TCR revision generates functional CD4+ T cells

Abstract

CD4(+)Vβ5(+) peripheral T cells in C57BL/6 mice respond to encounter with a peripherally expressed endogenous superantigen by undergoing either deletion or TCR revision. In this latter process, cells lose surface Vβ5 expression and undergo RAG-dependent rearrangement of endogenous TCRβ genes, driving surface expression of novel TCRs. Although postrevision CD4(+)Vβ5(-)TCRβ(+) T cells accumulate with age in Vβ5 transgenic mice and bear a diverse TCR Vβ repertoire, it is unknown whether they respond to homeostatic and antigenic stimuli and thus may benefit the host. We demonstrate in this study that postrevision cells are functional. These cells have a high rate of steady-state homeostatic proliferation in situ, and they undergo extensive MHC class II-dependent lymphopenia-induced proliferation. Importantly, postrevision cells do not proliferate in response to the tolerizing superantigen, implicating TCR revision as a mechanism of tolerance induction and demonstrating that TCR-dependent activation of postrevision cells is not driven by the transgene-encoded receptor. Postrevision cells proliferate extensively to commensal bacterial Ags and can generate I-A(b)-restricted responses to Ag by producing IFN-γ following Listeria monocytogenes challenge. These data show that rescued postrevision T cells are responsive to homeostatic signals and recognize self- and foreign peptides in the context of self-MHC and are thus useful to the host.

Figure 1. Post-revision T cells show an elevated steady-state proliferation in situ

Vβ5 Tg and nonTg mice 25–30 weeks of age were given BrdU by i.p. injection, followed by a 5 day administration of BrdU in the drinking water. Spleen and MLN cells were isolated and stained for surface markers and BrdU incorporation. A. Vβ5 and CD44 analysis of CD4⁺ gated splenocytes, showing further gating of CD44^high Vβ5⁻ and Vβ5⁺ T cells from nonTg and Vβ5 Tg mice. B. Representative BrdU incorporation in CD4⁺CD44^high Vβ5⁻ and Vβ5⁺ splenocytes from nonTg and Vβ5 Tg mice. C. Charts indicate the percent BrdU⁺ for CD4⁺CD44^high gated populations from spleen and MLN of all nonTg (filled circles) and Vβ5 Tg (open circles) mice analyzed. Data are compiled from 3 independent experiments (Vβ5 Tg mice: N=4, nonTg mice: N=5). Bars represent the mean percent of BrdU⁺ cells of the CD4⁺CD44^high populations. P values were calculated using a two-tailed nonparametric Student’s t test. Significant p values are shown; n.s. indicates non-significant p values of >0.05.

Figure 2. MHC class II and other factors drive extensive LIP of post-revision CD4⁺Vβ5⁻ T cells

3×10⁶ CFSE-labeled CD4⁺ T cells enriched from pooled spleen and lymph nodes of aged Vβ5 Tg mice were transferred into the indicated congenic recipients that had been sublethally irradiated (650 rads) 1 day previously to induce lymphopenia. Donor CD4⁺ T cells were analyzed 8 days later for the extent of proliferation as measured by CFSE dilution. For CFSE histograms, numbers represent the percent of gated cells that are undivided (upper right), have undergone at least one division (upper left), or have divided more than 5 times (lower left). A. Analysis of B6 recipient spleen with gate indicating Ly5.1⁺ donor CD4⁺ T cells (top left) and Vβ5 expression of gated donor CD4⁺Ly5.1⁺ T cells (top right). CFSE analysis of donor CD4⁺Vβ5⁻ (bottom left) and donor CD4⁺Vβ5⁺ (bottom right) T cells. Data are representative of 2 independent experiments analyzing a total of 4 recipients. B. Analysis of donor CD4⁺Vβ5⁻ (left) and CD4⁺Vβ5⁺ (right) cells after transfer into CD4^−/− (MHC II⁺ lymphopenic control) and I-Aβ^−/− recipients. Data are representative of 3 independent experiments, and a total of 5–6 recipients of each genotype. The percent of donor Vβ5⁻ post-revision cells that had fully diluted CFSE was 36.1±3.8 in CD4^−/− recipients and 7.1±1.6 in I-Aβ^−/− recipients; p=0.0001 as determined using a two-tailed nonparametric Student’s t test.

