Graft-versus-host disease (GVHD) remains a major complication of bone marrow transplantation. This report describes reversal of GVHD by infusion of stored recipient bone marrow following combined liver-bone marrow allotransplantation. Graft-versus-host disease developed at the end of the first postoperative week. The skin involvement progressively spread to approximately 80% of the body surface and was not affected by modification of the immunosuppressive treatment. On the 42nd and 43rd postoperative day 1.23 × 108 and 1.6 × 108 autologous bone marrow cells per kg of recipient body weight were infused. The skin rush began to dramatically improve and resolved within 2 wk from the autologous marrow infusion. Autologous bone marrow storage previous to allogeneic bone marrow transplantation for tolerance induction could constitute a safety net in case of occurrence of GVHD. © 1994

Adriana Zeevi

Anreas G. Tzakis

Anthony J. Demetris

Ball E.D.

Bhaduri B.R.

Billingham R.

Camillo Ricordi

Edward D. Ball

Edward E. Mullen

Ferdinand O. Ukah

Ignazio R. Marino

John J. Fung

Lerner K.G.

Massimo Trucco

Michael A. Nalesnik

Monchik G.J.

Paulo A.C. Fontes

Rosen C.B.

Thomas E. Starzl

Witold B. Rybka

PubMed

Ricordi, Camillo

Tzakis, Anreas G

Zeevi, Adriana

Rybka, Witold B

Demetris, Anthony J

Fontes, Paulo A.C

Nalesnik, Michael A

Trucco, Massimo

Ukah, Ferdinand O

Ball, Edward D

Mullen, Edward E

Marino, Ignazio R

Fung, John J

Starzl, Thomas E

University of Miami: Scholarship@Miami

Cell Transplantation

Reversal of Graft-Versus-Host Disease with Infusion of Autologous Bone Marrow

Ricordi, C

Tzakis, AG

Zeevi, A

Rybka, WB

Demetris, AJ

Fontes, PAC

Nalesnik, MA

Trucco, M

Ukah, FO

Ball, ED

Mullen, EE

Marino, IR

Fung, JJ

Starzl, TE

Name not available

Reversal of graft-versus-host disease with infusion of autologous bone marrow

D-Scholarship@Pitt

~ Pergamon Cell Transplantation, Vol. 3, No.2, pp. 187-192, 1994 Copyright © 1994 Elsevier Science Ltd Primed in the USA. All rights reserved 0963-6897/94 $6.00 + .00 0963-6897 (93)E0010-3 Original Contribution REVERSAL OF GRAFT-VERSUS-HOST DISEASE WITH INFUSION OF AUTOLOGOUS BONE MARROW CAMILLO RICORDI,* ANREAS G. TZAKIS, ADRIANA ZEEVI, WITOLD B. RYBKA, ANTHONY J. DEMETRlS, PAULO A.C. FONTES, MICHAEL A. NALESNIK, MASSIMO TRucco, FERDINAND O. UKAH, EDWARD D. BALL, EDWARD E. MULLEN, IONAZIO R. MARINO, JOHN J. FUNO AND THOMAS E. STARZL University of Pittsburgh Transplantation Institute, Pittsburgh, PA 15213, USA D Abstract - Graft-versus-host disease (GVHD) remains a major complication of bone marrow transplantation. This report describes reversal of GVHD by infusion of stored re-cipient bone marrow following combined liver-bone marrow allotransplantation. Graft-versus-host disease developed at the end of the first postoperative week. The skin involvement progressively spread to approximately 800/0 of the body sur-face and was not affected by modification of the immuno-suppressive treatment. On the 42nd and 43rd postoperative day 1.23 x 108 and 1.6 x 108 autologous bone marrow cells per kg of recipient body weight were infused. The skin rush began to dramatically improve and resolved within 2 wk from the autologous marrow infusion. Autologous bone marrow storage previous to allogeneic bone marrow transplantation for tolerance induction could constitute a safety net in case of occurrence of GVHD. o Keywords - Transplantation; Bone marrow, Human; Graft-versus-host. The concept of graft-versus-host disease (GVHD) as the recognition of the host by immunologically com-petent cells of the graft was first delineated by Bil-lingham and Brent (1), following spleen cell and bone marrow transplantation. However, it was soon recog-nized that the same problem could affect recipients of organs that are rich in lymphoid tissue (2-12). In this report, we describe reversal of GVHD by infusion of stored autologous bone marrow in a recipient of a combined liver-bone marrow transplant. CASE REPORT A 56-yr-old man underwent upper abdominal exen-teration and liver transplantation on July 16, 1992 for ACCEPTED 