bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 Title: T cell self-reactivity during thymic development dictates the timing of positive
2 selection
3
4
5 Authors:
6 Lydia K. Lutes1, Zoë Steier2, Laura L. McIntyre1, Shraddha Pandey1, James Kaminski3,#,
7 Ashley R. Hoover1,§, Silvia Ariotti1,†, Aaron Streets2,3,4, Nir Yosef2,3,4,5,6,, Ellen A. Robey1‡
8
9 Author Affiliations:
10 1 Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of
11 California, Berkeley
12 2 Department of Bioengineering, University of California, Berkeley
13 3 Center for Computational Biology, University of California, Berkeley
14 4 Chan Zuckerberg Biohub, San Francisco, California
15 5 Department of Electrical Engineering and Computer Sciences, University of California, Berkeley
16 6 Ragon Institute of MGH, MIT and Harvard
17 ‡ To whom correspondence may be addressed. E-mail: [email protected].
18
19 Present addresses: # Division of Pediatric Hematology/Oncology, Boston Children’s Hospital; Department of
20 Medical Oncology, Dana Farber Cancer Institute, and Harvard Medical School, Boston, MA; § Oklahoma
21 Medical Research Foundation, Oklahoma, United States; † Instituto de Medicina Molecular, João Lobo Atunes,
22 Faculdade de Medicina da Universidade de Lisboa, Av. Professor Egas Moniz, Lisbon, 1649-028, Portugal.
23
1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
24 Abstract
25 Functional tuning of T cells based on their degree of self-reactivity is established during
26 positive selection in the thymus, although how positive selection differs for thymocytes
27 with relatively low versus high self-reactivity is unclear. In addition, preselection
28 thymocytes are highly sensitive to low-affinity ligands, but the mechanism underlying their
29 enhanced TCR sensitivity is not fully understood. Here we show that murine thymocytes
30 with low self-reactivity experience briefer TCR signals and complete positive selection
31 more slowly than those with high self-reactivity. Additionally, we provide evidence that
32 cells with low self-reactivity retain a preselection gene expression signature as they
33 mature, including genes previously implicated in modulating TCR sensitivity and a novel
34 group of ion channel genes. Our results imply that thymocytes with low self-reactivity
35 down-regulate TCR sensitivity more slowly during positive selection, and suggest that
36 modulation of membrane ion channel function may play a role in regulating TCR tuning
37 throughout development.
38
39 Impact Statement
40 Developing T cells whose TCRs have relatively low reactivity experience very brief TCR
41 signaling events, delayed positive selection, and do not fully down regulate their TCR
42 sensitivity as they mature.
43
44 Introduction
45 T cell fate is dictated by the strength of the interaction between the T cell receptor (TCR)
46 and self-peptide major histocompatibility complexes (self-pMHCs) presented on thymic-
2 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
47 resident antigen presenting cells (APCs). While thymocytes whose TCRs interact too
48 weakly or too strongly with self-pMHC undergo death by neglect or negative selection
49 respectively, thymocytes whose TCRs react moderately with self-pMHC can survive and
50 give rise to mature CD4+ or CD8+ single positive (SP) T cells. Naïve CD4 and CD8 T cells
51 that emerge from the thymus were initially thought to be relatively homogeneous, with
52 differences emerging only after antigenic stimulation. However, recent studies have shown
53 that positively selected T cells span a range of self-reactivities, and that positive selection
54 results in functional tuning of T cells based on their degree of self-reactivity(Azzam et al.
55 1998; Fulton et al. 2015; Persaud et al. 2014; Weber et al. 2012; Mandl et al. 2013). On
56 Naïve T cells, surface expression levels of the glycoprotein CD5 serve as a reliable marker
57 for self-reactivity(Azzam et al. 2001; Azzam et al. 1998; Hogquist and Jameson 2014). CD5
58 also negatively regulates TCR signals, thereby serving as part of the tuning
59 apparatus(Azzam et al. 2001; Azzam et al. 1998; Hogquist and Jameson 2014; Persaud et al.
60 2014; Tarakhovsky et al. 1995). T cells with relatively high self-reactivity (CD5high) expand
61 more rapidly upon priming and exhibit greater sensitivity to cytokines(Fulton et al. 2015;
62 Persaud et al. 2014; Weber et al. 2012; Gascoigne and Palmer 2011), whereas T cells with
63 relatively low self-reactivity (CD5low) survive better in the absence of cytokines and exhibit
64 stronger proximal TCR signals upon stimulation(Persaud et al. 2014; Palmer et al. 2011; K.
65 Smith et al. 2001; Cho et al. 2016). How these distinct functional properties are imprinted
66 during positive selection in the thymus remains unknown.
67
68 Positive selection is a multi-step process in which CD4+CD8+ double positive (DP)
69 thymocytes experience TCR signals, migrate from the thymic cortex to the medulla, and
3 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
70 eventually downregulate either CD4 or CD8 co-receptor. During this process, thymocytes
71 remain motile and experience serial, transient TCR signaling events, to eventually reach a
72 threshold of signaling in order to complete positive selection(Au-Yeung et al. 2014; Ross et
73 al. 2014; Melichar et al. 2013). Thymocytes bearing MHC-II specific TCRs take 1-2 days to
74 complete positive selection and become mature CD4 SP thymocytes, whereas thymocytes
75 bearing MHC-I specific TCRs give rise to CD8 SP thymocytes about 2-4 days after the
76 initiation of positive selection(Saini et al. 2010; Lucas, Vasseur, and Penit 1993; Kurd and
77 Robey 2016). Interestingly, some studies report that CD8 T cells continue to emerge more
78 than one week after the beginning of positive selection(Saini et al. 2010; Lucas, Vasseur,
79 and Penit 1993). It remains unknown why some thymocytes move expeditiously through
80 positive selection, while others require more time to mature.
81
82 TCR signaling leads to a rise in cytosolic calcium concentration, and calcium flux during
83 positive selection induces a migratory pause that may prolong the interaction between
84 thymocyte and APC, thus promoting positive selection(Bhakta and Lewis 2005; Bhakta, Oh,
85 and Lewis 2005; Melichar et al. 2013). Calcium flux in thymocytes is also important for
86 activation of the calcium-dependent phosphatase calcineurin and the downstream
87 transcription factor NFAT, both of which are required for normal positive selection(Gallo et
88 al. 2007; Canté-Barrett, Winslow, and Crabtree 2007; Macian 2005; Neilson et al. 2004;
89 Hernandez, Newton, and Walsh 2010; Oh-hora and Rao 2008). Although the initial calcium
90 flux following TCR stimulation comes from the release of calcium from the endoplasmic
91 reticulum (ER) stores, calcium influx from the extracellular space is needed for prolonged
92 calcium signals(Oh-hora and Rao 2008). During mature T cell activation, depletion of ER
4 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
93 calcium stores triggers the influx of calcium via calcium release-activated calcium (CRAC)
94 channels. However, components of the CRAC channels are not required for positive
95 selection(Oh-Hora 2009; Gwack et al. 2008; Vig and Kinet 2009), and the ion channels
96 required for calcium influx during positive selection remain unknown.
97
98 Just after completing TCRαβ gene rearrangements and prior to positive selection, DP
99 thymocytes (termed preselection DP) are highly sensitive to low affinity ligands(Davey et
100 al. 1998; Lucas et al. 1999; Gaud, Lesourne, and Love 2018). This property is thought to
101 enhance the early phases of positive selection, but must be down-regulated to prevent
102 mature T cells from responding inappropriately to self(Hogquist and Jameson 2014). A
103 number of molecules have been identified which are involved in modulating TCR signaling
104 in DP thymocytes, and which are downregulated during positive selection. These include
105 the microRNA miR-181a: which enhances TCR signaling by downregulating a set of protein
106 phosphatases(Li et al. 2007), Tespa1: a protein that enhances calcium release from the
107 ER(Lyu et al. 2019; Liang et al. 2017), components of a voltage gated sodium channel
108 (VGSC): that prolongs calcium flux via an unknown mechanism(Lo, Donermeyer, and Allen
109 2012), and Themis: a TCR associated protein with controversial function(Kakugawa et al.
110 2009; Patrick et al. 2009; Lesourne et al. 2009; Johnson et al. 2009; Fu et al. 2009; Choi,
111 Cornall, et al. 2017; Fu et al. 2013; Choi, Warzecha, et al. 2017; Mehta et al. 2018). While a
112 diverse set of potential players have been identified, a clear understanding of the
113 mechanisms that render preselection DP thymocytes sensitive to low affinity self-ligands
114 remains elusive.
115
5 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
116 To understand how self-reactivity shapes TCR tuning during thymic development, we
117 compared the positive selection of thymocytes with MHC-I-restricted TCRs of low,
118 intermediate, or high self-reactivity. We show that positive selecting signals in thymocytes
119 with low self-reactivity occur with a more transient calcium flux, and without a pronounced
120 migratory pause. In addition, thymocytes with low self-reactivity proceed through positive
121 selection more slowly, with a substantial fraction taking over a week to complete positive
122 selection. Furthermore, we show that cells with lower self-reactivity retain higher
123 expression of a “preselection” gene expression program as they mature. This gene set
124 including genes known to modulate TCR signals and a novel set of ion channels genes.
125 These results indicate that T cell tuning during thymic development occurs via
126 developmentally layered sets of T cell tuning genes, maximizing the TCR signaling potential
127 of thymocytes with low self-reactivity throughout their development.
128
129 Results
130 Altered TCR signaling kinetics during positive selection of thymocytes with low self-
131 reactivity.
132 To investigate how self-reactivity impacts CD8 SP development, we used TCR transgenic
133 (TCRtg) mice expressing MHC-I-restricted TCRs of low, intermediate, or high self-reactivity.
134 Mice expressing rearranged TCR α and β transgenes from a T cell clone (TG6) with low self-
135 reactivity, exhibit a strong skewing to the CD8 lineage even in a Rag2 sufficient background
136 (Fig. S1). This is indicative of robust positive selection, and is in contrast to the HY TCR
137 transgenic model where low self-reactivity is accompanied by extensive endogenous
138 receptor rearrangement and weak skewing to the CD8 lineage(Azzam et al. 2001; Kisielow
6 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
139 et al. 1988). Compared to CD8SP from wild type mice, CD8SP thymocytes from TG6 mice
140 express low levels of CD5, a reliable surface marker of T cell self-reactivity (Fig. 1a,
141 b)(Azzam et al. 1998; Mandl et al. 2013; Fulton et al. 2015; Persaud et al. 2014; Azzam et al.
142 2001). CD8SP thymocytes from TG6 mice also have relatively low expression of Nur77, a
143 marker of recent TCR signaling that also correlates with self-reactivity (Fig. 1c)(Fulton et
144 al. 2015). In contrast, CD8SP from two well-characterized TCRtg models, OT-1 and F5, have
145 high and intermediate levels of these markers respectively (Fig. 1a-c)(Fulton et al. 2015;
146 Cho et al. 2016; Dong et al. 2017; Kieper, Burghardt, and Surh 2004; Palmer et al. 2011; Ge
147 et al. 2004). The relative levels of CD5 and Nur77 in lymph node T cells of TCRtg mice
Thymus LN a b **** **** **** ** **** TG6 **** 2.5 **** 2.0 **** F5 **** **** OT-1 2.0 1.5 B6 1.5 **** 1.0 * 1.0 % of Max CD5 gMFI gMFI CD5
CD5 gMFI gMFI CD5 0.5 0.5 (Relative to B6) to (Relative (Relative to B6) to (Relative 0.0 0.0 CD5 B6 TG6 F5 OT-1 B6 TG6 F5 OT-1
c ns d * ns ** TG6 * **** 2.0 ** 1.5 **** F5 *** ns OT-1 1.5 ** B6 ns 1.0 1.0
% of Max 0.5 0.5 Nur77 gMFI gMFI Nur77 Nur77 gMFI gMFI Nur77 (Relative to B6) to (Relative (Relative to B6) to (Relative 0.0 0.0 148 Nur77 B6 TG6 F5 OT-1 B6 TG6 F5 OT-1
149 Figure 1: TG6, F5, and OT-1 CD8SP cells express CD5 and Nur77 across the polyclonal
150 spectrum. CD8SP cells were harvested from the thymus and lymph nodes of wild-type (B6),
151 TG6, F5, and OT-1 TCRtg mice and analyzed by flow cytometry. (a and b) Representative (left)
152 and quantified by geometric mean fluorescence intensity (right) CD5 surface expression on (a)
153 CD8SP thymocytes and (b) CD8SP lymph node cells. (c and d) Representative (left) and
7 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
154 quantified (right) intracellular Nurr77 expression gated on (c) CD8SP thymocytes and (d)
155 CD8SP lymph node cells. Data are presented as average ± SD and analyzed using an ordinary
156 one-way ANOVA followed by a Tukey’s multiple comparisons (*P <0.05, **P <0.01, ***P <0.001,
157 ****P <0.0001) All data are compiled from 3 or more experiments.
158 follows a similar pattern, except that expression of Nur77 in F5 thymocytes decreases
159 somewhat in T cells relative to thymocytes (Fig. 1d). This may reflect weaker recognition
160 of the peripheral versus thymic self-peptides by the F5 TCR. Thus, these 3 TCRtg models
161 span the range of self-reactivity for CD8 T cell positive selection, with TG6 providing an
162 example of low self-reactivity coupled with relatively efficient positive selection.
