1. Immunology and Inflammation
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The somatically generated portion of T cell receptor CDR3α contributes to the MHC allele specificity of the T cell receptor

  1. Philippa Marrack  Is a corresponding author
  2. Sai Harsha Krovi
  3. Daniel Silberman
  4. Janice White
  5. Eleanor Kushnir
  6. Maki Nakayama
  7. James Crooks
  8. Thomas Danhorn
  9. Sonia Leach
  10. Randy Anselment
  11. James Scott-Browne
  12. Laurent Gapin
  13. John Kappler
  1. Howard Hughes Medical Institute, United States
  2. National Jewish Health, United States
  3. University of Colorado School of Medicine, United States
  4. La Jolla Institute for Allergy and Immunology, United States
Research Article
Cite this article as: eLife 2017;6:e30918 doi: 10.7554/eLife.30918
8 figures, 1 table, 1 data set and 3 additional files

Figures

CD4 selection in mice expressing single TCRβ chains and different MHC alleles.

Cells were isolated from the thymuses and lymph nodes of mice expressing a singl;e TCRβ, DOβWT or DOβ48A, and different MHC haplotypes and stained for expression of CD4 and CD8 and CD44. Results are the means and standard errors of the mean (SEMs) of three independently analyzed mice expressing the indicated TCRβs and MHC II alleles. Student t analyses were used to compare results between the DOβWT and DOβ48A paired samples. *p<0.05., **p<0.01, ***p<0.001.

https://doi.org/10.7554/eLife.30918.002
Figure 1—source data 1

Data from individual mice show that both CD4 and CD8 T cells appear in mice expressing a single TCRb chain regardless of the MHC allele expressed.

The numbers of thymus and lymph node cells were counted and analyzed for their expression of CD4 and CD8 and T cell receptor.

https://doi.org/10.7554/eLife.30918.003
TCRα contributes to the MHCII allele bias of selected naïve CD4 T cells.

Naïve CD4 T cells were isolated from the lymph nodes of DOβ48A H2b, f or s mice and incubated for 2 days in wells coated with anti-TCRβ and anti-CD28. Thus activated, the cells were spinfected with GFP-expressing retroviruses expressing also DOβWT or DOβ48A. The cells were cultured for a further 2 days and then challenged with spleen cells from mice expressing the indicated MHC alleles, or in the absence of added spleen cells. One day later the cells were stained for expression of CD69. Results were calculated as the (% of GFP+ T cells transduced with DOβWT-expressing retroviruses that were CD69+) – (the % of GFP+ T cells transfected with DOβ48A-expressing retroviruses that were CD69+) in wells containing the same challenge spleen cells. Shown are the average results ± standard error of the mean (SEM) from three independent experiments. *p<0.05, **p<0.01 by one way ANOVA followed by Neuman Keuls analyses.

https://doi.org/10.7554/eLife.30918.004
Figure 2—source data 1

After transduction with the DObWT chain, T cells from mice expressing DOb48A react with cells bearing the MHC allele that selected them.

CD4 T cells were isolated from individual DOb48A mice and transduced with retorviruses expressing DObWT or DOb48A as described in the Methods section. The cells were vultured with spoleen cells from mice expressing various MHC alleles and responses assessed one day later by measuring the % of cells that bore CD69. Data shown are from individual mice assayed on different days.

https://doi.org/10.7554/eLife.30918.005
Figure 3 with 1 supplement
Expression of a single TCRβ chain, DOβWT and, even more markedly, DOβ48A, reduces the number of different TCRα chains that can be positively selected, regardless of the selecting MHCII allele.

Naïve CD4 T cells were isolated from the spleens (B6) or lymph nodes of mice expressing MHC b, f or s, single TCRβ chains and heterozygous for expression of functional TCRα chains. Their expressed TCRα chains were sequenced and analyzed with species accumulation curves. Results were combined from three independently sequenced data sets from mice of each genotype except for those for H2s DOβWT animals, which were combined from only two independently sequenced animals. Data are shown together with an estimate (bracketed) of the total numbers of different TCRα protein sequences present in the naïve CD4 T cells of each type of mouse.

https://doi.org/10.7554/eLife.30918.006
Figure 3—figure supplement 1
The naïve CD4 T cells in mice expressing a single TCRa chain express a limited number of TCRα sequences regardless of the MHC allele involved in their selection in the thymus.

