Transplant Immunology 31 (2014) 125–133

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Transplant Immunology

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Hematopoietic chimerism following allotransplantation of the spleen,splenocytes or kidney in pigs

Hidetaka Hara a, Yih Jyh Lin a, Hao-Chih Tai a, Mohamed Ezzelarab a, Mubina A. Quader b, Stuart L. Houser c,Atsunori Nakao a, David K.C. Cooper a,⁎a Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USAb Department of Radiation Oncology, University of Pittsburgh Medical Center, Pittsburgh, PA, USAc Department of Pathology, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA

Abbreviations:GVHD, graft-versus-host disease;MHCplex;MLR,mixed leukocyte reaction; PBMCs, peripheral bllymerase chain reaction; Tx, transplantation;WBI,whole b⁎ Corresponding author at: Thomas E. Starzl Transplant

Biomedical Sciences Tower, Room W1543, University ofPittsburgh, PA 15261, USA. Tel.: +1 412 383 6961; fax: +

E-mail address: cooperdk@upmc.edu (D.K.C. Cooper).

http://dx.doi.org/10.1016/j.trim.2014.09.0060966-3274/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 24 July 2014Received in revised form 10 September 2014Accepted 11 September 2014Available online 20 September 2014

Keywords:Hematopoietic cell chimerismPigSplenocyteTransplantation, kidneyTransplantation, spleen

Background:Mixed chimerism is associated with donor-specific tolerance. Spleen or splenocyte allotransplanta-tion (Tx) is recognized as potentially tolerogenic. There is no definitive report comparing chimerism levelsfollowing spleen and splenocyte Tx in a large animal model. We have compared chimerism after spleen,splenocyte, or kidney Tx in pigs.Methods: Outbred (n = 5) and MHC-defined miniature (n = 1) pigs underwent orthotopic spleen Tx. Outbredpigs received splenocytes through a systemic vein (n= 1) or the portal vein (n= 3). Kidney Tx (n= 2) or con-comitant Tx of spleen + kidney (n= 2) was carried out. All except one recipient pigs were irradiated (700 cGythymic and 100–125 cGy whole body) on day− 2. Cyclosporine or tacrolimus was administered for 42 days. Alldonors were males and all recipients were females; chimerism in the blood was determined by Quantification-PCR for the donor Y chromosome. Mixed lymphocyte reaction (MLR) was performed before and after Tx.Results: One week after spleen Tx in outbred and MHC-defined pigs, chimerism ranged between 0.8 and 22.5%,

and 5.4–20.1%, respectively, and remained between 17.7 and 67.4%, and 2.2–7.4%, respectively, until day 28.One week after splenocyte Tx, chimerism ranged between 0.1 and 8.5%, and decreased to 0.1–0.8% at 3–4 weeks. There was no detectable chimerism 14 days after kidney Tx. The response on MLR of all recipientpigs to donor cells was decreased after Tx, except in one case of splenocyte Tx, indicating that this pig mighthave become sensitized. After discontinuation of immunosuppression, most isolated spleen or kidney graftswere not rejected, but the kidney was rejected after concomitant spleen + kidney Tx.Conclusions: There was a significantly higher level of blood chimerism following spleen Tx compared tosplenocyte or kidney Tx. However, concomitant Tx of spleen + kidney may be associated with acceleratedkidney graft rejection.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Immunological tolerance would eliminate the need for, and avoidcomplications of, chronic immunosuppressive drug therapy [1]. Suc-cessful spleen allotransplantation (Tx) in rodents can result in specifictolerance toward donor-matched organ allografts in the absence of ex-ogenous immunosuppressive therapy [2]. These studies indicated that,following spleen Tx, there exists a delicate balance between the donorand recipient immune responses that, if manipulated properly, canlead to a state of tolerance. The potential of a spleen to reconstitute

, major histocompatibility com-oodmononuclear cells; PCR, po-ody irradiation.ation Institute, Thomas E. StarzlPittsburgh, 200 Lothrop Street,1 412 624 1172.

the bone marrow of a lethally-irradiated animal (in the absence ofbone marrow Tx) has also been demonstrated [3], providing evidenceof its potential to result in hematopoietic cell chimerism under thecorrect circumstances.

In humans, a spleen allograft in an immunosuppressed host cancause graft-versus-host disease (GVHD) [4–7]. This observation indi-cates the spleen's potential in counteracting the host's immune re-sponse to obtain balanced immunity between donor and recipientthat may result in a chimeric and/or tolerogenic state.

Rejection of organ allografts is currently prevented by chronic non-specific immunosuppressive therapy. This therapy must balance therisks of immunosuppression (e.g., infection, lymphoproliferative dis-ease, and other drug-related complications) against those of rejection.Induction of specific Tx tolerance across complete MHC barriers wouldpermit organ Tx from deceased or living donors without the need forlong-term immunosuppression [1,8]. Since there are differences in theresponse to vascularized organ allografts between small and large

Fig. 1. Technique of orthotopic spleen Tx in the pig. (A) The spleen graft included celiacaxis to perfuse HTK solution ex vivo. (B) Orthotopic spleen Tx was carried out by end-to-end anastomoses between the donor and recipient splenic arteries and the donor andrecipient splenic veins. After reperfusion, the color of the spleen graft was similar to thatof the liver.

