Non-equilibrium and differential function between intraepithelial and lamina propria virus-specific TCRalphabeta(+) CD8alphabeta(+) T cells in the small intestinal mucosa

Abstract

The gastrointestinal mucosa regularly encounters commensal and pathogenic microbiota. Gut mucosal lymphocytes consist of two phenotypically different populations residing in the intestinal intraepithelial (IEL) compartment and lamina propria (LP). Little is known about compositional and functional differences of antigen-specific T cells from these mucosal compartments after mucosal infection, or the degree of trafficking between them. We here studied the B8R(20-27)-specific CD8 T-cell response in LP and IEL compartments after intrarectal immunization with modified vaccinia virus Ankara (MVA). CD8(+) T cells in the IEL compartment had much lower avidity than in the LP or spleen during acute and memory phases. Surprisingly, the TCR Vbeta-chain distribution of antigen-specific T cells and the length of the CDR3 region of the dominant Vbeta genes showed substantial dissimilarities between IEL and LP antigen-specific CD8alphabeta T cells in individual mice, increasing with time. We show functional and compositional differences between these mucosal compartments during the effector and memory phases of the immune response, indicating limited crosstalk and microenvironmental differences between the IEL, LP, and spleen. The restricted migration of cells from each of these mucosal compartments could partly account for a founder effect we observed in the IEL TCRalphabeta CD8alphabeta epitope-specific repertoire that might impact protective efficacy.

Figure 1

Expansion and contraction of B8R_{20 – 27}/H2-K^b tetramer ⁺ CD8 ⁺ T cells in the spleen, SI-IEL, and SI-LP. Relative percentages of antigen-specific CD8 ⁺ T cells after intrarectal immunization at 7 days (a) and 2 months (b) in the spleen, SI-IEL, and SI-LP are presented. Lymphocytes were isolated and stained with phycoerythrin-conjugated B8R_{20 – 27}/H2-K^b tetramer together with CD8β (SI-IEL and SI-LP) or CD8α (spleen) mAbs, and analyzed by flow cytometry. Magnitude of antigen-specific IFN-γ production at 7 days (c) and 2 months (d). Directly ex vivo isolated cells were in vitro stimulated with B8R_{20 – 27} peptide (1 μM). Number of IFN-γ – producing cells was counted on ELISpot Reader and presented as spots per 10⁶ cells in each tissue. Data from one of two representative experiments are shown as the mean and s.e.m. of four animals per interval. IEL, intraepithelial lymphocyte; IFN-γ, interferon-γ; LP, lamina propria; mAbs, monoclonal antibodies.

Figure 2

Functional avidity of B8R-specific CD8 ⁺ T cell response by ELISpot for IFN-γ production in the spleen, SI-IEL, and SI-LP. Data are normalized for comparison between compartments. Lymphocytes from the spleen and small intestine were isolated at 7 days (acute phase) and 2 months (memory phase) from the spleen, SI-IEL, and SI-LP and in vitro stimulated with different concentrations of B8R_{20 – 27} peptide as for Figure 1c and d. (a and b) ELISpot data on the number of IFN-γ-producing cells during acute and memory phases of MVA infection were normalized by GraphPad Prism software to a common scale. Data normalized to a common scale are shown to visualize differences in the avidity between compartments. Cells were in vitro stimulated with different B8R_{20 – 27} peptide concentrations as described in Methods. (c and d) Normalized net ELISpot data presented as the relative percentage of cells producing IFN-γ after in vitro stimulation with B8R_{20 – 27} peptide. To obtain net values, ELISpot data presented on a) and (b) were recalculated by the following formula: Net 1 pM responders = actual 1 pM responders; Net 100 pM responders = total 100 pM responders – actual 1 pM responders; Net 1 μM responders = total 1 μM responders – actual 100 pM responders. Data from one of two representative experiments are shown as the mean and s.e.m. of four animals per interval. IEL, intraepithelial lymphocyte; IFN-γ, interferon-γ; LP, lamina propria; MVA, Modified Vaccinia Ankara.

