Temporal Changes in Innate and Adaptive Immunity During Sepsis as Determined by ELISpot.

Background: The inability to evaluate host immunity in a rapid quantitative manner in patients with sepsis has severely hampered development of novel immune therapies. The ELISpot assay is a functional bioassay that measures the number of cytokine-secreting cells and the relative amount of cytokine produced at the single-cell level. A key advantage of ELISpot is its excellent dynamic range enabling a more precise quantifiable assessment of host immunity. Herein, we tested the hypothesis on whether the ELISpot assay can detect dynamic changes in both innate and adaptive immunity as they often occur during sepsis. We also tested whether ELISpot could detect the effect of immune drug therapies to modulate innate and adaptive immunity. Methods: Mice were made septic using sublethal cecal ligation and puncture (CLP). Blood and spleens were harvested serially and ex vivo IFN-γ and TNF-α production were compared by ELISpot and ELISA. The capability of ELISpot to detect changes in innate and adaptive immunity due to in vivo immune therapy with dexamethasone, IL-7, and arginine was also evaluated. Results: ELISpot confirmed a decreased innate and adaptive immunity responsiveness during sepsis progression. More importantly, ELISpot was also able to detect changes in adaptive and innate immunity in response to immune-modulatory reagents, for example dexamethasone, arginine, and IL-7 in a readily quantifiable manner, as predicted by the reagents known mechanisms of action. ELISpot and ELISA results tended to parallel one another although some differences were noted. Conclusion: ELISpot offers a unique capability to assess the functional status of both adaptive and innate immunity over time. The results presented herein demonstrate that ELISpot can also be used to detect and follow the in vivo effects of drugs to ameliorate sepsis-induced immune dysfunction. This capability would be a major advance in guiding new immune therapies in sepsis.

Regulation of antigen-specific CD8+ T cell homeostasis by perforin and interferon-gamma.

T cell memory depends on factors that regulate expansion and death of these cells after antigenic stimulation. Mice deficient in perforin and interferon-gamma (IFN-gamma) exhibited increased expansion, altered immunodominance, and decreased death of antigen-specific CD8+ T cells after infection with an attenuated strain of Listeria monocytogenes, which was cleared from these mice. Expansion of CD8+ T cells was controlled by perforin, whereas IFN-gamma regulated immunodominance and the death phase. Thus, perforin and IFN-gamma regulate distinct elements of CD8+ T cell homeostasis independently of their role as antimicrobial effector molecules.

Detection and analysis of antigen-specific CD8+ T cells.

Based on recent advances in techniques that can detect and enumerate antigen-specific CD8+ T cells, it is evident that these cells can differentially regulate CD8+ T cell effector mechanisms at the single-cell level. Interplay between effector mechanisms that are employed by antigen-specific CD8+ T cells during the immune response in vivo can be addressed with different techniques that "count" cells either directly (T cell receptor (TCR) expression) or indirectly (antigen-specific cytokine production).

Intracellular staining for TNF and IFN-gamma detects different frequencies of antigen-specific CD8(+) T cells.

CD8(+) T lymphocytes are important mediators of adaptive immunity against certain viral, protozoan and bacterial pathogens. Activated CD8(+) T cells are able to induce cytolysis of infected cells (perforin and CD95-CD95L mediated pathways) and also elaborate cytokines, including IFN-gamma and TNF after appropriate MHC class I-peptide recognition. New technologies for the detection of antigen-specific CD8(+) T cells, including tetrameric MHC class I-peptide complexes, intracellular IFN-gamma staining and IFN-gamma ELISPOT analysis have revised our understanding of the magnitude of the CD8(+) T cell response to infection. Here, using intracellular cytokine staining, we compare detection of IFN-gamma and TNF in the analysis of pathogen-specific CD8(+) T cell lines and CD8(+) T cells after primary viral infection (LCMV) or secondary bacterial infection (Listeria monocytogenes). Under multiple conditions and with multiple epitopes, we find that staining for intracellular IFN-gamma consistently detects a higher frequency of antigen-specific CD8(+) T cells than detection of intracellular TNF. However, (a) intracellular staining for TNF can be used to detect antigen-specific CD8(+) T cell responses and (b) intracellular staining for cytokines is a useful approach for in vitro characterization of antigen-specific CD8(+) T cell lines.

Adaptive immunity and enhanced CD8+ T cell response to Listeria monocytogenes in the absence of perforin and IFN-gamma.

