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The TCR's sensitivity to self peptide–MHC dictates the ability of naive CD8+ T cells to respond to foreign antigens

Nature Immunology volume 16, pages 107–117 (2015)Cite this article

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Abstract

The strength with which complexes of self peptide and major histocompatibility complex (MHC) proteins are recognized by the T cell antigen receptor (TCR) dictates the homeostasis of naive CD8+ T cells, but its effect on reactivity to foreign antigens is controversial. As expression of the negative regulator CD5 correlates with self-recognition, we studied CD5lo and CD5hi naive CD8+ T cells. Gene-expression characteristics suggested CD5hi cells were better poised for reactivity and differentiation than were CD5lo cells, and we found that the CD5hi pool also exhibited more efficient clonal recruitment and expansion, as well as enhanced reactivity to inflammatory cues, during the recognition of foreign antigen. However, the recognition of complexes of foreign peptide and MHC was similar for both subsets. Thus, CD8+ T cells with higher self-reactivity dominate the immune response to foreign antigens, with implications for T cell repertoire diversity and autoimmunity.

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Figure 1: CD5 expression by naive CD8+ T cells identifies stable populations with unique phenotypic traits.
Figure 2: Naive CD5lo and CD5hi CD8+ T cells have distinct gene-expression characteristics.
Figure 3: Naive CD5lo and CD5hi CD8+ T cell precursors differ in the capacity of their response to foreign antigen.
Figure 4: Analysis of the clonal responses of naive CD5lo and CD5hi CD8+ T cells to infection.
Figure 5: Early recruitment of CD5lo and CD5hi cells into the immune response after infection.
Figure 6: CD5hi and CD5lo cells show similar binding of the TCR to cognate antigen.
Figure 7: CD5hi cells 'preferentially' use proinflammatory signals during population expansion.

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Acknowledgements

We thank D. Masopust (University of Minnesota) for Il15−/− mice, P14 mice and LCMV Armstrong strain; L. Cauley (University of Connecticut) for F5 mice deficient in recombination-activating gene 1; J. Zhu (National Institute of Allergy and Infectious Diseases) for T-bet–ZsGreen reporter mice; J. Harty (University of Iowa) for L. monocytogenes strain ΔactA (DP-L1942) and OVA-expressing L. monocytogenes ΔactA; R. Kedl (National Jewish Medical Research Center) for LM-B8R (virulent and ΔactA); M. Prlic and M. Bevan (University of Washington) and J. Harty (University of Iowa) for Flt3L-expressing B16 cells; R. Kroczek (Robert Koch-Institute) for the conjugated monoclonal antibody MTAC-2; the University of Minnesota Flow Core personnel for flow-cytometry support and cell sorting; M. Jenkins and M. Mescher for critical review of the manuscript; G. Stritesky for insight on the analysis of Nur77 mice; J. Ding and S. Peery for technical assistance with mice; and members of the Jamequist laboratory for discussions. Supported by the US National Institutes of Health (R37 AI-38903 to S.C.J.; P30 CA77598, with the Biostatistics and Bioinformatics Core shared resource of the Masonic Cancer Center, University of Minnesota; UL1TR000114 from the National Center for Advancing Translational Sciences) and the Irvington Institute Fellowship Program of the Cancer Research Institute (R.B.F.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the US National Institutes of Health.

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Authors and Affiliations

  1. Department of Laboratory Medicine and Pathology, Center for Immunology, University of Minnesota Medical School, Minneapolis, Minnesota, USA

    Ross B Fulton, Sara E Hamilton, Yan Xing, Kristin A Hogquist & Stephen C Jameson

  2. Division of Biological Sciences, University of California San Diego, La Jolla, California, USA

    J Adam Best & Ananda W Goldrath

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  1. Ross B Fulton
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  2. Sara E Hamilton
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  3. Yan Xing
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Contributions

R.B.F. and S.C.J. designed the experimental approaches; R.B.F. and S.E.H. conducted experiments; Y.X. and K.A.H. provided mouse strains and bone marrow chimeras; J.A.B. and A.W.G. analyzed gene-expression data; and R.B.F. and S.C.J. wrote and edited the manuscript.

