to induce long-term graft survival and tolerance exist,including immunosuppressive drug treatment, induction of mixedchimerism by bone marrow transplantation, T cell depletion, T cellsignal blockade, and induction of Treg cells [1–6]. Current studies onTreg cells, particularly CD4+Foxp3+ Treg cells, strongly suggest thattransplant tolerance might be achievable via the manipulation of Tregcells [3,6–9]. In recent studies, we have reported that TCRαβ+CD3+

CD4−CD8− NK1.1− (double negative, DN)-Treg cells possess immuneregulatory functions and play an important role in the development ofpost-transplant tolerance [10–14]. Mouse DNT cells specifically caneliminate activated syngeneic anti-donor CD4+ T cells or CD8+ T cellsin allogeneic [10,11,13,15] as well as xenogeneic graft transplantation[16,17]. Furthermore, transfusion of DNT cells leads to significant

prolongation of immunogenic donor-specific skin and heart allograftsin a single MHC-mismatched mouse model [10,11,18]. In support ofour findings, others have reported that DNT cells can down-regulateCD8+ T cell-mediated immune responses in both autoimmune andinfectious disease models [19,20]. Interestingly, CD4+ T cell-convertedDNT cells are highly potent in suppressing alloimmunity both in vitroand in vivo in an antigen-specific manner and adoptively transferredcells can prolong islet graft survival [21]. Collectively, these findingsindicate that DNT cells can down-regulate immune responses both toself and to foreign antigens and importantly, that there is heterogeneityin the control of alloimmune responses by regulatory T cell phenotypes.DNT cells derived from humans, possess similar immune regulatoryfunctions as those we demonstrated, increasing the clinical relevance ofthese potent regulatory cells in patients [22].

Although DNT cells can induce long-term graft survival in a singleMHC-mismatched model [10,11,18], the capacity of DNT cells to inducelong-term graft survival and tolerance in a fully MHC-mismatchedheart transplantation model has not been tested. The potentialinteraction of DNT cells with CD4+Foxp3+ Treg cells has also notbeen studied as well. In this study, we have found that DNT cells incombination with short-term immunosuppression can induce long-term heart graft survival after BALB/c-to-C57BL/6 heart transplantationand that this is associated with enhanced CD4+Foxp3+ Treg cellaccumulation in vivo.

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2. Materials and methods

2.1. Animals

C57BL/6 (B6, H-2b), and BALB/c (H-2d), mice were purchased fromJackson Laboratories (Bar Harbor, ME) and Charles River Laboratories(Wilmington, MA). The animals weremaintained in the animal facilityat the University of Western Ontario using approved protocols andprocedures.

2.2. Antibodies and reagents

At various time points after activation, DNT cells were charac-terized with fluorescent-conjugated monoclonal antibodies thatspecifically recognize β-TCR, CD3, CD4, CD8, CD25, CD28, CD45R/B220, NK1.1, anti-CD25, and γδ-TCR (eBioscience, San Diego, CA).Data were acquired and analyzed on a Cytomics FC500 flow cytometer(Beckman Coulter, Fullerton, CA).

2.3. Generation of bone marrow-derived DCs as stimulators

Dendritic cells (DCs) were generated from bonemarrow progenitorcells. Briefly, bonemarrow cellswere flushed from the femurs and tibiasof BALB/c mice, and then washed and cultured in 24-well plates(2×106cells/ml) in 2 ml of complete medium: RPMI-1640 supple-mented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg strepto-mycin, 50 μM2-mercaptoethanol, and 10% foetal calf serum (InvitrogenCanada: Life Technologies, Burlington, Ontario, Canada), supplementedwith recombinant GM-CSF, 10 ng/ml (Peprotech, Rocky Hill, NJ) andrecombinant mouse IL-4, 10 ng/ml (Peprotech). All cultures wereincubated at 37 °C in 5% humidified CO2 for 8 days. Non-adherentgranulocyteswere removed after 48 h of culture, and freshmediumwasadded.

