The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: GJH PK CB JF. Performed the experiments: GJH ALS DHDG CC BHS AE SS JMP AJ DM. Analyzed the data: GJH DHDG JMP RM PK CB. Wrote the paper: CB JF.
The authors have declared that no competing interests exist.
A large correlation between variation in T cell subsets and hippocampal neurogenesis suggests that the immune system has an unexpectedly large influence on the brain.
Neurogenesis continues through the adult life of mice in the subgranular zone of the dentate gyrus in the hippocampus, but its function remains unclear. Measuring cellular proliferation in the hippocampus of 719 outbred heterogeneous stock mice revealed a highly significant correlation with the proportions of CD8+ versus CD4+ T lymphocyte subsets. This correlation reflected shared genetic loci, with the exception of the H-2Ea locus that had a dominant influence on T cell subsets but no impact on neurogenesis. Analysis of knockouts and repopulation of TCRα-deficient mice by subsets of T cells confirmed the influence of T cells on adult neurogenesis, indicating that CD4+ T cells or subpopulations thereof mediate the effect. Our results reveal an organismal impact, broader than hitherto suspected, of the natural genetic variation that controls T cell development and homeostasis.
In adult mice new neurons are produced in the hippocampus, where they are thought to influence learning, memory, and emotional regulation. The mechanisms and functions of this neurogenesis, however, remain unclear. Here we report that in different strains of mice, variation in cellular proliferation in the hippocampus (an index of neurogenesis) correlates with variation in the relative proportions of the ratio of CD4+ to CD8+ T cells (an immunology phenotype). We also show that T cells can influence neurogenesis (but that neurogenesis does not influence T cells) by analyzing knockouts, depleting mice of T cells, and repopulating alymphoid animals. The strong genetic correlation between T cells and cellular proliferation in the hippocampus contrasts with the weak, often non-significant, correlation with behavioral phenotypes. Of significance, the findings here suggest that modulation of the functions of the hippocampus to influence behavior is not the primary role of neurogenesis.
The discovery that neurogenesis occurs in the adult hippocampus has attracted considerable attention, yet its function remains unclear
While it is possible that the behavioural effects of neurogenesis ablation are subtle (a recent report argues that they include specific impairments in spatial discrimination
We decided to map variation in adult hippocampal neurogenesis in heterogeneous stock (HS) mice, a stock descended from eight inbred progenitor strains (A/J, AKR/J, BALB/cJ, C3H/HeJ, C57BL/6J, CBA/J, DBA/2J, and LP/J) and maintained for over 50 generations
We assessed cellular proliferation in the subgranular zone of the dentate gyrus by counting the absolute number of KI67-positive cells in 719 HS animals (KI67 is a marker of cell proliferation). The phenotypic distribution is shown in
(a) Correlations between hippocampal neurogenesis assayed by counting KI67 positive cells and phenotypes measured in outbred mice. Correlations are shown for all 97 phenotypes measured with the negative logarithm of the
We next asked whether cellular proliferation in the hippocampus was correlated with any of the other phenotypes measured in our HS screen, which were a priori unrelated to neurogenesis. Most unexpectedly, we found a highly significant positive correlation with changes in the relative proportions of CD4+ and CD8+ cells among blood CD3+ lymphocytes (
The same relationship was seen in analyses of inbred strains. Significant correlations were observed between KI67 counts and %CD8 in CD3+ blood T cells (
The striking correlation between QTLs influencing cellular proliferation in the hippocampus and variation in the distribution of T cell lineages suggests that one phenotype influences the other. We used a genetic approach to address this question and first investigated the extent to which the phenotypic correlation arises from common genetic determinants.
