Recall and propagation of allospecific memory T cells independent of secondary lymphoid organs

Abstract

The allospecifc T cell population responding to a transplanted organ consists of both naive and memory lymphocytes. Although it is established that naive T cells are activated by antigen within the organized structures of secondary lymphoid organs (the spleen, lymph nodes, and mucosal lymphoid tissues), it is not clear whether memory T cell activation and propagation depend on homing to these organs. To answer this question, we investigated whether allospecific naive or memory T cells can mediate acute cardiac allograft rejection in mutant mice that lack all of their secondary lymphoid tissues. The results of our experiments demonstrated that antigen-experienced memory T cells have two advantages over naive T cells: (i) memory T cells mount a vigorous immune response that leads to allograft rejection independent of secondary lymphoid organs; and (ii) memory T cells generate more memory T cells without homing to secondary lymphoid organs. These unique properties of memory T cells were further confirmed by showing that memory-like T cells that arise from the homeostatic proliferation of naive T cells in the absence of antigenic stimulation are suboptimal at rejecting allografts and do not generate memory T cells in mice devoid of secondary lymphoid tissues.

Figure 1

Memory T cells mediate allograft rejection independent of secondary lymphoid organs. Naïve and memory T cells were generated in wt hosts and adoptively transferred to splenectomized aly/aly heart transplant recipients as described in Methods. Identical enrichment methods were applied to naïve and memory T cells. (A) Phenotype of naive and memory allospecific T cells determined before adoptive transfer by four-color flow analysis after gating on the CD8⁺1B2⁺ (2C TCR-tg) cell population. Histograms shown are representative of three separate experiments. (B) CTL activity of naïve and memory T cells against target BALB/c splenocytes before adoptive transfer. CD8-enriched T cells from memory (immunized) mice were assayed for ex vivo CTL activity either immediately or 2 days after in vivo recall with BALB/c splenocytes. One representative assay is shown. (C) Cardiac allograft survival in splenectomized aly/aly mice that received either naive (n = 3) or memory (n = 3) T cell populations. (D) Histological (hematoxylin/eosin, H&E) and immunohistochemical (CD3) analysis of cardiac allograft tissue harvested 100 days after transplantation in the naive group and on the day of rejection in the memory group.

Figure 2

Memory-like T cells have intermediate in vivo function in the absence of secondary lymphoid organs. Memory-like (naive_Rag2^−/−) and antigen-experienced memory (memory_Rag2^−/−) T cells were generated in Rag2^−/− hosts and adoptively transferred to splenectomized aly/aly heart transplant recipients. The naïve group received T cells harvested from naïve wt and 2C mice as described in Methods. (A) Phenotype of naive, naive_Rag2^−/−, and memory_Rag2^−/− allospecific T cells determined before adoptive transfer by four-color flow analysis after gating on the CD8⁺1B2⁺ (2C TCR-tg) cell population. Histograms shown are representative of 4–5 experiments. (B) Cardiac allograft survival in splenectomized aly/aly mice that received naïve (●, n = 4), naive_Rag2^−/− (■, n = 4), or memory_Rag2^−/− (○, n = 5) T cell populations. (C) Histological (hematoxylin/eosin, H&E) and immunohistochemical (CD3) analysis of cardiac allograft tissue harvested 100 days after transplantation in the naive and naive_Rag2^−/− group and on the day of rejection in the memory_Rag2^−/− group. (Magnification = ×200.)

Figure 3

CD8 memory T cells generate more memory T cells in the absence of secondary lymphoid organs. Heart transplantation and adoptive transfer of T cells to splenectomized aly/aly mice were performed as in Figs. 1 and 2. CD8⁺1B2⁺ (2C TCR-tg) cells present in the blood of splenectomized aly/aly hosts at the time of cardiac allograft rejection (naive_Rag2^−/−, memory_Rag2^−/−, and memory) or at 100 days after transplantation in mice that accepted their allografts [naive and naive_Rag2^−/−(a)] were quantitated (A), phenotyped by flow analysis (B), and assayed for immediate ex vivo CTL activity (C). The bar graph compares the number of CD8⁺1B2⁺ T cells injected to the number of CD8⁺1B2⁺ cells retrieved in seven individual mice. The number of CD8⁺1B2⁺ cells retrieved was extrapolated based on the number of CD8⁺1B2⁺ T cells present per ml of blood.