163
164 Previous time-lapse studies of OT-1 thymocytes undergoing positive selection in situ
165 revealed serial TCR signals lasting about 4 minutes and associated with a migratory
166 pause(Ross et al. 2014; Melichar et al. 2013). To investigate how thymocyte self-reactivity
167 impacts TCR signaling kinetics during positive selection, we compared the pattern of
168 calcium changes and speed for TG6 or OT-1 thymocytes at 3 and 6 hours after the initiation
169 of positive selection. We isolated thymocytes from TCRtg mice on a non-selecting
170 background (herein referred to as preselection thymocytes), labeled the cells with the
171 ratiometric calcium indicator dye Indo-1LR, overlaid the labeled cells onto selecting or
172 non-selecting thymic slices, and imaged using two-photon, time-lapse microscopy as
173 previously described (Fig. 2a)(Bhakta, Oh, and Lewis 2005; Ross et al. 2014; Melichar et al.
174 2013; Dzhagalov et al. 2012; Ross et al. 2015). For both TG6 and OT-1, the frequency of
175 time points with elevated calcium was greater on selecting, compared to nonselecting
176 thymic slices, a difference that was statistically significant at the 6 hour timepoint (Fig. 2b
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177 and c). This confirmed that calcium flux under these conditions is dependent on the
178 presence of positive selecting ligands.
a Indo-labeled b High Ca2+ TG6 or OT-1 TG6 6hrs OT-1 6hrs Low Ca2+ pre-selection thymocytes
3 or 6hrs 2P Microscopy - frequency selecting or - duration Tracks non-selecting
0 10 20 0 10 20 Time (min) c 3hrs 6hrs ns ns 10 ns 10 * ** ns
5 5
% Signaling Timepoints Signaling % 0 0
TG6 sel TG6 sel OT-1 sel OT-1 sel TG6 nonsel 179 TG6 nonsel OT-1 nonsel OT-1 nonsel
180 Figure 2: TG6 thymocytes experience less frequent TCR signaling than OT-1 during positive
181 selection. (a) Experimental schematic. (b) Individual tracks (1 track = 1 cell; each track is a
182 horizontal line) over time from TG6 (left) and OT-1 (right) representative runs (movies) 6 hours
183 post addition to the slice. Grey indicates low Ca2+ timepoints; black indicates high Ca2+
184 timepoints. (c) Percentage of signaling timepoints (high Ca2+ ratio) out of total timepoints for
185 each run. Each dot represents a single run. All data are complied from 2 or more experiments,
186 except OT-1 3 hours data, which is from one experiment. Preselection thymocyte populations
187 were obtained from TG6 Rag2KO H2b mice and irradiated B2MKO mice reconstituted with OT-1
188 Rag2KO bone marrow. Data are presented as average ± SD and analyzed using an ordinary
189 one-way ANOVA followed by a Tukey’s multiple comparisons (*P<0.05, **P<0.01).
9 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
190 In line with their lower self-reactivity, TG6 cells in positively selecting slices spent
191 significantly less time signaling than did OT-1 cells (Fig. 2b, 2c, S2 and Table S1). To
192 further explore this difference, we identified individual signaling events from multiple
193 imaging runs, aligned the events based on the first timepoint with elevated calcium, and
194 averaged the calcium and speed over time as previously described(Melichar et al. 2013)
195 (see Materials and Methods section). At both 3 and 6 hours after addition to the slice, the
196 average signaling event for TG6 thymocytes is about one minute shorter than OT-1 (Fig. 3a
197 and b; Video S1 and S2). Moreover, OT-1 thymocytes pause during signaling events, as
198 reflected in a significant decrease in speed at signaling compared to non-signaling
199 timepoints (Fig. 3c and S3). In contrast, TG6 thymocytes have a much less pronounced
200 pause during signaling events, particularly at the 6-hour time point (Fig. 3c and S3).
201 Although reduced signal duration contributes to the reduced time TG6 cells spend
202 signaling, this difference could also be due to differences in TCR signaling frequency.
203 Indeed, the frequency of signaling events for TG6 thymocytes was approximately half that
204 for OT-1 thymocytes (1 signaling event every 2 hours for TG6 thymocytes, versus 1
205 signaling event every 75 minutes for OT-1: Table S2). Despite differences in signaling
206 frequency, the percentage of signaling tracks per run was comparable between the two
207 transgenics (Fig. S3). Thus, low self-reactivity is associated with less pronounced TCR-
208 induced pausing and a reduction in both the duration and frequency of TCR signals during
209 positive selection.
10 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
a 160s160s (2.67m) (2.67m) TG6TG6 3hrs 3hrs OT-1OT-1 3hrs 3hrs Ca Ca Ca 160s160s (2.67m) (2.67m) Ca 110s110s (1.83m) (1.83m) 1818 110s110s (1.83m) (1.83m) 1.21.2 1818 1.21.2 1.21.2 2+ 2+ 2+2+ 2+ 2+2+ 2+ Ratio (hi / lo) (hi Ratio / lo) (hi Ratio CaCa Ratio Ratio / lo) (hi Ratio CaCa Ratio Ratio / lo) (hi Ratio OT-1OT-1 3hrs 3hrs 1212 1.01.0 1212 1.01.0 1.01.0 SpeedSpeed SpeedSpeed TG6TG6 3hrs 3hrs 0.80.8 0.80.8 66 66 0.80.8 Ratio (hi / lo) Ratio (hi / lo) 2+ 0.60.6 0.60.6 2+ Speed (um/min)Speed (um/min)Speed Speed (um/min)Speed 00 (um/min)Speed 00 0.60.6 Ca -5-5 001122334455 1010 -5-5 001122334455 1010 Ca -5-5 001122334455 1010 TimeTime Relative Relative to to Trigger Trigger Event Event (min) (min) b TG6 6hrs OT-1 6hrs 120s (2m) Ca 120s (2m) Ca 18 60s (1m) 1.2 18 1.2 1.2 60s (1m) 2+ 2+ Ratio (hi / lo) (hi Ratio 12 1.0 12 1.0 / lo) (hi Ratio 1.0 OT-1 6hrs TG6 6hrs 0.8 0.8 6 6 0.8 Ratio (hi / lo)
0.6 0.6 2+ Speed (um/min)Speed 0 (um/min)Speed 0 0.6 -5 0 1 2 3 4 5 10 -5 0 1 2 3 4 5 10 Ca -5 0 1 2 3 4 5 10 Time Relative to Trigger Event (min)
c 3hrs 6hrs * **** ns **** 10 10 nonsig. tp sig. tp
5 5
Average (um/min) Speed 0 0 TG6 OT-1 TG6 OT-1 210 # cells: 20 39 24 23
211 Figure 3: TG6 thymocytes experience TCR signals of shorter duration and a less pronounced
212 signal-associated migratory pause compared to OT-1 during positive selection. Average calcium
213 (Ca2+) ratio and speed of signaling tracks (cells) for the indicated conditions, aligned by
214 signaling trigger event (time=0), over time, displayed separately for each TCRtg (left and middle)
215 and overlaid (right). (a) Average aligned Ca2+ ratio and speed 3 hours post addition to the slice
216 (TG6 n=27 cells, OT-1 n=37 cells). (b) Average aligned Ca2+ ratio and speed 6 hours post
217 addition to the slice (TG6 n=25 cells, OT-1 n=23 cells). Straight horizontal line at Ca2+
218 ratio=0.785 indicates the Ca2+ ratio of the end of the event following the trigger event. (c)
219 Average speed of signaling (sig) and nonsignaling (nonsig) timepoints for tracks with at least
220 one signaling event. In each column, a dot represents a single track. Between columns, lines
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221 connect data from the same track. All data are compiled from two or more experiments, except
222 OT-1 3hrs data, which is from one experiment. Preselection thymocyte populations were
223 obtained from TG6 Rag2KO H2b mice and irradiated B2MKO mice reconstituted with OT-1
224 Rag2KO bone marrow. Data are presented as average ± SD and analyzed using a two-way
225 ANOVA with Sidak’s multiple comparisons (*P<0.05, ****P<0.0001).
226
227
228 Self-reactivity correlates with time to complete positive selection.
229 Reduced TCR signals observed in TG6 thymocytes could mean that thymocytes with low
230 self-reactivity need more time to accumulate sufficient signals to complete positive
231 selection(Au-Yeung et al. 2014). To address this possibility, we examined the timing of
232 different stages of positive selection using thymic tissue slice cultures(Ross et al. 2015). We
233 previously showed that MHC-I specific preselection DP thymocytes undergo a synchronous
234 wave of maturation after introduction into positive selecting thymic tissue slices. This
235 includes transient upregulation of the TCR activation marker CD69, followed by a switch in
236 chemokine receptor expression from cortical (CXCR4) to medullary (CCR7) in DP
237 thymocytes, and eventually CD4 down-regulation(Ross et al. 2014; Melichar et al. 2013).
238 To examine the impact of self-reactivity on the timing of these landmarks in positive
239 selection, we overlaid preselection TG6, F5, or OT-1 thymocytes onto selecting and non-
240 selecting thymic slices, and analyzed progress through positive selection by flow cytometry
241 after 2, 24, 48, 72 and 96 hours of thymic slice culture. Slice-developed TG6, F5, or OT-1
242 CD8SPs exhibit the same relative CD5 levels as their in vivo counterparts (Fig. 4a),
12 20 **** 1.0 **** **** 40 **** 40 *** 15 *** **** **** 10 0.5 20 * 20 % CD69+ % 5 %CD69+ (Relative to Max) to (Relative 0 0 0 0.0 2 24 48 72 96 2 24 48 72 96 2 24 48 72 96 2 24 48 72 96
**** 15 20 **** **** *** 1.0 40 15 ** 10 10 0.5 20 *** * 5 5 Relative to Max to Relative % CXCR4-CCR7+% 0 0 0 %CCR7+CXCR4- 0.0 2 24 48 72 96 2 24 48 72 96 2 24 48 72 96 2 24 48 72 96
OT-1 1.5 **** 1.5 1.5 **** 1.0 F5 **** **** TG6 1.0 1.0 **** 1.0 ** 0.5 bioRxiv preprint0.5 doi: https://doi.org/10.1101/2021.01.18.427079* 0.5 ; this version0.5 posted January 21, 2021. The copyright holder for this preprint
(which was Max) notto (Relative certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Max) to (Relative 0.0 0.0 0.0 0.0 %CXCR4-CCR7+CD8SP 2 24 48 72 96 2 24 48 72 96 2 24 48 72 96 %CXCR4-CCR7+CD8SP 2 24 48 72 96 Time (Hours) Time (Hours) a 4 **** **** 3 **** 2
1
Relative CD5 gMFI CD5 Relative 0 TG6 F5 OT-1
b TG6 F5 OT-1 20 **** 1.0 **** **** 40 **** 40 *** 15 *** **** **** 10 0.5 20 * 20 % CD69+ % 5 %CD69+ (Relative to Max) to (Relative 0 0 0 0.0 2 24 48 72 96 2 24 48 72 96 2 24 48 72 96 2 24 48 72 96 c **** 15 20 **** **** *** 1.0 40 15 ** 10 10 20 *** * 0.5 5 5 Relative to Max to Relative % CXCR4-CCR7+% 0 0 0 %CCR7+CXCR4- 0.0 d 2 24 48 72 96 2 24 48 72 96 2 24 48 72 96 2 24 48 72 96 nonselecting nonselecting nonselecting OT-1 1.5 **** 1.5 1.5 **** 1.0 selecting selecting selecting F5 **** **** TG6 1.0 1.0 **** 1.0 ** 0.5 0.5 * 0.5 0.5 (Relative to Max) to (Relative (Relative to Max) to (Relative 0.0 0.0 0.0 0.0 %CXCR4-CCR7+CD8SP 2 24 48 72 96 2 24 48 72 96 2 24 48 72 96 %CXCR4-CCR7+CD8SP 2 24 48 72 96 243 Time (Hours) Time (Hours)
244 Figure 4: Thymocytes with lower self-reactivity exhibit delayed in situ positive selection kinetics 4 **** **** 3 245 relative to those**** with high self-reactivity. (a) Expression of CD5 on CXCR4-CCR7+ CD4-CD8+ 2
246 TG6 (96h),1 F5 (72h and 96h), and OT-1 (72h and 96h) donor cells developed in thymic slices.
Relative CD5 gMFI CD5 Relative 0 247 Values for eachTG6 experimentF5 OT-1 are normalized to the average CD5 expression of the CXCR4-
248 CCR7+ CD4-CD8+ polyclonal slice resident (SR) population at the same timepoints. (b-d)
249 Preselection TG6 (white bars, triangle), F5 (grey bars, square), or OT-1 (dark grey bars, circle)
250 thymocytes were overlaid onto selecting or non-selecting thymic slices, harvested at 2, 24, 48,
251 72 or 96 hours post thymocyte overlay, and analyzed using flow cytometry. Left three graphs
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252 show individual values for each transgenic. Horizontal lines in a and b indicate the average
253 value for non-selecting slices. Right line graph shows the average for all three transgenics
254 overlaid. (b) Percent of CD69+ cells within the CD4+CD8+ and CD4-CD8+ populations. (c)
255 Percentage of CXCR4-CCR7+ cells within CD4+CD8+ and CD4-CD8+ populations. (d)
256 Percentage of CXCR4-CCR7+ CD4-CD8+ cells out of the donor population. Values for each
257 experiment are normalized to the average at the timepoint with the maximum CD8SP
258 development (72h or 96h). For (a) data are presented as average ± SD and analyzed using an
259 ordinary one-way ANOVA with Tukey’s multiple comparisons (****P <0.000). For (b-d) data are
260 presented as average ± SD and analyzed using an ordinary one-way ANOVA with Dunnett’s
261 multiple comparisons (**P <0.01, ***P <0.001, and ****P <0.0001) comparing to 2 hour
262 timepoint. All data are compiled from 3 or more experiments. Preselection thymocyte
263 populations were obtained from TG6tg Rag2KO H2b mice, irradiated B2MKO mice reconstituted
264 with F5tg Rag1KO bone marrow, and OT-1tg Rag2KO B2MKO mice. See also Figure S4 and
265 S5.
266
267
268 indicating that thymic slices culture provides a good model for examining the impact of
269 self-reactivity on positive selection. In all three models, thymocytes exhibited a transient
270 increase in CD69 expression, however cells with lower self-reactivity reached peak CD69
271 expression later than cells with higher self-reactivity (Fig. 4b). In addition, the switch in
272 cortical to medullary chemokine receptor expression (CXCR4-CCR7+) and the appearance
273 of CD8SP were delayed in thymocytes with lower self-reactivity (Fig. 4c, d, S4, and S5).
