Cloned LYT-2+ cytolytic T lymphocytes destroy allogeneic tissue in vivo.

The long-accepted notion that alloimmune cytolytic T cells (CTL) mediate transplantation immunity has recently been called into question. In order to ascertain directly whether alloimmune CTL can mediate destruction of foreign tissue, we tested the ability of mouse CTL expanded as cloned populations in vitro to destroy allogeneic skin in vivo. The results of these studies prove unequivocally that cloned Lyt-2+ CTL can perform this task in an immunologically specific, H-2-restricted, and dose-dependent fashion.

Immunization with skin isografts taken from tolerant mice.

Skin isografts from mice that were immunologically tolerant to allogeneic tissue had the ability to immunize isogeneic recipients against subsequent skin allografts. The immunizing isografts showed no gross signs of rejection themselves and appeared to be only the vehicles for transplantation antigen. It seems likely that allogeneic leukocytes derived from the spleen and bone marrow cells used to confer tolerance were contained in the skin of the tolerant mice and were transferred by the skin isografts in sufficient numbers to stimulate transplantation immunity.

The role of iron in the pathogenesis of porphyria cutanea tarda. II. Inhibition of uroporphyrinogen decarboxylase.

Porphria cutanea tarda is characterized biochemically by excessive hepatic synthesis and urinary excretion of uroporphyrin I and 7-carboxylporphyrins. This pattern of excretion suggest an impaired ability to decarboxylate uroporphyrinogen to the paired ability to decarboxylate uroporphyringen to the 4-carboxyl porphyrinogen, coproporphyrinogen, a reaction catalyzed by the enzyme uroporphyringen decarboxylase. Because clinical evidence has implicated iron in the pathogenesis of porphyria cutanea tarda, these experiments were designed to study the effect of iron on uroporphyrinogen decarboxylase in procine crude liver extracts. Mitochondria-free crude liver extracts were preincubated with ferrous ion and aliquots were assayed for uroporphyrinogen decarboxylase activity. Uroporphyrinogens I and III, the substrates for the decarboxylase assay, were prepared enzymatically from (3H)porphobilinogen. The products of the decarboxylase reaction were identified and quantitated by three methods: (a) extraction into 1.5 N HCl and spectrophotometric quantitation; (b) adsorption onto talc, esterification, paper chromatographic identification, and quantitation by liquid scintillation counting; and (c) adsorption onto talc, esterification, thin-layer chromatographic identification on silica gel, and quantitation by liquid scintillation counting. The thin-layer scinllation method proved most sensitive as it was the only method which accurately identified and quantitated the 7-carboxyl porphyrin reaction product. Uroporphyrinogens I and III were decarboxylated at the same rate by porcine hepatic uroporphyrinogen decarboxylase, and the addition of iron induced marked inhibition of the decarboxylase activity. Ortholpehanthroline blocked the inhibitory effect of iron. The inhibition of uroporphyrinogen decarboxylase by ferrous ion, coupled with its previously reported inhibitory effect on uroporphyrinogen III cosynthetase, provides a possible biochemical explanation for the pattern of urinary porphyrin excretion observed in patients with porphyria cutanea tarda and the clinical association with disordered iron metabolism.

Experimental glomerulonephritis in the isolated perfused rat kidney.

The development of immune deposits on the subepithelial surface of the glomerular capillary wall was studied in isolated rat kidneys perfused at controlled perfusion pressure, pH, temperature, and flow rates with recirculating oxygenated perfusate containing bovine serum albumin (BSA) in buffer and sheep antibody to rat proximal tubular epithelial cell brush border antigen (Fx1A). Control kidney were perfused with equal concentrations of non-antibody immunoglobulin (Ig)G. Renal function was monitored by measuring inulin clearance, sodium reabsorption, and urine flow as well as BSA excretion and fractional clearance. Perfused kidneys were studied by light, immunofluorescence, and electron microscopy. All kidneys perfused with anti-Fx1A developed diffuse, finely granular deposits of IgG along the glomerular capillary wall by immunofluorescence. Electron microscopy revealed these deposits to be localized exclusively in the subepithelial space and slit pores. Similar deposits were produced in a nonrecirculating perfusion system, thereby excluding the formation of immune complexes in the perfusate caused by renal release of tubular antigen. Control kidneys perfused with nonantibody IgG did not develop glomerular immune deposits. Renal function and BSA excretion were the same in experimental and control kidneys. Glomerular deposits in antibody perfused kidneys were indistinguishable from deposits in rats injected with anti-Fx1A or immunized with Fx1A to produce autologous immune complex nephropathy. These studies demonstrate that subepithelial immune deposits can be produced in the isolated rat kidney by perfusion with specific antibody to Fx1A in the absence of circulating immune complexes. In this model deposits result from in situ complex formation rather than circulating immune complex deposition.

