Martha A. Alexander-Miller

Peptide Requirement for CTL Activation Reflects the Sensitivity to CD3 Engagement: Correlation with CD8αβ Versus CD8αα Expression

In our previous studies, CTL that were sensitive to low concentrations of peptide Ag were found to be far superior to those requiring high concentrations of Ag for reducing viral burden when adoptively transferred into SCID mice. Thus it is important that we understand the mechanisms that control the requirement for peptide Ag with the long-term goal of selectively expanding these exquisitely sensitive cells in vivo. Although TCR affinity is one parameter that can affect the CTL sensitivity for Ag, we investigated whether additional mechanisms may also be involved. In studies using a TCR transgenic mouse model, we successfully generated CTL with identical TCR affinity that possess distinctly different activation requirements. Using both peptide Ag and anti-CD3 Ab to activate the CTL lines of high vs low avidity, we found that the variations in activation threshold are the result of differences in the required number of engaged TCR. Additionally, we have observed that the ratio of CD8αβ to CD8αα is significantly greater in CTL lines that are more sensitive to TCR engagement, which may contribute to the lower activation threshold of these CTL following CD3 engagement. These studies identify a novel mechanism by which the activation requirements of Ag-specific CTL are determined by demonstrating a direct correlation between the sensitivity to TCR engagement, the expression of levels CD8αβ vs αα, and the amount of peptide Ag required to reach the threshold for activation.

Optimal Colocalization of TCR and CD8 as a Novel Mechanism for the Control of Functional Avidity

The improved efficacy of high avidity CTL for clearance of virus has been well-documented. Thus, elucidation of the mechanisms that confer the increased sensitivity to peptide ligand demonstrated by high avidity CTL is critical. Using CTL lines of high and low avidity generated from a TCR transgenic mouse, we have found that functional avidity can be controlled by the expression of CD8αα vs CD8αβ and the ability of CTLs to colocalize the TCR and CD8 in the membrane. Colocalization of these molecules was mediated by lipid rafts and importantly, raft disruption resulted in the conversion of high avidity CTL into a lower functional avidity phenotype. These novel findings provide insights into the control of functional avidity in response to viral infection.

High-avidity CD8+ T cells: optimal soldiers in the war against viruses and tumors.

The primary goal of vaccination is the establishment of protective immunity. Thus there has been significant effort put toward the identification of attributes of the immune response that are associated with optimal protection. Although the number of virus-specific cells elicited is unquestionably important, recent studies have identified an additional parameter, functional avidity, as critical in determining the efficiency of viral clearance. T-cell avidity is a measure of the sensitivity of a cell to peptide antigen. High-avidity cells are those that can recognize antigen-presenting cells (APC) bearing very low levels of peptide antigen, whereas low-avidity cells require much higher numbers of peptide major histocompatibility complex (MHC) complexes in order to become activated or exert effector function. We are only now beginning to gain insights into the molecular control of avidity and the signals required for the optimal activation, expansion, and retention of high-avidity cells in vivo. This review summarizes the current knowledge regarding CD8+ T-cell avidity and explores some of the important issues that are, as of yet, unresolved.

Omega-3 Fatty Acids Ameliorate Atherosclerosis by Favorably Altering Monocyte Subsets and Limiting Monocyte Recruitment to Aortic Lesions

