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We have previously shown that 12 days of high‐dose calcineurin inhibition induced tolerance in MHCinbred miniature swine receiving MHC‐mismatched lung, kidney, or co‐transplanted heart/kidney allografts. However, if lung grafts were procured from donation after brain death (DBD), and transplanted alone, they were rejected within 19‐45 days. Here, we investigated whether donor brain death with or without allograft ischemia would also prevent tolerance induction in kidney or heart/kidney recipients. Four kidney recipients treated with 12 days of calcineurin inhibition received organs from donors rendered brain dead for 4 hours. Six heart/kidney recipients also treated with calcineurin inhibition received organs from donors rendered brain dead for 4 hours, 8 hours, or 4 hours with 4 additional hours of cold storage. In contrast to lung allograft recipients, all isolated kidney or heart/kidney recipients that received organs from DBD donors achieved long‐term survival (>100 days) without histologic evidence of rejection. Proinflammatory cytokine gene expression was upregulated in lungs and hearts, but not kidney allografts, after brain death. These data suggest that the deleterious effects of brain death and ischemia on tolerance induction are organ‐specific, which has implications for the application of tolerance to clinical transplantation. Donor brain death prevents tolerance induction in lung, but not isolated kidney or heart plus kidney, allograft recipients, suggesting that the deleterious effects of brain death on tolerance induction are organ specific.

A 12‐day course of high‐dose tacrolimus induces tolerance of major histocompatibility complex–mismatched lung allografts in miniature swine but does not induce tolerance of heart allografts unless a kidney is cotransplanted. To determine whether lungs share with kidneys the ability to induce cardiac allograft tolerance, we investigated heart–lung cotransplantation using the same induction protocol. Hearts (n = 3), heart–kidneys (n = 3), lungs (n = 6), and hearts–lungs (n = 3) were transplanted into fully major histocompatibility complex–mismatched recipients treated with high‐dose tacrolimus for 12 days. Serial biopsy samples were used to evaluate rejection, and in vitroassays were used to detect donor responsiveness. All heart–kidney recipients and five of six lung recipients demonstrated long‐term graft survival for longer than 272 days, while all heart recipients rejected their allografts within 35 days. Tolerant recipients remained free of alloantibody and showed persistent donor‐specific unresponsiveness by cell‐mediated lympholysis/mixed‐lymphocyte reaction. In contrast, heart–lung recipients demonstrated rejection of both allografts (days 47, 55, and 202) and antidonor responsiveness in vitro. In contrast to kidneys, lung cotransplantation leads to rejection of both heart and lung allografts, indicating that lungs do not have the same tolerogenic capacity as kidneys. We conclude that cells or cell products present in kidney, but not heart or lung allografts, have a unique capacity to confer unresponsiveness on cotransplanted organs, most likely by amplifying host regulatory mechanisms. Lungs, unlike kidneys, do not induce cardiac allograft tolerance, suggesting that a cell or cell product unique to the kidney confers unresponsiveness upon cotransplanted organs.

Tolerance of allografts achieved in mice via stable mixed hematopoietic chimerism relies essentially on continuous elimination of developing alloreactive T cells in the thymus (central deletion). Conversely, while only transient mixed chimerism is observed in nonhuman primates and patients, it is sufficient to ensure tolerance of kidney allografts. In this setting, it is likely that tolerance depends on peripheral regulatory mechanisms rather than thymic deletion. This implies that, in primates, upsetting the balance between inflammatory and regulatory alloimmunity could abolish tolerance and trigger the rejection of previously accepted renal allografts. In this study, six monkeys that were treated with a mixed chimerism protocol and had accepted a kidney allograft for periods of 1–10 years after withdrawal of immunosuppression received subcutaneous injections of IL‐2 cytokine (0.6–3 × 106IU/m2). This resulted in rapid rejection of previously tolerated renal transplants and was associated with an expansion and reactivation of alloreactive pro‐inflammatory memory T cells in the host's lymphoid organs and in the graft. This phenomenon was prevented by anti‐CD8 antibody treatment. Finally, this process was reversible in that cessation of IL‐2 administration aborted the rejection process and restored normal kidney graft function. Low dose injections of IL‐2 break tolerance of kidney allografts induced via transient mixed hematopoietic chimerism in nonhuman primates.

