The TCR's sensitivity to self peptide-MHC dictates the ability of naive CD8(+) T cells to respond to foreign antigens - PubMed (original) (raw)

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

Ross B Fulton et al. Nat Immunol. 2015 Jan.

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 CD5(lo) and CD5(hi) naive CD8(+) T cells. Gene-expression characteristics suggested CD5(hi) cells were better poised for reactivity and differentiation than were CD5(lo) cells, and we found that the CD5(hi) 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.

PubMed Disclaimer

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

None

Figures

Figure 1

Figure 1. CD5 expression by naïve CD8+ T cells identifies stable populations with unique phenotypic traits

Flow cytometry of cells combined from spleen and lymph nodes of wild-type (a) or _Il15_−/− (b) mice were stained for CD44 and CXCR3 and transcription factors T-bet and Eomes. Data were gated on naive (CD44lo CD122lo) CD8+ T cells in the lowest 20% (red) and highest 20% (blue) with respect to CD5 expression. Memory phenotype (MP) cells (CD44hiCD122hi) are indicated as gray shaded histograms. Data represent expression of indicated molecules and forward scatter (as a measure of cell size). (c,d) Naïve CD8+ T cells were sorted on CD5 expression as indicated and congenic populations co-transferred into normal recipient mice, which were analyzed 4–8 weeks later without immunization. Representative data are shown for the histograms (c,d); d, right, summary of compiled data (each symbol represents an individual mouse). (e,f) Flow cytometry of CD4+ CD25– and CD8+ T cells from Nur77gfp transgenic mice. T cells were gated on the lower or upper CD5 expression and analyzed for GFP expression (e). MP cells (gray) are included for comparison. In (f) flow cytometry of HY or OT-I TCR transgenic CD8+ T cells, gated on the CD44lo population, for CD5 and Nur77gfp expression as compared to polyclonal naïve CD8+ T cells (gray). Data in (a,c,d) are representative of 4 independent experiments (a n=10; c,d n=9), in (b,f) 2 independent experiments (b n=4; f n=6–7) and in (e) 3–4 independent experiments (n=6–11).

Figure 2

Figure 2. Naïve CD5lo and CD5hi CD8+ T cells have distinct gene-expression characteristics

(a,b) Expression of XCL1, CXCR3 and T-bet by naïve (CD44lo) CD8+ T cells in the highest 20% (CD5hi) or lowest 20% (CD5lo) with respect to CD5 expression and memory phenotype (CD44hi) CD8+ T cell populations. (a) Splenocytes were stimulated with PMA/ionomycin in the presence of brefeldin A and then stained intracellularly for XCL1. Grey histograms represent unstimulated cells, solid lines indicate stimulated cells. (b) Frequencies of indicated subsets expressing XCL1, CXCR3 or T-bet. Expression of XCL1 was determined following activation (as in (a)), while CXCR3 and T-bet expression was determined in unstimulated B6 or T-bet reporter mice. (c) CD44lo CD8+ T cells were flow sorted on the lower/upper 20% of CD5 expression and mRNA from 3 individual sorts was isolated and analyzed using an Affymetrix gene array. Differences between CD5lo and CD5hi transcription were compared against gene clusters associated with stages of the in vivo CD8+ T cell response. Histograms represent fold-change ratio for expression in CD5lo versus CD5hi cells of genes from clusters II, III and X, revealing preferential expression in the CD5hi population (indicated as a fold change ratio <1). Data in (a) are representative of 4 experiments (9 mice); data in (b) were compiled from: 4 experiments (n=9) for XCL1; 3 experiments (n=7) for T-bet; 5 experiments (n=13) for CXCR3. Data in (c) derive from analysis of gene array analysis from 3 independent cell sorting experiments. * indicates p<0.001

Figure 3

Figure 3. Naïve CD5lo and CD5hi CD8+ T cell precursors differ in their capacity to respond to foreign antigen

