T cell self-reactivity during thymic development dictates the timing of positive selection
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Evaluation Summary:
This study is of interest to immunologists as it fills a key knowledge gap in understanding factors involved in determining the duration of intrathymic positive selection of T cells. The findings come from a series of both in vitro and in vivo experiments implicating the self-reactivity of thymocytes in the time to completion of positive selection. An RNA-sequencing analysis suggests that gene expression differences from the pre-selection to the single-positive thymocyte stage is self-reactivity dependent, correlating in particular the level of ion channel expression with positive selection completion rates.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
Functional tuning of T cells based on their degree of self-reactivity is established during positive selection in the thymus, although how positive selection differs for thymocytes with relatively low versus high self-reactivity is unclear. In addition, preselection thymocytes are highly sensitive to low-affinity ligands, but the mechanism underlying their enhanced T cell receptor (TCR) sensitivity is not fully understood. Here we show that murine thymocytes with low self-reactivity experience briefer TCR signals and complete positive selection more slowly than those with high self-reactivity. Additionally, we provide evidence that cells with low self-reactivity retain a preselection gene expression signature as they mature, including genes previously implicated in modulating TCR sensitivity and a novel group of ion channel genes. Our results imply that thymocytes with low self-reactivity downregulate TCR sensitivity more slowly during positive selection, and associate membrane ion channel expression with thymocyte self-reactivity and progress through positive selection.
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Author Response:
Reviewer #2 (Public Review):
In their study, Lutes et al examine the fate of thymocytes expressing T cell receptors (TCR) with distinct strengths of self-reactivity, tracking them from the pre-selection double positive (DP) stage until they become mature single positive (SP) CD8+ T cells. Their data suggest that self-reactivity is an important variable in the time it takes to complete positive selection, and they propose that it thus accounts for differences in timescales among distinct TCR-bearing thymocytes to reach maturity. They make use of three MHC-I restricted T cell receptor transgenics, TG6, F5, and OT1, and follow their thymic development using in vitro and in vivo approaches, combining measures at the individual cell-level (calcium flux and migratory behaviour) with population-level positive selection …
Author Response:
Reviewer #2 (Public Review):
In their study, Lutes et al examine the fate of thymocytes expressing T cell receptors (TCR) with distinct strengths of self-reactivity, tracking them from the pre-selection double positive (DP) stage until they become mature single positive (SP) CD8+ T cells. Their data suggest that self-reactivity is an important variable in the time it takes to complete positive selection, and they propose that it thus accounts for differences in timescales among distinct TCR-bearing thymocytes to reach maturity. They make use of three MHC-I restricted T cell receptor transgenics, TG6, F5, and OT1, and follow their thymic development using in vitro and in vivo approaches, combining measures at the individual cell-level (calcium flux and migratory behaviour) with population-level positive selection outcomes in neonates and adults. By RNA-sequencing of the 3 TCR transgenics during thymic development, Lutes et al make the additional observation that cells with low self-reactivity have greater expression of ion channel genes, which also vary through stages of thymic maturation, raising the possibility that ion channels may play a role in TCR signal strength tuning.
This is a well-written manuscript that describes a set of elegant experiments. However, in some instances there are concerns with how analyses are done (especially in the summaries of individual cell data in Fig 2 and 3), how the data is interpreted, and the conclusions from the RNA-seq with regard to the ion channel gene patterns are overstated given the absence of any functional data on their role in T cell TCR tuning. As such the abstract is currently not an accurate reflection of the study, and the discussion also focuses disproportionately on the data in the final figure, which forms the most speculative part of this paper.
(1) As the authors themselves point out (discussion), one of the strengths of this study is the tracking of individual cells, their migratory behaviour and calcium flux frequency and duration over time. However, the single-cell experiments presented (Figure 2 and 3) do not make use of the availability of single-cell read-outs, but focus instead on averaging across populations. For instance, Figure 3a,b provides only 2 sets of examples, but there is no summary of the data providing a comparison between the two transgenics across all events imaged. In Figure 3c, the question that is being asked, which is to test for between-transgenic differences is ultimately not the question that is being answered: the comparison that is made is between signaling and non-signaling events within transgenics. However, this latter question is less interesting as it was already shown previously that thymocytes pause in their motion during Ca flux events (as do mature T cells). Moreover, the average speed of tracks is probably not the best measure here in reading out self-reactivity differences between TCR transgenic groups.