274 Thus, the timing of all three major landmarks during positive selection is inversely
14 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
275 correlated with self reactivity, implying that cells with lower self-reactivity progress more
276 slowly through positive selection.
277
278 Mature CD8SP thymocytes appear around the end of gestation in mice and gradually
279 accumulate after birth. If thymocytes with low self-reactivity proceed more slowly through
280 positive selection, they may also appear more slowly during the neonatal period. To test
281 this prediction, we performed flow cytometry on the thymus and spleen from TG6, F5, and
282 OT-1 mice at 7, 14, and 21 days after birth. Consistent with thymic slice data, CD8SP
283 thymocytes accumulate slowly with age in TG6 mice, with the percent of CD8SP remaining
284 significantly below maximal adult levels at 21 days after birth (Fig. 5a and b). In contrast,
285 in OT-1 mice, CD8SP thymocytes reach their maximal levels at 7 days after birth, and F5
286 mice show an intermediate rate of accumulation (Fig. 5a and b). In addition, TG6 mice
287 were slowest to accumulate CXCR4-CCR7+ CD4+CD8+DP and CD8SP thymocytes (Fig. 5c),
288 and exhibited delayed appearance of CD8SP cells in the spleen (Fig. S6). These results are
289 consistent with results from thymic slice cultures, and provide in vivo confirmation that the
290 time to complete positive selection correlates inversely with thymocyte self-reactivity.
291
292 To examine the timing of positive selection in vivo in adult mice, we tracked a cohort of
293 thymocytes using pulse labeling with the thymidine analog 5-ethynyl-2’-deoxyuridine
294 (EdU). In vivo EdU injection labels a cohort of pre-positive selection thymocytes
295 proliferating after a successful TCRβ chain rearrangement at the time of the injection,
296 which then can be detected by flow cytometry, as they mature in vivo (Fig. 6a)(Lucas,
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TG6 a TG6 F5 OT-1 b F5 25 20 30 2.0 **** **** **** **** **** * OT-1 20 15 1.5 15 20 10 1.0 Adult 10 10 #CD8 SP %CD8SP 5 5 0.5
0 0 0 Adult) to (Relative 0.0 7 14 21 Adult 7 14 21 Adult 7 14 21 Adult 7 14 21 c
30 40 80 **** **** **** **** **** ** ** 30 60 20 20 40 10 10 20 0 0 0 %CXCR4-CCR7+ 7 14 21 Adult 7 14 21 Adult 7 14 21 Adult 297 Days Post Birth
298 Figure 5: Mature thymocytes with lower self-reactivity appear later post birth than those with
299 higher self-reactivity. Thymuses were harvested from TG6 (triangle, white bar), F5 (square, grey
300 bar), or OT-1 (circle, black bar) transgenic neonatal mice at 7, 14, and 21 days post birth and
301 from adult mice (6-9 weeks old), and analyzed using flow cytometry. (a) Percentage of CD4-
302 CD8+ cells. (b) Total number of CD4-CD8+ thymocytes at the indicated timepoint post birth,
303 relative to adult, for each transgenic. (c) Percentage of CXCR4-CCR7+ cells within CD4+CD8+
304 and CD4-CD8+ populations. Data are presented as average ± SD and analyzed using an
305 ordinary one-way ANOVA, Dunnett’s multiple comparisons (*P <0.05, **P <0.01, and ****P
306 <0.0001) comparing to adult. All data are compiled from 3 or more experiments.
307
308
309 Vasseur, and Penit 1993). EdU incorporated equally into the thymocytes of all three TCRtgs
310 (Fig. 6b). While the percent of DP cells was greater than the percent of CD8SP cells within
311 the EdU+ population in all three transgenics at 2 days post injection (p.i.) (Fig. 6c), EdU+
312 DP cells in TG6 mice remained higher than EdU+ CD8SP cells until day 6 p.i.. For F5
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a b TG6 EdU i.p. 30 F5 0 & 4hrs p.i. OT-1 20
Harvest Thymus %EdU+ 10 3-9 days TCR tg %EdU+ DP %EdU+ CD8SP 0 3 4 5 6 7 8 9 Days post EdU injection
c TG6 F5 OT-1 100 100 50 EdU+ SP 80 80 40 EdU+ DP 60 60 30 40 40 20 % cells% 20 20 10 0 0 0 3 4 5 6 7 8 9 3 4 5 6 7 3 4 5 6 7 Days post EdU injection d e EdU+ EdU- EdU i.p. 38.1 15.7 6.3 83.9 0 & 4hrs p.i. CD4 # Cells 36.8 3.37 Harvest Thymus 4,7,&10 days B6 CD5gMFI of EdU+ CD4 SP and TCRb+CD8 SP EdU CD8 CD8
EdU+ CD4 SP EdU-
f TCRβ+ CD8 CD4 1.5 * **** % of max ** * *** CD8 SP EdU+ EdU+ TCRb+ CD8 SP 1.0 1.0 EdU- EdU- Relative CD5 gMFI CD5 Relative 0.5 0.5 % of max 4 7 10 4 7 10 313 Days post EdU injection TCRβ CD5
314 Figure 6: Thymocytes with lower self-reactivity have delayed development in steady state TCRtg
315 and wildtype mice. TG6, F5, or OT-1 (a,b,c) or B6 (d,e,f) mice were injected with two doses of
316 1mg of EdU intraperitoneally (i.p.) at 0 and 4 hours post injection (p.i.). (a) TCRtg experimental
317 schematic. (b) EdU incorporation into TCRtg thymuses. (c) Percent CD8SP and DP thymocytes
318 out of gated EdU+ cells at various days p.i. in TG6, F5, and OT-1 tg thymuses (left to right). (d)
319 B6 experimental schematic. (e) Representative gating strategy and histograms showing B6 day
320 10 p.i. CD5 expression on EdU+ and EdU- mature SP cells. (f) CD5 expression of EdU+
17 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
321 TCRβ+CD8SP and EdU+ CD4SP thymocytes at 4, 7, and 10 days p.i.. CD5 expression shown
322 relative to total TCRb+CD8SP or CD4SP CD5 expression. For (f) data are presented as average
323 ±SD and analyzed using an ordinary one-way ANOVA, Tukey’s multiple comparisons (**
324 P<0.01, ***P<0.001, ****P<0.0001). All data are compiled from 3 or more experiments.
325
326
327 thymocytes, EdU+ CD8SP overtook the DPs one day earlier (day 5p.i.), and EdU+ CD8SP OT-
328 1 thymocytes accumulated even more quickly—by day 4p.i. (Fig. 6c). Though both F5 and
329 OT-1 thymocytes reach a plateau of EdU+ SP by day 5 or 6, TG6 DP thymocytes are still
330 developing into CD8SP thymocytes 7 to 9 days p.i. This data shows that increased time to
331 complete positive selection is also correlated with low self-reactivity in vivo. Furthermore,
332 these results show that some CD8SP thymocytes can take longer than 7 days to complete
333 positive selection, as also suggested by earlier studies(Saini et al. 2010; Lucas, Vasseur, and
334 Penit 1993).
335
336 To confirm the relationship between self-reactivity and time to complete positive selection,
337 we also performed EdU pulse experiments in non-TCRtg mice. We pulsed wild-type mice
338 with EdU and then examined CD5 levels on EdU+ mature TCRβ+CD8 and CD4SP
339 thymocytes at 4, 7, and 10 days p.i. (Fig. 6d). We observed that EdU+ mature thymocytes
340 found at earlier time points (and that therefore completed positive selection earlier) have
341 higher CD5 compared to EdU+ mature thymocytes found at later timepoints (Fig. 6e and f).
342 This is true for TCRβ+CD8SP thymocytes, however the trend is even more dramatic in
343 CD4SPs (Fig. 6f). These data indicate that self-reactivity and time to complete positive
18 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
344 selection are also inversely correlated in polyclonal mice, and this relationship exists for
345 both MHC-I-restricted and MHC-II-restricted thymocytes.
346
347 Thymocytes with low self-reactivity retain a preselection gene expression pattern marked
348 by elevated expression of ion channel genes
349 Positive selection is accompanied by large scale changes in gene expression, and delayed
350 progress through positive selection for thymocytes with low self-reactivity might lead to
351 synchronous or asynchronous delays in these gene expression changes. To investigate this
352 question, we performed bulk RNA sequencing (RNA-seq) on thymocytes from TG6, F5, and
353 OT-1 mice after sorting into three stages of development: early positive selection
354 (CD4+CD8+CXCR4+CCR7-), late positive selection (CD4+CD8+CXCR4-CCR7+), and mature
355 CD8SP (CD4-CD8+CXCR4-CCR7+). TG6 and OT-1 thymocytes showed significant
356 differences in gene expression at each developmental stage, with the number of
357 differentially expressed genes decreasing with maturity (Fig. S7). Gene set enrichment
358 analysis (GSEA) revealed that gene sets whose enrichment was associated with high self-
359 reactivity (i.e., OT-1>F5>TG6) were related to ribosome function, RNA processing, and
360 translation in early and late positive selection thymocytes (Fig. 7a, top, and S7, red dots).
361 Genes involved in cell division (Fig. S7, purple dots) and effector function (Fig. S7, blue
362 dots) were also upregulated in mature CD8SP thymocytes with high self-reactivity (Fig.
363 7a). Intriguingly, gene sets whose enrichment was associated with low self-reactivity (i.e.,
364 TG6>F5>OT-1) were related to transmembrane ion channel function—particularly voltage
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a Early Positive Selection Late Positive Selection Mature CD8 SP Structural Constituent Of Ribosome Structural Constituent Of Ribosome Chromosomal Part Translation Structural Molecule Activity Chromosome Structural Molecule Activity Translation RNA Processing RNP Complex Cellular Biosynthetic Process Mitosis RNA Binding Translational Initiation Spindle Cellular Biosynthetic Process Translation Initiation Factor Activity RNA Splicing RNP Complex Biogenesis And Assembly Ribosome mRNA Processing GO 0006397
OT-1 vs TG6 Translation Factor Activity NA Binding Regulation Of Translational Initiation mRNA Metabolic Process Up in high SR Translation Initiation Factor Activity Phagocytosis Spliceosome Translation Regulator Activity RNP Complex Actin Filament Based Process 0 1 2 3 0 1 2 3 0 1 2 b Voltage Gated Channel Activity Voltage Gated Potassium Channel Activity Voltage Gated Channel Activity Voltage Gated Cation Channel Activity Potassium Channel Activity Kinase Binding Voltage Gated Cation Channel Activity Delayed Rectifier Potassium Channel Activity Voltage Gated Potassium Channel Complex Delayed Rectifier Potassium Channel Activity TM Receptor Protein Phosphatase Activity Potassium Ion Transport Voltage Gated Potassium Channel Activity Voltage Gated Calcium Channel Complex Voltage Gated Cation Channel Activity Gated Channel Activity Voltage Gated Potassium Channel Activity Delayed Rectifier Potassium Channel Activity G Protein Coupled Receptor Activity Cation Channel Activity
TG6 vs OT-1 vs TG6 Voltage Gated Channel Activity Up in low SR Metal Ion TM Transporter Activity Metal Ion TM Transporter Activity Membrane Lipid Metabolic Process Calcium Channel Activity Ion Channel Activity 0 1 2 0 1 2 0 1 2 NES
Early Late Mature c Pos. Sel. Pos. Sel. CD8SP d DN4 vs DP CD69- DP CD69- vs DP CD69+ TG6 F5 OT-1 TG6 F5 OT-1 TG6 F5 OT-1
Glcci1 3000 3000 Themis Themis Glcci1 4 Ets2 Ets2 Rorc Plxnd1 Clk3 Rorc 2000 2000 Plxnd1
Cacng4 Clk3 3 Nox1
Kcna2 Wdr78 1000 CD69-) (DP 1000 Wdr78
Tdrd5 Expression Value Tdrd5 Spo11 Spo11 Kcna3 Cacna1e Slc7a11 Slc7a11 Scn4b Slc6a19 Slc6a19 Clcn2 0 0 2a -4 -3 -2 -1 1 2 3 4
Log2(Fold Change) Log (Fold Change) Cacnb3 2 2 Trpv6 Early Late Mature Pos. Sel. Pos. Sel. CD8SP TG6 F5 OT-1 TG6 F5 OT-1 TG6 F5 OT-1 2b Kcnh3 Kcnh2 Cacnb1
Scn5a 1 Scn2b
365
366 Figure 7: Thymocytes with low self-reactivity retain a preselection gene expression program
367 marked by elevated expression of ion channel genes. RNA-seq of OT-1, F5, and TG6
368 thymocytes at the early positive selection, late positive selection, and mature CD8SP stages. All
369 samples have three biological replicates except for TG6 late positive selection, which has two
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370 biological replicates. (a and b) Gene set enrichment analysis (GSEA) was performed on all
371 pairwise combinations of TCRtg samples and the gene sets were filtered for enrichment in the
372 order of self-reactivity (OT-1 > F5 > TG6) (a) or in the inverse order of self-reactivity (TG6 > F5 >
373 OT-1) (b). Normalized enrichment scores (NES) are from the OT-1 vs TG6 (a) or TG6 vs OT-1
374 (b) comparison. Abbreviations: self reactive (SR), ribonucleoprotein (RNP), nucleic acid (NA),
375 transmembrane (TM). (c) Heatmap of differentially expressed (padj<0.05 for OT-1 vs TG6
376 comparison) ion channel genes (see methods) across all three stages and all three transgenic
377 models. Gene expression values normalized by the DESeq2 variance stabilizing transformation
378 were averaged over biological replicates and scaled per row. Heatmap is hierarchically
379 clustered into four groups, numbered in the order of expression during development. Leading
380 edge genes from GSEA are in bold. (d) (Top) Plot of expression level in DP CD69- thymocytes
381 versus fold difference for DN4 versus DP CD69- (left side of X-axis) or DP CD69- versus DP
382 CD69+ (right side of X-axis) for ImmGen microarray data. The eleven genes chosen to
383 represent the “preselection DP” gene signature are indicated with red dots. (Bottom) Heatmap
384 of normalized expression (as in (c)) of the 11 representative preselection DP genes in TG6, F5,
385 and OT-1 thymocytes at the indicated stage of development.
