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The CD8 and CD4 T-cell response against Kaposi's sarcoma-associated herpesvirus is skewed towards early and late lytic antigens.

Robey RC, Lagos D, Gratrix F, Henderson S, Matthews NC, Vart RJ, Bower M, Boshoff C, Gotch FM - PLoS ONE (2009)

Bottom Line: Kaposi's sarcoma-associated herpesvirus (KSHV) is causally related to Kaposi's sarcoma (KS), the most common malignancy in untreated individuals with HIV/AIDS.Transduced moDCs naturally process the KSHV genes and present the resulting antigens in the context of MHC class I and II.This knowledge will be important to future immunological investigations into KSHV and may eventually lead to the development of better immunotherapies for KSHV-related diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Imperial College London, London, United Kingdom.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is causally related to Kaposi's sarcoma (KS), the most common malignancy in untreated individuals with HIV/AIDS. The adaptive T-cell immune response against KSHV has not been fully characterized. To achieve a better understanding of the antigenic repertoire of the CD8 and CD4 T-cell responses against KSHV, we constructed a library of lentiviral expression vectors each coding for one of 31 individual KSHV open reading frames (ORFs). We used these to transduce monocyte-derived dendritic cells (moDCs) isolated from 14 KSHV-seropositive (12 HIV-positive) and 7 KSHV-seronegative (4 HIV-positive) individuals. moDCs were transduced with up to 3 KSHV ORFs simultaneously (ORFs grouped according to their expression during the viral life cycle). Transduced moDCs naturally process the KSHV genes and present the resulting antigens in the context of MHC class I and II. Transduced moDCs were cultured with purified autologous T cells and the CD8 and CD4 T-cell proliferative responses to each KSHV ORF (or group) was assessed using a CFSE dye-based assay. Two pools of early lytic KSHV genes ([ORF8/ORF49/ORF61] and [ORF59/ORF65/K4.1]) were frequently-recognized targets of both CD8 and CD4 T cells from KSHV seropositive individuals. One pool of late lytic KSHV genes ([ORF28/ORF36/ORF37]) was a frequently-recognized CD8 target and another pool of late genes ([ORF33/K1/K8.1]) was a frequently-recognized CD4 target. We report that both the CD8 and CD4 T-cell responses against KSHV are skewed towards genes expressed in the early and late phases of the viral lytic cycle, and identify some previously unknown targets of these responses. This knowledge will be important to future immunological investigations into KSHV and may eventually lead to the development of better immunotherapies for KSHV-related diseases.

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Expression and multiplicity of infection in moDCs of each of the KSHV-gene-encoding lentiviruses.(a) KSHV ORF expression by mature moDCs 6 days after transduction with KSHV-gene-encoding lentiviruses. For each ORF, the left-hand lane shows the reverse transcription (RT)-PCR product and the right-hand lane shows the no reverse transcriptase control. (b) Multiplicity of Infection (MOI) of each of the KSHV-gene-encoding lentiviral preparations in one experiment. Average lentiviral copy number per cell and standard deviations of duplicates are shown.
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pone-0005890-g002: Expression and multiplicity of infection in moDCs of each of the KSHV-gene-encoding lentiviruses.(a) KSHV ORF expression by mature moDCs 6 days after transduction with KSHV-gene-encoding lentiviruses. For each ORF, the left-hand lane shows the reverse transcription (RT)-PCR product and the right-hand lane shows the no reverse transcriptase control. (b) Multiplicity of Infection (MOI) of each of the KSHV-gene-encoding lentiviral preparations in one experiment. Average lentiviral copy number per cell and standard deviations of duplicates are shown.

Mentions: We used RT-PCR to ensure that all KSHV ORFs were expressed by moDCs after lentiviral transduction (Figure 2a) and quantitative PCR to titre all KSHV-gene-encoding lentivirus preparations (Figure 2b). The volume of each lentivirus used in all subsequent experiments was then adjusted to achieve a uniform MOI of between 3.4 and 7.2 lentiviral copies per cell for all preparations (median 4.5; interquartile range 3.9 to 5.4; mean 4.75). This range was selected based on the results from experiments with our GFP construct discussed above.