We regret any lack of clarity in how we presented our analyses of the calcium imaging data. In the original submission, we did provide analyses of individual cells (Fig 2b, Fig 3c (Fig 2e in the revised manuscript), Suppl Tables 1 and 2, and supplemental videos S1 and S2). In the revised manuscript, we have added an additional analyses of individual cells (Figure 2—figure supplement 1a). In addition, Fig 3a and b (Fig 2c and d in the revised manuscript) provided information about the average behavior of thymocytes during signaling events by identifying numerous examples of individual signaling cells (23-37 individual cell signaling events per condition), aligning these multiple examples based on the start of their signaling events, and displaying the average changes in calcium and speed over time. Thus this data does take advantage of the single-cell measurements by providing information about the average behavior of signaling events, which could not be inferred from bulk measurements. Regarding Fig. 3c (Fig. 2e in the revised manuscript), we agree that a more direct comparison of pausing between TCR transgenic models was needed. To address this point, we have added a new panel (Fig 2f in the revised manuscript) that uses the difference in speed between the signaling and nonsignaling portions of the same track to define a “pause index” for each cell. The difference in pause index between the transgenic models is highly significant at both 3 and 6 hours into positive selection. In the revised manuscript we have added additional text to detail more precisely how we performed the analyses, and to make it clearer that individual tracks are being analyzed. We have also included a graph of the Calcium Ratio and the Average Speed for the individual cells shown in the supplemental videos.
(2) The authors conclude from their data that the self-reactivity of thymocytes correlates with the time to complete positive selection. However the definition of what this includes is blurry. It could be that while an individual cell takes the same amount of time to complete positive selection (ie, the duration from the upregulation of CD69 until transition to the SP stage is the same), but the initial 'search' phase for sufficient signaling events differs (eg. because of lower availability of selecting ligands for TG6 than for OT1), in which case at the population level positive selection would appear to take longer. Given that from Fig 2/3 it appears that both the frequency of events and their duration differ along the self-reactivity spectrum, this needs to be clarified. Moreover, whether the positive selection rate and positive selection efficiency can be considered independently is not explained. It appears that the F5 transgenic in particular has very low positive selection efficiency (substantially lower %CD69+ and of %CXCR4-CCR7+ cells than the OT1 and TG6) and how this relates to the duration of positive selection, or is a function of ligand availability is unclear.
(3) While the question of time to appearance of SP thymocytes of distinct self-reactivities during neonatal development presented (Figure 5) is interesting, it is difficult to understand the stark contrast in time-scales seen here compared with their in vitro thymic slice (Figure 4) and in vivo EdU-labelling data (Figure 6), where differences in positive selection time was estimated to be ~1-2 days between TCR transgenics of high versus low affinity. This would suggest that there may be other important changes in the development of neonates to adults not being considered, such as the availability of the selecting self-antigens.
Since, Reviewer #2’s comments 2 and 3 are related, we will discuss them together. In this study, we have used 3 independent approaches (the thymic slice system, the EdU labeling study, and analyses of neonatal transgenic mice) to estimate the relative time for thymocytes bearing different TCRs to complete positive selection, and all three confirm that OT1 is the most rapid and TG6 the slowest of the 3 transgenic models examined here. However, each approach relies on different start times and different read outs, so they are not directly comparable to each other. The thymic slice system tracks a cohort of preselection thymocytes over time. However, given the 4 day limit for this system, it is not possible to reach the theoretical maximum number of CD8SP. Thus, our estimates of the delay in positive selection are based on the timing of multiple phenotypic changes (CD69 induction, chemokine receptor switch, and CD8SP appearance) in this system. The EdU study (Fig 5 in the revised manuscript) allows us to track a cohort of thymocytes that have recently completed TCRb selection and follows them over a longer time period (up to 9 days). Because the number of OT1 and F5 CD8 SP thymocytes reached a clear plateau, this allows us to estimate the average time between the burst of cell division after TCRb selection and the downregulation of CD4 (3.5 days for OT1 and 4.5 days for F5). However, at 9 days the number of TG6 thymocytes is still increasing, and thus we have only a lower estimate (>6 days) of the average time after TCRb selection to the appearance of CD8SP thymocytes with this TCR. When we track the appearance of mature CD8SP after birth (Figure 4 in the revised manuscript), we are not tracking a synchronized cohort of positively selecting cells, but rather we are measuring the amount of time it takes for single positive cells to accumulate into a population size similar to what is seen in an adult. Thus, these experiments do not provide a direct measure of the time to complete positive selection, but rather provide an indirect measure of the number of cells that have successfully completed positive selection at the given timepoints post birth. The observation that OT-1 CD8 SP thymocytes reach their adult steady state numbers at one week whereas TG6 CD8 SP thymocytes are well below adult levels at 21 days is likely a reflection of lengthy positive selection of TG6, resulting in a much longer time to fill the adult niche for CD8SP thymocytes. We agree with the reviewer that there could be additional important differences in positive selection between neonatal vs adult. We explore this topic and relate our data to recent published in the discussion (line 574) of the manuscript.