While the induction of transient mixed chimerism has tolerized MHC‐mismatched renal grafts in nonhuman primates and patients, this approach has not been successful for more immunogenic organs. Here, we describe a modified delayed‐tolerance‐induction protocol resulting in three out of four monkeys achieving long‐term lung allograft survival without ongoing immunosuppression. Two of the tolerant monkeys displayed stable mixed lymphoid chimerism, and the other showed transient chimerism. Serial biopsies and post‐mortem specimens from the tolerant monkeys revealed no signs of chronic rejection. The tolerant recipients also exhibited T cell unresponsiveness and a lack of alloantibody. This is the first report of durable mixed chimerism and successful tolerance induction of MHC‐mismatched lungs in primates. The authors achieve nonhuman primate lung allograft tolerance using a mixed‐hematopoietic chimerism strategy.

Kidney‐induced cardiac allograft tolerance requires MHC‐matching between kidney allograft parenchyma and heart allografts, suggesting that cells intrinsic to the kidney enable cardiac allograft tolerance.

This study shows that belatacept promotes induction of mixed chimerism and renal allograft tolerance in nonhuman primates.

Cotransplantation of heart and kidney grafts in MHC‐disparate miniature swine recipients treated with 12 days of high‐dose tacrolimus leads to tolerance of both allografts.

IL‐2 is a known potent T cell growth factor that amplifies lymphocyte responses in vivo. This capacity has led to the use of high‐dose IL‐2 to enhance T cell immunity in patients with AIDS or cancer. However, more recent studies have indicated that IL‐2 is also critical for the development and peripheral expansion of regulatory T cells (Tregs). In the current study, low‐dose IL‐2 (1 million IU/m2BSA/day) was administered to expand Tregs in vivoin naïve nonhuman primates. Our study demonstrated that low‐dose IL‐2 therapy significantly expanded peripheral blood CD4+and CD8+Tregs in vivowith limited expansion of non‐Treg cells. These expanded Tregs are mainly CD45RA−Foxp3 highactivated Tregs and demonstrated potent immunosuppressive function in vitro. The results of this preclinical study can serve as a basis to develop Treg immunotherapy, which has significant therapeutic potential in organ/cellular transplantation.

We have previously shown that a short course of high‐dose tacrolimus induces long‐term tolerance to fully mismatched lung allografts procured from healthy MHC‐inbred miniature swine. Here, we investigate whether donor brain death affects tolerance induction. Four recipient swine were transplanted with fully mismatched lung grafts from donors that were rendered brain dead and mechanically ventilated for 4 h before procurement (Group 1). These recipients were compared to two control groups (Group 2: 4 h of donor ventilation without brain death [n = 5]; and Group 3: no donor brain death with <1 h of ventilation [n = 6]). All recipients were treated with a 12‐day course of tacrolimus. In contrast to both groups of control animals, the swine transplanted with lung allografts from brain dead donors all rejected their grafts by postoperative day 45 and showed persistent responsiveness to donor antigen by MLR. Several additional swine underwent brain death induction and/or mechanical ventilation alone to determine the effects of these procedures on the expression of proinflammatory molecules. Significant increases in serum concentrations of IL‐1, TNF‐α and IL‐10 were seen after brain death. Upregulation of IL‐1 and IL‐6 gene expression was also observed.

The presence of alloreactive memory T cells is a major barrier for induction of tolerance in primates. In theory, delaying conditioning for tolerance induction until after organ transplantation could further decrease the efficacy of the regimen, since preexisting alloreactive memory T cells might be stimulated by the transplanted organ. Here, we show that such “delayed tolerance” can be induced in nonhuman primates through the mixed chimerism approach, if specific modifications to overcome/avoid donor‐specific memory T‐cell responses are provided. These modifications include adequate depletion of CD8+ memory T cells and timing of donor bone marrow administration to minimize levels of proinflammatory cytokines. Using this modified approach, mixed chimerism was induced successfully in 11 of 13 recipients of previously placed renal allografts and long‐term survival without immunosuppression could be achieved in at least 6 of these 11 animals.