CD44lo CD8+ T cells in the lowest or highest 20% for CD5 expression were sorted and 1.25–1.5 × 106 each of congenically distinct CD5lo and CD5hi cells co-transferred into CD45.1/CD45.2 host animals, which were infected the next day with LM-B8R. B8R/Kb–specific cells were isolated from the spleen by B8R/Kb tetramer enrichment at the indicated time points. (a) shows the ratio of donor CD5hi and CD5lo B8R/Kb-specific cells at days 7 and 30 after primary LM-B8R infection or 5 days after re-challenge of memory recipients with virulent LM-B8R, while (b) shows total donor cells number for the d7 timepoint. Each dot is a host animal. Filled symbols in (a) indicate mice where a B8R/Kb-specific response was not detected for CD5lo donor cells – the CD5hi/CD5lo ratio was arbitrarily set to 100. The double dagger symbol (‡) indicates an outlier: exclusion of this group did not alter the statistical significance shown in (b). (c) The ratio of bulk LM-specific donor CD5hi/CD5lo CD8+ T cells from the experiments shown in (a). Data in (a,c) are shown as the geometric mean ± 95% confidence interval. (d,e) Total CD8+ CD44lo cells were sorted and combined with congenically distinct CD44lo CD5lo or CD44lo CD5hi populations and 1.25 – 1.5 × 106 of each population co-transferred into congenic recipients which were infected with LM-B8R 1d later. The B8R/Kb-specific (d) and bulk LM-specific (e) responses were determined 7d after infection. The graphs indicate mean ± SEM. (f) CD5hi naïve CD8+ T cells (CD44lo cells in the upper 20% for CD5 expression) were sorted into the lowest or highest 30% for CXCR3 expression. 1–2×106 of congenically distinct CXCR3lo and CXCR3hi CD5hi cells were co-transferred into CD45.1/45.2 donors, and infected 1d later with LM-B8R. B8R/Kb-specific cells were isolated from the spleen 7d after infection by tetramer enrichment. The upper panel shows the ratio of cells derived from CXCR3hi versus CXCR3lo donor cells in the B8R/Kb-specific and bulk LM-B8R responsive populations. The lower panel shows the number of B8R/Kb–specific CXCR3hi and CXCR3lodonor cells isolated. The geometric mean is indicated. (g) Mixed bone marrow chimeras were generated using congenic wild-type and Cd25 (IL-2Rα)-deficient bone marrow. Following T cell reconstitution, CD44lo CD5lo and CD5hi populations were sorted and congenically distinct combinations of WT and KO cells co-transferred into congenic recipients that were infected with LM-B8R 1d later. At d7 following infection, the spleen was recovered and the number of B8R/Kb–specific donor cells enumerated. Data show the geometric mean ± 95% confidence interval. In all figures, each symbol indicates data from a single animal. Data in (a-c) are compiled from four experiments at day 7 (n = 11), 3 experiments at day 30 (n = 9), and 2 experiments for day 5 recall (n = 6); (d) shows data compiled from 3 experiments (n = 11–12 mice), (e) from two experiments (n = 8); (f) is compiled from 3 experiments (n=10); (g) is compiled from 3 individual experiments (n = 6). For this figure, statistical analysis is indicated as follows: *** for p < 0.001; ** for p < 0.01; * for p < 0.05.

Figure 4

Figure 4. Analysis of clonal naïve CD5lo and CD5hi CD8+ T cell responses to infection

