Summary Repetitive antigen-stimulation by prime-boost vaccination or pathogen re-encounter increases memory CD8+ T cell numbers, however the impact on memory CD8+ T cell differentiation is unknown. Here we showed that repetitive antigen-stimulations induced accumulation of memory CD8+ T cells with uniform effector memory characteristics. However, genome-wide microarray analyses revealed that each additional antigen-challenge resulted in the differential regulation of several hundred new genes in the ensuing memory CD8+ T cell populations and therefore in stepwise diversification of CD8+ T cell transcriptomes. Thus, primary and repeatedly stimulated (secondary, tertiary, quaternary) memory CD8+ T cells differ substantially in their molecular signature while sharing expression of a small group of genes and biological pathways, which may constitute a core signature of memory differentiation. These results provide new insight into the complex regulation of memory CD8+ T cell differentiation and identify a spectrum of potential new molecular targets to dissect the function of memory cells generated by repeated antigen-stimulation.

Determining the magnitude and kinetics, together with the phenotypic and functional characteristics of responding CD8 T cells, is critical for understanding the regulation of adaptive immunity as well as in evaluating vaccine candidates. Recent technical advances have allowed tracking of some CD8 T cells responding to infection, and a body of information now exists describing phenotypic changes that occur in CD8 T cells of known Ag-specificity during their activation, expansion, and memory generation in inbred mice. In this study, we demonstrate that Ag but not inflammation-driven changes in expression of CD11a and CD8α can be used to distinguish naive from Ag-experienced (effector and memory) CD8 T cells after infection or vaccination. Interestingly and in contrast to inbred mice, tracking polyclonal CD8 T cell responses with this approach after bacterial and viral infections revealed substantial discordance in the magnitude and kinetics of CD8 T cell responses in outbred hosts. These data reveal limitations to the use of inbred mouse strains as preclinical models at vaccine development and suggest the same dose of infection or vaccination can lead to substantial differences in the magnitude and timing of Ag-specific CD8 expansion as well in differences in protective memory CD8 T cell numbers in outbred individuals. This concept has direct relevance to development of vaccines in outbred humans.

Patients who survive severe sepsis often display compromised immune function with impairment in innate and adaptive immune responses. These septic patients are highly susceptible to ‘secondary’ infections with intracellular pathogens that are usually controlled by CD8+ T-cells. It is unknown when and if this observed immunoparalysis of CD8+ T-cell immunity recovers and the long-term consequences of sepsis on the ability of naïve CD8+ T-cells to respond to subsequent infections are poorly understood. Here, using the CLP mouse model of sepsis we show that sepsis induces a rapid loss of naïve CD8+ T-cells. However, IL-15-dependent numerical recovery is observed a month after initial septic insult. Numerical recovery is accompanied by IL-15-dependent phenotypic changes where a substantial proportion of naïve (antigen-inexperienced) CD8+ T-cells display a ‘memory-like’ phenotype (CD44hi/CD11ahi). Importantly, the impairment of naïve CD8+ T-cells to respond to viral and bacterial infection was sustained for month(s) after sepsis induction. Incomplete recovery of naïve CD8+ T-cell precursors was observed in septic mice, suggesting that the availability of naïve precursors contributes to the sustained impairment in primary CD8+ T-cell responses. Thus, sepsis can result in substantial and long-lasting changes in the available CD8+ T-cell repertoire affecting the capacity of the host to respond to new infections.

Mortality from sepsis frequently results from secondary infections, and the extent to which sepsis affects pathogen-specific memory CD8 T cell responses remains unknown. Using the cecal-ligation and puncture (CLP) model of polymicrobial sepsis, we observed rapid apoptosis of pre-existing memory CD8 T cells after sepsis induction that led to a loss in CD8 T cell-mediated protection. Ag-sensitivity (functional avidity) and Ag-driven secondary expansion of memory CD8 T cells were decreased after sepsis, further contributing to the observed loss in CD8 T cell-mediated immunity. Moreover, Ag-independent bystander activation of memory CD8 T cells in response to heterologous infection was also significantly impaired early after sepsis induction. The reduced sensitivity of pre-existing memory CD8 T cells to sense inflammation and respond to heterologous infection by IFN-γ production was observed in inbred and outbred hosts and controlled by extrinsic (but not cell intrinsic) factors suggesting that sepsis-induced changes in the environment regulates innate functions of memory CD8 T cells. Taken together, the data in this study revealed a previously unappreciated role of sepsis in shaping the quantity and functionality of infection- or vaccine-induced memory CD8 T cells and will help further define the decline in T cell-mediated immunity during the sepsis-induced phase of immunosuppression.

Patients surviving the acute stages of sepsis develop compromised T cell immunity and increased susceptibility to infection. Little is known about the decreased CD4 T cell function after sepsis. We tracked the loss and recovery of endogenous Ag-specific CD4 T cell populations after cecal-ligation and puncture (CLP)-induced sepsis, and analyzed the CD4 T cell response to heterologous infection during or after recovery. We observed that the sepsis-induced early loss of CD4 T cells was followed by thymic-independent numerical recovery in the total CD4 T cell compartment. Despite this numerical recovery, we detected alterations in the composition of naïve CD4 T cell precursor pools, with sustained quantitative reductions in some populations. Mice that had experienced sepsis and were then challenged with epitope-bearing, heterologous pathogens demonstrated significantly reduced priming of recovery-impaired Ag-specific CD4 T cell responses, both in magnitude of expansion and functional capacity on a per-cell basis, which also correlated with intrinsic changes in Vβ clonotype heterogeneity. Our results demonstrate the recovery of CD4 T cells from sepsis-induced lymphopenia is accompanied by alterations to the composition and function of the Ag-specific CD4 T cell repertoire.