Alexandra M. Livingstone

Cutting Edge: CD4+ T Cell Help Can Be Essential for Primary CD8+ T Cell Responses In Vivo

Recent studies have shown that CD4+ T cell help is required for the generation of memory CD8+ T cells that can proliferate and differentiate into effector cells on Ag restimulation. The importance of help for primary CD8+ T cell responses remains controversial. It has been suggested that help is not required for the initial proliferation and differentiation of CD8+ T cells in vivo and that classical models of helper-dependent responses describe impaired secondary responses to Ag in vitro. We have measured primary CD8+ T cell responses to peptide-pulsed dendritic cells in mice by cytokine ELISPOT and tetramer staining. No responses were detected in the absence of help, either when normal dendritic cells were injected into MHC II-deficient mice or when MHC II-deficient dendritic cells were injected into normal mice. Thus, the primary in vivo CD8+ T cell response depends absolutely on help from CD4+ T cells in our experimental system.

Measuring the frequency of mouse and human cytotoxic T cells by the Lysispot assay: independent regulation of cytokine secretion and short-term killing

Antigen-specific T cells demonstrate several potent effector functions during immune responses. Direct killing of infected cells is crucial for clearing viruses and other intracellular pathogens, but it has been difficult to measure the frequency of cytolytic cells. We have now developed a single-cell assay to measure the number of cytotoxic cells in a population, using a herpes simplex virus amplicon vector to express Escherichia coli β-galactosidase in mouse or human target cells, and an Elispot to detect release of β-galactosidase from killed target cells. This antigen-specific, perforin-dependent Lysispot assay has been combined with a cytokine Elispot in a two-color assay to confirm that cytotoxicity and interferon-γ secretion are regulated independently. The simultaneous enumeration of cytokine-secreting and cytotoxic cells should be invaluable for ex vivo analysis of immune responses during infection and autoimmunity.

Cutting Edge: CD4+ T Cell-Derived IL-2 Is Essential for Help-Dependent Primary CD8+ T Cell Responses

CD4+ T cell help is essential for primary CD8+ T cell responses to noninflammatory Ags. IL-2 is one of the principal cytokines made by naive CD4+ T cells, and we show in this study that it is an essential component of help. Adoptively transferred naive CD4+ TCR-transgenic OT-II cells supported endogenous primary CD8+ T cell responses, but IL-2-deficient OT-II cells were unable to provide help, although they responded to Ag in vivo and up-regulated CD40 ligand in vitro. Wild -type OT-II cells helped endogenous CD8+ T cell responses in IL-2-deficient mice, but not in IL-2Rα-deficient mice. Thus, CD4+ T cell-derived IL-2 is essential for CD8+ T cell responses to noninflammatory, cell-associated Ags. We suggest that it is also a critical component of help for CD8+ T cell responses to pathogens, because protective memory also requires CD8+ T cell stimulation by IL-2 during priming.

Dendritic cells: the immune information management experts

Dendritic cells sense and respond to multiple signals that are then conveyed to lymphocytes to direct appropriate immune responses. This flow of information is bidirectional, as antigen-experienced T cells also influence dendritic cell function.

Regulation of heat-induced apoptosis by Mcl-1 degradation and its inhibition by Hsp70

Cellular stress eliminates irreversibly damaged cells by initiating the intrinsic death pathway. Cell stress is sensed by pro- and antiapoptotic members of the Bcl-2 protein family, which regulate the release of apoptogenic factors, such as cytochrome c, from mitochondria. Exposure of cells to hyperthermia results in the activation of the proapoptotic Bcl-2 family protein Bax, which plays an essential role in cytochrome c release. Heat directly affects Bax activity in vitro; however, antiapoptotic Bcl-2 family proteins, such as Bcl-xL, can suppress this activation, suggesting that a second heat-sensitive step must be breached before apoptosis ensues in cells exposed to hyperthermia. Here we show that heat shock causes the loss of Mcl-1 protein. Depletion of Noxa by short hairpin RNA protected cells from hyperthermia by preventing Mcl-1 degradation. Heat shock caused the dissociation of Noxa from Mcl-1, which allowed binding of the BH3-containing ubiquitin ligase Mule followed by Mcl-1 ubiquitination and degradation. Overexpression of Hsp70, which prevents heat-induced Bax activation, stabilized Mcl-1 protein levels in heat-shocked cells. This resulted from reduced Mule binding and ubiquitination as well as enhanced Mcl-1 expression compared with cells without Hsp70. Our results demonstrate that loss of Mcl-1 is a critical heat-sensitive step leading to Bax activation that is controlled by Hsp70.