386
387
388 gated ion channels (Fig. 7b, and Fig S7, green dots). Genes contributing towards the
389 enrichment scores of ion channel related gene sets, or leading-edge (LE) genes, included
390 components of calcium (Cacna1e, Cacnb1, Cacnb3, Cacng4), potassium (Kcna2, Kcna3,
391 Kcnh2, Kcnh3), sodium (Scn2b, Scn4b, Scn5a), and chloride (Clcn2) ion channels.
392
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393 To obtain a broader perspective on the relationship between thymocyte self-reactivity and
394 ion channel gene expression, we curated a list of 56 ion channel genes expressed in
395 thymocytes and examined expression changes associated with thymocyte maturation stage
396 and self-reactivity (Fig. 7c and S8a). At all three developmental stages, there were more
397 ion channel genes with higher gene expression in TG6 (green bars) compared to OT-1 (red
398 bars) thymocytes (S8a). Hierarchical clustering based on gene expression patterns across
399 all samples revealed a group of genes (group 2a) that shows a strong association with low
400 self-reactivity, and is also upregulated during early positive selection within each mouse
401 strain. This cluster includes Scn4b (Fig. S8b), which encodes the regulatory subunit of a
402 VGSC linked to positive selection(Lo, Donermeyer, and Allen 2012). Groups 1, 2b, and 3 are
403 also upregulated in cells with low self-reactivity but their expression peaks later during
404 positive selection. Eleven out of the 56 genes (group 4) have higher expression in cells
405 with high self-reactivity and also peak at the mature CD8SP stage. This group includes
406 P2rx7 and Trmp4, both of which have been linked to modulation of T cell effector
407 responses(Trebak and Kinet 2019; Feske, Wulff, and Skolnik 2015).
408
409 The observation that many of the ion channel genes that correlated with low self-reactivity
410 were also preferentially expressed at the early positive selection stage (Fig. 7c, group 2a)
411 suggested that thymocytes of low self-reactivity may retain an immature gene expression
412 pattern later into positive selection. To investigate this question, we used microarray data
413 from the Immunological Genome Project (ImmGen)(Heng, Painter, and Consortium 2008)
414 to identify a representative set of eleven genes preferentially expressed in non-TCR
415 transgenic, preselection (CD69-) DP thymocytes relative to postselection (CD69+) DP
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416 thymocytes, and to their immediate precursor (DN4 thymocytes) (see methods) (Fig. 7d,
417 top). We then examined expression of these genes in TG6, F5, and OT-1 thymocytes at
418 different developmental stages (Fig. 7d, bottom). Interestingly, all eleven genes correlated
419 with low self-reactivity, particularly at the early DP stage of development. This included
420 Themis (Fig. S8b), which encodes a protein required for modulating TCR signaling during
421 positive selection(Lesourne et al. 2009; Patrick et al. 2009; Kakugawa et al. 2009; Fu et al.
422 2009; Johnson et al. 2009; Choi, Warzecha, et al. 2017; Choi, Cornall, et al. 2017; Mehta et al.
423 2018). Moreover, expression of the curated set of ion channel genes in the ImmGen
424 microarray data set revealed that group 2 genes, whose expression correlated with low
425 self-reactivity, also displayed higher expression in preselection wildtype thymocytes (Fig
426 S8c). Furthermore, GSEA between CD69- DP compared to DN4 or CD69+ DP thymocytes
427 showed enrichment of gene sets relating to ion channels (DN4 < CD69- DP > CD69+ DP)
428 (Fig. S8d). Together, this data suggests that cells with low self-reactivity retain a
429 preselection DP phenotype later into development than those with high self-reactivity, and
430 that elevated expression of ion channel genes is a prominent feature of that phenotype.
431
432 Retention of a preselection gene expression program can account for some, but not all, of
433 the elevated ion channel gene expression by thymocytes of low self-reactivity. Specifically,
434 there is another set of ion channel genes whose expression peaks at the late positive
435 selection or CD8SP stage (Fig. 7c, group 3). Interestingly, two of these genes, Kcna2 and
436 Tmie (Fig. S8b), are also upregulated in mature peripheral CD5 low CD8SP T cells relative
437 to CD5 high CD8SP T cells, based on published data sets (Table S3 and S4)(Fulton et al.
438 2015; Matson et al. 2020; Barrett et al. 2013). Additionally, cells with low self-reactivity
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439 also have high expression of the sodium-dependent neutral amino acid transporter Slc6a19
440 (Fig. S7 and S8b, Table S3 and S4)(Fulton et al. 2015; Matson et al. 2020). Thus, elevated
441 expression of distinct sets of ion channel genes is observed throughout the development of
442 CD8 T cells with low self-reactivity.
443
444 Discussion
445 T cell positive selection is a multi-day process during which thymocytes experience
446 transient, serial TCR signals, triggering a series of phenotypic changes culminating in co-
447 receptor down regulation and lineage commitment. Preselection DP thymocytes are highly
448 sensitive to low affinity self-ligands, and gradually downmodulate their sensitivity as they
449 mature(Davey et al. 1998; Lucas et al. 1999). However the factors that modulate TCR
450 sensitivity during positive selection are not well understood. Moreover, there is a growing
451 appreciation that thymocytes differentially adjust their response to self-ligands based on
452 their degree of self-reactivity, although little is known about how positive selection differs
453 for thymocytes of relatively low versus relatively high self-reactivity. Here we show that
454 thymocytes with low self-reactivity experience briefer TCR signals and can take more than
455 twice as long to complete positive selection, compared to those with relatively high self-
456 reactivity. Thymocytes with low self-reactivity retain a preselection gene expression
457 program as they progress through development, including elevated expression of genes
458 previously implicated in modulating TCR signaling during positive selection, and a novel
459 set of ion channel genes. In addition, a separate set of ion channel genes that peaks at the
460 CD8SP stage is also selectively upregulated in mature CD8SP thymocytes of low self-
461 reactivity. These results indicate that the modulation of TCR sensitivity during T cell
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462 development occurs with distinct kinetics for T cells with low self-reactivity, and suggests
463 that changes in membrane ion channel activity may be an important component of T cell
464 tuning during both early and late stages of T cell development.
465
466 Our results contrast with an earlier study, which reported that the overall time for positive
467 selection was constant for different CD8 T cell clones, with an accelerated early phase
468 compensating for a delayed late phase for CD8 T cells of low self-reactivity(Kimura et al.
469 2016). Importantly, that study relied on the decrease in Rag-GFP reporter expression to
470 infer the timing of development, rather than directly tracking cohorts of positively selecting
471 cells as we have done in the current study. While the slow loss of Rag-GFP reporter due to
472 GFP protein turnover can provide an indication of the elapsed time after shutoff of rag
473 expression(McCaughtry, Wilken, and Hogquist 2007), reporter expression during the early
474 phase of positive selection may be impacted by the timing of rag shutoff and cell turnover,
475 making this an unreliable indicator of developmental time in this experimental setting.
476
477 A variety of molecules have been implicated in modulating the TCR sensitivity of DP
478 thymocytes, including the TCR-associating protein Themis, ER associating protein Tespa1,
479 microRNA mir-181a, and components of a VGSC (encoded by Scn4b and Scn5a)(Wang et al.
480 2012; Lo, Donermeyer, and Allen 2012; Fu et al. 2009; Johnson et al. 2009; Lesourne et al.
481 2009; Patrick et al. 2009; Kakugawa et al. 2009). Here we show that the VGSC is part of a
482 larger group of ion channel genes, that also includes components of voltage gated calcium
483 channels (Cacna1e, Cacnb3), that are both upregulated in preselection DP thymocytes, and
484 selectively retained during the positive selection of thymocytes with low self-reactivity. We
25 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
485 also identified another group of ion channels whose expression peaks at the CD8SP stage,
486 and that are also upregulated in CD8 T cells of low self-reactivity. This group includes
487 Kcna2, a component of a voltage gated potassium channel, and Tmie a gene essential for
488 calcium-dependent mechanotransduction in hair cells within the inner ear(Qiu and Müller
489 2018; Zhao et al. 2014). Thus, ion channel expression appears to be selectively modulated
490 based on the degree of self-reactivity at two distinct stages of T cell development, and may
491 help to enhance weak TCR signals during both the initial phase of positive selection and
492 during T cell homeostasis in the periphery.
493
494 Voltage gated ion channels are best known for their role in generating action potentials in
495 neurons and muscle cells, but they also play roles in non-electrically excitable cells such as
496 T cells and thymocytes(Feske, Wulff, and Skolnik 2015; Lo, Donermeyer, and Allen 2012;
497 Cahalan and Chandy 2009; Trebak and Kinet 2019). In these cells, voltage gated ion
498 channels have been proposed to be regulated by local fluctuations of membrane charge, or
499 alternative mechanisms such as phosphorylation(Rook et al. 2012). While the link between
500 ion channels and T cell tuning is not known, the modulation of calcium entry is a potential
501 mechanism. TCR triggering initially leads to a rise in cytosolic calcium via release of ER
502 stores, and the calcium rise is sustained in mature T cells when depletion of ER calcium
503 stores triggers the opening of calcium release activated channels (CRAC). CRAC channels
504 appear to be dispensable for positive selection(Oh-Hora 2009; Gwack et al. 2008; Vig and
505 Kinet 2009), and it remains unclear how thymocytes sustain calcium signals during the
506 days required to complete positive selection. Evidence suggests that the VGSC promotes
507 prolonged calcium flux in response to TCR triggering in DP thymocytes, although the
26 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
508 mechanism remains unclear(Lo, Donermeyer, and Allen 2012). Our data indicates that, not
509 only sodium, but also potassium, chloride, and calcium channels are upregulated in
510 preselection DP thymocytes and positive selecting thymocytes with low self-reactivity,
511 implying that positive selection leads to broad changes in transmembrane ion flux. In
512 addition to potentially promoting calcium influx, changes in membrane ion flux could also
513 directly impact TCR triggering by regulating local membrane charge at the receptor
514 complex(Ma et al. 2017). Altered ion flux in T cells with low self-reactivity may also impact
515 ion-coupled transport of nutrients. In this regard, it is intriguing that the sodium-
516 dependent neutral amino acid transporter gene Slc6a19 is also consistently upregulated in
517 thymocytes and CD8 T cells with low self-reactivity (Fig. 7c and d, S7 and Table S3 and
518 S4)(Fulton et al. 2015; Matson et al. 2020).
519
520 Taken together, our results support a model in which TCR tuning based on the degree of
521 self-reactivity occurs in two temporally-distinct phases (Fig. 8). Thymocytes with low self-
522 reactivity (Fig. 8, dashed lines) experience briefer TCR signals and accumulate TCR
523 signaling more slowly, resulting in a delayed time course of positive selection. During phase
524 1, a “preselection DP” gene expression program (including Themis, Scn4b, and Cacna1e) is
525 down-regulated in all thymocytes undergoing positive selection, but more gradually and
526 incompletely in thymocytes with low self-reactivity. During phase 2, a distinct set of genes
527 (including Kcna2 and Tmie) whose expression peaks in CD8 SP, are upregulated to a
528 greater extent in thymocytes with low self-reactivity. Higher expression of both sets of
529 genes help to sustain TCR signals throughout positive selection, allowing thymocytes to
530 continue to receive survival and differentiation signals in spite of their low self-reactivity.