2.4. Isolation of DNT cells and CD4+CD25+ Treg cells

Spleen and lymph node cells, obtained from B6 mice, were treatedwith anti-CD4 and anti-CD8 MACS beads (Miltenyi Biotec, Auburn,CA) to deplete CD4+ and CD8+ T cells. The remaining cells wereadded to anti-CD90 (Thy-1)-coated MACS beads (Miltenyi Biotec) topurify CD4−CD8− T cells. Purified B6 DNT cells were stimulated withBALB/c DCs or anti-CD3-dynale beads (Invitrogen) in RPMI-1640,supplemented with 10% FCS, penicillin (100 unit/ml), streptomycin(100 μg/ml), glutamin (2 mM), sodium pyruvate (1 mM), HEPES(10 mM), β-mercaptoethanol (0.5 mM), and 100 IU/ml IL-2 for 3–4 days. Viability and purity of the cells were assessed by flowcytometry. Any contaminating CD4+, CD8+ and NK1.1+ T cells wereremoved by MACS beads (Miltenyi Biotec) following activation. Thefinal purity of DNT cells used prior to adoptive transfer was N90% asdetermined by TCRβ+TCRγδ−CD3+CD4−CD8−NK1.1− expression inflow cytometry assays. Approximately 7–14×105 DNT cells (permouse) were obtained from this procedure. DNT cell do not expressFoxp3 gene before or after activation by PCR analysis (data notshown).

CD4+CD25+ Treg cells were purified from C57BL/6 spleens byusing MACS beads (Miltenyi Biotec) and purity was analyzed by anti-CD4, anti-CD25 and anti-Foxp3 in intracellular staining (eBioscience).

2.5. Suppression assays

DNT, CD4+ CD25+ Treg, and CD8+ cells were isolated as describedabove. DNT cells or CD4+Foxp3+Treg cells, used as putative suppressorcells, were plated in serial dilutions in 96-well, round-bottom plateseither alone or in the presence of T cell responders. Cultureswith only Tcell responders were used as controls for maximum proliferation.Each well was given irradiated (100 Gy) allogeneic BALB/c splenocytes

(H-2d) as stimulators. Cells were incubated at 37 °C in 5% CO2 for3.5 days before receiving 1 μCi H3-Thymidine, and 18 h later, cells wereharvested and counted in a Topcount® beta scintillation counter(Packard Instrument Company, Meriden, CT).

2.6. Heart transplantation and treatment

Intra-abdominal heterotopic BALB/c (H-2d)-to-C57BL/6 (H-2b)cardiac transplants were performed in our microsurgery laboratory,in accordance with an approved protocol. Briefly, donor hearts wereprocured through a butterfly thoracic incision. All major vessels wereligated, except the pulmonary artery and aorta, which were sharplytransected. The ascending aorta and pulmonary artery was anasto-mosed ‘end-to-side’ to the recipient, by way of the infrarenalabdominal aorta and inferior vena cava. Pulsation of the heart graftwas monitored daily. Cessation of beating was defined as the endpoint of rejection.

Recipientmicewere treatedwith the immunosuppressive rapamycin(Rapa, L C Laboratories, Woburn, MA) at 2 mg/kg i.p. from day 1 topostoperative day 9, according to a previously published protocol [23].DNT cells, obtained from C57Bl/6 mice as described above, wereintravenously transfused into each transplanted mouse on day 11.Some mice received anti-CD25 (clone PC61. 0.5 mg/mouse i.p.) orsubtype control rat IgG1onday-3 andday-1beforeheart transplantation.

2.7. Histology and immunohistochemistry

Formalin-fixed heart graft sections were stainedwith H&E. Criteriafor rejection included necrosis and immune cell infiltration. Forinfiltrating cell analysis, either formalin-fixed or snap-frozen sectionswere fixed in acetone and stained with antibody using a standardprotocol. All slides were stained by a streptavidin–biotin immunoper-oxidase and a substrate method as described in a standard protocol.

2.8. Statistical analysis

Data were compared using Student's t-test for unpaired values andone-way ANOVA formulti-group differences unpaired values. P valuesless than 0.05 were considered to be significantly different.

