Deepa Rai

Repetitive Antigen Stimulation Induces Stepwise Transcriptome Diversification but Preserves a Core Signature of Memory CD8+ T Cell Differentiation

Repetitive antigen stimulation by prime-boost vaccination or pathogen reencounter increases memory CD8(+) T cell numbers, but 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, and quaternary) memory CD8(+) T cells differed 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 reveal the complex regulation of memory CD8(+) T cell differentiation and identify potential new molecular targets to dissect the function of memory cells generated by repeated antigen stimulation.

Tracking the Total CD8 T Cell Response to Infection Reveals Substantial Discordance in Magnitude and Kinetics between Inbred and Outbred Hosts

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.

Immune unresponsiveness to secondary heterologous bacterial infection after sepsis induction is TRAIL dependent.

Sepsis is the leading cause of death in most intensive care units, and patients who survive the hyperinflammation that develops early during sepsis later display severely compromised immunity. Not only is there apoptosis of lymphoid and myeloid cells during sepsis that depletes these critical cellular components of the immune system, but also the remaining immune cells show decreased function. Using a cecal-ligation and puncture (CLP) model to induce intra-abdominal polymicrobial peritonitis, we recently established a link between the apoptotic cells generated during sepsis and induction of sepsis-induced suppression of delayed-type hypersensitivity. The present study extends this earlier work to include a secondary heterologous bacterial infection (OVA257-expressing Listeria monocytogenes [LM-OVA]) subsequent to sepsis initiation to investigate sepsis-induced alterations in the control of this secondary infection and the associated naive Ag-specific CD8 T cell response. We found that CLP-treated wild-type (WT) mice had a reduced ability to control the LM-OVA infection, which was paralleled by suppressed T cell responses, versus sham-treated WT mice. In contrast, CLP-treated Trail−/− and Dr5−/− mice were better able to control the secondary bacterial infection, and the Ag-specific CD8 T cell response was similar to that seen in sham-treated mice. Importantly, administration of a blocking anti-TRAIL mAb to CLP-treated WT mice was able to restore the ability to control the LM-OVA infection and generate Ag-specific CD8 T cell responses like those seen in sham-treated mice. These data further implicate TRAIL-dependent immune suppression during sepsis and suggest TRAIL neutralization may be a potential therapeutic target to restore cellular immunity in septic patients.

Sustained and Incomplete Recovery of Naive CD8+ T Cell Precursors after Sepsis Contributes to Impaired CD8+ T Cell Responses to Infection

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 not known when and if this observed immunoparalysis of CD8+ T cell immunity recovers, and the long-term consequences of sepsis on the ability of naive CD8+ T cells to respond to subsequent infections are poorly understood. In this study, using the cecal-ligation and puncture mouse model of sepsis, we show that sepsis induces a rapid loss of naive 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 naive (Ag-inexperienced) CD8+ T cells display a “memory-like” phenotype (CD44hi/CD11ahi). Importantly, the impairment of naive CD8+ T cells to respond to viral and bacterial infection was sustained for month(s) after sepsis induction. Incomplete recovery of naive CD8+ T cell precursors was observed in septic mice, suggesting that the availability of naive 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.

Division-linked generation of death-intermediates regulates the numerical stability of memory CD8 T cells

Infection or successful vaccination results in the formation of long-lived memory CD8 T-cell populations. Despite their numerical stability, memory CD8 T-cell populations are thought to completely turn over through proliferation within a 2- to 3-mo period. Therefore, steady-state memory cell proliferation must be balanced by a precisely regulated and equivalent death rate. However, the mechanisms regulating this balancing process remain completely undefined. Herein, we provide evidence for “death-intermediate memory cells” (TDIM) within memory CD8 T-cell populations generated by infection. Importantly, CD62LLo/CD27Lo TDIMs are functionally characterized by an inability to produce cytokines, the failure to internalize T-cell receptor following antigenic stimulation, and signatures of apoptotic death. Furthermore, we demonstrate that, mechanistically, TDIM are directly generated from dividing “central memory” T-cell populations undergoing memory turnover in vivo. Collectively, these results demonstrate that as central memory CD8 T cells proliferate, they continuously generate a population of CD8 T cells that are nonfunctional and apoptotic; thus, our data support a model wherein division-linked generation of TDIM contributes to numerically stable CD8 T-cell memory.

