Rapid acquisition of tissue-specific homing phenotypes by CD4(+) T cells activated in cutaneous or mucosal lymphoid tissues

Abstract

Effector and memory T cells can be subdivided based on their ability to traffic through peripheral tissues such as inflamed skin and intestinal lamina propria, a property controlled by expression of 'tissue-specific' adhesion and chemoattractant receptors. However, little is known about the development of these selectively homing T cell subsets, and it is unclear whether activation in cutaneous versus intestinal lymphoid organs directly results in effector/memory T cells that differentially express adhesion and chemoattractant receptors targeting them to the corresponding nonlymphoid site. We define two murine CD4(+) effector/memory T cell subsets that preferentially localize in cutaneous or intestinal lymphoid organs by their reciprocal expression of the adhesion molecules P-selectin ligand (P-lig) and alpha 4 beta 7, respectively. We show that within 2 d of systemic immunization CD4(+) T cells activated in cutaneous lymph nodes upregulate P-lig, and downregulate alpha 4 beta 7, while those responding to antigen in intestinal lymph nodes selectively express high levels of alpha 4 beta 7 and acquire responsiveness to the intestinal chemokine thymus-expressed chemokine (TECK). Thus, during an immune response, local microenvironments within cutaneous and intestinal secondary lymphoid organs differentially direct T cell expression of these adhesion and chemoattractant receptors, targeting the resulting effector T cells to the inflamed skin or intestinal lamina propria.

Figure 1.

α4β7 and P-lig define three CD4⁺ memory T cell populations that differentially localize in intestinal and cutaneous lymphoid organs. (Left) CD4 and CD45RB expression by lymphocytes isolated from the cutaneous PLNs of a >1-y-old BALB/c mouse. The gates used to define ‘naive’ (CD4⁺CD45RB⁺) and ‘memory’ (CD4⁺CD45RB⁻) T cells are shown. (Right) α4β7 and P-lig expression by gated naive and memory CD4⁺ T cells isolated from the indicated lymphoid tissues of a >1-y-old BALB/c mouse.

Figure 2.

P-lig expression does not distinguish Th1, Th2, and Th0 cells analyzed directly ex vivo. (A, top) IFN-γ and IL-4 expression by gated CD4⁺ T cells determined by intracellular cytokine staining and flow cytometry after 4 h of PMA plus ionomycin stimulation of lymphocytes isolated from the indicated organs. (Bottom) Staining of stimulated lymphocytes from PLNs with isotype control antibodies. (B) Percentage of P-lig⁺ cells among gated Th1, Th2, and Th0 CD4⁺ T cells isolated from the indicated organs. Each data point represents a measurement taken from an individual animal. A total of three >1-y-old BALB/c mice were analyzed.

Figure 2.

P-lig expression does not distinguish Th1, Th2, and Th0 cells analyzed directly ex vivo. (A, top) IFN-γ and IL-4 expression by gated CD4⁺ T cells determined by intracellular cytokine staining and flow cytometry after 4 h of PMA plus ionomycin stimulation of lymphocytes isolated from the indicated organs. (Bottom) Staining of stimulated lymphocytes from PLNs with isotype control antibodies. (B) Percentage of P-lig⁺ cells among gated Th1, Th2, and Th0 CD4⁺ T cells isolated from the indicated organs. Each data point represents a measurement taken from an individual animal. A total of three >1-y-old BALB/c mice were analyzed.

Figure 3.

CD4⁺ T cells activated in PLNs and MLNs differentially upregulate P-lig and α4β7. (A) Percentage of OVA-specific KJ1–26⁺ cells among gated CD4⁺ T cells isolated from peripheral blood at the indicated times after intraperitoneal injection of OVA plus LPS. Data are mean and SD of values obtained from five mice at each time point. (B) Representative flow cytometry data of cellular CFSE content and KJ1–26 staining on gated CD4⁺ T cells isolated from the indicated tissues 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS. The horizontal marker in the histograms represents the CFSE fluorescence intensity of naive CD4⁺KJ1–26⁺ T cells isolated from animals immunized with LPS alone (data not shown). (C) Expression of α4β7 and P-lig by gated CD4⁺KJ1–26⁺ cells isolated from PLNs (• and ○) and MLNs (▪ and □) 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS (black symbols) or LPS alone (white symbols). Each data point represents a measurement from an individual animal. (D) Representative flow cytometry data of α4β7 and P-lig staining on gated CD4⁺KJ1–26⁺ cells isolated from PLNs (top) and MLN (bottom) 2 d after immunization of DO11.10 adoptive transfer recipients with OVA plus LPS (left) or LPS alone (right). The quadrant gate used to define the P-lig⁺ and α4β7^hi populations in C is indicated. (E) Mean fluorescence intensity (MFI) of α4β7 (left) and P-lig (right) staining on gated CD4⁺KJ1–26⁺ cells isolated from the MLNs (▪) or PLNs (•) 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS as a function of cell division (as determined by CFSE content). Data are mean and SE of values obtained from four (α4β7) or five (P-lig) mice. N represents the MFI of α4β7 or P-lig staining on naive cells isolated from animals immunized with LPS alone. Dotted lines represent background MFI of cells stained with an isotype control (left) or unstained cells (right).

