The frequency of double-positive thymocytes expressing an αβ TCR clonotype regulates peripheral CD4 T cell compartment homeostasis

Abstract

The present study aimed to determine whether the frequency of double positive (DP) thymocytes expressing αβ T-cell receptor (TCR) clonotypes at the time of selection regulates peripheral CD4 T-cell compartment size. Scid recipients were inoculated with various ratios of TCR Cα^0/0 and wild-type bone marrow (BM) stem cells. Increasing the frequency of TCR Cα^0/0 thymocytes at steady-state introduced a graded decrease in the maturation probability of the total DP thymocyte pool. At 12–14 weeks following BM inoculation, the frequency of TCR Cα^0/0 DP thymocytes was inversely correlated with that of CD4 single positive (SP) thymocytes. Notwithstanding, a decreased frequency of wild-type DP thymocytes led to a marked increase in their transit efficiency from the DP to SP compartments. The frequency-dependent increase in thymocyte transit efficiency was associated with a CD4 SP cell surface phenotype indicative of increased antigenic experience. Importantly, the frequency of DP thymocytes capable of expressing TCR clonotypes dictated the steady-state size of the peripheral CD4 T cell compartment and its potential for homeostatic proliferation. Collectively, these results indicate that the efficiency of DP to CD4 SP transit is a frequency dependent process, which determines (1) the steady-state size of the peripheral T cell compartment and (2) the threshold for homeostatic expansion of peripheral CD4 T cells.

Introduction

It is well established that adoptive transfer of mature peripheral T cells into a T-cell deficient host causes them to undergo polyclonal expansion.^1–3 Irrespective of the initial number of cells transferred, the T-cell population eventually reaches a plateau. This self-renewal process has been termed ‘homeostatic proliferation’ and is thought to be an important parameter assuring the maintenance of a stable peripheral T cell compartment size over time.³ Homeostatic expansion of naive T cells under lymphopenic conditions occurs upon T-cell receptor (TCR) interaction with a similar range of self-major histocompatibility complex (MHC) peptide combinations driving positive selection in the thymus.^4,5 Moreover, the cytokines interleukin (IL)-7 and IL-15 are critical in the regulation of homeostatic T-cell proliferation, both by naive and antigen-experienced T cells.^6–10 Implicit to the phenomenon of homeostatic proliferation is that peripheral T cells possess the ability to sense their own population's density. There is strong evidence that the avidity of TCR–MHC interaction and the baseline level of available IL-7 and IL-15 are critical ‘resources’ regulating this process.³ Additionally, evidence from transgenic systems indicates that the specificity of any particular TCR clonotype influences the T cell's propensity to undergo homeostatic expansion.^4,11 Thus, it is logical to hypothesize that any parameter(s), which control the range of selectable TCR clonotypes during thymic development, would dictate peripheral compartment size and the repertoire's propensity for homeostatic proliferation under conditions of lymphopenia.

The present study was undertaken to determine whether the density of αβ TCR bearing double positive (DP) thymocytes during thymic selection influences (1) CD4 single positive (SP) thymocyte selection efficiency, (2) the steady state size of the peripheral CD4 T cell compartment and (3) the propensity of peripheral CD4 T cells for initiating homeostatic expansion. Thus, we generated bone marrow (BM) chimeric mice in which the frequency of αβ TCR bearing DP thymocytes was decreased within a defined range and determined the homeostatic properties of the resultant peripheral CD4⁺ T cell compartment at steady-state.

Materials and methods

Mice

Wildtype, Scid, Thy1.1 congenic, Ly5.1 congenic and TCR Cα^0/012 C57BL/6 mice were purchased from the Jackson Laboratories (Bar Harbor, ME). All mice were housed under specific pathogen-free barrier conditions.