With regard to point (2), our data suggest that the longer time for positive selection is a result of both a longer search phase and a longer progression phase. Specifically, the % of CD69+ cells (Fig 3b and Figure 3—figure supplement 2a) peaks at 24 hours for OT1 and F5, but is delayed until 48 hours for TG6, consistent with a 1-2 day delay in the “search phase” for TG6. However, if this initial search phase was the only factor contributing to delayed TG6 development then we might expect to see a 1-2 day lag in TG6 development compared to OT-1. However, as discussed above, the EdU data indicates a > 3 day lag in the appearance of TG6 CD8SP compared to OT1. Thus, there is evidence that both the search phase and the progression phase of positive selection are longer in thymocytes with low self reactivity.
(4) The conclusion that "ion channel activity may be an important component of T cell tuning during both early and late stages of T cell development" is not supported by any data provided. The authors have shown an interesting association between levels of expression of ion channels, their self-affinity and the thymus selection stage. However, some functional data on their expression playing a role in either the strength of TCR signaling or progression through the thymus (for instance using thymic slices and the level of CD69 expression over time), would be needed to make this assertion. Moreover, from how the data is presented it is difficult to follow the conclusion that a 'preselection signature' is retained by the low but not the high self-reactivity thymocytes.
We agree that a role for ion channel activity in T cell tuning is speculative at this point, and we have tempered our conclusions in the revised manuscript. With regard to the evidence that a preselection signature is retained by thymocytes with low self reactivity, this conclusion is based on 2 separate lines of evidence presented in Figure 6 (previously Figure 7 in the original submission) and Figure 6—figure supplement 2. To summarize: 1). We defined a “preselection” gene signature based on preselection (CD69-DP) wild type thymocytes from the ImmGen microarray data, and show that this set of genes is also tends to be more highly expressed in thymocytes of low vs high self reactivity (TG6>F5>OT1) at equivalent stages of development (Fig 6d). 2). We identify a set of ion channel genes (cluster 2a from Fig 6c) that are more highly expressed in thymocytes of low vs high self reactivity (TG6>F5>OT1), and are also more highly expressed in earlier stages of positive selection for each TCR. This trend can also be seen in Figure 6— figure supplement 2c when comparing the expression of all cluster 2 ion channel genes across the wild type thymocyte subsets from ImmGen microarray data. Again, expression of this gene set peaks in the DP CD69- (preselection) population compared to other stages, including the preceding (DN4) and following (DP CD69+) stages of thymocyte development. We have edited this part of the results section in the revised manuscript to improve clarity.
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Reviewer #3 (Public Review):
The differences in signaling and responses in the three different T cell receptor transgenics are shown by several different means. These include Nur77 and CD5 expression as markers for the strength of signaling, the frequency of calcium fluxes and length of signaling-induced pauses in movement, using 2 photon microscopy of thymic slices (comparing selecting and non-selecting thymus), time course of induction of markers of positive selection signaling, the time course of "arrival" of CD8 single positive cells and CCR7+ cells in the post-natal thymus, and a time course of development of SP thymocytes after injection of EdU. Each of these methods is fairly convincing on its own, but added up, they are very convincing.
The only issues that I could take issue with are about how we define self-reactivity. Because …
Reviewer #3 (Public Review):
The differences in signaling and responses in the three different T cell receptor transgenics are shown by several different means. These include Nur77 and CD5 expression as markers for the strength of signaling, the frequency of calcium fluxes and length of signaling-induced pauses in movement, using 2 photon microscopy of thymic slices (comparing selecting and non-selecting thymus), time course of induction of markers of positive selection signaling, the time course of "arrival" of CD8 single positive cells and CCR7+ cells in the post-natal thymus, and a time course of development of SP thymocytes after injection of EdU. Each of these methods is fairly convincing on its own, but added up, they are very convincing.
The only issues that I could take issue with are about how we define self-reactivity. Because it is not feasible to measure the affinities for self peptides on MHC (due to low affinity and the fact that we mostly don't know what they are), the authors have to rely on surrogate markers, the upregulation of CD69 and of Nur77. These are widely accepted in the field, so they are as good a surrogate as is possible at this time.
Similarly, 3 transgenic strains are taken as examples of high, medium and low self-reactivity. Two of the strains are positively selected on H2Kb, one on Db, one on Ld. Therefore, the experiments cannot be genetically controlled in the same manner. On balance, I accept that there aren't too many other ways to do the experiment, and that all the main points are supported by other types of experiment.