Figure 3.

CD4⁺ T cells activated in PLNs and MLNs differentially upregulate P-lig and α4β7. (A) Percentage of OVA-specific KJ1–26⁺ cells among gated CD4⁺ T cells isolated from peripheral blood at the indicated times after intraperitoneal injection of OVA plus LPS. Data are mean and SD of values obtained from five mice at each time point. (B) Representative flow cytometry data of cellular CFSE content and KJ1–26 staining on gated CD4⁺ T cells isolated from the indicated tissues 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS. The horizontal marker in the histograms represents the CFSE fluorescence intensity of naive CD4⁺KJ1–26⁺ T cells isolated from animals immunized with LPS alone (data not shown). (C) Expression of α4β7 and P-lig by gated CD4⁺KJ1–26⁺ cells isolated from PLNs (• and ○) and MLNs (▪ and □) 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS (black symbols) or LPS alone (white symbols). Each data point represents a measurement from an individual animal. (D) Representative flow cytometry data of α4β7 and P-lig staining on gated CD4⁺KJ1–26⁺ cells isolated from PLNs (top) and MLN (bottom) 2 d after immunization of DO11.10 adoptive transfer recipients with OVA plus LPS (left) or LPS alone (right). The quadrant gate used to define the P-lig⁺ and α4β7^hi populations in C is indicated. (E) Mean fluorescence intensity (MFI) of α4β7 (left) and P-lig (right) staining on gated CD4⁺KJ1–26⁺ cells isolated from the MLNs (▪) or PLNs (•) 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS as a function of cell division (as determined by CFSE content). Data are mean and SE of values obtained from four (α4β7) or five (P-lig) mice. N represents the MFI of α4β7 or P-lig staining on naive cells isolated from animals immunized with LPS alone. Dotted lines represent background MFI of cells stained with an isotype control (left) or unstained cells (right).

Figure 3.

CD4⁺ T cells activated in PLNs and MLNs differentially upregulate P-lig and α4β7. (A) Percentage of OVA-specific KJ1–26⁺ cells among gated CD4⁺ T cells isolated from peripheral blood at the indicated times after intraperitoneal injection of OVA plus LPS. Data are mean and SD of values obtained from five mice at each time point. (B) Representative flow cytometry data of cellular CFSE content and KJ1–26 staining on gated CD4⁺ T cells isolated from the indicated tissues 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS. The horizontal marker in the histograms represents the CFSE fluorescence intensity of naive CD4⁺KJ1–26⁺ T cells isolated from animals immunized with LPS alone (data not shown). (C) Expression of α4β7 and P-lig by gated CD4⁺KJ1–26⁺ cells isolated from PLNs (• and ○) and MLNs (▪ and □) 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS (black symbols) or LPS alone (white symbols). Each data point represents a measurement from an individual animal. (D) Representative flow cytometry data of α4β7 and P-lig staining on gated CD4⁺KJ1–26⁺ cells isolated from PLNs (top) and MLN (bottom) 2 d after immunization of DO11.10 adoptive transfer recipients with OVA plus LPS (left) or LPS alone (right). The quadrant gate used to define the P-lig⁺ and α4β7^hi populations in C is indicated. (E) Mean fluorescence intensity (MFI) of α4β7 (left) and P-lig (right) staining on gated CD4⁺KJ1–26⁺ cells isolated from the MLNs (▪) or PLNs (•) 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS as a function of cell division (as determined by CFSE content). Data are mean and SE of values obtained from four (α4β7) or five (P-lig) mice. N represents the MFI of α4β7 or P-lig staining on naive cells isolated from animals immunized with LPS alone. Dotted lines represent background MFI of cells stained with an isotype control (left) or unstained cells (right).