Generation of BM chimeric mice

BM cells were isolated from Thy1.1 congenic and TCR Cα^0/0 C57BL/6 mice. T and B lymphocyte depleted mixed inocula of Thy1.1 congenic and TCR Cα^0/0 bone marrow cells at various ratios, ranging from 1 : 0 to 1 : 30, were prepared. Various cohorts of Scid recipient mice were intravenously (i.v.) inoculated with a cell preparation containing the different ratio cell preparations. The recipients were allowed to reconstitute their immune systems for 12–16 weeks prior to analysis.

Flow cytometry

Cells (1 × 10⁶) were surface stained with various combinations of the following the following antibodies: 2C11 (anti-CD3), 53-7.1 (anti-CD5), GK1.5 (anti-CD4), 53-6.7 (anti-CD8a), IM7 (anti-CD44), A20 (anti-CD45.1), HIS51 (anti-CD90·1), and F23.1 (anti-TCRVβ8) (BD Biosciences Pharmingen, San Diego, CA). All samples were analysed on a FACSCalibur (Becton Dickinson, Mountain View, CA) using Cellquest software. 50 000–500 000 events were collected within a live lymphoid gate set based on forward and side scatter.

BrdU labelling and analysis of DP→CD4 SP thymocyte transit efficiency

BM chimeric mice were intraperitoneally (i.p.) injected with 200 µl of 5-bromodeoxyuridine (BrdU) (Sigma Chemical Co., St. Louis, MO) in phosphate-buffered saline (PBS) every 12 hr for 3 days. The mice were then killed and thymocytes were isolated and their absolute number determined. Cells were then subjected to surface staining with anti-CD4 and anti-CD8 monoclonal antibodies (mAbs). The cells were then fixed, permeabilized and stained with anti-BrdU fluoroscein isothiocyanate (FITC) using the FITC BrdU Flow Kit (BD Biosciences Pharmingen). Following the staining procedure, 1 × 10⁵−1 × 10⁶ events were acquired using a FACSCalibur and analysed using the CellQuest Software. The following ratio was calculated as a measure of DP→CD4 SP transit efficiency over the 3-day labelling period:

The advantage of using this ratio is that it delineates transit efficiency within the subset of thymocytes generated in each compartment within the 3-day BrdU labelling window; thereby eliminating any artefacts introduced as a result of changes in the duration of CD4 SP thymocyte persistence in the BM chimeric thymi.

Homeostatic proliferation assay

Lymph node cells from the various BM chimeric mice were isolated and labelled with 5-(and 6-) Carboxyflourescein diacetate succinimidyl ester (CFSE), as previously described.¹³ CFSE labeled lymph node cells (10 × 10⁶) from an individual mouse were then i.v. injected into 600 rad irradiated wild-type C57BL/6 recipients. 11–12 days later the recipients were killed, their splenocytes were isolated, stained with anti-CD90.1, anti-CD4 and anti-CD8 and analysed using flow cytometry, as described above. By using CFSE to track lymphocyte division history, this approach allowed quantification of the extent of homeostatic CD4 T-cell division in the recipient mice.

In vitro proliferation assay

Lymph node cells from the various BM chimeric mice and Ly5.1 congenic C57BL/6 were mixed at a 1 : 1 ratio and CFSE labelled, as above. The various cell preps were then plated in 24-well flat-bottom plates at a density of 2 × 10⁶ total cells per well and cultured with soluble anti-CD3 (2C11) at concentrations ranging from 0 to 0·5 µg/ml. After a 65–70 hr incubation period at 37°, the cells were harvested and stained with anti-CD45.1, anti-CD8 and anti-CD4 to distinguish between the chimeric (Ly5.2⁺) and wild-type (Ly5.1⁺) T cells. The cells were then analysed by flow cytometry as before, in order to quantify the extent of cell division in the CD4⁺ gate.

Statistical analyses

In the present study, both raw and derived data points are presented as a function of Cα^0/0 or wild-type DP thymocytes. Each point is representative of data obtained from a single mouse. The trend lines presented are the ones best fit to each data spread and are calculated using the statistical software included with Microsoft Excel version X. The best-fit line was selected based on an r² value closest to 1.