11112/93. "'Correspondence should be addressed to Camillo Ricordi, M.D., Diabetes Research Institute, University of Miami 187 a gastric leiomyosarcoma with liver metastases. Using standard techniques autologous bone marrow cells were harvested before the surgical procedure and stored in liquid nitrogen (14). A few hours before op-eration, the patient was treated with a single dose of 550 rads thoraco-abdominal lymphoid irradiation (TLI). Bone marrow cells from the same donor as the liver allograft were separated from vertebral bodies ob-tained at the time of the multiorgan donor procedure. Then, 2 X 108 bone marrow cells per kg of recipient body weight were infused IV through a central line im-mediately after the orthotopic liver transplant. Post-operative immunosuppression was with conventional doses of FK 506 and prednisone (Fig. 1). A skin rash developed at the end of the first post-operative week that was initially mild and confined to the areas exposed to the preoperation irradiation. The rash worsened during the second postoperative week, and on the 15th postoperative day (POD), the diagno-sis of GVHD that was Grade 2 by the criteria of Lerner et al. (13) was made on a skin biopsy. On days 21 and 32 progressively more florid changes compatible with acute GVHD were seen in second and third skin biop-sies. The skin biopsy obtained on POD 15 showed a mild, predominantly T-Iymphocyte infiltrate localized to the upper dermis and associated with focal exocyto-sis and spongiosis. Occasional keratinocyte necrosis was present. Immunoperoxidase studies disclosed that the infiltrating cells were of donor HLA type. The sec-ond biopsy on POD 21 revealed more florid changes which included acantholysis and focal cleft formation School of Medicine, PO Box 016960 (R-134), Miami, FL 33101. 188 Cell Transplantation. Volume 3, Number 2, 1994 50 40 30 20 10 l~----~~~~--~------KKKgi~i---~------~fA-~ -...J -::J I-(J) « • 650 550 450 350 250 150 550 450 a » 350 , ~ 250 150 C -, -~l RMi-~~~~~~~~~as~~~~~ __ --t c: 4.5 :g =- 3.5 :EJ2 ~ ~ 2.5 n:J -(5 1.5 l-• 0.5 >: 0.8 n:J :g, 0.6 ~ 0) 0.4 .s <0 0.2 0 II) ~ 0 LL Q) 50 c: -0>' K~ ~ 100 c:--£ .§: 150 • 200 ~---:-::------=~i--P-M---4-MKKKuKK--R-M---S-M---j1TR 96 Days 550 a 450 » , 350 ?' 250 iJ ;::r 150 f: 50 .c: 6 a 5 ," 4 (1) A <U'1 3 (1) 0 _0) -iJ 2 cStii' -en 1 3 3 0 -I» Fig. 1. Clinical course of the combined bone marrow-liver allograft recipient (AST = aspartate aminotransferase; AL T = alanine aminotransferase). (Fig. 2a), compatible with Grade 3 acute GVHD ac-cording to the criteria of Lerner et al. (13). The biopsy on POD 32 showed similar changes with continued damage to keratinocytes and adnexal cells (Fig. 2b) and a significantly more conspicuous inflammatory infil-trate of the upper dermis than previously. Immunoper-oxidase stains showed the cells to be T-lymphocytes of donor origin. Stains for Epstein-Barr virus were neg-ative. Grossly, the skin involvement spread to approx-imately 80070 of the body surface, including the palms, soles, and face. Its progress was not altered by increases or decreases of FK 506 or prednisone. Bouts of eosin-ophilia (2,040/mm 3 ) and diarrhea were recorded. On the 42nd and 43rd POD posttransplantation, 1.23 x 108 and 1.6 x 108 autologous BMC/kg were infused. The skin rash began to dramatically improve 1 wk af-ter the autologous cell infusion and had largely re-solved after 2 wk. At skin biopsy, four days after the autologous marrow infusion (POD 47), the donor cell infiltrate was less. Focal exocytosis of mononuclear Reversal of graft-vs.