Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation

Abstract

In a depleted lymphoid compartment, naive T cells begin a slow proliferation that is independent of cognate antigen yet requires recognition of major histocompatibility complex-bound self-peptides. We have followed the phenotypic and functional changes that occur when naive CD8(+) T cells undergo this type of expansion in a lymphopenic environment. Naive T cells undergoing homeostasis-driven proliferation convert to a phenotypic and functional state similar to that of memory T cells, yet distinct from antigen-activated effector T cells. Naive T cells dividing in a lymphopenic host upregulate CD44, CD122 (interleukin 2 receptor beta) and Ly6C expression, acquire the ability to rapidly secrete interferon gamma, and become cytotoxic effectors when stimulated with cognate antigen. The conversion of naive T cells to cells masquerading as memory cells in response to a homeostatic signal does not represent an irreversible differentiation. Once the cellularity of the lymphoid compartment is restored and the T cells cease their division, they regain the functional and phenotypic characteristics of naive T cells. Thus, homeostasis-driven proliferation provides a thymus-independent mechanism for restoration of the naive compartment after a loss of T cells.

Figure 1

Naive T cells undergoing homeostasis-driven proliferation acquire a memory phenotype. Expression of CD44, Ly6C, and CD122 on OT-I RAG^−/− T cells transferred 17 d earlier into an irradiated B6 host compared with naive, memory, and effector OT-I RAG^−/− CD8⁺ T cells. Histogram plots show expression of the indicated marker for Vα2⁺Vβ5⁺CD8⁺ gated cells. Before transfer, OT-I CD8⁺ cells were depleted of CD44^hi and Ly6C^hi cells. Memory cells were recovered from irradiated B6 hosts that had received 3 × 10⁶ OT-I RAG^−/− cells, followed by infection with vaccinia virus encoding OVA >90 d earlier. OT-I effectors were analyzed on day 5 after in vitro stimulation with OVAp-coated, irradiated splenocyte feeder cells.

Figure 2

Kinetics of homeostasis-driven proliferation. Irradiated B6 hosts received 3 × 10⁶ OT-I RAG^−/− T cells. (A) Total numbers of OT-I T cells were estimated by determining the number of Vα2⁺Vβ5⁺CD8⁺ T cells recovered from the pooled spleen and lymph nodes (four mice per time point). At day 28 of transfer, 25%⁺/−3.4 of recovered cells are OT-I, 6.8%⁺/−0.5 are host-derived CD8⁺ T cells, and 21%⁺/−1.1 are host-derived CD4⁺ T cells. (B) The level of proliferation by transferred OT-I cells was determined at each time point by BrdU incorporation. Mice were given BrdU for 7 d before each time point (two mice per time point). The percentage of Vα2⁺CD8⁺ cells that had incorporated BrdU is shown. The insets show BrdU staining for Vα2⁺CD8⁺ gated cells 12 and 49 d after transfer.

Figure 3

OT-I T cells revert to a naive phenotype after homeostasis-driven proliferation. Expression levels of CD44, Ly6C, and CD122 for Vα2⁺Vβ5⁺CD8⁺ gated naive OT-I T cells analyzed on the same day (▴) or OT-I RAG^−/− cells recovered from two different irradiated B6 hosts (circles) at the indicated time points are graphed. (A) Percentage of CD44^hi of Vα2⁺Vβ5⁺CD8⁺ gated T cells. Histogram insets show CD44 expression for transferred OT-I cells at days 20, 49, and 120 (filled histogram) overlayed with naive OT-I cells (open histogram). (B) Percentage of Ly6C⁺ of Vα2⁺Vβ5⁺CD8⁺ gated T cells. Histogram insets show Ly6C expression for transferred OT-I cells at days 20, 45, and 120 (filled histogram) overlayed with naive OT-I T cells (open histogram). (C) Expression of CD122 on naive OT-I cells (open histogram) and OT-I cells 20 and 68 d after transfer (filled histogram).

Figure 4

OT-I cells undergoing homeostasis-driven proliferation transiently acquire ex vivo CTL activity. Percentage of specific lysis of OVAp-pulsed syngeneic targets was determined in a 6-h CTL assay. Lysis of target cells in the absence of OVAp was <6%. (A) Cytotoxic activity is shown for in vitro–stimulated OT-I effectors, in vivo–derived memory cells, and naive OT-I cells. (B) Cytotoxic activity is shown for OT-I cells recovered from spleen and lymph nodes of irradiated B6 hosts at the indicated time points after transfer. The E/T ratios were normalized for the number of OT-I (Vα2⁺Vβ5⁺CD8⁺) cells in all samples. Fresh, naive OT-I RAG^−/− control cells and OT-I effector cells were run at every time point. Naive cells never gave lysis >10% at the highest E/T, and effector cells always gave lysis >60% at 11:1. CTL activity of memory OT-I cells was assayed on four occasions and was always intermediate between naive and effector activity.

Figure 5

OT-I cells undergoing homeostasis-driven proliferation rapidly make IFN-γ at early time points. Pooled spleen and lymph node cells were incubated with (bold lines) or without (dotted lines) OVAp for 8 h, and were stained with anti–IFN-γ. Histogram overlays are shown for CD8⁺ gated cells, as the cells cannot be stained for either Vα2 or Vβ5 expression because the antigen pulse induces TCR downregulation. (A) IFN-γ levels for naive, memory, and effector OT-I cells. The inset shows tetramer staining, K^b-OVAp (bold line), or a control K^b-peptide (dotted line), of CD8⁺ gated memory cells before peptide stimulation. (B) IFN-γ staining for CD8⁺ gated cells recovered from irradiated B6 hosts at the indicated time points.

Figure 6

Proliferation of OT-I cells in RAG^−/− recipients. (A) BrdU incorporation is shown for Vα2⁺CD8⁺ gated cells recovered from pooled spleen and lymph nodes of RAG^−/− hosts given BrdU for 7 d before the indicated time point. (B) Phenotype of Vα2⁺Vβ5⁺CD8⁺ gated T cells from pooled spleen and lymph nodes of naive OT-I RAG^−/− mice or OT-I RAG^−/− cells recovered from RAG^−/− hosts at the indicated time points. (C) IFN-γ production by CD8⁺ cells recovered from RAG^−/− hosts at the indicated time points after 8 h of stimulation with OVAp (bold lines) or media alone (dotted lines).