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BRIEF DEFINITIVE REPORT |
CORRESPONDENCE Steven L. Reiner: sreiner{at}mail.med.upenn.edu
 Top ABSTRACT RESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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and drives differentiation of effector and effector–memory CD8+ T cells at the expense of central–memory cells. We also found T-bet to be overexpressed in CD8+ T cells that differentiated in the absence of CD4+ T cell help, a condition that is associated with defective central–memory formation. Finally, deletion of T-bet corrected the abnormal phenotypic and functional properties of "unhelped" memory CD8+ T cells. T-bet, thus, appears to function as a molecular switch between central– and effector–memory cell differentiation. Antagonism of T-bet may, therefore, represent a novel strategy to offset dysfunctional programming of memory CD8+ T cells. When a naive CD8+ T cell encounters antigen, it undergoes vigorous clonal expansion and differentiation into a population comprised primarily of short-lived, cytotoxic effectors that undergo cell death after pathogen is cleared (1). The elimination of the antigen-experienced progeny of a selected T cell clone, however, is generally incomplete, ensuring preservation of a portion of daughter cells to provide a "memory" of the pathogen encounter. At least two functionally distinct classes of memory cells have been described, based on their pattern of tissue homing in the absence of antigen. Effector–memory CD8+ T cells provide protection against reinfection by patrolling peripheral tissues, but have a poor capacity for homeostatic renewal and secondary proliferation. Central–memory CD8+ T cells, in contrast, recapitulate the surveillance behavior of their naive predecessor by migrating through secondary lymphoid organs; they are distinguished by efficient homeostatic renewal and rapid secondary proliferative responses to generate cytotoxic effectors upon reencounter with pathogen (2, 3). It remains controversial whether commitment to the self-renewing, central–memory lineage occurs before or after adoption of the effector fate (3–6).
The intrinsic molecular program that directs commitment to the effector– versus central–memory CD8+ T cell lineage is not fully understood. We found that deficiency of the transcription factor T-bet resulted in defective generation of effector–memory CD8+ T cells and excessive generation of central–memory CD8+ T cells. The enhanced central–memory formation resulting from T-bet deficiency directly contrasted with the defective central–memory formation occurring in the absence of CD4+ T cell help. Deletion of T-bet prevented the defective phenotypic and functional characteristics of unhelped CD8+ T cells. These results suggest that T-bet may represent a novel target for manipulating the balance between terminal differentiation and self-renewal of pathogen-specific CD8+ T cells.
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RESULTS AND DISCUSSION |
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TopABSTRACTRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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in effector CD8+ T cells marks pathogen-specific CD8+ T cells destined for elimination, whereas the cells that will give rise to self-renewing memory CD8+ T cells seem to be contained within the IL-7R–expressing subset (7, 8). When we examined the lymphocytic choriomeningitis virus (LCMV)–specific CD8+ T cell response in T-bet–deficient mice, we observed defective repression of IL-7R at day 8 after infection (Fig. 1 A).
Because this finding suggested that T-bet may function as a repressor of IL-7R, we tested whether T-bet was sufficient to repress IL-7R in T cells. Retroviral-mediated expression of T-bet in CD8+ or CD4+ T cells stimulated in vitro resulted in repression of IL-7R mRNA (Fig. 1 B). In addition, ectopic expression of T-bet in developing Th2 cells in which T-bet is not normally expressed resulted in repression of IL-7R surface expression (Fig. 1 C).
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prompted us to test whether T-bet expression is associated with the IL-7Rlo subset of effector CD8+ T cells. We sorted LCMV-specific P14 CD8+ T cells 8 d after infection on the basis of IL-7R expression and examined T-bet mRNA. T-bet was enriched in the IL-7Rlo subset of effector CD8+ T cells, whereas the IL-7Rhi subset had reduced expression of T-bet mRNA (Fig. 1 D) and protein (not depicted). A similar pattern of gene expression was observed in IL-7Rhi and IL-7Rlo cells from the endogenous LCMV-specific CD8+ T cell response (not depicted).
