Dynamic T cell migration program provides resident memory within intestinal epithelium

Abstract

Migration to intestinal mucosa putatively depends on local activation because gastrointestinal lymphoid tissue induces expression of intestinal homing molecules, whereas skin-draining lymph nodes do not. This paradigm is difficult to reconcile with reports of intestinal T cell responses after alternative routes of immunization. We reconcile this discrepancy by demonstrating that activation within spleen results in intermediate induction of homing potential to the intestinal mucosa. We further demonstrate that memory T cells within small intestine epithelium do not routinely recirculate with memory T cells in other tissues, and we provide evidence that homing is similarly dynamic in humans after subcutaneous live yellow fever vaccine immunization. These data explain why systemic immunization routes induce local cell-mediated immunity within the intestine and indicate that this tissue must be seeded with memory T cell precursors shortly after activation.

Figure 1.

Only early effector CD8 T cells migrate to intestinal epithelium and express α4β7. (A) Dynamics of P14 response to LCMV. The day 37 time point represents 12 mice analyzed from days 31 to 50 after infection. (B) Experimental design consists of transferring P14 at different stages of differentiation and harvesting tissues the next day. (C) Numbers of naive (N), effector (isolated 4.5 or 7 d after infection), and memory (isolated 60 d after infection) cells isolated from recipient spleen and intestinal epithelium (IEL). ns, not significant, *, P < 0.05; **, P < 0.01, unpaired Student’s t test. Error bars indicate SEM. (D) Virus-specific P14 CD8 T cells were analyzed for expression of α4β7. gmfi, geometric mean fluorescence intensity of α4β7 staining. All plots are gated on Thy1.1⁺ CD8⁺ lymphocytes and are representative of at least three independent experiments totaling >10 mice/time point.

Figure 2.

Antigen-dependent reexpression of α4β7 by spleen-derived transgenic and endogenous memory CD8 T cells upon infection with virus or bacteria. (A–D) Splenocytes isolated from P14 immune chimeras (>30 d after LCMV Arm infection) were transferred to naive recipients. (A and B) The next day, recipients were challenged with high-dose LCMV Arm or left unchallenged. (A) α4β7 expression was monitored in blood among donor P14 (Thy1.1⁺/gp33 tetramer⁺), nontransgenic gp33-specific cells (Thy1.1⁻/gp33 tetramer⁺), and CD44^lo (naive) CD8 T cells. Representative flow cytometry data are shown. (B) Change in GMFI of α4β7 expression relative to α4β7 GMFI of memory P14 transferred to unchallenged mice that were analyzed on the same day. (C and D) As in A and B, except mice were challenged with LCMV Cl⁻13 (C) or LM-gp33 (D), or the noncognate antigen bearing inflammation control LM-WT. (E) 19 d after infection, small intestine IEL of these mice were examined for the presence of donor P14. (F) Splenocytes from LCMV Arm–immune C57BL/6J mice (which did not contain P14) were transferred to naive CD45.1 recipients. Recipients were challenged the next day with LM-gp33, and α4β7 expression among CD45.1⁻ gp33-tetramer⁺ CD8 T cells was monitored in blood. At least three mice were analyzed at each time point in each experiment. Error bars indicate SEM. One of two experiments with similar results is shown.

Figure 3.

Memory CD8 T cells do not retain α4β7 expression regardless of anatomical location or immunization route. Naive Thy1.1⁺ P14 were transferred to naive mice. (A and B) The next day, mice were infected intranasally with 500 pfu of recombinant influenza virus that expresses gp33. 5 (A) or 86 (B) d later, lymphocytes were isolated from the indicated tissues and stained with α-Thy1.1, CD8, and α4β7 or CD44. Plots are gated on CD8⁺ lymphocytes. (C and D) Alternatively, 200 µg DNA that expresses the glycoprotein of LCMV under control of the CMVie promoter was administered intramuscularly into both anterior tibialis muscles. (C) 9 and 12 d later, Thy1.1⁺ cells were examined for expression of α4β7 (gated on CD8⁺ Thy1.1⁺ lymphocytes). (D) 12 d after immunization, lymphocytes were isolated from spleen and IEL and stained with α-CD8α, Thy1.1, and CD44 antibodies. Plots are gated on CD8α⁺ lymphocytes. (E) Naive Thy1.1⁺ P14 were transferred to naive C57BL/6J mice. Control mice received normal drinking water, whereas treated mice were exposed to 2 µg/ml FTY720 in the drinking water ad libitum for the duration of the experiment. The next day, both groups of mice were immunized with LCMV, and α4β7 expression among Thy1.1⁺ P14 in spleen was compared among control mice (black line), FTY720-treated mice (dashed line), and CD44^lo CD8 T cells isolated from spleens of naive mice (gray histogram). Plots are gated on Thy1.1⁺ CD8⁺ lymphocytes. All data are representative of two experiments with at least three mice per group in each experiment.