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Correspondence to Stephen C Jameson.

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Integrated supplementary information

Supplementary Figure 1 Phenotypic characteristics of naive CD5lo and CD5hi CD8+ T cells in wild-type and Il15−/− mice.

Peripheral lymphocytes were isolated from WT (a and c) or IL-15-/- (b and c) mice. Naive (CD44lo) CD8 T cells were gated on the lower (red) and upper (blue) 20% of CD5 expression and compared to memory phenotype (MP) cells (CD44hiCD122hi, gray shaded). Flow plots are representative of 2 separate experiments. (d) CD5hi and CD5lo naïve CD8 T cells were sorted and transferred into normal hosts (as in Fig 1c). The graph shows the percentage of each population that acquired CD44hi (memory)-phenotype, presented as mean ± SEM). Data in (a,c) are representative of 4 experiments and those in (b) 2 experiments. * indicates p<0.05.

Supplementary Figure 2 Naive CD5hi CD8+ T cells exhibit rapid production of the chemokine XCL1.

Splenocytes from T-bet-GFP reporter mice were left untreated or stimulated with PMA/ionomycin in the presence of brefeldin A. Cells were then stained for CD8, CD44 and CD5, and then fixed, permeabilized and stained intracellularly for XCL1 and also CXCR3 (since detectable surface expression of CXCR3 was reduced by stimulation). T-bet expression was determined using the T-bet-ZsGreen (enhanced GFP) reporter (note – some quenching of the ZsGreen reporter is apparent in stimulated cells). In other experiments (including those shown in Fig. 1a,b), T-bet was detected by intracellular staining, with similar results. (a) Expression of CXCR3 and T-bet (T-bet-ZsGreen reporter) was determined in unstimulated cells, while staining for XCL1 was assessed after PMA/Ionomycin stimulation. The cells were counterstained for CD5 and the data gated on CD44lo CD8+ T cells. (b,c) Co-expression of XCL-1, CXCR3 and T-bet in (b) CD44hi memory phenotype CD8+ T cells or (c) CD44lo CD5hi CD8+ T cells. Cells were stimulated with PMA/Ionomycin or not, as indicated. Using Boolean gating, the average frequency of (stimulated) CD44hi cells positive for CXCR3, T-bet AND XCL1 was 36.5%, while that for CD44lo, CD5hi cells was 1.2% (data not shown) in this experiment. These data are representative of 3 experiments (n=7), utilizing T-bet reporter cells.

Supplementary Figure 3 Additional characterization of the expansion potential of naive CD5hi and CD5lo CD8+ T cell populations.