3. Results

3.1. Adoptive transfer of DNT cells with short-term RAPA treatment induces long-termheart allograft survival

In previous studies, we found that DNT cells could suppress anti-donor T cellresponses and prevent graft rejection in a single MHC-mismatched transplantationmodel [10,11,18]. Preventing heart graft rejection using DNT cells in a fully MHC-mismatched model had not been previously tested. Hence, we studied the role of DNTin a stringent fully mismatched rejection model, BALB/c-to-C57BL/6 heart transplan-tation. DNT cells enriched from C57BL/6 mice were activated for 3–4 days before finalpurification and adoptive transfer. DNT cells were analyzed by flow cytometry andwere βTCR+γδTCR− CD3+CD4−CD8−CD25+CD45R+NK1.1− (Fig. 1).

Before the treatment of DNT cells, recipientswere given a short course of RAPA (2 mg/kg) from day 1 to day 9 after BALB/c heart transplantation. A short course use of RAPA canprevent acute immune rejectionbut doesnot induce long-termgraft survival [23]. C57BL/6DNT cells (2 or 10×106) were prepared as described in the methods and transferred onday 10 or 11. Heart graft survival results are shown in Fig. 2a. Treatment with RAPA aloneslightly prolonged vascular graft survival (mean survival in days=39±13 vs. 10±1 daysin non-treated mice, Pb0.01). In contrast, 107 DNT cells along with RAPA treatment,further prolonged survival (101±31 days vs. 39±13 days in RAPA alone treatment,pb0.01).

To determine the effect of DNT cells at early time points after transplantation, fouradditional mice that received RAPA and DNT cell treatment were sacrificed on day 40for histopathology. Representative results of a BALB/c heart in C57BL/6 mouse areshown in Fig. 2b demonstrating massive cell infiltration and tissue damage in therejecting grafts. In contrast, mice that were treated with RAPA plus DNT cells had muchfewer graft-infiltrating cells on day 40 than RAPA alone (Fig. 2b). Mice with RAPA onlytreatment and sacrificed on day 40 were found to have massive graft infiltration withCD8+ T cells, which was substantially reduced in DNT/RAPA treated recipients (Fig. 2c).In summary, adoptive transfer of DNT cells in combination of short term of RAPA

Fig. 1. Characterization of DNT cells. DNT cells were purified from C57BL/6mice activated as described in theMethods. DNT cells were stained by using antibodies to β-TCR, CD3, CD4,CD8, CD25, CD45R/B220, NK1.1, anti-CD25, and γδ-TCR (eBioscience, San Diego, CA) then analyzed by flow cytometry (Cytomics FC500, Beckman Coulter, Fullerton, CA).

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induced long-term heart graft survival and inhibited anti-donor responses at earlystage of transplantation.

3.2. DNT cells augment CD4+Foxp3+ Treg accumulation in transplant recipients, but donot alter their function

CD4+ Treg cells have been shown to play an important role in induction andmaintaining transplantation tolerance in a broad range of studies. Following ourobservation that DNT cells could induce long-term heart allograft survival, we testedwhether CD4+Foxp3+ Treg cells were affected by the adoptive transfer of DNT cells as apotential mechanism. BALB/c-to-C57BL/6 heart transplant recipients were treated withRAPA alone or RAPA combined with DNT cell transfers as described in Fig. 2. On day 40,micewere sacrificed and spleenswere analyzedbyflowcytometry after antibody staining.CD4+Foxp3+ Treg cells were significantly increased in RAPA treatment alone comparedwith naïve mice (8.9±2.3%, n=4 vs. 6.4+2.0%, n=6, Pb0.05, Fig. 3a and b).Interestingly, the percentage of CD4+Foxp3+ Treg cells was further increased in micethat received an adoptive transfer DNT cells on day 40 (12.8±2.8% n=4 vs. 8.9±2.3%,n=4, RAPA alone, Pb0.05, Fig. 3a and b). This high percentage of CD4+Foxp3+ Treg cellsin DNT cell treated mice was persistent in long-term allograft survivors that weresacrificed between day 80 and day 150 (13.6±4.1%, n=6, vs. 8.9±2.3% in RAPA alone,pb0.01). Taken together, these data suggest that DNT cells transfer can augment CD4+

Foxp3+ Treg accumulation in transplant recipient as a possible mechanism to prolongsurvival.

