Early chimerism threshold predicts sustained engraftment and NK-cell tolerance in prenatal allogeneic chimeras

Abstract

The failure of engraftment in human cases of in utero hematopoietic cell transplantation (IUHCT) in which no immunodeficiency exists suggests the presence of an unrecognized fetal immune barrier. A similar barrier in murine IUHCT appears to be dependent on the chimerism level and is poorly explained by a lack of T-cell tolerance induction. Therefore, we studied the effect of the chimerism level on engraftment and host natural killer (NK)-cell education in a murine model of IUHCT. The dose of transplanted cells was found to exhibit a strong correlation with both the engraftment rate and chimerism level. More specifically, a threshold level of initial chimerism (> 1.8%) was identified that predicted durable engraftment for allogeneic IUHCT, whereas low initial chimerism (< 1.8%) predicted a loss of engraftment. NK cells taken from chimeras above the "chimerism threshold" displayed durable calibration of alloresponsive Ly49A receptors and tolerance to donor antigens. Depletion of recipient NK cells stabilized engraftment in low-level chimeras (< 1.8%). These studies illustrate the importance of the early chimerism threshold in predicting long-term engraftment and host NK-cell tolerance after in utero transplantation.

Figure 1

Initial chimerism in recipients of allogeneic IUHCT. Initial PB hematopoietic chimerism was measured at 3 weeks of age in B6Ly5.2 recipients of prenatal Balb/c donor fetal liver cell transplants. Recipients are grouped according to cell dose at transplantation (2 × 10⁴-2 × 10⁶ cells/fetus). (A) The engraftment prevalence (> 0.3%) at the initial PB analysis for each recipient group. (B) Chimerism level at initial PB analysis for each recipient group. Data points represent the mean value plus or minus standard error of the mean (SEM).

Figure 2

Long-term analysis of allogeneic prenatal chimeras. (A) Engraftment prevalence at 6 months of age is plotted for each recipient group. Recipients are grouped according to cell dose at transplantation (2 × 10⁴-2 × 10⁶ cells/fetus). (B) PB chimerism level by cell dose in recipients at 6 months of age. Data points represent the mean value plus or minus SEM. (C) Surviving recipients who remained engrafted were killed at 1 year of age (n = 11), and a bone marrow lineage analysis for both donor and host hematopoiesis was performed (*P < .05). Data points represent the mean value plus or minus SEM.

Figure 3

Evidence of chimerism threshold in allogeneic chimeras necessary for long-term engraftment. (A,B) The drop in the engraftment prevalence and chimerism level between 3 and 24 weeks of age for each group of prenatal chimeras is depicted (*P < .05, **P < .001). Data points for the chimerism level represent the mean value plus or minus SEM. (C) Sample flow cytometry dot-plots for donor chimerism assessment of 2 chimeric littermates (no. 8442 and no. 8424) with initial chimerism levels either above or below 1.8% at 3 and 6 weeks of age. (D) Plot of serial chimerism measurements in low-level chimeras (PB chimerism < 3.5%) illustrating significance of initial chimerism threshold in predicting the durability of long-term engraftment. Data for high-level chimeras (PB chimerism > 3.5%) are not plotted; however, all remained stable chimeras throughout the study period of 1 year. (E) Chimerism summary for congenic and allogeneic strain combinations demonstrating that the significance of the chimerism threshold is limited to the setting of immunologic disparity. Both allogeneic and congenic data are pooled from similar transplant dose cohorts. Prevalence of detectable chimerism throughout the study period is listed for animals either above or below 1.8% (*P < .01).

Figure 4

Ly49A receptor calibration in allogeneic recipients with chimerism levels above and below threshold. The expression of Ly49A on host NK cells was studied because B6 recipient mice are responsive to the MHC class Ia antigen H-2D^d that is highly expressed on the donor Balb/c hematopoietic cells. (A) Relationship between the frequency of Ly49A expression and the level of chimerism in B6 recipients. Y-axis values represent the frequency of receptor-bearing cells relative to reference values for age-matched noninjected controls (rFreq). (B) Relationship between the intensity Ly49A expression and the level of chimerism in B6 recipients. Y-axis represents the relative mean fluorescence intensity relative to naive animals (rMFI). Reference values for the frequency and intensity of Ly49A expression for naive animals is indicated by a dotted line.

Figure 5

Kinetics of Ly49A calibration and development of tolerance in prenatal chimeras above and below chimerism threshold. (A) The relative intensity (rMFI) of Ly49A expression at 3, 6, 9, and 12 weeks is plotted for subthreshold chimeras (0.3%-1.8% PB chimerism) and chimeras above threshold (> 1.8% PB chimerism), and comparisons are made relative to nonchimeric littermate controls (< 0.3% PB chimerism; *P < .05, **P < .01). Data points represent the mean rMFI plus or minus SEM at each time point. (B) NK-cell tolerance as a function of CD107a cell surface expression is shown reported as percentage specific expression. Comparison is made between prenatal allogeneic chimeras above and below chimerism threshold in response to syngeneic (B6.Ly5.1), donor-specific allogeneic (Balb/c), and third-party allogeneic (C3H) target cells (*P < .05).

Figure 6

Prevalence of engraftment and PB chimerism levels after NK-cell depletion in allogeneic recipients. (A) Level of circulating NK cells in control or NK1.1-depleted subthreshold chimeras expressed as a percentage of normal levels (nonchimeric controls). (B) Prevalence of engraftment is reported as a function of time for subthreshold prenatal allogeneic chimeras subject to mAb-mediated NK-cell depletion and chimerism-matched untreated subthreshold chimeras. (C) PB chimerism levels were measured at 3, 6, and 12 weeks of age in NK1.1-depleted and untreated chimeras. Data represent mean PB chimerism level of the group plus or minus SEM at each time point (*P < .05).