Dynamic T cell migration program in spleen
To examine the dynamics of CD8 T cell homing, we used the lymphocytic choriomeningitis virus (LCMV) and P14 transgenic CD8 T cell system, which provides access to large numbers of naive, effector, and memory CD8 T cells of known specificity. To generate effector and memory CD8 T cells, naive Thy1.1+ P14 were transferred to naive mice, which were then challenged i.p. with the Armstrong strain of LCMV. In response to infection, P14 underwent clonal expansion, which peaked 7 d after challenge, followed by contraction of the population and formation of stable memory by ~1 mo later (). We first examined the trafficking potential of cells isolated from spleen. We transferred 2 × 106 naive P14 splenocytes or 2 × 106 P14 splenocytes isolated 4.5, 7, or 60 d after LCMV Armstrong infection into naive Thy1.2+ recipients. It should be noted that day-4.5 CD8 T cells represent early effectors that are still undergoing clonal expansion. The next day, lymphocytes were harvested from recipient tissues ().
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 (more ...)
As expected, naive CD8 T cells populated blood and secondary lymphoid tissues but were excluded from the IEL compartment of the small intestine ( and Fig. S1
). When early effector P14 splenocytes isolated from spleen (or blood; Fig. S2
) were transferred, they were recovered from spleen and the epithelium of the intestinal mucosa. These data demonstrated that early effector CD8 T cells were capable of homing from spleen to gut. Pertussis toxin treatment of donor cells inhibited effector cell homing to intestinal epithelium, demonstrating that this migration was chemokine dependent (Fig. S1).
Remarkably, CD8 T cells lost the ability to migrate to the IEL compartment by 7 d after infection (). Memory CD8 T cells were entirely excluded from migrating to the intestinal epithelium. Thus, CD8 T cells were endowed with the ability to migrate to intestinal epithelium only early during the response.
We then tested whether effector cells were contaminated with a cofactor that permitted T cells, regardless of differentiation state, to migrate to the intestinal epithelium. When day-4.5 and memory P14 splenocytes were mixed and transferred to the same naive recipient, both populations of cells were equivalently recovered from spleen. However, memory CD8 T cells were ~20-fold more efficient at trafficking to LN and early effectors were >50-fold more efficient at migrating to the small intestinal epithelium (Fig. S3
). This indicated that effector and memory CD8 T cells exhibit intrinsically different homing properties.
To test this conclusion further, we examined the dynamics of α4β7 expression, which is required for homing to the small intestine. shows that α4β7 expression is transiently up-regulated on all early effector LCMV-specific CD8 T splenocytes and is then rapidly down-regulated. Thus, the timing of α4β7 expression reflected the transient ability of T cells to traffic to the gut epithelium.
We then tested whether reactivation of memory CD8 T cells would result in transient reexpression of α4β7. Splenocytes isolated from LCMV-immune Thy1.1+ P14 chimeras () were transferred to naive mice. Recipients were boosted the next day with pathogens that contained cognate gp33 antigen, including LCMV Armstrong strain, LCMV clone 13 strain, or recombinant Listera monocytogenes (LM-gp33). In each case, secondary donor CD8 T cell responses were coupled with transient reexpression of α4β7 and dissemination into the small intestinal epithelium (). Endogenous H-2Db/gp33-specific reactivated memory CD8 T cells also transiently up-regulated α4β7 upon boosting (), so this phenomenon was not unique to transgenic CD8 T cells. In contrast, challenge with LM that lacked recombinant cognate gp33 antigen (LM-WT) failed to induce α4β7 expression among memory P14, suggesting that the infectious milieu alone was insufficient and that cognate antigen was required.
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) (more ...)
It should be noted that LCMV infects the spleen, almost 100% of early effector splenocytes expressed α4β7, and there are ~20-fold more early effectors in spleen than GALT (7.2 × 106 early effector P14 in spleen versus 3.4 × 105 in mLNs). Thus, it is unlikely that activation in GALT was required for α4β7 expression. To examine this issue more stringently, mice were infected intranasally with influenza virus. 5 d later, almost no activated virus-specific CD8 T cells could be detected within GALT, yet flu-specific CD8 T cells in spleen- and lung-associated tissues expressed α4β7 and memory CD8 T cells were established within small intestinal epithelium (). Intramuscular injection of a DNA vaccine into the anterior tibialis (located within lower hind limb) also led to transient α4β7 expression and the establishment of CD8 T cells within intestinal epithelium (). Moreover, disruption of lymphocyte egress from GALT via treatment of mice with FTY720 did not affect α4β7 expression among effector CD8 T cells in spleen (). Collectively, these data demonstrate that activation in GALT is not required for α4β7 expression among CD8 T cells.
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 (more ...)
Homing potential among distinct lymphoid tissues
We next tested whether memory CD8 T cells in mucosal tissues also lacked α4β7, and we determined the dynamics of CCR9 expression, which is also involved in migration to gut (Zabel et al., 1999
; Kunkel et al., 2000
; Johansson-Lindbom and Agace, 2007
). demonstrates that neither α4β7 nor CCR9 expression is maintained on memory CD8 T cells isolated from any lymphoid tissue. Although IEL memory CD8 T cells expressed CCR9, α4β7 was down-regulated by day 7 after infection ( and Fig. S4
). Thus, LCMV-specific memory CD8 T cells, regardless of location, did not coexpress the essential homing molecules required for entry into the intestinal mucosa.
