John D. Altman

Phenotypic Analysis of Antigen-Specific T Lymphocytes

Identification and characterization of antigen-specific T lymphocytes during the course of an immune response is tedious and indirect. To address this problem, the peptide-major histocompatability complex (MHC) ligand for a given population of T cells was multimerized to make soluble peptide-MHC tetramers. Tetramers of human lymphocyte antigen A2 that were complexed with two different human immunodeficiency virus (HIV)-derived peptides or with a peptide derived from influenza A matrix protein bound to peptide-specific cytotoxic T cells in vitro and to T cells from the blood of HIV-infected individuals. In general, tetramer binding correlated well with cytotoxicity assays. This approach should be useful in the analysis of T cells specific for infectious agents, tumors, and autoantigens.

Initiation of Signal Transduction through the T Cell Receptor Requires the Multivalent Engagement of Peptide/MHC Ligands

While much is known about intracellular signaling events in T cells when T cell receptors (TCRs) are engaged, the mechanism by which signaling is initiated is unclear. We have constructed defined oligomers of soluble antigen-major histocompatibility complex (MHC) molecules, the natural ligands for the TCR. Using these to stimulate specific T cells in vitro, we find that agonist peptide/MHC ligands are nonstimulatory as monomers and minimally stimulatory as dimers. Similarly, a partial-agonist ligand is very weakly active as a tetramer. In contrast, trimeric or tetrameric agonist ligands that engage multiple TCRs for a sustained duration are potent stimuli. Ligand-driven formation of TCR clusters seems required for effective activation and helps to explain the specificity and sensitivity of T cells.

INDUCTION OF RAPID T CELL ACTIVATION AND TOLERANCE BY SYSTEMIC PRESENTATION OF AN ORALLY ADMINISTERED ANTIGEN

To understand how orally introduced antigen regulates peripheral immune responses, we fed cytochrome c protein to mice transgenic for the beta chain of a cytochrome c-specific TCR and followed the antigen-specific T cell responses with a cyt c/I-Ek tetramer staining reagent. We find that within 6 hr of cytochrome c administration, antigen-specific systemic T cell activation is induced, and spleen cells gain the ability to stimulate cytochrome c-specific T cell responses. Feeding multiple low doses of cytochrome c down-regulates the systemic immune response, which can be correlated with a reduction of antigen-specific T cells and not with immune deviation. These results suggest that systemic distribution of antigen contributes significantly to oral tolerance induction.

Dominance of CD8 Responses Specific for Epitopes Bound by a Single Major Histocompatibility Complex Class I Molecule during the Acute Phase of Viral Infection

ABSTRACT Cytotoxic T-lymphocyte (CTL) responses are thought to control human immunodeficiency virus replication during the acute phase of infection. Understanding the CD8+ T-cell immune responses early after infection may, therefore, be important to vaccine design. Analyzing these responses in humans is difficult since few patients are diagnosed during early infection. Additionally, patients are infected by a variety of viral subtypes, making it hard to design reagents to measure their acute-phase immune responses. Given the complexities in evaluating acute-phase CD8+ responses in humans, we analyzed these important immune responses in rhesus macaques expressing a common rhesus macaque major histocompatibility complex class I molecule (Mamu-A*01) for which we had developed a variety of immunological assays. We infected eight Mamu-A*01-positive macaques and five Mamu-A*01-negative macaques with the molecularly cloned virus SIVmac239 and determined all of the simian immunodeficiency virus-specific CD8+ T-cell responses against overlapping peptides spanning the entire virus. We also monitored the evolution of particular CD8+ T-cell responses by tetramer staining of peripheral lymphocytes as well as lymph node cells in situ. In this first analysis of the entire CD8+ immune response to autologous virus we show that between 2 and 12 responses are detected during the acute phase in each animal. CTL against the early proteins (Tat, Rev, and Nef) and against regulatory proteins Vif and Vpr dominated the acute phase. Interestingly, CD8+ responses against Mamu-A*01-restricted epitopes Tat28-35SL8 and Gag181-189CM9 were immunodominant in the acute phase. After the acute phase, however, this pattern of reactivity changed, and the Mamu-A*01-restricted response against the Gag181-189CM9 epitope became dominant. In most of the Mamu-A*01-positive macaques tested, CTL responses against epitopes bound by Mamu-A*01 dominated the CD8+ cellular immune response.