126 H. Hara et al. / Transplant Immunology 31 (2014) 125–133

animal species with regard to tolerance induction [9,10], if a tolerance-inducing protocol is to be practicable for human organ Tx, then it mustat least be successful in a large animal model across a full MHC barrier.FromDor's initial studies, it would seem that spleen Tx ismuch less like-ly to induce GVHD than is bonemarrow Tx [11,12]. Spleen Tx, therefore,offers potential as an approach to the induction of allograft tolerance inthe clinic.

Dor, Gollackner and their colleagues have previously reported thatheterotopic spleen Tx in miniature swine is technically feasible [13],and that, in appropriate models, it results in hematopoietic cell chime-rism and tolerance to the transplanted spleen [13]. Pigs with function-ing spleen grafts had multi-lineage chimerism in blood, thymus, andbone marrow for N6 months, without GVHD, and developed in vitrodonor-specific hyporesponsiveness. The chimerism results because thespleen in adult pigs and primates has been demonstrated to be arelatively rich source of very primitive hematopoietic progenitor cells[14]. In two pigs tolerant to the spleen graft, subsequent donor MHC-matched kidney grafts survived for N4 and N7 months in the absenceof exogenous immunosuppression. In contrast, in two asplenic pigs, kid-ney grafts were rejected on days 4 and 15 [11].

We have continued these studies by measuring the extent of hema-topoietic cell chimerism after spleen or kidney Tx in pigs. Furthermore,if spleen Tx is to play any role when organs are to be procured from adeceased donor, we have investigated the clinical outcome afterconcomitant Tx (rather than the sequential Tx) of the spleen and kidney.We have also investigated the effect on the immune system of the intra-venous (i.v.) infusion of donor splenocytes.

2. Materials and methods

2.1. Animals

Spleen, splenocyte, and kidney donors (n= 17) and recipients (n=17) were selected from either (i) outbred Landrace large white pigs(n = 24; Wally Whippo, Enron, PA), or (ii) a herd of specificpathogen-free partially-inbred, MHC-defined Yucatan miniature swine(n = 10; Sinclair, St. Louis, MO). Donors and recipients were matchedfor weight (approximately 20–25 kg). The Yucatanminiature swine do-nors and recipientswere reported to be fully-mismatched forMHC classI and class II antigens [15].

All animal care procedures were in accordancewith the Principles ofLaboratory Animal Care formulated by the National Society for MedicalResearch and the Guide for the Care and Use of Laboratory Animals pre-pared by the Institute of Laboratory Animal Resources and published bythe National Institutes of Health (NIH publication No. 86–23, revised1996). All protocolswere approved by theUniversity of Pittsburgh Insti-tutional Animal Care and Use Committee (Protocol #0409338-2).

2.2. Surgical procedures

All recipient pigs had catheters inserted into jugular veins for blooddraw and drug infusion [16]. Procurement of the spleens and kidneyswas by standard techniques [13,16]. The donor spleen was procuredwith a Carrel patch of aorta for ex vivo perfusion using histidine–tryptophan–ketoglutarate (HTK) solution (Essential Pharmaceuticals,Newtown, PA) (Fig. 1A). Heterotopic spleen Txwas carried out as previ-ously described [11,13]. In addition, in some cases orthotopic (ratherthan heterotopic) spleen Tx was carried out, as it was hypothesizedthat this approach might be beneficial in that leukocytes migratingfrom the donor spleen would initially pass through the recipient liver,where they are possibly more likely to induce a state of tolerance [17].This involved end-to-end anastomoses between the donor and recipi-ent splenic arteries and the donor and recipient splenic veins, using anoperating microscope (Fig. 1B).

The technique of kidney Tx involved end-to-side anastomoses be-tween the cuff of donor aorta/renal artery to the recipient abdominal

aorta, and between the cuff of donor inferior vena cava/renal vein tothe recipient inferior vena cava [16]. The donor ureter was implantedinto the wall of the recipient bladder using a ‘tunnel’ technique, usingan extravesical ureterovesical anastomosis (according to Lich–Gregoir[18]). One native kidney was excised and the ureter of the other kidneywas ligated. In order to identify donor hematopoietic cells (by detectionof the Y chromosome) in the recipient, all transplants were from maledonors into female recipients. All recipient pigs in all groups underwentnative splenectomy.

2.3. Splenocyte preparation and infusion

After perfusion with HTK solution, the spleen was cut into smallpieces, and mashed. Splenocytes were sequentially passed throughmeshes (100, 70 and 40 μm, Fisher Scientific, Waltham, MA), andwashed with PBS (Invitrogen, Carlsbad, CA). In some experiments,mononuclear splenocytes were isolated. Diluted whole splenocyteswith PBS were placed onto Ficoll-Paque PLUS (Amersham Bioscience,Piscataway, NJ) followed by centrifugation (600 g, 30 min). The buffy

127H. Hara et al. / Transplant Immunology 31 (2014) 125–133

coat was collected and washed with PBS. Isolated mononuclearsplenocytes were passed through 70 μm and 40 μmmeshes. All isolatedsplenocytes from the entire spleenwere placed into a 0.9%NaCl solution(250 mL). Total splenocytes (1–2 × 109 cells, approximately 5 × 107

cells/kg) were infused into the recipient through either the superiorvena cava (through an indwelling central catheter; n = 1) or the portalvein (through a mini-laparotomy; n = 3).

