Mathematical Modeling Reveals the Biological Program Regulating Lymphopenia-Induced Proliferation

Mice

C57Bl6/J wild-type (WT), Ly5.1 C57Bl/6J, RAG-1-deficient (Rag1^−/−) mice, Il7^−/− (The Jackson Laboratory), F5 Rag1^−/− mice, and breeding combinations thereof were bred in a conventional colony free of pathogens at the National Institute for Medical Research (London, U.K.). All lines used were of H-2^b haplotype. Animal experiments were done according to institutional guidelines and Home Office regulations.

Flow cytometry

Flow cytometry was conducted using 2–5 × 10⁶ lymph node or spleen cells. Cell concentrations were determined using a Scharf Instruments Casy Counter. Cells were incubated with saturating concentrations of Abs in 100 μl of PBS-BSA (0.1%)-azide (1 mM) for 1 h at 4°C followed by three washes in PBS-BSA-azide. Monoclonal Ab used in this study were as follows: allophycocyanin-TCR (H57–597) (BD Pharmingen), allophycocyanin-CD44 (Leinco Technologies), bio-CD44 (BD Pharmingen), and PerCP- and PE-CD8 (BD Pharmingen). Biotinylated mAb staining was detected using PerCP steptavidin (BD Pharmingen) and PE-TR steptavidin (Caltag Laboratories). Four- and five-color cytometric staining was analyzed on a FACSCalibur and LSR Instruments (BD Biosciences), respectively, and data analysis was performed using FlowJo V8.1 software (TreeStar).

Labeling and adoptive transfer of T cells

Lymphocytes were teased from lymph nodes and spleen of donor mice and single cell suspensions were prepared. Cells were labeled with 2 μM CFSE (Molecular Probes) in Dulbecco’s PBS (Invitrogen Life Technologies) for 10 min at 37°C and washed twice. Cells were transferred into various recipient mice via tail vein injections. Mice further challenged with influenza virus (A/NT/60-68) were injected i.v. with 10⁷ hemagglutinating units of virus.

Cellular expansion of CFSE-labeled cells was calculated by determining the frequency (F_i) of recovered donor cells that had undergone i divisions, ΣF_i = 1. Adjusted frequencies, or precursor frequencies, f_i were calculated by dividing F_i by 2ⁱ to remove the effects of expansion (Σf_i ≤ 1). The fold expansion was then given by the quantity 1/Σf_i. Mean divisions were calculated as Σ_i f_i/Σf_i.

In vivo killing assay

Splenocytes from Ly5.1 C57BL/6J were incubated with either 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, or no Ag as control for 2 h at 37°C, then labeled with 4 μM, 1 μM, 250 nM, 62 nM, and 16 nM CFSE, respectively, for 10 min at 37°C and washed twice. Four × 10⁶ of each population of cells were mixed and injected into Rag1^−/− recipients. Rag1^−/− and F5 Rag1^−/− recipients received targets alone as controls. Twenty-four hours later, recipients were killed and splenocytes isolated. Target cells were stained with Ly5.1 and analyzed by FACS. Percent killing was determined by comparison of target populations remaining in experimental hosts with control hosts that received targets alone and normalized to the unpulsed control population.

Mathematical modeling of homeostatic proliferation

Lymphopenia and Ag induce distinct programs of cell division and differentiation in F5 T cells

A mouse model extensively used in our laboratory for studying the processes that govern homeostatic survival and proliferative responses is the F5 TCR transgenic mouse strain that expresses a class I restricted TCR specific for a peptide of influenza nucleoprotein (27). F5 T cells make relatively weak proliferative responses under conditions of T cell deficiency (12) as compared with other TCR transgenic strains (14). However, to directly compare the proliferative response of F5 T cells in lymphopenia with that induced by Ag, we transferred F5 T cells labeled with the cell dye CFSE into Rag1^−/− T cell-deficient hosts either in the presence or absence of influenza (Flu) virus. At various days post transfer, cells were recovered from host mice and analyzed by FACS. In the absence of Flu virus, T cells underwent a sustained but slow process of cell division. In hosts additionally challenged with flu virus, F5 T cells underwent a rapid and robust proliferation with most cells losing CFSE dye by day 7 (Fig. 1⇓A). In fact, in lymphopenic hosts it took approximately 2 wk for F5 T cells to proliferate to a similar degree as observed in only 3 days in the presence of Ag (Fig. 1⇓B). Furthermore, F5 T cells failed to up-regulate activation markers such as CD44 compared with the same cells challenged with Flu virus (Fig. 1⇓B) and failed to elicit any effector function when challenged with Ag pulse targets in vivo (Fig. 1⇓C).