27 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
a High Self Reactivity Low Self Reactivity 2+ 2+ Ca Ca
Time (min) Time (min)
High Self Reactivity Low Self Reactivity Cumulative TCR Signaling Threshold Accumulated Signal
b
Low Self Reactivity TCR Sensitivity High Self Reactivity c
Phase 2 genes low self reactivity
Phase 2 genes high self reactivity
Phase 1 genes low self reactivity
Relative Gene Expression Phase 1 genes high self reactivity
531 Time (days)
532 Figure 8: A model for temporal signal accumulation and TCR sensitivity during development in
533 cells with relatively high and low self-reactivity. (a) Positively selected cells with relatively high
534 self-reactivity (SR) receive serial, brief (minutes) TCR signals, allowing for signal accumulation
535 over a period of days, to eventually complete positive selection. Those with relatively low self-
536 reactivity (dashed lines) experience briefer TCR signals leading to slower signal accumulation
537 and a longer time to achieve the cumulative TCR signaling threshold required for positive
28 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
538 selection. (b) All DP thymocytes begin positive selection with high TCR sensitivity, and gradually
539 downregulate their sensitivity as they accumulate TCR signals. Thymocytes with low self-
540 reactivity downregulate their sensitivity more gradually and less completely than those with high
541 self-reactivity. (c) An early (phase 1: black lines) and a late (phase 2: grey lines) set of gene
542 expression changes modulate TCR signaling during positive selection. Elevated expression of
543 phase 1 genes (including Themis, Scn4b, and Cacna1e) help to confer high TCR sensitivity on
544 preselection DP thymocytes. Phase 1 genes are downregulated more slowly in thymocytes with
545 low self-reactivity, allowing them to retain high TCR sensitivity later into development, and aiding
546 in signal accumulation from weak ligands. Later during positive selection, phase 2 genes
547 (including Kcna2 and Tmie) are upregulated to a greater extent in thymocytes with low self-
548 reactivity.
549
550
551 Previous work has shown that CD8 T cells can take 3 or more days to complete
552 development, but it was unclear why positive selection takes longer for some cells than
553 others(Saini et al. 2010; Lucas, Vasseur, and Penit 1993; Kurd and Robey 2016). Our data
554 show that positive selection can take considerably longer for cells with low self-reactivity
555 compared to those with high self-reactivity. In particular, EdU pulse labeling in adult TG6
556 transgenic mice shows that some thymocytes are still converting from DP to CD8SP after
557 more than 1 week of positive selection. It has been previously noted that CD8 T cells that
558 arise in the neonatal period have higher CD5 levels, and are more proliferative and
559 responsive to cytokines compared to CD8 T cells that arise in adult mice(Dong et al. 2017;
560 Smith et al. 2018). This phenomenon may be explained in part by the shorter time
561 required for T cells with high self-reactivity to complete positive selection. However,
29 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
562 distinct mechanisms independent of TCR specificity also contribute to this phenomenon.
563 For example, T cells that arise in the neonatal period are derived from fetal stem cells, and
564 are intrinsically programmed to be more proliferative than those derived from adult stem
565 cells(Smith et al. 2018; Wang et al. 2016; Mold et al. 2010; Ikuta et al. 1990; Bronevetsky,
566 Burt, and McCune 2016). There are also indications that age-related changes in the thymic
567 environment may alter the TCR affinity thresholds for positive and negative selection(Dong
568 et al. 2017). Thus, three distinct mechanisms may all contribute to a bias toward high self-
569 reactivity T cells in young individuals, with T cells of lower self-reactivity accumulating
570 with age. It is tempting to speculate that this arrangement is evolutionarily advantageous,
571 since highly self-reactive T cells can provide a rapid response against acute infections,
572 which are the biggest threat to a young individual. As the organism ages, however, it may
573 become advantageous to have a more diverse, specialized T cell repertoire, particularly to
574 fight chronic infections. In line with this notion, we recently reported that TG6 T cells are
575 resistant to exhaustion and provide a protective response to T. gondii chronic infection,
576 properties that are linked to limited binding of self-peptides to the restricting MHC-1
577 molecule Ld (Blanchard et al. 2008; Chu et al. 2016; Tsitsiklis et al. 2020).
578
579 Overall, the work presented here provides insight on the differences in the development of
580 T cells with low and high self-reactivity that may impact their distinct peripheral functions.
581 The question of how a lengthy positive selection process and differences in ion channel
582 expression help to functionally tune T cells with low self-reactivity remains an important
583 area for future investigations.
30 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
584 Materials and Methods
585 Mice and bone marrow chimeras
586 All mice were bred and maintained under pathogen-free conditions in an American
587 Association of Laboratory Animal Care-approved facility at the University of California,
588 Berkeley. The University of California, Berkeley Animal Use and Care Committee approved
589 all procedures. B6 (C57BL/6, Stock No.: 000664), B6.C (B6.C-H2d/bByJ, Stock No.: 000359),
590 and Rag2-/- (B6(Cg)-Rag2tm1.1Cgn/J, Stock No.: 008449), Rag1-/- (B6.129S7-Rag1tm1Mom, Stock
591 No.: 002216), and B2M-/- (B6.129P2-B2mtm1Unc/DcrJ, Stock No.: 002087) mice were from
592 Jackson Labs. OT-1 Rag2-/- (B6.129S6-Rag2tm1Fwa Tg(TcraTcrb)1100Mjb, Model No.: 2334)
593 mice were from Taconic. F5 Rag1-/- and B6xB6C (H2b/d) mice were generated by crossing
594 as previously described(Au-Yeung et al. 2014; Chu et al. 2016). TG6 transgenic mice were
595 generated as previously described by Chu et al., 2016(Chu et al. 2016), and further crossed
596 in house to generate TG6 H2b/d (used in neonatal, EdU pulse experiments, and RNA-seq),
597 and TG6 H2b/d Rag2-/- (used to measure self-reactivity), and TG6 H2b Rag2-/- mice (used for
598 overlay onto thymic slices). Preselection OT-1 Rag2-/- thymocytes were generated by
599 crossing onto a non-selecting MHC-I deficient background (B2M-/-), or by transferring 1 ×
600 106 OT-1 Rag2-/- bone marrow cells intravenously (i.v.) into lethally irradiated (1,200 rad)
601 β2M-/- recipients (used for imaging experiments). Preselection F5 Rag1-/- thymocytes were
602 generated by transferring 1 × 106 bone marrow cells i.v. into lethally irradiated (1,200 rad)
603 β2M-/- recipients. Preselection TG6 Rag2-/- thymocytes were generated by crossing onto a
604 non-selecting background lacking H2d MHC-I (B6, H2b haplotype). Bone marrow chimeras
605 were analyzed 5–7 weeks following reconstitution. For all experiments, mice were used
31 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
606 from three to ten weeks of age, with the exception of neonatal experiments where mice
607 were used one to nine weeks of age.
608
609 Experimental Design
610 Sample size in this study was consistent with previous studies, and was not determined
611 based on a prior statistical test. All experiments were completed with at least 3 biological
612 replicates unless otherwise stated in the figure legend. Consistent with previous studies, 3-
613 8 thymic slices (technical replicates) per condition were used for thymic slice experiments
614 analyzed by flow cytometry. Samples were excluded if their thymic phenotype indicated
615 signs of stress (<107 total cells or less than 20% DP thymocytes out of live thymocytes).
616 Samples for thymic slice experiments were excluded if the culture had very low viability
617 (<15%) or <0.2% of donor derived thymocytes of total live cells in the final sample. For
618 EdU experiments, samples that did not incorporate detectable EdU (<0.001% of live cells
619 that were EdU+) were excluded. For experiments where thymic slices from the same
620 genotypic background received multiple treatments, slices were randomly allocated to
621 treatment conditions.
622
623 Thymic slices
624 Preparation of thymic slices has been previously described(Dzhagalov et al. 2012; Ross et
625 al. 2015). Briefly, thymic lobes were gently isolated and removed of connective tissue,
626 embedded in 4% agarose with a low melting point (GTG-NuSieve Agarose, Lonza), and
627 sectioned into slices of 400-500µm using a vibratome (VT1000S, Leica). Slices were
628 overlaid onto 0.4µm transwell inserts (Corning, Cat. No.: 353090) set in 6 well tissue
32 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
629 culture plates with 1ml cRPMI under the insert. 2x106 thymocytes in 10µl cRPMI were
630 overlaid onto each slice and allowed to migrate into the slice for 2-3 hours. Afterwards,
631 excess thymocytes were removed by gentle washing with PBS and cultured at 37°C 5%CO2
632 until harvested for flow cytometry or two-photon analysis. For flow cytometry, thymic
633 slices were dissociated to single-cell suspensions and then stained with fluorophore-
634 conjugated antibodies. For two-photon imaging, thymic slices were glued onto a glass
635 coverslip and imaged as described below.
636
637 Thymocyte labeling for overlay onto thymic slices
638 Thymuses collected from TCRtg preselection mice and dissociated through a 70µm cell
639 strainer to yield a cell suspension. For overlay onto thymic slices followed by flow
640 cytometry, TCRtg preselection thymocytes were labeled with 1µM Cell Proliferation Dye
641 eFluor450 or 0.5µM Cell Proliferation Dye eFluor670 as previously described(Kurd et al.
642 2019). For two-photon imaging of thymic slices, thymocytes were labeled with 2 mM
643 leakage-resistant Indo-1 (Thermo Fisher Scientific, Cat. No.: I1226) as previously described
644 followed by overlay onto slices(Melichar et al. 2013).
645
646 Flow cytometry
647 Thymic slices, whole thymuses, and spleens were dissociated into FACS buffer (0.5% BSA
648 in PBS) or RPMI and passed through a 70µm filter before staining. Splenocytes were then
649 red blood cell lysed using ACK lysis buffer (0.15M NH4Cl, 1mM KHCO3, 0.1mM Na2EDTA)
650 for 5 minutes at room temperature prior to staining. For thymic slices, cells were first
651 stained for 60 minutes in a 37°C water bath with CXCR4 (L276F12 or 2B11) and CCR7
33 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
652 (4B12) antibodies in 2.4G2 supernatant. Cells were then surfaced stained for 15 minutes on
653 ice in 2.4G2 supernatant containing the following antibodies: CD4 (clone GK1.5 or RM4-5),
654 CD8α (53-6.7), CD5 (53-7.3), and CD69 (H1.2F3). Finally, cells were washed in PBS and
655 stained in Ghost Dye Violet 510 (Tonbo Biosciences, Cat. No.: 13-0870-T100) for 10
656 minutes on ice. The following antibodies were used for flow cytometric analysis of whole
657 thymuses and spleens: CD4 (GK1.5 or RM4-5), CD8α (53-6.7), CD5 (53-7.3), and CD69
658 (H1.2F3), CD24 (M1/69), TCRb (H57-597), CXCR4 (L276F12 or 2B11), CCR7 (4B12), Vα2
659 (B20.1), Vβ8.1/8.2 (KJ16-133.18), and Vβ2 (B20.6). Cells were then washed in PBS and
660 stained in Ghost Dye Violet 510 as described above. For intracellular staining, cells were
661 then fixed and permeabilized using Invitrogen’s Transcription Factor Staining Buffer Set
662 (Thermo Fisher Scientific; Cat. No.: 00-5523-00) according to manufacturer’s instructions
663 and stained with Nur77 (12.14) antibody (Thermo Fisher Scientific, Cat. No.: 12-5965-82).
664 All antibodies were from Thermo Fisher Scientific, Biolegend, or Tonbo Biosciences. Cells
665 were analyzed using an LSRII, LSR, or Fortessa X20 flow cytometer (BD Biosciences) and
666 data analyzed using FlowJo software (Tree Star).
667
668 EdU pulse labeling
669 TCRtg and non-transgenic selecting mice, aged 5-8 weeks, received two intraperitoneal
670 (i.p.) injections of 5-ethynyl-2’-deoxyuridine (EdU) (Thermo Fisher Scientific, Cat. No.:
671 A10044) 1mg each and 4 hours apart, as previously described for BrdU(Lucas, Vasseur, and
672 Penit 1993). Thymuses were harvested from pulsed mice 3-10 days after the second
673 injection and dissociated into a single-cell suspension and were surface stained with CD4,
674 CD8α, CD5, CD69, CD24, and TCRβ antibodies as described above. Cells were then
34 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
675 processed using Click-iT™ EdU Alexa Fluor™ 488 Flow Cytometry Assay Kit from Invitrogen
676 according to the manufacturer’s instructions (Thermo Fisher Scientific, Cat. No.: C10420).
677 Flow cytometry and data analysis was performed as described above.
678
679 Two-photon imaging
680 Two-photon imaging of thymic slices has been previously described(Melichar et al. 2013;
681 Dzhagalov et al. 2012; Ross et al. 2014; Dzhagalov et al. 2013; Au-Yeung et al. 2014). All
682 imaging was performed 3 to 6 hours after the addition of thymocytes to thymic slices.
683 Thymic slices were glued onto glass cover slips and fixed to the bottom of a petri dish being
684 perfused at a rate of 1mL/min with 37°C oxygenated, phenol red–free DMEM during
685 imaging using an MPII Mini-Peristaltic Pump (Harvard Apparatus) and a 35mm Quick
686 Exchange Platform (QE-1) in conjunction with a TC-324B temperature controller (Warner
687 Instruments). Samples were imaged in the cortex of the thymus; determined by proximity
688 to the thymic capsule. Images were collected using a Zeiss LSM 7 MP upright, two-photon
689 microscope with a 20x/1.0 immersive Zeiss objective and a Coherent Chameleon laser
690 tuned to 720 nm for imaging Indo-1. Ratiometric Ca2+ signals were collected with 440nm
691 long pass dichroic mirror and 400/45 and 480/50 bandpass filters. Ca2+ hi signals
692 (~401nm) were collected using a bialkali PMT; Ca2+ lo signals (~475nm) were collected
693 using a GaASP PMT. Image areas of up to 212 x 212 µm to a depth of up to 55 µm were
694 acquired every 10 sec for 15 or 20 min with 3-µm z steps starting from beneath the cut
695 surface, using Zen 2010 software from Zeiss.