2.4. Blood transfusion

To increase the blood volume (as blood perfused into thetransplanted spleen), all recipient pigs, including those that receivedonly splenocytes or kidney grafts, received a transfusion of donor unir-radiated packed whole blood cells (~200 mL) through a central venouscatheter during Tx (day 0). No allergic reactionswere observed (unirra-diated packed whole blood cells were transfused as we felt that the in-clusion of leukocytes might increase the graft-versus-host effect of thedonor spleen and thus increase the level of chimerism, but would be in-sufficient to have any significant effect in the absence of the spleen).

2.5. Recipient irradiation

Whole body irradiation (WBI; 100–125 cGy on day− 2) and thymicirradiation (TI; 700 cGy on day − 2) were administered using a 6 MVphoton beam from a Varian CLINAC 600C linear accelerator (VarianMedical System, Palo Alto, CA), as previously described [16].

2.6. Immunosuppressive/supportive therapy

Cyclosporine (10–30 mg/kg × 2/day i.v., Novartis, East Hanover, NJ),or tacrolimus (0.05 mg/kg × 2/day i.m., generously provided byAstellas, Chicago, IL) was administered on days 0–42 to maintain awhole blood trough level of 400–800 ng/mL or 10–15 ng/mL, respec-tively. (As the cyclosporine available to us could only be administeredi.v. or p.o., which proved difficult, we switched to tacrolimus simply be-cause it could be administered i.m.). Methylprednisolone (Pfizer, NewYork, NY) was administered at 5 mg/kg i.m. on day 0 only. All pigs re-ceived prophylactic cefazolin (10 mg/kg/day i.v./day) (West-Ward,Eatontown, NJ) and dalteparin (100 IU/kg/day subcutaneously) (Eisai,Woodcliff Lake, NJ) while indwelling venous catheters were present.

2.7. Monitoring

Complete blood cell count, serum chemistry, and cyclosporine ortacrolimus levels were determined daily or ×3 weekly by routine

Fig. 2. After orthotopic spleen Tx, the graft vessels (artery and vein) remained patent long-termartery to the graft 14 days after Tx (P36-06). (B) Both splenic artery (red) and vein (blue) rem

methods [16]. Bloodwas drawn at regular intervals tomonitor hemato-poietic cell chimerism by detection of the donor Y chromosome.Rejection of the transplanted spleen wasmonitored by sequential mea-surements of lactate dehydrogenase and platelet count. Kidney graftfunction was determined by sequential measurement of serumcreatinine. Blood flow of splenic and kidney vessels was monitored bycolor Doppler ultrasound (Biosound Genesis 3000 AP, Esaote NorthAmerica, Indianapolis, IN) (Fig. 2A and B). Monitoring for GVHD includ-ed daily inspection of the skin and stools, and regular determination ofliver enzymes.

2.7.1. Flow cytometry for T and B cells in recipient bloodT cell depletion (as a result of irradiation) was assessed by flow

cytometry before and after WBI and TI by staining with anti-pig-CD3εantibody (clone BB22-8E6-8C8), anti-pig CD4a (clone 74-12-4), anti-pig CD8a (clone 76-2-11), and anti-human CD21 (clone B-ly4) (allfrom BD Biosciences, San Jose, CA), as previously described [19]. Appro-priate isotype antibodies were used as negative controls. Data were ac-quired using a FACScan (BD), and analyzed with WinMDI analysissoftware (version 2.8, Joseph Trotter; Scripps Research Institute, LaJolla, CA).

2.7.2. Polymerase chain reaction (PCR) analysis of chimerismPCR was performed to detect the sex-determining region of the Y

(male donor) chromosome in the blood or tissues from female (recipi-ent) pigs, as reported previously [20]. Briefly, DNA obtained fromblood taken before and after Tx was extracted by using QIAamp DNAMini Kit (Qiagen, Chatsworth, CA). Genomic DNA was amplified withthe sex-determining region of the Y chromosome or control primers(listed below). Cycling was performed on a GeneAmp 9700 (AppliedBiosystems, Foster, CA). Samples were taken after every 3 cycles, runon a 2% agarose gel stained with ethidium bromide, and photographed[19].

2.7.3. Quantitative-PCR analysis of chimerismQuantitative-PCR was also performed to assess the level of chime-

rism, as reported previously [20]. The quality of the DNA was assessedby control PCRwith pig S100C primers [20]. Each samplewas comparedwith a standard curve of 0%, 0.16%, 0.8%, 4%, 20% and 100%male DNAdi-luted in female DNA using SYBR Green two-step, real-time PCR [19].Primers used were MSR upper 5′-CCA TCG GCC ATT GTT TTC CTG TTCA-3′, MSR lower 5′-CCT CTG TGC CCA CCT GCT CTC TAC A-3′, S100Cupper 5′-ATG CTG GAA GGG ACG GTA ACA ACA-3′, and S100C lower5′-GCT CAG CTG CTG TCT TTC ACT CGT-3′. In 4 pigs (spleen Tx[n = 2]; splenocyte Tx [n = 2]), chimerism in the spleen graft, and in

. (A) Color Doppler ultrasound demonstrated good blood flow (red) through the splenicained patent 42 days after Tx (P262-05).

