Early allogeneic immune modulation after establishment of donor hematopoietic cell-induced mixed chimerism in a nonhuman primate kidney transplant model

BackgroundMixed lymphohematopoietic chimerism is a proven strategy for achieving operational transplant tolerance, though the underlying immunologic mechanisms are incompletely understood.MethodsA post-transplant, non-myeloablative, tomotherapy-based total lymphoid (TLI) irradiation protocol combined with anti-thymocyte globulin and T cell co-stimulatory blockade (belatacept) induction was applied to a 3-5 MHC antigen mismatched rhesus macaque kidney and hematopoietic cell transplant model. Mechanistic investigations of early (60 days post-transplant) allogeneic immune modulation induced by mixed chimerism were conducted.ResultsChimeric animals demonstrated expansion of circulating and graft-infiltrating CD4+CD25+Foxp3+ regulatory T cells (Tregs), as well as increased differentiation of allo-protective CD8+ T cell phenotypes compared to naïve and non-chimeric animals. In vitro mixed lymphocyte reaction (MLR) responses and donor-specific antibody production were suppressed in animals with mixed chimerism. PD-1 upregulation was observed among CD8+ T effector memory (CD28-CD95+) subsets in chimeric hosts only. PD-1 blockade in donor-specific functional assays augmented MLR and cytotoxic responses and was associated with increased intracellular granzyme B and extracellular IFN-γ production.ConclusionsThese studies demonstrated that donor immune cell engraftment was associated with early immunomodulation via mechanisms of homeostatic expansion of Tregs and early PD-1 upregulation among CD8+ T effector memory cells. These responses may contribute to TLI-based mixed chimerism-induced allogenic tolerance

Guidelines for the management of a brain death donor in the rhesus macaque: A translational transplant model.

The development of a translatable brain death animal model has significant potential to advance not only transplant research, but also the understanding of the pathophysiologic changes that occur in brain death and severe traumatic brain injury. The aim of this paper is to describe a rhesus macaque model of brain death designed to simulate the average time and medical management described in the human literature.Following approval by the Institutional Animal Care and Use Committee, a brain death model was developed. Non-human primates were monitored and maintained for 20 hours after brain death induction. Vasoactive agents and fluid boluses were administered to maintain hemodynamic stability. Endocrine derangements, particularly diabetes insipidus, were aggressively managed.A total of 9 rhesus macaque animals were included in the study. The expected hemodynamic instability of brain death in a rostral to caudal fashion was documented in terms of blood pressure and heart rate changes. During the maintenance phase of brain death, the animal's temperature and hemodynamics were maintained with goals of mean arterial pressure greater than 60mmHg and heart rate within 20 beats per minute of baseline. Resuscitation protocols are described so that future investigators may reproduce this model.We have developed a reproducible large animal primate model of brain death which simulates clinical scenarios and treatment. Our model offers the opportunity for researchers to have translational model to test the efficacy of therapeutic strategies prior to human clinical trials

Urine output throughout brain death.

<p>Although there was variability among animals (Fig 6B), the average peak UO occurred 5–8 hour after BD induction (Fig 6A). All but one animal exceeded the 4ml/kg/hr of urine output at some time point in the experiment.</p

Pancreas H&E analysis.

<p>Biopsy A shows H&E of naïve rhesus, age 21 years, diagnosed mild apoptosis, minimal lobular atrophy. Biopsy B shows H&E of brain dead rhesus, age 17 years, diagnosed mild apoptosis, minimal lobular atrophy.</p

Hemodynamics of brain death: Systolic blood pressure and heart rate.

<p>When examining the change is systolic blood pressure (SBP), there was a predicable hypertension, followed by hypotension before return to baseline. Fig 3B shows the individual SBP response to brain death and Fig 3A shows the average response for all animals. When change in HRs with BD there was a trend of slightly delayed tachycardia at 15 minutes after brain death, followed by slow return to a HR approximately 10–15 bpm above the animal’s original baseline by 1 hour post brain death. Fig 3C shows the individual HR response to brain death and Fig 3D shows the average response for all animals.</p

Histological analysis of brain dead vs. naïve primates.

<p>Histological analysis of brain dead vs. naïve primates.</p

Algorithm for management of a hemodynamically unstable primate.

<p>An algorithm for vasoactive infusions and isotonic IV fluid boluses needed to augment the animal’s blood pressure is outlined above.</p

Vasoactive drug use by animal.

<p>Vasoactive drug use by animal.</p

Liver H&E analysis.

<p>Biopsy A shows Liver H&E of naïve rhesus, age 15 years, diagnosed 5–10% macrovesicular fatty infiltrates, moderate/marked vacuolization. Biopsy B shows Liver H&E of brain dead rhesus, age 18 years, diagnosed 5–10% macrovesicular fatty infiltrates, moderate vacuolization.</p

Management and severity of diabetes insipidus.

<p>Management and severity of diabetes insipidus.</p