P09-15. Selection of higher avidity HLA-restricted T cell responses as a viral adaptation strategy

Loss of immune reactivity due to HIV mutational escape is well described. Data generated from a large population-based study (n>800) suggested that certain CD8 T cell epitopes are created as a result of HIV adaptation and are associated with enhanced viral replication. Here we sought to investigate the HLA-restricted T-cell responses associated with seven such adaptations

DR*W201/P65 Tetramer Visualization of Epitope-Specific CD4 T-Cell during M. tuberculosis Infection and Its Resting Memory Pool after BCG Vaccination

In vivo kinetics and frequencies of epitope-specific CD4 T cells in lymphoid compartments during M. tuberculosis infection and their resting memory pool after BCG vaccination remain unknown.Macaque DR*W201 tetramer loaded with Ag85B peptide 65 was developed to directly measure epitope-specific CD4 T cells in blood and tissues form macaques after M. tuberculosis infection or BCG vaccination via direct staining and tetramer-enriched approach. The tetramer-based enrichment approach showed that P65 epitope-specific CD4 T cells emerged at mean frequencies of approximately 500 and approximately 4500 per 10(7) PBL at days 28 and 42, respectively, and at day 63 increased further to approximately 22,000/10(7) PBL after M. tuberculosis infection. Direct tetramer staining showed that the tetramer-bound P65-specific T cells constituted about 0.2-0.3% of CD4 T cells in PBL, lymph nodes, spleens, and lungs at day 63 post-infection. 10-fold expansion of these tetramer-bound epitope-specific CD4 T cells was seen after the P65 peptide stimulation of PBL and tissue lymphocytes. The tetramer-based enrichment approach detected BCG-elicited resting memory P65-specific CD4 T cells at a mean frequency of 2,700 per 10(7) PBL.Our work represents the first elucidation of in vivo kinetics and frequencies for tetramer-bound epitope-specific CD4 T cells in the blood, lymphoid tissues and lungs over times after M. tuberculosis infection, and BCG immunization

Identification and HLA-Tetramer-Validation of Human CD4(+) and CD8(+) T Cell Responses against HCMV Proteins IE1 and IE2

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4+ and CD8+ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8+ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4+ and CD8+ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4+ and CD8+ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4+ and 44 CD8+ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy

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Document(en) uit de collectie Chemische ProcestechnologieDelftChemTechApplied Science

Identification of multiple novel HIV-specific T-cell epitopes using HLA-associated HIV polymorphisms

HIV adapts to T-cell immunity by mutating within or near HLA-restricted CD8-T cell epitopes and therefore HLA allele-specific HIV polymorphisms in a human population mark the immunogenic targets of CD8 T-cell responses relevant to that population in-vivo. We used the imprints of HLA-associated T-cell selection generated from the analysis of a combined cohort of 800 anti-retroviral naïve, HLA-diverse, predominantly subtype B-infected individuals derived from national populations from the USA and Western Australia. The cohort consensus sequence spanning 13 amino acid windows around HLA associations was scanned with the ‘Epi-pred’ epitope prediction programme and was used to identify putative in-vivo selection CD8 T-cell targets (non-adapted epitopes) as well as HLA-adapted variants capable of inducing a de novo immune response (adapted epitopes). PBMC IFN-responses to predicted optimal length epitopes were quantified in ELISpot assays, taking into account the autologous HLA genotype and the autologous viral sequence of 200 individuals from a North American cohort. In the analysis of the first 29 individuals, we identified nine possible novel epitopes with the predicting HLA restriction matching the genotype of the reacting PBMC. In many instances, the HLA-driven change led to loss of reactivity as predicted for classical CD8 T-cell escape, however more complex patterns of reactivity were seen, particularly for epitopes in the Nef region. The creation of an escape variant was shown to elicit IFN- responses, including equivalent or higher responses compared with the non-adapted epitopes, suggesting that HIV adaptation may create ineffective immune recognition in some cases. These results provide further insights into CD8 T cell responses against the virus and have implications for HIV vaccine design

Translation of HLA-HIV associations to the cellular level: HIV adapts to inflate CD8 T cell responses against Nef and HLA-adapted variant epitopes

Strong statistical associations between polymorphisms in HIV-1 population sequences and carriage of HLA class I alleles have been widely used to identify possible sites of CD8 T cell immune selection in vivo. However, there have been few attempts to prospectively and systematically test these genetic hypotheses arising from population-based studies at a cellular, functional level. We assayed CD8 T cell epitope-specific IFN-gamma responses in 290 individuals from the same cohort, which gave rise to 874 HLA-HIV associations in genetic analyses, taking into account autologous viral sequences and individual HLA genotypes. We found immunological evidence for 58% of 374 associations tested as sites of primary immune selection and identified up to 50 novel HIV-1 epitopes using this reverse-genomics approach. Many HLA-adapted epitopes elicited equivalent or higher-magnitude IFN-gamma responses than did the nonadapted epitopes, particularly in Nef. At a population level, inclusion of all of the immunoreactive variant CD8 T cell epitopes in Gag, Pol, Nef, and Env suggested that HIV adaptation leads to an inflation of Nef-directed immune responses relative to other proteins. We concluded that HLA-HIV associations mark viral epitopes subject to CD8 T cell selection. These results can be used to guide functional studies of specific epitopes and escape mutations, as well as to test, train, and evaluate analytical models of viral escape and fitness. The inflation of Nef and HLA-adapted variant responses may have negative effects on natural and vaccine immunity against HIV and, therefore, has implications for diversity coverage approaches in HIV vaccine design

Progression to CMV end-organ disease in HIV-1-infected individuals despite abundance of highly differentiated CMV-specific CD8(+) T-cells

Since CMV-specific T-cells have been shown to generally express an advanced state of differentiation, we investigated whether these mature CMV-specific T-cells are sustained in HIV-infected patients, who are not treated with HAART, receive no CMV medication, but do progress to AIDS with CMV end-organ disease (AIDS-CMV). CD8(+) and CD4(+) T-cell phenotype was studied in these patients in comparison with long-term asymptomatic HIV-infected individuals, progressors to AIDS without CMV end-organ disease as well as CMV-seropositive HIV-negative controls. CMV-specific CD8(+) T-cells from progressors to AIDS-CMV expressed markers typical of highly differentiated effector T-cells, being CCR7(-), CD27(-) CD45RO(+/-), with high CD57 expression and increased Ki67 expression, compatible with functional effector cell capabilities. In addition, CD4(+) T-cells with the characteristic CD27(-)CD28(-) phenotype previously shown to be induced by CMV infection specifically, were found in very high numbers in the HIV+ individuals, but the highest in progressors to AIDS-CMV just before onset of disease. Also the normally rare CD45RO(-)CD27(-)CD4(+) subset increased significantly, whereas the CD45RO(-)CD27(+)CD4(+) subset decreased. Our data show that in patients progressing to AIDS-CMV, CMV-specific CD8(+) T-cells have expanded and are fully differentiated to mature functional effector T-cells. These cells are not protective apparently, but may contribute to tissue-associated immunopathology characteristic of these clinical conditions. (c) 2004 Elsevier B.V. All rights reserve