The most interesting aspect of the work consists of analysis of gene expression by RNASeq from cells from each of the three TCR transgenic mice from early positive selection, late positive selection, and mature CD8 SP. Perhaps unsurprisingly, the more strongly self-reactive cells showed increased expression of genes involved in protein translation, RNA processing, etc. However, genes associated with lower self-reactivity were enriched for lots of different ion channels. These included calcium, potassium, sodium and chloride channels. One of these was Scn4b, part of a voltage gated sodium channel previously shown by Paul Allen's lab to be involved in positive selection. These types of genes were associated with the stage of development before selection, and were retained through selection in the weakly self-reactive thymocytes. Other ion channel genes that typically came on at the end of selection were also upregulated earlier in the lower self-reactivity cells, and may be involved in allowing long-term signaling for these cells to undergo the whole positive selection program.
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Reviewer #2 (Public Review):
In their study, Lutes et al examine the fate of thymocytes expressing T cell receptors (TCR) with distinct strengths of self-reactivity, tracking them from the pre-selection double positive (DP) stage until they become mature single positive (SP) CD8+ T cells. Their data suggest that self-reactivity is an important variable in the time it takes to complete positive selection, and they propose that it thus accounts for differences in timescales among distinct TCR-bearing thymocytes to reach maturity. They make use of three MHC-I restricted T cell receptor transgenics, TG6, F5, and OT1, and follow their thymic development using in vitro and in vivo approaches, combining measures at the individual cell-level (calcium flux and migratory behaviour) with population-level positive selection outcomes in neonates and …
Reviewer #2 (Public Review):
In their study, Lutes et al examine the fate of thymocytes expressing T cell receptors (TCR) with distinct strengths of self-reactivity, tracking them from the pre-selection double positive (DP) stage until they become mature single positive (SP) CD8+ T cells. Their data suggest that self-reactivity is an important variable in the time it takes to complete positive selection, and they propose that it thus accounts for differences in timescales among distinct TCR-bearing thymocytes to reach maturity. They make use of three MHC-I restricted T cell receptor transgenics, TG6, F5, and OT1, and follow their thymic development using in vitro and in vivo approaches, combining measures at the individual cell-level (calcium flux and migratory behaviour) with population-level positive selection outcomes in neonates and adults. By RNA-sequencing of the 3 TCR transgenics during thymic development, Lutes et al make the additional observation that cells with low self-reactivity have greater expression of ion channel genes, which also vary through stages of thymic maturation, raising the possibility that ion channels may play a role in TCR signal strength tuning.
This is a well-written manuscript that describes a set of elegant experiments. However, in some instances there are concerns with how analyses are done (especially in the summaries of individual cell data in Fig 2 and 3), how the data is interpreted, and the conclusions from the RNA-seq with regard to the ion channel gene patterns are overstated given the absence of any functional data on their role in T cell TCR tuning. As such the abstract is currently not an accurate reflection of the study, and the discussion also focuses disproportionately on the data in the final figure, which forms the most speculative part of this paper.
(1) As the authors themselves point out (discussion), one of the strengths of this study is the tracking of individual cells, their migratory behaviour and calcium flux frequency and duration over time. However, the single-cell experiments presented (Figure 2 and 3) do not make use of the availability of single-cell read-outs, but focus instead on averaging across populations. For instance, Figure 3a,b provides only 2 sets of examples, but there is no summary of the data providing a comparison between the two transgenics across all events imaged. In Figure 3c, the question that is being asked, which is to test for between-transgenic differences is ultimately not the question that is being answered: the comparison that is made is between signaling and non-signaling events within transgenics. However, this latter question is less interesting as it was already shown previously that thymocytes pause in their motion during Ca flux events (as do mature T cells). Moreover, the average speed of tracks is probably not the best measure here in reading out self-reactivity differences between TCR transgenic groups.
(2) The authors conclude from their data that the self-reactivity of thymocytes correlates with the time to complete positive selection. However the definition of what this includes is blurry. It could be that while an individual cell takes the same amount of time to complete positive selection (ie, the duration from the upregulation of CD69 until transition to the SP stage is the same), but the initial 'search' phase for sufficient signaling events differs (eg. because of lower availability of selecting ligands for TG6 than for OT1), in which case at the population level positive selection would appear to take longer. Given that from Fig 2/3 it appears that both the frequency of events and their duration differ along the self-reactivity spectrum, this needs to be clarified. Moreover, whether the positive selection rate and positive selection efficiency can be considered independently is not explained. It appears that the F5 transgenic in particular has very low positive selection efficiency (substantially lower %CD69+ and of %CXCR4-CCR7+ cells than the OT1 and TG6) and how this relates to the duration of positive selection, or is a function of ligand availability is unclear.
