Frances M. Gotch

The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides

A proportion of cytotoxic T lymphocytes (CTL) responding to infection by influenza recognize target cells that express the viral nucleoprotein. Recent work showed that CTL can recognize short overlapping regions of large nucleoprotein fragments expressed in transfected L cells. This led to the suggestion that CTL recognize segmental epitopes of denatured or degraded proteins in a similar way to helper T cells. One corollary of this idea is that CTL should recognize appropriate short peptides on the target cell surface. We demonstrate that the epitopes of nucleoprotein recognized by CTL in association with class I molecules of the major histocompatibility complex in both mouse and man can be defined with short synthetic peptides derived from the nucleoprotein sequence.

Cytotoxic T cells recognize fragments of the influenza nucleoprotein

Recent work has shown that a major population of murine influenza A specific cytotoxic T lymphocytes (CTL) recognize the viral nucleoprotein. In order to investigate the mechanism by which this nonglycoprotein component of the virus is recognized by CTL, a series of deletion mutants of an A virus NP gene were studied. The results showed that CTL recognize three distinct epitopes of the NP molecule. Both N- and C-terminal fragments of the protein are transported, independently of each other, to the site of recognition by CTL. These findings imply that a mechanism may exist for transport to the cell surface and presentation to CTL, of viral proteins and protein fragments that lack defined signal sequences.

HIV-specific cytotoxic T-cell activity in an HIV-exposed but uninfected infant

The factors necessary for protective immunity against HIV-1 are unknown. Important information about these factors should come from study of people at high risk of HIV infection who have not apparently become infected. Among these are the estimated 60-85% of children who may be exposed in utero or perinatally to HIV-1 but do not become infected. We observed the transient appearance of HIV-specific cytotoxic T-lymphocyte (CTL) activity in a baby born to HIV-1-infected parents, in whom all standard markers of infection remained negative. These findings suggest that HIV-specific CTLs may be a marker for recently exposed, but uninfected, individuals.

Identification of conserved antigenic components for a cytotoxic T lymphocyte-inducing vaccine against malaria

Several cellular and humoral mechanisms probably play a role in natural immunity to Plasmodium falciparum malaria, but the development of an effective vaccine has been impeded by uncertainty as to which antigens are targeted by protective immune responses. Experimental models of malaria have shown that cytotoxic T lymphocytes (CTL) which kill parasite-infected hepatocytes can provide complete protective immunity against certain species of Plasmodium in mice, and studies in The Gambia have provided indirect evidence that CTL play a protective role against P falciparum in humans. By using an HLA-based approach, termed reverse immunogenetics, we have previously identified peptide epitopes for CTL in liver-stage antigen-1 and the circumsporozoite protein of P falciparum. We have extended this work to identify CTL epitopes for HLA class I antigens that are found in most individuals from Caucasian and African populations. Most of these epitopes are in conserved regions of P falciparum. CTL peptide epitopes were found in a further two antigens, thrombospondin-related anonymous protein and sporozoite threonine and asparagine rich protein, indicating that a subunit vaccine designed to induce a protective CTL response may need to include parts of several parasite antigens. However, CTL levels in both children with malaria and in semi-immune adults from an endemic area were low suggesting that boosting these low levels by immunisation might provide substantial or even complete protection against infection and disease.

Recognition of influenza A virus nucleoprotein by human cytotoxic T lymphocytes

A recombinant vaccinia virus (NP-VAC) containing cDNA corresponding to segment 5, the nucleoprotein (NP) gene of influenza A/PR/8/34 virus was used to examine the specificity of human influenza virus immune cytotoxic T lymphocytes (CTL). Effector cell preparations from two donors recognized autologous lymphocytes that had been infected with NP-VAC. Lysis was specific because cells infected with vaccinia virus were not killed and recognition was HLA-restricted. In one donor, the influenza virus-specific CTL response changed with time so that his effector cells no longer recognized autologous lymphocytes infected with NP-VAC. However, a component that was NP-specific remained because these CTL lysed the more sensitive autologous B lymphoblastoid cells that had been infected with NP-VAC. In four other donors, no NP-specific CTL response could be detected using autologous lymphocyte targets. Thus NP, an internal virus protein, is one antigen that is recognized by human influenza A virus-specific CTL, but it is likely that other individual virus components contribute to the total CTL response.

