In this study we have used cross-species gene-expression analysis to identify a molecular signature of memory CD8 differentiation that is conserved between humans and mice. We found that elements of this CD8 memory differentiation signature were shared by CD4 and B cell memory differentiation suggesting that coordinated upregulation of this set of genes is common to all memory lymphocyte lineages. Alterations in this signature could distinguish T cells from functional and dysfunctional responses to viral infection. These data indicate a central role for this common differentiation signature in memory development in each lymphoid lineage, and suggest that the genomic classification of memory differentiation could be used to assess functional immunity.
It is surprising that lymphocytes with such distinct functions and diverse transcriptional regulation as T cells and B cells should share a common transcriptional program during memory development. Indeed the formation of discrete populations of mature lymphocytes depends on discrete, lineage-specific transcription factors that impart functions unique to that lineage (10
). However, our data suggest that in addition to these lineage-specific mechanisms, memory lymphocytes in multiple lineages employ a common transcriptional program during differentiation. These findings differ from previous studies that have evaluated the genome-wide changes during memory differentiation. Working in the mouse, Luckey et al showed a similarity between the signature of genes upregulated in CD8 memory T cells and those in hematopoietic stem cells (HSC), and also between memory B cells and HSC. However, that study found only limited similarity in the genes upregulated during memory differentiation in both T and B cell lineages.(15
). Appay et al, studying human CD4 and CD8 memory-phenotype cells in humans showed similarity in gene expression profiles between terminally differentiated cells in each lineage but none in earlier stages (16
). Our study, in contrast, which encompassed both functionally defined mouse memory CD8 T cells and phenotypically-defined human CD4, CD8 T cells and B cells (sorted with different markers in T and B lineages), identified a transcriptional program upregulated during memory differentiation in both species and all three lineages.
Several reasons may account for the difference between the previous studies and our own. First, we used a cross-species genomic comparison of memory differentiation. Focusing only on the signature of genes upregulated during CD8 differentiation in two species would be more likely to identify genes critical to the differentiation process by virtue of their evolutionary conservation. Second, we used a sensitive analytic technique (GSEA) well suited to detecting the coordinate upregulation of correlated sets of genes that could have been missed by other analyses, and to determining its statistical significance. GSEA has proven a powerful analytic tool to evaluate coordinately regulated patterns of genes occurring in cellular differentiation in stem cell biology (38
), and in immunology (31
The transcripts identified in our analysis include many which are not known to be involved in memory differentiation. However, many have functions consistent with the functional characteristics common to the memory state in each lineage. For example, the kinetics of proliferation in memory lymphocytes is different than in their naive precursors: memory lymphocytes show a higher rate of division and shorter lag time after antigen stimulation than do naive cells (39
). This finding has been attributed to the increased expression of cell-cycle components necessary for G0 - G1 transition (39
). How memory cells remain quiescent but "poised" to divide rapidly remains unclear. Our data show that a common feature of memory lymphocytes in all lineages is the elevated expression of transcription factors that could serve to enforce quiescence e.g. KLF10 and BHLHB2. Several of the Kruppel-like transcription factors have been implicated blocking cell-cycle passage (41
), and the absence of KLF2 leads to spontaneous T cell activation and abnormal trafficking (42
). Similarly, mice deficient in the transcription factor Stra-13/BHLHB2 also show spontaneous activation of T cells and develop autoimmunity (45
). Increased expression of these transcription factors in memory cells compared to naive cells may be a critical component of the quiescence that is a common feature of memory differentiation. The ability of memory lymphocytes to migrate to sites of inflammation is crucial to their function, a finding that is consistent with the increased expression of transcripts for S100 family members (47
), MYO1F (48
) and chemokine receptors by both T and B memory lymphocytes. S100A4, a member of the calcium-binding EF-hand motif superfamily, enhances motility of cancer cells through its interaction with myosin-IIA (47
), suggesting that it may have a similar role in augmenting the ability of memory T and B lymphocytes to migrate. However, it is more than upregulation of individual genes that characterizes memory differentiation in each lineage. Rather, the coordinated upregulation of a set
of genes suggests the existence of a common developmental program shared by memory cells. Such a program would provide an efficient mechanism by which common attributes such as quiescence and migratory potential could be acquired in different lineages.
Some of the genes contained in the memory differentiation signature are not uniquely expressed by memory cells, (e.g. GZMA and KLRG1) and are known to be upregulated in effector T cells (18
). We found that expression levels of these effector genes is greatly reduced in memory cells compared to effector cells, consistent with previous observations, allowing clear distinction of effector and memory states () (18
). This illustrates that the differentiation signature of memory cells represents a composite of genes uniquely expressed by memory cells and those retained from prior stages of differentiation, possibly to allow rapid re-expression on re-exposure to antigen. Thus it is not necessarily the genes themselves that are unique to the memory T cells as it is the expression pattern of the signature of a whole that defines the memory state.
Our findings have implications for the diagnosis of T cell dysfunction in humans. Impressive progress has been made on defining the phenotypic and functional characteristics of memory T cells in different infectious settings, but it has been difficult to use these properties as a measure of protective immunity in humans (7
). There is significant need for accurate correlates of immunity in order to develop effective immunotherapies for chronic viral diseases and cancer. We used the “gold-standard” mouse model of memory differentiation as a biological filter to identify the corresponding differentiation signature in human memory-phenotype CD8 T cells. Alterations in the conserved memory signature distinguished between T cells specific from effective and ineffective viral responses in mice (acute versus chronic LCMV) and in humans (influenza versus HIV). This suggests that the pattern of gene expression in this memory differentiation signature correlated with T cell functional status. Ultimately, larger studies of clinical outcomes in viral infection or vaccination will be required to refine this signature and determine how well the presence of this optimal differentiation signature predicts clinical endpoints such as viral load or protection from infection in humans. However, measuring the integrity of a validated signature of genes corresponding to a defined differentiation state in antigen-specific T cells may be a useful tool for interrogating the human immune response.
Our data used cross-species genomic analysis to identify, for the first time, a common signature of genes upregulated during memory differentiation in CD4, CD8 and B cell lineages, which is conserved between humans and mice. These findings support the hypothesis that the shared attributes of memory cells are achieved in different lineages by common transcriptional programs. The ability to identify these critical differentiation signatures in antigen-specific T cells could allow the genome-wide assessment of memory differentiation as a correlate of functional immunity in humans, and as a target for therapeutic intervention.