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. 2015 Jun;21(6):647-53.
doi: 10.1038/nm.3860. Epub 2015 May 11.

Common clonal origin of central and resident memory T cells following skin immunization

Olivier Gaide  1 , Ryan O Emerson  2 , Xiaodong Jiang  1 , Nicholas Gulati  3 , Suzanne Nizza  1 , Cindy Desmarais  2 , Harlan Robins  4 , James G Krueger  3 , Rachael A Clark  1 , Thomas S Kupper  1
Affiliations

Common clonal origin of central and resident memory T cells following skin immunization

Olivier Gaide et al. Nat Med. 2015 Jun.

Abstract

Central memory T (TCM) cells in lymph nodes (LNs) and resident memory T (TRM) cells in peripheral tissues have distinct roles in protective immunity. Both are generated after primary infections, but their clonal origins have been unclear. To address this question, we immunized mice through the skin with a protein antigen, a chemical hapten, or a non-replicating poxvirus. We then analyzed antigen-activated T cells from different tissues using high-throughput sequencing (HTS) of the gene encoding the T cell receptor (TCR) β-chain (Trb, also known as Tcrb) using CDR3 sequences to simultaneously track thousands of unique T cells. For every abundant TRM cell clone generated in the skin, an abundant TCM cell clone bearing the identical TCR was present in the LNs. Thus, antigen-reactive skin TRM and LN TCM cell clones were derived from a common naive T cell precursor after skin immunization, generating overlapping TCR repertoires. Although they bore the same TCR, TRM cells mediated rapid contact hypersensitivity responses, whereas TCM cells mediated delayed and attenuated responses. Studies in human subjects confirmed the generation of skin TRM cells in allergic contact dermatitis. Thus, immunization through skin simultaneously generates skin TRM and LN TCM cells in similar numbers from the same naive T cells.