696
697 Image analysis
35 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
698 Two-photon movies were processed and rendered using Imaris software (Bitplane
699 Scientific Software) to collect x, y, and z coordinates and fluorescence intensity as
700 previously described(Melichar et al. 2013; Ross et al. 2014; Au-Yeung et al. 2014). Imaging
701 data were then analyzed using a custom MATLAB script (MathWorks; as previously
702 described (Melichar et al. 2013; Ross et al. 2014)), ImageJ, and Microsoft Excel. GraphPad
703 Prism software was used for graphing and statistical analysis. For calculation of the percent
704 of signaling timepoints, data were converted into flow cytometry-like files using custom
705 DISCit software and further analyzed using FlowJo software (TreeStar)(Moreau et al.
706 2012). The background calcium ratio is average Ca2+ ratio under non-selecting conditions
707 (0.675 for OT-1 and TG6) from the Ca2+ ratio of the raw fluorescence values at each
708 timepoint per run. To calculate the average event length, cells were considered to be
709 signaling when the average Ca2+ ratio was ≥0.2 above the background calcium ratio for ≥2
710 consecutive timepoints on a cell track. A trigger event was defined by the first of the
711 consecutive timepoints to have a Ca2+ ratio ≥0.2 above background. The end of a signaling
712 event was defined by when the Ca2+ ratio returned to <0.1 above background for two
713 consecutive timepoints. Only events that had a defined beginning and end were included in
714 calculating signal duration. For tracks with multiple events, only the first signaling event
715 was used to calculating signal duration. For calculating speed over time, we averaged speed
716 values from Imaris over two timepoints (20sec). The speed of signaling and nonsignaling
717 timepoints was measured by averaging the instantaneous speed for each timepoint. The
718 frequency of events was calculated by dividing the number of events compiled from all
719 runs by the cumulative track imaging time (the sum of all of the track durations for all
36 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
720 compiled runs), for each condition. For this calculation, events were defined as ≥2
721 consecutive timepoints of high Ca2+ ratio bound by periods ≥4min of nonsignaling.
722
723 RNA preparation, sequencing and analysis
724 Thymocytes from OT-1 Rag2KO, F5 Rag1KO, and TG6 H2b/d mice were sorted using a BD
725 Influx or BD FACSAria Fusion into CD4+CD8+CXCR4+CCR7- (early positive selection),
726 CD4+CD8+CXCR4-CCR7+ (late positive selection), and CD4-CD8+CXCR4-CCR7+ (Mature
727 CD8SP) populations for RNA sequencing. RNA was harvested using a Quick-RNA Microprep
728 Kit from Zymo Research (Cat. No. R1050) according to the manufacturer’s instructions.
729 RNA integrity was confirmed via Bioanalyzer and Qbit. RNA was sent to BGI Genomics for
730 library generation and RNA sequencing. RNA was sequenced on an Illumina-
731 HiSeq2500/4000 to a depth of 20 million reads.
732
733 Sequencing reads were processed with Trimmomatic(Bolger, Lohse, and Usadel 2014) to
734 remove adapter sequences and trim low-quality bases. Reads were aligned to the mm10
735 genome using Bowtie 2(Langmead and Salzberg 2012) and transcripts were quantified
736 using RSEM(Li and Dewey 2011). Differential expression testing (OT-1 vs TG6, OT-1 vs F5,
737 and F5 vs TG6 at each stage of development) was performed using DESeq2(Love, Huber,
738 and Anders 2014), which produced adjusted p-values corrected by the Benjamini-
739 Hochberg procedure. Gene set enrichment analysis (GSEA) was performed for each DESeq2
740 comparison using FGSEA(Korotkevich and Sukhov 2019) with gene sets downloaded from
741 MSigDB(Liberzon et al. 2011), including the C5 collection (Gene Ontology (GO) gene sets).
742 Following GSEA, we identified gene sets that were enriched in the order of self-reactivity
37 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
743 (OT-1>F5>TG6 or TG6>F5>OT-1) by normalized enrichment score (NES). Normalized
744 counts plotted in Figure S8b were derived by DESeq2’s median of ratios method(Love,
745 Huber, and Anders 2014).
746
747 The curated list of ion channel genes was created by first identifying ion channels
748 previously known to be expressed in T cells (207 genes)(Trebak and Kinet 2019; Feske,
749 Wulff, and Skolnik 2015). We narrowed this list to include only genes that were well
750 expressed in our RNA-seq data set (>20 reads in at least one sample), and also significantly
751 different by DEseq2 (padj<0.05) for OT-1 vs TG6 in at least one stage of development. We
752 also included Tmie, an ion channel gene differentially expressed by cells with low self-
753 reactivity. This resulted in a list of 56 genes.
754
755 ImmGen microarray analysis
756 Microarray data (RMA normalized signal intensities) were downloaded from the NCBI GEO
757 database(Edgar, Domrachev, and Lash 2002) from accession number GSE15907(Heng,
758 Painter, and Consortium 2008) and loaded with the GEOquery package(Davis and Meltzer
759 2007). Samples were filtered to include T.DN4.Th, T.DP.Th, and T.DP69+.Th (three
760 replicates each). Differential expression testing between cell types was performed using
761 limma(Ritchie et al. 2015). For the analysis in Figure 7d, genes representative of a
762 preselection gene expression program were identified from ImmGen microarray data
763 based on differential expression between preselection CD69- DP compared to DN4 and
764 CD69+ DP (log2 fold change >1), and either combined expression of >2000 (arbitrary units)
765 (Clk3, Ets2, Glcci1, Rorc, Plxd1, Spo11, Themis) or log2 fold change of >2 (Slc6a19, Slc7a11,
38 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
766 Tdrd5, Wdr78). Rag1 and Rag2 were excluded. ImmGen populations were relabeled in
767 Figure S8c: T.DN4.Th (DN4), T.DP.Th (DP CD69-), T.DP69+.Th (DP CD69+), T.4SP24-.Th
768 (Mature CD4SP (T)), T.8SP24-.Th (Mature CD8SP (T)), T.4Nve.Sp (Naïve CD4SP (S)), and
769 T.8Nve.Sp (Naïve CD8SP (S)). The gene set enrichment analysis in Figure S8d was
770 performed using FGSEA(Korotkevich and Sukhov 2019) with gene sets downloaded from
771 MSigDB(Liberzon et al. 2011), including the C5 collection (Gene Ontology (GO) gene sets).
772
773 Matson et al. RNA-seq analysis
774 RNA-seq count data were downloaded from the NCBI GEO database(Edgar, Domrachev,
775 and Lash 2002) from accession number GSE151395(Matson et al. 2020). The data
776 contained four biological replicates for each of four cell types (CD4+CD5lo, CD4+CD5hi,
777 CD8+CD5lo, CD8+CD5hi). Differential expression testing was performed using
778 DESeq2(Love, Huber, and Anders 2014).
779
780 Heatmap Plotting
781 Heatmaps (Fig. 7b, c, and S8c) were plotted using pheatmap with hierarchical clustering of
782 rows using default settings. Data was scaled per row. For Figure 7b, rows (genes) were
783 divided into four clusters based on hierarchical clustering. Genes in Figure S8c are
784 arranged by the same ordering as in Figure 7b.
785
786 Data Availability
39 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
787 RNA-seq data have been deposited in NCBI's Gene Expression Omnibus(Edgar, Domrachev,
788 and Lash 2002) and are accessible through GEO Series accession number GSE164896
789 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE164896).
790
791 Statistical Analysis
792 Statistical analysis was performed using Prism software (GraphPad) or as explained above
793 for the RNA-seq dataset. Specific statistical tests are indicated in the figure legends. Sample
794 sizes were chosen based on previous publications of similar studies. P values of <0.05 were
795 considered significant.
796
797 Acknowledgements
798 We would like to thank all the members of the Robey lab for insightful conversation and
799 scientific advice. We would also like to thank Diana Bautista, Nadia Kurd, and Derek Bangs
800 for critical reading of the manuscript. 2-photon imaging experiments were conducted at the
801 CRL Molecular Imaging Center, and we would like to thank Paul Herzmark, Holly Aaron,
802 and Feather Ives for their microscopy training and assistance. This work benefitted from
803 data assembled by the ImmGen consortium. This work was funded by NIH RO1AI064227
804 (E.A.R.). L.L.M. and A.R.H were supported by NIH T32AI100829, and S.A. was supported by
805 a Human Frontiers Fellowship. Z.S. is supported by the National Science Foundation
806 Graduate Research Fellowship.
807
808 Competing Financial Interests
809 The authors declare no competing financial interests.
40 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
810
811 Author Contributions
812 L.K.L., L.L.M., and E.A.R. designed research; L.K.L., L.L.M., S.P., A.R.H., and S.A. performed
813 research; L.K.L., Z.S., L.L.M., J.K. and E.A.R. analyzed data; and L.K.L. and E.A.R. wrote the
814 paper.
41 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
815 Supplemental Figures: a b B6xB6C (H2b/d) TG6tg Rag2+ H2b/d TG6tg Rag2-/- H2b/d 300 ) 6 10 x
( 200
s e t CD4 cy o 100 m y h T 0 CD8 CD8 CD8 C + -
Rag 100 10 15 B6xB6 6 TG TG6 Rag 80 8
P P 10 S S
P 60 6 4 8 D % 40 CD 4 CD
% % 5 20 2
0 0 0 C + - C + - C + -
Rag Rag Rag B6xB6 6 B6xB6 6 B6xB6 6 TG TG6 Rag TG TG6 Rag TG TG6 Rag
c CD8SP 6 20 100 8+ 2+ 2+ 15 β β α 4 V V V
+ + + β β β 10 50 CR CR CR T 2 T T 5 % % %
0 0 0 - - C + - C + C + Rag Rag Rag 6 B6xB6 6 B6xB6 6 B6xB6 816 TG TG6 Rag TG TG6 Rag TG TG6 Rag
817 Supplemental Figure 1: TG6 mice have efficient positive selection and allelic exclusion in the
818 thymus. Thymuses from B6xB6C (H2b/d), TG6 H2b/d Rag+/+, and TG6 H2b/d Rag-/- mice were
819 harvested and analyzed via flow cytometry. (a) Total thymocyte numbers. (b) Representative
820 flow plots and quantification of the percent of DP, CD4SP, and CD8SP thymocytes for the
821 indicated genotypes. (c) Allelic exclusion measured by percent TCRβ and Vα2, Vβ8, or Vβ2
822 double positive out of CD8SP thymocytes from the indicated genotypes. Data are compiled from
823 two experiments.
42 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
3hrs 6hrs ns 60 60 ns ns ns ns ns 40 40
20 20
% Signaling Tracks Signaling % 0 0
TG6 sel OT-1 sel TG6 sel OT-1 sel 824 TG6 nonsel OT-1 nonsel TG6 nonsel OT-1 nonsel
825 Supplemental Fig. 2: TG6 and OT1 thymocytes have a similar proportion of signaling cells per
826 run. Percentage of signaling tracks (i.e. tracks with at least one signaling timepoint) out of total
827 tracks for each run. Each dot represents a single run. Data are presented as average ± SD and
828 analyzed using an ordinary one-way ANOVA with Tukey’s multiple comparisons (ns=not
829 significant).
830
3hrs 6hrs
8 ns * 8 ns ** nonsig. tp sig. tp 6 6
4 4
2 2
Average (um/min) Speed 0 0 831 TG6 OT-1 TG6 OT-1
832 Supplemental Figure 3: TG6 thymocytes have a less pronounced pause while TCR signaling
833 compared to OT-1 thymocytes. Average instantaneous speed of signaling (sig.) and
834 nonsignaling (nonsig.) timepoints for runs with at least one signaling track. In each column, a
835 dot represents a single run. Between columns, lines connect data from the same run. All data
836 are complied from 2 or more experiments, except OT-1 3hrs data, which is from one
837 experiment. Data are presented as average ± SD and analyzed using an ordinary two-way