Figure 3.

CD4⁺ T cells activated in PLNs and MLNs differentially upregulate P-lig and α4β7. (A) Percentage of OVA-specific KJ1–26⁺ cells among gated CD4⁺ T cells isolated from peripheral blood at the indicated times after intraperitoneal injection of OVA plus LPS. Data are mean and SD of values obtained from five mice at each time point. (B) Representative flow cytometry data of cellular CFSE content and KJ1–26 staining on gated CD4⁺ T cells isolated from the indicated tissues 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS. The horizontal marker in the histograms represents the CFSE fluorescence intensity of naive CD4⁺KJ1–26⁺ T cells isolated from animals immunized with LPS alone (data not shown). (C) Expression of α4β7 and P-lig by gated CD4⁺KJ1–26⁺ cells isolated from PLNs (• and ○) and MLNs (▪ and □) 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS (black symbols) or LPS alone (white symbols). Each data point represents a measurement from an individual animal. (D) Representative flow cytometry data of α4β7 and P-lig staining on gated CD4⁺KJ1–26⁺ cells isolated from PLNs (top) and MLN (bottom) 2 d after immunization of DO11.10 adoptive transfer recipients with OVA plus LPS (left) or LPS alone (right). The quadrant gate used to define the P-lig⁺ and α4β7^hi populations in C is indicated. (E) Mean fluorescence intensity (MFI) of α4β7 (left) and P-lig (right) staining on gated CD4⁺KJ1–26⁺ cells isolated from the MLNs (▪) or PLNs (•) 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS as a function of cell division (as determined by CFSE content). Data are mean and SE of values obtained from four (α4β7) or five (P-lig) mice. N represents the MFI of α4β7 or P-lig staining on naive cells isolated from animals immunized with LPS alone. Dotted lines represent background MFI of cells stained with an isotype control (left) or unstained cells (right).

Figure 3.

CD4⁺ T cells activated in PLNs and MLNs differentially upregulate P-lig and α4β7. (A) Percentage of OVA-specific KJ1–26⁺ cells among gated CD4⁺ T cells isolated from peripheral blood at the indicated times after intraperitoneal injection of OVA plus LPS. Data are mean and SD of values obtained from five mice at each time point. (B) Representative flow cytometry data of cellular CFSE content and KJ1–26 staining on gated CD4⁺ T cells isolated from the indicated tissues 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS. The horizontal marker in the histograms represents the CFSE fluorescence intensity of naive CD4⁺KJ1–26⁺ T cells isolated from animals immunized with LPS alone (data not shown). (C) Expression of α4β7 and P-lig by gated CD4⁺KJ1–26⁺ cells isolated from PLNs (• and ○) and MLNs (▪ and □) 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS (black symbols) or LPS alone (white symbols). Each data point represents a measurement from an individual animal. (D) Representative flow cytometry data of α4β7 and P-lig staining on gated CD4⁺KJ1–26⁺ cells isolated from PLNs (top) and MLN (bottom) 2 d after immunization of DO11.10 adoptive transfer recipients with OVA plus LPS (left) or LPS alone (right). The quadrant gate used to define the P-lig⁺ and α4β7^hi populations in C is indicated. (E) Mean fluorescence intensity (MFI) of α4β7 (left) and P-lig (right) staining on gated CD4⁺KJ1–26⁺ cells isolated from the MLNs (▪) or PLNs (•) 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS as a function of cell division (as determined by CFSE content). Data are mean and SE of values obtained from four (α4β7) or five (P-lig) mice. N represents the MFI of α4β7 or P-lig staining on naive cells isolated from animals immunized with LPS alone. Dotted lines represent background MFI of cells stained with an isotype control (left) or unstained cells (right).

Figure 4.

CD4⁺ T cells activated in PLNs or MLNs differentially respond to the intestinal chemokine TECK. Migration of CD4⁺KJ1–26⁺ cells isolated from the MLNs or PLNs 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS to medium alone (white bars) or to the indicated chemokines (gray bars). Data are mean and SD of multiple measurements taken in one experiment, which is representative of 4.