Migration matters: regulatory T-cell compartmentalization determines suppressive activity in vivo

Abstract

Regulatory T cells (Tregs) play a fundamental role in the suppression of different immune responses; however, compartments at which they exert suppressive functions in vivo are unknown. Although many groups have described the presence of Tregs within inflammatory sites, it has not been shown that inflamed tissues are, indeed, the sites of active suppression of ongoing immune reactions. Here, by using alpha(E)+ effector/memory-like Tregs from fucosyltransferase VII-deficient animals, which lack E/P-selectin ligands and fail to migrate into inflamed sites, we analyzed the functional importance of appropriate Treg localization for in vivo suppressive capacity in an inflammation model. Lack of suppression by Tregs deficient in E/P-selectin ligands demonstrates that immigration into inflamed sites is a prerequisite for the resolution of inflammatory reactions in vivo because these selectin ligands merely regulate entry into inflamed tissues. In contrast, control of proliferation of naive CD4+ T cells during the induction phase of the immune response is more efficiently exerted by the naive-like alpha(E)-CD25+ Treg subset preferentially recirculating through lymph nodes when compared with its inflammation-seeking counterpart. Together, these findings provide the first conclusive evidence that appropriate localization is crucial for in vivo activity of Tregs and might have significant implications for anti-inflammatory therapies targeting recruitment mechanisms.

Figure 1.

The α_E⁺ Tregs efficiently suppress committed Th1 cells in vivo. (A) FACS analysis of pooled spleen and lymph node CD4⁺ T cells before and after subset sorting. (B) In vitro generated Th1 cells (5 × 105) were injected intravenously together with 5 × 105 FACS-sorted and preactivated T cells into naive BALB/c mice. Twenty-four hours later the DTH response was induced by OVA peptide injection into the footpad. Shown is progression of the inflammatory response monitored by the thickness of footpads injected with OVA/IFA (♦, •, ▴) and control footpads injected with PBS/IFA (♦,○, ▵; mean ± SD; n = 6). The α_E⁺ Tregs showed a significantly higher suppressive capacity than α_E^–CD25+ cells (P < .01, repeated measure analyses). The insert shows the same data set expressed as percent suppression by indicated Treg subsets, in relation to the footpad swelling (OVA/IFA-injected footpad minus PBS/IFA-injected footpad) of mice adoptively transferred with α_E^–CD25^– control cells. (C) In vitro suppressive capacity of indicated T-cell subsets on naive T-cell proliferation was determined after 72 hours of coculture with CFSE-labeled responder cells at a 1:1 ratio. Culture of CFSE-labeled responder cells alone (medium) served as control. Representative histogram plots of CFSE profiles from CFSE⁺CD4⁺ T cells are shown. Quantification of suppressive capacity is based on the CFSE geometric mean of total CFSE⁺CD4⁺ T cells (n = 3; mean ± SD; 1 representative of 2 independent experiments).

Figure 2.

FucTVII^{–/ –} α_E⁺ Tregs cannot enter the inflamed footpad and show no suppression of the Th1-mediated DTH reaction. (A) Expression of P-selectin–binding ligands on indicated CD4⁺ T-cell subsets from DO11.10 (WT) and DO11.10×FucTVII^–/–mice is shown. Representative histogram plots from 3 independently analyzed mice were selected. Numbers indicate frequency of P-selectin–binding cells. (B) In vitro preactivated Treg subsets from DO11.10 (WT) and DO11.10×FucTVII^–/– mice were radioactively labeled with 111In and injected intravenously into BALB/c mice, in which 24 hours before a DTH response had been induced, followed by the determination of radioactivity in the indicated organs after 24 hours using a γ counter. Percentage of total recovered radioactivity is shown (n = 12; mean ± SD; data pooled from 2 independent experiments; **P < .01). (C) In vitro generated Th1 cells (5 × 105) were injected intravenously together with 2.5 × 105 FACS-sorted and preactivated α_E⁺ Tregs from DO11.10 (WT) and DO11.10 × FucTVII^–/– mice. Twenty-four hours later the DTH response was induced. Shown is the progression of the inflammatory response monitored by the thickness of footpads injected with OVA/IFA (mean ± SD; n = 6). Two individual experiments are depicted. The α_E⁺ Tregs from FucTVII^–/– mice showed a significantly reduced suppressive capacity compared to α_E⁺ Tregs from WT mice (P < .01, repeated measure analyses).

