T cell receptor contact to restricting MHC molecules is a prerequisite for peripheral interclonal T cell competition

Abstract

T cell survival and homeostatic proliferation in the periphery requires T cell receptor (TCR) binding to restricting major histocompatability complex (MHC)-encoded molecules, as well as the availability of certain lymphokines. However, the exact mechanisms by which these signals interrelate and contribute to homeostasis are not understood. By performing T cell transfers into TCR transgenic hosts we detected a hierarchical order of homeostatic proliferation for T cells differing in MHC restriction, such that OT1 cells (K(b) restricted) proliferated in P14 (D(b)-restricted TCR) recipients, but not vice versa. Using K(b) mutant mice, we demonstrated that proliferation of OT1 cells in P14 recipients, as well as the ability of host OT1 cells to hinder the proliferation of donor P14 cells, were dependent on OT1-TCR binding to K(b) molecules. However, interclonal T cell competition was not mediated simply by competition for physical access to the MHC-bearing cell. This was shown in parabiotic pairs of OT1 and K(b) mutant mice in which P14 cells failed to proliferate, even though the OT1 cells could not interact with half of the APCs in the system. Thus, we conclude that the interaction between the TCR and restricting MHC molecule influences the ability to compete for trophic resources not bound to the stimulating APC. This mechanism allows a local competitiveness that extends beyond a T cell's specificity.

Figure 1.

Hierarchical competitiveness in homeostatic proliferation of OT1 and P14 T lymphocytes. (A) Equal numbers of CFSE-labeled lymphocytes from OT1 Rag-2^−/− and P14 Rag-2^−/− donors were cotransferred into B6.Rag-2^−/− recipient mice. Proliferation of donor CD8⁺ cells expressing the OT1 or P14-TCR, stained for Vβ₅ or Vβ₈, respectively, is shown for the indicated time points after transfer. Two mice were analyzed for each time point and dose; an independent experiment using OT1 donor cells was also performed. (B) CFSE-labeled lymphocytes from OT1 Rag-2^−/− and P14 Rag-2^−/− Ly-5.1⁺ mice were cotransferred into P14 Rag-2^−/− and B6.Rag-2^−/− (not depicted) recipients. Note that some CD8⁺ non–T cells of the P14 recipient will be included in the population of Vβ₈⁻ CD8⁺ Ly-5.1⁻ cells used to define OT1 donor T cells. However, division of OT1 cells was clearly evident at all time-points when focusing on cells that are labeled with CFSE and thus donor derived, or by staining for Vβ₅ to positively identify OT1 cells (as Vβ₅⁺ CD8⁺ Ly-5.1⁻) as performed from week 2 onward (not depicted). For each time point, two mice per genotype were analyzed. Proliferation of OT1 cells in P14 Rag-2^−/− recipients was also detected in further experiments (Fig. 2 B, Fig. 3 A, and Fig. 5, B and C). (C and D) Equal numbers of CFSE-labeled lymphocytes from OT1 Rag-2^−/− Ly-5.1⁺ and P14 Rag-2^−/− Ly-5.1⁺ mice were cotransferred into OT1 Rag-2^−/− (C), F5 Rag-1^−/− (D), or B6.Rag-2^−/− (not depicted) recipients. For each time point, two mice per genotype were analyzed. A similar experiment, without the Ly-5 allotype marker, was also performed. A, B and C and D were derived from independent experiments.

Figure 2.

Hierarchical competitiveness in homeostatic proliferation of naive OT1, P14, and F5 T lymphocytes. (A) Total lymphocyte numbers in the same pool of lymph nodes and from spleen of individual animals of the indicated genotype were determined. The proportion of CD8⁺ TCR-β⁺ or CD8⁺ CD44⁺ TCR-β⁺ cells was determined by FACS. Individual animals are each indicated by a symbol; the horizontal bar indicates the arithmetic mean. (B) Sorted CD44⁻122⁻ naive T cells, or unseparated cells, from OT1 Rag-2^−/− mice were mixed with cells from P14 Rag-2^−/− mice, labeled with CFSE, and cotransferred into OT1 Rag-2^−/−, P14 Rag-2^−/−, or B6.Rag-2^−/− recipients. Lymphocytes were analyzed 3 wk after transfer as previously described in Fig. 1. Two individual P14 Rag-2^−/− recipients were analyzed for each of the donor cell mixtures. *, there is a predominant CFSE-negative population within the gate of OT1 T cells that is host derived in OT1 recipients (shaded area) that was excluded from the calculation of percentages. (C) Sorted CD44⁻ naive T cells from P14 Rag-2^−/− Ly-5.1⁺ and F5 Rag-1^−/− mice were labeled with CFSE and cotransferred into P14 Rag-2^−/−, F5 Rag-1^−/−, or B6.Rag-2^−/− recipients. Analysis was performed after 2 (not depicted) and 4 wk after transfer as previously described in Fig. 1. *, there is a predominant CFSE-negative population within the gate of F5 cells that is host derived in F5 recipients.

Figure 3.