Impairment of Gag-Specific CD8+ T-Cell Function in Mucosal and Systemic Compartments of Simian Immunodeficiency Virus mac251- and Simian-Human Immunodeficiency Virus KU2-Infected Macaques

ABSTRACT The identification of several simian immunodeficiency virus mac251 (SIVmac251) cytotoxic T-lymphocyte epitopes recognized by CD8+ T cells of infected rhesus macaques carrying the Mamu-A*01 molecule and the use of peptide-major histocompatibility complex tetrameric complexes enable the study of the frequency, breadth, functionality, and distribution of virus-specific CD8+ T cells in the body. To begin to address these issues, we have performed a pilot study to measure the virus-specific CD8+ and CD4+ T-cell response in the blood, lymph nodes, spleen, and gastrointestinal lymphoid tissues of eight Mamu-A*01-positive macaques, six of those infected with SIVmac251 and two infected with the pathogenic simian-human immunodeficiency virus KU2. We focused on the analysis of the response to peptide p11C, C-M (Gag 181), since it was predominant in most tissues of all macaques. Five macaques restricted viral replication effectively, whereas the remaining three failed to control viremia and experienced a progressive loss of CD4+ T cells. The frequency of the Gag 181 (p11C, C→M) immunodominant response varied among different tissues of the same animal and in the same tissues from different animals. We found that the functionality of this virus-specific CD8+ T-cell population could not be assumed based on the ability to specifically bind to the Gag 181 tetramer, particularly in the mucosal tissues of some of the macaques infected by SIVmac251 that were progressing to disease. Overall, the functionality of CD8+ tetramer-binding T cells in tissues assessed by either measurement of cytolytic activity or the ability of these cells to produce gamma interferon or tumor necrosis factor alpha was low and was even lower in the mucosal tissue than in blood or spleen of some SIVmac251-infected animals that failed to control viremia. The data obtained in this pilot study lead to the hypothesis that disease progression may be associated with loss of virus-specific CD8+ T-cell function.

Effects of Cytotoxic T Lymphocytes (CTL) Directed against a Single Simian Immunodeficiency Virus (SIV) Gag CTL Epitope on the Course of SIVmac239 Infection

ABSTRACT Vaccine-induced cytotoxic T lymphocytes (CTL) have been implicated in the control of virus replication in simian immunodeficiency virus (SIV)-challenged and simian-human immunodeficiency virus-challenged macaques. Therefore, we wanted to test the impact that vaccine-induced CTL responses against an immunodominant Gag epitope might have in the absence of other immune responses. By themselves, these strong CTL responses failed to control SIVmac239 replication.

Enumeration and Characterization of Memory Cells in the TH Compartment

There is a great deal of interest in understanding how helper T cells differentiate in vivo and exert their regulatory role on a developing, immune response. Essential to development of protective immunity is the development of memory T cells. To study memory T cells in vivo we first need the means to identify and characterize these cells as they develop in their complex microenvironments. We have developed a method which allows us to directly purify both primary and memory helper T cells from the draining lymph nodes of mice as they respond to pigeon cytochrome c in vivo. Junctional sequences from these populations and from individual T cells show a strong selection for CDR3 length and residues characteristic of antigen binding. Overall these studies support a model of progressive clonal maturation with the memory T cell repertoire being more homogeneous than that of the primary response. There is some suggestion that affinity maturation may take place after repeated immunization, but on a more modest scale than that seen for antibodies. Finally we present the use of two new technologies that promise to greatly expand the analysis of immune responses in vivo. The use of flow cytometry with simultaneous detection of five and six fluorescence parameters helps to reliably resolve rare subsets of antigen-specific cells in order to understand the progression of their differentiation in vivo. Lastly, we have developed peptide/MHC tetramers as a new class of staining reagent that has wide applicability in the study of T cell responses in vivo.

TRACKING ANTIGEN-SPECIFIC HELPER T CELL RESPONSES

T-cell receptor transgenic animals provide an excellent source of T cells for the analysis of antigen-specific helper T-cell development. Alternatively, studies in normal animals continue to focus on specific immune responses dominated by T cells using restricted sets of antigen receptors. These complementary strategies provide direct access to the dynamics of helper T-cell differentiation in vivo.

Design and In Vivo Immunogenicity of a Polyvalent Vaccine Based on SIVmac Regulatory Genes

Most vaccine modalities for human immunodeficiency virus type 1 (HIV-1) tested for immunogenicity and efficacy in the SIVmac (simian immunodeficiency virus) macaque model do not include the viral regulatory proteins. Because viral regulatory proteins are expressed early during the virus life cycle and represent an additional source of antigens, their inclusion as a vaccine component may increase the overall virus-specific immune response in vaccinees. However, at least two of the early proteins, Tat and Nef, may be immunosuppressive, limiting their usefulness as components of an SIV vaccine. We have constructed a polyvalent chimeric protein in which the open reading frames for Tat and Nef have been reassorted and the nuclear localization sequence for Tat and Rev and the myristoylation site for Nef have been removed. The resulting DNA plasmid (pDNA-SIV-Retanef) (pDNA-SIV-RTN) encodes a protein of 55 kDa (Retanef) that localizes at the steady state in the cytoplasma of transfected cells. Both the DNA-SIV-RTN and the highly attenuated recombinant poxvirus vector NYVAC-SIV-RTN were demonstrated to be immunogenic in SIVmac251-infected macaques treated with ART as well as in naive macaques. An equivalent strategy may be used for the generation of polyvalent antigens encoding the regulatory proteins in a HIV-1 vaccine candidate.