Cd45.1 Cells
Arnaude Kubiak
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Allelic variants of the pan-haematopoietic cell marker CD45, identified as CD45.1 and CD45.2, have been established as a marker system to track haematopoietic cells following congenic mouse bone marrow transplants. Despite the frequent use of this model for studying the impact of genetic modifications on relative differentiation potential, it is now evident that a bias exists in CD45.1 versus CD45.2 cell reconstitution. While this bias has been demonstrated by reduced reconstitution potential in B cells of CD45.1 origin, differences in the development of other lymphocytes, as well as the impact of sex on this bias, remain uncertain. We performed bone marrow transplants with wild-type CD45.1 and CD45.2 donor cells, and characterised haematopoietic cell reconstitution in dual-expressing CD45.1/2 host mice. We report an increase in CD45.2 reconstitution in the bone marrow that persists in the spleen, thymus and blood. Through the use of CD45.1/2 hosts, we demonstrate the intrinsic bias towards CD45.2 reconstitution is independent of an immunogenic response to the CD45.1 epitope. Furthermore, we identify a sex-specific difference in reconstitution efficiencies, with female mice exhibiting a greater bias towards CD45.2 reconstitution than males. This work sheds new light on the limitations of the CD45.1/CD45.2 congenic system for tracking lymphocyte development.
CD45, also known as protein tyrosine phosphatase, receptor type C, is encoded by the Ptprc gene and is expressed on the surface of all haematopoietic cells with the exception of erythrocytes and platelets1. In addition to its use as a marker for cells of the haematopoietic lineage, the surface protein also acts as a costimulatory molecule for cell activation and signalling2,3,4. Different isoforms of CD45 have been identified in mice; the common form is CD45.2, which is expressed by C57BL/6 mice, and is encoded by the Ptprc b allele. However, an additional allelic variant, Ptprc a, which translates to the CD45.1 form of the surface protein, has been identified in the SJL mouse strain. This CD45.1 allele has been successfully backcrossed on to the BL/6 background5 and has been widely used in immunological studies to track the contribution of specific genes to haematopoietic cell development6. In a competitive congenic bone marrow transplant model, irradiated host mice are reconstituted with a chimeric 1:1 bone marrow mix from CD45.1 and CD45.2 donors, enabling the independent tracking and quantification of reconstituted haematopoietic cells from each donor. When these assays include genetically modified mice, these animals are typically available on a BL/6 background, allowing for the identification of genetically modified cells by the CD45.2 marker when reconstituted alongside wild-type cells bearing the CD45.1 epitope.
Historically, the competitive congenic model involves the use of CD45.2+ BL/6 mice as wild-type hosts, due to the ease of their availability. Although CD45.1 and CD45.2 have been accepted as being functionally equivalent, this use of CD45.2 hosts raises the possibility of bias in the model secondary to alloreactivity towards the foreign CD45.1 epitope7, 8. There have been conflicting reports of the presence or absence of alloreactivity between CD45 epitopes9, depending on the study design. As demonstrated by Basu et al., the CD45 type of the irradiated host influences the relative reconstitution of CD45.1 and CD45.2 donor cells10. While the use of CD45.1 hosts and higher dose of total body gamma radiation reportedly results in lower alloreactivity9, 11, the presence of residual host CD45.2 or CD45.1 cells following irradiation may skew results by as much as 10% in either direction, as these cells are indistinguishable from donor-derived cells bearing the same CD45 epitope. The use of CD45.1 or CD45.2 host mice may be especially poorly-suited for studies involving the T cell lineage, as these cells are particularly resistant to irradiation, enhancing the skewing of data caused by residual host cells12.
Beyond these issues of alloreactivity, functional differences in the relative homing efficiency and haematopoietic cell reconstitution potential of CD45.1 and CD45.2 have also been reported in the competitive congenic model11, 12. These differences were originally linked to a congenic interval containing over 300 genes11. More recently, Mercier et al. identified a single exon region responsible for the difference in CD45.1 and CD45.2 reconstitution12. Considering the continued popularity of this model in studies of haematopoietic differentiation potential, a more complete picture of the differences between CD45.1 and CD45.2 reconstitution efficiency is required.
In the current study, we performed competitive congenic bone marrow transplants utilising CD45.1/2 host mice, thereby eliminating any potential bias associated with alloreactivity and allowing for the clear distinction of donor cells from cells of host origin. T cell, B cell and natural killer (NK) cell populations were characterized, with the goal of clarifying the intrinsic bias in development between cells of CD45.1 and CD45.2 origins in each of these subsets. Unlike previous work in the field, we present data on CD45.1 versus CD45.2 development of lymphocyte stages in both primary and secondary lymphoid tissues, including the bone marrow, thymus and spleen. Furthermore, our use of sex-matched donor-host trios allowed for the identification of a previously unappreciated sex bias in the relative reconstitution efficiency of bone marrow progenitors from CD45.1 and CD45.2 mice, with female mice demonstrating an exaggerated bias when compared to males.