128 H. Hara et al. / Transplant Immunology 31 (2014) 125–133

native lung, liver, lymph nodes, thymus, and bone marrow was investi-gated [20].

2.7.4. Mixed lymphocyte reaction (MLR)MLR was carried out before and after spleen, splenocyte, or kidney

Tx, as previously described [19,21]. Briefly, peripheral blood mononu-clear cells (PBMCs) were isolated [19]. Recipient PBMCs as responders(4 × 105 cells/well) were co-cultured with irradiated (2500 cGy) autol-ogous, donor or third-party PBMCs (at responder–stimulator ratios 1:1)for 5 days. All assays were performed in AIM V Medium (Invitrogen).The mean of triplicate results was expressed as 3H-thymidine incorpo-ration (cpm). Stimulation index was also calculated by dividing themagnitude of the donor-specific or third-party proliferative responseby the magnitude of the autologous proliferative response [21].

2.8. Microscopic examination of spleen and kidney grafts

Tissue was fixed in 10% buffered formalin and processed for exami-nation by light microscopy [22]. Sections were stained with hematoxy-lin and eosin to assess overall morphology and presence or absence ofacute rejection, as described by Dor et al. [22]. Spleen [22] and kidney[23] rejection was diagnosed according to standardized gradingsystems.

2.9. Experimental groups (Table 1)

Group 1: Heterotopic spleen Tx in outbred pigs (n = 3).Group 2: Orthotopic spleen Tx in outbred pigs (n = 5).Group 3: Whole splenocyte (n = 2) or mononuclear splenocyte

(n = 2) Tx in outbred pigs.Group 4A: Orthotopic spleen Tx in a MHC-mismatched inbred pig

(n = 1).Group 4B: Concomitant orthotopic spleen and kidney Tx in MHC-

mismatched inbred pigs (n = 2).Group 5: Kidney Tx in MHC-mismatched inbred pigs (n = 2).

2.10. Statistical methods

Statistical differences between the groups were determined by theStudents' t test using GraphPad Prism version 4 (GraphPad Software,San Diego, CA). Values are presented as mean ± SD. Differences wereconsidered to be significant at p b 0.05.

Table 1Summary of individual experiments.

Group Type of Tx Pig no. Immunosuppression Gra(D

WBI TI CyA/TAC Days

1 Heterotopic spleen Tx P395-04 + + CyA 4 4P1-05 + + CyA 4 4P11-05 + + CyA 5 5

2 Orthotopic spleen Tx P190-05 + + TAC 31 32P200-05 + + TAC 28 28P222-05 + + TAC 31 32P247-05 + + TAC 10 10P262-05 − − TAC 42 42

3 Whole splenocyte Tx P83-05 + + CyA 0 N/AP89-05 + + CyA 42 N/A

Mononuclear splenocyte Tx P147-05 + + TAC 26 N/AP243-05 + + TAC 21 N/A

4A Orthotopic spleen Tx P36-06a + + TAC 42 144B Orthotopic spleen + kidney Tx P150-06 + + TAC 42 86

P211-06a + + TAC 9 95 Kidney Tx P151-06a + + TAC 42 16

P213-06a + + TAC 42 16

CyA = cyclosporine. N/A = not applicable. PV = portal vein. TAC = tacrolimus. TI = thym125 cGy). % of chimerism in blood was shown as %Y (male donor) chromosome in the blood f

a MHC-mismatched pig.b The level of chimerism in blood was recorded at either the time of euthanasia or 42 days a

3. Results

3.1. Clinical course and donor chimerism

Therewas a high incidence of complications in locally-outbred pigs inGroups 1, 2, and3 (Table 1). Radiation/infectious pneumonitis developed in 4 of these pigs undergoingspleen Tx, and in 2 after splenocyte Tx. Infectious agents were almost certainly carriedby the pigs before the transplant, but caused overt infection only after pre-Tx irradiationand post-Tx immunosuppressive therapy. In total, 9 recipients were euthanized fortreatment-related complications, e.g., radiation/infectious pneumonitis (n = 6), graftthrombosis (n= 2), and intussusception (n= 1) (Table 1). The single pig that did not re-ceive irradiation (P262-05) survived for 158 days after spleen Tx.

In contrast, specific pathogen-free partially-inbred MHC-defined pigs (Groups 4 and5) did not experience complications, except one recipient (P211-06) that was euthanizedfollowing internal bleeding 9 days after Tx.

After irradiation, all pigs showed significantly reduced leukocyte, lymphocyte, andmonocyte numbers in the blood on the day of Tx (day 0) (Fig. 3A). Analysis of lymphocytesubsets by flow cytometry showed significantly reduced CD3+T cells, including CD4+ andCD8+, and CD21+B cells (Fig. 3B). No features of GVHDwere seen in anypigs in any group.

3.1.1. Group 1: heterotopic spleen Tx in outbred pigs (n = 3)All recipientswere euthanizedwithin 5 days of Tx due to complications (Table 1), and

therefore have been excluded from immunological analysis (e.g.,MLR). However, all dem-onstrated significant levels of hematopoietic chimerism in the blood (Table 1). Histopath-ologic abnormalities were seen in 2 of 3 spleens (Table 2).

