Naïve T cells re-distribute to the lungs of selectin ligand deficient mice

Abstract

Background: Selectin mediated tethering represents one of the earliest steps in T cell extravasation into lymph nodes via high endothelial venules and is dependent on the biosynthesis of sialyl Lewis X (sLe(x)) ligands by several glycosyltransferases, including two fucosyltransferases, fucosyltransferase-IV and -VII. Selectin mediated binding also plays a key role in T cell entry to inflamed organs.

Methodology/principal findings: To understand how loss of selectin ligands (sLe(x)) influences T cell migration to the lung, we examined fucosyltransferase-IV and -VII double knockout (FtDKO) mice. We discovered that FtDKO mice showed significant increases (approximately 5-fold) in numbers of naïve T cells in non-inflamed lung parenchyma with no evidence of induced bronchus-associated lymphoid tissue. In contrast, activated T cells were reduced in inflamed lungs of FtDKO mice following viral infection, consistent with the established role of selectin mediated T cell extravasation into inflamed lung. Adoptive transfer of T cells into FtDKO mice revealed impaired T cell entry to lymph nodes, but selective accumulation in non-lymphoid organs. Moreover, inhibition of T cell entry to the lymph nodes by blockade of L-selectin, or treatment of T cells with pertussis toxin to inhibit chemokine dependent G-coupled receptor signaling, also resulted in increased T cells in non-lymphoid organs. Conversely, inhibition of T cell egress from lymph nodes using FTY720 agonism of S1P1 impaired T cell migration into non-lymphoid organs.

Conclusions/significance: Taken together, our results suggest that impaired T cell entry into lymph nodes via high endothelial venules due to genetic deficiency of selectin ligands results in the selective re-distribution and accumulation of T cells in non-lymphoid organs, and correlates with their increased frequency in the blood. Re-distribution of T cells into organs could potentially play a role in the initiation of T cell mediated organ diseases.

Figure 1. Increased numbers of naïve T cells in lungs of FtDKO mice.

Lymphocytes were isolated from WT and FtDKO mice. A and B, Representative FACS analysis of organs from WT or FtDKO mice. Cells were stained with CD4, CD8 and CD44 mAbs. Numbers indicate percent of CD4, CD8, or CD44^Lo CD8 T cells. C, Enumeration of CD8 and CD4 T cells in indicated organs. D, Enumeration of CD44 ^Lo CD8 or CD44 ^Lo CD4 T cells in indicated organs. E, Photomicrographs of representative H & E staining of WT and FtDKO lungs at ×40 magnification. F. Photomicrographs of representative immuno-histology staining of T cells using Thy1 of WT and FtDKO lungs at ×200 magnification. Data was collected from n = 6 WT and n = 7 FtDKO in two independent experiments. For O-NALT, one experiment is shown where cells were pooled from n = 4 mice/group. The unpaired Student t test was used to compare groups. (*p<0.05, ** p<0.01, and *** p<0.001).

Figure 2. Ag-specific CD8 T cells are reduced in inflamed lung of FtDKO mice.

WT or FtDKO mice were infected with LCMV-Armstrong i.p and lymphocytes were isolated on day 8 or day 60 p.i. A, Representative FACS analysis of single cell suspensions from indicated organs on day 8 p.i. Cells were stained with CD8, CD44 and D_bNP396-404 tetramer. Gating on CD8 T cells, numbers in FACS panels indicate percent of Ag-specific CD8 T cells in organs. B, Representative FACS analysis of single cell suspensions from indicated organs on day 60 p.i. Cells were stained with CD8, CD44 and D_bGP33-41 tetramer. C, Enumeration of Ag-specific, CD44^high, and total CD8 T cells in WT or FtDKO mice at day 8. D, Enumeration of Ag-specific, CD44^high, and total CD8 T cells in WT or FtDKO mice at day 60. For day 8, n = 2 WT and n = 3 FtDKO. For day 60, n = 4 WT and n = 4 FtDKO. For O-NALT, organs were pooled from each group. (*p<0.05, ** p<0.01, and *** p<0.001).

Figure 3. T cells preferentially accumulate in non-lymphoid organs of FtDKO mice.

CD8 T cells were purified from naïve Thy1.1xP14 Tg mice (CFSE⁺) or immune (day 200 p.i.) Thy1.1xP14 Tg chimeric mice (CFSE^-), transferred into WT or FtDKO recipient mice, and recipient mice were sacrificed 18 hours later. Thy1.1 was used to differentiate endogenous from transferred T cell populations from indicated organs. A, Experimental design and phenotype of transferred cell populations. B, Enumeration of transferred CD8 T cell populations in indicated organs. C, Pie charts showing the distribution of Thy1.1 cells in WT and FtDKO recipient mice in spleen, PBMC, liver, lung, CLN, ILN, and MedLN. Of the indicated organs, ∼80% of transferred cells are located in spleen. The smaller pie chart shows the distribution of the remaining transferred cells. Data was collected from n = 5 WT and n = 6 FtDKO from two independent experiments. (*p<0.05, ** p<0.01, and *** p<0.001).

Figure 4. Inhibition of T cell trafficking to LN by pertussis toxin treatment results in selective re-distribution of naïve T cells to non-lymphoid organs.

Splenocytes from Thy1.1xP14 Tg mice were treated with either pertussis toxin (Ptx) or PBS control and transferred into WT recipient mice. 18 hours post transfer, T cells were isolated from indicated organs and analyzed by FACS. A, Experimental design. B, Representative FACS analysis of indicated organs gating on CD8 T cells. Numbers indicate percentage of transferred cells in the indicated organ. FACS panels from animals that received untreated cells, Ptx-treated cells, or received no transferred cells are shown. C, Enumeration of transferred cells in organs. Data was analyzed from n = 3 control untreated and n = 4 Ptx treated groups. (*p<0.05, ** p<0.01, and *** p<0.001)

Figure 5. Retention of naïve T cells in LN by FTY720 treatment depletes naïve T cell populations in non-lymphoid organs.

Splenocytes from naïve Thy1.1xP14 Tg mice were adoptively transferred into WT recipient. Recipient mice were treated for three days with FTY720 and lymphocytes in organs analyzed by FACS. A, Experimental design. B, Representative FACS analysis of indicated organs. Gating on CD8 T cells, numbers indicate percentage of transferred cells. C, Total lymphocyte numbers isolated from indicated organs. D, Transferred Thy1.1⁺ CD8⁺ T cells from indicated organs. Data from one representative experiment is shown, with n = 2 control and n = 3 FTY720 treated mice. (*p<0.05, ** p<0.01, and *** p<0.001).

Figure 6. Naïve T cell migration to non-lymphoid organs is influenced by T cell concentration in the blood.

Proportions of transferred naïve T cells in selectin ligand deficient mice (FtDKO), Gαi signal disruption (Ptx), CD62L blockade, and FTY720 treated mice are shown. A, Total number of transferred naïve CD8 T cells in CLN, MedLN, ILN, liver, and lung was calculated, and the percent of transferred naïve CD8 T cells in LNs versus non-lymphoid organs is shown for each treatment group. B, The relationship of naïve CD8 T cells in the blood compared to naïve CD8 T cells in organs (LN versus non-lymphoid organs). Concentration of naïve CD8 T cells in PBMCs was determined for each animal and the ratio of treatment versus control for each treatment group is shown (R = 1 for control, untreated group). Data is shown from 5 independent experiments for a total of n = 28 mice.