Healthy rhesus macaques underwent mobilization of PBPCs with G-CSF and SCF followed by leukapheresis. They were conditioned with myeloablative TBI and transplanted with PBMCs, purified CD34+ PBPCs, or purified CD34+ PBPCs that had been retrovirally transduced with a vector containing the bacterial neomycin resistance gene (neo) and cultured for 4 days in the presence of Flt-3L, MDGF, SCF, and Retronectin (; ). The number of CD34+ cells within each graft was adjusted to be the same for the three groups. For the animals receiving selected-transduced cells, the number of CD34+ cells placed into culture was adjusted. The total expansion during culture was 1.3−1.5-fold. The purity of the grafts for the CD34-selected and CD34-selected/transduced groups was 88% or higher, with less than 3% residual T-cells. The culture conditions used did not include any cytokines capable of supporting viability or proliferation of T-cells, and during similar cultures of rhesus CD34+ cells, we have found a 6.5 ± 2.6-fold decrease (n = 6) in T-cells by day 4 of culture.
All animals engrafted (leukocytes >1,000/μ
l, neutrophils >500/μ
l) between day 8 and day 14 post-transplantation. Up to 10% of circulating granulocytes and mononuclear cells contained the vector early after transplantation in the selected-transduced group. This level stabilized thereafter to a level of 0.1%−2%, comparable to levels found previously [14
]. One animal in this group required euthanasia 6 months after transplantation due to radiation-induced lung fibrosis.
The absolute numbers of total CD3+ T-cells in the animals combined from all groups prior to transplantation were 1,622 ± 234 CD3+ T-cells per μl of blood (mean ± SEM). One month after transplantation, all animals showed dramatic depletion of T-cells (). Animals that received selected-transduced PBPCs had 424 ± 82 CD3+ T-cells per μl 1 month after transplantation, whereas animals receiving selected PBPCs or unselected leukocytes had 936 ± 533 T-cells per μl and 970 ± 337 T-cells per μl, respectively. Even though the selected-transduced group showed the lowest number of CD3+ T-cells 1 month after transplantation, they had the fastest recovery of CD3+ T-cells compared with the other two groups (). Numbers of CD3+ T-cells on the square root scale from month 1 to month 5 suggest differences between the selected-transduced group and the other groups (one-way ANOVA p value = .09).
Figure 2 Phenotypic T-cell reconstitution. Absolute numbers of T-cells in peripheral blood prior and post-transplantation using three different sources of CD34+ peripheral blood progenitor cell (PBPCs). The gray lines with triangles represent cell counts (mean (more ...)
Faster Reconstitution of Naïve T-Cells with Transduced and Cultured PBPCs
In all groups, there was a rapid expansion of CD8+
T-cells, whereas the recovery of CD4+
T-cells showed slower kinetics. This led to an inversion of the CD4+
ratio found during the first 3 months after transplantation, as previously reported in humans [16
]. Detailed phenotyping of CD8+
T-cell subsets was performed. Optimal delineation of CD4+
naive and memory T-cells was achieved by identifying naive cells as a uniform CD95low
population as previously described [18
] (). Effector memory CD4+
T-cells were identified as CD95high β7
, and the rest of the CD4+
T-cells were defined as central memory T-cells (). Naïve CD8+
T-cells showed a homogeneous phenotype of CD95low
, and the remaining CD8+
T-cells were defined as memory phenotype [18
Figure 3 Definition of naïve and memory T-cells. Flow plots show T-cells gated based on expression of CD4 or CD8β, respectively, in a representative rhesus macaque prior to transplantation. (A): Putative naive CD4+ T-cells are apparent as a uniform (more ...)
