1. Immunology and Inflammation
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Gamma delta T cells recognize haptens and mount a hapten-specific response

Research Article
Cite this article as: eLife 2014;3:e03609 doi: 10.7554/eLife.03609
3 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
Cy3 is a γδ T cell antigen.

(A) Chemical structure of Cyanine 3 (Cy3). FACS analysis of (B) Cy3 tetramer (Cy34-SAv) staining of splenic γδ T cells in the presence of 10-fold molar excess of moth cytochrome c peptide coupled SAv (MCC4-SAv); (C) NX6/58α-β- cells stained with Cy3-MCC-SAv or PE-MCC-SAv; (D) NX6/58α-β- cells stained with Cy3-MCC-SAv in the absence (left), or presence of anti-Cy3 Fab (right). (E) IL-2 production by NX6/58α-β- cells activated by the indicated amount of plate-bound Cy3-OVA, OVA, PE, anti-CD3 for 16 hr. (F) The saturating binding curves of Cy34-SAv and un-conjugated SAv to a soluble form of NX6 as determined by surface plasmon resonance. No detectable binding was observed for 1 mM applications of PE or BSA (not shown). (G) Kinetics of Cy34SAv binding to NX6/58α-β- cells. t1/2 was determined using real time flow cytometry in the presence of anti-Cy3 antibody Fab fragments (left). KD was determined from Scatchard analysis (right). All results are representative of at least three independent experiments.

https://doi.org/10.7554/eLife.03609.003
Figure 1—figure supplement 1
NX6/58α-β- cells stained with different fluorescently labeled ovalbumin preparations.

Flow cytometry analysis of NX6/58α-β cells stained with Cy3- OVA, FITC-OVA and APC-OVA.

https://doi.org/10.7554/eLife.03609.004
Figure 1—figure supplement 2
Correlation between the mean fluorescence intensities of PE-SAv and Cy34SAv on red blood cells.
https://doi.org/10.7554/eLife.03609.005
Cy3-specific γδ T cell response after immunization.

(A) CD44 expression on Cy3-OVA+ (red) and Cy3-OVA γδ T cells in the draining lymph nodes of mice immunized with Cy3-CGG-alum or CGG-alum 24 hr prior. (B) BioMark analysis of CD62LloCD44hi Cy3+ and CD62LhiCD44lo Cy3 γδ T cells isolated from the draining lymph nodes of C57BL/6 mice immunized with Cy3-CGG 60 hr prior (5 cells/sample). The heatmap, where rows are individual genes and columns are individual samples, indicates the expression or non-expression of a gene/sample pair (relative to the β2m expression). Upper panel shows genes expressing higher (p < 0.001) in Cy3+ cells than that in Cy3 cells. Middle panel shows non-varying genes. Bottom panel shows genes expressing lower (p < 0.001) in Cy3+ cells than that in Cy3 cells. (C) Thy1.1 expression on γδ T cells from IL-17fThy1.1/Thy1.1 mice immunized with Cy3-CGG-alum 60 hr prior, representative of three independent experiments.

https://doi.org/10.7554/eLife.03609.007
NP is a γδ T cell antigen.

(A) Chemical structure of 4-hydroxy-3-nitrophenyl acetyl (NP). Flow cytometry analysis of (B) NP67-PE staining of γδ T cells from C57BL/6 or G8/Rag2−/− mouse splenocytes and PE staining of γδ T cells from B6 splenocytes; (C) staining of 58α-β- cells expressing an NP-specific γδ TCR, 1G9, with NP43-CGG-Cy5 or CGG-Cy5, showing staining in relation to γδ TCR expression (left) or as a histogram (right); (D) staining of 58α-β- cells expressing an NP-specific γδ TCR, 1E3, with NP43-CGG-Cy5, NP26-BSA-Cy5, or BSA-Cy5 (left) and NP67-PE alone, NP67-PE with a 20-fold molar excess of anti-NP Fab, or PE (right). (E) IL-2 production by 1E3/58α-β- cells activated by the indicated amount of plate-bound NP25-KLH, KLH (light gray bars), or 0.1 μg/ml anti-CD3. (F) Sensorgram and steady state analysis of NP43-CGG (0–7 μM) binding to soluble 1G9 TCR measured by surface plasmon resonance. Apparent KD was determined by steady state analysis of SPR measurements (circles). Equal concentrations of un-modified CGG were tested (squares), as well as NP43-CGG with a PE-specific γδ TCR, MA2 (triangles).

https://doi.org/10.7554/eLife.03609.008

Tables

Table 1

TCR sequences of Cy3 and NP-specific γδ TCRs

https://doi.org/10.7554/eLife.03609.006
ND1ND2NN
Cy3NX6Vδ8C A A SAT D KVγ1C A V WS RS G T S W V K
C5Vδ6AC A L W E LGG G I RA SD KVγ1C A V WT RG T S W V K
NP1G9Vδ4C A L M E RRG YR R D TR AD KVγ4C S Y G SYS S G F H K
1E3Vδ6BC A L S E LG GG GS AT D KVγ1C A V WK K TG T S W V K
1B2Vδ4C A L M E RVGL YR R D TS L AT D KVγ1C A VFS G T S W V K
  1. Each pair of γ and δ chain sequences were identified from a single Cy3 or NP-specific γδ T cell derived from mouse splenocytes and verified by their ability to confer NP- or Cy3-specific binding to 58α-β- cells expressing the TCR.

Additional files

Supplementary file 1

Primers used in this study.

https://doi.org/10.7554/eLife.03609.009

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