In addition to elevated expression of T-bet, the IL-7Rlo subset of effector CD8+ T cells exhibited substantial enrichment for KLRG1 mRNA (Fig. 1 D) and protein (not depicted). KLRG1 is an NK-like inhibitory receptor that marks replicative senescence in CD8+ T cells (7, 9) and whose expression in NK cells is dependent on T-bet (10). In contrast, IL-7Rlo effectors had reduced expression of CCR7 mRNA (Fig. 1 D), which is a chemokine receptor that represents a defining feature of central–memory CD8+ T cells (2, 3). In addition, we discovered that the IL-7Rlo subset exhibited elevated expression of Blimp-1 mRNA (Fig. 1 D), a transcriptional repressor that promotes terminal differentiation of plasma cells (11) and that may also function in T lymphocytes (12, 13). Thus, enhanced expression of T-bet appears to selectively mark IL-7Rlo effector CD8+ T cells, which exhibit features of terminal differentiation and which have previously been shown to fail to give rise to self-renewing memory cells (7, 8).
Cell transfer experiments have suggested that the IL-7Rlo subset of effector CD8+ T cells preferentially gives rise to effector–memory cells (7). Consistent with this potential lineage relationship, we found that T-bet was enriched in effector–memory CD8+ T cells (Fig. 1, E and F). The skewing of T-bet expression in effector– versus central–memory cells was also recently reported in human memory CD8+ T cells (14). Given the variety of haploinsufficient phenotypes resulting from hemizygous deletion of T-bet, it is possible that the differences in the amount of T-bet between IL-7Rlo versus IL-7Rhi effectors and central– versus effector–memory CD8+ T cells could be functionally relevant (15–19). Expression of Blimp-1 paralleled that of T-bet, with elevated expression in the effector–memory CD8+ T cell subset (Fig. 1, E and F). Eomes expression, in contrast, did not vary substantially between IL-7Rhi and IL-7Rlo (Fig. 1 D) or central– and effector–memory subsets (Fig. 1, E and F).
T-bet deficiency results in enhanced generation of central–memory CD8+ T cells
The expression pattern of T-bet and its role in repressing IL-7R suggested that T-bet might negatively regulate the development of central–memory CD8+ T cells. We found that loss of T-bet, in addition to causing derepression of IL-7R, resulted in effector CD8+ T cells that acquired several characteristics of central–memory cells, including high expression of CD27, low expression of KLRG1, and robust IL-2 production (Fig. 2, A and C) (16, 20).
30 d after infection, LCMV-specific CD8+ T cells from wild-type mice exhibited substantial heterogeneity (Fig. 2 B), which is consistent with the presence of both central– and effector–memory CD8+ T cells (2, 3, 5). In contrast, T-bet–deficient mice exhibited a predominance of central–memory CD8+ T cells (Fig. 2, B and C). T-bet–deficient memory CD8+ T cells also exhibited a gene expression pattern similar to central–memory cells, with increased CCR7, reduced Blimp-1, and elevated Eomes mRNA (Fig. 2, D and E).
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Two features of central–memory CD8+ T cells are their robust capacity for secondary proliferation and an ability to confer heightened resistance to reinfection. Therefore, we assessed these characteristics in Tbx21–/– memory CD8+ T cells by transferring equal numbers of GP33-specific wild-type or Tbx21–/– memory CD8+ T cells into naive wild-type recipients and challenging with Listeria monocytogenes expressing GP33-41. T-bet–deficient memory CD8+ T cells exhibited severalfold greater reexpansion than wild-type cells upon rechallenge (Fig. 3 A) and were found to express IL-2 at a greater frequency (Fig. 3, A and B). Mice that received Tbx21–/– memory CD8+ T cells also showed substantially reduced bacterial burdens in both spleen and liver compared with recipients of wild-type cells (Fig. 3 C). Thus, deletion of T-bet promotes the development of highly functional memory CD8+ T cells. Presently, we are unsure why the defect in effector–memory CD8+ T cells resulting from deficiency of T-bet appears more permanent in this study compared with prior findings (16), although the operational definitions of effector–memory differ (phenotypic/functional versus chemokine receptor) in the two studies.
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, CD27, and IL-2, and elevated expression of KLRG1 (Fig. 4 A).
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lo, CD27lo, KLRG1hi; Fig. 4 C and Fig. S4, available at http://www.jem.org/cgi/content/full/jem.20070841/DC1). In addition to exhibiting elevated T-bet mRNA (Fig. 4 D), unhelped memory CD8+ T cells were found to express more Blimp-1 mRNA, but less CCR7 and Eomes mRNA (Fig. 4, D and E). Lack of CD4+ T cell help, thus, appears to impair central–memory and/or promote effector–memory CD8+ T cell development.
