Low-affinity ligands for the TCR drive proliferation of mature CD8+ T cells in lymphopenic hosts

Abstract

In the absence of thymic emigration, the peripheral T cell pool is maintained by division of mature lymphocytes. We have examined the molecular interactions required for peripheral CD8+ T cell expansion in lymphopenic mice without conventional antigenic stimulation. Expansion of CD8+ T cells in lymphopenic hosts was found to be peptide specific. An antagonist peptide known to serve as a ligand for positive selection of these T cells promoted expansion; however, a control peptide that binds the same class I molecule did not. Surprisingly, the cells undergoing proliferation in lymphopenic hosts did not mature to cytotoxic effectors and displayed a partially activated surface phenotype. These data suggest that division of T cells in the periphery of lymphopenic hosts requires specific recognition of self-peptide/MHC complexes, similar to the signal for thymocyte maturation.

Figure 1. Proliferation of Donor Cells in Lymphopenic Hosts Is MHC Class I Dependent

CD8⁺ T cells were enriched from pooled spleen and lymph node of OT-1 or OT-1 Thy1.1⁺ mice, labeled with CFSE, and 2 × 10⁶ CD8⁺Vα2⁺ cells were injected per recipient. Splenocytes from hosts were analyzed 5 days after transfer for detection of CFSE⁺ donor OT-1 cells by staining with anti-CD8 and anti-Vα2. (A) CFSE expression is shown for OT-1 CD8⁺Vα2⁺ cells transferred into unirradiated B6, irradiated B6, or irradiated TAP° hosts. Similar results were observed with OT-1 RAG° donor cells and on days 3 and 6 post transfer in irradiated hosts. (B) CFSE expression is shown for OT-1 Thy1.1⁺ CD8⁺Vα2⁺ cells transferred into untreated B6, Thy1.2-depleted B6, or Thy1.2-depleted TAP° hosts.

Figure 2. Expression of ss-Peptide Transgenes

(A) Expression of H-2K^b is not upregulated in TAP° mice expressing ss-peptide transgenes. Expression levels of the MHC class I molecule K^b were compared on splenocytes from ss-OVApTAP° (thin black line), ss-R4TAP° (dashed line), ss-VSVTAP° (dotted line), TAP° (bold gray line), and B6 (bold black line) mice. (B) ss-peptide transgenic APC stimulate proliferation of peptide-specific CTL. Filled symbols represent ss-peptide transgene positive and open symbols represent control transgene negative APC. R4-specific CTL were incubated with ss-R4TAP° (filled triangles) and control TAP° (open triangles) splenocytes (left panel). VSV-specific CTL were incubated with ss-VSV (filled circles) and control B6 (open circles) splenocytes (right panel). Peptide-specific CTL (5 × 10⁴) were cultured with increasing numbers of irradiated APC for 72 hr and were pulsed with [³H]thymidine for 18 hr.

Figure 3. Proliferation of OT-1 CD8⁺ T Cells in Irradiated TAP° Hosts Is Restored by Expression of an Antagonist Peptide

Donor cells were pooled spleen and lymph node from OT-1 (A) or OT-1 RAG° mice (B). CD8⁺Vα2⁺ CFSE-labeled cells (2 × 10⁶) were injected per indicated host. CFSE expression is shown for CD8⁺Vα2⁺ cells from spleen on day 5 (A) or day 6 (B) post transfer. Data are representative of at least five experiments.

Figure 4. Exogenous R4 and ss-R4 Transgene-Expressing Cells Do Not Stimulate Proliferation or CTL Targeting by OT-1 CD8⁺ T cells

(A) OT-1 splenocytes were cultured with OVAp (circles), R4 (diamonds), or VSV (squares) peptides and irradiated B6 splenocytes and were pulsed with [³H]thymidine for 6 hr after 36 hr of culture. (B) OT-1 CTL were used at 10:1 effector to target ratio with OVAp (circles), R4 (diamonds), or VSV (squares) peptide coated and EL4 target cells. (C) OT-1 splenocytes were cultured with increasing numbers of irradiated splenocytes from the following mice: ss-OVATAP° (circles), B6 (triangles), ss-R4TAP° line 1 (open diamonds), ss-R4TAP° line 2 (filled diamonds), and ss-VSVTAP° (squares). The assay was pulsed with [³H]thymidine for 18 hr after 36 hr of culture. (D) OT-1 CTL were used at the indicated effector to target ratio with ⁵¹Cr-labeled Con A blasts as targets made from the following mice: ss-OVATAP° (circles), TAP° (triangles), ss-R4TAP° line 1 (open diamonds), and ss-VSVTAP° (squares).

Figure 5. Surface Phenotype of CD8⁺ T Cells Proliferating in Various Hosts

CD8⁺Vα2⁺ cells (2 × 10⁶) from OT-1 RAG° mice were CFSE labeled, injected into irradiated hosts, and splenocytes were analyzed 6 days post transfer. (A) This panel shows Vα2 versus CFSE levels for CD8⁺ cells transferred into irradiated TAP°, B6, ss-R4TAP°, and ss-OVApTAP° hosts. (B) This panel compares levels of activation markers on the transferred OT-1 CD8⁺ T cells. Donor cells were gated on CD8⁺Vα2⁺ from TAP° (bold gray line), B6 (thin black line), ss-R4TAP° (dotted line), and ss-OVApTAP° (bold black line). Similar results were obtained 4 and 7 days post transfer.

Figure 6. OT-1 Cells Proliferating in Irradiated B6 and ss-R4TAP° Mice Do Not Become Cytotoxic Effector Cells

CD8⁺Vα2⁺ cells (2 × 10⁶) from OT-1 RAG° mice were CFSE labeled and injected into irradiated B6 (triangles), ss-R4TAP° (diamonds), or ss-OVApTAP° (circles) hosts. Spleen cells were harvested on day 4 and assayed for lysis of OVAp-coated EL4 targets. Flow cytometry revealed that transferred OT-1 Vα2⁺CD8⁺ cells represented 15%, 6%, and 75% of splenocytes recovered from B6, ss-R4TAP°, or ss-OVApTAP° hosts, respectively (data not shown). The E:T was based on the number of CD8⁺Vα2⁺ cells used as effectors. The wells were normalized for total cell number by diluting cells recovered from B6 (triangles) and ss-OVApTAP° hosts (filled circles) with fresh B6 splenocytes to 6% CD8⁺Vα2⁺ cells, or cells recovered from ss-OVApTAP° hosts were used undiluted (open circles). For all effectors at all E:T ratios, lysis of EL4 targets in the absence of peptide was less than 3% (data not shown).