3.1.2. Group 2: orthotopic spleen Tx in outbred pigs (n = 5)Spleen graft survival persisted for approximately one month except in one recipient

(P247-05), at which time the recipients were euthanized for complications (Table 1).Throughout follow-up, both splenic artery and vein remained patent, confirmed by colorDoppler ultrasound (Fig. 2). Importantly, we saw no features of GVHD. The level of chime-rismwas relatively high in 2 recipients (Fig. 4), indicating that donor cells were migratingfrom the spleen into the host's blood, but low in 3 (including the one in which no pre-Txirradiation was administered). Macrochimerism was documented for the entire periodduringwhich exogenous immunosuppressionwas being administered, but in some recip-ients the level of chimerism gradually fell and no donor cells could be detected at the timeof euthanasia (which was carried out for complications) (Table 1).

The cells in the donor spleen were steadily replaced by host cells in 3 cases withoutany microscopic evidence of rejection (Fig. 5) (Table 2). In P222-05, macroscopic chime-rism was also measurable in the native liver, lung, lymph nodes, thymus, and bone mar-row at necropsy (Fig. 6A). On MLR, there was a reduced proliferative response to bothdonor-specific and third-party PBMCswhile immunosuppressionwas being administered(Fig. 6C). Donor-specific immunological unresponsiveness continued even afterdiscontinuing immunosuppressive therapy, whereas the anti-third party responserecovered.

3.1.3. Group 3: splenocyte Tx in outbred pigs (n = 4)Donor splenocytes were infused into either the superior vena cava (n = 1) or portal

vein (n= 3). The slow infusion ofwhole splenocytes into the portal vein caused hemody-namic shock that rapidly progressed to cardiac arrest (P83-05) (Table 1); resuscitation at-tempts were unsuccessful. Whole splenocyte infusion into the superior vena cava was

ft survivalays)

Chimerismb inblood (%)

Day ofeuthanasia

Complications and other

50 4 Pneumonitis48 4 Intussusception12 5 Graft thrombosis0.01 32 Pneumonitis

68 28 Pneumonitis18 32 Pneumonitis0.2 10 Graft thrombosis1.8 158 None

22 0 Died (splenocyte Tx into PV)0 69 None (splenocyte Tx into systemic vein)0.1 26 Pneumonitis (splenocyte Tx into PV)0.8 21 Pneumonitis (splenocyte Tx into PV)

5 6.9 145 None10 86 Kidney graft rejection5.4 9 Extraperitoneal bleeding

2 0.3 162 None0 0.1 160 None

ic irradiation (700 cGy). Tx = transplantation. WBI = whole body irradiation (100–rom female (recipient) pigs.

fter transplantation.

Fig. 3. Leukocyte, leukocyte subset, and T and B cell subset counts in the blood after irra-diation. Recipient pigs received WBI (100–125 cGy) and TI (700 cGy) on day − 2.(A)Mean numbers of leukocytes, lymphocytes, andmonocyteswere significantly reduced48 h after irradiation (day 0) (*p b 0.01). (B) CD3+T cell counts, including CD3+CD4+ andCD3+CD8+ subsets, and CD21+ B cell counts were significantly decreased within 48 h(day 0) compared to before irradiation (*p b 0.01). Datawere obtained from all groups ex-cept one pig (P262-05) that did not receive irradiation, and are expressed as the mean ±SD (n = 16).

129H. Hara et al. / Transplant Immunology 31 (2014) 125–133

uncomplicated (Table 2). When isolated mononuclear splenocytes were infused slowlyinto the portal vein, the recipient tolerated the infusion, but required inotropic supportfor a falling blood pressure.

Table 2Histopathological findings.

Group Pig # Tissues Histopathological findings

1 P395-04 Spleen Hemorrhage and necrosis, extensive; consistent wP1-05 Spleen No abnormality.P11-05 Spleen Hemorrhage and necrosis, diffuse; consistent wit

2 P190-05 Spleen Replacement fibrosis, granulation tissue, focal dyinflammation; consistent with post-rejection rem

P200-05 Spleen No abnormality.P222-05 Spleen Patchy congestion of red pulp.P247-05 Spleen Hemorrhage and necrosis, diffuse; consistent witP262-05 Spleen No abnormality.

3 P83-05 N/A Not testedP89-05 Liver Nonspecific chronic inflammation in portal triadsP147-05 Liver Nonspecific chronic inflammation in portal triadsP243-05 Liver No abnormality.

4A P36-06 Spleen Congestion of red pulpLiver No abnormality.

4B P150-06a Spleen No abnormality.Kidney Acute cellular rejection, type I: mild (diffuse inte

peritubular capillaries). Arteriopathy, consistentLiver No abnormality.

P211-06a Spleen No abnormality.Kidney Focal mononuclear cell interstitial infiltrates (butLiver No abnormality.

5 P151-06a Kidney Focal, small mononuclear cell interstitial infiltrateP213-06a Kidney Acute cellular rejection, type 2; moderate. Glome

a MHC-mismatched pig.