The potential influence of the CD45 variants on T cell development was also assessed through a cytometric analysis of the various stages of thymocyte development (Fig. 5A). As with the bone marrow, spleen and blood, analysis of thymocytes revealed an increased proportion of cells of CD45.2 origin compared to CD45.1 in both male and female mice that appeared to be more pronounced in females (Fig. 5B). However, as with our assessment of B cell development in the bone marrow, an examination of the proportions of T cells at each developmental stage among cells of CD45.1 and CD45.2 origin revealed no significant differences, suggesting no specific influence of these allelic variants on the efficiency of T cell development in the thymus.
We report that cells of CD45.1 and CD45.2 origins are intrinsically non-identical in their lymphocyte development potential following bone marrow transplant into CD45.1/2 hosts. We further show that the degree of this bias is different between male and female mice, with female mice showing a more dramatic difference in CD45.2 versus CD45.1 reconstitution potential than males. This sex-specific difference is primarily evident among circulating lymphocyte populations, which exhibit the greatest CD45.2 versus CD45.1 reconstitution bias. Additionally, among the increased CD45.2 cells, we have observed a preferential expansion of CD45.2+ B cells in females that results in a relative reduction in the proportion of circulating T cells. This skewing of T lymphocyte proportion is not observed to a significant degree in male mice.
Our data builds on previous findings by Waterstrat and colleagues, as well as Basu and colleagues, both of which reported a differential capacity of CD45.1 and CD45.2 cells to reconstitute transplanted mice10, 11. Earlier studies examining this issue used host mice of CD45.2 origin, which have the potential to skew results through the persistence of residual host cells following irradiation. The use of CD45.2 hosts may also influence results through immunogenic responses, resulting in increased CD45.2 cell repopulation. In the current study, this issue of immunogenicity was addressed through the use of dual-expressing CD45.1/2 mice as hosts.
Our assessment of the stages of haematopoietic progenitor cell development in the bone marrow failed to identify significant differences in the relative proportion of any particular haematopoietic progenitor cell population. This finding suggests that a large part of the observed CD45.2 versus CD45.1 reconstitution bias originates at the earliest stage of haematopoiesis and is likely a result of differential homing of donor cells with long-term repopulation potential to the bone marrow. Similarly, the populations representing the stages of B cell development in the bone marrow and T cell development in the thymus were found to be proportionally equivalent between cells of CD45.1 and CD45.2 origins, despite the overall increase in CD45.2 reconstitution. In contrast, the overall bias towards CD45.2+ cells in the circulation was enhanced by what appears to be a direct effect of the CD45 variant on the differentiation of mature B cell populations in the spleen. This difference was demonstrated by the increased proportion of follicular B cells and relative reduction in marginal zone B cells among cells of CD45.2 origin. These findings in the spleen help to explain our observation of proportionally increased B cells in the circulation, as splenic follicular B cells represent the population of B cells that enter the blood stream.
In summary, our report of this sex bias in the competitive congenic transplant model provides new insight into this experimental system and helps to clarify the specific stages of reconstitution that are impacted by variation in CD45 alleles. Of note, the current study was primarily focused on the impact of this transplant model on lymphocyte subsets, including T, B and NK cells. Although an assessment of myeloid cell types was not included in the current study, a similar strategy to the one shown here could be used to examine the impact of this model on specific myeloid subsets. This work also highlights the need for appropriate controls and careful analysis in the execution of congenic bone marrow transplant experiments. Specifically, control transplants with wild-type donor cells of both CD45.1 and CD45.2 origins are required, against which transplants using CD45.2 donor cells from genetically modified mice can be normalized. As the bias toward CD45.2 would be present in both test and control animals, calculation of the impact of specific genetic manipulations on haematopoietic potential can be determined relative to these wild-type only controls. Unfortunately, such controls are not a consistent feature of all studies making use of this transplant model. Without these controls, this system of bone marrow cell transplantation is unsuitable to study the effect of specific genetic manipulations on T and B cell development, due to the intrinsic reconstitution bias between CD45.1 and CD45.2 donors. It is notable that the NK cell subset is least influenced by the use of the congenic CD45 marker system, particularly in studies utilizing male mice, as the long-term repopulation efficiency of these cells as a proportion of donor-matched CD45+ cells remains unaltered in both male and female models.
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