The MHC E locus in macaques is polymorphic and is conserved between macaques and humans

Although the functions of the molecules encoded by the classical MHC class I loci are well defined, no function has been ascribed to the molecules encoded by the non-classical MHC class I loci. To investigate the evolution and conservation of the non-classical loci, we cloned and sequenced HLA-E homologues in macaques. We isolated four E locus alleles from five rhesus monkeys and two E locus alleles from one cynomolgus monkey, which indicated that the E locus in macaques is polymorphic. We also compared the rate of nucleotide substitution in the second intron of the macaque and human E locus alleles with that of exons two and three. The rate of nucleotide substitution was significantly higher in the introns, which suggested that the E locus has evolved under selective pressure. Additionally, comparison of the rates of synonymous and non-synonymous substitutions in the peptide binding region versus the remainder of the molecule suggested that the codons encoding the amino acids in the peptide binding region had been conserved in macaques and humans over the 36 million years since macaques and humans last shared a common ancestor.

Characterization of the HLA-A2.2 Subtype: T Cell Evidence for Further Heterogeneity

Five blood donors were identified whose HLA-A2 is different from the common HLA-A2. Their A2 molecule (A2.2) had a more basic isoelectric point than normal A2 (A2.1). Cytotoxic T lymphocytes (CTL) restricted. by HLA-A2.1, specific for influenza A and Epstein-Barr viruses, failed to lyse virus-infected target cells with HLA-A2.2. Identical patterns were obtained with both viruses. CTL from four of the A2.2-positive donors recognized target cells prepared from others in the group that shared only the HLA-A2.2 antigen. The A2.2 antigen from one donor seemed to be different in that target cells were not recognized by CTL from donors with the normal A2.1 nor with basic A2.2. There seems, therefore, to be heterogeneity within the HLA-A2.2 subtype.

Recognition of the PB1, neuraminidase, and matrix proteins of influenza virus A/NT/60/68 by cytotoxic T lymphocytes

We have investigated the recognition of the PB1, neuraminidase, and matrix (M1) proteins of influenza virus A/NT/60/68 (H3N2 subtype) by secondary in vitro stimulated polyclonal cytotoxic T lymphocyte (CTL) populations. While these three proteins have different functions and cellular locations, they can all be recognized as target antigens. However, the immunogenicity of these proteins for CTLs is under strict genetic control. Thus, PB1 protein is recognized as a cross-reactive target antigen by CTLs raised in CBA (H-2k) but not BALB/c (H-2d) mice. CBA, but not BALB/c mice, also generate a low-level CTL response to the neuraminidase. This latter response was only detectable following in vivo priming of CBA mice with a recombinant vaccinia virus expressing neuraminidase (N2-VACC). The matrix protein, expressed from recombinant vaccinia virus M-VACC, was not recognized as an antigen by CTL generated from either CBA or BALB/c strains of mice. By contrast, human HLA-A2-restricted influenza virus-specific CTLs were shown to recognize this matrix protein as a target antigen. Endogenous expression of as little as 90 amino acids of the matrix protein was sufficient to render target cells susceptible to lysis by such CTLs.

Recognition of Influenza A Virus by Human Cytotoxic T Lymphocytes

Cytotoxic T lymphocytes (CTL) are an important component of the immune system carrying the CD8 surface glycoprotein and forming a significant part of the total lymphoid population. The role of CTL appears to be to kill cells of the body that differ from normal and this occurs during infection with intracellular parasites, particularly viruses. It is likely that CTL have evolved as a major defence mechanism against virus infections. CTL are detected in vitro by their ability to kill infected target cells that are labelled with chromium-51. There is evidence that CTL lyse infected cells in vivo (1) and they may also act by releasing lymphokines such as Interferon-Gamma (2). When infected cells are killed they can no longer replicate new virus particles, and if the cell is already doomed this is an effective way of controlling a virus infection. However the constraints on CTL antigen receptors preclude them from recognising and neutralising virus particules directly. The natural role of CTL therefore is likely to be quite distinct from that of the antibody response.

Binding and Functional Analysis of the Workshop T Cell Specific Set of Monoclonal Antibodies

One hundred and fifty-nine samples were studied in the T cell set of monoclonal antibodies. The first part of this report analyzes the binding data obtained following the protocol of the workshop. The second part describes the use of these antibodies in blocking virus-specific HLA-restricted cytotoxic T lymphocyte activity and natural killer cell-mediated lysis, stimulation of T cell proliferation, co-capping with a standard CD3 specific antibody, and blocking of E-rosette formation. Combining the two types of analysis has allowed most of the antibodies to be placed into known CD clusters. Although no new clusters were clearly defined, a number of individual antibodies gave unusual patterns of reactivity or functional properties.