Figure 3.

The α_E⁺ Tregs from WT and FucTVII^–/– mice show similar Foxp3 expression and in vitro suppressive capacity. (A) Intracellular expression of Foxp3 in α_E⁺ Tregs from DO11.10 (WT) and DO11.10 × FucTVII^–/– mice is shown. Representative histogram plots from 3 independently analyzed mice were selected. (B) In vitro suppressive capacity of α_E⁺ Treg subsets from DO11.10 and DO11.10 × FucTVII^–/– mice on naive T-cell proliferation was determined after 72 hours of coculture with CFSE-labeled responder cells at a 1:1 ratio. Culture of CFSE-labeled responder cells alone (medium) served as control. Representative histogram plots of CFSE profiles from CFSE⁺CD4⁺ T cells are shown. Quantification of suppressive capacity is based on the CFSE geometric mean of total CFSE⁺CD4⁺ T cells (n = 3; mean ± SD; 1 representative of 2 independent experiments).

Figure 4.

The α_E^–CD25⁺ Tregs efficiently prevent naive T-cell proliferation in vivo. (A) In vitro preactivated Treg subsets from DO11.10 mice were radioactively labeled with 111In and injected intravenously into BALB/c mice, in which 24 hours before a DTH response had been induced, followed by the determination of radioactivity in the control and the antigen-draining lymph node (dr LN) after 24 hours using a γ counter. Percentage of total recovered radioactivity is shown (n = 12; mean ± SD; data pooled from 2 independent experiments; **P < .01). (B-D) CFSE-labeled naive CD4⁺ T cells (5 × 105) derived from DO11.10 mice were adoptively transferred into BALB/c recipients, which 48 hours before had received 5 × 105 non-preactivated FACS-sorted Tregs followed by subcutaneous OVA immunization. Three days later the effect of the transferred Tregs on naive T-cell proliferation was assessed by measurement of footpad swelling and by FACS. (B) Measurement of footpad thickness shows the suppressive effect of indicated Treg subsets on the development of effector cells. Significance was determined by one-tailed unpaired Student t test (n = 3; mean ± SD; 1 representative of 2 independent experiments; *P < .05). (C) Representative FACS plots of gated CD4⁺ cells from antigen-draining popliteal lymph nodes show the effect of indicated Treg subsets on the proliferation of CFSE-labeled KJ1.26⁺ responder cells. Numbers indicate the frequency of CFSE⁺KJ1.26⁺ cells, which have undergone 3 or more cell divisions, and the frequency of CFSE⁺KJ1.26⁺ cells in the undivided fraction. Interestingly, during the 5-day in vivo period more than 95% of adoptively transferredα_E^–CD25⁺ Tregs (CFSE^–KJ1.26⁺) did not acquire α_E expression, indicating relatively stable phenotypes under these conditions. (D) The quantification of suppressive capacity is based on the CFSE geometric mean of total CFSE⁺CD4⁺KJ1.26⁺ T cells (n = 3; mean ± SD; 1 representative of 2 independent experiments; **P < .01).

Figure 5.

Compartmentalization of immune regulation by distinct migration behavior of α_E^–CD25⁺ and α_E⁺ Tregs. High expression levels of CD62L enable both α_E^–CD25⁺ Tregs and naive T cells to efficiently enter the antigen-draining lymph node from the bloodstream. Due to preferential recirculation of α_E^–CD25⁺ Tregs through lymph nodes these naive-like Tregs most efficiently inhibit activation and expansion of naive T cells. In contrast, both Th1 effector cells and α_E⁺ Tregs efficiently enter inflamed sites via binding of E/P-selectin ligands (E/P-Lig) to E/P-selectin being up-regulated on inflamed endothelial cells. Due to the preferential accumulation of inflammation-seeking α_E⁺ Tregs the Th1-mediated inflammatory reaction is most efficiently controlled by these effector/memory-like Tregs.