Results

Bone marrow chimeric mice with varying frequencies of αβ TCR bearing DP thymocytes

C57BL/6 scid mice were injected with a mixed inoculum containing 1 × 10⁷ BM stem cells from syngeneic TCR Cα^0/0,¹² and wild-type C57BL/6.Thy1.1 congenic donors at different ratios (i.e. 0 : 1, 5 : 1, 10 : 1, 20 : 1, 30 : 1 and 1 : 0). By introducing increasing frequencies of TCR Cα^0/0 thymocytes, a graded decrease in the maturation probability of thymocytes at the population level was engineered at the DP stage. These mice were allowed to reconstitute for up to 4 months prior to analysis. Thymocytes from these mice were isolated, stained, and analysed by flow cytometry to determine the frequency of wild-type Thy1.1⁺ DP thymocytes in each group (Fig. 1a). The frequency of TCR wild-type DP thymocytes correlated with the frequency of their BM progenitors in the reconstituting inoculum. All CD4 and CD8 single positive (SP) thymocytes present in the thymus were Thy1.1⁺, as expected (data not shown). As expected, reducing the frequency of wild-type DP thymocytes led to a decrease in the frequency of CD4 SP thymocytes (Fig. 1b), presumably reflecting a decreased steady state production rate.

Figure 1.

Analysis of thymocyte subsets in mixed bone marrow chimeric mice. (a) Flow cytometric analysis of thymocytes stained with anti-Thy1.1 (CD90.1), anti-CD4 and anti-CD8. The ratios above each dot plot are that of Wild-type (Thy1.1⁺) : TCR Cα^0/0 BM cells in the reconstituting inoculum used to generate the chimera. The histograms show the frequency of wild-type, Thy1.1⁺ thymocytes present in the DP thymocyte gate. The presented data are representatives from a group of 50 total chimeric mice generated in the various ratios. (b) Frequency of CD4 SP thymocytes as a function of the percentage of wild-type, Thy1.1⁺ DP thymocytes present at 12–16 weeks following reconstitution. Each dot represents data collected from a single BM chimeric mouse.

Thymocyte DP→CD4 SP transit efficiency as a function of TCR αβ DP thymocyte frequency

We next asked whether modulating the frequency of wild-type DP thymocytes has an effect on the overall efficiency of CD4 SP T-cell selection. The ratio of DP : CD4 SP thymocytes can be utilized as one measure of selection efficiency. An increase in this ratio from that seen normally indicates a relative inefficiency in the DP→SP transition, assuming that persistence time for individual CD4 SP cells in the thymus does not vary drastically as a result of our experimental manipulation in the chimeras. This issue can be addressed using BrdU labelling. The ratios calculated in our chimeric mice were standardized against the average ratio calculated for a group of 10 wild-type C57BL/6 mice and the data presented as percentage of wild-type. As expected, the presence of developmentally arrested Cα^0/0 DP thymocytes increased the total DP : CD4 SP ratio (Fig. 2a) relative to that seen in wild-type mice. This result indicates a graded inefficiency in DP→CD4 SP transit as a function of the frequency of TCR Cα^0/0 DP thymocytes. Gating on the wild-type, Thy1.1⁺ thymocytes only (i.e. excluding the TCR Cα^0/0 derived thymocytes from the analysis of transit efficiency using the DP : CD4 SP ratio) demonstrated that as the frequency of wild-type DP thymocytes decreased, the ratio of wild-type DP : CD4 SP thymocytes also decreased relative to that seen in wild-type mice (Fig. 2b). This result indicated that transit efficiency from the wild-type DP→CD4 SP thymocyte compartments increased as wild-type DP thymocytes became less frequent. In order to directly examine the transit efficiency of recently generated wild-type DP thymocytes to the CD4 SP compartment, we performed a 3 day BrdU labelling of a cohort of the BM chimeric mice. The ratio of wild-type DP:CD4 SP thymocytes in the BrdU⁺ gate was calculated. Figure 2(c) demonstrates that this ratio decreases as the frequency of TCR Cα^0/0 DP thymocytes is increased, confirming an enhanced efficiency of transit. Importantly, the potential confounding effect of homeostatic proliferation of CD4 SP thymocytes in the ‘lymphopenic’ setting on the use of this ratio was considered. Twenty-four hr BrdU pulsing of three chimeric mice at the 1 : 10 ratio demonstrated no appreciable difference in the proportion of labelled CD4 SP cells in the chimeras as compared to controls (12 ± 3% in chimeric versus 14 ± 4% in control mice; n = 3). Therefore, we were reassured that homeostatic proliferation of newly emerging CD4 SP T cells was unlikely to be a confounding factor in utilizing the ratio of BrdU⁺, wild-type DP:CD4 SP over the 3-day labelling period in which we compared the efficiency of thymic selection in the various chimeric mice.