Figure 3

TCR Vβ-chain profile expressed by B8R_{20 – 27}/H2-K^b tetramer ⁺ CD8 ⁺ T cells in the spleen, SI-IEL, and SI-LP during acute and memory phases of MVA infection. (a and b) Lymphocytes from the spleen and small intestine were isolated from the spleen, SI-IEL, and SI-LP at acute (7 days) and memory (2 months) phases of MVA infection. Cells were stained with phycoerythrin-conjugated B8R_{20 – 27}/H2-K^b tetramer ⁺ and CD8α mAbs and gated on tetramer ⁺ CD8α ⁺ population. Then, additionally stained with anti-TCR Vβ-chain mAbs, we assessed the relative percentage of each TCR Vβ-chain inside gated cells. Data represent TCR Vβ-chain profile in individual mice from two independent experiments (mean±s.d.). (c and d) To measure the variation in a TCR Vβ-chain data distribution in different tissues in individual mice, we calculated coefficient of variation (CV) = s.d.×100%/mean value. A high CV corresponds to the high degree of variation in the sample. IEL, intraepithelial lymphocyte; LP, lamina propria; mAbs, monoclonal antibodies; MVA, Modified Vaccinia Ankara.

Figure 4

Correlative analysis of TCR Vβ-chain profile expressed by B8R_{20 – 27}/H2-K^b tetramer ⁺ CD8 ⁺ T cells in the spleen, SI-IEL, and SI-LP at acute and memory phases of MVA infection. Selective correlative analysis for TCR Vβ2, −5.1/5.2, −7, −8.1/8.2, −8.3, and −13 chain carrier distribution. Data represent the original values shown on Figure 3a and b. Acute phase: IEL vs. spleen: Vβ2 (R² = 0.2708), Vβ5.1/5.2 (R² = 0.1392), Vβ7 (R² = 0.0136), Vβ8.1/8.2 (R² = 0.212), Vβ8.3 (R² = 0.02099), Vβ13 (R² = 0.0089); IEL vs. LP: Vβ2 (R² = 0.0839), Vβ5.1/5.2 (R² = 0.0849), Vβ7 (R² = 0.0046), Vβ8.1/8.2 (R² = 0.0091), Vβ8.3 (R² = 0.0089), Vβ13 (R² = 0.5364); LP vs. spleen: Vβ2 (R² = 0.0039), Vβ5.1/5.2 (R² = 0.0762), Vβ7 (R² = 0.1109), Vβ8.1/8.2 (R² = 0.0368), Vβ8.3 (R² = 0.0032), Vβ13 (R² = 0.3505). Memory phase: IEL vs. spleen: Vβ2 (R² = 0.1447), Vβ5.1/5.2 (R² = 0.0662), Vβ7 (R² = 0.1825), Vβ8.1/8.2 (R² = 0.087), Vβ8.3 (R² = 0.0004), Vβ13 (R² = 0.2366). IEL, intraepithelial lymphocyte; LP, lamina propria; MVA, Modified Vaccinia Ankara.

Figure 5

Immunoscope analysis of the B8R tetramer ⁺ -sorted CD8 ⁺ T cells from IEL and LP compartments. CDR3 length analysis of the IEL and LP B8R tetramer ⁺ CD8 ⁺ -sorted T cells from the same mouse is shown for d7 (acute phase) and d60 (memory phase) after infection. CDR3 analysis only for dominant Vβ-chain genes is shown. Strong oligoclonality is found in all samples. Memory B8R tetramer ⁺ CD8 ⁺ IELs are represented by significantly fewer clones than memory LP T cells. Representative data of N = 4 are presented. IEL, intraepithelial lymphocyte; LP, lamina propria.

Figure 6

Adoptively transferred CD8α ⁺ IELs poorly migrate into small intestinal mucosa. (a and b) Lymphocytes from the spleen and IEL from naive donor C57Bl/6 mice were isolated on CD8 magnetic beads (viability >99%), then CD8 ⁺ IEL cells were labeled with red fluorescent linker, and splenocytes labeled with CFSE, mixed together at a 1:1 ratio, and total 5×10⁶ CD8 ⁺ T cells were transferred i.v. into naive wild-type mice. Twenty-four hours later, lymphocytes were isolated from the small intestine IEL (a) and LP, large intestine IEL and LP, mesenteric lymph nodes, spleen, (b) and lungs and analyzed by flow cytometry. (c) The number of donor spleen and IEL cells recovered in different tissues of recipient animals is shown. (d and e) Cells from naive donor C57Bl/6 mice were isolated from IEL or spleen as described for (a) and (b), labeled with CFSE, and separately transferred i.v. into naive recipient SCID mice. Seven days after the transfer, the presence of donor CFSE ⁺ IEL (d) or donor splenocytes (e) in the corresponding recipient tissue was analyzed by flow cytometry. (f) Number of donor spleen and IEL cells recovered in different tissues of recipient animals is shown. CFSE, carboxyfluorescein succinimidyl ester; IEL, intraepithelial lymphocyte; LP, lamina propria; SCID, severe combined immunodeficiency.