T-cell responses to KSHV infection: a systematic approach

Abstract

Prior studies of T-cell responses to KSHV have included relatively few participants and focused on relatively few KSHV antigens. To provide a more comprehensive analysis, we investigated T-cell responses to the whole KSHV proteome using IFN-γ ELISpot. Using ∼7,500 overlapping 15mer peptides we generated one to three peptide pools for each of the 82 KSHV ORFs. IFN-γ ELISpot analysis of PBMCs from 19 patients with a history of KSHV-associated disease and 24 healthy donors (11 KSHV seropositive) detected widely varied responses. Fifty six of the 82 ORFs were recognized by at least one individual but there was little overlap between participants. Responses to at least one ORF pool were observed in all 19 patients and in 7 seropositive donors. Four seropositive donors and 10 seronegative donors had no detectable responses while 3 seronegative donors had weak responses to one ORF. Patients recognised more ORFs than the donors (p=0.04) but the response intensity (spot forming units: SFU per million cells) was similar in the two groups. In four of the responding donors, individual peptides eliciting the predominant responses were identified: three donors responded to only one peptide per ORF, while one recognized five. Using intracellular cytokine staining in four participant samples, we detected peptide-induced IFN-γ, MIP1-β, and TNF-α as well as CD107a degranulation, consistent with multifunctional effector responses in CD8+ and CD4+ T cells. Sequence analysis of TCRs present in peptide specific T-cell clones generated from two participants showed both mono- and multi-clonotypic responses. Finally, we molecularly cloned the KSHV specific TCRs and incorporated the sequences into retroviral vectors to transfer the specificities to fresh donor cells for additional studies. This study suggests that KSHV infected individuals respond to diverse KSHV antigens, consistent with a lack of shared immunodominance and establishes useful tools to facilitate KSHV immunology studies.

Keywords: ELISpot; KSHV; T-cells; cell-mediated immunity.

Figure 1. Summary of IFN-γ ELISpot responses to the KSHV proteome

A heat map plot of IFN-γ ELISpot responses detected by our KSHV proteome analysis is presented with ORF pools examined in indicated in columns and donor codes identified in rows. Responses are denoted by blue-coloured squares with the intensity of each response proportional to the intensity of its corresponding square. A reference intensity scale is proved below the heat map.

Figure 2. Presence of CD4+ and CD8+ specific IFN-γ ELISpot T-cell responses

A graph of IFN-γ responses obtained by CD8⁺ and CD4⁺ sorting followed by type-specific ELISpot assay from three healthy donors is presented. The number of spots is presented on they-axis and the donors and respective ORF pools under the x-axis.

Figure 3. Multifunctional effector responses in donor PBMC

A summary of flow cytometry for peptide-induced effector responses in PBMC from selected KSHV responding donors identified by whole proteome ELISpot analysis is presented. Donors and their respective ORFs are indicated on the y-axis and the percent of responding CD3⁺ cells out of >250,000 gated CD8⁺ or CD4⁺ T-cell events are displayed on the axis.

Figure 4. Multifunctional effector response profile of CD8+ ORF25/p88 Clone 8 T cells from donor R23

Flow cytometry analysis of ORF25-peptide 88-stimulated Clone 8⁺ T cells for IFN-γ, CD107a, and MIP-1β responses are presented.

Figure 5. Multifunctional effector response profile of CD4+ ORF37/p49 Clone 1 T cells from donor R20

Flow cytometry analysis from ORF37/peptide 49-stimulated Clone 1 T cells for IFN-γ, CD107a, and MIP-1β responses are presented.

Figure 6. Induced cytokine response profiles and TCR clonotype analysis of ORF37 T-cell clones

IFN-γ, and MIP-1β flow cytometry analysis of four ORF37/peptide 49-stimulated CD4⁺ T-cell clones are presented with their α and β CDR3 sequences displayed under their corresponding dot plots. Shared CDR3 sequences are indicated in red.

Figure 7. Multifunctional effector responses from Clone 1 TCR transductions

IFN-γ, CD107a, and MIP-1β flow cytometry for analysis of donor R20 CD4⁺ T cells transduced with the ORF37/p49 TCR that were stimulated with the ORF37-peptide 49 are presented.

Figure 8. MHC restriction of ORF37/p49 TCR

IFN-γ, and MIP-1β flow cytometry results for cognate peptide induced responses from CD4⁺ T cells transduced with the Clone 1 ORF37/peptide 49 TCR in the presence of HLA-blocking antibodies are presented. Treatment of each culture is indicated above their respective dot plots.