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Long-term acceptance of heart allografts in a UVB rat model
3rd lntl Congress of Cell Transplant Society • ABSTRACTS
9.23
Evidence for the Presence of Multilineage Donor Cell Chimerism and Dendritic Cell Progenitors in Bone MarrowAugmented Transplant Recipients Aitouche A, Rugeles M, Zeevi A, Thomson A, Logar A, Banas R, Fung JJ, Starzl TE, Rao AS; Pittsburgh USA
9.24
TRANSPLANTATION OF CULTURED BONE MARROW CELLS (eBMC) INDUCES DONOR-SPECIFIC TOLERANCE Abou EI-Ezz AY, Becker YT, Harmon G, Tarter J, Riehie RE, Johnson HK, Miller HC; Nashville USA We previously reported that transplantation of rat cBMC (WF) caused prolongation of heart allograft survival in 50% of Lew recipients who received Immunosuppressive Regimen (IR) consisting of CyA for 3 days & rabbit Anti-Rat Thymocyte Serum (ATS) for 11 days. BMC were cultured with murine rGM-CSF for 6-12 days, then 1x 10v cells were injected into recipients' portal vein immediately post-transplant. The mean survival for heart allogratLs in this group was prolonged (mean=98_W.25d), compared to animals receiving IR alone (mean=45+16d). In the present study, we examined whether a state of donor-specific tolerance was induced. Heart allografl recipient was sacrificed on POD#143 with a beating heart. The heart showed no gross signs of rejection despite a lymphocytic infiltration on tissue sections comparative to moderate rejection. Splenocytes were harvested for MLR assays. Proliferation was assessed at 96 hours by [3H]-thymidine uptake. MLR data are expressed as mean cpm+SD with (stimulation index). RESPONDERS
MEDIA
cx-WF
ct-BUF
SYNGENEIC (LEW--*LI~W)
2,515 + 1841 (1)
10,922 + 899 (4.3)
27,715 + 1956 (11)
EXPERIMENTAL (WF-+LEW)
4,980 + 631 (1)
6,543 + 899 (1.3)
29,875 + 3324 (6)
These in-vitro results suggest a specific tolerance to the donor strain (WF) with intact reactivity to 3rd-party (But') stimulator in the experimental group but not the syngeneic control.
An Attempt to Induce Tolerance with Infusion of Donor Bone Marrow in Organ Aliograft Recipients Rao AS, Fontes P, Shapiro R, Zeevi A, Iyengar A, Corry R, Pham S, Keenan R, Fang JJ, Starzl TE; Pittsburgh USA The persistence of donor cell chimerism after organ transplantation (Tx) has been postulated to play an important role in allograft acceptance and in the induction of donorspecific hyporeactivity (DSH). To augment this phenomenon, since December 1992, we have infused 3-6x108 unmodified donor bone marrow (BM) cells/kg body weight into 150 (liver" n--44; kidney n=64; heart n=15; lungs n=15; small bowel n--O; multiorgan n=3) non-conditioned primary allograft recipients. The mean recipient age was 44+13 with a mean follow-up of 475+248 days. Additionally, 95 recipients were monitored as controls. Immunosuppression (IS) was with Taerolimus and steroids. The infusion of BM was uneventful and all patients have excellent graft function except for 17 (11%), 11 of whom succumbed to causes unrelated to BM infusion; whereas, the remaining 6 grafts were lost to rejection. Of the controls 9, (9.5%) have died over the course of follow-up; one graft has been lost to rejection whereas, the remaining have adequate graft function. Forty percent of the study and 36% of controls are off steroids. Mild to moderate rejection was witnessed in 641150 (43%) of study and 48•95 (51%) of controls. Serial detection of chimerism in the peripheral blood by flow and/or PCR confirmed the presence of donor cells in 97% of evaluable study but in only 50% of controls. Serial immunological monitoring revealed a higher incidence of induction of DSH in study (48%) as compared to that of controls (30%). These data suggest that infusion of BM is safe and it leads to augmentation of donor cell chimerism and increased incidence of DSH. However, it remains to be seen if this augmented level of microchimerism will enhance allograft survival and allow for reduction or withdrawal of non-specific IS.