Single Ag-specific CD8+ T cells from IFN-gamma-deficient (GKO) or perforin-deficient (PKO) mice provide substantial immunity against murine infection with Listeria monocytogenes. To address the potential for redundancy between perforin and IFN-gamma as CD8+ T cell effector mechanisms, we generated perforin/IFN-gamma (PKO/GKO) double-deficient mice. PKO/GKO-derived CD8+ T cells specific for the immunodominant listeriolysin O (LLO91-99) epitope provide immunity to LM infection similar to that provided by Ag-matched wild-type (WT) CD8+ T cells in the liver but reduced in the spleen. Strikingly, polyclonal CD8+ T cells from immunized PKO/GKO mice were approximately 100-fold more potent in reducing bacterial numbers than the same number of polyclonal CD8+ T cells from immunized WT mice. This result is probably quantitative, because the frequency of the CD8+ T cell response against the immunodominant LLO91-99 epitope is >4.5-fold higher in PKO/GKO mice than WT mice at 7 days after identical immunizations. Moreover, PKO/GKO mice can be immunized by a single infection with attenuated Listeria to resist >80,000-fold higher challenges with virulent organisms than naive PKO/GKO mice. These data demonstrate that neither perforin nor IFN-gamma is required for the development or expression of adaptive immunity to LM. In addition, the results suggest the potential for perforin and IFN-gamma to regulate the magnitude of the CD8+ T cell response to infection.

Cutting edge: OFF cycling of TNF production by antigen-specific CD8+ T cells is antigen independent.

Although they are known for their capacity to kill infected cells, Ag-specific CD8(+) T cells elaborate other effector mechanisms, including TNF and IFN-gamma, that contribute to defense against infection. Ag-specific CD8(+) T cells rapidly turn ON and turn OFF IFN-gamma production in direct response to Ag contact, presumably to minimize the potential immunopathology that could result from inappropriate secretion of this inflammatory mediator. In this study, we show, using in vitro propagated and directly ex vivo-analyzed Ag-specific CD8(+) T cells, that in contrast to Ag-dependent ON/OFF cycling of IFN-gamma production, the cessation of TNF production by the same IFN-gamma producing cells is rapid and Ag independent.

Adaptive immunity against Listeria monocytogenes in the absence of type I tumor necrosis factor receptor p55.

Tumor necrosis factor (TNF) and the type I TNF receptor (TNFRI), p55, are critical for resistance against primary infections with the intracellular bacterial pathogen Listeria monocytogenes. Importantly, however, susceptibility to primary listeriosis in cytokine-deficient mice does not preclude the development or expression of effective adaptive immunity against virulent L. monocytogenes. We used TNFRI(-/-) mice to study adaptive antilisterial immunity in the absence of interactions between TNF and TNFRI. Our experiments indicate that TNFRI(-/-) mice survive and clear high-dose challenges with an attenuated strain of L. monocytogenes that is incapable of cell-to-cell spread. Furthermore, TNFRI(-/-) mice immunized with attenuated L. monocytogenes go on to develop potent adaptive immunity to subsequent high-dose challenges with virulent L. monocytogenes. Interestingly, CD8(+) T-cell depletion in vivo inhibits immunity to L. monocytogenes in the spleen but not in the liver of TNFRI(-/-) mice. The adaptive immune response in these animals is characterized by activation of listeriolysin O-specific CD8(+) T cells, which are capable of transferring antilisterial immunity to naive wild-type C57BL/6 host mice. These experiments demonstrate the development and expression of potent CD8(+) T-cell-mediated antilisterial immunity in the absence of TNFRI.

Cutting edge: antilisterial activity of CD8+ T cells derived from TNF-deficient and TNF/perforin double-deficient mice.

The mechanisms by which CD8+ T cells mediate immunity against bacterial pathogens remain largely unknown. Perforin-dependent cytolysis plays a role, but is not required for CD8+ T cell-mediated immunity against Listeria monocytogenes. TNF is essential for CD8+ T cell immunity to L. monocytogenes, but the cellular source of TNF is undefined. TNF-deficient and TNF/perforin double-deficient mice were used to generate CD8+ T cells specific for an L. monocytogenes-derived Ag. Wild-type and TNF-deficient CD8+ T cells mediated antilisterial immunity in wild-type but not TNF-deficient host mice, revealing that CD8+ T cell-derived TNF is not required for CD8+ T cell-mediated antilisterial immunity, but demonstrating a role for TNF derived from other cell types. TNF/perforin double-deficient CD8+ T cells mediated antilisterial immunity in the liver, but not in the spleen, of wild-type recipient mice, suggesting that perforin-independent immunity in the spleen requires CD8+ T cell-derived TNF.