Cim: an MHC class II-linked allelism affecting the antigenicity of a classical class I molecule for T lymphocytes

Two alleles at the major histocompatibility complex (MHC)-linked locus cim determine “gain and loss” changes in the rat RT1.Aa class I molecule which affect its structure both as an alloantigen and as a restriction element. Alleles at the cim locus also influence the post-translational modification of RT1.Aa. These effects may reflect the participation of the cim gene product in the processes of peptide loading or assembly of RT1.Aa. In this study we have used the discriminating RT1.Aa-specific monoclonal antibody JY3/84, as well as cytotoxic T cells raised in appropriate combinations, to determine the cim alleles of eight haplotypes in 15 independent inbred strains of rat. We have also employed the same techniques to analyse a panel of F1 hybrid animals derived from various MHC recombinant strains. These experiments map the cim locus to the class II region of RT1, probably between the DP-related genes (RT1.H) and the DQ-related RT1.Bα.

Antigen Presentation by Neoclassical MHC Class I Gene Products in Murine Rodents

A number of cytotoxic T cell responses in rats and mice are restricted by medial or non- classical class I antigens of the major histocompatibility complex (MHC). The class I heavy chain encoded by the H-2M3 gene presents MTF, an N-formylated peptide derived from the amino terminus of the mitochondrially-encoded ND1 protein, which is polymorphic in the sixth residue. H-2M3 shows minimal polymorphism in mice, and it is more similar to its rat ortholog, RT1.M3, than to any other H-2 class I gene. In rats, a minor antigen is presented by an RT1.C-encoded restriction element. Other examples of antigen presentation by medial class I antigens are reviewed. Whereas the majority of medial class I genes have ill-defined, if any, function, we propose that some may serve to present special antigens, and that these neo-classical class I genes are highly conserved between species and may be localized at the distal end of the MHC.

Dendritic cells need T cell help to prime cytotoxic T cell responses to strong antigens

Peptide‐pulsed mouse dendritic cells (DC) primed peptide‐specific CD8+ cytotoxic T cell responses very effectively if they expressed minor histocompatibility antigens, which could stimulate a CD4+ T helper cell response. These DC could also prime most syngeneic mice, although there was no deliberate immunization for help (the DC were prepared in syngeneic mouse serum, to avoid any response to fetal calf serum antigens). In contrast, DC were unable to prime MHC class II‐deficient mice for cytotoxic responses to the classical helper‐dependent antigens Qa1a and HY. More strikingly, Balb.B DC failed to prime B6 MHC class II‐deficient mice for cytotoxic responses to Balb minor antigens, even though these two strains differ at more than 40 minor histocompatibility loci. When peptide‐pulsed DC were prepared without enzymes (used to release DC from lymphoid tissues), they failed to prime the majority of normal syngeneic mice, even though they expressed high levels of B7 and ICAM‐1 co‐stimulatory molecules, suggesting that help was provided by responses to antigens in the enzyme cocktail. The enzyme treatment itself did not provide signals that could substitute for help, since DC prepared with enzymes could not prime MHC class II‐deficient mice. The observation that highly immunogenic minor‐incompatible DC failed to prime MHC class II‐deficient mice suggests that in the absence of inflammatory signals, even strong antigens cannot stimulate CD8+ T cell responses without help.

Modeling of Influenza-Specific CD8+ T Cells during the Primary Response Indicates that the Spleen Is a Major Source of Effectors

The biological parameters that determine the distribution of virus-specific CD8+ T cells during influenza infection are not all directly measurable by experimental techniques but can be inferred through mathematical modeling. Mechanistic and semimechanistic ordinary differential equations were developed to describe the expansion, trafficking, and disappearance of activated virus-specific CD8+ T cells in lymph nodes, spleens, and lungs of mice during primary influenza A infection. An intensive sampling of virus-specific CD8+ T cells from these three compartments was used to inform the models. Rigorous statistical fitting of the models to the experimental data allowed estimation of important biological parameters. Although the draining lymph node is the first tissue in which Ag-specific CD8+ T cells are detected, it was found that the spleen contributes the greatest number of effector CD8+ T cells to the lung, with rates of expansion and migration that exceeded those of the draining lymph node. In addition, models that were based on the number and kinetics of professional APCs fit the data better than those based on viral load, suggesting that the immune response is limited by Ag presentation rather than the amount of virus. Modeling also suggests that loss of effector T cells from the lung is significant and time dependent, increasing toward the end of the acute response. Together, these efforts provide a better understanding of the primary CD8+ T cell response to influenza infection, changing the view that the spleen plays a minor role in the primary immune response.

Unravelling the mechanisms of help for CD8+ T cell responses

CD8+ T cells are critically important for immune defense against many viral and bacterial pathogens, and are also key components of cancer immunotherapy. Help from CD4+ T cells is usually essential for optimal CD8+ T cell responses, driving the primary response, the survival of memory cells, and the generation of protective and therapeutic immunity. Understanding the mechanisms of help is thus essential for vaccine design, and for restoring protective immunity in immunosuppressed individuals. Our laboratory has developed an immunization protocol using peptide-pulsed dendritic cells to stimulate help-dependent primary, memory, and secondary CD8+ T cell responses. We have used gene-targeted and T cell receptor transgenic mice to identify two distinct pathways that generate help-dependent and help-independent CD8+ T cell responses, respectively, and are now starting to define the molecular mechanisms underlying these two pathways.