To determine if these CD4+Foxp3+Treg cells have a regulatory capacity, CD4+CD25+Tcells were purified from spleens of mice sacrificed on day 40 using MACS beads (MylteyniBioTec). By thismethod75–80%of these CD4+CD25+T cellswere determined to be Foxp3+

which were then used in a suppression assay to determine their function. As showed inFig. 3c, CD4+CD25+ Treg cells purified from naïvemice, RAPA alone treated mice or RAPA/DNT treated mice suppressed T cell proliferation at similar levels and in a dose-dependentmanner.

3.3. Depletion of CD4+Foxp3+ T cells inhibited DNT cells-induced long-term heart graftsurvival

Our above data indicated that DNT cells transfer induce a long-term graft survival andenhanced CD4+Foxp3+ Treg cell accumulation in transplant recipients. We then tested ifthe accumulation of CD4+Foxp3+ Treg cells byDNT cell transfer in thismodel contributedto graft survival. CD4+CD25+Foxp3+ Treg cells were depleted by two injections of anti-CD25 (Clone PC61). The capacity of two doses of anti-CD25 antibody (day-3, day-1) todeplete CD4+CD25+Foxp3+ cells prior to transplant was confirmed in non-transplanta-tion/naive mice (n=3) by intracellular staining of Foxp3 and CD4+ T cell numbers inC57BL/6 spleen. Depletion was tested 24 h after the last injection, corresponding to day 0in transplanted mice (Fig. 4a). Identical antibody treatment was applied to transplantrecipient C57BL/6 mice (day-3 and day-1) before receiving BALB/c heart transplants(day 0) and RAPA/DNT cell transfers. Depletion CD4+CD25+Foxp3+ Treg cells clearlylimited the benefit of DNT cell transfer on long-term heart graft survival and resulted in anaccelerated heart graft loss (46.6±6.7 days, n=4 vs. 101±31 days, n=12, pb0.001,Fig. 4b). In control groups, RAPA treatment alone and depletion CD4+CD25+Foxp3+

Treg cells plus RAPA treatment had a similar effect on graft survival (42±7.9, n=4 vs.39.5±3.8, n=4, pN0.05). These data suggest that CD4+Foxp3+ Treg cells may play a rolein DNT cell-induced long-term heart graft survival.

Fig. 2. Adoptive transfer of DN-Treg cells induces long-term heart graft survival. a) C57BL/6mice recipients were treated with 2 mg/kg RAPA from day 1 to day 9 after receiving BALB/c heart transplants. DNT cells (107) purified from C57B/6 mice were intravenously transferred into transplanted mice on day 11. Phosphate buffered saline or RAPA-treated alonewas used as controls (n=4–12). Heart pulsation was monitored daily and cessation of beating was defined as the end point of rejection. b). Mice were sacrificed on day 0 and 40 andheart grafts were collected for routine H&E staining and for immunohistochemistry staining with anti-CD8 (c). Representative microscopic photos from each group are depicted(n=4/each group). Positive staining areas were shown by arrows. Original magnification ×200. **:Pb0.01.

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4. Discussion

Treg cells have been consistently shown to be critical componentsin the induction and maintenance of transplantation tolerance inrodent models [3,6–9]. Adoptive transfer of Treg cells as animmunotherapy approach would be an ideal strategy for clinical

transplantation but has not been practically implemented due to ourlack of insight into the function as well as phenotypes of relevantregulatory cells in vivo. In this study, we have shown that adoptivetransfer of DNT cells, in combination with early, short-termimmunosuppression, can induce long-term heart graft survival aswell as the accumulation of functional CD4+Foxp3+ Treg cells.

