Geraldine Gillespie

Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections

The viruses HIV-1, Epstein–Barr virus (EBV), cytomegalovirus (CMV) and hepatitis C virus (HCV) are characterized by the establishment of lifelong infection in the human host, where their replication is thought to be tightly controlled by virus-specific CD8+ T cells. Here we present detailed studies of the differentiation phenotype of these cells, which can be separated into three distinct subsets based on expression of the costimulatory receptors CD28 and CD27. Whereas CD8+ T cells specific for HIV, EBV and HCV exhibit similar characteristics during primary infection, there are significant enrichments at different stages of cellular differentiation in the chronic phase of persistent infection according to the viral specificity, which suggests that distinct memory T-cell populations are established in different virus infections. These findings challenge the current definitions of memory and effector subsets in humans, and suggest that ascribing effector and memory functions to subsets with different differentiation phenotypes is no longer appropriate.

Large clonal expansions of CD8+ T cells in acute infectious mononucleosis.

Primary infection with Epstein–Barr virus often results in the clinical syndrome of acute infectious mononucleosis (glandular fever). This illness is characterized by a striking lymphocytosis, the nature of which has been controversial. We show that large monoclonal or oligoclonal populations of CD8+ T cells account for a significant proportion of the lymphocytosis and provide molecular evidence that these populations have been driven by antigen. The results suggest that the selective and massive expansion of a few dominant clones of CD8+ T cells is an important feature of the primary response to this virus.

Functional Heterogeneity and High Frequencies of Cytomegalovirus-Specific CD8+ T Lymphocytes in Healthy Seropositive Donors

ABSTRACT Human cytomegalovirus (HCMV) infection is largely asymptomatic in the immunocompetent host, but remains a major cause of morbidity in immunosuppressed individuals. Using the recently described technique of staining antigen-specific CD8+ T cells with peptide-HLA tetrameric complexes, we have demonstrated high levels of antigen-specific cells specific for HCMV peptides and show that this may exceed 4% of CD8+ T cells in immunocompetent donors. Moreover, by staining with tetramers in combination with antibodies to cell surface markers and intracellular cytokines, we demonstrate functional heterogeneity of HCMV-specific populations. A substantial proportion of these are effector cytotoxic T lymphocytes, as demonstrated by their ability to lyse peptide-pulsed targets in “fresh” killing assays. These data suggest that the immune response to HCMV is periodically boosted by a low level of HCMV replication and that sustained immunological surveillance contributes to the maintenance of host-pathogen homeostasis. These observations should improve our understanding of the immunobiology of persistent viral infection.

Tetrameric HLA class I-minor histocompatibility antigen peptide complexes demonstrate minor histocompatibility antigen-specific cytotoxic T lymphocytes in patients with graft-versus-host disease.

Graft-versus-host disease (GvHD) is a chief complication of allogeneic bone marrow transplantation. In HLA-identical bone marrow transplantation, GvHD may be induced by disparities in minor histocompatibility antigens (mHags) between the donor and the recipient, with the antigen being present in the recipient and not in the donor. Cytotoxic T lymphocytes (CTLs) specific for mHags of the recipients can be isolated from the blood of recipients with severe GvHD (ref. 3). A retrospective study demonstrated an association between mismatch for mHags HA-1, -2, -4 and -5 and the occurrence of GvHD in adult recipients of bone marrow from HLA genotypically identical donors. Tetrameric HLA-peptide complexes have been used to visualize and quantitate antigen-specific CTLs in HIV-infected individuals and during Epstein-Barr virus and lymphocytic choriomeningitis virus infections. Here we show the direct ex vivo visualization of mHag-specific CTLs during GvHD using tetrameric HLA-class and I-mHag HA-1 and HY peptide complexes. In the peripheral blood of 17 HA-1 or HY mismatched marrow recipients, HA-1- and HY-specific CTLs were detected as early as 14 days after bone marrow transplantation. The tetrameric complexes demonstrated a significant increase in HA-1- and HY-specific CTLs during acute and chronic GvHD, which decreased after successful GvHD treatment. HLA class I–mHag peptide tetramers may serve as clinical tools for the diagnosis and monitoring of GvHD patients.