838 ANOVA with Sidak’s multiple comparisons (* P<0.05, **P<0.01).
43 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Live Donor a All Events Lymphocytes Single Cells Thymocytes
250K 250K 5 5 10 10
200K 200K
4 4 10 10
150K 150K Single Cells CXCR4- CCR7+ 97.5 15.1 SSC-A FSC-H CXCR4
Live/Dead 3
SSC 3 100K 100K 10 10 FSC-H CXCR4
Live/Dead 0 Lymphocytes 0 50K 54.0 50K 3 live slice live donor -10 3 64.6 2.45 -10 0 0 3 4 5 3 4 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 10 10 10 0 10 10 b FSC-AFSC-A FSC-AFSC-A eFluor679eFluor670 Cell Cell Dye Dye CCR7CCR7 2hrs 24hrs 48hrs 72hrs 96hrs
DP DP DP DP DP 83.583.5 80.080.0 76.076.0 77.777.7 44.044.0
4 4 4 4 4 10 10 10 10 10
3 3 3 3 3 10 DP & SP 10 DP & SP 10 DP & SP 10 DP & SP 10 DP & SP TG6 85.785.7 83.883.8 82.082.0 86.286.2 78.578.5 CD4
0 0 0 0 0
CD8SP CD8SP CD8SP CD8SP CD8SP 0.94 1.60 2.99 3.75 28.7 0.94 1.60 2.99 3.75 28.7
3 3 4 3 3 4 3 3 4 3 3 4 3 3 4 -10 0 10 10 -10 0 10 10 -10 0 10 10 -10 0 10 10 -10 0 10 10
5 DP 5 DP 5 DP 5 DP 5 DP 10 95.5 10 91.3 10 86.2 10 75.0 10 64.9 95.5 91.3 86.2 75.0 64.9
4 4 4 4 4 10 10 10 10 10
DP & SP DP & SP DP & SP DP & SP DP & SP F5 98.2 98.2 96.4 95.6 93.1 CD4 3 98.2 3 3 96.4 3 95.6 3 93.1 10 10 98.2 10 10 10
0 0 0 0 0 CD8 SP CD8 SP CD8 SP CD8 SP CD8 SP 2.02 5.05 6.41 13.5 24.6
3 3 3 3 3 -10 2.02 -10 5.05 -10 6.41 -10 13.5 -10 24.6
3 4 5 3 4 5 3 4 5 3 4 5 3 4 5 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10
DP DP DP DP DP 93.1 86.1 74.4 68.3 71.4 93.1 86.1 74.4 86.1 71.4 4 4 4 4 4 10 10 10 10 10
DP & SP DP & SP DP & SP DP & SP DP & SP OT-1 97.197.1 95.695.6 89.789.7 95.686.3 92.492.4
CD4 3 3 3 3 3 10 10 10 10 10
CD8 SP CD8 SP CD8 SP CD8 SP CD8 SP 0 2.90 0 7.53 0 10.7 0 14.0 0 19.2 2.90 7.53 10.7 7.53 19.2
3 4 3 4 3 4 3 4 3 4 0 10 10 0 10 10 0 10 10 0 10 10 0 10 10 CD8
c 2hrs 24hrs 48hrs 72hrs 96hrs
4 4 4 4 4 10 10 10 10 10
3 3 3 3 3 TG6 10 10 10 10 10 CD4
0 0 0 0 0
CD8SP CD8SP CD8SP CD8SP CD8SP 1.231.23 4.004.00 5.745.74 9.579.57 57.257.2
3 3 4 3 3 4 3 3 4 3 3 4 3 3 4 -10 0 10 10 -10 0 10 10 -10 0 10 10 -10 0 10 10 -10 0 10 10
5 5 5 5 5 10 10 10 10 10
4 4 4 4 4 10 10 10 10 10 F5
3 3 3 3 3 CD4 10 10 10 10 10
0 0 0 0 0 CD8 SP CD8 SP CD8 SP CD8 SP CD8 SP 0.000 0.000 30.230.2 72.072.0 87.687.6 3 3 3 3 3 -10 -10 -10 -10 -10
3 4 5 3 4 5 3 4 5 3 4 5 3 4 5 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10
4 4 4 4 4 10 10 10 10 10 OT-1
CD4 3 3 3 3 3 10 10 10 10 10
CD8 SP CD8 SP CD8 SP CD8 SP CD8 SP 0 0.000 0 9.019.01 0 30.030.0 0 52.552.5 0 86.086.0
3 4 3 4 3 4 3 4 3 4 0 10 10 0 10 10 0 10 10 0 10 10 0 10 10 839 CD8
44 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
840 Supplemental Figure 4: TG6 thymocytes exhibit delayed development in thymic slices. (a)
841 Representative flow plots illustrating the gating strategy for thymic slice experiments. (b)
842 Representative CD4 vs CD8 flow plots of TG6, F5, and OT-1 live donor thymocytes (bolded red
843 gate in a). (c) CD4 vs CD8 representative flow plots for TG6, F5, and OT-1 live donor CXCR4-
844 CCR7+ thymocytes (bolded blue gate in a). Bolded numbers within plots indicate the percentage
845 of cells within a given gate.
45 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
a 2hrs 24hrs 48hrs 72hrs 96hrs 100 100 100 100 100
80 80 80 80 80
60 60 60 60 60 TG6 40 40 40 40 40
CD69+ CD69+ CD69+ CD69+ CD69+ 0.92 12.2 33.6 13.5 7.93 % of Max 0.92 12.2 33.6 13.5 7.93 20 20 20 20 20
0 0 0 0 0
3 4 5 3 4 5 3 4 5 3 4 5 3 4 5 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10
100 100 100 100 100
80 80 80 80 80
60 60 60 60 60 F5 40 40 40 40 40 CD69+ CD69+ CD69+ CD69+ CD69+ 4.494.49 15.115.1 13.113.1 9.709.70 6.136.13 % of Max 20 20 20 20 20
0 0 0 0 0
3 4 3 4 3 4 3 4 3 4 0 10 10 0 10 10 0 10 10 0 10 10 0 10 10
100 100 100 100 100
80 80 80 80 80
OT-1 60 60 60 60 60
40 40 40 40 40
CD69+ CD69+ CD69+ CD69+ CD69+ 3.90 42.2 17.0 8.98 5.40
% of Max 3.90 42.2 17.0 8.98 5.40 20 20 20 20 20
0 0 0 0 0
3 4 3 4 3 4 3 4 3 4 0 10 10 0 10 10 0 10 10 0 10 10 0 10 10 CD69
b 2hrs 24hrs 48hrs 72hrs 96hrs
5 5 5 5 5 10 10 10 10 10
CXCR4-CCR7+ CXCR4-CCR7+ CXCR4-CCR7+ CXCR4-CCR7+ CXCR4-CCR7+ 0.570.57 0.260.26 4.334.33 17.917.9 35.935.9 4 4 4 4 4 10 10 10 10 10 TG6 3 3 3 3 3 10 10 10 10 10 CXCR4
0 0 0 0 0
3 3 3 3 3 -10 -10 -10 -10 -10
3 3 4 5 3 3 4 5 3 3 4 5 3 3 4 5 3 3 4 5 -10 0 10 10 10 -10 0 10 10 10 -10 0 10 10 10 -10 0 10 10 10 -10 0 10 10 10
4 4 4 4 4 10 10 10 10 10 CXCR4-CCR7+ CXCR4-CCR7+ CXCR4-CCR7+ CXCR4-CCR7+ CXCR4-CCR7+ 0.0750.075 0.0540.054 0.550.55 4.074.07 10.710.7
3 3 3 3 3 F5 10 10 10 10 10 CXCR4 0 0 0 0 0
3 4 3 4 3 4 3 4 3 4 0 10 10 0 10 10 0 10 10 0 10 10 0 10 10
4 4 4 4 4 10 10 10 10 10 CXCR4-CCR7+ CXCR4-CCR7+ CXCR4-CCR7+ CXCR4-CCR7+ CXCR4-CCR7+ 0.130.13 6.536.53 15.115.1 26.526.5 27.027.0
3 3 3 3 3 10 10 10 10 10 OT-1
2 2 2 2 2 10 10 10 10 10 CXCR4
0 0 0 0 0
2 2 2 2 2 -10 -10 -10 -10 -10
2 3 4 2 3 4 2 3 4 2 3 4 2 3 4 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10 846 CCR7
847 Supplemental Figure 5: TG6 thymocytes have delayed expression of positive selection markers
848 in thymic slices. Representative flow plots for TG6, F5, and OT-1 thymocytes developing in
849 slices at the indicated timepoints post addition to the slice (timepoint indicated above each
46 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
850 column) for (a) CD69 expression and (b) CXCR4 and CCR7 expression. Numbers within plots
851 indicate the percentage of cells within a given gate.
852
853
TG6 2.0 F5 1.5 OT-1 1.0 Adult
#CD8 SP 0.5 (Relative to Adult) to (Relative 0.0 854 7 14 21
855 Supplemental Figure 6: T cells with lower self-reactivity appear later post birth than those with
856 higher self-reactivity in the spleen. Total number of CD4-CD8+ splenocytes relative to adult for
857 each transgenic. Data compiled from 3 or more experiments.
47 Early Pos. Sel. Early Pos. Sel. 2538 2538 3116 3116 Gtf2h4 Gimap4Ddr1 Gtf2h4 Gimap4 Ddr1 Rps18 Ddr1 Rps18 Gimap4 Rpsa Rpsa Rps18 100 100 Slc30a4 Slc30a4 Rpsa-ps10100 P2rx7 Slc30a4 Rpsa-ps10 P2rx7 Cacna1e Rpsa-ps10 Cacna1e Cacna1eMme Rpl36a Mme ) Rpl36a Rpl36a Mme ) Slc6a19 ) Slc6a19 Rpl3 Rpl3Gm10260 Slc6a19 Rpl3 Gm10260 Rps2Gm10260 Rps2 P2rx1 P2rx1 Rps2 Cd5 Cacnb3 Cd5 Cacnb3 Cd5 Rpl12-ps3 Tmie Rpl12-ps3 Tmie Rpl12-ps3 Kcnd2 10 Kcnd2
10 value Padj Kcnd2 Kcna2
Padj value Padj Kcna2 10 (
Padj value Padj Kcna2 ( ( 10
10 Pagr1a 10 Pagr1a Pagr1a Scn4b
Scn4b -log
-log Scn4b -log Rpl17-ps8 Rpl17-ps8
bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079Nox1; this version posted JanuaryNox1 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a licenseNox1 to display the preprint in perpetuity. It is made available1 under aCC-BY 4.0 International1 license. -15 -10 -5 0 5-15 10-10 15-520 25 0 5 10 15 20 25 TG6 OT-1TG6 OT-1
Early Pos. Sel. Late Pos. Sel.1103 Late Pos. Sel. 1454 2538 1103 3116 1103 1454 Late Pos. Sel. 1454 100 Gtf2h4 100Gimap4 100 Slc6a19 Ddr1 Slc6a19 Slc6a19 Plac8 Rpsa Rps18 Il6ra Plac8 Il6ra Plac8 Kcna2 Kcna2 Il6ra 100 Kcna2 Slc30a4 Rpsa-ps10 P2rx7 Cacnb3 Cacnb3 Cacna1e Cacnb3 Rps18 Mme ) Rps18 Rpsa ) Rpl36a Rpsa Rps18 Mme ) ) TmieMme Rpsa Mme Slc6a19 Rpl3 Tmie Tmie Gm10260 Rpsa-ps10 Rpsa-ps10 Rps2 Kcnh2 Kcnh2 P2rx1 Rps8 Kcnh2 Rps8 Cacnb3 Cd5 Slc30a4 Slc30a4 Slc30a4 P2rx7 Rpl12-ps310 Cd510 P2rx7 Kcnh3 Cd5 P2rx7 Tmie Kcnd2 Kcnh3 10 Kcnd2 Kcnh3 Cd5
Kcnd2 value Padj Rps12-ps3 Gm14288
10 value Padj Rps12-ps3 Scnn1aGm14288 ( Padj value Padj Padj value Padj Kcna2 Scnn1a Rps12-ps3 Gm14288 ( Scnn1a ( ( 10 10 10 10 Pagr1a Rpl17-ps5 -log Rpl17-ps5 Scn4b -log Rpl17-ps5 -log -log Rps6-ps4 Rpl17-ps8Gm10260 Gm10260Rps6-ps4 Rps6-ps4
Rps4l Rps4l Nox1 Rps4l 1 1 1 -15 -10 -5 0 5 -1510 -1015 20 25-5 0 5-15 10-10 15-520 25 0 5 10 15 20 25 TG6 OT-1 TG6 OT-1TG6 OT-1
Mature CD8SP Mature CD8SP Late Pos. Sel. Mature CD8SP 1103 635 1454 635 620 620 100 5830411N06Rik 100 Slc6a19 100 5830411N06Rik100 5830411N06Rik Il6ra Plac8 Ccr8 Ccr8 Kcna2 Mme Ccr8 Mme Ccr4 Ccr4 Slc30a4 Slc30a4 Ccr4 Cacnb3 Gm10260 Tmie Gm10260 Tmie
) Tmie ) Plch1 Rps18 ) ) Rpsa Mme Plch1 Plch1 Tmie Kcnh2 Kcnh2 Rpsa-ps10 Rps18 Il17re Kcnh2 Rps18 Il17re Gm13443 Kcna2 Gm13443 Kcna2 Rps18 Kcnh2 Rps8 P2rx7 Kcna2 P2rx7 Slc30a4 Cd40lg Cd40lg P2rx7 Slc6a19 10 Slc6a19 10 Kcnd2 Kcnh3 Cd5 10 10 Kcnd2 Slc6a19 Kcnd2 Cd5 Kcnd2Il17rc Cd5 Il17rc (Padj value Cd5 Padj value Padj Rps12-ps3(Padj value Gm14288
Scnn1a (Padj value ( Il1r1 Mcoln2 Il1r1 Mcoln2 10 Il1r1 10
10 Mcoln2 Kcnh3 Cit10 Kcnh3 Cit Rpl17-ps5 Il23r Kcnh3 Cit Il23r Hist1h2ah -log Hist1h2ah Il23r -log Hist1h2ah -log Rps6-ps4
Gm10260 -log Gm10260 Cacnb3 Gm10260 Cacnb3 Gm10260Hist1h4f Hist1h4f Cdc25b Cdc25b Rps4l Scnna1 Scnna1 Foxm1 Scnna1 Foxm1 1 1 1 -15 -10 -5 0 5 -1510 -1015 20-525 0 5 -15 10 -10 15 -5 0 5 10 15 TG6 OT-1 TG6 OT-1 TG6 OT-1 Log (Fold Change) Log2(Fold Change) Log2(Fold Change) 858 Mature CD8SP 2 635 620 859 Supplemental100 Figure 7:5830411N06Rik Gene expression differences between TG6 and OT-1 thymocytes at Ccr8 Mme Slc30a4 Ccr4 860 differentGm10260 stagesTmie of development. Plots of significance versus fold change for the OT-1 vs TG6 ) Plch1 Kcnh2 Gm13443 Rps18 Il17re 861 comparison atKcna2 the early positiveP2rx7 selection, late positive selection, and mature SP stages of Cd40lg 10 Slc6a19 Kcnd2 Cd5 Il17rc 862 development.(Padj value The numbers in boxes denote the number of genes significantly upregulated for Mcoln2 Il1r1 10 Kcnh3 Cit Il23r Hist1h2ah 863 TG6-log (upper left)Cacnb3 and for OT-1 (upper right). Genes discussed in this study are indicated in color: Gm10260 Hist1h4f Cdc25b Scnna1 Foxm1 864 green1 (ion channel genes), red (translation & ribosome genes), blue (T cell activation/effector -15 -10 -5 0 5 10 15 865 function genes),TG6 purple (cell OT-1division genes), and orange (other). Other genes with very
Log (Fold Change) 866 significant p values2 and/or high fold change are labeled in grey.