We mapped QTLs contributing to variation in cellular proliferation in the hippocampus
Phenotype | Chr | Start | End | RMIP |
%CD8+ in CD3 | 1 | 59.2 | 59.9 | 0.31 |
KI67 | 1 | 132.7 | 137.7 | 0.4 |
%CD8+ in CD3 | 3 | 126.9 | 129.5 | 0.59 |
KI67 | 4 | 40.4 | 49.8 | 0.26 |
%CD8+ in CD3 | 5 | 113.6 | 114.7 | 0.94 |
KI67 | 6 | 127.1 | 127.8 | 0.27 |
%CD8+ in CD3 | 7 | 47.9 | 51.8 | 0.25 |
%CD8+ in CD3 | 7 | 118.4 | 122.6 | 0.36 |
%CD8+ in CD3 | 12 | 26.6 | 30.9 | 0.77 |
KI67 | 13 | 110.6 | 119.2 | 0.49 |
%CD8+ in CD3 | 17 | 33.7 | 34.5 | 1 |
%CD8+ in CD3 | 17 | 43.6 | 49.6 | 0.58 |
The table gives the start and end coordinates of the 95% confidence intervals of each QTL (these coordinates are in megabases and are from Build 37 of the mouse genome). Entries in bold indicate QTLs where there is overlap between the two phenotypes. Our measure of probability that the locus is correctly identified is a resample model inclusion probability (RMIP) in the last column. An RMIP is the expected proportion of times a locus is included in a multilocus model. A value of 1 means that the locus would be included in all repeated analyses (estimated by re-sampling the data) and an RMIP of 0.5 means the locus is included in 50% of such analyses. Assessing false positive rates by simulation indicates that at an RMIP threshold of 0.25 about one false positive QTL occurs every four genome scans.
QTLs with the same location for both phenotypes are found on five chromosomes, 5, 6, 10, 13, and 16. However, plots of the logPs frequently identify the same peaks for both phenotypes (
The horizontal scale is the megabase (Mb) distance along the chromosome (build 37 of the mouse genome) and the horizontal scale the negative logarithm of the
There is also evidence for loci with specific effects: the single largest QTL effect on CD4+ versus CD8+ T cell subsets scarcely contributes to KI67 counts. This latter QTL lies on chromosome 17 within the Major Histocompatibility Complex (MHC) and is consistent with a strong impact of variation in the MHC in determining the relative distribution of CD4+ and CD8+ cells
Comparison between QTLs influencing behavioural measures putatively related to hippocampal neurogenesis and KI67 showed little overlap in location (results for all phenotypes are presented online at
For each pair of phenotypes we calculated the total length of the QTLs and the total length of the overlap and used a distance measure (Sørensen's similarity coefficient) to construct a tree of the relationships between phenotypes (using a clustering algorithm). Analysis of all 97 individual phenotypes shows that cellular proliferation in the hippocampus is genetically most closely related to variation in T cell subsets. We show this result in
The distance matrix is based on the degree of QTL overlap. In order to simplify the presentation, the 97 phenotypes were grouped into 16 trait sets according to the disease models that they were designed to test, or on their physiological functions (for instance, combining all QTLs for T cell function or for hippocampal function). Species typical behaviour, burrowing, has been shown to be affected by hippocampal lesions yet does not reflect emotional reactivity
Our genetic data are consistent with a model in which adult neurogenesis influences T cells and also with one in which T cells influence neurogenesis (where our measure of neurogenesis is cellular proliferation in the hippocampus). We tested the first model by ablating adult neurogenesis and then looking for changes in T cells. We did this using i.c.v. ganciclovir (GCV) in both GFAP-tk
We tested the second hypothesis, an effect of T cell function on neurogenesis, by examining a series of mouse mutants with alterations in T cell differentiation. As well as the cell proliferation marker KI67, we used DCX to detect hippocampal neurogenesis in 10-wk-old mice. We first investigated Rag-deficient mice, which are devoid of any B or T lymphocytes: mutant mice showed a marked reduction in both readouts of neurogenesis (KI67 and DCX) (
(A) Effect of RAG knockout and corresponding littermate wild types (
To determine which of the CD4+ or CD8+ lineages of αβT cells had the most influence, we tested knockout mice affecting MHC-I or MHC-II molecules (which are largely devoid of CD8+ and CD4+ T cells, respectively) as well as double-knockout animals. A reduction in KI67 and DCX was present for both MHC-II (
We performed two independent tests of the functional involvement of CD4+ and CD8+ T cells. First, we depleted either population by injection of anti-CD4 or anti-CD8 monoclonal antibodies, reducing their numbers to less than 5% of untreated mice at 10 d post-treatment. Despite the successful depletion of T-cell subsets, there was no significant change of KI67 levels in the dentate gyrus of the hippocampus (
(A) The KI67 positive cell counts in the dentate gyrus of the hippocampus 10 d after CD4, CD8, or CD4 and CD8 T cell depletion by antibody injection. Each group consisted of eight animals. (B) The KI67 positive cell counts in the hippocampus after 2 wk of reconstitution of TCRα knockout mice with CD4 or CD8 lymphocytes by intraperitoneal injection of 2×107 cells/mouse. Each group consisted of six animals. Values shown are means ± SEM.
