Abstract

MATERIALS AND METHODS

RESULTS

Evidence of Chimerism

ICC

The donor HLA specificity of the renal allografts after 27–29 years was most easily demonstrated in the vascular endothelial cells (Fig. 1A). In contrast, the Y probe in case LD 52 showed the male marker in the renal tubules (Fig. 2A).

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FIGURE 1

Kidney allograft (A, 450×) and recipient lymph node (B, 100×) stained with anti-HLA B 7,40 mAb using an avidin-biotin complex method, The rust-colored endothelial cells in the kidney and scattered cells in the lymph node are of donor origin, (C) (l00×) The negative control in which anti-A2, 28 (irrelevant class-matched antibody) was used as the primary antibody.

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FIGURE 2

Fluorescent in situ hybridization for the Y chromosome. (A) Kidney allograft showing the male (Y) marker in over 50% of the tubular epithelial cells. (B) Y marker (arrow) in a skin biopsy (both photomicrographs 1000×).

All 5 of the recipients had small numbers of donor cells in their lymph nodes (Fig. 1B), skin (Fig. 2B), or both (Table 3). The smallest number of donor cells was in the female recipient of a paternal kidney. The number of donor cells in recipient tissues never exceeded 1 % and usually was estimated to be < 0.1%.

PCR

In the 4 cases in which blood was available from both the donor and recipient (LD17, 33, 51, 90), genomic DNA was extracted from the peripheral blood lymphocytes of the recipient. In addition, the blood of the respective donors, together with the biopsies of lymph nodes and skin of the recipients, were used for DNA extraction. The amplification with HLA-DRB “generic” primers was successful in all cases and allowed us to chose the primers for the allele-“specific” amplifications (Figs. 3 and ​and4).4). Tissue chimerism was demonstrated in all 4 cases, and in 2 there also was blood chimerism (Table 4).

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FIGURE 3

LD51: Chimerism demonstrated with PCR. After PCR amplification of HLA-DRB genes with “generic” oligo primers and hybridization of this material with allele-specific oligo probes, the recipient tissues were typed as DR4 (probe 1004) and DR7 (probe 1006). The donor blood was positive for probes 1004 (DR4), shared with the recipient, and 7011 (DR2). The amplified DNAs from donor blood (DB), recipient lymph nodes (RL), and recipient skin (RS) obtained using DR2 “specific” oligo primers were successfully hybridized with the allele-specific oligo probes 7011 and 8603, which recognize the DR2 allele of the donor. Probes 7002, 7003, and 8601 were negative. C is the negative control. In this experiment, the blood of the recipient (RB) was not positive.

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FIGURE 4

LD 90: PCR demonstration with Southern blot technique of chimerism in blood (RB), lymph node (RL), and skin (RS) of the recipient as detected with DR₇ (donor) specific oligo probes after preliminary steps described in Figure 3. C is the negative control. DB is donor blood DNA diluted 100 times to avoid overwhelming the other bands.

TABLE 4

PCR

Patient	Donor-specific allele	Donor-specific probe	Tissue distribution
			Blood	L-node	Skin
17	DR7	0701	−	+	−
33	DR4	0409	+	+	−
51	DR2	1501	−	+	+
52	Y chromosome	SRY	−	−	+
90	DR7	0701	+	+	+

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In the woman whose paternal donor had died (LD 52), the detection of the Y chromosome in her skin showed the presence of donor (male) cells in the recipient (female) tissues. As in all the other cases, the allograft also was a chimeric structure.

DISCUSSION

Biopsies in all 5 of the long-surviving renal allograft recipients showed low level chimerism. This was identified by immunostaining of discrete cells of donor type in host lymph nodes and skin, and by confirmation in all 5 cases of donor DNA in the tissues with PCR techniques. In addition, 2 of the patients were blood chimeras by PCR. The subtlety of the chimerism in the skin and lymph nodes versus much more overt findings in liver transplant recipients (2,3) apparently reflected the relative paucity of the migratory leukocytes in the kidney compared to the liver. This could explain why tolerance of the renal allograft is harder to induce (1).