Results were calculated as described in Figure 3. Data shown are for individual mice.

https://doi.org/10.7554/eLife.30918.007
Figure 4 with 1 supplement
TCRβ sequences on naïve CD4 T cells are determined by the selecting MHCII allele and the co-selected TCRβ.

TCRαs on naïve CD4 T cells from the lymph nodes of TCRα+/- mice expressing a single TCRα and various MHC alleles were sequenced and analyzed as described in Figure 3. Results are the means and SEMs of three independently sequenced animals of each genotype except for H2s DOβWT animals, of which only two mice were analyzed. ***p<0.001 by one way ANOVA with Newman-Keuls post analysis.

https://doi.org/10.7554/eLife.30918.008
Figure 4—figure supplement 1
TCRα sequences are somewhat more likely to be shared between T cells selected on the same MHCII allele but differing in TCRβ than between T cells sharing TCRβ but selected on different MHCII alleles.

TCRα sequences were obtained and analyzed as described in Figure 4. Data shown are combined as indicated between all the mice illustrated in Figure 4. Results are the means and SEMs of the data. ***p<0.001 by one way ANOVA with Newman Keuls post analysis.

https://doi.org/10.7554/eLife.30918.009
Figure 5 with 3 supplements
The frequency with which TRAVs are used on naïve CD4 T cells in TCRβ transgenic mice depends on their selecting MHCII and their partner TCRβ.

TCRαs on preselection thymocytes or naïve T cells from the lymph nodes of TCRα+/- mice expressing a single TCRβ and various MHC alleles were sequenced and analyzed as described in Figure 3. (A) Shown are the Euclidean distances for TRAV use between the data for individual mice. Samples are hierachically ordered. Individual mice of the same genotype are numbered m1-3. (B) The average % use of each TRAV in mice expressing the indicated MHCII allele and TCRβ. Results are the means ± SEMs of 3 identical mice, except for H2s DOβWT animals, for which results are the averages of 2 mice. TRAVs are ordered by family, not by position on the chromosome.

https://doi.org/10.7554/eLife.30918.010
Figure 5—figure supplement 1
Different TRAVs are detected at different frequencies in preselection thymocytes.

Preselection DP thymocytes were obtained from individual mice expressing the indicated MHCII alleles and TCRβs. Shown are the %s with which individual TRAVs were detected. Subfamily members of each family are color coded as shown. Note, the TRAVs are ordered by family and not by their position in the TCRα locus.

https://doi.org/10.7554/eLife.30918.011
Figure 5—figure supplement 2
TRAV usage by naive CD4 T cells depends on the selecting MHCII allele and partner TCRβ.

Pairwise scatter plots of individual TRAV frequencies between all replicates of mature naïve CD4 T cells from mice expressing different MHCII alleles and different TCRβ chains. Data are plotted as log2 transformed TRAV counts per 1 × 10^4 total TCRα sequences in each mouse noted on the x axis versus each mouse noted on the y axis. Values distributed along the diagonal are equivalent between the two mice compared in the plot. Individual mice of each genotype were numbered m1-m3, numbers for each mouse are used consistently throughout.

https://doi.org/10.7554/eLife.30918.012
Figure 5—figure supplement 3
TRAVs are used to different extents by naïve CD4 T cells in mice expressing different MHCII alleles and/or different TCRβs.

Data were obtained and analyzed as described in Figures 3 and 5. DESeq2 was used to compare the frequencies with which different TRAVs were used by naïve CD4 T cells in mice expressing different MHCII alleles and/or different TCRβs. Differential expression analyses were performed using the DESeq2 package (v1.8.1) in the R language (v3.2.2). DESeq2 fit negative binomial regression models to each feature to compare between groups. Corrections for size factors for each sample (animal) to account for differences in repertoire size were applied. The negative binomial dispersion parameter for each feature was then calculated, sharing information across features with similar expression levels to moderate extreme empirical dispersion estimates. Wald tests were applied to each feature to test for differential expression between groups. Features were considered differentially expressed if they had a Benjamini-Hochberg adjusted p-value (i.e., false discovery rate)<0.05.

https://doi.org/10.7554/eLife.30918.013
Figure 6 with 2 supplements
Naïve CD4 T cells in DOβWT or DOβ48A mice preferentially use TRAJs from the distal end of the TRAJ locus.