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FIGURE 1.

F5 T cells maintain their naive state following lymphopenia induce proliferation. CD8⁺ T cells from F5 Rag1^−/− mice were labeled with CFSE and transferred i.v. to Rag1^−/− recipients (2 × 10⁶/mouse) with or without influenza virus. At days 3, 7, and 14, splenocytes were recovered from recipient mice and analyzed by FACS for expression of CD8, TCR, CD44, and CFSE labeling. A, Histograms show CFSE labeling of CD8⁺ TCR^high cells 3 and 7 days post transfer, either in the absence (top) or presence (bottom) of challenge with influenza virus. B, Dot plots show CD44 expression vs CFSE labeling of F5 T cells either 3 days after transfer and influenza challenge, or 14 days post transfer in the absence of influenza challenge. C, 1.5 × 10⁷ F5 T cells were transferred to Rag1^−/− hosts or 2 × 10⁶ F5 T cells were transferred to Rag1^−/− hosts and challenged with flu. Eleven days later, killing capacity of F5 T cells in these mice was determined by injecting hosts with a mixture of target splenocyte populations pulsed with varying concentrations of NP68 peptide. Dot plots show the frequency of F5 T cells in the lymph nodes of recipient mice. The graph shows the percentage killing of targets by F5 T cells undergo homeostatic (○) or Ag-induced (•) proliferation. D, CD8 T cells were purified from WT mice, labeled with CFSE and transferred to Rag1^−/− hosts. At days 6 and 13, lymph node cells from recipients were stained for CD8, CD44, TCR and analyzed by FACS. Dot plots show CFSE vs CD44 expression by CD8⁺TCR^high cells. Percentages indicate frequency of CD44^high and CD44^low cells. Histograms show CFSE labeling of CD44^high and CD44^low populations from the dot plots. Numbers on CFSE histograms indicate mean division within the precursor population. Percentage of input indicates the proportion of the precursor population that gave rise to the corresponding CD44^high and CD44^low populations on the dot plots as determined by current frequency and CFSE labeling.

Because of the wide variation in lymphopenic proliferative responses reported in different TCR transgenic systems, we more closely examined homeostatic proliferation of WT CD8⁺ T cells to collectively quantify the variety of responses made within a polyclonal repertoire. Purified CD8⁺ T cells from WT mice were labeled with CFSE and transferred into Rag1^−/− hosts. At 6 and 13 days post transfer, cells were recovered and the lymphopenia-induced proliferation assessed by FACS. By day 13, it appeared the majority of cells recovered were CFSE negative and CD44^high (Fig. 1⇑D) as described previously (9, 13, 28). However, a significant proportion of T cells were still labeled with varying levels of CFSE and remained CD44^low. Examination of responses at day 6 allowed more detailed quantitative analysis because all cells were still CFSE positive. More than 40% of T cells had up-regulated CD44 at this stage and had undergone more divisions than the CD44^low population. However, using the CFSE profile to calculate the precursor frequency (see Materials and Methods) revealed that the CD44^high population arose from only 17% of the precursor population. The remaining 83% of the precursor pool remained CD44^low and divided more slowly or not at all. In fact, 37% of the injected T cells were still undivided by day 6. Therefore, it appears that the abundance of the CD44^high population at day 13 reflects the extensive proliferation of a small proportion of the starting population rather than a majority response by the entire initial cohort. These data suggest that the ability of T cells to undergo extensive proliferation and phenotypic conversion is a feature of a relatively small proportion of the normal WT repertoire. Rather, the majority of CD8 T cells divided slowly, or not at all, without up-regulating activation markers. Therefore, the slow division rate of F5 TCR transgenic T cells in Rag1^−/− hosts closely resembles the slow dividing population of WT CD8 T cells, further highlighting the suitability of F5 T cells for the present analysis.