Renal failure limiting antihypertensive therapy as an indication for renal revascularization. A case report.

Although surgical repair of renal artery stenosis occasionally improves renal function, it is not yet known when revascularization is indicated for that reason. We report the results observed in a patient with renovascular hypertension and additional stenosis in the contralateral kidney whose renal function deteriorated on repeated occasions during antihypertensive therapy. Renal hemodynamic studies during sodium nitroprusside infusion showed severely impaired autoregulation of blood flow, and glomerular filtration rate was corrected after revascularization of the contralateral kidney alone. After surgery, normal BPs were tolerated without loss of function. These findings demonstrate a specific clinical indication for renal revascularization to preserve kidney function.

Domination by epidermal cell-reactive autoantibodies in attempts to raise antibodies to Epa-1 alloantigens.

Despite extensive efforts to produce polyclonal sera and monoclonal antibodies (MoAbs) to Epa-1, a non-H-2 alloantigen expressed by murine epidermal cells (EC) but not lymphoid cells (LC), we were unsuccessful. In total, we screened nearly 3000 hybridoma culture supernatants--a sampling of B cells from 37 mice in 27 fusions--without finding significant evidence of Epa-1-reactive antibodies, an effort greater than that made to produce antibodies to other histocompatibility (H) antigens. Although mice immunized with Epa-1+ contained high levels of EC-reactive antibodies that showed little crossreactivity with LC targets, these were autoantibodies rather than alloantibodies because they reacted as strongly with syngeneic as with allogeneic EC targets. In the course of these studies, we found that even sera of normal mice contain EC-reactive autoantibodies, and that these could be further boosted by immunization with allogeneic EC. These data indicate that the humoral response to allogeneic EC is dominated by the response to a group of tissue-restricted autoantigens expressed on EC. These findings are discussed in the light of the general inability to produce antibodies to minor H antigens.

Expression of cell-defined H-Y antigen on mouse epidermal cells.

Cytotoxic T lymphocytes (CTL)5 of female origin that readily lysed syngeneic male lymphoid cells in specific, dose-dependent, H-2 restricted fashion had little or no activity against syngeneic male epidermal cells (EC) in short-term or long-term chromium-release assays. Moreover, although male EC were quite capable of priming syngeneic female lymphocytes in vivo for the accelerated rejection of male-specific skin grafts and for the subsequent generation of H-Y-specific CTL by exposure of primed female spleen cells (SC) to irradiated, syngeneic male SC in vitro, male EC themselves were incapable of stimulating the development of H-Y CTL when cocultured with primed female SC. Tests of EC from reciprocal male-female radiation chimeras revealed that keratinocytes, not marrow-derived EC (Langerhans cells), were responsible for the priming ability of EC in vivo. Moreover, H-Y antigen was serologically defined on EC that failed to express H-Y-specific CTL target-cell determinants. Alternative explanations of these findings are discussed, including the possibility that the inability of H-2-restricted T cells to lyse male EC results from the lack of association of H-Y antigen and H-2 restricting elements on the EC membrane.

Establishment of cytolytic T lymphocyte clones to epidermal alloantigen Epa-1.

