Immunology and Inflammation

Commensal Bacteria Maintain a Qa-1^b-restricted Unconventional CD8⁺ T Population in Gut Epithelium

Jian Guan author has email address
J. David Peske
Michael Manoharan Valerio
Chansu Park
Ellen A. Robey
Scheherazade Sadegh-Nasseri author has email address

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
Institute of Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
Division of Immunology and Molecular Medicine, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA

https://doi.org/10.7554/eLife.90466.2

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Figures and data

Abundant Vα3.2⁺ QFL T cells in both the spleen and siIEL compartment of naïve WT mice.
(a) Definition of QFL T cells by flow cytometry. Cells from the spleen or siIEL compartment were stained with Qa-1^b-FL9 (QFL) dextramers labeled with phycoerythrin (QFL-Dex-PE) or allophycocyanin (QFL-Dex-APC) and analyzed for before (Unenriched) or after (Enriched) magnetic enrichment of dextramer-positive cells. QFL T cells are defined as the CD45⁺CD19⁻TCRβ⁺CD4⁻QFL-Dex-PE⁺APC⁺ population. Plots representing siIELs from naïve WT mice after enrichment for QFL-Dex⁺ cells. (b) Flow cytometry of cells from the spleen (Sp) and siIEL compartment of naïve WT mice ‘Unenriched’ or ‘Enriched’ for dextramer-positive cells. Numbers in plots indicate absolute numbers of QFL-Dex⁺ cells detected after enrichment. (c) Absolute numbers (left) and frequencies (right) of QFL-Dex⁺ cells detected (as in b) among TCRβ⁺CD4⁻ population in the spleen or siIEL compartment. **P=0.0069 (d, f) Analysis of Vα3.2 expression on QFL-Dex⁺(QFL) or total CD8⁺ T (Total) cells from the spleen (d) or the siIEL compartment (f). Numbers in plots indicate average percentages of Vα3.2⁺ cells within the indicated populations. (e) Frequencies of Vα3.2⁺ cells detected as in d. ****P<0.0001 (g) Frequencies of Vα3.2⁺ cells detected as in f. **P=0.0058 Representative data are shown in flow plots (a, b, d, f) and the number of replicates is specified in the bar graphs (c, e, g). Each symbol represents data collected from the indicated tissue isolated from an individual mouse. Data from ≧ 5 independent experiments were pooled for statistical analysis. P values were calculated with Student’s t test. ‘ns’ indicates the comparison was not significant.

The unconventional phenotype of Vα3.2⁺ QFL T cells.
(a) Analysis of CD44 expression on Vα3.2⁺ or Vα3.2⁻ QFL-Dex⁺ cells enriched from the spleen of naïve WT mice. Numbers in plots indicate average percentages of CD44^hi cells detected among the Vα3.2⁺ or Vα3.2⁻ QFL-Dex⁺ populations. (b) Frequencies of CD44^hi cells detected as in a.****P<0.0001 (c) Analysis of CD8α and CD8β expression on Vα3.2⁺ or Vα3.2⁻ QFL-Dex⁺ cells enriched from the siIEL compartment of naïve WT mice. Numbers in plots indicate average percentages of CD8αα⁺ cells detected among the Vα3.2⁺ or Vα3.2⁻ QFL-Dex⁺ populations. (d) Frequencies of CD8αα⁺ cells detected as in c. **P=0.0065 Samples of <20 cells in the Vα3.2⁺QFL-Dex⁺ gate were excluded in the phenotype analysis. Representative data are shown in flow plots (a, c) and the number of replicates is specified in the bar graphs (b, d). Each symbol represents data collected from the indicated tissue isolated from an individual mouse. Data from ≧11 independent experiments were pooled for statistical analysis. P values were calculated with Student’s t test. ‘ns’ indicates the comparison was not significant.