48 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Early Positive Selection a Scn4b Themis 10 b OT-1OT-1 150 20000 F5F5 TG6TG6 5 15000 100 10000 0
(Fold Change) (Fold 50 2 5000 log Normalized Reads Normalized Normalized Reads Normalized -5 0 0
Mcu Itpr2 Itpr1 Nox1Tmie P2rx7 Orai1 Kcna3Clcn6 Tpcn1 Clcn7Clcn2 Orai2Trps1 Kcnh2 Scn5aKcnh3Scn2b Kcna2P2rx1 Scn4b Kcnd2 Kcnab2 Mcoln1 Cacnb1 Gabbr1Slc41a1 Scnn1aAtp2b4 Slc30a4Cacnb3 Slc39a4Cacna1d Slc39a8 Slc39a13Trpc4ap Cacna1e Mature SP Mature SP Cacna2d4 Early PosLate Sel Pos Sel Early PosLate Sel Pos Sel
Late Positive Selection Mature CD8 SP Tmie Kcna2 10 10 1500 5000 4000 5 5 1000 3000 2000 0 0 500 (Fold Change) (Fold 2 1000 Normalized Reads Normalized Reads Normalized log -5 -5 0 0
Mcu Orai3 Itpr1 Tmie Tmie P2rx7Hvcn1 Trpv2 Lrrc8bSlc4a2 Trps1 Kcna3Kcnh3Kcnh2Trpv6 Kcna2 Kcnd2 P2rx7Trpm4 Kcnh3 P2rx1Kcnh2 Kcna2 Kcnd2 Mature SP Mature SP Mcoln3 Kcnab2Slc39a6 Cacnb1 Slc30a4Scnn1aCacnb3 Kcnab2Cacng4Slc30a4Mcoln2Cacnb3 Scnn1a Early PosLate Sel Pos Sel Early PosLate Sel Pos Sel
c Slc6a19 Cacna1e 4 1500 500 400 1000 3 300 200 500 100 Normalized Reads Normalized Reads Normalized Normalized Reads 0 0 2
Early PosLate Sel Pos SelMature SP Early PosLate Sel Pos SelMature SP
Cacnb3 Tespa1 1 DN4 800 3000
DP CD69-DP CD69+ 600 2000 Mat. CD4SPMat. CD8SP (T) (T) 400 Naïve CD4SPNaïve CD8SP (S) (S) 1000 Early and Late 200 Normalized Reads Normalized Reads Normalized Normalized Reads Preselection Positive Selection 0 0 DP DP Mature DN4 Mature SP Mature SP CD69- CD69+ SP Early PosLate Sel Pos Sel Early PosLate Sel Pos Sel Relative Expression Time
d DP (CD69-) vs DN4 DP (CD69-) vs DP CD69+ Collagen Microtubule Motor Activity Extracellular Matrix Part Integrin Binding Voltage Gated Channel Activity Spindle Pole Proteinaeous Extracellular Matrix Voltage Gated Calcium Channel Complex Gated Channel Activity Spindle Voltage Gated Cation Channel Activity Spindle Organization and Biogenesis Substrate Specific Channel Activity Cell Cycle Process Extracellular Matrix Mitotic Spindle Organization And Biogenesis G Protein Coupled Receptor Activity Microtubule Organizing Center Part Ion Channel Activity Voltage Gated Calcium Channel Activity Potassium Ion Transport Protein Complex Binding Calcium Channel Activity Cell Cycle Phase Basement Membrane Organic Anion Transmembrane Transporter Activity Cation Channel Activity Neutral AA Transmembrane Transporter Activity Voltage Gated Calcium Channel Activity Monocarboxylic Acid Transport 0 1 2 3 0 1 2 867 NES NES
49 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
868 Supplemental Fig. 8: Thymocytes with low self-reactivity retain a preselection gene expression
869 program marked by elevated expression of ion channel genes. (a) Ion channel genes from the
870 manually curated list in Figure 7b that exhibit significantly different (padj<0.05, fold change>0.5)
871 expression between OT1 versus TG6 thymocytes at the indicated stages of development.
872 Positive values (green) indicate genes enriched in TG6; negative values (red) indicate genes
873 enriched in OT-1. (b) Normalized read counts (see methods) for the indicated genes at each
874 stage of development. Red circles: OT-1; Blue squares: F5; Green triangles: TG6. (c) Left:
875 Heatmap of normalized expression of the ion transport genes (Group 2 genes from Figure 7d) in
876 wild thymocytes at the indicated stages of development (scaled per row). Data are from the
877 ImmGen microarray database. Bottom: Graphic representation of the average gene expression
878 pattern. (d) Normalized enrichment score (NES) for the top 15 gene sets enriched in DP CD69-
879 cells compared to DN4 and DP CD69+ thymocytes, using gene set enrichment analysis (GSEA)
880 and the Gene Ontology database. Black bars indicate gene sets relating to ion channels.
50 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
881 Supplemental Tables:
TG6 OT-1 TG6 OT-1 3h 3h 6h 6h Total Signaling Timepoints 172 409 177 163 Total Timepoints 16928 21973 32367 14553 Percent Time Signaling 1.02 1.86 0.55 1.12 882
883 Supplemental Table 1: TG6 thymocytes spend less time signaling compared to OT-1 cells.
884 Percent time signaling was calculated by dividing the number of signaling timepoints by total
885 timepoints.
886
TG6 3h OT-1 3h TG6 6h OT-1 6h Total Signaling Events 42 61 44 32 Cumulative Track Imaging Time (h) 47.022 61.036 89.908 40.425 Signaling Events/Hour 0.89 1.00 0.49 0.79 887
888 Supplemental Table 2: TG6 thymocytes experience less frequent TCR signaling than OT-1
889 thymocytes. The frequency of signaling events was calculated by dividing the total number of
890 signaling events by the cumulative track imaging time (the sum of all of the track durations for all
891 runs) for each condition.
51 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Expression Rank Gene (fold) P value 1 Dntt 9.27 0.0110 2 Slc6a19 4.18 0.0025 Slc6a19 4.10 0.0143 3 Slc16a5 2.79 0.0257 4 Ddc 2.55 0.0035 5 A130038J17Rik 2.30 0.0175 6 Grik4 2.20 0.0034 7 Tmem154 2.10 0.0322 8 4930513N10Rik 2.08 0.0453 9 Tubb2a 2.03 0.0200 10 Itk 2.01 0.0411 11 Tmie 2.00 0.0385 12 Nipa1 1.97 0.0218 13 Sc4mol 1.96 0.0425 14 D14Abb1e 1.96 0.0236 15 Akap13 1.89 0.0062 16 A130004G07Rik 1.88 0.0028 17 Bambi-ps1 1.86 0.0182 ENSMUSG00000074335 18 (Kcna2) 1.86 0.0209 19 Art4 1.81 0.0367 20 Ccr9 1.78 0.0018 21 D5Wsu152e 1.78 0.0080 22 Satb1 1.71 0.0241 23 Tsepa 1.70 0.0287 24 Smarca5 1.70 0.0055 25 Pvr 1.69 0.0100 26 Arhgef6 1.69 0.0252 27 Scfd1 1.67 0.0291 28 Gpr97 1.66 0.0211 29 Tmem136 1.66 0.0176 30 Morc3 1.66 0.0063 31 Vps35 1.66 0.0083 32 Dstyk 1.65 0.0313 33 Tbrg3 1.64 0.0078 34 Socs3 1.63 0.0312 35 Frmd4b 1.63 0.0026 36 A130091K22Rik 1.63 0.0207 37 Senp6 1.62 0.0287 38 Dnajc6 1.62 0.0235 39 Avl9 1.61 0.0060 Ccr9 1.61 0.0077 40 Dennd5a 1.61 0.0317 41 AI854517 1.61 0.0350 42 Jun 1.60 0.0302 43 Pacs2 1.60 0.0253 Tmie 1.60 0.0177 44 Ntrk3 1.59 0.0104 45 Rrad 1.58 0.0103 46 BC065397 1.58 0.0379 47 1700019D03Rik 1.57 0.0002 892
893 Supplemental Table 3: Genes upregulated in CD5lo compared to CD5hi naïve polyclonal
894 CD8SP T cells by microarray analysis. The top 47 significantly different (P<0.05, >1.45 fold
52 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
895 difference) genes upregulated in sorted CD5lo naive polyclonal CD8SP T cells, by microarray
896 analysis, and ranked by their expression (fold) difference. Gene that appear multiple times
897 represent multiple probe sets for the same gene, and a number in the left-most column is
898 included only for the first listing. Genes of interest, Kcna2 and Tmie, are in bold. Data was
899 analyzed by Fulton et al (GSE62142).
53 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Rank Gene Expression (fold) padj 1 Col2a1 5.95 7.25E-05 2 Irs1 5.91 6.68E-04 3 Meg3 5.87 2.58E-02 4 Col9a2 4.81 1.13E-02 5 Igf2 4.28 1.17E-03 6 Lect1 4.25 1.06E-02 7 P3h3 4.00 3.99E-02 8 Mir6934 3.62 2.65E-02 9 Thbs1 3.61 3.01E-02 10 Pcsk1 3.52 2.74E-07 11 5930430L01Rik 3.35 4.95E-05 12 Slc6a19 3.20 3.39E-14 13 Col12a1 3.12 1.29E-02 14 Grm6 3.00 6.58E-10 15 Dntt 2.94 3.83E-21 16 Lrrc3b 2.93 1.08E-05 17 Adamts14 2.67 7.45E-21 18 Tdrd5 2.55 5.10E-06 19 Zfp36l3 2.52 4.89E-02 20 Nrg2 2.30 2.22E-03 21 Fbxl21 2.22 2.17E-04 22 Kcnj16 2.20 4.85E-06 23 Btc 2.17 1.60E-02 24 Islr 2.14 5.34E-12 25 Slc6a19os 2.09 7.51E-07 26 P4ha2 2.01 5.01E-09 27 Nphs2 1.98 7.03E-04 28 A130077B15Rik 1.96 4.63E-06 29 4930447K03Rik 1.94 2.69E-02 30 Fbln2 1.92 4.58E-05 31 Arpp21 1.91 6.49E-07 32 Osgin1 1.89 1.61E-09 33 Gpr34 1.89 1.50E-05 34 4930512J16Rik 1.82 4.43E-02 35 Pomgnt2 1.81 2.95E-03 36 Slc16a5 1.72 5.13E-29 37 Cyp2s1 1.70 1.19E-13 38 Dmbt1 1.65 1.76E-18 39 Slc6a9 1.61 1.41E-02 40 Gm10516 1.61 1.84E-02 41 Prdm1 1.53 9.84E-08 42 Prss50 1.52 3.91E-03 43 Ntn5 1.51 4.42E-02 44 Rmrp 1.48 2.13E-03 45 Tmie 1.47 4.08E-04 46 Igsf23 1.47 4.61E-15 47 Smyd1 1.42 1.03E-03 48 Zp3r 1.41 1.81E-02 49 Angptl2 1.41 3.47E-06 50 Bmf 1.38 3.00E-07 51 Kcna2 1.35 1.92E-13
54 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
900 Supplemental Table 4: Genes upregulated in CD5lo compared to CD5hi naïve polyclonal
901 CD8SP T cells by RNA-seq analysis. The top 51 significantly different (padj<0.05) upregulated
902 genes in sorted CD5lo naive polyclonal CD8+ T cells and ranked by their expression (fold)
903 difference, from the Matson, et al. RNA-seq dataset (GSE151395). Genes of interest, Kcna2
904 and Tmie, are in bold. Data was analyzed by DEseq2.
905
906 Supplemental Movies: (please see Supporting Files)
907 Movie S1: Representative TG6 thymocyte signaling event. Preselection TG6 thymocytes were
908 loaded with the ratiometric calcium indicator dye Indo1LR and allowed to migrate into selecting
909 thymic slices for 6hrs, then imaged using two-photon-microscopy. Video is a z-projection of the
910 ratio of fluorescent signal in the calcium-bound channel over calcium-unbound channel for
911 Indo1LR displayed as a heatmap (red=calcium high, purple=calcium low). Arrow indicates
912 timepoints in which the cell displays elevated calcium (130 seconds). Frames were collected
913 every 10s for 10.3min. Dimensions: 45.7μm (width) by 75.0μm (height) by 24.9μm (depth).
914 Scale bar is 10μm.
915
916 Movie S2: Representative OT-1 thymocyte signaling event. Preselection OT-1 thymocytes were
917 loaded with the ratiometric calcium indicator dye Indo1LR and allowed to migrate into selecting
918 thymic slices for 6hrs, then imaged using two-photon-microscopy. Video is a z-projection of the
919 ratio of fluorescent signal in the calcium-bound channel over calcium-unbound channel for
920 Indo1LR displayed as a heatmap (red=calcium high, purple=calcium low). Arrow indicates
921 timepoints in which the cell displays elevated calcium (230 seconds). Frames were collected
922 every 10s for 14.3min. Dimensions: 68.9μm (width) by 49.8μm (height) by 18μm (depth). Scale
923 bar is 10μm.
55 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427079; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
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