The role of beta7 integrins in CD8 T cell trafficking during an antiviral immune response

Abstract

The requirement of beta7 integrins for lymphocyte migration was examined during an ongoing immune response in vivo. Transgenic mice (OT-I) expressing an ovalbumin-specific major histocompatibility complex class I-restricted T cell receptor for antigen were rendered deficient in expression of all beta7 integrins or only the alphaEbeta7 integrin. To quantitate the relative use of beta7 integrins in migration in vivo, equal numbers of OT-I and OT-I-beta7(-/-) or OT-I-alphaE-/- lymph node (LN) cells were adoptively transferred to normal mice. Although OT-I-beta7(-/-) LN cells migrated to mesenteric LN and peripheral LN as well as wild-type cells, beta7 integrins were required for naive CD8 T cell and B cell migration to Peyer's patch. After infection with a recombinant virus (vesicular stomatitis virus) encoding ovalbumin, beta7 integrins became critical for migration of activated CD8 T cells to the mesenteric LN and Peyer's patch. Naive CD8 T cells did not enter the lamina propria or the intestinal epithelium, and the majority of migration of activated CD8 T cells to the small and large intestinal mucosa, including the epithelium, was beta7 integrin-mediated. The alphaEbeta7 integrin appeared to play no role in migration during a primary CD8 T cell immune response in vivo. Furthermore, despite dramatic upregulation of alphaEbeta7 by CD8 T cells after entry into the epithelium, long-term retention of intestinal intraepithelial lymphocytes was also alphaEbeta7 independent.

Figure 1

Analysis of β7 integrin expression in OT-I T cells lacking either β7 or αE chains. Lymphocytes from MLN of OT-I, OT-I-αE^−/−, or OT-I-β7^−/− mice were examined for expression of Vα2 and Vβ5 or for CD8 and β7 by fluorescence flow cytometry. Similar staining was observed with PLN cells (data not shown). Top panel, analysis of Vα2 and Vβ5 expression of gated CD8⁺ LN cells from OT-I mice. Similar results were obtained from analysis of CD8⁺ cells from all three mouse strains. MFC, mean fluorescence channel.

Figure 2

Tracking in secondary lymphoid organs of cotransferred OT-I and OT-I-β7^−/− cells before and after virus infection. Equal numbers of LN cells from OT-I (Ly5.1⁺5.2⁺) and OT-I-β7^−/−(Ly5.1⁺5.2⁻) mice were transferred to normal C57BL/6-Ly5.2 mice. 48 h later, mice were infected with VSV-ova (+VSV) or not (for naive LN, MLN cells are shown; PLN cells were similar). 3 d later, MLN and PLN cells were isolated and analyzed for the presence of transferred cells by flow cytometric detection of Ly5.1 and Ly5.2 expression.

Figure 3

β7 integrins are required for trafficking of naive lymphocytes to PP. Equal numbers of LN cells (∼60% CD8 T cells and 40% B cells) from OT-I (Ly5.1⁻5.2⁺) and OT-I-β7^−/− (Ly5.1⁺5.2⁻) mice were transferred to normal C57BL/6-Ly5.1⁺5.2⁺ mice. 2 d later, cells from PP were isolated and analyzed for presence of donor populations by detection of CD8, Ly5.1, and Ly5.2 by flow cytometry. Cells were positively gated for CD8⁺ cell analysis and negatively gated (CD8⁻) for B cell analysis. Separate analysis revealed that the CD8⁻ donor cells were >95% B cells (data not shown).

Figure 4

Migration of activated CD8 T cells to intestinal mucosa is dependent on β7 integrins. Equal numbers of LN cells from OT-I (Ly5.1⁺5.2⁺) and OT-I-β7^−/− (Ly5.1⁺5.2⁻) mice were transferred to normal C57BL/6-Ly5.1⁻5.2⁺ mice. 48 h later, mice were infected with VSV-ova or not. 3 d after infection, mice were killed and lymphocyte populations were isolated and examined for the presence of donor populations by detection of Ly5.1/Ly5.2 expression by fluorescence flow cytometry.

Figure 5

Upregulation of β7 integrins on antigen-specific CD8 T cells after immunization with VSV-ova. Equal numbers of LN cells from OT-I (Ly5.1⁺5.2⁺) and OT-I-β7^−/− (Ly5.1⁺5.2⁻) mice were transferred to normal C57BL/6-Ly5.1⁻5.2⁺ mice. 2 d after transfer, mice were infected with VSV-ova. 3 d later, lymphocytes from the indicated tissues were analyzed for expression of CD8, Ly5.1, Ly5.2, and β7 integrin by four-color flow cytometry. Analysis shown is of donor CD8⁺ cells. Open histogram, OT-I-β7^−/− cells; filled histograms, OT-I cells. Top, naive MLN cells; naive PLN cells had a similar expression pattern.

Figure 6

Analysis of migration in LNs of CD8 T cells lacking αEβ7. Equal numbers of LN cells from OT-I (Ly5.1⁺5.2⁺) and OT-I-αE^−/− (Ly5.1⁺5.2⁻) mice were transferred to normal C57BL/6-Ly5.1⁻5.2⁺ mice. 48 h later, mice were infected with VSV-ova (+VSV) or not (naive LN). 3 d later, MLN and PLN cells were isolated and analyzed for the presence of transferred cells by flow cytometric detection of Ly5.1 and Ly5.2 expression.

Figure 7

αEβ7 integrin is not required for entry of activated CD8 T cells into the intestinal mucosa. Equal numbers of LN cells from OT-I (Ly5.1⁺5.2⁺) and OT-I-αE^−/− (Ly5.1⁺5.2⁻) mice were transferred to normal C57BL/6-Ly5.1⁻5.2⁺ mice. 48 h later, mice were infected with VSV-ova or not (naive). 3 d later, IEL and LP cells were isolated and analyzed for the presence of transferred cells by flow cytometric detection of  Ly5.1 and Ly5.2 expression.

Figure 8

Long-term retention of IEL is not dependent on αEβ7 integrin. Equal numbers of LN cells from OT-I (Ly5.1⁻5.2⁺) and OT-I-αE^−/− (Ly5.1⁺5.2⁺) mice were transferred to normal C57BL/6-Ly5.1⁺5.2⁻ mice. 48 h later, mice were infected with VSV-ova or not (naive). 3 wk later, IELs were isolated and analyzed for expression of CD8, Ly5.1, Ly5.2, and β7 integrin by four-color flow cytometry. Top panel, β7 integrin expression of CD8⁺OT-I-αE^−/− IEL (open histogram) and CD8⁺OT-I-αE^+/+ IEL (filled histogram).