Figure 3. Post-revision T cells undergo LIP when competing against TCR nonTg effector/memory phenotype cells

CD4⁺ T cells from 25–30-week-old Thy-1.2⁺ nonTg (Ly5.2⁺) and Vβ5 Tg (Ly5.1⁺) mice were enriched by negative selection, stained with CD62L, and sorted to isolate untouched CD62L⁻ CD4⁺CD44^high T cells. An equal number of sorted CD4⁺ Tg and nonTg cells were mixed, labeled with CFSE, and a total of 2×10⁶ CD4⁺ T cells adoptively transferred into sublethally irradiated Thy-1.1⁺Thy-1.2⁻ B6 recipient mice. Thy-1.2⁺ donor CD4⁺CD44^high splenocyte populations were analyzed for the extent of proliferation as measured by CFSE dilution. A. CD62L and CD44 analysis of pre- and post-sort CD4⁺ T cells from nonTg and Vβ5 Tg mice. B. CFSE analysis of donor CD44^high nonTg and Vβ5 Tg CD4⁺Vβ5⁻ (left) and CD4⁺Vβ5⁺ (right) cells 8 days after transfer into sublethally irradiated recipients. Numbers in CFSE histograms represent the percent of gated cells that are undivided (upper right), have undergone at least one division (upper left), or have divided more than 5 times (lower left). Data are representative of 2 independent experiments, for a total of 4 recipients. The percent of donor Vβ5⁻ cells that had undergone at least 1 cell division was 62.3±4.0 for nonTg cells and 72.1±4.2 for Vβ5⁻ post-revision cells; p=0.1391 as determined using a two-tailed nonparametric Student’s t test.

Figure 4. Post-revision T cells are tolerant to Mtv superantigen

Splenocytes from aged Ly5.1⁺ nonTg and Ly5.2⁺ Vβ5 Tg mice were mixed, CFSE labeled, and cultured for 3 days in the presence of either 3×10⁵ irradiated Mtv-overexpressing PN53HI tumor cells (A) or solubleαCD3 + αCD28 (B). 3 days later, cells were analyzed for proliferation as measured by CFSE dilution. Numbers in CFSE histograms represent the percent of gated cells that have undergone at least one division. Data are representative of 4 independent experiments.

Figure 5. Post-revision T cells undergo rapid proliferation to bacterial Ag following transfer into chronically lymphopenic recipients

2.6×10⁶ CFSE-labeled Ly5.1⁺ CD4⁺ T cells (comprised of 12% Vβ5⁻ post-revision cells and 88% Vβ5⁺ cells) enriched from pooled spleen and lymph nodes of aged Vβ5 Tg mice were transferred into chronically lymphopenic Rag^−/− recipient mice that were either untreated or treated (+ Abx) with a cocktail of antibiotics in their drinking water beginning 6 days prior to adoptive transfer. Donor CD4⁺ T cells were analyzed 6 days post-transfer for the extent of proliferation as measured by CFSE dilution and the percent recovery of each population. A. Analysis of live gated splenocytes and MLN cells with gate indicating Ly5.1⁺ donor CD4⁺ T cells. B. CD4 and Vβ5 analysis of donor Ly5.1⁺CD4⁺ gated cells from the MLN. C. CFSE analysis of donor CD4⁺Vβ5⁻ (left) and CD4⁺Vβ5⁺ (right) T cells from the MLN. Numbers within CFSE histograms represent the percent of gated cells that have fully diluted CFSE (left), or have undergone 0–4 cell divisions (right). D. Charts indicate the percent recovery of donor Vβ5⁻ and Vβ5⁺ CD4⁺ T cells in spleen and MLN.

Figure 6. Post-revision T cells include IFNγ-producing cells

Splenocytes from 25–34-week-old nonTg and Vβ5 Tg mice were stimulated with PMA + ionomycin (iono) for 6 hours or left untreated (unstim), followed by surface CD4 and Vβ5 and intracellular IFNγ staining. A. Representative Vβ5 and IFNγ analysis of CD4⁺ T cells. B. Chart indicates the percent of stimulated Vβ5⁻ and Vβ5⁺ CD4⁺ T cells from nonTg and Vβ5 Tg mice that produce IFNγ. Bars represent the mean percent of each indicated cell population with error bars indicating the SD (Vβ5 Tg: N=3; nonTg: N=3). No significant differences were obtained using a two-tailed nonparametric Student’s t test.

Figure 7. Post-revision Vβ5⁻ T cells produce IFNγ in a primary response to Listeria infection

Aged nonTg and Vβ5 Tg mice were infected with Listeria monocytogenes and 7 days later, CD8-depleted splenocytes were cultured for 5 hours with or without LLO_190–201 peptide, followed by surface staining for CD4, Vβ5, and TCRβ, and intracellular staining for IFNγ. A. Intracellular IFNγ staining of CD4⁺ gated splenocytes. B. Vβ5 expression by total CD4⁺ and Ag-specific CD4⁺IFNγ⁺ gated splenocytes. C. Surface panTCRβ expression by CD4⁺ gated and Ag-specific CD4⁺IFNγ⁺ gated splenocytes.