Neither whole splenocytes nor mononuclear splenocytes were associated withprolongedmacroscopic chimerism (Table 1 and Fig. 4C). At necropsy after 4 weeks, no chi-merism was detected in the bone marrow or any major organ (Fig. 6B). Rather thanresulting in unresponsiveness to donor cells, one recipient demonstrated an increased re-sponse on MLR (Fig. 6D); 42 days after splenocyte infusion, there was a significant in-creased proliferative response to donor-specific PBMCs (stimulation index: preTx; 5.7,42 days after Tx; 30), though little increase in the response to third-party PBMCs (stimu-lation index: preTx; 7.4, 42 days after Tx; 8.6) (Fig. 6D). These results strongly suggestedthat splenocytes alone would not induce a state of chimerism or tolerance in the host.

3.1.4. Group 4A: spleen Tx in a MHC-mismatched inbred pig (n = 1)The recipient survived for 145 days after Tx without complication, at which time it

was electively euthanized. A significant level of chimerism (6.9%) was detected in theblood 42 days after Tx (Fig. 4D) although the level was lower than in the Group 2 (out-bred) pigs (Fig. 4B). The level of chimerism gradually decreased, and was minimal(0.1%) at the time of euthanasia (145 days). On MLR, there was no increase in the prolif-erative response to donor-specific and third-party PBMCs after Tx (data not shown) (de-spite theMHCmismatchbetween donor and recipient, the initial proliferative response onMLR was weak [as it was also in Groups 4B and 5]).

3.1.5. Group 4B: spleen + kidney Tx in MHC-mismatched inbred pigs (n = 2)Two concomitant transplants of spleen + kidney from the same donor were carried

out. One recipient (P211-06)was euthanized 9 days after Tx due to extraperitoneal bleed-ing, atwhich time chimerismwas low (5.4%) (Table 1). The source of the bleedingwas un-certain, but was not from a rejected or ruptured spleen, which was macroscopically andmicroscopically normal (Table 2). The serum creatinine on day 7 had been 2.2 mg/dL,which was not significantly different from other pigs with renal grafts at this time, sug-gesting that acute rejection was not developing.

In contrast, in the other recipient (P150-06), a significant level of chimerism (10%)was detected in the blood 42 days after Tx (Fig. 4D), but was not detectable at euthanasia(day 86). On MLR, there was no increase in proliferative response to donor-specific orthird-party PBMCs after Tx (data not shown). The serum creatinine in this recipient(P150-06) was stable until 42 days after Tx, when immunosuppressionwas discontinued;the kidneywas completely rejected by day 86 (Fig. 7). Histopathologic examination of thekidney graft demonstrated acute cellular rejection with features of chronic rejection, butno histopathologic features of rejection were seen in the spleen graft (Table 2).

3.1.6. Group 5: kidney Tx in MHC-mismatched inbred pigs (n = 2)Serum creatinine, as an assessment of early graft function (within 7 days of Tx), was

lower in Group 5 than in Group 4 (possibly related to a longer period of cold ischemia inGroup 4). In both recipients, the donor kidneys survived long-term (N4 months) beforeeuthanasia, andwere not rejected (Fig. 7). However, histopathologic examination demon-strated features of acute cellular rejection with glomerulitis and endothelialitis in one pig(P213-06) (Table 2). Although the kidney survived long-term, the degree of chimerismwas much less than that associated with spleen Tx (Fig. 4E). Forty-two days after kidneyTx, on MLR there was no increase in the proliferative response to donor-specific or tothird-party PBMCs (data not shown).

ith acute rejection.

h graft venous outlet obstruction.strophic calcification, and lymphohistiocyticodeling.

h acute rejection or graft venous outlet obstruction.

.

.

rstitial infiltrate; glomerulitis; congestedwith chronic rejection (No vasculitis identified.)

insufficient to diagnose acute cellular rejection).

s, insufficient for acute cellular rejection; No vasculitis or glomerulopathy identified.rulitis and endothelialitis identified.

Fig. 4. Spleen Tx, but not splenocyte or kidney Tx, was associated with a significant level of chimerism in the blood. Detection of the pig male-specific repeat DNA on the Y chromosome.PCRwas carried out to detect the Y chromosome inmale pig blood. Female pig bloodwas used as a negative control. The Y chromosome bandwas detected inmale pig blood at 201 bp, butnot in female pig blood. The kinetics of chimerism (Y chromosome) by quantitative-PCR in (B) outbred pigs with orthotopic spleen transplants (Group 2, n = 5), (C) outbred pigs withsplenocyte transplants (Group 3, n = 3), (D) MHC-full mismatched pigs with orthotopic spleen transplants ± kidney transplants (Group 4A, n = 1 and 4B, n = 2), (E) MHC-full mis-matched pigs with kidney transplants (Group 5, n = 2). Chimerism was consistently detected when orthotopic spleen Tx was performed (Groups 2 and 4). Unfortunately, the majorityof outbred pigs (Group 2) died frompneumonitis/infectionwithin 5 weeks. Outbredpigswith spleen transplants had relatively high levels of chimerism,whileMHC-mismatched pigs hadlow levels. After splenocyte Tx (Group 3), chimerism had decreased by 3 weeks. Chimerism was absent or minimal within 7 days after kidney Tx (Group 5).

130 H. Hara et al. / Transplant Immunology 31 (2014) 125–133

4. Discussion

There are several limitations to our studies. The numbers of experi-ments in each group vary considerably; this is because in the earlyexperiments we persisted with the use of WT pigs despite a high inci-dence of infectious complications before switching to SPF miniatureswine. Furthermore, toward the end of the study, we were limited

with regard to the number of experiments we could carry out. Never-theless, we believe some tentative conclusions can be cautiouslydrawn from our data.

