Both exogenous commensal and endogenous self antigens stimulate T cell proliferation under lymphopenic conditions

Abstract

Within lymphopenic recipients, naïve T cells undergo proliferation that is induced by homeostatic mechanisms. Earlier studies have demonstrated that commensal antigens play a key role in inducing the proliferation. However, a relative contribution of endogenous self antigens in this process has not been formally investigated. In this study, we utilized a pharmacologic inhibitor that blocks T cell egress from the lymphoid tissues, antibiotics, and germ-free animals to examine the role of commensal and self antigens. The results suggest that T cell proliferation under lymphopenic conditions is a heterogeneous process triggered by both exogenous commensal and endogenous self antigens.

Figure 1. The effect of FTY720 treatment on the proliferation of adoptively transferred naive CD4 and CD8 T cells

FACS sorted naive Ly5.1 T cells were transferred into TCRβ−/− mice. Mice were treated with FTY720 (0.5 mg/kg) or water for control group every day. (A) Total donor cell recovery, (B) CFSE profiles, and (C) BrdU incorporation of the transferred T cells were analyzed 7 days post transfer. Mice were injected with 1mg BrdU i.p 24 hr prior to sacrifice. Shown are the mean ± SD of individually tested mice (n =3). Each symbol shown in (A) represents individually tested recipients. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 2. Treatment of FTY 720 does not alter the TCR repertoire of adoptively transferred naive CD4 and CD8 T cells

Sorted naive Ly5.1 T cells were transferred into TCRβ−/− recipients that were treated with FTY720 or water every day. TCR repertoire was examined 7 days post transfer. (A) The distribution of each Vβ expressing T cells was compared between different lymphoid tissues. (B) The distribution of each Vβ expressing T cells in mLN tissue was compared between FTY720- and water-treated recipients. Data shown are the mean ± SD of individually tested mice (n=3~4).

Figure 3. The effects of commensal microflora on the proliferation of adoptively transferred naïve CD4 and CD8 T cells

CFSE labeled Thy1.1 naïve T cells were transferred TCRβ−/− mice as described above. (A) Donor T cells expansion, (B) the absolute numbers, and (C) CFSE profiles were analyzed 7 days post transfer. Recipient mice were treated with water or antibiotic water containing ampicillin, neomycin sulfate, vancomycine and metronidazole in drinking water for 4–5 weeks prior to T cell transfer. Each symbol shown in (B) represents individually tested recipients. Shown are the mean ± SD of individually tested mice (n =3). *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 4. The effects of both FTY720 and antibiotic treatment on the proliferation of adoptively transferred naïve T cells

CFSE labeled Thy1.1 naive T cells were transferred TCRβ−/− mice. CFSE profiles (A) and the total donor cell recovery (B) were examined 7 days post transfer. Data shown are the mean ± SD of individually tested mice (n=3~4). *, p<0.05.

Figure 5. The effect of splenectomy and lymphadenectomy on the proliferation of adoptively transferred naïve T cells

Sorted naive Ly5.1 T cells were transferred into TCRβ−/− recipients that were either spenectomized (Spx) or mesenteric lymphadenectomized (Lnx). (A) Donor cells expansion and (B) the total donor cell recovery were examined 7 days post transfer. Results are representative of two independent experiments. Each symbol shown in (B) represents individually tested recipients. **, p<0.01; ***, p<0.001. (C) CFSE profiles of donor CD4 T cells from TCRβ−/− recipients with Spx or Lnx are shown. Proliferation of donor CD8 T cells was similar to that of CD4 T cells (data not shown).

Figure 6. T cell expansion and differentiation under GF conditions

(A) FACS sorted Thy1.1 CD4 T cells were transferred into CTX-treated SPF or GF recipients as described in the Materials and Methods. The recipients were sacrificed 2 weeks post transfer, and the total donor T cell number was calculated from the spleen. Each symbol represents individually tested recipient. (B) CD44^high memory phenotype CD4 T cells were isolated from 7-day old SPF or GF neonates. TCR repertoire distribution was determined using the immunoscope analysis as described in the Materials and Methods. ns, not significant. TCR repertoires shown are representative of three independent experiments.