In utero transplantation of hematopoietic stem cells is a promising treatment for immune and hematologic diseases of fetuses and newborns. Unfortunately, there are limited data from nonhuman primates and humans describing optimal transplantation conditions. The purpose of this investigation was to determine the effect of T-cell number on engraftment and the level of chimerism after in utero transplantation in nonhuman primates. CD34 + allogeneic adult bone marrow cells, obtained from the sire after G-CSF and stem cell factor administration, were transplanted into female fetal recipients. The average CD34 + cell dose was 3.0 × × 10 9 /kg (range, 9.9 × × 10 8 to 4.4 × × 10 9 ) and the T-cell dose ranged from 2.6 × × 10 5 to 1.1 × × 10 8 /kg. Chimerism was determined in peripheral blood subsets (CD2, CD13, and CD20) and in progenitor cell populations by using polymerase chain reaction. Chimerism was noted in seven of eight live-born animals. The level of chimerism in the progenitor population was related to the fetal T-cell dose (r = 0.64, p < 0.02). At the lowest T-cell dose (2.6 × × 10 5 /kg), no chimerism was detected. As the T-cell dose increased to 10 6-7 /kg, the level of chimerism increased. Adjusting the T-cell dose to 1.1 × × 10 8 /kg resulted in fatal graft-versus-host disease (GVHD). The results of this study emphasize the importance of T cells in facilitating donor cell engraftment and in producing GVHD in fetal nonhuman primates. Some animals achieved levels of chimerism in the marrow hematopoietic progenitor cell population that would likely have clinical relevance. However, the levels of chimerism in peripheral blood were too low for therapeutic benefit. Further studies are needed to test methods that are likely to enhance donor cell engraftment and peripheral blood levels of donor cells.

In utero hematopoietic stem cell transplantation could potentially be used to treat many genetic diseases but rarely has been successful except in severe immunodeficiency syndromes. We explored two ways to potentially increase chimerism in a nonhuman primate model: (a) fetal immune suppression at the time of transplantation and (b) postnatal donor stem cell infusion. Fetal Macaca nemestrina treated with a combination of the corticosteroid betamethasone (0.9 mg/kg) and rabbit thymoglobulin (ATG; 50 mg/kg) were given haploidentical, marrow‐derived, CD34+‐enriched donor cells. Animals treated postnatally received either donor‐derived T cell–depleted or CD34+‐enriched marrow cells. Chimerism was determined by traditional and real‐time polymerase chain reaction from marrow, marrow progenitors, peripheral blood, and mature peripheral blood progeny. After birth, the level of chimerism in the progenitor population was higher in the immune‐suppressed animals relative to controls (11.3% ± 2.7% and 5.1% ± 1.5%, respectively; p = .057). Chimerism remained significantly elevated in both marrow (p = .02) and fluorescence‐activated cell sorted and purified CD34+ cells (p = .01) relative to control animals at ≥ 14 months of age. Peripheral blood chimerism, both at birth and long term, was similar in immune‐suppressed and control animals. In the animals receiving postnatal donor cell infusions, there was an initial increase in progenitor chimerism; however, at 6‐month follow‐up, the level of chimerism was unchanged from the preinfusion values. Although fetal immune suppression was associated with an increase in the level of progenitor and marrow chimerism, the total contribution to marrow and the levels of mature donor progeny in the peripheral blood remained low. The level of long‐term chimerism also was not improved with postnatal donor cell infusion.

Natural killer (NK) cells from nonhuman primates have not been completely characterized, and methods for expanding nonhuman primates NK cells in vitro have been described only in rhesus species. The purpose of this report was to characterize NK cells in pigtail macaques (Macaca nemestrina), a species that is frequently used in studies of transplantation biology/immunology, virology, vaccine development, and reproductive biology. NK cells from Macaca nemestrina peripheral blood were best defined by the expression of CD16 and CD8alpha, and the absence of CD3. Subsets of these cells express CD56, NKp30, and NKp46. An enhanced ability to kill K562 cells was not present in fluorescence activated cell sorted (FACS)-purified CD16-/CD3+ and CD16-/CD56+ cells isolated from fresh peripheral blood. However, FACS-purified CD16+/CD3- and CD16+/CD56- cells were highly efficient killers of K562 cells. Macaca nemestrina NK cells can be expanded by in vitro culturing of FACS-purified CD16+/CD2-/CD3-/CD56- cells, or from peripheral blood cells depleted of cells expressing CD3, CD4, and HLA-DR. Cells in these cultures expand 70-fold after 21 days of culturing. After culturing, these cells express high levels of natural cytotoxicity receptors (NCRs) NKp30 and NKp46. NK cell populations obtained from FACS-purified CD16+/CD3-, CD16+/CD56- cells and CD3/CD4/HLA-DR-depleted cells were highly efficient killers of K562 cells. These data suggest that a population of highly enriched cytolytic NK cells can be obtained from purified CD16+/CD3- and CD16+/CD56- cells obtained from peripheral blood, as well as from cells that have been cultured and expanded from peripheral blood that is depleted of CD3/CD4/HLA-DR-expressing cells.

The ability to discriminate and further quantify the proportion of donor and host cells is essential in hematopoietic stem cell transplant protocols. In human sex-mismatched transplants, this can be easily accomplished by the use of commercially available fluorescent in situ hybridization (FISH) probes. In many animal models, including non-human primates, this methodology is not possible due to the lack of commercially available FISH probes. In animal models, donor cell detection could be accomplished if there is a known species-specific sex determining region Y (SRY) (male) or other unique DNA sequence using either semiquantitative or quantitative real-time polymerase chain reaction (PCR). The use of real-time quantitative PCR has the obvious advantage of providing detailed enumeration of the percentage of donor cells present. We report the development of extremely sensitive primer and probe combinations for male (SRY) and major histocompatibility complex (MHC)-DQA sequences in the macaque and baboon non-human primate models. This assay has a sensitivity of a five-log range and can detect less than four target cells in the presence of 10(5) background cells (approximately 0.001%) and fetal DNA obtained from maternal serum from Macaca nemestrina. The SRY (male) primer and probe combination has similar sensitivity in Macaca fasicularis, Macaca mulatta, and Papio cynocephalus anubis.