As all animals studied were older juveniles at the time of study entry, naïve T-cells were the most prevalent T-cell phenotype both in the CD4+ and CD8+ T-cell populations prior to transplantation. Naïve, memory, and effector CD4+ T-cell counts were 722 ± 154 cells per μl, 260 ± 34 cells per μl, and 17 ± 4 cells per μl, respectively (mean ± SEM), whereas there were 251 ± 29 CD8+ naïve T-cells per μl and 110 ± 15 memory CD8+ T-cells per μl. After transplantation, there was a dramatic change in the ratio of naïve to memory T-cell pheno-type in all animals. One month after transplantation, memory T-cells were predominant in both the CD4+ and CD8+ T-cell populations (). The numbers of T-cells with a naïve phenotype increased gradually over time in all groups. The fastest recovery for both naïve CD4+ and CD8+ T-cells was observed in the animals that received a selected-transduced graft compared with the two other groups (p = .0364 for CD4 and p = .0356 for CD8) (). There were no apparent differences in T-cell recovery between the sexes included in the respective groups. However, due to the limited numbers of subjects in each group, a solid evaluation of this is not possible.
Thymic-Dependent CD4+ T-Cell Reconstitution Is Promoted by Transduction and Culture of CD34+ PBPC
To estimate thymic output, we measured TRECs within sorted CD4+ and CD8+ T-cell populations from all animals. Irrespective of the graft type, 1 month after transplantation, there were very low numbers of TRECs, with a median reduction of 22-fold compared with pretransplant levels in all animals combined (). By 3 months post-transplant, TREC levels had increased by a median of 18-fold compared with the first month. Although at the majority of early time points post-transplantation (<7 months), the selected-transduced animals had the highest numbers of TRECs in CD4+ T-cells (), only the 4-month time point reached a statistically significant difference (p = .0055). At 5−7 months, TREC levels in all groups were shown to exceed the TREC levels found at baseline, suggesting preferential colonization by recent thymic emigrants for naïve T-cell production.
Figure 4 Thymic output. Proportion of T-cells containing TRECs, as well as T-cells undergoing division pre- and post-transplantation. Gray lines represent the selected group, dotted lines represent the nonselected group, and solid lines represent the selected-transduced (more ...)
To address this further, we examined the numbers of dividing peripheral blood T-cells by staining for the nuclear cell cycle-associated antigen Ki-67. The numbers of Ki-67+ CD4+ and Ki-67+ CD8+ T-cells were less than 10% prior to transplantation in all groups (). At 1 month after transplantation, the mean Ki-67 expression levels in CD4+ and CD8+ T-cells were 37.8% and 33.9%, respectively, in the group that had received selected PBPC, 17.1% and 23.2% in the group that received an unselected graft, and 9.1% and 8% in the selected-transduced group. After the peak of Ki-67+ T-cells observed at 1−2 months in the selected and unselected groups, the numbers of Ki-67+ T-cells declined and reached low levels similar to those found in the selected-transduced group. No significant differences were found for numbers of Ki-67+ CD4+ T-cells between the groups () (p = .1099 using a random effects model). However, there were significantly fewer Ki-67+ CD8+ T-cells within the first 4 months of transplantation in the selected-transduced group compared with the two other groups combined () (p = .0356 using a random effects model). As expected, the vast majority of Ki-67+ T-cells had a memory phenotype. The lowest frequency of Ki-67+ T-cells together with the highest TREC levels observed in the selected-transduced group suggest that this group had greater thymic output of de novo-generated T-cells and thus less peripheral T-cell expansion.
Preserved Thymic Architecture in Animals Receiving Transduced/Cultured CD34+ PBPC
Histological analysis was performed on tissues obtained at necropsy 13−18 months post-transplantation to evaluate the integrity of the lymphoid organs without having to disrupt their architecture. There were no discernible differences in the architecture or cellularity of lymph nodes, tonsils, spleen, bone marrow, or Peyer's patches between the groups. Lymph nodes showed follicular and paracortical hyperplasia in all animals, and the white pulp of the spleen was hyperplastic with prominent follicles.