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 (more ...)
We then determined whether α4β7 expression was maintained on memory CD8 T cells after oral infection. OT-I transgenic CD8 T cells specific for ovalbumin were transferred to mice before oral infection with recombinant LM expressing ovalbumin (LM-ova; Masopust et al., 2001a
; Pope et al., 2001
). As shown in , memory CD8 T cells did not retain α4β7 after oral infection in any tissue analyzed, including the IEL compartment.
Expression of gut homing molecules on early effector CD8 T cells varied by location (). Those isolated from skin-draining iLN up-regulated less α4β7 than cells in spleen and mLN. And early effectors in mLN expressed higher levels of CCR9 than those isolated from spleen or iLN. We compared the homing potential of each population on a per cell basis by transferring 106 P14 isolated from each lymphoid tissue 4.5 d after LCMV infection to separate recipients (). With regard to homing to intestinal epithelium, P14 cells derived from mLN were the most efficient, and iLN-derived cells migrated poorly. P14 derived from spleen exhibited an intermediate ability to traffic to mucosal epithelium. When one considers the fact that there are ~20-fold more early effector T cells in spleen than mLN, it is likely that more IEL effectors are derived from spleen than from mLN after an LCMV infection. To test this hypothesis, all lymphocytes derived from one spleen versus all mLNs derived from one mouse were transferred into separate recipients. Although recovery varied among different experiments, transfer of spleen consistently resulted in 2.5–4-fold more P14 within the gut epithelium than the transfer of mLN on a per-organ basis (, n = 5 per group for the experiment shown).
Memory IELs are resident
FTY720 treatment causes an accumulation of recirculating lymphocytes within lymphoid tissue and a corresponding decrease within other tissues that contain recirculating lymphocytes (Schwab and Cyster, 2007
). To further test whether IELs recirculate, we treated LCMV-immune mice with FTY720 and examined whether treatment induced a reduction in memory CD8 T cells within the intestinal epithelium. LCMV-immune P14 chimeras (generated as described in ) were treated with FTY720 for 2 or 30 d in drinking water. As shown in , treatment led to a rapid depletion of memory CD8 T cells in blood within 2 d, and this reduction was maintained for 30 d with continuous FTY720 treatment (). As expected, a reciprocal increase in P14 was observed in iLN, suggesting that lymphocyte recirculation through the iLN was interrupted. However, there was no reduction in the number of P14 IEL, even after 30 d of treatment (). These data support the hypothesis that IELs do not recirculate via afferent lymphatics.
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 (more ...)
We wished to independently test whether memory CD8 T cells recirculated through gut epithelium over a period of several weeks. To this end, we removed 7 cm of small intestine from naive C57BL/6J mice and transplanted this tissue into C57BL/6J mice that had been immunized with LCMV 2 mo previously and contained large numbers of Thy1.1+ memory P14 CD8 T cells (). Blood vessels were ligated upon transplantation and donor gut remained viable and appeared healthy by visual inspection upon cessation of the experiment. This method allowed us to test whether memory CD8 T cells, which are present in all tissues of the recipient mouse, would equilibrate with donor gut epithelium. Association of donor small intestine with donor mLN remained intact and served as a positive control for recirculation between graft and host. 42 d after transplantation, host and donor tissues were removed and assessed for the presence of host memory P14. As shown in , donor mLNs have similar frequencies of memory P14 CD8 T cells compared with host mLNs, as expected. Within the donors’ IEL compartment, there were far fewer host memory P14 CD8 T cells compared with host epithelium, indicating minimal recirculation.
Dynamic α4β7 and CLA expression after human s.c. yellow fever vaccination
Our observations in mice revealed that α4β7 was expressed by early effector CD8 T cells but not memory CD8 T cells. Expression of the putative skin homing molecule, functional P-selectin ligand, was similarly dynamic and was coexpressed with α4β7 on effector splenocytes shortly after LCMV infection (Fig. S5
). We wished to examine whether expression of peripheral homing molecules was also dynamic after a primary immunization of humans. To follow a primary CD8 T cell response in humans, we examined the response to the live attenuated yellow fever virus (YFV)–17D vaccine (Miller et al., 2008
). We identified an HLA-A2–restricted immunodominant epitope that we used to construct MHC class I tetramers. Volunteers were vaccinated s.c. and a tetramer+
population in PBMC could be visualized by day 11. Because immunization was delivered s.c., we examined expression of CLA, which is involved in skin homing. CLA expression on YFV-specific T cells was remarkably transient, peaking on day 11, decreasing substantially by day 14, and was not detectable by day 30 after immunization (). When CD8 T cells were costained for both CLA and α4β7 molecules, early effectors expressed detectable levels of both homing molecules (). However, by day 30, YFV-specific effector CD8 T cells had predominantly down-regulated CLA and α4β7, and this phenotype persisted among memory CD8 T cells (90 d after infection; ). Thus, human virus-specific CD8 T cells also express a very dynamic pattern of homing molecule expression and support the hypothesis that CD8 T cell homing to body surfaces occurs predominantly upon recent activation.
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. (more ...)