-host disease. C. RICORDI ET AL. 189 (a) (b) Fig. 2. (a) Skin biopsy obtained on POD 21. A mononuclear infiltrate of the superficial dermis and epidermis is associated with spongiosis, acantholysis and an area of confluent keratinocyte necrosis. Disruption of the dermal-epidermal junction is also apparent (x250). (b) Skin biopsy on postoperative day (POD) 32 showing an area of dense monoculear inflammation (lower left corner) with involvement and single cell necrosis of hair follicle (upper right corner). cells was found in association with ballooning of ba-sal cells and aggregates of necrotic keratinocytes in the upper layers of the epidermis (Fig. 2c). Seven days af-ter this (POD 54), skin biopsies had less evidence of acute GVHD ranging from Grade 0 to 2 (Fig. 2d). By this time, the rash caused by GVHD had largely re-solved. No additional biopsies were obtained. The patient, who had been seriously ill, was restored to a sense of well being and was discharged. How-ever, on October 13 (90 POD), he was readmitted with jaundice. A needle biopsy of the liver was diag-nosed as acute viral hepatitis. Because of the additional diagnosis of partial biliary obstruction from ampullary dysfunction, the biliary reconstruction of choledocho-dochostomy was converted to a Roux-y-choledocho-jejunostomy on October 22, 1992. Another liver biopsy was obtained intraoperatively with which the diagno-sis of C-virus hepatitis (HCV) was confirmed by iden-tification of viral RNA using the polymerase chain reaction (PCR). A recipient lymph node and a segment of bowel excised for the biliary reconstruction were taken for immunocytochemical staining and histopath-ologic staining. After immunocytochemical staining, both the intraabdominallymph node and segment of jejunum contained sparsely distributed donor cells. This degree of chimerism has been associated with graft acceptance and donor specific nonreactivity (15). For the 1.5 mo before as well as after the biliary re-construction, minimum immunosuppression was given with 1 mg FK 506, every other day and 5 mg/day pred-nisone. Alpha-interferon therapy was started on Oc-tober 27. The patient was discharged on November 20. On February 24,1993, when the bilirubin was 1.2 mgOJo, immunosuppression with FK 506 was discontinued with continuation of 2.5 mg prednisone. On May 4, 1993, he was admitted with a bilirubin of l.9 mgOJo 190 Cell Transplantation. Volume 3, Number 2, 1994 (c) Fig. 2(c). Inflammation and epidermal damage is still present in the biopsy on postoperative day (POD) 47 (four days after autologous bone marrow infusion) (x250). which rose over the next 13 days to 9.7 mgmOJo. Re-transplantation was performed on May 17, 1993, but the patient died 4 rna later after repeated intraabdom-inal and systemic infections. Histopathologically, the primary allograft had findings of hepatitis but there also was evidence of rejection with obliterative arteri-apathy and diffuse small duct loss. Flow cytometry (FACS II, Becton Dickinson & Co., Mountain View, CA) was used to determine the relative percentages of PBL bearing HLA Class I specific for the donor or recipient. Blood was collected and RBC were ACK-Iysed. The remaining cells were stained with anti-HLA Class I monoclonal antibodies (roAbs) (gen-erously provided by Dr. Paul Terasaki) for 30 min at 4°C and counterstained with sandwich when required. Donor cells were detected in the peripheral blood from the time of the first analysis, 11 days following liver transplantation. These accounted for 22-3411,70 of the total cell number out to POD 43 (Fig. 1), but then dra-matically decreased following the autologous bone marrow infusion, to 3% at 96 days following trans-plantation. On POD 227, cytospin examination of a buffy coat preparation stained with donor MHC reac-tive monoclonal antibodies showed < 1 % donor cells. The cytospin during the week before retransplantation was negative for donor cells. Donor specific proliferation was assessed in MLR pretransplant and monthly intervals postoperatively for 8 mo. Before surgery, the recipient's lymphocytes exhibited significant proliferative responses to both donor and third party HLA unrelated stimulators (Ta-ble 1). For the first month postoperatively, the patient's PBL showed no response to either donor or third party. However, the PBL blood sample from day 32 at the time of florid GVHD was stimulated by the stored autologous pre-transplant PBL (3,946 cpm vs. 269 cpm background). Three days after infusion on POD 42-43 of the autologous marrow, the patient's PBL showed highly significant spontaneous prolifer-POD Table 1. MLR Responses of Peripheral Blood Lymphocytes from Combined Liver-Bone Marrow Transplant Recipient Proliferative Responses (CPM) Backg. Recipient* Donor* T.P.