The indicated number of sorted CD5lo or CD5hi cells was transferred into congenic recipients and the B8R/Kb-specific donor-derived response was determined in the spleen 7 days after infection with LM-B8R. (The congenic marker scheme used is illustrated in Fig. S4). (a) Response rate for the indicated number of transferred donor cells for which a B8R/Kb-specific response was observed above the limit of detection (5 cells). Error bars are calculated standard deviation. Statistical analysis using a one-sided binomial test showed that the response rate for 2.5×104 and 1×105 CD5lo cells (0/40 and 18/125, respectively) were significantly different from that expected (25% and 100%, respectively) (p<0.0001 for both) while the response rate for 2.5–3.0 × 104 CD5hi cells was not significantly different from average expected rate (~27.5%) (p=0.2). (b) Numbers of B8R/Kb-specific CD8+ T cells derived from each donor population, shown as the geometric mean ± 95% confidence interval. Each symbol represents cell numbers from an individual donor cohort. Donor populations that did not mount a detectable response are shown on the limit of detection line. Two very large clonal responses are indicated by arrows. Host responses are shown for comparison. Statistical significance was assessed only on responses above the limit of detection, using the Mann-Whitney test on log-transformed values. The numbers of transferred cohorts for each condition is given in the text, and the data derive from 2–4 separate experiments. The symbol * indicates p<0.05.

Figure 5

Figure 5. Early recruitment of CD5lo and CD5hi cells into the immune response after infection

(a) Congenically distinct CD44lo CD5lo and CD5hi cells were sorted and co-transferred into congenic recipients as in Fig. 3. Donor-derived B8R/Kb-specific responses were analyzed from the spleen on day 3 or 4 after infection with LM-B8R. Data are shown as the geometric mean ± 95% confidence interval. (b,c) Nur77gfptransgenic mice were injected i.v. with 50 μg anti-CD3 or PBS and spleens harvested 5 hrs later. Representative data is shown for expression of Nur77GFPand CD69 (b) and CD5 levels (c) on CD44lo CD8+ T cells. (d-f) Nur77gfp transgenic mice were infected with 1 × 107CFU LM-B8R and spleens harvested 5h later. B8R/Kb- and M57/Kb-specific CD8+ T cells were isolated by tetramer based enrichment. (d) Nur77gfp and CD69 expression was assessed on B8R/Kb- and M57/Kb-specific CD8+ T cells, and (e) B8R/Kb-specific cells with activated (Nur77gfp-hi, CD69+) and non-activated (Nur77gfp-lo, CD69−) phenotype assessed for CD5 expression. (d,e) are representative data. (f) shows normalized geometric mean fluorescence intensity (gMFI) for CD5 expression on non-activated and activated B8R/Kb specific naïve CD8+ T cells. (a) shows data compiled from a total of 3 experiments (1 at day 3; n=5, 2 at day 4; n=9); Data in (b,c) are from 2 separate experiments (n=3); (d–f) show representative or compiled data from 3 experiments (n=13) for B8R/Kb–specific cells and 2 experiments (n=9) for M57/Kb–specific cells. * indicates p < 0.01.