TCRαs on preselection thymocytes or naïve CD4 T cells from the lymph nodes of TCRα±mice expressing a single TCRβ and various MHC alleles were sequenced and analyzed as described in Figure 3. (A) Shown are the Euclidean distances for TRAJ use between the data for individual mice. Samples are hierachically ordered. Individual mice of the same genotype are numbered m1-3. (B-L) The % use of each TRAJ in mice expressing the indicated MHCII allele and TCRβ. Results are the means and SEMs of 3 identical mice, except for H2s DOβWT animals, for which results are the averages of 2 identical mice. TRAJs are ordered by position on the chromosome. Also shown are the means and SEMs of TRAJ use by five independently sequenced preselection thymocytes and three independently sequenced naïve CD4 T spleen T cells from B6 mice.

https://doi.org/10.7554/eLife.30918.014
Figure 6—figure supplement 1
TRAJ usage by naive CD4 T cells depends on the selecting MHCII allele and partner TCRβ.

Pairwise scatter plots of individual TRAJ frequencies between all replicates of mature naïve CD4 T cells from mice expressing different MHCII alleles. Each point is a TRAJ. Data are plotted as log2 transformed TRAV counts per 1 × 10^4 total TCRαsequences in each mouse noted on the x axis versus each mouse noted on the y axis. Values distributed along the diagonal are equivalent between the two mice compared in the plot. Individual mice of each genotype were numbered m1-m3, numbers for each mouse are used consistently throughout.

https://doi.org/10.7554/eLife.30918.015
Figure 6—figure supplement 2
TRAJs are used to different extents by naïve CD4 T cells in mice expressing different MHCII alleles and/or different TCRβs.

Data were obtained and analyzed as described in Figures 3 and 5. DESeq 2 was used as described in Figure 5—figure supplement 3 to compare the frequencies with which different TRAJs were used by naïve CD4 T cells in mice expressing different MHCII alleles and/or different TCRbs.

https://doi.org/10.7554/eLife.30918.016
Figure 7 with 1 supplement
CDR3α sequences on naïve CD4 T cells are determined by the selecting MHCII allele and the co-selected TCRβ.

TCRαs on naïve T cells from mice expressing a single TCRβ and various MHCII alleles were sequenced and analyzed for their CDR3α sequences as described in Figures 3 and 4. CDR3α sequences were defined as the amino acids between and including the conserved cysteine at the C terminal end of the TRAV and the conserved phenyl alanine, tryptophan or leucine in the TRAJ region. Shown are the means and SEMs of 3 independently sequenced identical mice except for H2s DOβWT mice, in which case only two mice were analyzed. ***p<0.001, **p<0.01 by one way ANOVA with Newman-Keuls post analysis.

https://doi.org/10.7554/eLife.30918.017
Figure 7—figure supplement 1
CDR3α length on naïve CD4 T cells depends upon the selecting MHC allele and the co-selected TCRβ.

Data were obtained as described in Figures 3 and 4. Shown are the average lengths of CDR3α and N region bases on naïve CD4 T cells coexpressed with the indicated TCRβ. Results are the means and SEMs of 3 mice of the same haplotype except for s DOβWT mice, in which case the results from two mice were averaged. Errors are calculated with one way ANOVA with the Bonferroni post test comparing all pairs. *p<0.05, **p<0.01, ***p<0.001. Statistical results are omitted for pairs which were not significantly different from each other.

https://doi.org/10.7554/eLife.30918.018
Figure 8 with 2 supplements
N region amino acids in CDR3α of naïve CD4 T cells are determined by the selecting MHCII allele and the co-selected TCRβ.