* Pre-Tx 32 302 269 32,178 1,915 1,890 204 167 3,946 28,058 15,543 173 37,217 27,212 42,665 10,842 205 41,024 11,387 24,362 46 62 97 108 223 227 305 975 729 3045 2,415 813 225 431 448 666 *Stimulator cells were irradiated 2000R. 8,006 3,994 8,656 10,704 5,775 12,982 17,723 13,730 Reversal of graft-vs.-host disease. C. RICORDI ET AL. 191 (d) Fig. 2(d). Biopsy on postoperative (POD) 54 from the resolving area of rash shows only a modest mononuclear in-filtrate associated with isolated necrotic keratinocytes. Changes of acute GVHD ranged from 0 to 2 in the four sam-ples taken at this time (x250). ation (Table 1) which was not markedly altered by the addition of stored autologous, donor, or recipient cells. However, by POD 62, (20 days after autologous bone marrow infusion) spontaneous proliferation had died down and MLR responses to donor and third party were restored. For the next 2 mo, the patient's proliferative response to donor pretransplant lympho-cytes was higher than that to third party. By 7 mo post-surgery, the MLR response to the donor had dropped to only 250/0 of the response to third party. However, by 10 mo, 6 wk before retransplantation, antidonor party responses had recovered and were similar to those before the primary transplantation. Significant donor-specific CML activity was ob-served with the blood sample on POD 62, 20 days fol-lowing autologous bone-marrow infusion (Fig. 3). Even at a low effector:target ratio (10: 1), recipient lym-phocytes exhibited 20% donor specific CML activity 40 30 ~ ~ u < ...I 20 ::i 0 "# 10 .. o 40:1 Donor-Specific Cytotoxic Activity Of l.iver-Bone Marrow Recipient • 20:1 Effector:Target Ratio -10:1 POD 62 POD 97 Fig. 3. Donor-specific cytotoxic activity of the combined bone marrow-liver allograft recipient postoperative day (POD). with no CML reactivity generated towards third party stimulator cells then or later. This donor-specific CML response was not evoked by the donor cells in a sub-sequent sample on day 97 at which time a significant proliferative response in MLR still was present. DISCUSSION These results indicate that stored autologous bone marrow could constitute a safety net for GVHD in splanchnic organ recipients or in clinical trials of bone marrow transplantation that are designed to induce do-nor specific unresponsiveness for whole organ or non-hematolymphopoietic (i. e., islet) cell transplantation. Neither temporary reduction of immunosuppression nor augmentation of treatment affected the GVHD. After the autologous cell infusion, the skin biopsy was better within 4 days, and dramatic clinical improve-ment was evident by the end of the week. Ten days before the bone marrow infusion, the MLR showed no response against donor or third party, indicating the profoundly immunocompromised status of the recipient. This could explain why the recipient peripheral leukocytes were ineffective in control of the GVHD, despite their quantitative advantage relative to the infused bone marrow cells which had been removed and cryopreserved preoperatively, before the patient underwent radiation treatment or was exposed to phar-macologic immunosuppression. The infused autologous cells retained their ability to reverse the GVHD, and ultimately, the alloreactivity of the circulating recipient cells to those of the donor was restored to levels that 192 Cell Transplantation. Volume 3, Number 2, 1994 were even higher than those observed pre-transplanta-tion. Eventually, the recipient whose immunosup-pression had been stopped regained enough immune competence to reject the graft which also was afflicted with severe HeV. The autologous cell treatment could be considered analogous to the "tolerance breaking" experiments described by Billingham, Brent, and Me-dawar (16). REFERENCES 1. Billingham, R; Brent, L. Quantitative studies on trans-plantation immunity. IV. Induction of tolerance in new-born mice and studies on the phenomenon of runt disease. Philos. Trans. R. Soc. Lond. (BioI.) 242:439-477; 1956. 