Figure 6

Figure 6. CD5hi and CD5lo cells show similar TCR binding to cognate antigen

(a) B8R/Kb and M57/Kb specific CD8+ T cells were enriched from unimmunized mice and CD44lo precursors were gated on the lower and upper 20% of CD5 expression. Tetramer staining intensity (gMFI) was determined for both groups and expressed as a ratio, to normalize between experiments. (b) B8R/Kb tetramer staining (gMFI) and burst size of expanded clonal populations derived from CD5lo and CD5hi donor cells at d7 following LM-B8R infection. Data derive from transfers of 1×105 CD5lo and 2.5–3.0 × 104 CD5hi cells (see Fig. 4). Tetramer gMFI on donor cells was normalized to tetramer gMFI on host B8R/Kb-specific cells from the same mice, to yield relative fluorescence intensity (RFI). (c) Spleen and lymph node cells from unimmunized mice were subjected to tetramer enrichment. CD5 expression as gMFI on B8R/Kb-specific and M57/Kb-specific CD44lo CD8+ T cell populations was compared to normalized CD5 expression on bulk CD44loCD8+ T cells. (d) CD5 expression levels on indicated MHC class I-restricted TCR transgenic CD8+ T cells. The histograms shows representative data while the bar graph shows CD5 relative fluorescence intensity (RFI), CD5 staining on polyclonal B6 CD44lo CD8+ T cells being set at “100”. (e) MHC class I tetramer binding to TCR transgenic cells after staining with the indicated range of tetramer concentrations. RFI reflects the geometric MFI of tetramer staining normalized to maximum intensity, which was set at “100”. Similar data were obtained in a second experiment. (f) In vitro activation of TCR transgenic CD8+ T cells. T cells were incubated with titrated doses of peptide for 6 hrs and CD69 expression was assessed by flow cytometry. Data are normalized to maximum CD69 expression. Similar data were obtained in a repeat experiment. (g) Expression of Kb or Db on RMA-S cells following incubation with indicated doses of the peptides recognized by studied TCR transgenic T cells. RFI reflects the MFI of Class I MHC molecule staining, normalized to maximum intensity, which was set at “100”. Data are representative of three experiments. (h) OT-I bone marrow chimeras were generated by transferring OT-I bone marrow (RAG+) into irradiated WT or β2m−/− hosts. Flow plots showing CD5 expression is representative of 4 separate sets of chimeras. Congenically distinct CD44loOT-I CD8+ T cells were enriched from each chimera source and 103 cells of each population co-transferred into congenic recipients that were untreated or infected 1-3d later with attenuated ΔActA LM-OVA. (i) Donor cell engraftment (“take”) was determined uninfected recipients, and compared to ratios of donor-derived populations at 7d and 12d after LM-OVA infection. Data are shown as mean ± SEM. In (a) data are compiled from 4 experiments (n = 14 for B8R/Kb, n = 9 for M57/Kb); (b) data are from 18 (CD5lo) and 46 (CD5hi) clonal responses from at least 3 experiments. * indicates p<0.05. Statistical significance was not changed by exclusion of the outlier (marked as ‡); In (c) the data is compiled from 6 experiments (n = 21) for B8R/Kb-specific cells and two experiments (n = 8) for M57/Kb-specific cells. **p < 0.001. In (d) data are compiled from 2–3 experiments (n=4–6); (e,f,g) are representative of 2–3 independent experiments; (h,i) Data are representative or compiled from 4 independent sets of chimeras.

Figure 7

Figure 7. CD5hi cells preferentially utilize pro-inflammatory signals during expansion

Congenically distinct CD44lo H-Y and OT-I TCR transgenic CD8+ T cells were co-transferred into congenic recipients. Mice were co-immunized the next day with single peptide-pulsed matured DCs, with or without collateral infection with ΔactA−/ − LM (a,b) or LCMV Armstrong (c,d). (a,c) Show total numbers of splenic H-Y and OT-I CD8+T cells 7d post-immunization with antigen pulsed DC, with or without indicated infections, while (b,d) shows ratios of OT-I/H-Y normalized to the “take” ratio observed in unimmunized animals 1d after adoptive transfer. All graphs show mean ± SEM. Data in (a,b) are compiled from 3 independent experiments (n=10); data in (c.d) are compiled from 2 experiments (n=6 for DC alone and n=7 for DC+LCMV). * = p<0.01; ** = p <0.001.

Comment in

References

    1. Zehn D, Lee SY, Bevan MJ. Complete but curtailed T-cell response to very low-affinity antigen. Nature. 2009;458:211–214. - PMC - PubMed
    1. Tubo NJ, et al. Single Naive CD4(+) T Cells from a Diverse Repertoire Produce Different Effector Cell Types during Infection. Cell. 2013;153:785–796. - PMC - PubMed
    1. Fazilleau N, McHeyzer-Williams LJ, Rosen H, McHeyzer-Williams MG. The function of follicular helper T cells is regulated by the strength of T cell antigen receptor binding. Nature immunology. 2009;10:375–384. - PMC - PubMed
    1. Plumlee CR, Sheridan BS, Cicek BB, Lefrancois L. Environmental cues dictate the fate of individual CD8(+) T cells responding to infection. Immunity. 2013;39:347–356. - PMC - PubMed
    1. Dorfman JR, Stefanova I, Yasutomo K, Germain RN. CD4+ T cell survival is not directly linked to self-MHC-induced TCR signaling. Nature immunology. 2000;1:329–335. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources