Stephen J. Turner

Influenza and the challenge for immunology

The continued westward dissemination of H5N1 influenza A viruses in avian populations and the nearly 50% mortality rate of humans infected with H5N1 are a source of great international concern. A mutant H5N1 virus with the capability to spread rapidly between humans could cause a global catastrophe. Governments have reacted by developing national response plans, stockpiling antiviral drugs and speeding up the development and approval of vaccines. Here we summarize what is known about the interaction between influenza A viruses and the mammalian host response, specifically emphasizing issues that might be of interest to the broader immunology community.

Measuring the diaspora for virus-specific CD8+ T cells.

The CD8+ T cell diaspora has been analyzed after secondary challenge with an influenza A virus that replicates only in the respiratory tract. Numbers of DbNP366- and DbPA224-specific CD8+ T cells were measured by tetramer staining at the end of the recall response, then followed sequentially in the lung, lymph nodes, spleen, blood, and other organs. The extent of clonal expansion did not reflect the sizes of the preexisting memory T cell pools. Although the high-frequency CD8+ tetramer+ populations in the pneumonic lung and mediastinal lymph nodes fell rapidly from peak values, the “whole mouse” virus-specific CD8+ T cell counts decreased only 2-fold over the 4 weeks after infection, then subsided at a fairly steady rate to reach a plateau at about 2 months. The largest numbers were found throughout in the spleen, then the bone marrow. The CD8+DbNP366+ and CD8+DbPA224+ sets remained significantly enlarged for at least 4 months, declining at equivalent rates while retaining the nucleoprotein > acid polymerase immunodominance hierarchy characteristic of the earlier antigen-driven phase. Lowest levels of the CD69 “activation marker” were detected consistently on virus-specific CD8+ T cells in the blood, then the spleen. Those in the bone marrow and liver were intermediate, and CD69hi T cells were very prominent in the regional lymph nodes and the nasal-associated lymphoid tissue. Any population of “resting” CD8+ memory T cells is thus phenotypically heterogeneous, widely dispersed, and subject to broad homeostatic and local environmental effects irrespective of epitope specificity or magnitude.

Structural determinants of T-cell receptor bias in immunity.

Antigen-specific T-cell responses induced by infection, transplantation, autoimmunity or hypersensitivity are characterized by cells expressing biased profiles of T-cell receptors (TCRs) that are selected from a diverse, naive repertoire. Here, we review the evidence for these TCR biases, focusing on crystallographic analysis of the structural constraints that determine the binding of a TCR to its ligand and the persistence of certain TCRs in an immune repertoire. We discuss the ways in which diversity in a selected TCR repertoire can contribute to protective immunity and the implications of this for vaccine design and immunotherapy.

Differential Antigen Presentation Regulates the Changing Patterns of CD8+ T Cell Immunodominance in Primary and Secondary Influenza Virus Infections

The specificity of CD8+ T cell responses can vary dramatically between primary and secondary infections. For example, NP366–374/Db- and PA224–233/Db-specific CD8+ T cells respond in approximately equal numbers to a primary influenza virus infection in C57BL/6 mice, whereas NP366–374/Db-specific CD8+ T cells dominate the secondary response. To investigate the mechanisms underlying this changing pattern of immunodominance, we analyzed the role of antigen presentation in regulating the specificity of the T cell response. The data show that both dendritic and nondendritic cells are able to present the NP366–374/Db epitope, whereas only dendritic cells effectively present the PA224–233/Db epitope after influenza virus infection, both in vitro and in vivo. This difference in epitope expression favored the activation and expansion of NP366–374/Db-specific CD8+ memory T cells during secondary infection. The data also show that the immune response to influenza virus infection may involve T cells specific for epitopes, such as PA224–233/Db, that are poorly expressed at the site of infection. In this regard, vaccination with the PA224–233 peptide actually had a detrimental effect on the clearance of a subsequent influenza virus infection. Thus, differential antigen presentation impacts both the specificity of the T cell response and the efficacy of peptide-based vaccination strategies.