Induction of Multifunctional Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T Cells Capable of Proliferation in Healthy Subjects by Using a Prime-Boost Regimen of DNA- and Modified Vaccinia Virus Ankara-Vectored Vaccines Expressing HIV-1 Gag Coupled to CD8+ T-Cell Epitopes

ABSTRACT A double-blind randomized phase I trial was conducted in human immunodeficiency virus type 1 (HIV-1)-negative subjects receiving vaccines vectored by plasmid DNA and modified vaccinia virus Ankara (MVA) expressing HIV-1 p24/p17 gag linked to a string of CD8+ T-cell epitopes. The trial had two groups. One group received either two doses of MVA.HIVA (2× MVA.HIVA) (n = 8) or two doses of placebo (2× placebo) (n = 4). The second group received 2× pTHr.HIVA followed by one dose of MVA.HIVA (n = 8) or 3× placebo (n = 4). In the pTHr.HIVA-MVA.HIVA group, HIV-1-specific T-cell responses peaked 1 week after MVA.HIVA vaccination in both ex vivo gamma interferon (IFN-γ) ELISPOT (group mean, 210 spot-forming cells/106 cells) and proliferation (group mean stimulation index, 37), with assays detecting positive responses in four out of eight and five out of eight subjects, respectively. No HIV-1-specific T-cell responses were detected in either assay in the 2× MVA.HIVA group or subjects receiving placebo. Using a highly sensitive and reproducible cultured IFN-γ ELISPOT assay, positive responses mainly mediated by CD4+ T cells were detected in eight out of eight vaccinees in the pTHr.HIVA-MVA.HIVA group and four out of eight vaccinees in the 2× MVA.HIVA group. Importantly, no false-positive responses were detected in the eight subjects receiving placebo. Of the 12 responders, 11 developed responses to previously identified immunodominant CD4+ T-cell epitopes, with 6 volunteers having responses to more than one epitope. Five out of 12 responders also developed CD8+ T-cell responses to the epitope string. Induced T cells produced a variety of anti-viral cytokines, including tumor necrosis factor alpha and macrophage inflammatory protein 1β. These data demonstrate that prime-boost vaccination with recombinant DNA and MVA vectors can induce multifunctional HIV-1-specific T cells in the majority of vaccinees.

Mature CD8(+) T lymphocyte response to viral infection during fetal life.

Immunization of newborns against viral infections may be hampered by ineffective CD8(+) T cell responses. To characterize the function of CD8(+) T lymphocytes in early life, we studied newborns with congenital human cytomegalovirus (HCMV) infection. We demonstrate that HCMV infection in utero leads to the expansion and the differentiation of mature HCMV-specific CD8(+) T cells, which have similar characteristics to those detected in adults. High frequencies of HCMV-specific CD8(+) T cells were detected by ex vivo tetramer staining as early as after 28 weeks of gestation. During the acute phase of infection, these cells had an early differentiation phenotype (CD28(-)CD27(+)CD45RO(+), perforin(low)), and they acquired a late differentiation phenotype (CD28(-)CD27(-)CD45RA(+), perforin(high)) during the course of the infection. The differentiated cells showed potent perforin-dependent cytolytic activity and produced antiviral cytokines. The finding of a mature and functional CD8(+) T cell response to HCMV suggests that the machinery required to prime such responses is in place during fetal life and could be used to immunize newborns against viral pathogens.