Quantifying the F5 T cell response to lymphopenia

F5 T cells proliferate in response to lymphopenia in a manner quite distinct from that induced by cognate Ag (Fig. 1⇑A). Previous studies have demonstrated that T cell stimulation with cognate Ag results in the induction of a predetermined program of cell division with distinct characteristics, such as time to first division and subsequent interdivisional durations. To gain an insight into the cell cycle regulation controlling homeostatic cell division and how it compares to that described following T cell activation, we aimed to use models to describe the cell division processes quantitatively. To do this, we required a precise experimental description of lymphopenia-induced proliferation by F5 T cells to underpin any modeling. Therefore, we first attempted to measure the F5 T cell response to lymphopenia in detail.

F5 T cells were labeled with CFSE and transferred into T cell-deficient Rag1^−/− hosts. At various time points, cohorts of mice were taken and the proliferative and expansive behavior of the F5 T cells determined by cellular enumeration in lymph nodes and spleen and analysis of CFSE labeling by FACS. Over the duration of the experiments, F5 T cells were seen to undergo steady progressive divisions (Fig. 2⇓A). Calculating the mean number of divisions undergone revealed a brief lag followed by a near linear increase in mean division with time corresponding to roughly exponential growth (Fig. 2⇓B), confirmed by measurement of absolute cell numbers (Fig. 2⇓C). The F5 T cell precursor number, calculated by removing the effects of expansion predicted by the cells’ CFSE profile from the cell recovery, did not decline during the course of the experiment, suggesting that there was negligible cell death of F5 T cells in the lymphopenic hosts (Fig. 2⇓C).

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FIGURE 2.

Kinetics of F5 T cells homeostatic proliferation. CD8⁺ T cells from F5 Rag1^−/− mice were labeled with CFSE and transferred to Rag1^−/− hosts (10⁶/mouse). At different time points, lymph nodes were recovered from recipient mice and analyzed by FACS for expression of CD8, TCR, and CFSE labeling. A, Histograms show CFSE labeling of CD8⁺TCR^high cell at the days indicated. B, The line graphs show the mean divisions by CD8⁺TCR^high cells in lymph node at different days post transfer (left) and the population expansion predicted by the profile of cell divisions revealed by the CFSE (right). C, In a similar experiment described in A, cell recoveries and CFSE labeling of F5 T cells transferred to Rag1^−/− hosts (n = 5) was determined at different times post transfer. The graph shows total cell recoveries (•) and cell recoveries of the precursor population that excludes the expansive effects of cell division (○). D, CD8⁺ T cells from F5 Rag1^−/− mice were labeled with CFSE and transferred to Rag1^−/− hosts at a range of doses. The graph shows the mean divisions of CFSE-labeled F5 T cells at day 14 after transfer of the cell doses indicated. E, CFSE-labeled F5 T cells (2 × 10⁶/mouse) were transferred to either Il7^+/+ Rag1^−/− or Il7^+/−Rag1^−/− recipients. Two, 7, and 13 days later, lymph node cells were harvested from groups of mice and analyzed by FACS for expression of CD8, TCR, and CFSE labeling. The graph shows mean division of F5 T cells with time in Il7^+/+ Rag1^−/− (filled squares) or Il7^+/−Rag1^−/− (half filled squares) hosts. Data are representative of at least three experiments.

We next assessed the sensitivity of the homeostatic proliferative response to host environment. First, we compared the proliferative response when different numbers of F5 T cells were transferred to Rag1^−/− recipients. Hosts were injected with cell doses between 5 × 10⁵ and 1.5 × 10⁷ CFSE-labeled F5 T cells and cell division assessed 14 days post transfer. F5 T cell proliferation as measured by mean division number was inversely proportional to inoculum size and the response was indeed exquisitely sensitive to initial cell dose, with reproducible differences observed between as little as 2-fold variations in initial cell dose (Fig. 2⇑D). Since IL-7 is critically required for homeostatic cell division, we asked whether varying IL-7 availability would have a quantitative effect on F5 T cell proliferation. We tested this by transferring CFSE-labeled F5 T cells into control Rag1^−/− hosts or into Rag1^−/− Il7^+/− hosts that have only one functional Il7 allele. F5 T cells in Il7^+/− hosts proliferated more slowly than in controls as measured by mean division number versus time (Fig. 2⇑E), further illustrating that homeostatic proliferation was very sensitive to the availability of resources in the host.