The CD8 and CD4 T-cell response against Kaposi's sarcoma-associated herpesvirus is skewed towards early and late lytic antigens.

Robey RC, Lagos D, Gratrix F, Henderson S, Matthews NC, Vart RJ, Bower M, Boshoff C, Gotch FM - PLoS ONE (2009)

Expression and multiplicity of infection in moDCs of each of the KSHV-gene-encoding lentiviruses.(a) KSHV ORF expression by mature moDCs 6 days after transduction with KSHV-gene-encoding lentiviruses. For each ORF, the left-hand lane shows the reverse transcription (RT)-PCR product and the right-hand lane shows the no reverse transcriptase control. (b) Multiplicity of Infection (MOI) of each of the KSHV-gene-encoding lentiviral preparations in one experiment. Average lentiviral copy number per cell and standard deviations of duplicates are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691989&req=5

pone-0005890-g002: Expression and multiplicity of infection in moDCs of each of the KSHV-gene-encoding lentiviruses.(a) KSHV ORF expression by mature moDCs 6 days after transduction with KSHV-gene-encoding lentiviruses. For each ORF, the left-hand lane shows the reverse transcription (RT)-PCR product and the right-hand lane shows the no reverse transcriptase control. (b) Multiplicity of Infection (MOI) of each of the KSHV-gene-encoding lentiviral preparations in one experiment. Average lentiviral copy number per cell and standard deviations of duplicates are shown.
Mentions: We used RT-PCR to ensure that all KSHV ORFs were expressed by moDCs after lentiviral transduction (Figure 2a) and quantitative PCR to titre all KSHV-gene-encoding lentivirus preparations (Figure 2b). The volume of each lentivirus used in all subsequent experiments was then adjusted to achieve a uniform MOI of between 3.4 and 7.2 lentiviral copies per cell for all preparations (median 4.5; interquartile range 3.9 to 5.4; mean 4.75). This range was selected based on the results from experiments with our GFP construct discussed above.

Bottom Line: Kaposi's sarcoma-associated herpesvirus (KSHV) is causally related to Kaposi's sarcoma (KS), the most common malignancy in untreated individuals with HIV/AIDS.Transduced moDCs naturally process the KSHV genes and present the resulting antigens in the context of MHC class I and II.This knowledge will be important to future immunological investigations into KSHV and may eventually lead to the development of better immunotherapies for KSHV-related diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Imperial College London, London, United Kingdom.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is causally related to Kaposi's sarcoma (KS), the most common malignancy in untreated individuals with HIV/AIDS. The adaptive T-cell immune response against KSHV has not been fully characterized. To achieve a better understanding of the antigenic repertoire of the CD8 and CD4 T-cell responses against KSHV, we constructed a library of lentiviral expression vectors each coding for one of 31 individual KSHV open reading frames (ORFs). We used these to transduce monocyte-derived dendritic cells (moDCs) isolated from 14 KSHV-seropositive (12 HIV-positive) and 7 KSHV-seronegative (4 HIV-positive) individuals. moDCs were transduced with up to 3 KSHV ORFs simultaneously (ORFs grouped according to their expression during the viral life cycle). Transduced moDCs naturally process the KSHV genes and present the resulting antigens in the context of MHC class I and II. Transduced moDCs were cultured with purified autologous T cells and the CD8 and CD4 T-cell proliferative responses to each KSHV ORF (or group) was assessed using a CFSE dye-based assay. Two pools of early lytic KSHV genes ([ORF8/ORF49/ORF61] and [ORF59/ORF65/K4.1]) were frequently-recognized targets of both CD8 and CD4 T cells from KSHV seropositive individuals. One pool of late lytic KSHV genes ([ORF28/ORF36/ORF37]) was a frequently-recognized CD8 target and another pool of late genes ([ORF33/K1/K8.1]) was a frequently-recognized CD4 target. We report that both the CD8 and CD4 T-cell responses against KSHV are skewed towards genes expressed in the early and late phases of the viral lytic cycle, and identify some previously unknown targets of these responses. This knowledge will be important to future immunological investigations into KSHV and may eventually lead to the development of better immunotherapies for KSHV-related diseases.

Show MeSH
Related in: MedlinePlus