To increase the tolerogenicity of organs such as the kidney and heart that have a small migratory leukocyte constituency, the therapeutic possibility has been discussed elsewhere of augmenting cell migration to the level naturally enjoyed by the immunologically privileged liver (1). The most obvious expedient is the concomitant infusion of bone marrow from the organ donor, as has been advocated by Monaco et al. (12–15) and Thomas et al. (16, 17) for tolerance induction of kidneys and carried out clinically by Barber et al. (18), Van Twuyver et al. (19) have speculated that the tolerogenic effects of blood transfusion for conditioning of patients for kidney transplant recipiency could be due under certain conditions to the homing in of transfused lymphocytes to privileged sites, resulting in mixed allogeneic chimerism. All 5 of our kidney recipients had undergone splenectomy. However, the presence or absence of a spleen was not an important factor in the chimeric state of a series of long-surviving liver recipients, all of whom were chimeras (2, 3,20).

The critical migratory cells may be the dendritic cells (21) that previously were known as passenger leukocytes (22, 23). However, there are very few clues about how these cells of bone marrow orgin, whose life expectancy after maturation has been said to be days or weeks (24), have disseminated from the grafts and perpetuated themselves for nearly 30 years in their new environment. Inaba et al. (25) recently demonstrated stem cells in mouse blood that could be grown selectively in culture enriched with GM/CSF. However, it is unlikely that residual blood in allografts deliberately perfused to an asanguinous state could be a consistent source of stem cells that replicate in the recipients. The migratory cells, which generate progeny, could be hematopoetic stem cells that come in small numbers from the interstitium of the transplanted organ under conditions that are propitious in the perioperative period for migration, engraftment, and self-renewal. It was noteworthy that 2 of our 5 patients had circulating donor DNA demonstrated by PCR 27 and 29 years post-transplantation.

In the past, the clinical meaning of tolerance has been construed as permanent engraftment in the absence of continuing therapy. We have suggested that such drug-free classical tolerance and the “graft acceptance” achieved with chronic immunosuppression are both expressions of the cell repopulation (1). The achievement of a potentially drug-free state probably is not rare after kidney transplantation, particularly if well-matched family donors are used. Of the first 64 patients in the original University of Colorado series (26), 10, including the first 4 in the present study, still bear their original consanguineous transplants, which are among the 16 longest continuously functioning allografts in the world (27). Two of these 10 recipients who had HLA-compatible donors have perfect renal function after stopping all medications more than 26 years ago (28). In addition, Patient LD 52 of this report, who has a one haplotype mismatched graft, has been only on 7.5 mg/day PRED for more than 12 years.

The practical problem is how to determine when the need for treatment ends and the drug-free state begins. Strober et a1. (29) documented tolerance by discontinuance of treatment soon after transplantation in 3 cadaveric kidney recipients whose immunosuppression was with 20 Gy total lymphoid irradiation, a perioperative course of antithymocyte globulin, and maintenance PRED that was gradually weaned and eventually stopped. Donor-specific nonreactivity of lymphocytes from their drug-free patients was demonstrated with MLR and CML. In 4 of our 5 cases, the availability for blood sampling of the original kidney donors allowed these same in vitro immunologic tests to be done. The results showed absolute donor-specific nonresponsiveness with MLR in 2 cases, relative nonresponsiveness in the 2 others, and an inability in all 4 cases of the donor lymphocytes to stimulate a CML response. Unfortunately, donor-specific nonresponsiveness with either MLR or CML (or both) does not provide complete assurance of stable graft acceptance without immunosuppression in experimental animals (30) or humans (10, 11). Conversely, the demonstration of in vitro antigraft reactivity does not necessarily imply instability (31). This means that the late discontinuance of therapy still has an element of trial and error.

Footnotes

REFERENCES