Phenotype of Vα3.2⁺ QFL T cells in mice of various genotypes.
(a) Absolute numbers of Vα3.2⁺QFL-Dex⁺ cells detected in the spleen (left) or siIEL compartment (right) of Qa-1^b, ERAP1 or TAP deficient (Qa-1^b-KO, ERAP1-KO or TAP-KO) mice in comparison with WT mice.** P<0.009 ***P=0.0006 Symbols on x-axis indicate that the cells were undetectable in TAP-KO mice. (b) Analysis of CD44 expression on splenic Vα3.2⁺QFL-Dex⁺ cells enriched from naïve WT, Qa-1^b-KO or ERAP1-KO mice. Numbers in plots indicate average percentages of CD44^hi cells. (c) Frequencies of CD44^hi cells detected among Vα3.2⁺(as in b) or Vα3.2⁻ QFL T cells.***P=0.0004 *P=0.0388 (d) Flow cytometry analysis of CD8α and CD8β expression on the Vα3.2⁺QFL-Dex⁺ cells enriched from the siIEL compartment of naïve WT, Qa-1^b-KO or ERAP1-KO mice. Numbers in plots indicate average percentages of CD8αα⁺ cells. (e) Percentages of CD8αα⁺ cells detected among Vα3.2⁺(as in d) or Vα3.2⁻ QFL T cells. **P=0.0082 *P=0.0335 Representative data are shown in flow plots (b, d) and the number of replicates is specified in the bar graphs (a, c, e). Each symbol represents data collected from the indicated tissue isolated from an individual mouse. Data from ≧4 independent experiments were pooled for statistical analysis. P values were calculated with Student’s t test. ‘ns’ indicates the comparison was not significant.

Distinct functional features of QFLTg cells in the spleen and gut.
(a, c) Proliferation of splenic (a) or siIEL (c) QFLTg or OT-1 cells from QFLTg or OT-1 mice in response to IL-15 (left) or a combination of IL-7 and IL-18 (right) stimulation. Cells were tracked using Cell Tracing Violate (CTV). Numbers in plots indicate average percentages of proliferated QFLTg cells. (b) Percentages of proliferated splenic QFLTg or OT-1 cells as detected in a. ****P<0.0001 **P=0.0052 (d) Percentages of proliferated siIEL QFLTg or OT-1 as detected in c. ***P=0.0009 *P=0.0101 (e) Generation of QFLTg_WT or OT-1_WT partial hematopoietic chimera mice. 2×10⁶ of the bone marrow (BM) cells from GFP⁺QFLTg or GFP⁺OT-1 mice were transferred into WT neonates at 3~5 days of age. The chimera mice were analyzed at 8~10 weeks of age. (f) Gating strategy for the QFLTg cells originated from donor bone marrow cells in QFLTg_WT chimera mice. Plots representing donor-derived QFLTg population in the spleen being gated as GFP⁺CD45⁺CD19⁻TCRβ⁺CD4⁻Vα3.2⁺ cells. (g, i) Flow cytometry measurement of IFN-ɣ production by QFLTg or OT-1 cells isolated from the spleen (g) or siIEL compartment (i) of naïve chimera mice stimulated with or without 2µM FL9 or SL8 peptide respectively for 4.5h. Numbers in plots indicate average percentages of IFN-ɣ⁺ cells. (h) Percentages of IFN-ɣ⁺ cells detected as in g. ****P<0.0001 ***P=0.0001 (j) Percentages of IFN-ɣ⁺ cells detected as in i. ***P=0.0004 *P=0.0247 Representative data are shown in flow plots (a, c, f, g, i) and the number of replicates is specified in the bar graphs (b, d, h, j). Data from 2(b, d) or 3 (h, j) independent experiments were pooled for statistical analysis. Each symbol represents data collected from the indicated tissue isolated from an individual mouse. P values were calculated with Student’s t test. ‘ns’ indicates the comparison was not significant.