Polymicrobial Sepsis Alters Antigen-Dependent and -Independent Memory CD8 T Cell Functions

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 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 regulate 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.

Polymicrobial Sepsis Increases Susceptibility to Chronic Viral Infection and Exacerbates CD8+ T Cell Exhaustion

Patients who survive sepsis display suppressed immune functions, often manifested as an increased susceptibility to secondary infections. Recently, using a cecal-ligation and puncture (CLP) model of sepsis, we showed that sepsis induces substantial and long-lasting changes in the available naive CD8+ T cell repertoire affecting the capacity of the host to respond to newly encountered acute infections. However, the extent to which sepsis changes the host susceptibility to chronic infection and affects CD8+ T cell responses is currently unknown. In this study, we demonstrate that inbred and outbred mice recovering from a septic event are more susceptible to lymphocytic choriomeningitis virus (LCMV) clone-13 infection exhibited by mortality and viral burden. Primary virus-specific CD8+ T cells in LCMV clone-13–infected septic mice displayed exacerbated CD8+ T cell exhaustion illustrated by increased inhibitory molecule expression (e.g., programmed cell death 1, lymphocyte-activation gene 3, and 2B4) and diminished Ag-driven cytokine production (e.g., IFN-γ, TNF-α) compared with similarly infected sham-treated mice. Importantly, therapeutic inhibitory molecule dual blockade (anti–PD-L1 and anti–lymphocyte-activation gene 3) increased the number of circulating LCMV-specific CD8+ T cells, and improved CD8+ T cell function and pathogen control in chronically infected septic mice. Together, these results illustrate that polymicrobial sepsis compromises the overall health of the host leading to increased vulnerability to chronic infection and exacerbated CD8+ T cell exhaustion. Collectively, our findings suggest that septic survivors may be more susceptible and at greater risk for developing exhaustible CD8+ T cells upon encountering a subsequent chronic infection.

Alterations in Antigen-Specific Naive CD4 T Cell Precursors after Sepsis Impairs Their Responsiveness to Pathogen Challenge

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–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 naive 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, with regard to both magnitude of expansion and functional capacity on a per-cell basis, which also correlated with intrinsic changes in Vβ clonotype heterogeneity. Our results demonstrate that 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.

The Longevity of Memory CD8 T Cell Responses after Repetitive Antigen Stimulations

In experimental models in which the Ag-stimulation history of memory CD8 T cell populations was clearly defined (adoptive transfer of a known number of TCR-transgenic memory CD8 T cells), all facets of the ensuing CD8 T cell responses, including proliferative expansion, duration and extent of contraction, diversification of memory CD8 T cell transcriptomes, and life-long survival, were dependent on the number of prior Ag encounters. However, the extent to which sequential adoptive-transfer models reflect the physiological scenario in which memory CD8 T cells are generated by repetitive Ag challenges of individual hosts (no adoptive transfer involved) is not known. Direct comparison of endogenous memory CD8 T cell responses generated in repetitively infected hosts revealed that recurrent homologous boosting was required to preserve the numbers and increase the phenotypic and functional complexity of the developing memory CD8 T cell pool. Although life-long survival of the memory CD8 T cells was not impacted, phenotype (i.e., upregulation of CD62L) and function (i.e., homeostatic turnover, Ag-stimulated IL-2 production) of repeatedly stimulated memory CD8 T cells were dependent on time after last Ag encounter. Therefore, repetitive Ag challenges of individual hosts can substantially influence the numerical and functional attributes of polyclonal memory CD8 T cells, a notion with important implications for the design of future vaccination strategies aimed at increasing the number of protective memory CD8 T cells.