Based on in vivo studies, it has been suggested that the extreme susceptibility of skin to the deleterious effects of transplantation immunity reflects the presence of skin-specific histocompatibility antigens. Immunization of C3H/He mice with purified epidermal cells from H-2-compatible CBA mice leads to the generation of cytolytic T lymphocytes (CTLs) that recognize the epidermal-specific alloantigen Epa-1 in H-2-restricted fashion. In this report we describe four CTL lines that have been maintained in vitro for over 9 months that possess Epa-1 and H-2 restriction specificity identical to that observed in bulk CTL populations, as well as two CTL lines with novel specificities. The predominant cell surface phenotype of all of the CTL lines is Lyt-1-, Lyt-2+, and Thy-1+ as determined by indirect immunofluorescence. We could detect no evidence for overlap between these CTL lines and CTLs mediating lysis of target cells bearing foreign H-2 antigens. From these observations, we conclude that the CTLs in each of these long-term lines represent the progeny of a single clone whose specificity is directed toward tissue-specific alloantigens.

Blocking of interleukin-2 production, but not the tissue destruction induced by cytotoxic T cells, by cyclosporine.

Cytotoxic T lymphocytes generated against epidermal alloantigen-1 (Epa-1), a tissue-restricted, non-H-2 alloantigen that is a target-cell determinant of both skin allograft rejection and cutaneous graft-versus-host reactions, directly produce full-thickness ulcerative skin lesions in Epa-1+ mice. Anti-Epa-1 CTL also indirectly cause extensive damage of "innocent bystander" tissue when injected admixed with Epa-1+ target cells into the skin of Epa-1- hosts. Unlike the direct destruction of host tissue by CTL in "immune lymphocyte transfer reactions" (TrR), "bystander reactions" (ByR) apparently are initiated by the release of lymphokines that recruit host inflammatory cells to the injection site. Treatment of CTL with cyclosporine in vitro prevents their production of lymphokines like interleukin 2 but has no effect on cell-mediated cytotoxicity. However, little is known about the function of CsA-treated CTL in vivo. We confirmed the differential effect of CsA on CTL function in vitro with bulk-culture anti-Epa-1 CTL-CsA pretreatment of CTL abrogated IL-2 production but did not affect CMC. Moreover, we found that CsA pretreatment did not affect the ability of CTL to evoke TrR, nor did it significantly impair their ability to mediate ByR. Therefore, when CTL are treated with CsA in such a way that they lose their capacity to produce IL-2, their cytotoxic activity in vitro as well as their ability to directly and indirectly mediate tissue destruction in vivo are left intact. These results suggest that the ability of CTL to mediate allograft rejection is not dependent on their ability to produce IL-2 and that CMC plays a role in the rejection process.

Evidence that epidermal alloantigen Epa-1 is an immunogen for murine heart as well as skin allograft rejection.

Epa-1 is a non-H-2 mouse alloantigen defined by MHC-restricted, CD8+ cytotoxic T cells. In vitro it is a strong determinant for the lysis of epidermal cells, fibroblasts, and macrophages but not lymphocytes, and in vivo it functions as a target for skin allograft rejection and cutaneous graft-versus-host reactions. Genetically, Epa-1 appears to be the nonpolymorphic manifestation of a loss mutation. The establishment of C3H.Epa-1 (Epa), an Epa-1+ congenic strain on the Epa-1- C3H/HeJ (C3H) inbred strain background, facilitated the investigation of the role of Epa-1 in skin and heart allograft rejection. C3H females and males rejected first-set Epa skin grafts with median survival times (MSTs) of 20 and 30 days, respectively. However, there was a strong factor of immunization, because all second-set skin allografts were rejected by hosts of both sexes within 10 days. In contrast, all Epa hosts of both sexes permanently accepted C3H skin allografts, consistent with Epa-1 arising from a loss mutation. C3H hosts of both sexes rejected primarily vascularized first-set Epa heart allografts in similar tempo to first-set Epa skin allografts, with MSTs of about 30 days. However, in contrast to the accelerated rejection of skin allografts, sensitized C3H hosts rejected Epa heart allografts in chronic fashion, with some transplants showing very prolonged survival. Thus, Epa-1 is a relatively strong determinant of skin allograft rejection but a weaker determinant of heart allograft rejection.

Serum-blocking factors versus specific cellular tolerance in long-term survival of rat heart allografts.