In the absence of restricting K^b molecules, OT1 cells cannot compete successfully, resulting in homeostatic proliferation of P14 cells. (A) CFSE-labeled lymphocytes from OT1 Rag-2^−/− and P14 Rag-2^−/− mice were cotransferred into P14 Rag-2^−/−, P14 Rag-2^−/− K^b−/−, and OT1 Rag-2^−/−, B6.Rag-2^−/−, or K^b−/− Rag-2^−/− (not depicted) recipients. Lymphocytes were recovered after 15 d and analyzed as in Fig. 1. *, the prominent population of recipient P14 cells (shaded) was excluded from the calculation of percentages of cells that had not divided or divided 1–4 times. Results presented in Fig. 5 (A and B) were obtained from the same mice. Additional experiments comparing P14 Rag-2^−/− (n = 3), P14 Rag-2^−/− K^b−/− (n = 3), OT1 Rag-2^−/− (n = 2), and B6.Rag-2^−/− (n = 2) recipients analyzed donor cell division 2 and 4 wk after transfer. (B) CFSE-labeled lymphocytes from OT1 Rag-2^−/− and P14 Rag-2^−/− mice were cotransferred into OT1 Rag-2^−/− K^b+/−-heterozygote or OT1 Rag-2^−/− K^b−/−-homozygote recipients. Of the latter, some mice had earlier (−1 mo) received 5–10 fetal (day 14.5) thymus lobes of K^b-sufficient origin (B6.Rag-2^−/−) to allow for the reconstitution of CD8⁺ peripheral T cells. As judged upon analysis, reconstitution of host CD8⁺ OT1 cell numbers was up to > 70% compared with host CD8⁺ OT1 cell numbers observed in K^b-sufficient OT1 Rag-2^−/− recipients; the accumulation of host OT1 cells in thymus grafted OT1 Rag-2^−/− K^b−/− recipients reduces the relative proportion of donor P14 and OT1 cells (identified as CFSE⁺ cells) to < 1%, similar to their proportion in OT1 Rag-2^−/− K^b+ recipients, compared with 38% in nongrafted recipients. Histograms show the CFSE profile of OT1 and P14 donor cells that were gated for as shown in the dot-plots. Mice were analyzed 14 d after cell transfer (for each group n = 2). A similar experiment was performed using OT1 K^b-/bm1 mice, not grafted (n = 2) or B6.Rag-2^−/− fetal thymus grafted (n = 2).

Figure 4.

Homeostatic dominance of OT1 cells operates beyond the MHC-bearing cell, indicating a graded, TCR-based competition for a limiting ligand not bound to APCs. The following parabiotic couples of mice were made: [OT1 Rag-2^−/− and K^b−/−Rag-2^−/−] (n = 5), [OT1 Rag-2^−/− and K^b+ B6.Rag-2^−/−] (n = 2), and [K^b+ B6.Rag-2^−/− and K^b−/−Rag-2^−/−] (n = 2). 3 wk after surgery, CFSE-labeled lymphocytes from OT1 Rag-2^−/− and P14 Rag-2^−/− mice were cotransferred into the parabionts. Analyses shown were performed 14 d later. Detailed analysis of donor T cell and host DC exchange for individual pairs of parabiotic mice is shown in Fig. S2. All parabiotic couples were prepared at the same time and were injected with the same mixture of donor cells. The experiment was repeated using K^bm1Rag-2^−/− mice in place of K^b−/−Rag-2^−/− mice (in the same order as above: n = 7, 3, 2 parabiotic pairs, respectively).

Figure 5.

Homeostatic dominance of OT1 cells is controlled by multiple soluble factors. (A) CFSE-labeled lymphocytes from OT1 Rag-2^−/− mice (filled symbols) and P14 Rag-2^−/− mice (open symbols) were transferred into B6.Rag-2^−/− (triangle), OT1 Rag-2^−/− (circles), or P14 Rag-2^−/− (squares) recipients. On day 15, lymph node cells were stained to determine the MFI for CD122 and CD127 on each subpopulation of donor cells. (B) CD122 expression on proliferating splenic OT1 cells in P14 Rag-2^−/− (left) and B6.Rag-2^−/− (right) recipients. (A and B) Analysis was performed in individual mice to allow for the direct comparison between cells, within the same recipient, that had undergone one to five divisions, as tracked by CFSE dilution; the experiment was conducted twice. (C) Mice were treated on day −3, 8, and 15 after transfer with 2 mg and on day −2, 0, 2, 4, 6, 11, 13, and 18 with 1 mg blocking antibodies toward the IL-7Rα chain (A7R34, CD127) or isotype-matched control antibodies toward CD23 (B3B4) or metallophilic macrophages (MOMA-1, not depicted) i.p. OT1 cell proliferation in the spleen was analyzed on day 19 after transfer as in Fig. 1. Two more experiments, comparing nontreated, control rat IgG-injected, and A7R34-injected P14 Rag-2^−/− mice that received OT1 cells, gave similar results (two to four mice per experimental group).