Cutting Edge: Allele-Specific and Peptide-Dependent Interactions between KIR3DL1 and HLA-A and HLA-B

Although it is clear that KIR3DL1 recognizes Bw4+ HLA-B, the role of Bw4+ HLA-A allotypes as KIR3DL1 ligands is controversial. We therefore examined the binding of tetrameric HLA-A and –B complexes, including HLA*2402, a common Bw4+ HLA-A allotype, to KIR3DL1*001, *005, *007, and *1502 allotypes. Only Bw4+ tetramers bound KIR3DL1. Three of four HLA-A*2402 tetramers bound one or more KIR3DL1 allotypes and all four KIR3DL1 allotypes bound to one or more HLA-A*2402 tetramers, but with different binding specificities. Only KIR3DL1*005 bound both HLA-A*2402 and HLA-B*5703 tetramers. HLA-A*2402-expressing target cells were resistant to lysis by NK cells expressing KIR3DL1*001 or *005. This study shows that HLA-A*2402 is a ligand for KIR3DL1 and demonstrates how the binding of KIR3DL1 to Bw4+ ligands depends upon the bound peptide as well as HLA and KIR3DL1 polymorphism.

High viral burden in the presence of major HIV-specific CD8(+) T cell expansions: evidence for impaired CTL effector function.

To investigate the effect of HIV‐specific CD8+ T cells on viral plasma load and disease progression, we enumerated HLA‐A2‐, B8‐ and B57‐restricted CD8+ T cells directed against several HIV epitopes in a total of 54 patients by the use of tetrameric HLA‐peptide complexes. In patients with high CD4+ T cell numbers, HIV‐specific tetramer+ cells inversely correlated with viral load. Patients with CD4+ T cell numbers below 400/μ l blood, however, carried high viral load despite frequently having high tetramer+ T cell numbers. This lack of correlation between viral load and tetramer+ cells did not result from viral escape variants, as in only 4 of 13 patients, low frequencies of viruses with mutated epitopes were observed. In 15 patients we measured CD8+ T cell antigen responsiveness to HIV peptide stimulation in vitro. FACS analyses showed differential IFN‐γ production of the tetramer+ cells, and this proportion of IFN‐γ‐producing tetramer+ cells correlated with AIDS‐free survival and with T cell maturation to the CD27– effector stage. These data show that most HIV‐infected patients have sustained HIV‐specific T cell expansions but many of these cells seem not to be functional, leaving the patient with high numbers of non‐functional virus‐specific CD8+ T cells in the face of high viral burden.

Interleukin 10–Mediated Immunosuppression by a Variant CD4 T Cell Epitope of Plasmodium falciparum

The immunodominant CD4 T cell epitope region, Th2R, of the circumsporozoite protein of Plasmodium falciparum is highly polymorphic. Such variation might be utilized by the parasite to escape from or interfere with CD4 T cell effector functions. Here, we show that costimulation with naturally occurring altered peptide ligands (APL) can induce a rapid change from IFNgamma production to the immunosuppressive mediator interleukin 10 (IL-10). This mechanism may contribute to the low levels of T cell responses observed to this pathogen in malaria-endemic areas.

Cross-reactive cytotoxic T lymphocytes against a HIV-1 p24 epitope in slow progressors with B*57.

Objectives To determine whether CD8 T lymphocytes from HIV-1-infected patients expressing B*5701 and B*5703 show broad cross-reactivity against different variants of a conserved p24 epitope, which might account for the good prognosis of HIV-1-infected individuals with HLA-B*57. Design B*5701+ and B*5703+ were recruited from Nairobi, Kenya and from Oxford, UK. All patients had been HIV positive for at least 8 years and could be categorized as slow progressors. Methods CD8 cytotoxic T cell clones were generated from B*5701+ and B*5703+ donors and tested for their ability to recognize clade variants of an index p24 epitope in standard cytolytic assays. Cross-reactive responses in freshly isolated peripheral blood mononuclear cells (PBMC) were assessed by interferon-γ (IFNγ) production and tetramer binding. Results Broad cross-clade reactivity for both cytolysis and tetramer binding was observed in CD8 T cell clones from patients harbouring the index epitope sequence. Patterns of cross-reactivity were similar in freshly isolated PBMC but varied between individuals in terms of strength and breath of responses generated. One common variant induced an unusual response with tetramer binding but often failed to induce IFNγ production, and another was a weak stimulator of both IFNγ and cytolytic activity. Conclusion B*5701+ and B5703+ donors demonstrate broad functional cross-reactivity to both common and rare variants of a dominant p24 epitope, which could be relevant to the association of B*57 alleles with slow progression to AIDS.