Figure 2.

The effect of decreasing the frequency of wild-type DP thymocytes on CD4 SP T cell selection. The ratio of total DP (i.e. both TCR Cα^0/0 and wild-type derived) (a) and wild-type, Thy1.1⁺ DP (b) thymocytes to total CD4 SP thymocytes in the various BM chimeric mice was calculated as a percentage of that normally seen in 8–10 week old C57BL/6 thymi. These variables were plotted as a function of the frequency of wild-type DP thymocytes (a and b). (c) The ratio of the absolute number of wild-type (Thy1.1⁺), BrdU⁺, DP to CD4 SP thymocytes as a function of the frequency of TCR Cα^0/0 DP thymocytes in the BM chimeric mice. Individual dots represent values obtained from the analysis of a single chimeric mouse injected with BrdU for 3 days.

Cell surface TCR complex expression by CD4 SP thymocytes as a function of wild-type DP thymocyte frequency

We determined that modulating the frequency of wild-type thymocytes in the DP compartment leads to appreciable changes in the level of TCR complex expression in the CD4 SP compartment. Figure 3 demonstrates that the level of CD3ε and TCR Vβ8 expression on the cell surface of CD4 SP thymocytes decreases upon decreasing the frequency of wild-type DP thymocytes. In these analyses the mean fluorescence intensity (MFI) obtained for these markers on each population analysed was standardized against that seen in a group of control wild-type C57BL/6 mice, allowing us to present the data as a percentage of wild-type.

Figure 3.

Relative TCR complex expression levels on the CD4 SP thymocyte in BM chimeric mice. Using flow cytometry, the mean fluorescence index (MFI) for CD3ε and TCR Vβ8 was determined for CD4 SP thymocytes in the chimeric mice. In order to compare this variable among mice analysed in different experiments, the MFIs obtained for each of the chimeric mice were standardized as a percentage of that seen in a group of two to three wild-type mice analysed in each experiment. This percentage was then plotted as a function of the frequency of wild-type (Thy1.1⁺) DP thymocytes seen in each chimera. Individual dots represent each of the analysed mice.

Steady state peripheral CD4 T cell compartment size and phenotype as a function of wild-type DP thymocyte frequency

Early in the analyses, the frequency of mature Thy1.1⁺ T cells found in the peripheral lymphoid organs of chimeras starting at 8 weeks following BM inoculation was found to vary depending on the input ratio of the BM inoculum. This finding was initially attributed to a decreased T-cell production rate requiring a longer period for the immune system to achieve its steady state. However, when the chimeric mice were examined at 12–20 weeks of age an identical pattern was observed. Figure 4(a) is a representative profile of peripheral CD4 T cell frequencies in the spleen and lymph nodes of mice reconstituted with the various BM inocula at approximately 15 weeks following reconstitution. The frequency of peripheral CD4 T cells present at steady-state was correlated with the frequency of wild-type DP thymocytes in the same mice (Fig. 4b). Here we used the ratio of CD4:non-T cells (using a CD4^– CD8^– gate to define the non-T cells) and standardized this ratio to that seen in a group of 10 age- and sex-matched C57BL/6 mice in order to express the data as a percentage of wild-type. This parameter was directly correlated with the frequency of wild-type DP thymocytes in each chimeric mouse analysed.