The major messages from the study are (i) that spleen Tx resultsin a greater level of chimerism in the blood when comparedwith that which follows either splenocyte or kidney Tx, and (ii)concomitant Tx of the spleen + kidney does not appear to result in

Fig. 5. No features of rejection after orthotopic spleen Tx. The pigs that had significantlevels of chimerism in blood showed no features of rejection [22] after orthotopic spleenTx. Hematoxylin and eosin staining showed no histopathological features of rejection ina spleen graft at 28 days (P200-05).

Fig. 6.Chimerism inmultiple organs and long-term immunological unresponsiveness to donor-a Group 2 pigwith an orthotopic spleen transplant (P222-05), on day 32macrochimerismwas dlymph nodes (LN), thymus, and bonemarrow (BM) (representative of 2 different experiments)the portal vein 26 days after Tx (P147-05) (representative of 2 different experiments). (C & D)(C) In a Group 2 pig with a spleen graft (P262-05), in the absence of immunosuppressive thera42 days after Tx; 1.0, and at the time of euthanasia; 0.9). (D) In contrast, one recipient in Grouincrease in the donor-specific immune response even while immunosuppressive therapy waseuthanasia; 7.7), suggesting sensitization (each sample was tested in triplicate to determine th

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tolerance of the kidney as has been reported after sequential Tx of thespleen and kidney [11].

Tx tolerance without long-term immunosuppressive therapy is theultimate goal in alloTx. The spleen has long been recognized aspotentially tolerogenic and, in some species, can act as a source ofhematopoietic cell restoration [2]. Spleen allografting in certain straincombinations of rats and guinea pigs, either in the absence of any im-munosuppressive therapy or in association with a short course of im-munosuppression, has been followed not only by survival of thespleen, but also by the induction of donor-specific tolerance to othertransplanted organs. In humans, Kato et al. demonstrated that spleenalloTx with a multivisceral graft showed a protective effect on smallbowel graft rejection without increasing the risk of GVHD comparedwith those who did not receive a spleen [24].

Tolerance may be associated with the development of regulatorycells in the recipient, which have developed some 6 weeks after spleenTx [25,26]. In some studies, even when the spleen was excised or be-came involuted, the state of tolerance persisted. Dor et al. showed theprolonged presence of chimerism and donor-specific hyporesponsive-ness on MLR after spleen Tx in pigs [11]. In the present study, the in-creased chimerism seen in some pigs with spleen Tx rather than

specific antigens in pigswith spleen transplants, but notwith splenocyte transplants. (A) Inetected in the peripheral blood (PB) and in several tissues, including liver, lung,mesenteric. (B) In contrast, chimerismwas absent orminimal in a Group 3 pig after splenocyte Tx intoMLR was carried out before the transplant, 42 days after Tx, and at the time of euthanasia.py, anti-donor-specific hyporesponses were demonstrated (stimulation index: preTx; 5.8,p 3 that received splenocyte Tx (P89-05) into the superior vena cava showed a significantbeing administered (stimulation index: preTx; 5.7, 42 days after Tx; 30, and at the time ofe mean ± SD); *p b 0.05, **p b 0.01: vs preTx.

Fig. 7. Concomitant spleen + kidney Tx was associated with accelerated rejection. Serumcreatinine levels were compared between Group 4B (spleen + kidney Tx) and Group 5(kidney Tx alone). At early time-points (less than 7 days after Tx), serum creatinine washigher in Group 4B than Group 5 (possibly because in Group 4B the kidney underwent alonger cold ischemic period while the spleen was transplanted first). Thereafter, levelswere stable until day 42. However, there was an increase in serum creatinine in the oneremaining pig in Group 4B (P211-06) after discontinuation of tacrolimus. In contrast,the two recipients of kidneys alone showednormal serum creatinine levels until electivelyeuthanized (more than100 days after discontinuation of tacrolimus), although one kidneygraft showed some features of acute rejection (Table 2).

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splenocyte Tx suggests that donor hematopoietic cell engraftment hadoccurred. The levels of chimerism in some Group 2 (outbred) pigswere higher than that in the single Group 4A MHC-mismatched pig,and this could be associated with MHC compatibility since the outbredpigs could have been related.

Using splenectomized outbred pigs and the same tacrolimus-basedregimen, the infusion of undifferentiated donor splenocytes intothe portal vein proved rapidly fatal to the recipient as a result of hemo-dynamic collapse, possibly associated with release of cytokines(e.g., TNF-α) and reactive oxygen species by granulocytes (e.g. neutro-phils). The infusion of undifferentiated donor splenocytes into the supe-rior vena cava resulted in a marked increase in the MLR against donorstimulator cells, indicating sensitization rather than hyporesponsive-ness. When isolated donor splenocytes (consisting of mononuclearcells, but no polymorphonuclear cells) were infused intraportally, theywere tolerated relatively well. However, in all pigs with splenocyte Tx,the level of donor-specific chimerism achieved was low and transient(with no chimerism present after day 21).

These results suggest that splenocyte infusion does not reproducethe immunological unresponsiveness that can be achieved with Tx ofthe whole spleen. This is an important observation that has not beenmade previously in a large animal model. However, in rats, the infusionof donor splenocytes has been reported to prolong donor-specific heartgraft survival [27,28].

