One naive T cell, multiple fates in CD8+ T cell differentiation - PubMed (original) (raw)

One naive T cell, multiple fates in CD8+ T cell differentiation

Carmen Gerlach et al. J Exp Med. 2010.

Abstract

The mechanism by which the immune system produces effector and memory T cells is largely unclear. To allow a large-scale assessment of the development of single naive T cells into different subsets, we have developed a technology that introduces unique genetic tags (barcodes) into naive T cells. By comparing the barcodes present in antigen-specific effector and memory T cell populations in systemic and local infection models, at different anatomical sites, and for TCR-pMHC interactions of different avidities, we demonstrate that under all conditions tested, individual naive T cells yield both effector and memory CD8+ T cell progeny. This indicates that effector and memory fate decisions are not determined by the nature of the priming antigen-presenting cell or the time of T cell priming. Instead, for both low and high avidity T cells, individual naive T cells have multiple fates and can differentiate into effector and memory T cell subsets.

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Figures

Figure 1.

Figure 1.

Genetic tagging of naive T cells. (A) Experimental setup for the generation and use of naive, barcode-labeled T cells. Donor thymocytes are transduced with a barcode library. Transduced cells are sorted and injected intrathymically into primary (1°) recipient mice. 3 wk later, mature T cells are isolated from 1° recipients, pooled, and transferred into 2° recipients. (B) Total CD45.1+ OT-I thymocytes were transduced with the retroviral barcode library. 1 d after transduction, cells were stained and analyzed by flow cytometry. CD4 and CD8 expression is shown for the GFP+ (barcode-labeled) population. Numbers indicate percentages. Data are representative of two independent experiments. (C and D) Sorted GFP+ CD45.1+ OT-I thymocytes were injected into the thymi of CD45.2+ B6 1° recipient mice (n = 3). 3 wk later, spleen and lymph node cells were isolated, pooled, enriched for CD8+ cells, stained for the indicated surface markers, and analyzed by flow cytometry. Data are representative of two independent experiments. (C) Expression of CD45.1 and GFP on gated CD8+ cells from unmanipulated (control) B6 or 1° recipient mice. Numbers indicate percentages. (D) Expression of CD44 and CD62L on unmanipulated (CD45.1+) and barcode-labeled (GFP+ CD45.1+) CD8+ T cells.

Figure 2.

Figure 2.

Barcode-labeled T cells can be used for lineage relationship analysis. (A) CD45.1+ B6 2° recipient mice (n = 5) received unmanipulated CD8+CD45.2+ OT-I cells plus naive barcode-labeled CD8+CD45.1+ OT-I cells and subsequent i.v. LM-OVA infection. Average responses of transferred OT-I cells are depicted; error bars indicate SD. (B) Naive, barcode-labeled or unmanipulated CD8+CD45.1+ OT-I cells were injected i.v. into CD45.2+ B6 2° recipient mice (n = 7 and 2, respectively). Mice were subsequently infected i.v. with LM-OVA. At day 7 after infection, blood cells were stained for the indicated surface markers and analyzed by flow cytometry. Shaded histograms represent total endogenous CD45.1−CD8+ T cells; black lines represent transferred CD45.1+ OT-I cells. One representative mouse per group is shown. Data are representative of two independent experiments. (C and D) Naive, barcode-labeled CD8+CD45.1+ OT-I cells were injected into CD45.2+ B6 2° recipient mice. Mice were subsequently challenged with WSN-OVA (i.n.) and EL-4 OVA tumor cells (s.c.) the next day. At day 5 after infection, lung- and tumor-draining lymph node cells were isolated, and each lymph node sample was split into two half-samples (samples A and B). Each half-sample was cultured separately for 3–4 d in vitro in the presence of 10 µg/ml IL-7 and 20 U/ml IL-2. Barcode analysis was performed independently on samples A and B of both lymph nodes (LN1 and LN2). Barcodes with a p-value of <0.001 were considered to be present above background. Rectangular dividers indicate which barcodes are present above background. Note that rather than fixing the absolute position of dividers in terms of intensity, it is the statistical probability of barcode presence that is kept constant for all samples in a given experiment. Data are representative of two independent experiments. (C) Representative 2-D plots of barcode analyses. Numbers indicate the correlation between signals from samples A and B. (D) Correlation analysis of barcodes present in T cells from the same or from distinct lymph nodes. Data are shown for three mice from two independent experiments. Mice from which barcodes in one out of the two draining lymph nodes could not be sampled reliably were excluded from further analysis.

Figure 3.

Figure 3.

CD8+ T cells that are present during effector and resting memory phases are derived from the same naive T cells. CD45.2+ B6 2° recipient mice (n = 4) were injected with naive, barcode-labeled CD8+CD45.1+ OT-I cells and subsequently infected i.v. with LM-OVA. Barcode analysis was performed on a 250–300-µl blood sample drawn at day 8 after infection (effector phase), and on a blood sample as well as on a spleen sample isolated at day 28 after infection (memory phase) from the same mice. Blood and spleen samples were divided into two halves (samples A and B) that were independently analyzed for barcode content. Barcodes with a p-value <0.005 were considered to be present above background. On average, 150 barcodes were detected per mouse. Data are representative of two independent experiments. (A and B) Representative 2-D plots of barcode comparisons. Numbers indicate the correlation between signals from samples A and B. (C) Correlation analysis of barcodes present in samples A and B from effector phase blood and memory phase blood (left plot) or effector phase blood and memory phase spleen (right plot). Data from four mice are depicted.