Figure 4.

The frequency of CD4⁺ T cells in secondary lymphoid organs of the chimeric mice at 12–16 weeks following reconstitution. (a) Histograms exhibit representative CD4⁺ T-cell frequency distributions found within live lymphoid cell gates of lymph node and splenocyte populations isolated from the various chimeras. The ratios noted are those of wild-type (Thy1.1) : TCR Cα^0/0 BM stem cell preps used to reconstitute the chimeric mice. (b) The ratio of CD4⁺ T cells : non-T cell in live lymphoid gates of lymph node cells and splenocytes from the BM chimeric mice presented as a percentage of that seen in wild-type C57BL/6 mice. The percentages are plotted as a function of the frequency of wild-type DP thymocytes.

We also analysed the surface phenotype of the wild-type lymph node CD4 T cells in the chimeric mice in order to determine the impact of modulating the frequency of wild-type DP thymocytes on the phenotype of the peripheral CD4 compartment (Fig. 5). A similar pattern as that shown in Fig. 5 for lymph node cells was observed in the case of splenic CD4 T cells (data not shown). Similarly to our approach in presenting Fig. 3, we standardized the mean fluorescence intensity (MFI) for each marker (i.e. CD3ε, TCR Vβ8, CD5 and CD44) against that seen in a group of control C57BL/6 mice. Figure 5(a) demonstrates that as the frequency of wild-type DP thymocytes decreases, the level of TCR complex (as measured by CD3ε and TCR Vβ8) present on the cell surface of lymph node CD4 T cells at steady state is diminished. Moreover, the level of cell surface CD5 expression by peripheral CD4 T cells appears to be inversely correlated with the frequency of wild-type DP thymocytes (Fig. 5b). Finally, the level of CD44 expression by peripheral CD4 T cells is also inversely correlated with the frequency of wild-type DP thymocytes (Fig. 5c). Indeed, as the frequency of wild-type DP thymocytes decreases in our chimeric system a greater proportion of the peripheral CD4 T cells at steady state exhibit a CD44^hi surface phenotype. Collectively, these data indicate that decreasing the frequency of wild-type DP thymocytes might impact the baseline stimulation experienced by the steady state CD4 T cell compartment.

Figure 5.

Phenotypic analysis of lymph node CD4+ T cells in the bone marrow chimeric mice. Using flow cytometry, the mean fluorescence index (MFI) for CD3ε (a), CD5 (b), CD44 (c) and TCR Vβ8 (a) were determined for lymph node CD4⁺ T cells in the chimeric mice. In order to compare each of these variables in the various mice analysed in different experiments, the MFIs obtained for each of the chimeric mice were standardized as a percentage of that seen in a group of two to three control wild-type mice analysed in each experiment. This percentage was then plotted as a function of the frequency of wild-type (Thy1.1⁺) DP thymocytes seen in each chimera. Individual dots represent each of the analysed mice. The histograms presented in (c) are representative profiles of CD44 expression by lymph node CD4⁺ T cells from chimeric mice reconstituted at the stated ratio. The dotted line is drawn as a reference to allow comparison of CD44 expression levels exhibited by CD4⁺ T cells from the various chimeric mice.