Figure 4.

Memory CD8 T cells do not retain α4β7 expression regardless of anatomical location or immunization route. (A and B) Expression of α4β7 and/or CCR9 by P14 isolated from various tissues 4.5, 7, or 60 d after i.p. LCMV infection (A) or by OT-I after oral LMova infection (B). All plots are gated on Thy1.1⁺ CD8⁺ lymphocytes. (C) 10⁶ Thy1.1⁺ P14 isolated from spleen (red), iLN (blue), or mLN (green) 4.5 d after LCMV infection was transferred to naive mice. The next day, lymphocytes were harvested from recipient spleen and small intestinal epithelium, and the proportion of CD8⁺ lymphocytes that were Thy1.1⁺ was determined. Only p-values of <0.05 are shown. (D) Recipient mice received the entire single cell suspension derived from either one spleen or the complete cluster of mLN derived from one mouse isolated 4.5 d after LCMV infection. The next day, the proportion of CD8⁺ lymphocytes that were Thy1.1⁺ was determined. Error bars indicate SEM. All data are representative of at least two experiments with at least three mice per group in each experiment.

Figure 5.

Memory CD8 T cells in intestinal epithelium do not recirculate. Naive P14 cells were transferred to naive C57BL/6J mice, and recipients were infected with LCMV. 90 d later, 2 µg/ml FTY720 was dissolved in drinking water (white bars) or mice were maintained on normal drinking water (black bars). (A and B) 2 (A) or 30 (B) d after FTY720 treatment, the number of LCMV-specific P14 memory CD8 T cells was determined in blood (PBL), iLNs, lung, or small intestinal epithelium (IEL). Data shown is one of five experiments with three mice per group with similar results. Immune mice were generated by transferring naive Thy1.1⁺ P14 into naive C57BL/6J mice and infecting recipients with LCMV. Error bars indicate SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001, unpaired Student’s t test. (C) 2 mo later, 7 cm of small intestine, along with associated mesentery and mLN, were transplanted from naive mice into immune mice. (D) 42 d after transplantation, lymphocytes were isolated from host spleen, blood (PBL), mLN, and intestinal epithelium (IEL), as well as donor mLN and IEL. The presence of host memory P14 was determined in each tissue by Thy1.1 staining and flow cytometry. All plots are gated on CD8⁺ lymphocytes and are representative of one of a total of three mice examined in two independent experiments. NA, not applicable.

Figure 6.

Primary human CD8 T cell response to s.c. yellow fever vaccine results in only short-term expression of α4β7 and CLA. Blood was isolated from HLA-A2–positive volunteers 11, 14, 30, and 90 d after s.c. vaccination with YFV-17D. (A) Expression of CLA versus staining with MHC class I tetramers that recognize YFV-specific CD8 T cells from three representative patients. (B) CLA versus α4β7 expression. HLA2-YFV tetramer⁺ cells are blue and HLA2-YFV tetramer⁻ cells are gray. Numbers indicate percentage of tetramer⁺ cells in each quadrant. All plots are gated on CD3⁺ CD8⁺ lymphocytes. (C) Summary of CLA and α4β7 expression among YFV tetramer⁺ cells. Longitudinal analysis is shown, although some patients were not examined on days 30 and 90. Horizontal bars show the mean. Day 11, n = 7; day 14, n = 7; day 30, n = 4; day 90, n = 6.