Modeling the homeostatic proliferation of F5 T cells

To test different hypotheses for the regulation of LIP, we fitted a series of mathematical models of cell division to these time series data. For each model, we searched for values of the model parameters that gave predicted patterns of cell division over time that best fitted our experimental time course data. In all cases we assumed that cell death among the cohort of cells that survived the transfer was negligible. This seemed reasonable on biological grounds as the lymphopenic host is likely to present an abundance of T cell survival signals or resources, but this assumption was also validated by the observation that the precursor number calculated from cells recovered from spleen and lymph nodes was approximately constant during the first 14 days after transfer (Fig. 2⇑C).

We attempted to describe the data with two classes of stochastic model. The formulation of the models and the fitting procedure are described in detail in Materials and Methods. The first class of model assumed that the mode of proliferation observed was analogous to the programmed proliferation of Ag-stimulated T cells observed in vitro, a model first studied quantitatively by Gett and Hodgkin (18) and extended in further studies (19, 21). We refer to it here as the GH model. To apply this model in the context of lymphopenia-induced proliferation in vivo we assume cells receive a proliferative stimulus from their environment soon after transfer to the lymphopenic host. For each cell, the time between receipt of this stimulus and first mitosis is assumed to be a random variable drawn from a left-bounded unimodal distribution. We used log-normal, gamma, and Weibull distributions as candidates. Cells that have divided once then embark on a program of divisions each of duration Δ before returning to a quiescent state (Fig. 3⇓A).

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FIGURE 3.

Models of lymphopenia-induced proliferation of T cells. A model of asynchronous “programmed” T cell proliferation based on the GH model (18 ) (upper panel). Cells transferred to the lymphopenic host take a time to their first mitosis drawn from a probability distribution (shown schematically) and subsequently divide deterministically. Our model includes the additional assumption that division is not possible during a time interval T immediately following transfer. Two cells are illustrated here: cell 1 undergoes its first division at time t1, cell 2 at a later time t2. Both continue to divide with each subsequent division taking a fixed time Δ. An alternative model of homeostatic proliferation, based on the stochastic SM model (24 ) (lower panel). T cells in a lymphopenic environment receive signals that induce cell division. However, not all cells divide at once. As in the model above, entry into division (here denoted B phase) is a process of chance, and in this model within a given time interval all cells have an equal probability of receiving the necessary signals to stimulate division. However, cells that receive the necessary stimulus only divide once (taking a time Δ) before returning to the resting state (A phase). In this figure, we illustrate a single cell dividing, but recruitment into B phase is continuous and at any given moment, many cells may progress through a single division. The process is memoryless in the sense that cells that have divided have the same chance as other resting T cells of receiving further division signals.

The free parameters in this model were the mean and variance specifying the probability distribution of times to first division, the division time Δ, and a delay before any cell could divide at all (T). The Weibull distribution provided the best fit of the three candidates (ΔAIC values; log-normal-Weibull = 75; gamma-Weibull = 35). The best fit parameters were as follows: distribution shape parameter = 2.26, scale parameter = 8.27 (mean time to first mitosis = 7.33 days, SD = 3.43 days), division time Δ = 2.88 days, lag time T = 0. On comparing the time course of cell division predicted by this GH model to the response observed experimentally, the model appeared to provide a very poor description of the data (Fig. 4⇓A), both in terms of the kinetics of mean division for the whole population and the predicted profiles of cell divisions at the various time points.

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FIGURE 4.

Models of lymphopenia-induced proliferation. The upper graphs show the mean and variance of division number in each animal (solid triangles); these quantities averaged over independent experiments at each time point (gray lines); and the best-fit model predictions of the mean and variance of division number (black lines). Histograms indicating the proportion of F5 T cells that have undergone different numbers of divisions at the days indicated. Gray bars show the observed experimental profiles, each bar corresponding to one CFSE peak and with height equal to the proportion of cells observed in each division (averaged over several independent experiments and not adjusted for expansion). Black bars indicate the corresponding model predictions using the best-fit parameters. In A, we show the best fit from a programmed divisions model, a variant of the GH model in which times to first division are Weibull-distributed and subsequent divisions are deterministic, each with duration Δ. B and C, Variants of the SM model. In B, we assumed constant mean rate of entry into division (λ). An extended SM model (C) assumed an exponential decline in λ with time as the cells divide to fill the host, and provided a significantly better fit.