CD4 T cell compartment homeostatic proliferation potential as a function of the frequency of wild-type DP thymocytes

We determined whether the frequency of wild-type DP thymocytes impacts the potential of the peripheral CD4 T-cell compartment to undergo homeostatic proliferation upon adoptive transfer into sublethally irradiated mice. CFSE-labelled lymph node T cells from the various chimeras (5 × 10⁶) were adoptively transferred into sublethally irradiated syngeneic, Thy1 disparate (i.e. Thy1·2), C57BL/6 recipients. After 11 days, splenocytes from these mice were isolated and analysed by flow cytometry gating on the adoptively transferred CD4⁺/Thy1.1⁺ cells. As shown in Fig. 6(a), the percentage of the adoptively transferred CD4 T cells found in the divided gate (divisions 1–4) decreases appreciably as a function the frequency of wild-type DP thymocytes. Figure 6(b) summarizes the ratio of homeostatically activated (i.e. divisions 1–4) daughter cells to undivided CD4 T cells as a function of the frequency of wild-type DP thymocytes in the mice from which the transferred cells were derived. It is important to note that the relative frequency of cells found in the rapidly dividing CD4 T cell population (i.e. those in divisions >4) appeared variable and did not follow a trend as a function of the frequency of wild-type DP thymocytes. The daughter cells at divisions >4 were, thus, excluded from this analysis.

Figure 6.

Homeostatic proliferation by CD4⁺ T cells from BM chimeric mice adoptively transferred into 600 rad irradiated wild-type C57BL/6 recipients. (a) Representative division profiles of homeostatically proliferating CD4⁺ T cells isolated from BM chimeric mice reconstituted at the indicated ratios. (b) Ratio of homeostatically divided to undivided CD4⁺ T cells as a function of the frequency of wild-type DP thymocytes.

Peripheral CD4 T-cell activation threshold is not modulated by the frequency of wild-type DP thymocytes

We next determined whether the frequency of DP thymocytes capable of expressing a TCR clonotype impacts the activation threshold of peripheral CD4 T cells. To this end, we used soluble anti-CD3 (2C11) to stimulate mixed cultures containing CFSE-labelled lymph node cells isolated from wild-type, Ly5.1 congenic B6 mice and the various chimeric mice. Following a 72 hr culture period, we analysed the cultured cell populations by flow cytometry and compared the extent of CD4 T cells in the Ly5.1⁺ (i.e. wild-type derived) and Ly5.2⁺ (i.e. chimeric derived) populations. Figure 7 demonstrates that no appreciable difference in the dose responsiveness of chimeric versus wild-type CD4 T cells to soluble anti-CD3 was detectable, as exemplified here in the case of 1 : 5 and 1 : 20 chimeras.

Figure 7.

In vitro activation of peripheral CD4⁺ T cells from BM chimeric mice. Dose–response of cocultured CD4⁺ lymph node T cells from Ly5.1 congenic C57BL/6 and the various BM chimeric mice stimulated with soluble anti-CD3 for 65 hr. The division profiles of CD4 T cells from a representative 1 : 5 and 1 : 20 BM chimera is compared to that of cocultured wild-type CD4 T cells. The dotted line is drawn after division 1 and provides a visual aide for comparison.