T-bet deletion prevents dysfunctional programming of unhelped memory CD8+ T cells
To discriminate whether elevated T-bet expression in unhelped memory CD8+ T cells plays a causal role in, or is simply a consequence of, their altered differentiation, we performed antibody depletion of CD4+ T cells from wild-type or Tbx21–/– mice, followed by infection with LCMV. Again, we found that unhelped memory CD8+ T cells in wild-type mice exhibited a predominance of effector–memory–like cells (CD62Llo, IL-7Rlo, CD27lo, and KLRG1hi), with impaired production of IL-2 and CD40L (Fig. 5, A and B, and Fig. S3). Deletion of T-bet, however, prevented the phenotypic and functional defects associated with the lack of CD4+ T cell help (Fig. 5, Fig. S3, and Fig. S5, available at http://www.jem.org/cgi/content/full/jem.20070841/DC1), resulting in enhanced generation of central–memory CD8+ T cells, regardless of the availability of CD4+ T cell help. The rescue was apparent even when T-bet deficiency was confined to antigen-specific CD8+ T cells (Fig. S5). Moreover, delayed antagonism of T-bet after unhelped memory CD8+ T cell development was capable of reversing several aspects of their dysfunctional phenotype (Fig. S6). Thus, T-bet appears to be required for the aberrant differentiation of memory CD8+ T cells that occurs in the absence of CD4+ T cell help.
How T-bet and CD4+ T cells execute their opposing effects on the balance between central– versus effector–memory CD8+ T cells remains to be determined. In several different systems, transcription factors function as intrinsic modulators of self-renewal or terminal differentiation. In B lymphocytes, the transcriptional repressor Blimp-1 promotes terminal differentiation (plasma cell) at the expense of self-renewal (memory B cell) (11). In the Drosophila melanogaster neural stem cells, the homeodomain transcription factor Prospero functions as a similar switch by repressing genes required for self-renewal, such as stem cell and cell-cycle genes, while activating genes involved in terminal differentiation (26). Parallels from other biological systems may help elucidate whether T-bet similarly functions to promote terminal differentiation at the expense of self-renewal, which has been proposed to represent an essential distinction between effector– versus central–memory CD8+ T cells, respectively (2–7, 27).
The potential ability of T-bet to switch between two polar states of differentiation may explain numerous aspects of viral pathogenesis. Clonal deletion, for example, could be regarded as a "self-renewal deficiency state," associated with extreme excess of T-bet expression resulting from prolonged antigenic stimulation. Likewise, the association of high precursor frequency with enhanced central–memory formation might be considered a "disorder of excessive self-renewal," explained by diminished T-bet expression as a consequence of excessive competition for antigen (5). The potential role of T-bet in these processes is currently being investigated.
The excessive differentiation of effector–memory CD8+ T cells in the absence of CD4+ T cell help could involve direct effects, such as loss of soluble or membrane-bound signals typically delivered by CD4+ T cells to dendritic cells or CD8+ T cells, including IL-2, chemokines, or CD40L (28–30). Alternatively, loss of CD4+ T cell help could act more indirectly, for example, by altering kinetics of pathogen clearance and necessitating prolonged effector function (3). Although the abnormalities arising in the absence of CD4+ T cell help appear to be at least partially T-bet–dependent, it is anticipated there could be T-bet–independent derangements contributing to the aberrant differentiation of unhelped memory CD8+ T cells. Nonetheless, antagonism of T-bet might represent a novel therapeutic approach for preventing the dysfunctional programming of CD8+ T cells in patients with compromised CD4+ T cell function.