Gut microbiota is associated with retention but not homing of QFL T cells in the siIEL compartment.
(a, c) Flow cytometry of QFL-Dex⁺ cells enriched from the spleen (a) or siIEL compartment (c) of specific-pathogen-free (SPF) or germ-free (GF) WT mice at 10 or 18 weeks of age. Numbers in plots indicate absolute numbers of QFL-Dex⁺ cells. (b) Absolute numbers (left) and frequencies (right) of QFL-Dex⁺ cells detected within the total splenic CD8⁺ population in SPF or GF WT mice of 8~17 weeks or 18~22 weeks of age.***P=0.009 ****P<0.0001 (d) Absolute numbers and frequencies of QFL-Dex⁺ cells detected among the total siIEL TCRβ⁺CD4⁻ population in SPF or GF WT mice of 8~17 weeks or 18~22 weeks of age. **P=0.005 *P=0.037 Representative data are shown in flow plots (a, c) and the number of replicates is specified in the bar graphs (b, d). Each symbol represents data collected from the indicated tissue isolated from an individual mouse. Data from ≧5 independent experiments were pooled for statistical analysis. P values were calculated with Student’s t test. ‘ns’ indicates the comparison was not significant.

Identification of a QFL T cross-reactive FL9 homolog peptide expressed in commensal bacterium P. pentosaceus.
(a) Structure model of the QFL TCR:FL9:Qa-1^b complex predicted by AlphaFold2. Detailed view of the binding groove is shown in the right panel. (b) Conformation alignment of FL9 (red) and Qdm (periwinkle). (c) Alanine screen of the predicted key residues (P4 and P6) in FL9. Activation of QFL T cells was assessed by measuring the LacZ response of BEko8Z hybridoma cells to the Qa-1^b-expressing Lmtk⁻ (L-Qa-1^b) cells pulsed with mutant FL9 peptides including FYAEAAPML (FL9-P6A) and FYAAATPML(FL9-P4A) or the wild-type FYAEATPML(FL9) using spectrophotometry. (d) Workflow for identification of commensal bacterial FL9 homolog peptide which cross-activate QFL T cells. (e) Identification of the QFL T cross-reactive peptide FYAEDGTPIL(FL10). BEko8Z response to the L-Qa-1^b cells or the non-Qa-1^b-expressing Lmtk⁻ cells pulsed with 50nM of the FL9 or the candidate homolog peptides. (f) Response of BEko8Z hybridoma to L-Qa-1^b cells pulsed with FL9, FL10 or FYAEDDTPIV (FV10) peptide of various concentrations. (c, e, f) Data are from one experiment representative of three independent experiments.

Association between Vα3.2⁺QFL T cells and the commensal bacterium P. pentosaceus.
(a) Colonization of GF mice with commensal bacteria. GF QFLTg_WT chimera mice received oral gavage of P. pentosaceus at 8 weeks of age and were analyzed for both the donor-derived QFLTg cells and the endogenous Vα3.2⁺QFL T cells at 16 weeks of age. GF WT mice received oral gavage of P. pentosaceus or L. johnsonii at 8 and 10 weeks of age and were analyzed for the endogenous Vα3.2⁺QFL T cells at 16 weeks of age. (b) Absolute numbers of donor-derived QFLTg cells in the spleen (left) or siIEL compartment (right) of 16-week GF QFLTg_WT chimera mice colonized with P. pentosaceus in comparison with SPF or uncolonized GF chimera mice. Symbols on x-axis indicate that QFLTg cells were undetectable in the indicated group. *P<0.03 (c) Relative abundance of P. Pentosaceus or L. Johnsonii determined by whole shotgun metagenomic sequencing of microbiota composition in pooled fecal pallets collected from each cage. (d) Absolute numbers of endogenous Vα3.2⁺QFL T cells in the spleen (left) or siIEL compartment (right) of 16-week colonized GF mice compared with SPF or uncolonized GF chimera mice. Endogenous Vα3.2⁺QFL T data collected from the GF QFLTg_WT chimeric mice and the non-chimeric WT mice were pooled for this analysis. **P<0.008 The number of replicates is specified in the bar graphs with each symbol representing data collected from the indicated sample/tissue isolated from an individual cage (c) or mouse (b, d). Data from ≧3 independent experiments were pooled for statistical analysis. P values were calculated with Student’s t test. ‘ns’ indicates the comparison was not significant.

Characteristics of Vα3.2⁺ QFL T cells in the spleen and IEL
Summary of the characteristic features of the splenic and IEL Vα3.2⁺ QFL T cells including their cell type, expression of cell surface markers, dependence on microbiota for tissue homing/retention and the critical molecules required for population establishment or phenotype imprinting.

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