Discussion

The total number of peripheral T cells is maintained at a relatively constant level throughout the life of mammalian organisms. This constancy in the absolute number of T lymphocytes is regulated by a combination of thymic production¹⁴ and self-renewal.³ With respect to thymic production, unlike erythropoiesis, it has been determined that the rate of T-cell production is not under tight feedback regulation from the periphery.^14,15 Furthermore, irrespective of the number of T cells produced per day, the periphery can only retain a relatively constant number of newly emerging T cells.¹⁴ As the rate of T-cell production decreases consequent to thymic atrophy, it is thought that the peripheral T-cell compartment's ability to self-renew permits the maintenance of a steady-state T-cell compartment size. Indeed, it is established that through a combination of TCR/MHC and IL-7/IL-15 interactions the T lymphocyte compartment has the ability to sense ‘space’ and homeostatically expand in the setting of lymphopenia.³ It is now clear that both TCR/MHC interaction and IL-7/IL-15 are necessary for the initiation of homeostatic proliferation by naive peripheral CD4 T cells. Nevertheless, studies utilizing transgenic T cells have demonstrated that certain TCR clonotypes do not undergo homeostatic proliferation.^4,11,16 These results indicate that the sensitivity of the peripheral T-cell compartment to homeostatic expansion may be a function of the range of selected TCR clonotypes. Therefore, it is possible that microenvironmental cues, which modulate TCR clonotype selection efficiency would, by extension, regulate peripheral T-cell compartment size and the propensity for homeostatic proliferation. An elegant recent study by Canelles et al.^17,18 demonstrated that the frequency of αβ TCR-bearing DP thymocytes is a parameter, which at the population level, dictates the efficiency of transgenic TCR clonotype selection and CD4 versus CD8 lineage commitment. Two related studies by the Rudensky and von Boehmer groups demonstrated that the frequency of a transgenic TCR clonotype present during selection modulates the efficiency with which these cells mature.^19,20 Our study also aimed to determine whether the frequency of αβ TCR clonotype bearing DP thymocytes impacts CD4 T cell selection efficiency, in addition to peripheral compartment size and homeostatic proliferation potential; albeit, in the case of a non-transgenic T cell compartment.

The presented results demonstrate that decreasing the frequency of αβ TCR bearing DP thymocytes precipitates a marked increase in SP thymocyte selection efficiency. This finding is correlated with a CD4 SP T-cell surface phenotype indicative of increased antigenic stimulation as a function of DP thymocyte frequency. Indeed, lower TCR and increased CD5 cell membrane levels on mature CD4 SP, as a function of decreasing intrathymic competition, may indicate that on a per cell basis a stronger signal is delivered to developing thymocytes. Furthermore, that this observation correlates with an increased efficiency of selection may be caused by a lowered threshold for selection in the face of decreasing interclonotype competition at the DP stage. Importantly, this latter parameter also seems to regulate the steady state size of the peripheral T-cell compartment, with lower DP thymocyte frequencies dictating a correspondingly smaller peripheral T-cell compartment size, despite ongoing thymic production up to 12–16 weeks following BM reconstitution. This observation was surprising given the expectation that naive CD4 T cells would undergo homeostatic proliferation to fill the empty peripheral niche to a normal level. That a CD4 T cell compartment size comparable to that of wild-type mice was not attained, despite ongoing T-cell production, suggested that the frequency of TCR bearing DP thymocytes might ‘imprint’ the peripheral compartment's size and propensity for undergoing homeostatic proliferation. Indeed, peripheral CD4 T cells from the lymphopenic chimeras underwent fewer total divisions to generate daughter cells when adoptively transferred into sublethally irradiated syngeneic hosts. Whether this effect resulted from a shift in the range of selectable TCR clonotypes under low frequency selection or due to the parallel decrease in the level of cell surface TCR complex expression remains unclear. Notwithstanding, despite its effect in modulating thymocyte selection efficiency, peripheral T-cell compartment size and cell surface TCR complex expression level, thymocyte frequency does not appear to ‘tune’ the activation threshold of peripheral CD4 T cells when stimulated with soluble anti-CD3 in vitro. This finding suggests that, on a per-cell basis, the intrinsic activation threshold of peripheral CD4 T cells is not a function of thymocyte frequency at the time of selection and may be ‘hard-wired’.

Overall, this study demonstrates that the efficiency of thymic clonotype selection is not a cell-autonomous event but, rather, occurs in a frequency dependent fashion. Moreover, the steady state frequency of DP thymocytes appears to regulate peripheral T cell compartment size and its propensity for self-renewal in the face of lymphopenia. These results suggest that interclonal DP thymocyte competition for thymic stromal elements (e.g. MHC-peptide ligands, IL-7, etc.) regulates the probability of successful clonotype selection and governs the overall size and self-renewal capacity of the peripheral repertoire.