T Cell Antigen Receptor Recognition of Antigen-Presenting Molecules

The Major Histocompatibility Complex (MHC) locus encodes classical MHC class I and MHC class II molecules and nonclassical MHC-I molecules. The architecture of these molecules is ideally suited to capture and present an array of peptide antigens (Ags). In addition, the CD1 family members and MR1 are MHC class I-like molecules that bind lipid-based Ags and vitamin B precursors, respectively. These Ag-bound molecules are subsequently recognized by T cell antigen receptors (TCRs) expressed on the surface of T lymphocytes. Structural and associated functional studies have been highly informative in providing insight into these interactions, which are crucial to immunity, and how they can lead to aberrant T cell reactivity. Investigators have determined over thirty unique TCR-peptide-MHC-I complex structures and twenty unique TCR-peptide-MHC-II complex structures. These investigations have shown a broad consensus in docking geometry and provided insight into MHC restriction. Structural studies on TCR-mediated recognition of lipid and metabolite Ags have been mostly confined to TCRs from innate-like natural killer T cells and mucosal-associated invariant T cells, respectively. These studies revealed clear differences between TCR-lipid-CD1, TCR-metabolite-MR1, and TCR-peptide-MHC recognition. Accordingly, TCRs show remarkable structural and biological versatility in engaging different classes of Ag that are presented by polymorphic and monomorphic Ag-presenting molecules of the immune system.

Hierarchies in Cytokine Expression Profiles for Acute and Resolving Influenza Virus-Specific CD8+ T Cell Responses: Correlation of Cytokine Profile and TCR Avidity

The development and resolution phases of influenza-specific CD8+ T cell cytokine responses to epitopes derived from the viral nucleoprotein (DbNP366) and acid polymerase (DbPA224) were characterized in C57BL/6J mice for a range of anatomical compartments in the virus-infected lung and lymphoid tissue. Lymphocyte numbers were measured by IFN-γ expression following stimulation with peptide, while the quality of the response was determined by the intensity of staining and the distribution of CD8+ T cells producing TNF-α and IL-2. Both the levels of expression and the prevalence of TNF-α+ and IL-2+ cells reflected the likely Ag load, with clear differences being identified for populations from the alveolar space vs the lung parenchyma. Irrespective of the site or time of T cell recovery, IL-2+ cells were consistently found to be a subset of the TNF-α+ population which was, in turn, contained within the IFN-γ+ set. The capacity to produce IL-2 may thus be considered to reflect maximum functional differentiation. The hierarchy in cytokine expression throughout the acute phase of the primary and secondary response tended to be DbPA224 > DbNP366. Both elution studies with the cognate tetramers and experiments measuring CD8β coreceptor dependence for peptide stimulation demonstrated the same DbPA224 > DbNP366 profile for TCR avidity. Overall, the quality of any virus-specific CD8+ T cell response appears variously determined by the avidity of the TCR-pMHC interaction, the duration and intensity of Ag stimulation characteristic of the particular tissue environment, and the availability of CD4+ T help.

Analysis of clonotype distribution and persistence for an influenza virus-specific CD8+ T cell response.

The spectrum of TCR V beta usage is compared for primary and recall CD8(+)D(b)PA(224)(+) T cell responses in mice with influenza pneumonia. Single-cell RT-PCR established that the same clonotypes were present in the lymphoid tissue and in the virus-infected lung. Longitudinal analysis indicated that the memory TCR repertoire reflects the primary response, with no decrease in diversity prior to (or after) secondary challenge. The re-engagement of memory T cells looked to be stochastic in this localized, transient infection. Analysis of clonotypes from the blood, spleen, regional lymph nodes, bone marrow, lung, and liver over a 200 day interval showed no evidence of selective localization or loss. The long-term distribution of memory T cells seemed to be essentially random.