TCRαs on naïve CD4 T cells of mice expressing a single TCRβ and various MHCII alleles were sequenced as described in Figures 3 and 4. Mice were as listed in those Figures. Comparisons were made of N regions derived from the same TRAV TRAJ pair providing that all mice in the comparisons expressed at least five different sequences involving the chosen TRAV TRAJ pair. Results shown are the means ± SEMs of the data from identical mice. Statistical analyses involved one way ANOVA tests with Newman-Keuls post test analyses (A, B) or Student t tests (C–F). *p<0.05, **p<0.01, ***p<0.001.

https://doi.org/10.7554/eLife.30918.019
Figure 8—figure supplement 1
N region amino acids in CDR3α of naïve CD4 T cells are determined by the selecting MHCII allele and the co-selected TCRβ.

TCRαs on naïve CD4 T cells of mice expressing a single TCRβ and various MHCII alleles were sequenced as described in Figures 3 and 4. Mice were as listed in those Figures. The results of samples expressing the same TRAV TRAJ pairs for all comparisons in which each mouse expressed at least five different sequences involving the chosen TRAV/TRAJ pair were averaged. Results shown are the means ± SEMs of the data from mice of identical genotypes. Statistical analyses for comparisons involving three different types of mice used one way ANOVA tests with Newman-Keuls post test analyses. Student t tests were used to analyze statistically the differences between pairs of mice. **p<0.01, ***p<0.001.

https://doi.org/10.7554/eLife.30918.020
Figure 8—figure supplement 2
The frequency of amino acid use in CDR3α use on naïve CD4 T cells depends on the selecting MHCII allele, the coselected TCRβ and the length of the CDR3α.

Data from TCRα sequences obtained as in Figure 4 were used to analyze the frequency with which different amino acids were used at different positions.

https://doi.org/10.7554/eLife.30918.021

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
T cell receptor beta chain from
the DO11.10 hybridoma
(mus musculus musculus)
DObWTHaskins, K., Kubo, R., White, J., Pigeon, M.,
Kappler, J. and Marrack, P. The major
histocompatibility complex-restricted
antigen receptor on T cells. I. Isolation
with a monoclonal antibody. J. Exp. Med.
157:1149, 1983.
T cell receptor beta chain from
the DO11.10 hybridoma (mus
musculus musculus) with the
amino acid at position 48 of
the chain mutated from a
tyrosine to an alanine
DOb48A375. Scott-Browne, J.P., White, J., Kappler,
J.W., Gapin, L. and Marrack, P. Germline-
encoded amino acids in the abT cell
receptor control thymic selection. Nature
458:1043–1046, 2009. PMC2679808
C57BL/6J (mouse)B6The Jackson Laboratory
Mice congenic with B6 but
expressing H2f
This publication
Mice congenic with B6 but
expressing H2s
This publication
Mice with one TCRa
gene inactivated
a+/-The Jackson Laboratory; Mombaerts et al.
Nature 360:225–231,1992
B6 mice with the TCRb
genes inactivated
b-/-The Jackson Laboratory; Creation of a
large genomic deletion at the T-cell
antigen receptor beta-subunit locus in
mouse embryonic stem cells by gene
targeting. Mombaerts et al. PNAS 88
3084–7, 1991
Software for analysis and
correction of TCRalpha
sequences
https://www.nationaljewish.org/research-science/programs-depts/biomedical-research/labs/kappler-marrack-research-lab/protocols
B6 TRAV and
TRAJ sequences
MOVA-B6.VDB
MOJA.JDB
https://www.nationaljewish.org/research-science/programs-depts/biomedical-research/labs/kappler-marrack-research-lab/protocols
TCRa sequences
used in analyses
GEO accession GSE105129

Data availability

The following data sets were generated
  1. 1

Additional files

Supplementary file 1

In normal mice, a significant number of TCRα sequences appear on naïve CD4 T cells regardless of the selecting MHCII allele.

Naïve CD4 T cells were isolated from the lymph nodes of normal mice of the indicated strains and their TCRα sequences identified as described in the Materials and methods section. Shown are the %s of unique sequences and the %s of total sequences that were shared between pairs of mice of the indicated strains. Data were obtained from three independently sequenced B6 mice and one each B6.AKR and B6.NOD animals and are the means and standard errors of the means of the comparisons.

https://doi.org/10.7554/eLife.30918.022
Supplementary file 2

Sequences of TCRβ transgenes

https://doi.org/10.7554/eLife.30918.023
Transparent reporting form
https://doi.org/10.7554/eLife.30918.024

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