Migration of passenger leukocytes from transplanted organs into the recipient has been proposed as the first step towards allograft acceptance and the induction of donor-specific tolerance (DST). Furthermore, the comparative tolerogenieity of various organs is thought to hinge on the quantity and quality of resident migratory cells of which, those of dendritic lineage are thought to play an important role. For detection of multilineage chimerism, recipient PBMC, stained with various lineage-specific (CD3, CD22, CD56, etc.) mAbs were sorted and within this population the presence of donor cells was confirmed by PCR. In all evaluated patients, the presence of donor T and B cells was confirmed; whereas, that of macrophages and NK cells was variable. To document the presence of donor dendritic cells (DC), recipient PBMC were cultured in rhGM-CSF and rhIL-4-enriched medium, a condition known to give distinct advantage to growth of progenitors of the myeloid lineage. Cells harvested from 10-14 d cultures had a distinct DC morphology and potent allostimulatory activity. Interestingly, phenotypic analysis of cultured cells of BMaugmented patients, unlike that of controls, exhibited a marked down-regulation of B7-2 (CD86) while retaining normal levels of expression of B7-1 (CD80) cell surface molecules. In 4/5 patients, the presence of donor DNA was also verified within lineage-/class II + sorted putative DC. Taken together, these data suggest that chimerism in BM-augmented patients is of a multi-lineal character, with the documentation of the presence of the progenitors of donor DC providing unequivocal evidence for the engraftment of infused cells. It is therefore tempting to speculate that the variable expression of the co-stimulatory molecules may endow these cells in selected patients with the capacity to induce DST.
9.25
69
9.26
LONG-TERM ACCEPTANCE OF HEART ALLOGRAFTS IN A UVB RAT MODEL. Abou EI-Ezz AY, Becker YT, Harmon G, Richie RE, Johnson IlK, Miller HC, Van Buren D; Nashville USA Chronic immunosuppression is associated with serious side effects. In the present study, we investigated whether complete cessation of immunosuppression after sensitization of the recipient with cultured bone marrow cell (cBMC) can induce indefinite acceptance of heart allogratLs. Lewis rats received 4 x 104kj total body UVB irradiation on day 0, followed by sensitization with 107 WF cBMC, injected SC on day 3. WF beterotopic heart transplant was carried out on day 11, and CyA was given on days 11,12,13 then weekly for at least 3 months before it was stopped completely. Allografl recipients (n=3) were sacrificed at least 150 days after CyA stoppage with still beating hearts. The transplanted hearts showed no gross or histologic signs of rejection. Splanocytes were harvested for MLR assays. Proliferation was assessed at 96 hours by [3H]-thymidine uptake. MLR data are expressed as mean cpm + SD with (stimulation index). RESPONDERS
or-SELF
¢t-WF
ot-BUF
LEW CONTROL
5,039 + 1,758 (1)
93,004 + 7,331 (18.5)
59,167 + 6,669 (11.7)
EXPERIMENTAL (WF-,LEW)
6,682 + 1,739 (1)
34,486 + 3,335(5.2)
28,736 + 2,564 (4.3)
This approach provided long-term unresponsiveness to heart allografts. Despite a good alloresponse to 3rd-party (BUF) stimulator, no donor-specific tolerance was identified in-vitro, suggesting a possible state of "split tolerance".
9.23
Evidence for the Presence of Multilineage Donor Cell Chimerism and Dendritic Cell Progenitors in Bone MarrowAugmented Transplant Recipients Aitouche A, Rugeles M, Zeevi A, Thomson A, Logar A, Banas R, Fung JJ, Starzl TE, Rao AS; Pittsburgh USA
9.24
TRANSPLANTATION OF CULTURED BONE MARROW CELLS (eBMC) INDUCES DONOR-SPECIFIC TOLERANCE Abou EI-Ezz AY, Becker YT, Harmon G, Tarter J, Riehie RE, Johnson HK, Miller HC; Nashville USA We previously reported that transplantation of rat cBMC (WF) caused prolongation of heart allograft survival in 50% of Lew recipients who received Immunosuppressive Regimen (IR) consisting of CyA for 3 days & rabbit Anti-Rat Thymocyte Serum (ATS) for 11 days. BMC were cultured with murine rGM-CSF for 6-12 days, then 1x 10v cells were injected into recipients' portal vein immediately post-transplant. The mean survival for heart allogratLs in this group was prolonged (mean=98_W.25d), compared to animals receiving IR alone (mean=45+16d). In the present study, we examined whether a state of donor-specific tolerance was induced. Heart allografl recipient was sacrificed on POD#143 with a beating heart. The heart showed no gross signs of rejection despite a lymphocytic infiltration on tissue sections comparative to moderate rejection. Splenocytes were harvested for MLR assays. Proliferation was assessed at 96 hours by [3H]-thymidine uptake. MLR data are expressed as mean cpm+SD with (stimulation index). RESPONDERS
MEDIA
cx-WF
ct-BUF
SYNGENEIC (LEW--*LI~W)
2,515 + 1841 (1)
10,922 + 899 (4.3)
27,715 + 1956 (11)
EXPERIMENTAL (WF-+LEW)
4,980 + 631 (1)
6,543 + 899 (1.3)
29,875 + 3324 (6)
These in-vitro results suggest a specific tolerance to the donor strain (WF) with intact reactivity to 3rd-party (But') stimulator in the experimental group but not the syngeneic control.