Our analysis of a large stock of outbred mice makes two novel observations about adult neurogenesis in the hippocampus. First, we find a correlation between variation in cellular proliferation in the hippocampus and the relative proportions of CD4+ and CD8+ subsets of αβT cells. The correlation is much larger than that found for over 90 other phenotypes. Second, the correlation is driven by genetics: QTLs that contribute to variation in cell proliferation in the hippocampus also contribute to variation in the relative proportions of CD4+ and CD8+ T cells. Importantly, the genetic correlation is due to natural sequence variants that differentiate inbred strains of laboratory mice.
Our findings extend previous observations that T cells contribute to variation in adult neurogenesis
Results obtained from the HS indicate that the relationship between T cells and cellular proliferation in the hippocampus is not merely an artefact of transgenic or knockout mice, or of the very unusual conditions created in alymphoid mice upon immune reconstitution, but arises from the far more subtle cues of natural genetic variation: loci that contribute to variation in cell proliferation in the hippocampus also contribute to variation in the relative proportions of CD4+ and CD8+ T cells. Naturally occurring genetic variation is a very different setting (and obviously more physiogically relevant) than the gross abnormalities of knockout mice.
Two questions follow: why do T cells influence neurogenesis in the hippocampus, and how do they do it? Our data suggest a broader role for T cell function on adult neurogenesis than previously suspected
How do T cells influence neurogenesis in the hippocampus? This is difficult to explain, given that T cells are very rare in the brain under normal circumstances. Moreover genetic mapping experiments, and those performed in knockout or reconstituted mice, appear to give different clues about mechanism: analysis of the HS mice pointed to the involvement of CD8+ T cells, as their frequency correlated with increased neurogenesis, while the analysis of knockouts and repopulation experiments implicated CD4+ T cells, here and in the work of Wolf et al.
An important clue comes from the delineation of the QTLs that affect both T cell and neurogenesis phenotypes. All QTLs that control the proportions of CD4+ and CD8+ T cells also have an impact on neurogenesis, with the glaring exception of the strongest of them, the H2-Ea locus
In regards to the apparent discrepancy between the impact of natural genetic variation and the results of experimental manipulation, it is important to realize that different populations are being affected in the different modalities. CD4+ T cells are a complex mix of cells. Some of them have positive effector functions, while others exhibit regulatory potential over other cells of the immune system and also over non-lymphoid organs such as the adipose tissue (in particular FoxP3+ “Treg” cells
In cellular terms, how A-restricted effector or regulatory T-cells influence hippocampal activity remains conjectural. One might hypothesize direct interactions occurring in the hippocampus between CD4+ T cells and MHC-II+ microglia. CD4+ T cells are very rare but not completely absent in this anatomical location. Alternatively, the hippocampus may be responding to long-range mediators released by T cells, for instance the cytokines released during T cell activation in lymphoid organs.
