E-cadherin promotes accumulation of a unique memory CD8 T-cell population in murine salivary glands

Abstract

The salivary glands are important effector sites for IgA-mediated humoral immunity to protect oral surfaces. Within murine submandibular glands (SMG), we identified a memory CD8 T-cell population that exhibited a unique cell-surface phenotype distinct from memory CD8 T cells in spleen but similar to memory T cells resident in the intraepithelial lymphocyte compartment of the intestinal mucosa. In mice immune to lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus(VSV), virus-specific memory CD8 T cells with this unusual phenotype were present in SMG at remarkably high frequencies. LCMV-specific memory CD8 T cells in SMG showed potent functional activities in vivo, including cytokine-induced bystander proliferation, antigen-triggered IFNγ production, and viral clearance. Adoptive transfer experiments further revealed that the capacity to accumulate in SMG decreased during CD8 T-cell differentiation and that SMG CD8 T cells were poorly replenished from the circulation, indicating that they were tissue-resident. Moreover, they preferentially relocalized within their tissue of origin after adoptive transfer and antigen rechallenge, thus revealing an imprinted differentiation status. Accumulation of memory CD8 T cells within SMG did not require local antigen presentation but was promoted by the epithelial differentiation molecule E-cadherin intrinsically expressed by these CD8 T cells. This finding extends the epithelial-restricted function of E-cadherin to an impact on lymphocyte accumulation within epithelial tissues.

Fig. 1.

Accumulation of CD8 T cells in SMG of LCMV-immune mice. (A) Absolute numbers of CD45 leukocytes per SMG in noninfected vs. LCMV-immune B6 mice (4–16 wk p.i.). *P < 0.05. (B) Percentage of corresponding cell populations within CD45 cells of SMG in noninfected vs. LCMV-immune B6 mice. Dots represent values of SMG pools from three to five mice. *P < 0.05; **P < 0.01.

Fig. 2.

High frequencies of antigen-specific memory CD8 T cells in SMG. (A) Percentages of CD8 memory T cells specific for LCMV gp33 peptide in spleen (SP), SMG, and IEL of LCMV-immune (4–16 wk p.i.) B6 mice determined by MHC class I tetramer (gp33/H-2D^b) staining. (B) Percentages of CD8 memory T cells specific for VSV np52 in spleen (SP), SMG, and IEL of VSV-immune B6 mice determined by MHC class I tetramer (np52/H-2K^b) staining 28 wk p.i. (C) Percentages of CD8 memory T cells carrying the P14 TCR (Thy1.1) in spleen (SP), SMG, and IEL determined in LCMV-immune (4–16 wk p.i.) P14 chimeric mice. Representative dot plots and statistical graphs are shown. All samples were gated on CD45 cells. Percentages of CD8 T cells specific for the indicated markers are depicted. Dots in statistical graphs represent values from individual mice. **P < 0.01; ***P < 0.001.

Fig. 3.

Unique cell-surface phenotype of SMG CD8 memory T cells. P14 memory T cells from SMG (red line) and spleen (SP, blue line) of LCMV-immune (week 14 p.i.) P14 chimeric mice were analyzed with the markers indicated. Representative flow cytometry profiles are depicted. All samples were pregated on CD45 cells. Histograms were further gated on P14 T cells (Thy1.1/CD8). Isotype controls are shown in gray.

Fig. 4.

Functional activities of SMG CD8 memory T cells. (A) Poly I:C (250 μg) was administered i.p. into LCMV-immune B6 mice, and intranuclear Ki-67 expression was evaluated 72 h later. Controls are derived from untreated LCMV-immune B6 mice. Percentages of splenic total CD8 T cells or CD44^high CD8 T cells and total SMG CD8 T cells positive for Ki-67 are depicted. Dots represent values from individual mice. **P < 0.01; ***P < 0.001. (B) LCMV-immune P14 chimeric mice were injected (i.v.) with gp33 peptide (200 μg). Six hours later, P14 memory T cells from spleen (SP) and SMG were isolated and immediately stained for intracellular IFNγ. Shown are representative dot plots and a statistical graph depicting percentages of P14 memory T cells positive for IFNγ. Dots represent values from individual mice. Horizontal dashed black line represents mean percentage of P14 memory T cells positive for IFNγ from untreated LCMV-immune P14 chimeric mice. (C) The indicated groups of mice were infected i.g. with LCMV-Docile, and viral titers in SMG were determined on day 3 p.i. Dots represent titers in plaque-forming units per organ from individual mice. Horizontal gray line marks the detection limit (5 × 10³ pfu/organ).

Fig. 5.

Only P14 effector but not resting P14 memory T cells accumulate in SMG in the absence of a LCMV infection. P14 effector T cells (day 8 p.i., 5 × 10⁶), P14 late effector T cells (day 18 p.i., 4 × 10⁶), or resting P14 memory T cells (day 110 p.i., 2 × 10⁶) from spleen of P14 chimeric mice were transferred (i.v.) into naive B6 mice. Percentages of CD8 T cells carrying the P14 TCR (Thy1.1) recovered from spleen (SP) and SMG of the recipient mice were determined 1 wk after adoptive transfer. Dots in statistical graphs represent values from individual mice. *P < 0.05.

Fig. 6.

Tissue-specific accumulation of SMG P14 memory T cells after LCMV rechallenge. (A) B6 mice were seeded with 10⁵ P14 memory T cells from spleen (SP) or SMG, and proliferative capacity was assessed in blood of the recipient mice 6 d after LCMV rechallenge. (B) Tissue distribution of P14 memory T cells derived from spleen (SP) or SMG determined 3 wk after retransfer and rechallenge. Dots in statistical graphs represent values from individual mice. *P < 0.05; ***P < 0.001.

Fig. 7.

Antigen-independent accumulation of P14 T cells in SMG and tissue-specific induction of E-cadherin and CD103 expression. P14 T cells (5 × 10⁶) activated in vitro with LCMV gp33 peptide for 3 d were transferred into naive B6 mice. Percentage of CD8 T cells carrying the P14 TCR (Thy1.1) recovered from spleen (SP) and SMG (A) and from IEL (B) were determined 1 and 4 wk after adoptive cell transfer. (C) E-cadherin expression by in vitro-activated and transferred P14 T cells (Thy1.1) recovered from spleen (SP), SMG, and IEL. (D) Representative dot plots depicting CD103 expression of P14 T cells (Thy1.1) collected from spleen (SP), SMG, and IEL 3 wk after adoptive transfer of in vitro-activated P14 T cells. All samples were pregated on CD45. Dots in statistical graphs represent values from individual mice. *P < 0.05; **P < 0.01.

Fig. 8.

E-cadherin expressed by SMG CD8 T cells promotes their tissue accumulation. (A) Expression of E-cadherin and KLRG1 by SMG CD8 T cells of LCMV-immune CD4-Cre⁺E-cad^fl/fl and CD4-Cre⁻E-cad^fl/fl mice. Representative dot plots of CD45 pregated samples are shown. (B) Percentage of CD8 T cells within CD45 cells in spleen (SP) and SMG of LCMV-immune CD4-Cre⁺E-cad^fl/fl (E-cad−) and CD4-Cre⁻E-cad^fl/fl (E-cad+) mice. Dots in statistical graphs represent values from individual mice. **P < 0.01.