Figure 4.

Figure 4.

Effector phase CD8+ T cells present in blood, spleen, bone marrow, and lymph nodes are derived from the same naive T cells. CD45.2+ B6 2° recipient mice (n = 3) were injected with naive, barcode-labeled CD8+CD45.1+ OT-I cells and subsequently infected i.v. with LM-OVA. Barcode analysis was performed at day 8 after infection on cells recovered from blood, spleen, bone marrow, and lymph nodes. All samples were divided into two halves (samples A and B) that were independently analyzed for barcode content. Barcodes with a p-value <0.02 were considered to be present above background. On average, 150 barcodes were detected per mouse. S versus B comparisons are representative of two independent experiments; LN and BM barcodes were analyzed in one experiment. (A) Representative 2-D plots of barcode comparisons. Numbers indicate the correlation between signals from samples A and B. (B) Correlation analysis of barcodes present in samples A and B. Data from three mice are depicted.

Figure 5.

Figure 5.

Naive T cell clones that contribute to the resting memory population also have progeny that respond to secondary antigen encounter. B6 2° recipient mice (n = 3) were injected with naive, barcode-labeled CD8+ OT-I cells and subsequently infected i.v. with LM-OVA. 28 d after infection, blood, spleen, lymph node, and bone marrow cells were isolated, pooled per mouse, and divided into three parts. Two parts were used for barcode analysis (samples A and B, resting memory phase), and the third part was transferred into a B6 3° recipient mouse that was then infected with LM-OVA. 5 d later, barcodes were analyzed from spleen half-samples (2° expansion). Barcodes with a p-value <0.01 were considered to be present above background. On average, 1,080 barcodes were detected per mouse. (A and B) Representative 2-D plots of barcode comparisons. Numbers indicate the correlation between signals from samples A and B. (C) Correlation analysis of barcodes present in samples A and B. Data are derived from three 2° recipient and three 3° recipient mice analyzed within one experiment. These data confirm results obtained in an independent pilot experiment in which 1° expansion (effector phase) and 2° expansion memory populations could be compared for one mouse.

Figure 6.

Figure 6.

Kinship of effector and memory phase CD8+ T cells upon local infection. B6 2° recipient mice (n = 4) were injected with naive, barcode-labeled CD8+ OT-I cells and subsequently infected i.n. with WSN-OVA. Barcode analysis was performed on a one-fourth spleen sample obtained by partial splenectomy at day 9 after infection (effector phase), and on a spleen sample isolated at day 25 after infection (memory phase) from the same mice. Effector and memory phase samples were divided into two halves (samples A and B) that were independently analyzed for barcode content. Barcodes with a p-value <0.001 were considered to be present above background. On average, 800 barcodes were detected per mouse. (A and B) Representative 2-D plots of barcode comparisons. Numbers indicate the correlation between signals from samples A and B. (C) Correlation analysis of barcodes present in samples A and B. Data from four mice are depicted. These data confirm results obtained in an independent pilot experiment in which effector and memory phase populations could be compared for one mouse.

Figure 7.

Figure 7.

Relatedness of effector and memory T cells is independent of TCR avidity. B6 2° recipient mice (n = 5) were injected with naive, barcode-labeled CD8+ OT-I cells and subsequently infected i.v. with LM-Q4-OVA. 27 d later, mice were rechallenged with LM-Q4-OVA. Barcode analysis was performed on a one-fourth spleen sample obtained by partial splenectomy at day 7 after infection (effector phase), and on a spleen sample isolated at day 5 after rechallenge (2° expansion) from the same mice. Effector and 2° expansion samples were divided into two halves (samples A and B) that were independently analyzed for barcode content. Barcodes with a p-value <0.005 were considered to be present above background. On average, 250 barcodes were detected per mouse. (A and B) Representative 2-D plots of barcode comparisons. Numbers indicate the correlation between signals from samples A and B. (C) Correlation analysis of barcodes present in samples A and B. Data from five mice analyzed within one experiment are depicted. Data were confirmed in a second experiment (five mice) performed with LM-A2-OVA, an L. monocytogenes strain containing another lower functional avidity variant of the OVA epitope (SAINFEKL).

Figure 8.

Figure 8.

Relatedness of different T cell subsets. Results from barcoding experiments in Figs. 2, 3, 4, 5, 6, and 7 are depicted as the percentage of maximal attainable correlation. 100% reflects the mean correlation of all intrasample correlations for each individual experiment. 0% reflects the mean correlation of all between-mice comparisons for each individual experiment. Note that the average correlation of two sampling controls forms a reasonable estimate and possibly a slight overestimate of the maximal attainable correlation in case the two cell populations would be fully related (

Fig. S4 B

). From the effector versus memory phase comparison during LM-OVA infection, only the effector blood versus memory blood comparison is depicted. Circles represent correlations within individual mice; bars indicate group averages. B, blood; BM, bone marrow; LN, lymph nodes; S, spleen.

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