Chronic allograft vasculopathy (CAV) in murine heart allografts can be elicited by adoptive transfer of donor specific antibody (DSA) to class I MHC antigens and is independent of complement. Here we address the mechanism by which DSA causes CAV. B6.RAG1−/−or B6.RAG1−/−C3−/−(H‐2b) mice received B10.BR (H‐2k) heart allografts and repeated doses of IgG2a, IgG1 or F(ab’)2fragments of IgG2a DSA (anti‐H‐2k). Intact DSA regularly elicited markedly stenotic CAV in recipients over 28 days. In contrast, depletion of NK cells with anti‐NK1.1 reduced significantly DSA‐induced CAV, as judged morphometrically. Recipients genetically deficient in mature NK cells (γ‐chain knock out) also showed decreased severity of DSA‐induced CAV. Direct NK reactivity to the graft was not necessary. F(ab’)2DSA fragments, even at doses twofold higher than intact DSA, were inactive. Graft microvascular endothelial cells responded to DSA in vivoby increased expression of phospho‐extracellular signal‐regulated kinase (pERK), a response not elicited by F(ab’)2DSA. We conclude that antibody mediates CAV through NK cells, by an Fc dependent manner. This new pathway adds to the possible mechanisms of chronic rejection and may relate to the recently described C4d‐negative chronic antibody‐mediated rejection in humans.

The high frequency of memory T cells present in primates is thought to represent a major barrier to tolerance induction in transplantation. Therefore, it is crucial to characterize these memory T cells and determine their functional properties. High numbers of memory T cells were detected in peripheral blood and all lymphoid tissues except lymph nodes, which were essentially the site of naïve T cells. The majority of CD8memory T cells were effector memory cells located in the blood and bone marrow while most CD4memory T cells were central memory cells present in the spleen. Next, memory T cells from over 100 monkeys were tested for their response to alloantigens by ELISPOT. Memory alloreactivity mediated via direct but not indirect allorecognition was detected in all animals. The frequency of allospecific memory T cells varied dramatically depending upon the nature of the responderstimulator monkey combination tested. MHC gene matching was generally associated with a low-memory alloreactivity. Nevertheless, low anamnestic alloresponses were also found in a significant number of fully MHC-mismatched monkey combinations. These results show that selected donorrecipient combinations displaying a low memory alloresponsiveness can be found. These combinations may be more favorable for transplant tolerance induction.

Complement fixation, as evidenced by C4d in the microvasculature, is a widely accepted criterion of antibody-mediated rejection. Complement fixation has been shown to be essential in acute antibody-mediated rejection, but its role in chronic rejection has not been addressed. Previous studies showed that passive transfer of complement fixing monoclonal IgG2a anti-H-2Kkinto B6.RAG1??KO recipients of B10.BR hearts led to progressive chronic transplant arteriopathy (CTA) over 14-28 days, accompanied by C4d deposition. The present studies were designed to test whether complement was required for these lesions. We report that a noncomplement fixing donor-specific alloantibody (DSA, monoclonal IgG1 anti-H-2Kk) injected into B6.RAG1--KO recipients of B10.BR hearts also promotes CTA, without C4d deposition. Furthermore, a passive transfer of DSA (monoclonal IgG2a anti-H-2Kk) initiated endarteritis followed by CTA in B6.RAG1?-mice genetically deficient in the third component of complement (RAG1??C3??). These studies indicate that antibody to class I MHC antigens can trigger chronic arterial lesions in vivowithout complement participation, in contrast to acute antibody-mediated rejection. This pathway may be relevant to C4d-negative chronic rejection sometimes observed in patients with DSA, and argues that lack of C4d deposition does not exclude antibody-mediated chronic rejection.

Viral infections including those due to cytomegalovirus have been associated with accelerated cardiac allograft vasculopathy (CAV) in clinical trials and some animal models. Evidence demonstrating a direct causal relationship between such infections and de novoformation of coronary vascular lesions is lacking. Heterotopic murine cardiac transplants were performed in a parental to F1 combination in animals lacking both T- and B-lymphocytes (RAG??). Coronary vasculopathy developed almost exclusively in the presence of recipient infection with lymphocytic choriomeningitis virus but not in uninfected controls. This process was also dependent upon the presence of natural killer (NK) cells as depletion of NK cells abrogated the process. These data show that a viral infection in its native host, and not previously implicated in the production of CAV, can contribute to the development of advanced coronary vascular lesions in cardiac allotransplants in mice. These data also suggest that virus-induced CAV can develop via an NK-cell-dependent pathway in the absence of T- and B-lymphocytes.