Fig. 3. CD4+Foxp3+ Treg cells accumulation in transplant recipients. a) Spleen cells frommice received BALB/c heart transplantation and RAPA or DNT cells on day 40 or on day rejectionwere used for anti-CD4, anti-CD25 and anti-Foxp3 intracellular staining. b) Percentage of CD4+Foxp3+ T cells were pooled from 4–6 mice from each group. c) CD4+CD25+ T cells werepurified from each group and used for suppression assay. 2×104 CD8+ T cells were purified byMACS beads (MiltenyiBiotec) from naïve C57BL/6mice and stimulated by 5 fold irradiatedBALB/c spleen cells. Various dose of CD4+CD25+T cellswere added in96u-bottomwells and co-cultured for 3 day and [3H]-Thymidinewas added to eachwell tomeasure proliferation ofT cells. Eighteen hours later, cells were harvested and counted in a β-scintillation counter. Percentage of suppression was calculated based on CPM values: (T cells alone-mixture)/T cellsalone. The experiment has been repeated twice by using CD4+CD25+ T cells purified from two additional mice, and similar results were obtained. *: Pb0.05.

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Furthermore, the depletion of CD4+ Treg cells inhibited DNT cell-induced heart graft survival, implying that there is a cooperative rolefor different Treg phenotypes in transplant recipients.

Various approaches to induce long-term graft survival and toleranceexist, including immunosuppressive drug treatment, inductionofmixedchimerism by bone marrow transplantation, T cell depletion, T cellsignal blockade, and induction of Treg cells. In the clinical setting,immunosuppressive treatment improves graft and patient survival, buttheir continuous use has numerous side effects [24–26]. As well whileadministrationof immunosuppressive drugsmaypreventor delayacutegraft rejection, their use does not guarantee immune tolerance or long-term survival in patients. There is considerable evidence that Treg cells

play a key role in achieving and maintaining graft tolerance [1–3,27].Thus, a clinical approach for the induction of transplantation tolerancemay combine both depletion/inhibition strategies and regulatory T celltherapy [2,28]. Indeed numerous studies have combined immunosup-pressive drugs with either tolerogenic DCs or Treg cells to induce long-term graft survival [2,23,29–31]. Immunosuppressive drug treatment incombination with Treg cells or DCs has shown significant progress withinduction of transplant tolerance in murine models [23,32–35].

In addition to CD4+Foxp3+ Treg cells, other regulatory cells maycontribute to graft survival and may be affected by currentimmunosuppression. In previous studies, DNT cells have been foundto suppress anti-donor T cell responses and prevent heart and skin

Fig. 4. Depletion of naïve CD4+Foxp3+ T cells inhibits DNT cells-induced long-term heart graft survival. a) Depletion of CD4+ Treg cells was tested in three naïve C57BL/6 mice byinjection of anti-CD25 (PC61, 0.5 mg/mouse/injection) on day-3 and day-1 before harvesting spleens. Depletion of Treg cells was confirmed by anti-CD4, anti-CD25 and anti-Foxp3intracellular staining. Flow data from one of three mice was shown. Same depletion result was seen in all mice. b) Similar depletion procedure was applied in transplantation group.Anti-CD25 (0.5 mg/mouse) was i.p. injected into C57BL/6 mice 1 and 3 days before BALB/c heart transplantation. Mice were received RAPA treatment and DNT cell transfer asdescribed in Fig. 2. Heart pulsation was monitored daily and cessation of beating was defined as the end point of rejection. ***: Pb0.001.

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graft rejection in a single MHC-mismatched model [10,11,18]. In thisstudy, we examined whether DNT cells are able to prevent heart graftrejection in a fully MHC-mismatched model, a more robust represen-tation of clinical transplants. When treated with a short course ofRAPA treatment, transfer of DNT cells could indeed induce long-termheart graft survival (Fig. 2a). Our results thus support the rationale foran effective clinical approach to transplantation tolerance bycombining depletion/suppression and regulation therapy.