Unfortunately, because of a high incidence of pneumonitis, we couldnot carry out an MLR in all pigs (in Groups 2 and 3) at 42 days or afterdiscontinuation of immunosuppressive therapy. However, one pigwith a spleen transplant (P262-05) showed donor-specific hyporespon-siveness on MLR. In contrast, one pig with splenocyte Tx (P89-05)showed a significantly increased anti-donor response at 42 days. Al-though the number of studies is very small, this suggests that successfulspleen Tx results in donor-specific hyporesponsiveness, at least to thespleen itself. Although it would be more practical to achieve tolerance

through donor splenocyte infusion rather than spleen Tx, splenocyteTx may induce sensitization rather than unresponsiveness.

In Dor's studies, in ‘control’ pigs that underwent native splenectomyand received only an allogeneic blood transfusion, but with an identicalimmunosuppressive protocol, low levels of chimerism (±1%) weredetectable on day 1, but chimerism was lost within 11 days [11].These observations correlate with those in the present experimentswhere donor splenocyte Tx and a whole blood transfusion did notachieve a high level of chimerism on day 1 in two of three pigs inGroup 3. Furthermore, the data suggest that the concomitant bloodtransfusion did not contribute significantly to the chimerism seenafter spleen Tx.

The immunosuppressive regimen used in the present study wasbased on that of Dor et al. [11]. Whole body irradiation (WBI) of100 cGy can reduce the host-versus-graft response without affectingthe graft-versus-host response of the transplanted spleen [2]. In thepresent study, irradiation prior to Tx resulted in a significant reductionin lymphocytes, including T and B cell subsets, in the blood. This effect,however, can probably be duplicated by pharmacologic agents adminis-tered pre-Tx [29], although it should be noted that certain agentsthat are highly effective clinically have no or little effect in pigs,e.g., cyclophosphamide [30]. Thymic irradiation (TI)was probably benefi-cial in depleting residual T cells in the thymus [31] and in creating thymic‘space’ [32], thus ensuring exposure ofmaturing thymocytes to donor an-tigen that would subsequently be recognized as ‘self’. This regimen wasnonmyeloablative and minimally myelosuppressive (although it was as-sociated with significant morbidity, particularly in local outbred pigs,which was probably related to infectious microorganisms carried by thepigs before therapy was initiated).

The Tx of a kidney alone, using the same regimen, resulted in onlyminimal chimerism that was lost within 1 week, although the kidneysfunctioned for N4 months. There have been several previous studies inwhich tolerance to kidneys has been induced fairly readily in the pig,and our findings of kidney graft survival in the absence of significantchimerism throw doubt on the value of the pig as an experimentalmodel for this type of study even though MHC-full mismatched pigswere selected in this study. Although spleen Tx was followed by signif-icant chimerism in the blood, neither of the two spleen+ kidney trans-plants carried out was a complete success.

In view of the limitations of the model and of the study, it is difficultto draw any firm conclusions. However, we cautiously suggest that con-comitant Tx of the spleen + kidney may induce a state of acceleratedrejection of the kidney. The cause of this is unknown, but we hypothe-size that spleen Tx might be associated with release of cytokines thatmay be detrimental to survival of the kidney, though we have no evi-dence for this. Perhaps of relevance, in a previous study in baboons,the i.v. administration of a liver cell suspension was associated with ac-celerated rejection of a concomitant cardiac allograft, even in the pres-ence of immunosuppressive therapy [33]. We speculate that cells froma spleen graft may produce inflammatory mediators (e.g., cytokines)resulting in kidney graft injury after reperfusion, that may contributeto the development of accelerated renal graft rejection. This may ac-count for the short kidney graft survival in the present study and theprolonged survival when the kidney was transplanted several weeksafter the spleen [11]. The long survival of the kidney when it alonewas transplanted suggested to us that further spleen + kidney trans-plants in pigs would not provide us with any further data of value.

In summary, a higher level of hematopoietic cell chimerism can beobtained by spleen Tx compared to splenocyte Tx or kidney Tx. SpleenTx, therefore, might have the potential to induce tolerance to a kidneytransplant. Nevertheless, the concomitant Tx of the spleen + kidneydid not provide the long-term success we anticipated, and the reasonsfor this remain uncertain. The long-term survival of MHC-mismatchedkidney grafts when transplanted alone suggests that tissue typing inpigs is not yet a fully-developed science and the pig may not be anideal model for this type of study, as this species appears to be

133H. Hara et al. / Transplant Immunology 31 (2014) 125–133

particularly malleable with regard to the induction of tolerance. Studiesin the nonhuman primate model would appear to be worth pursuingsince there remains considerable evidence that the spleen may be a“tolerogenic” organ, and spleen Tx may well be of clinical relevance.

Conflict of interest

No authors have a conflict interest.

Acknowledgments

Hidetaka Hara, M.D., Ph.D. was a recipient of a postdoctoral fellow-ship grant from the Uehara Memorial Foundation, Japan. The authorsthank Stacy Cashman and Michael Nakon for their invaluable technicalassistance. This work was carried out with a grant from the AmericanDiabetes Association #1-04-RA-15 (DKCC).

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