We studied the effects of indirect allorecognition on the induction and maintenance phases of tolerance in miniature swine cotransplanted with heart and kidney allografts. MHC class I-mismatched heart and kidney grafts were cotransplanted in recipients receiving CyA for 12 days. Recipients were unimmunized or immunized with a set of donor-derived or control third-party MHC class I peptides either 21 days prior to transplantation or over 100 days after transplantation. T-cell proliferation, delayed type hypersensitivity reaction (DTH) and antibody production were assessed. All animals injected with donor MHC class I peptides developed potent indirect alloresponses specific to the immunizing peptides. While untreated recipients developed stable tolerance, all animals preimmunized with donor allopeptides rejected kidney-heart transplants acutely. In contrast, when peptide immunization was delayed until over 100 days after kidney-heart transplantation, no effects were observed on graft function or in vitromeasures of alloimmunity. Donor peptide immunization prevented tolerance when administered to recipients pre transplantation but did not abrogate tolerance when administered to long-term survivors post transplantation. This suggests that the presence of T cells activated via indirect allorecognition represent a barrier to the induction but not the maintenance of tolerance.

Cardiac allograft vasculopathy (CAV) is a major source of late posttransplant mortality. Although numerous cell types are implicated in the pathogenesis of CAV, it is unclear which cells actually induce the vascular damage that results in intimal proliferation. Because macrophages are abundant in CAV lesions and are capable of producing growth factors implicated in neointimal proliferation, they are leading end-effector candidates. Macrophages were depleted in a murine heterotopic cardiac transplant system known to develop fulminant CAV lesions. C57BL6 hearts were transplanted into (C57BL6 × BALBc)F1recipients, which then received anti-macrophage therapy with intraperitoneal carrageenan or i.v. gadolinium. Intraperitoneal carrageenan treatment depleted macrophages by 30-80 with minimal effects upon T, B or NK cells as confirmed by flow cytometry and NK cytotoxicity assays. Carrageenan treatment led to a 70 reduction in the development of CAV, as compared to mock-treated controls (p 0.01), which correlated with the degree of macrophage depletion. Inhibition of macrophage phagocytosis alone with gadolinium failed to prevent CAV. Macrophages may represent the end-effector cells in a final common pathway towards CAV independent of T-cell or B-cell alloreactivity and exert their injurious effects through mechanisms related to cytokinegrowth factor production rather than phagocytosis.

A vertical grating that sinusoidally reverses contrast can be synthesized from two identical component gratings that move with equal velocities in opposite directions (leftward and rightward). Such a counterphase grating is used as a suprathreshold masking pattern. When the mask is of low spatial frequency and is modulated rapidly, a test pattern consisting of an increment of the rightward component and an equivalent simultaneous decrement of the leftward component is highly detectable compared with simultaneous increments or decrements of both components. The visibility of the opponent-movement test signal is strongly facilitated by high-contrast masks. This facilitation is accompanied by a high sensitivity for judging the direction of motion of the test. These results show that certain detection mechanisms are highly sensitive to the difference of the rightward and leftward components. However, when the mask is of threshold contrast, the rightward- and leftward-moving test components appear to be detected independently. A high-contrast grating that rapidly moves in one direction strongly masks gratings moving in the same or opposite direction; this shows that moving patterns are not detected by unidirectional mechanisms when contrast is clearly suprathreshold. The results may be explained by a model with mechanisms that are excited by one direction of motion and inhibited by the opposite direction.

Evidence for motion-selective mechanisms sensitive to high spatial frequencies (e.g., 15 c/deg) was obtained via direction-specific adaptation and measurements of the threshold ratios for moving and counterphase flickering gratings.

Zusammenfassung Es wird eine Methode zur Proportionalitätsverstärkung beschrieben, bei der nur rein Ohmsche Widerstände als Koppelungselemente und eine gemeinsame Spannungsquelle benutzt werden. Das spezielle Spannungsteilerdiagramm erlaubt die Anwendung einer Rückkoppelung.

A vertical, sinusoidal adapting grating of 0.4 cycle/degree that moved laterally at 3 Hz produced a considerably greater threshold rise for a subsequently viewed test grating that moved in the same direction as the adapting grating than in the opposite direction. The test grating moved at 0.25 or 0.12 Hz. The result shows that very slowly moving test patterns that move in the direction opposite to the adapting pattern are detected in part by direction-selective mechanisms.

Zusammenfassung Es wird an Zählrohren versucht, die Zählrohrspannung mit rhythmischen Spannungsstößen zu überlagern; dadurch kann die maximale Stoßzahl der Zählrohre festgestellt werden. Die übereinstimmung mit den Resultaten verschiedener Zählrohrschaltungen wird diskutiert.