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Figures

Figure 1
Figure 1. Skin immunization generates skin TRM and TCR-identical TCM in LN
(a), (b) and (c) Dot plots of the frequency (number of a given sequence divided by the total number of sequences observed in the given sample) of TCRβ CDR3 sequences shared in antigen-exposed (ear, green vertical axis) and distant (tail, red horizontal axis) skin after OVA+CT immunization (a), DNFB sensitization (b), and MVA skin scarification (c). Each blue dot represents a T cell clone with a unique CDR3 sequence, and the most abundant expanded clones are enclosed in the blue rectangle. In (a), the OT-I Vβ CDR3 sequence (CASSRANYEQYF) is highlighted (n=3 mice per antigen). (d), (e), (f), Tracking of selected abundant TCRβ CDR3 sequences (d[OVA+CT], e[DNFB], f[MVA]) identified in (a), (b) and (c), respectively. I: inguinal LN; T: tail skin; E: Ear skin; Draining LN (ear-draining); T: tail skin; I: inguinal LN. Data for CDR3 sequences (TCR percentage) are shown as the percentage (specific/total) of CDR3 sequences (n=3 mice per antigen) from T cell clones present at n≥5 copies in skin assessed in other tissues. (g), (h), (i) Unique expanded clones (g[OVA+CT], h[DNFB], i[MVA])are indicated by colored contiguous rectangles, and the corresponding clones (each color is a unique clone) present in draining, and distant LN are shown in the same fashion. The average clone size in each compartment is shown to the right in the colored circle. (j) total number of T cell clones ( n≥5 per clone, horizontal bar graphs) and mean clone size (colored circle) in the tissues of all mice combined (immunized with OVA+CT, DNFB, or MVA). (n=9 mice).
Figure 2
Figure 2. Repetitive sensitization increases the abundance of TRM in skin
(a) Temporal scheme of the repetitive DNFB sensitizations and challenges. (n=3 mice for each condition, all experiments performed 2-3 times, representative data is shown). (b) Frequency of respective beta chain V and J α T cell populations in the skin and LN of the repetitively immunized mice at day 91. The x-axis shows the TCR Vβ subtype (described in detail in methods), the y-axis their frequency, and the z-axis shows the TCR Jβ subtype. The VβJβ repertoire observed in different skin areas (upper) exposed to DFNB as well as the respective draining LN (lower graphs; inguinal, right and left submandibular LN respectively). (n=3 mice). (c) The left pie chart represents the frequency of the highly expanded V13.3/J2.2 clones in the left ear of DNFB sensitized, boosted and challenged mice, both in the left ear and left cervical (draining) LN. The right pie chart represents the three most abundant individual TCR clones (unique CDR3 sequences) comprising this specific VJ subgroup, plus all other minor clones from this VJ subgroup in pale blue. (n=3 mice). (d) Clone frequency of the three most abundant TCR CDR3 sequences (depicted in c) skin and tail after 0,2, and 6 DNFB exposures. (e) The single most abundant T cell clone in skin exposed 0×, 2×, and 6× to DNFB in each of three mice, expressed as T cells/400ng gDNA . The copy number of the same clone in LN is shown in parentheses (in the 6× circle), n=3 mice. (f) Number of highly expanded clones present in at least 10 copies per 400ng of tissue in the skin and LN of the immunized mice, showing skin-specific (TRM) increase (n=3 mice).
Figure 3
Figure 3. TRM mediate rapid CHS responses, while TCM mediate delayed attenuated CHS responses
(a) Experimental plan for the subsequent experiments (n=3 pairs of parabiotic mice; n=3 for naive and sensitized non-parabiotic controls, respectively). Experiments were performed 2-4 times. (b) Ear swelling kinetics in parabiotic sensitized and parabiotic naive mice, with non-parabiotic sensitized and naive mice as controls. (*p=0.001; § p=0,005, n=3 in each group). (c) An identical experiment to that shown in (b) was performed, but this time mice were pre-treated with FTY-720 (*p=0.002; § p=0.002, n=3 per group). (d) Comparison of the ear swelling kinetics after DNFB challenge at the initial (local) site of immunization (Left) and at a distant site (Right) in parabiotic sensitized mice. In parabiotic naive mice, the Left and Right ears were measured after DNFB challenge (these parabiotic naive mice had not been sensitized) ( *p=0.001; § p=0.01; θp=0.02, n=5 mice in each group). (e) Kinetics of left and right ear swelling after DNFB challenge in parabiotic naive mice, with or without FTY-720 treatment. (*p=0.001; § p=0.001, n=5 mice in each group). (f) Left ear swelling after the final (2nd) DNFB challenge in parabiotic naive and parabiotic sensitized mice, treated as shown in (a), with non-parabiotic naive and sensitized mice as controls (see panel d) (§ p=0.04; θp=0.001, n=5 mice in each group). (g) Left (local) vs. right (distant) ear swelling after the final DNFB challenge parabiotic naive and parabiotic sensitized mice (*p=0.04, n=3 in each group). (h) Ratio (%) of the left vs right maximal ear swelling after the second challenge in the sensitized vs. naïve parabiotic mice. (p=0.029, n=3 in each group). All results show mean +/− SD. Statistical analysis using two way ANOVA with Holm-Bonferroni post hoc analysis where appropriate, or one way ANOVA only, were used (see Materials and Methods). P>0.05 was considered not significant (ns).
Figure 4
Figure 4. Contact dermatitis to DPCP induces TRM cells in human skin
(a) Schematic of DPCP sensitization and sample recovery. (b) Clinical photos, hematoxylin-eosin histology and CD3+ T cell staining at day 3 (placebo and DPCP challenged), 14 (DPCP challenged) and 120 (DPCP challenged) of the skin of subjects, at indicated time points. (Representative data are shown from a total of 11 subjects entered in the study). (c) Dot plots of the frequency (number of a given sequence divided by the total number of sequences observed in the given sample) of TCRβ CDR3 sequences shared (or not) in non-exposed or DPCP-exposed skin from a representative individual. The left panel shows clones in 4 day post DPCP challenge site skin (vertical axis) vs. the same time point in placebo challenged skin (horizontal axis). Clones present in both sites are shown as blue dots. Five clones are identified (1-5) for subsequent reference. The right panel shows clones 14 days post DPCP challenge (vertical axis) vs clones 4 months after DPCP challenge. The same five clones are identified as being present in both samples. (d) quantification of the T cell clone size observed in c over time. (n=3 out of 11 subjects: F, 48; M, 52; M, 55; biopsies were subjected to HTS; data from one representative patient shown).
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