2. Starzl, TE; Kaupp, HA, Jr; Brock, DR; Butz, GW, Jr; Linman, JW. Homotransplantation of multiple visceral organs. Am. J. Surg. 103:219-229; 1962. 3. Monchik, GJ; Russell, PS. Transplantation of the small bowel in the rat: Technical and immunologic consider-ations. Surgery 70:693-702; 1971. 4. Burdick, JF; Vogelsang, GB; Smith, WJ; Farmer, ER; Bias WB; Kaufmann, SH; Horn, J; Colombani, PM; Pitt, HA; Perler, BA. Severe graft-versus-host disease in a liver transplant recipient. N. Engl. J. Med. 319:689-691; 1988. 5. Ramsey, G; Nusbacher, J; Starzl, TE; Lindsay, GD. Iso-hemagglutinins of graft origin after ABO-unmatched liver transplantation. N. Eng!. J. Med. 311:1167-1170; 1984. 6. Marubashi, S; Matsuzaka, C. Fatal generalized acute graft recipient. Transplantation 50:709-711; 1990. 7. Bhaduri, BR; Tan, KC; Humphreys S; Williams, R; Donaldson, P; Vergani, D; Mowat, AP; Mieli-Vergani, G. Graft-versus-host disease after orthotopic liver trans-plantation in a child. Transplant. Proc. 22:2378-2380; 1990. 8. Roberts, JP; Ascher, NL; Lake, J; Capper, J; Purohit, S; Carovoy, M; Lynch, R; Ferrell, L; Wright, T. Graft vs. host disease after liver transplantation in humans: A report of four cases. Hepatology 14:274-281; 1991. 9. Jamieson, NY; Joysey, V; Friend, PJ; Marcus, R; Rams-bottom, S; Baglin, T; Johnston, PS; Williams, R; Calne, RY. Graft-versus-host disease in solid organ transplan-tation. Transplant. Int. 4:67-71; 1991. 10. Rosen, CB; Moore, SB; Batts, KP; Santranch, PJ; Noel, P; Wiesner, RH; Krom, RAP. Clinical and patho-logical features of graft-versus-host disease after liver transplantation: A case report and review of the litera-ture. Clin. Transplantation. (in press). 11. Comenzo, RL; Malachowski, ME; Rohrer, RJ; Free-man, RB; Rabson, A; Berkman, EM. Anomalous ABO phenotype in a child after an ABO-incompatible liver transplantation. N. Eng!. J. Med. 326:867-89; 1992. 12. Starzl, TE; Demetris, AJ; Trucco, M; Murase,. N; Ri-cordi, C; Ildstad, S; Ramos, H; Todo, S; Tzakis, A; Fung, 11; Nalesnik, M; Zeevi, A; Rudert, WA; Kocova, M. Cell migration and chimerism after whole organ transplantation: The basis of graft acceptance. Hepatol-ogy 17:(June); 1993. 13. Lerner, KG; Kao, GF; Storb, R; Buckner, CD; Clift, RA; Thomas, ED. Histopathology of Graft-vs-Host Re-action (GVHR) in human recipients of marrow from HLA-matched sibling donors. Transplant. Prec. 6(4): 367-371; 1974. 14. Ball, ED; Mills, L; Coughlin, C; Beck, R; Cornwell, GG, III. Autologous bone marrow transplantation in AML: In vitro treatment with myeloid cell-specific monoclonal antibodies. Blood 68:1311-15; 1986. 15. Starzl, TE; Demetris, AJ; Murase, N; Thomson, AW; Trucco, M; Ricordi, C. Cell chimerism permitted by im-munosuppressive drugs is the basis of organ transplant acceptance and tolerance. Immunology Today, 14(6): 326-332, 1993. 16. Billingham, R; Brent, L; Medawar, P. Quantitative stud-ies on tissue transplantation immunity. III. Activelyac-quired tolerance. Philos. Trans. R. Soc. Lond. (Bio!.) 239:357-412; 1956. 

 Graft-versus-host disease (GVHD) remains a major complication of bone marrow transplantation. This report describes reversal of GVHD by infusion of stored recipient bone marrow following combined liver-bone marrow allotransplantation. Graft-versus-host disease developed at the end of the first postoperative week. The skin involvement progressively spread to approximately 80% of the body surface and was not affected by modification of the immunosuppressive treatment. On the 42nd and 43rd postoperative day 1.23 × 108 and 1.6 × 108 autologous bone marrow cells per kg of recipient body weight were infused. The skin rush began to dramatically improve and resolved within 2 wk from the autologous marrow infusion. Autologous bone marrow storage previous to allogeneic bone marrow transplantation for tolerance induction could constitute a safety net in case of occurrence of GVHD. </jats:p

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Reversal of graft-versus-host disease with infusion of autologous bone marrow

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