(3) While the question of time to appearance of SP thymocytes of distinct self-reactivities during neonatal development presented (Figure 5) is interesting, it is difficult to understand the stark contrast in time-scales seen here compared with their in vitro thymic slice (Figure 4) and in vivo EdU-labelling data (Figure 6), where differences in positive selection time was estimated to be ~1-2 days between TCR transgenics of high versus low affinity. This would suggest that there may be other important changes in the development of neonates to adults not being considered, such as the availability of the selecting self-antigens.
(4) The conclusion that "ion channel activity may be an important component of T cell tuning during both early and late stages of T cell development" is not supported by any data provided. The authors have shown an interesting association between levels of expression of ion channels, their self-affinity and the thymus selection stage. However, some functional data on their expression playing a role in either the strength of TCR signaling or progression through the thymus (for instance using thymic slices and the level of CD69 expression over time), would be needed to make this assertion. Moreover, from how the data is presented it is difficult to follow the conclusion that a 'preselection signature' is retained by the low but not the high self-reactivity thymocytes.
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Reviewer #1 (Public Review):
The work by Lutes et al. addresses how thymocytes undergo positive selection during their differentiation into mature T cells. The authors make use of several in vitro and in vivo model systems to the test whether developing thymocytes at the critical preselection CD4+CD8+ stage, expressing T cell receptors (TCRs) with different levels of putative self-reactivity, undergo different or similar differentiation events, in terms of migration, thymic epithelial cell engagement and temporal kinetics, and gene expression changes.
The authors selected three TCR-transgenes, which have increasing levels of self-reactivity, TG6, F5 and OT1, respectively, to test their hypothesis, that TCR signals during positive selection are not only sensed differently but lead to different outcomes that then define the functional …
Reviewer #1 (Public Review):
The work by Lutes et al. addresses how thymocytes undergo positive selection during their differentiation into mature T cells. The authors make use of several in vitro and in vivo model systems to the test whether developing thymocytes at the critical preselection CD4+CD8+ stage, expressing T cell receptors (TCRs) with different levels of putative self-reactivity, undergo different or similar differentiation events, in terms of migration, thymic epithelial cell engagement and temporal kinetics, and gene expression changes.
The authors selected three TCR-transgenes, which have increasing levels of self-reactivity, TG6, F5 and OT1, respectively, to test their hypothesis, that TCR signals during positive selection are not only sensed differently but lead to different outcomes that then define the functional status of mature T cells. The author's conclusions that thymocytes with low self-reactivity differentiate with distinct kinetics (migration, engagement and temporal) and express a different suite of genes than thymocytes that experience high self-reactivity is well supported by several elegant approaches, and convincing findings.
The authors clearly established that low to high TCR signaling outcomes affect the timing of positive selection, which is beautifully illustrated in Figures 3-6, and extend that work to non-TCR transgenic mice as well. Lastly, their findings from RNA-seq analyses shed light into the different genetic programs experienced by high-reactivity fast differentiating CD8 T cells as compared to low-reactivity slower differentiating cells, which appear to retain the expression a unique set of ion channels during later stages of their differentiation process.
However, what the expression of these ion channels means in terms of either supporting the slow progression or perhaps responsible for the slow progression is not directly addressed, and likely beyond the scope. Nevertheless, the authors posit as to the potential role(s) for the differently expressed gene subsets. Overall, the work is crisply executed, and the findings reveal new aspects as to how positive selection can be achieved by thymocytes expressing very different TCR reactivities.
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Evaluation Summary:
This study is of interest to immunologists as it fills a key knowledge gap in understanding factors involved in determining the duration of intrathymic positive selection of T cells. The findings come from a series of both in vitro and in vivo experiments implicating the self-reactivity of thymocytes in the time to completion of positive selection. An RNA-sequencing analysis suggests that gene expression differences from the pre-selection to the single-positive thymocyte stage is self-reactivity dependent, correlating in particular the level of ion channel expression with positive selection completion rates.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 …
Evaluation Summary:
This study is of interest to immunologists as it fills a key knowledge gap in understanding factors involved in determining the duration of intrathymic positive selection of T cells. The findings come from a series of both in vitro and in vivo experiments implicating the self-reactivity of thymocytes in the time to completion of positive selection. An RNA-sequencing analysis suggests that gene expression differences from the pre-selection to the single-positive thymocyte stage is self-reactivity dependent, correlating in particular the level of ion channel expression with positive selection completion rates.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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