Objective—Fish oil, containing omega-3 fatty acids, attenuates atherosclerosis. We hypothesized that omega-3 fatty acid–enriched oils are atheroprotective through alteration of monocyte subsets and their trafficking into atherosclerotic lesions. Methods and Results—Low–density lipoprotein receptor knockout and apolipoprotein E−/− mice were fed diets containing 10% (calories) palm oil and 0.2% cholesterol, supplemented with an additional 10% palm oil, echium oil (containing 18:4 n-3), or fish oil. Compared with palm oil–fed low–density lipoprotein receptor knockout mice, echium oil and fish oil significantly reduced plasma cholesterol, splenic Ly6Chi monocytosis by ≈50%, atherosclerosis by 40% to 70%, monocyte trafficking into the aortic root by ≈50%, and atherosclerotic lesion macrophage content by 30% to 44%. In contrast, atherosclerosis and monocyte trafficking into the artery wall was not altered by omega-3 fatty acids in apolipoprotein E−/− mice; however, Ly6Chi splenic monocytes positively correlated with aortic root intimal area across all diet groups. In apolipoprotein E−/− mice, fish oil reduced the percentage of blood Ly6Chi monocytes, despite an average 2-fold higher plasma cholesterol relative to palm oil. Conclusion—The presence of splenic Ly6Chi monocytes parallels the appearance of atherosclerotic disease in both low–density lipoprotein receptor knockout and apolipoprotein E−/− mice. Furthermore, omega-3 fatty acids favorably alter monocyte subsets independently from effects on plasma cholesterol and reduce monocyte recruitment into atherosclerotic lesions.

Functional Divergence among CD103+ Dendritic Cell Subpopulations following Pulmonary Poxvirus Infection

ABSTRACT A large number of dendritic cell (DC) subsets have now been identified based on the expression of a distinct array of surface markers as well as differences in functional capabilities. More recently, the concept of unique subsets has been extended to the lung, although the functional capabilities of these subsets are only beginning to be explored. Of particular interest are respiratory DCs that express CD103. These cells line the airway and act as sentinels for pathogens that enter the lung, migrating to the draining lymph node, where they add to the already complex array of DC subsets present at this site. Here we assessed the contributions of these individual populations to the generation of a CD8+ T-cell response following respiratory infection with poxvirus. We found that CD103+ DCs were the most effective antigen-presenting cells (APC) for naive CD8+ T-cell activation. Surprisingly, we found no evidence that lymph node-resident or parenchymal DCs could prime virus-specific cells. The increased efficacy of CD103+ DCs was associated with the increased presence of viral antigen as well as high levels of maturation markers. Within the CD103+ DCs, we observed a population that expressed CD8α. Interestingly, cells bearing CD8α were less competent for T-cell activation than their CD8α− counterparts. These data show that lung-migrating CD103+ DCs are the major contributors to CD8+ T-cell activation following poxvirus infection. However, the functional capabilities of cells within this population differ with the expression of CD8, suggesting that CD103+ cells may be divided further into distinct subsets.

Cutting Edge: CD8 + T Cell Clones Possess the Potential to Differentiate into both High- and Low-Avidity Effector Cells

The property of functional avidity is recognized to be of critical importance in determining pathogen clearance. An unresolved question with regard to this property is whether distinct naive subsets exist that display inherent differences in their peptide sensitivity requirements for activation, i.e., functional avidity, or whether differences in peptide sensitivity are induced following peptide encounter. In this study, we demonstrate that naive populations that can give rise to both high- and low-avidity cells do not contain subsets that exhibit differences in the amount of peptide required for activation. Furthermore, we show that an individual T cell clone can generate both high- and low-avidity effectors. The work presented here provides the first formal demonstration that an individual cell can give rise to both high- and low-avidity progeny, suggesting that avidity modulation at the level of an individual cell may play an important role in the CD8+ T cell response generated in vivo.

IFNγ-producing, virus-specific CD8+ effector cells acquire the ability to produce IL-10 as a result of entry into the infected lung environment

It has become clear that T cells with the potential to negatively regulate the immune response are normal constituents of the immune system. These cells often mediate their effects through the production of immunosuppressive factors. At present our understanding of how these cells are generated is limited. Here we report the presence of a population of IL-10-producing, virus-specific CD8+ T cells in the lungs of mice following acute respiratory infection. These cells were only found at minimal levels in the spleen and draining lymph node; instead they were restricted primarily to the infected lung tissue. A major finding from this study is demonstration that the ability to produce IL-10 can be acquired by IFNgamma-producing effector cells following entry into the infected lung. These studies suggest IL-10 production is the result of further differentiation of an antigen-specific CD8+ T cell that is governed by signals present in infected lung tissue.