The second class of model assumed that cells circulate and compete for homeostatic division signals such that a given cell has a constant probability of entering the cell cycle in any short time interval and dividing once. We used a version of the canonical SM model of the cell cycle (24), illustrated schematically in Fig. 3⇑B, in which cells spend exponentially distributed times in a quiescent A phase (corresponding approximately to the G₀/G₁ states of the cell cycle) and then progress through a B phase (S, G₂, and M) before dividing and returning to the A phase. The B phase was taken to be of fixed duration. The rate of transition from A to B phase, i.e., the rate at which T cells are triggered into cell division, is represented by the term λ, and duration of the B phase by Δ. The rate λ is defined such that the probability of any given cell entering B phase in a short time interval δt is simply λδt. We also estimated a lag time T after transfer during which no entry into B phase is possible.

The assumption of a constant mean rate of entry into division (λ) after the lag time T gave a reasonable fit to the data (Fig. 4⇑B). This model predicts a linear increase in the mean and variance of division number with time after a brief transient. The best-fit parameter estimates with 95% confidence intervals were λ = 0.210 day⁻¹ (0.157, 0.266), Δ = 6.65 h (3.29, 18.8), and T = 2.03 days (0.172, 2.72). Therefore we estimate the mean duration of the A phase, λ⁻¹, to be around 4.8 days, and the average time between divisions for each cell (λ⁻¹ + Δ) as just over 5 days. The trend in the mean division number averaged over all animals at each time point (Fig. 4⇑) suggested a progressive slowing of the division rate with time. To investigate this possibility we extended the model to allow a time-dependent change in division rate, defined by an additional parameter μ such that λ = λ₀ exp(−μt). This improved the fit significantly (partial F-test, p < 10⁻⁴; ΔAIC = 14; Fig. 4⇑C). Best-fit parameter estimates were λ₀ = 0.455 day⁻¹ (0.228, 3.58), μ = 0.115 day⁻¹ (0.020, 0.683), Δ = 6.19 h (2.26, 15.1), and T = 2.44 days (1.99, 2.90). The slowing of the division rate means that at day 3 the mean interdivision time was 3.4 days, increasing to 11.3 days at day 14.

Both the simple and modified SM model provided a significantly better fit to the data than the best fitting “programmed divisions” GH model (ΔAIC values; GH − basic SM model = 214; GH − modified SM model = 227). Despite the poor fit of the GH model, however, we clearly cannot rule out this model with other distributions for the times to first division. In particular the assumption that all cells receive their first division stimulus at the same time, as is likely in in vitro Ag-driven proliferation assays, may be questioned in vivo. Nevertheless we note that the gradient of the curve of mean division number with time is the inverse of the average time between divisions, which is roughly 5 days for the linear fit predicted by the simple SM model (the quantity λ⁻¹ + Δ) and between 3 and 11 days for the extended SM model with slowing division. Irrespective of the precise form of the distribution of times to first division, the GH model predicts that a plot of mean division number versus time tends to a straight line with slope 1/Δ after all cells have entered the proliferative program and are dividing asynchronously but uniformly with constant division time Δ. Crucially, the slope we observe indicates an interdivision >10-fold slower than that observed in Ag-driven T cell responses (18) and such slow proliferation is unprecedented in programmed responses. In summary, the SM model is a good description of the data, with even the more parsimonious three-parameter version describing proliferation better than the GH autopilot model.

Testing the biological predictions of the mathematical model in vivo

Although the SM-based model of stochastic cell divisions appears to provide the best description of the observed response by F5 T cells to lymphopenia, this observation neither proves the model is correct nor indeed excludes the possibility that other models might equally well or better describe the data. The model did, however, make several specific predictions about the nature of the F5 T cell response to lymphopenia. Therefore, we sought to further validate the model by directly testing its predictions experimentally.