Although current studies strongly support the hypothesis that Tregcells play a key role in achieving and maintaining tolerance, theinteraction of different subsets of Treg cells and their contribution totolerance in similar experimental models has not been well studied.The existence of immunosuppressive CD4+, CD8+, NKT, and γδ-TCRcells has been described in various studies (reviewed in [36–38].These cells may be required to cooperate in vivo to achieve tolerance.A recent study has indicated that the effective function of CD4+ Tregcells requires sufficiently high levels of CD8+ Treg cells in anautoimmune disease mouse model [39]. Several studies have shownthat NKT cells can enhance CD4+Treg cells function and help CD4+

Treg cells expansion in various models including GVHD, autoimmune

disease, or type 1 diabetes in mouse models [40–44]. In our currentstudy, we have found that adoptive transfer of DNT cells can augmentCD4+ Treg cell accumulation in transplant recipient (Fig. 3). Toenhance long-term graft survival, our results clearly supports animportant role for both CD4+ Treg cells as well as cells that regulatetheir function or numbers. Indeed, depletion of CD4+CD25+Foxp3+

Treg cells by anti-CD25 limited the capacity of DNT cells to inducelong-term graft survival (Fig. 4). As RAPA alone only minimallyincreased the percentage of CD4+ Treg cells in the spleen (Fig. 3b),and anti-CD25 alone did not affect graft survival in the RAPA alonegroup (Fig. 4b), our results suggest that the benefit of DNT cell transferwas related to CD4+Foxp3+ Treg cells. The mechanism of expansionof CD4+ Treg as well as the location of expansion was not addressedby our studies, as expansion of CD4+ Treg may have occurred withinthe graft along with DNT cells which have been shown to increase inskin and heart transplants with prolonged survival [12,14]. Futurestudies will be required to address these mechanistic questions.Importantly however, our data support the concept that differentsubsets of Treg cells may be required to control anti-donorimmunoresponses in transplant recipients. Given the loss of effect

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using anti-CD25 antibody in the DNT transfer model, our resultshighlight the complex biology of regulation in the transplant recipientand the potential for unintended harmful effects of immunosuppres-sion as well.

Previous studies have suggested that human NKT cells do not alterCD4+ Treg cells function, but help their proliferation throughproduction of IL-2 [40]. A recent study has suggested that host NKTcells induce bone marrow donor CD4+ Treg cells expansion throughIL-4 in a GVHD model [43]. In our study, the mechanism(s) by whichadoptive transfer of DNT cells helped accumulation of CD4+Foxp3+

Treg cells in transplant recipient is unclear. We have analyzedcytokine expression by real time PCR and TGF-β, IL-2 and IL-4 werenot detected by PCR in naïve and activated DNT cells (data notshown). Previous studies have demonstrated that DNT cell them-selves also need IL-2 and IL-4 for their activation and function[10,11,45]. Therefore, other as yet unidentified factor(s) may result inthe accumulation of CD4+Foxp3+ Treg cells after DNT cells transfer.This will be of interest for further investigation.

In summary, adoptive transfer of DNT cells can induce long-termgraft survival in fully MHC-mismatched heart transplantation. Moreimportantly, our data indicate that DNT cell transfer augments theaccumulation of CD4+Foxp3+ Treg cells as a potential mechanism andthat depletion of CD4+ Treg cells by anti-CD25 antibody blocks thebenefit of DNT cells on long-term graft survival. These results furthersuggest that the development and interaction of different types ofTreg cells are required for controlling immune responses in vivo.Greater insights on the interaction of different phenotypes of Tregcells in vivo may be required to develop robust immune tolerance inthe clinic.

Funding sources

This study was supported by the Heart and Stroke Foundation ofCanada, the Canadian Institutes of Health (CIHR), the Program ofExperimental Medicine (POEM) at University of Western Ontario,London, Ontario, Canada.

Conflict-of-interest disclosure

There are no financial disclosures or conflict of interest in thisstudy.

Acknowledgement

The authors would like to thank Ms. Cate Abbott for the editorialassistance and Dr. Hao Wang for surgery arrangement.

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