An Attempt to Induce Tolerance with Infusion of Donor Bone Marrow in Organ Aliograft Recipients Rao AS, Fontes P, Shapiro R, Zeevi A, Iyengar A, Corry R, Pham S, Keenan R, Fang JJ, Starzl TE; Pittsburgh USA The persistence of donor cell chimerism after organ transplantation (Tx) has been postulated to play an important role in allograft acceptance and in the induction of donorspecific hyporeactivity (DSH). To augment this phenomenon, since December 1992, we have infused 3-6x108 unmodified donor bone marrow (BM) cells/kg body weight into 150 (liver" n--44; kidney n=64; heart n=15; lungs n=15; small bowel n--O; multiorgan n=3) non-conditioned primary allograft recipients. The mean recipient age was 44+13 with a mean follow-up of 475+248 days. Additionally, 95 recipients were monitored as controls. Immunosuppression (IS) was with Taerolimus and steroids. The infusion of BM was uneventful and all patients have excellent graft function except for 17 (11%), 11 of whom succumbed to causes unrelated to BM infusion; whereas, the remaining 6 grafts were lost to rejection. Of the controls 9, (9.5%) have died over the course of follow-up; one graft has been lost to rejection whereas, the remaining have adequate graft function. Forty percent of the study and 36% of controls are off steroids. Mild to moderate rejection was witnessed in 641150 (43%) of study and 48•95 (51%) of controls. Serial detection of chimerism in the peripheral blood by flow and/or PCR confirmed the presence of donor cells in 97% of evaluable study but in only 50% of controls. Serial immunological monitoring revealed a higher incidence of induction of DSH in study (48%) as compared to that of controls (30%). These data suggest that infusion of BM is safe and it leads to augmentation of donor cell chimerism and increased incidence of DSH. However, it remains to be seen if this augmented level of microchimerism will enhance allograft survival and allow for reduction or withdrawal of non-specific IS.
Migration of passenger leukocytes from transplanted organs into the recipient has been proposed as the first step towards allograft acceptance and the induction of donor-specific tolerance (DST). Furthermore, the comparative tolerogenieity of various organs is thought to hinge on the quantity and quality of resident migratory cells of which, those of dendritic lineage are thought to play an important role. For detection of multilineage chimerism, recipient PBMC, stained with various lineage-specific (CD3, CD22, CD56, etc.) mAbs were sorted and within this population the presence of donor cells was confirmed by PCR. In all evaluated patients, the presence of donor T and B cells was confirmed; whereas, that of macrophages and NK cells was variable. To document the presence of donor dendritic cells (DC), recipient PBMC were cultured in rhGM-CSF and rhIL-4-enriched medium, a condition known to give distinct advantage to growth of progenitors of the myeloid lineage. Cells harvested from 10-14 d cultures had a distinct DC morphology and potent allostimulatory activity. Interestingly, phenotypic analysis of cultured cells of BMaugmented patients, unlike that of controls, exhibited a marked down-regulation of B7-2 (CD86) while retaining normal levels of expression of B7-1 (CD80) cell surface molecules. In 4/5 patients, the presence of donor DNA was also verified within lineage-/class II + sorted putative DC. Taken together, these data suggest that chimerism in BM-augmented patients is of a multi-lineal character, with the documentation of the presence of the progenitors of donor DC providing unequivocal evidence for the engraftment of infused cells. It is therefore tempting to speculate that the variable expression of the co-stimulatory molecules may endow these cells in selected patients with the capacity to induce DST.
9.25
69
9.26
LONG-TERM ACCEPTANCE OF HEART ALLOGRAFTS IN A UVB RAT MODEL. Abou EI-Ezz AY, Becker YT, Harmon G, Richie RE, Johnson IlK, Miller HC, Van Buren D; Nashville USA Chronic immunosuppression is associated with serious side effects. In the present study, we investigated whether complete cessation of immunosuppression after sensitization of the recipient with cultured bone marrow cell (cBMC) can induce indefinite acceptance of heart allogratLs. Lewis rats received 4 x 104kj total body UVB irradiation on day 0, followed by sensitization with 107 WF cBMC, injected SC on day 3. WF beterotopic heart transplant was carried out on day 11, and CyA was given on days 11,12,13 then weekly for at least 3 months before it was stopped completely. Allografl recipients (n=3) were sacrificed at least 150 days after CyA stoppage with still beating hearts. The transplanted hearts showed no gross or histologic signs of rejection. Splanocytes were harvested for MLR assays. Proliferation was assessed at 96 hours by [3H]-thymidine uptake. MLR data are expressed as mean cpm + SD with (stimulation index). RESPONDERS
or-SELF
¢t-WF
ot-BUF
LEW CONTROL
5,039 + 1,758 (1)
93,004 + 7,331 (18.5)
59,167 + 6,669 (11.7)
EXPERIMENTAL (WF-,LEW)
6,682 + 1,739 (1)
34,486 + 3,335(5.2)
28,736 + 2,564 (4.3)
This approach provided long-term unresponsiveness to heart allografts. Despite a good alloresponse to 3rd-party (BUF) stimulator, no donor-specific tolerance was identified in-vitro, suggesting a possible state of "split tolerance".