β1 integrin is critical for the maintenance of antigen-specific CD4 T cells in the bone marrow but not long-term immunological memory

Abstract

The long-term maintenance of memory CD4 T cells promotes protective immunity against future pathogen reinfection. As a site rich in survival cytokines, the bone marrow is proposed to be a critical niche for the survival of memory CD4 T cells. We demonstrate that endogenous, polyclonal Ag-specific CD4 T cells rapidly enter and are recovered long-term from the bone marrow following i.v. infection with Listeria monocytogenes. β(1) integrin-deficient CD4 T cells also populate the bone marrow early following an infection, but their numbers in this site rapidly decline. This decline was not caused by increased death of T cells lacking β(1) integrin but rather by reduced retention in the bone marrow after the primary immune response. The loss of memory CD4 T cells from the bone marrow does not lead to a loss of the predominant source of memory CD4 T cells in the spleen or the ability to mount a memory response. Thus, β(1) integrin-dependent maintenance of memory CD4 T cells in the bone marrow is not required for long-term CD4 T cell memory.

Figure 1. CD4 T cells rapidly enter the bone marrow following i.v bacterial infection

(A) Tracking the 2W1S-specific population following i.v. infection with A⁻Lm-2W1S in wt mice. Numbers indicate calculated cell number from gate (day 5 are averages, n=9). (B) Spleen and bone marrow histograms of β1 integrin expression on 2W1S-specific CD44^high CD4 T cells day 5 post-infection from wt and β1^−/− mice. (C) Spleen and bone marrow histograms of CCR7 expression on 2W1S-specific CD44^high CD4 T cells day 5 post-infection from wt and β1^−/− mice. Gray histogram represents naïve CD44^low CD4 T cells from the spleen. Numbers indicate percent of cells in the drawn gate (n=3, mean +/− s.d.).

Figure 2. Maintenance, but not entry, of CD4 T cells in the bone marrow is dependent on β1 integrin

2W1S-specific CD44^high CD4 T cells recovered from the bone marrow of wt, β1^−/−, and β1β7^−/− mice following A⁻Lm-2W1S infection. Data are from ten experiments (n=3–11, mean +/– s.e.m.). (ns) p > 0.05, *p < 0.05, **p=0.009 compared to wt, two-tailed unpaired t test.

Figure 3. Loss of CD4 T cells from the bone marrow is not related to increased cell death

(A) Number of 2W1S-specific CD44^high CD4 T cells recovered from the spleen and bone marrow 12 days post-infection with A⁻Lm-2W1S in wt and β1^−/− mice (mean). * p < 0.05, two-tailed unpaired t test. (B) Staining for active caspases 3 & 7 (FLICA 3&7) in 2W1S-specific CD44^low CD4 T cells from the spleen of uninfected mice or 2W1S-specific CD44^high CD4 T cells from the spleen and bone marrow 12 days post-infection. Bottom graphs show replicates (mean). (ns) p > 0.05, ** p<0.01, *** p<0.001, one-way ANOVA, followed by Tukey’s multiple comparison test.

Figure 4. Loss of CD4 T cells from the bone marrow is due to decreased ability to recirculate

(A) Bar graphs showing the percentage of transferred 2W1S-specific CD44^high CD4 T cells recovered from the bone marrow of host mice at 2 and 18 hours post-transfer. Percentage of input was calculated by dividing recovered cell number by transferred cell number (mean +/− s.e.m., n=4–6). (B) Representative dot plots of spleen and bone marrow of mice infected 5 days prior to co-transfer of day 5 post-infection wt and β1^−/− 2W1S-specific CD44^high CD4 T cells. Transferred cells are distinguished from the host population by labeling with CTG (wt) and CTO (β1^−/−). (C) Bar graphs showing the percentage of transferred CD44^high CD4 T cells recovered from the bone marrow of host mice at 2 and 18 hours post-transfer (n=4–5, mean +/− s.e.m.). * p=0.02, ** p=0.008, two-tailed unpaired t test. (D) Representative dot plots of spleen and bone marrow of host mice infected 20 days prior to co-transfer of day 20 post-infection wt and β1^−/− CD44^high CD4 T cells. Transferred cells are distinguished from the host population by labeling with CTO (wt) and CTG (β1^−/−). The 2W1S-specific host population (negative for CTO and CTG) has been excluded from all dot plots.

Figure 5. Expression of LFA-1 and functional PSGL-1 is decreased between day 5 and 20 post-infection

2W1S-specific CD44^high CD4 T cells from the spleen and bone marrow of wt and β1^−/− mice following i.v. infection with A⁻Lm-2W1S stained for (A) LFA-1 or (B) functional PSGL-1. Dark gray histograms represent staining of CD44^low CD4 T cells (naïve) from the spleen. Numbers indicate percent of cells in the drawn gate (n=3–4, mean +/− s.d.). (C) Median Fluorescence intensity (MFI) of LFA-1 staining on PSGL-1^hi 2W1S-specific CD44^highCD4 T cells from the spleen and bone marrow of wt and β1^−/− mice following i.v. infection with A⁻Lm-2W1S. (D) Percentage of LFA-1+ PSGL-1^hi 2W1S-specific CD44^high CD4 T cells from the spleen and bone marrow of wt and β1^−/− mice following i.v. infection with A⁻Lm-2W1S. Bar represents the mean. * p<0.02, ** p<0.0002, two-tailed unpaired t test.

Figure 6. Retention of CD4 T cells in the bone marrow is not required for survival or response of memory CD4 T cells

2W1S-specific CD44^high CD4 T cells recovered from the spleen (A) and bone marrow (B) of wt and β1^−/− mice following i.v. infection with A⁻Lm-2W1S. At day 325 post-infection, mice were re-challenged with 2W1S-LPS. Spleen and bone marrow were harvested three days following re-challenge (n=5, mean+/− s.e.m.). The primary (1°) response of naïve (uninfected) mice to 2W1S-LPS was also characterized three days following challenge. Horizontal dotted lines on graphs indicate the average number of 2W1S-specific CD44^low CD4 T cells in naïve mice. (ns) p > 0.05, two-tailed unpaired t test.