A key biological prediction of the model was that TCR stimulation by self-peptide MHC should induce independent, single stochastic cell divisions. That being the case, ongoing homeostatic cell division should rely on constant signals from the lymphopenic environment to maintain continued cell division. Reversal of lymphopenia should, therefore, bring about a cessation of cell division, which would not be the case if the divisions were programmed or on autopilot. To test this directly, we injected groups of Rag1^−/− mice with CFSE-labeled F5 T cells. Three days later, when cell division of F5 T cells had already started and was at its highest rate, we injected cohorts of mice with very high doses of WT T cells (10⁸/mouse) to quickly reverse the lymphopenia. Strikingly, such treatment abruptly halted the observed cell division of F5 T cells in these hosts, whereas the same cells in untreated Rag1^−/− hosts continued to divide (Fig. 5⇓A). Although F5 T cells had divided when examined 2 days later in treated hosts, they had undergone fewer divisions than F5 T cells from untreated control hosts and there was no further cell division detected in the treated hosts up to 17 days later. Only 27% of cells in treated hosts underwent any cell division, compared with 83% in controls. Were a deterministic program of division at work, then cells triggered into division before treatment would be expected to complete their program of divisions. Rather, in treated mice, the vast majority of the dividing population (80%) underwent only a single division.

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FIGURE 5.

Validation of predictions made by the mathematical model. A, F5 T cells were labeled with CFSE and injected (10⁶/mouse) into Rag1^−/− hosts. At day 3, one group of mice was injected with 10⁸ WT T cells. Mice were bled at days 3, 5, 6, and 11 and expression of CD8 and CFSE labeling analyzed by FACS. The graph shows the mean divisions of F5 T cells in the two different groups at the days indicated. B, Rag1 ^−/− hosts were injected with 10⁶ F5 T cells. Two weeks later, CFSE-labeled F5 T cells (10⁶/mouse) were injected into the pretreated hosts or empty Rag1^−/− mice as control. Groups of these mice were taken at different times and the mean division of CFSE-labeled F5 T cells determined. The graph shows the mean divisions with time of CFSE-labeled F5 T cells in empty Rag1^−/− controls (•) and in Rag1^−/− mice pretreated with unlabeled F5 T cells (^). Data are representative of three independent experiments. C, F5 T cells were injected (2 × 10⁶/mouse) into either Rag1^−/− or C57BL/6 Ly5.1 WT hosts. At days 1, 2, and 3 post transfer, cells were recovered and labeled for CD8, TCR, Ly5.2, and CD5. Histograms are of FSc (top row) and CD5 (bottom row) for CD8⁺ Ly5.2⁺ TCR^high gated F5 T cells from either Rag1^−/− hosts (solid lines), WT hosts (broken line) or from F5 control mice (gray filled). Data are representative of two independent experiments with 4 mice per group.

Although the simple SM model described the data well (Fig. 4⇑B), introducing a time-dependent reduction in division rate resulted in a significantly better fit (Fig. 4⇑C). The slowing in division rate indicated by this analysis was also supported by our observation that F5 T cell homeostatic division is very sensitive to cell numbers in the host environment (Fig. 2⇑D). It seems reasonable that the rate of cell division should slow as the T cells expand in number, either due to the effects of competition or adaptation to the host. This slowing was not always evident in longer time courses because at later time points many cells had become CFSE negative, and mean cell divisions could not be accurately determined. To overcome this limitation and examine the cell kinetics of expansion over longer time periods, we measured the division rate by injecting hosts first with unlabeled F5 T cells and then transferring CFSE-labeled F5 sensor cells 2 wk later and measuring their cell division over time compared with the same cells in untreated Rag1^−/− hosts. As the data show, F5 T cells injected into the pretreated hosts proliferated at a significantly reduced rate compared with cells transferred to empty hosts (Fig. 5⇑B). The division rate also further reduced with time in both hosts.

Finally, another prediction of the model was that there is a lag phase of ∼2 days before cells started to respond to lymphopenia. To investigate whether there was evidence of such a delay in responsiveness of F5 T cells to the lymphopenic environment, we assessed cell size and CD5 expression by F5 T cells following transfer to lymphopenic hosts. Previous studies have shown that environmental signals in lymphopenia have trophic affects on T cells (29), resulting in increases in cell size, while the level of CD5 expression by peripheral T cells has been shown to be positively regulated by contact with self-peptide-MHC complexes (5, 30). Upon transfer to Rag1^−/− hosts, F5 T cells did indeed increase their cell size and up-regulated CD5 expression compared with the same cells transferred to C57Bl6 controls, indicative of enhanced contact with self-peptide-MHC that is required to induce LIP (Fig. 5⇑C). However, these changes in F5 T cell phenotype were not evident until 2 days post transfer and not maximal until day 3. By 24 h post transfer, the phenotype of F5 T cells in lymphopenic Rag1^−/− hosts was almost indistinguishable from the same cells in replete C57BL/6 controls.