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MATERIALS AND METHODS |
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TopABSTRACTRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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5 x 104 CD8+ T cells from naive P14 TCR transgenic mice (Thy1.1/1.1) were transferred intravenously into C57BL/6 or Cd4–/– recipients (Thy1.2/1.2). For P14 cotransfer experiments, 5 x 104 wild-type P14 cells (Thy1.1/1.1) were mixed with 5 x 104Tbx21–/– P14 cells (Thy1.1/1.2) and transferred to wild-type C57BL/6 recipients (Thy1.2/1.2). 1 d after transfer, recipient mice were infected with LCMV. For CD4 depletion experiments, wild-type or Tbx21–/– mice received intraperitoneal injection of GK1.5 antibody (0.2 mg in PBS) 1 d before and 1 d after infection with LCMV. GK1.5 treatment resulted in efficient depletion of CD4+ T cells for >1 wk (not depicted). For Listeria monocytogenes experiments, mice were challenged with 2.5 x 105 CFU of L. monocytogenes with transgenic expression of the LCMV-derived peptide GP33-41. 4 d after bacterial challenge, spleens and livers were homogenized in 1% Triton X-100 in PBS, followed by limiting dilution culture on blood heart infusion agar.
Flow cytometry, cell culture, stimulation, and retroviral transduction.
Surface staining, intracellular cytokine staining, and flow cytometry were performed as previously described (18). For LCMV-derived peptide stimulations, splenocytes or lymph node cells were stimulated for 6 h with 0.2 µg/ml GP33-41 or NP396-404 peptide in the presence of 1 µg/ml brefeldin A. Antibodies used for flow cytometry were purchased from BD Biosciences. Intranuclear T-bet staining (Santa Cruz Biotechnology) was performed by fixation with 4% paraformaldehyde in PBS, followed by permeabilization and staining in 0.1% Triton X-100 and 1% FBS in PBS. Splenocytes from DO11.10 or P14 TCR transgenic mice were stimulated and transduced as previously described (18).
Quantitative RT-PCR.
RNA extraction, cDNA synthesis, and quantitative real-time RT-PCR were performed as previously described (18). Primer and probe sets used for HPRT and Eomes detection were previously described (18). Presynthesized Taqman Gene Expression Assays (Applied Biosystems) were used to amplify the following sequences (gene symbols and Applied Biosystems primer set numbers in parentheses): Blimp-1 (Prdm1; Mm00476128_m1), CCR7 (Ccr7; Mm01301785_m1), KLRG1 (Klrg1; Mm00516879_m1), and T-bet (Tbx21; Mm00450960_m1). ‘Test’ gene values are expressed relative to that of HPRT, with the lowest experimental value standardized at 1.
Online supplemental material.
Fig. S1 shows that T-bet–deficient antigen-specific memory CD8+ T cells preferentially accumulate in lymph nodes. Fig. S2 shows that T-bet represses central–memory development in a CD8+ T cell–intrinsic manner. Fig. S3 shows that deletion of T-bet rescues the phenotype and function of unhelped memory CD8+ T cells. Fig. S4 shows that unhelped memory CD8+ T cells have elevated expression of T-bet. Fig. S5 shows that T-bet deficiency restricted to antigen-specific CD8+ T cells is capable of correcting abnormalities associated with unhelped memory cells. Fig. S6 shows that preexisting dysfunction in unhelped memory CD8+ T cells can be reversed by antagonism of T-bet. The online version of this article is available at http://www.jem.org/cgi/content/full/jem.20070841/DC1.
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Acknowledgments |
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These studies were supported by National Institutes of Health grants (AI042370, AI061699, AI071309, and AI007532) and the Abramson Family Cancer Research Institute.
The authors have no conflicting financial interests.
Submitted: 25 April 2007
Accepted: 24 July 2007
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and IL-2. Results are representative of three experiments with multiple mice per time point. (D and E) Q-PCR of H-2DbGP33+ plus H-2bNP396+ CD8+ T cells from spleens of wild-type or Tbx21–/– mice. (D) CCR7 and Blimp-1 mRNA 60 d after infection. Naive represents CD44loCD8+ T cells from uninfected wild-type mice. (E) Eomes mRNA 60 and 100 d after infection. Day 0 represents CD44loCD8+ T cells from uninfected wild-type or Tbx21–/– mice. Results are representative of three similar experiments. (F) Quantification of LCMV-specific CD8+ T cells from wild-type or Tbx21–/– mice 8, 30, and 60 d after infection. Left graph shows H-2DbGP33+ cells as the percentage of CD8+ in blood. Middle and right graphs show numbers of CD8+ T cells from spleen or lymph node (pooled axillary, inguinal, cervical, paraaortic, and mesenteric), respectively, producing IFN-