Long-term survival of Ag-B compatible rat heart allografts was obtained by short-term treatment of the recipients with antilymphocytic serum (ALS). Graft survival apparently was based on a specific change in the hosts rather than on persistent nonspecific effects of ALS. The hosts were not fully tolerant in that they were able to reject secondary skin allografts from the heart donor strain, although in a delayed fashion. The long-surviving heart allografts retained their immunogenicity as they were rejected when retransplanted to new hosts. The passive transfer of serum from long-term heart graft acceptors to new hosts receiving fresh allografts delayed rejection by several days. This effect was seen only with the serum from long-term acceptors suggesting that serum-blocking factors were involved in long-term survival of the heart allografts. However, the ability of adoptively transferred lymphoid cells to break tolerance to a heart allograft residing in a classically tolerant host was tested. In contrast to normal lymphoid cells, cells from the long-term acceptors were unable to break tolerance, suggesting that a specific cellular tolerance had been induced in this cell population. Moreover, a serum from the long-term acceptors failed to block the breakage of tolerance by normal lymphoid cells.

Participation of H-2 regions in heart-transplant rejection.

Hearts of newborn mice were cut into small pieces, the fragments transplanted under the ear skin of adult recipients, and the graft survival followed visually (pulsating fragments were considered viable). Donor-recipient combinations were chosen from H-2 congenic (recombinant and mutant) strains in such a way as to provide differences in the entire H-2 complex or in only a small portion of it. The data obtained indicate that a difference between the donor and the recipient in either K, D, or I regions suffices for the rejection of the heart fragments. The rejection is often accompanied by the production of antibodies against classical H-2 antigens (in the case of K- or D-region disparities) or Ia antigens (in the case of I region disparities). In some instances, the antibodies persist in the recipient for more than 50 days. We conclude from these experiments that the same loci that cause acute skin graft rejection (H-2K, H-2D, and H-2I) are responsible for heart graft rejection. Furthermore, we also conclude that serologically Ia-negative tissues may carry Ia antigens in sufficient quantities to stimulate the production of Ia antibodies.

The use of duplex Doppler ultrasonography to evaluate renal allograft dysfunction.

This study evaluated the utility of duplex Doppler sonograms (DS) and the resistive index (RI) in the identification and differential diagnosis of various causes of renal allograft dysfunction. The efficacy of DS and RI was studied either during acute episodes of allograft dysfunction or during periodic posttransplantation longitudinal analyses. The unique features of each renal allograft results in poor correlative value for single isolated measurements of RI. We observed that the highest RIs were in ATN and that an RI of 0.9 was not specific for acute vascular rejection. Also, an RI of 0.9 was rare in acute cellular rejection. RI could not distinguish acute rejection, chronic rejection, CsA toxicity, or obstruction, although the mean RI was significantly different from normal in these groups. Serial studies of RI did document a change at the time of a clinical event compared to baseline. It is concluded that RI is not specific to any one clinical entity.

In vitro and in vivo cytotoxicity of T cells cloned from rejecting allografts.

Epa-1 is a tissue-restricted, non-major histocompatibility (MHC) antigen that may be responsible for the extreme sensitivity of skin to allograft rejection and graft-versus-host disease (GVHD), especially with MHC-compatible donors and recipients. To confirm that Epa-1 serves as a target in allograft rejection and GVHD, we isolated Epa-1-specific cytotoxic T lymphocyte (CTL) clones completely in vivo from sponge-matrix allografts and from lymph nodes draining rejecting skin allografts. These clones induced GVHD-like skin lesions in antigen-specific, MHC-restricted fashion following intradermal inoculation into appropriate hosts. The in vivo-derived clones are conventional CTL since they are IL-2-dependent and express the Thy-1.2+, Lyt-1-, Lyt-2+, L3T4- phenotype. The results of this study also are pertinent to the controversy over which T-cell subset actually mediates allograft immunity, since the intragraft isolation and subsequent cloning of conventional CTL that induce necrotizing skin lesions are direct evidence that CTL are the proximal mediators of allograft rejection.