One candidate for the molecular mechanism set in motion by T cells is the Wnt pathway
We expect clues to their identity to emerge from identifying the molecules under the QTLs that co-regulate T cells and cellular proliferation in the hippocampus. Mapping resolution in the HS is approximately 3 Mb, insufficient to formally identify single genes. However, it is worth noting that the locus on chromosome 6 contains the
The interaction between neurogenesis and T cell subsets is another example of neural-immune communication across the blood brain barrier: it is now clear that this happens outside of disease states
Our insights into the role of T cells in adult neurogenesis depended upon access to multiple phenotypes obtained in a set of genotyped animals. The delineation of biological control pathways is hard to achieve through experiments that test one phenotype, and one gene, at a time. The approach used here is likely to become more common with the availability of genetic reference populations, such as the collaborative cross
Original Northport HS mice were obtained from Dr. Robert Hitzemann at the Oregon Health Sciences Unit. Inbred strains (A/J, AKR/J, BALB/cJ, C3H/HeJ, C57BL/6 J, CBA/J, DBA/2 J, and LP/J) were obtained from Harlan (UK) or the Jackson Laboratory (Bar Harbor, ME). TCRα (Philpott PMID: 1604321), RAG (PMID: 1547488), and MHC (Aβ and β2M, Cosgrove PMID: 1909605 and Koller PMID: 2682666, respectively) deficient mice were from our custom-breeding colony housed at the Jackson; wild-type controls used as comparators were littermates from the same crosses. Neurogenesis parameters were analysed in 10-wk-old animals. All mice were maintained in a specific pathogen free facility under barrier conditions.
All behavioural and other assays performed in the HS animal are described in
Ablation of adult neurogenesis was performed with both cellular and temporal specificity using the nestin-tk mouse
For KI67 staining, sections were mounted on the superfrost slide (BDH, UK), dried overnight, incubated in the 0.01 mol/L citric buffer for 40 min at 90, incubated in 3% H2O2 for 10 min, rinsed, and incubated overnight at room temperature with rabbit anti-KI67 antibody (1∶4000, Vector Lab). Next day, a standard rabbit IgG ABC kit procedure was used and reacted 5–10 min with Sigma DAB tablet. Sections were then counterstained with cresyl violet and cover-slipped under DPX. KI67-labeled cells were counted on every eighth section through the entire rostrocaudal extent of the granule cell layer bilaterally. Section thickness is 40 µm. For DCX staining, free floating sections were incubated with goat anti-DCX antibody (1∶400, Santa Cruz Labs), then following the same ABC kit procedure, reacted with Sigma DAB tablet. DCX-labeled cells were counted on every sixteenth section through the entire rostrocaudal extent of the granule cell layer bilaterally. Section thickness is 40 µm.
Blood samples (100–150 µl) were gathered in EDTA via tail-vein phlebotomy. Erythrocytes were lysed in 5 rounds of incubation with ACK lysis buffer followed by centrifugation. Blocking in Fc solution was followed by incubation with CD3-PE, CD4-APC, and CD8 FITC (eBiosciences) and two rinses in FACS buffer. Flow cytometry was then performed on a BD FacsCanto II instrument with FACS Diva software (BD Biosciences). Counts of CD4 and CD8 positive cells were gated on the CD3 population, and all percentages are expressed as a fraction of CD3+ cells.
C57BL6/J mice (
Single cell suspensions of splenocytes from C57BL6/J donors were subjected to magnetic bead cell sorting using anti-CD4 or anti-CD8α paramagnetic beads (Miltenyi-Biotech) and positively sorted with the AutoMACs cell separation equipment (Miltenyi-Biotech) according to the manufacturer's instructions. Briefly, cells were incubated with beads on ice for 20 min in PBS 2 mM EDTA washed and applied to the AutoMACS, and positive and negative fractions were collected and tested for purity prior to transfer (all transferred populations were >96% pure as judged by FACS). Sorted cell populations were washed by centrifugation in DMEM, resuspended to 8×107/ml and administered to TCRα KO mice (
ganciclovir
heterogeneous stock
Major Histocompatibility Complex
quantitative trait loci
resample-based model inclusion probability