Primary CTL response magnitude in mice is determined by the extent of naive T cell recruitment and subsequent clonal expansion

CD8+ T cell responses to viral infection are characterized by the emergence of dominant and subdominant CTL populations. The immunodominance hierarchies of these populations are highly reproducible for any given spectrum of virus-induced peptide-MHCI complexes and are likely determined by multiple factors. Recent studies demonstrate a direct correlation between naive epitope-specific CD8+ T cell precursor (CTLp) frequency and the magnitude of the response after antigen challenge. Thus, the number of available precursors in the naive pool has emerged as a key predictor of immunodominance. In contrast to this, we report here no consistent relationship between CTLp frequency and the subsequent magnitude of the immune response for 4 influenza virus-derived epitopes following intranasal infection of mice with influenza A virus. Rather, the characteristic, antigen-driven T cell immunodominance hierarchy was determined by the extent of recruitment from the available pool of epitope-specific precursors and the duration of their continued expansion over the course of the infection. These findings suggest possibilities for enhancing protective immune memory by maximizing both the size and diversity of typically subdominant T cell responses through rational vaccine design.

Protection and compensation in the influenza virus-specific CD8+ T cell response

Influenza virus-specific CD8+ T cells generally recognize peptides derived from conserved, internal proteins that are not subject to antibody-mediated selection pressure. Prior exposure to any one influenza A virus (H1N1) can prime for a secondary CD8+ T cell response to a serologically different influenza A virus (H3N2). The protection afforded by this recall of established CD8+ T cell memory, although limited, is not negligible. Key characteristics of primary and secondary influenza-specific host responses are probed here with recombinant viruses expressing modified nucleoprotein (NP) and acid polymerase (PA) genes. Point mutations were introduced into the epitopes derived from the NP and PA such that they no longer bound the presenting H2Db MHC class I glycoprotein, and reassortant H1N1 and H3N2 viruses were made by reverse genetics. Conventional (C57BL/6J, H2b, and Ig+/+) and Ig-/- (μMT) mice were more susceptible to challenge with the single NP [HKx31 influenza A virus (HK)-NP] and PA (HK-PA) mutants, but unlike the Ig-/- mice, Ig+/+ mice were surprisingly resistant to the HK-NP/-PA double mutant. This virus was found to promote an enhanced IgG response resulting, perhaps, from the delayed elimination of antigen-presenting cells. Antigen persistence also could explain the increase in size of the minor KbPB1703 CD8+ T cell population in mice infected with the mutant viruses. The extent of such compensation was always partial, giving the impression that any virus-specific CD8+ T cell response operates within constrained limits. It seems that the relationship between protective humoral and cellular immunity is neither simple nor readily predicted.

Combined NKT cell activation and influenza virus vaccination boosts memory CTL generation and protective immunity

Current influenza A virus vaccines do not generate significant immunity against serologically distinct influenza A virus subtypes and would thus be ineffective in the face of a pandemic caused by a novel variant emerging from, say, a wildlife reservoir. One possible solution would be to modify these vaccines so that they prime cross-reactive CD8+ cytotoxic T lymphocytes (CTL) cell-mediated immunity directed at conserved viral epitopes. A further strategy is to use novel adjuvants, such as the immunomodulatory glycolipid α-galactosylceramide (α-GalCer). We show here that giving α-GalCer with an inactivated influenza A virus has the paradoxical effect of diminishing acute CTL immunity via natural killer T (NKT) cell-dependent expression of indoleamine 2,3-dioxygenase (IDO), an important mediator of immune suppression, while at the same time promoting the survival of long-lived memory CTL populations capable of boosting protection against heterologous influenza A virus challenge. This enhancement of memory was likely due to the α-GalCer-induced upregulation of prosurvival genes, such as bcl-2, and points to the potential of α-GalCer as an adjuvant for promoting optimal, vaccine-induced CD8+ T cell memory.