Notable differences were observed in the thymus between the three groups (). In the selected-transduced group, all animals showed preserved lobular architecture and well-defined cortical and medullary areas (). The thymus was composed of CD3+ T-cells, a high proportion of which were proliferating in the cortex as determined by Ki-67 staining, whereas no or few proliferating cells were noted in the medulla (data not shown). In the other two groups, the majority of the animals instead showed various degrees of thymic atrophy, that is, fat replacement, decreased thickness of the cortex, and cystic changes of the thymic epithelium (). These findings were particularly prominent in two out of three animals in the unselected group. In the latter group, the number of CD3+ T-cells was reduced, and so was the proliferative rate. The degree of thymic atrophy could not be assessed in one animal in the selected group that died of sepsis, with pleuritis and pericarditis that also incorporated the thymus 11 months after transplantation.
Figure 5 Thymic histology. H&E stains of thymus at necropsy 13−18 months after transplantation. (A, B): Representative example from selected transduced group. (C, D): Representative example from group transplanted with selected CD34+ cells. (E, (more ...)
B-Cell and NK-Cell Immune Reconstitution
B-cells, defined in our study as CD20+
cells (supplemental online Fig. 1), recovered quickly within a 2-month time period after transplantation using any of the graft regimens (supplemental online Fig. 1). For all groups, we observed supranormal absolute B-cell numbers throughout the year post-transplantation, as described earlier in clinical studies [19
]. There were no statistical significant differences in the reconstitution pattern between the groups after transplantation.
We did not detect differences in NK-cell reconstitution after transplantation between the groups. Here, we defined NK-cells as either CD3− CD14− CD56+ or CD3− CD14− CD16+ (supplemental online Fig. 1). In our study, overall, the CD56+ NK-cell population was smaller than the CD16+ NK-cell population (p = .0017 using a t test on the baseline values, pooling all the data across groups; supplemental online Fig. 1).
Immune Reconstitution of Dendritic Cell Subsets and Monocytes
We assessed the recovery of DCs and monocytes after transplantation. The two subsets of DCs were identified as CD11c+
myeloid DCs (MDCs) and CD11c−
plasma-cytoid DCs (PDCs), which are both HLA-DR+
() Here, we found that CD11c+
MDCs were the most predominant subset in blood, which has been described previously [21
] (). The absolute numbers of circulating MDCs and PDCs in all the animals combined prior to transplantation were 70 ± 20 cells per μ
l of blood and 8 ± 2.4 cells per μ
l of blood, respectively. These levels were not found to be changed at 1 month after irradiation and transplantation in any of the groups of transplanted animals (). However, 2−4 months post-transplantation, an increase in the numbers of both MDCs and PDCs was observed. The animals that received selected-transduced PBPCs showed the highest peak value of both subsets of DCs. However, this was not found to be statistically different from the other groups.
Figure 6 Fluctuations of DCs and monocytes pre- and post-transplantation. Gray lines represent the selected group, dotted lines represent the nonselected group, and solid lines represent the selected-transduced group. (A–C): Graphs show absolute numbers (more ...)
The ratio of MDCs and PDCs was reversed in lymph nodes compared with blood. Irrespective of time point pre- or post-transplant, PDCs were the predominant DC subset in lymph nodes. At 1 month post-transplantation, there was an increase in proportions of both DC subsets in the lymph nodes compared with the levels found prior to transplantation in all groups (). The frequencies of DCs were found to return to values observed pre-transplantation 3−6 months post-transplantation (). CD14+ monocytes, both in blood and in lymph nodes, showed a pattern similar to that of the DC subsets (). There was an increase above baseline in the absolute numbers of circulating monocytes within the first 5 months post-transplantation () and an increase in the percentage of monocytes residing in the lymph node within the first 3 months. This early recruitment of antigen-presenting cells (APCs) after transplantation may be important in driving T-cell development. The highest levels of DCs in blood and lymphoid tissue, as well as monocytes in lymph nodes, were found in animals that had received the selected-transduced grafts. This may account in part for the superior T-cell recovery found in this group.