(A and B) Detection of CD5lo/hi B8R/Kb-specific (A) and bulk LM-specific cells (B) by IFN-γ. Bulk splenocytes were stimulated with 1 μM B8R20-27 peptide (A) or PMA/ionomycin (B) for 5 hrs in the presence of BFA and then stained for intracellular IFN-γ. (C-E) The preferential expansion of naive CD5hi CD8 T cells is not unique to B8R/Kb specific cells or to LM infection. (C) shows the ratio of donor CD5hi: CD5lo OVA/Kb-specific cells on day 7 after primary LM-B8R infection or 5 days after re-challenge of memory recipients with virulent LM-B8R. Red symbols indicate mice where OVA/Kb-specific responses were not detected within the CD5lo donor population and the ratio was arbitrarily set to 100. (D and E) Ratio of donor CD5hi:CD5lo gp33/Db (D) or bulk pathogen-specific cells (E) on days 7 and 30+ after primary acute infection with LCMV Armstrong. (F) In vitro stimulation of CFSE-labeled polyclonal CD5lo/hi cells with plate-bound αCD3/αCD28 (2.5ug/mL) for 6 days. (G) CD5hi donor cells still have an advantage over CD5lo cells in RAG-/- recipients. Donor cells were transferred into RAG-/- recipients, infected the next day with LM-B8R, and the splenic responses were analyzed 7 days later. (H) CD5lo/hi cells were sorted on equally low CD44 expression and adoptively transferred into congenic wild type recipients. Mice were infected with LM-B8R and responses in peripheral blood were analyzed 7 days later. Red symbol indicates mice were the B8R/Kb-specific response was not detected within the CD5lo donor population and the ratio was arbitrarily set to 100. (I) CD5 expression levels on WT and CD25-/- naïve CD8+ T cells derived from bone marrow chimeras (described in Fig. 3g). Data in (a,b) are compiled from 1 to 4 experiments (n=4-11), (c) compiled from 2-4 experiments (n=6-11), (d) compiled from 2 experiments (n=4-7) (f) is representative of 2 experiments (g,h) from 1 experiment (n=4) (i) is representative of 3 chimera sets (n=6).

Supplementary Figure 4 Schematic for the simultaneous adoptive transfer of multiple congenic naive CD5lo or CD5hi CD8+ T cells.

Peripheral CD8 T cells from 4-8 congenic donors were negatively enriched and then equal numbers from each donor were combined. CD44lo CD5lo and CD5hi cells were flow sorted and then the indicated number of each individual donor population was transferred into congenic recipients. Mice were infected the next day with LM-B8R and the B8R/Kb-specific response was assessed 7 days later using tetramer enrichment

Supplementary Figure 5 Skewed CD5 distribution on naive LM-B8R–H-2Kb–specific CD8+ T cell precursors.

The gates demarcating the lower and upper 20% for CD5 expression on bulk CD44lo CD8+ T cells cells were applied to CD44lo B8R/Kb tetramer stained CD8+ T cells from the same sample. The data indicate what percentage of B8R/Kb specific cells are contained in the gates used for sorting CD5hi and CD5lo cells. Values above 20% indicate the B8R/Kb specific naïve CD8+ T cells was overrepresented in that sorted population, values less than 20% indicate B8R/Kb specific population were underrepresented in that group. Data are cumulative from 5 separate experiments (n=17) and are shown as mean ± SEM. Median values were used to calculate the fold-change from the bulk population (i.e. by how much the B8R/Kb-specific population is under- or overrepresented in that population).

Supplementary Figure 6 Characterization of the C2A peptide, and effect of CpG on the response of OT-I and H-Y T cells.

(a) Relative stabilization of H-2Db on RMA-S cells following incubation with titrated doses of the HY Smcy/Db peptide with or without a C2A substitution. Data are shown as mean ± SEM. (b) H-Y TCR transgenic MHC class I tetramer binding intensities (geometric MFI normalized to maximum intensity) over a range of tetramer concentrations. Data are representative of one out of two experiments. (c) In vitro activation of H-Y CD8 T cells. T cells were incubated with titrated doses of wild-type or altered C2A peptide for 6 hrs and CD69 expression was assessed by flow cytometry. Data is normalized to maximum CD69 expression and representative of two experiments. (d) HY and OT-I T cells were adoptively co-transferred and primed with peptide-pulsed DC +/- CpG administration, and the response of each population was measured on day 7 after priming. Data are presented in the same way as Fig. 7a,b. Data in (a) are compiled from 3 separate experiments, (b,c) are representative of 2 separate experiments; (d) is from a single experiment (n=3 for DC alone and n=4 for DC+CpG).

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Fulton, R., Hamilton, S., Xing, Y. et al. The TCR's sensitivity to self peptide–MHC dictates the ability of naive CD8+ T cells to respond to foreign antigens. Nat Immunol 16, 107–117 (2015). https://doi.org/10.1038/ni.3043

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