Dissociation of tissue destruction induced by cytolytic T cells in vivo and cytotoxicity as measured in vitro.

Two forms of local cutaneous graft-versus-host reactions were used to examine the in vivo activity of cytolytic T cells in a large number of antigen systems and mouse strain combinations. In immune lymphocyte transfer reactions (TrRs), CTL were injected intradermally into allogeneic hosts to which they were sensitized; in bystander reactions (ByRs), CTL were mixed with target cells and the mixture injected into hosts syngeneic to the CTL. Both reactions frequently culminate in full-thickness skin destruction. However, CTL highly active in cell-mediated lympholysis assays in vitro sometimes failed to induce significant reactions in vivo, and CTL with negligible CML activity often induced severe, necrotizing lesions. In addition, Clone 58, a non-MHC-specific CD8+ clone that originated from cells extracted from a sponge matrix allograft, lost its CML activity but continued to induce necrotizing TrRs and ByRs. Insofar as these reactions may exemplify the specific (TrR) and nonspecific (ByR) tissue injury that occurs in the rejection process, these findings question the reliability of CML for predicting the ability of CTL to induce the tissue destruction seen in allograft rejection.

The development of proteinuria and focal-segmental glomerulosclerosis in recipients of pediatric donor kidneys.

Several reports in animals, and sporadic case reports in humans, have suggested that kidneys with decreased nephron mass may be more susceptible to the development of focal-segmental glomerosclerosis. This prompted a reexamination of our previously reported group of pediatric donor-adult recipient renal transplant combinations. Data were analyzed from 31 adult recipients who had received renal transplants from cadaver pediatric donors (less than 6 years) with graft function for greater than 6 months and no evidence of chronic rejection. These were compared with a control group transplanted during the same period with adult donor kidneys. Immunosuppression consisted of azathioprine/prednisone or quadruple therapy in 16 and 15 patients respectively. End-stage renal disease (ESRD) was secondary to chronic glomerulonephritis (n = 9), diabetes mellitus (n = 6), polycystic kidney disease (n = 5), and miscellaneous causes (n = 11). Twenty patients had radiographic documentation of renal hypertrophy posttransplant. All patients had serial 24-hr urinalysis for protein and creatinine after transplantation during periods of stable renal function. Ten patients had renal biopsies performed at a mean time from transplant to biopsy of 10.4 +/- 1.6 months. Seven recipients had biopsies that revealed glomerulosclerosis at 13 +/- 6 months posttransplant. Protein excretion and serum creatinine in these patients were significantly higher than in control patients (1.6 +/- 0.37 vs. 0.49 +/- 0.15 g/24 hr and 1.96 +/- 0.11 vs. 1.64 +/- 0.09 mg%; P less than 0.03 and P less than 0.01, respectively). Only 3 of 25 control adult donor recipients developed proteinuria greater than 0.8 g/24 hr within 2 years of transplantation vs. 15/31 pediatric donor recipients. No correlations with the etiology of ESRD, age (greater than or less than 40 years), weight, sex, diabetes, hypertension, or the number of acute rejection episodes could be found. Our data suggest that adult recipients of pediatric donor renal transplants may be at greater risk for the development of glomerulosclerosis than those recipients receiving adult donor kidneys.

A prospective analysis of the accuracy and cost-effectiveness of digital subtraction angiography for living-related renal donor evaluation.

From 1982 to 1984, we conducted a prospective study to evaluate the usefulness of i.v. renal digital subtraction angiography (DSA) for living-related donor (LRD) evaluation. Twenty-eight LRDs were evaluated with the traditional approach of intravenous pyelography (IVP) and standard catheter arteriography (SCA) (group 1). During the same period, 33 LRDs underwent renal DSA and IVP from a single i.v. contrast injection (group 2). If renal arterial imaging with DSA was considered satisfactory, no further radiographic studies were done (group 2-A, n = 23). If renal arterial imaging with DSA was not satisfactory, SCA was then obtained (group 2-B, n = 10). DSA alone accurately defined the number and location of renal arteries in 21 of 23 patients from group 2-A, and in 5 of 10 patients from group 2-B. The major limitation of DSA was in patients with multiple renal arteries; accurate imaging was obtained in only 7 of these 13 patients (54%). In group 2 overall, preoperative renal imaging was not accurate in 2 of 33 patients (6%); in both cases, an unsuspected polar artery was found at nephrectomy. The mean cost per patient of all radiographic renal imaging studies was $953.00 for group 2 and $1721.00 for group 1. These data suggest that the approach of preferentially evaluating LRDs with DSA-IVP, and obtaining SCA only if DSA yields poor visualization, is more cost-effective but not as accurate as the traditional policy of obtaining SCA and IVP in all cases.