A novel CD8-independent high-avidity cytotoxic T-lymphocyte response directed against an epitope in the phosphoprotein of the paramyxovirus simian virus 5.

ABSTRACT Adoptive transfer studies have shown that cytotoxic T lymphocytes (CTL) of high avidity, capable of recognizing low levels of peptide-MHC I molecules, are more efficient at reducing viral titers than are low-avidity CTL, thus establishing CTL avidity as a critical parameter for the ability of a CTL to clear virus in vivo. It has been well documented that CTL of high avidity are relatively CD8 independent, whereas low-avidity CTL require CD8 engagement in order to become activated. In this study we have analyzed the antiviral CTL response elicited following infection with the paramyxovirus simian virus 5 (SV5). We have identified the immunodominant and subdominant CTL responses and subsequently assessed the avidity of these responses by their CD8 dependence. This is the first study in which the relationship between immunodominance and CTL avidity has been investigated. The immunodominant response was directed against an epitope present in the viral M protein, and subdominant responses were directed against epitopes present in the P, F, and HN proteins. Similarly to other CTL responses we have analyzed, the immunodominant response and the subdominant F and HN responses were comprised of both high- and low-avidity CTL. However, the subdominant response directed against the epitope present in the P protein is novel, as it is exclusively high avidity. This high-avidity response is independent of both the route of infection and expression by recombinant SV5. A further understanding of the inherent properties of P that elicit only high-avidity CTL may allow for the design of more efficacious vaccine vectors that preferentially elicit high-avidity CTL in vivo.

Dose-dependent modulation of CD8 and functional avidity as a result of peptide encounter

The generation of an optimal CD8+ cytotoxic T lymphocyte (CTL) response is critical for the clearance of many intracellular pathogens. Previous studies suggest that one contributor to an optimal immune response is the presence of CD8+ cells exhibiting high functional avidity. In this regard, CD8 expression has been shown to contribute to peptide sensitivity. Here, we investigated the ability of naive splenocytes to modulate CD8 expression according to the concentration of stimulatory peptide antigen. Our results showed that the level of CD8 expressed was inversely correlated with the amount of peptide used for the primary stimulation, with higher concentrations of antigen resulting in lower expression of both CD8α and CD8β. Importantly the ensuing CD8low and CD8high CTL populations were not the result of the selective outgrowth of naive CD8+ T‐cell subpopulations expressing distinct levels of CD8. Subsequent encounter with peptide antigen resulted in continued modulation of both the absolute level and the isoform of CD8 expressed and in the functional avidity of the responding cells. We propose that CD8 cell surface expression is not a static property, but can be modulated to ‘fine tune’ the sensitivity of responding CTL to a defined concentration of antigen.

Altered Function in CD8+ T Cells following Paramyxovirus Infection of the Respiratory Tract

ABSTRACT For many respiratory pathogens, CD8+ T cells have been shown to play a critical role in clearance. However, there are still many unanswered questions with regard to the factors that promote the most efficacious immune response and the potential for immunoregulation of effector cells at the local site of infection. We have used infection of the respiratory tract with the model paramyxovirus simian virus 5 (SV5) to study CD8+ T-cell responses in the lung. For the present study, we report that over time a population of nonresponsive, virus-specific CD8+ T cells emerged in the lung, culminating in a lack of function in ∼85% of cells specific for the immunodominant epitope from the viral matrix (M) protein by day 40 postinfection. Concurrent with the induction of nonresponsiveness, virus-specific cells that retained function at later times postinfection exhibited an increased requirement for CD8 engagement. This change was coupled with a nearly complete loss of functional phosphoprotein-specific cells, a response previously shown to be almost exclusively CD8 independent. These studies add to the growing evidence for immune dysregulation following viral infection of the respiratory tract.