Low-dose maintenance prednisone and antilymphoblast globulin for the treatment of acute rejection. A steroid-sparing approach to immunosuppressive therapy.

The purpose of this prospective randomized trial was to evaluate an immunosuppressive protocol involving reduced maintenance and antirejection steroid dosages in cadaver renal transplantation. The study comprises 23 first cadaver graft recipients who experienced an acute rejection episode. All patients received an initial 14-day course of antilymphocyte globulin (ALG) and azathioprine 1.5 to 2.0 mg/kg/day. In 11 patients (group 1), a low maintenance dose of prednisone (30 mg/day) was administered and first rejection episodes were treated with a second 10-day course of ALG. The remaining 12 patients (group 2) received high maintenance doses of prednisone (2 mg/kg/day with tapering) and intravenous methylprednisolone (IVMP) for first rejection episodes. Subsequent rejections in both groups were treated with high doses of steroids. In group 1, all first rejection episodes were reversed with ALG alone, 6 patients experienced no subsequent rejection, and 10 patients currently have a functioning graft. In Group 2, the first rejection episode was reversed with IMVP alone in 10 patients; in two patients in whom IVMP therapy was unsuccessful, ALG was then administered, and subsequent rejection reversal was effected. In group 2, 4 patients experienced no subsequent rejection, and 9 patients currently have a functioning graft. Patients in group 1 received significantly lower (P less than .01) cumulative steroid doses in the first six months following transplantation, which resulted in a reduced number of major infections, as compared with patients in group 2. We conclude that the steroid-sparing regimen of low maintenance prednisone and ALG for first rejection is as effective immunologically as the established high steroid protocol.

A controlled randomized double-blind study of antilymphoblast globulin in cadaver renal transplantation.

Herein are presented the results of a controlled prospective randomized double-blind evaluation of antilymphoblast globulin as an immunosuppressive adjunct to azathioprine and prednisone in cadaver renal transplantation. There were 31 patients and 36 patients randomly assigned to therapeutic and control groups, respectively. ALG-treated patients experienced no major side-effects, a delayed onset of rejection following transplantation (P less than .005), a reduced total number of rejection episodes (P less than .05), fewer days in the hospital (P less than .05), a reduced cost of transplantation (P less than .02), improved graft survival (P less than .05), and patient survival equivalent to that of the control group. These data indicate that ALG is safe, cost-effective, and of immunologic benefit in cadaver renal transplantation.

Functional capacity and rehabilitation of recipients with a functioning renal allograft for ten years or more.

Forty-nine renal transplant recipients who had a single functioning allograft for ten or more years are reviewed. There were 17 cadaver recipients and 32 living-related recipients. Most patients have enjoyed excellent long-term renal function with stable mean daily dosages of azathioprine and prednisone. Fifty-three percent of patients never experienced a rejection episode, and 24% of patients experienced only one rejection episode. Five recipients (10%) developed malignancy following transplantation. Based on the Karnofsky activity scale, 80% of patients enjoyed unrestricted activity at ten years posttransplant. The two major factors contributing to declining activity were progression of systemic diseases such as atherosclerosis or diabetes, and declining allograft function. Following transplantation, all patients developed renewed interest in sexual activity, all men were potent, and all women experienced regular menses. Nine men achieved fatherhood and five women underwent successful pregnancy. Currently, 46 recipients are alive with a functioning allograft. These data confirm the ability of recipients with a long-term functioning renal allograft to return to the work force, participate in preillness levels of activity, and enjoy sexual activity and parenthood.