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. 2020 Mar 17;94(7):e01901-19.
doi: 10.1128/JVI.01901-19. Print 2020 Mar 17.

Human Cytomegalovirus Decreases Major Histocompatibility Complex Class II by Regulating Class II Transactivator Transcript Levels in a Myeloid Cell Line

Praneet K Sandhu  1 , Nicholas J Buchkovich  2
Affiliations

Human Cytomegalovirus Decreases Major Histocompatibility Complex Class II by Regulating Class II Transactivator Transcript Levels in a Myeloid Cell Line

Praneet K Sandhu et al. J Virol. .

Abstract

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that encodes many proteins to modulate the host immune response. Extensive efforts have led to the elucidation of multiple strategies employed by HCMV to effectively block NK cell targeting of virus-infected cells and the major histocompatibility complex (MHC) class I-primed CD8+ T cell response. However, viral regulation of the MHC class II-mediated CD4+ T cell response is understudied in endogenous MHC class II-expressing cells, largely because the popular cell culture systems utilized for studying HCMV do not endogenously express MHC class II. Of the many cell types infected by HCMV in the host, myeloid cells, such as monocytes, are of particular importance due to their role in latency and subsequent dissemination throughout the host. We investigated the impact of HCMV infection on MHC class II in Kasumi-3 cells, a myeloid-progenitor cell line that endogenously expresses the MHC class II gene, HLA-DR. We observed a significant reduction in the expression of surface and total HLA-DR at 72 h postinfection (hpi) and 120 hpi in infected cells. The decrease in HLA-DR expression was independent of the expression of previously described viral genes that regulate the MHC class II complex or the unique short (US) region of HCMV, a region expressing many immunomodulatory genes. The altered surface level of HLA-DR was not a result of increased endocytosis and degradation but was a result of a reduction in HLA-DR transcripts due to a decrease in the expression of the class II transactivator (CIITA).IMPORTANCE Human cytomegalovirus (HCMV) is an opportunistic herpesvirus that is asymptomatic for healthy individuals but that can lead to severe pathology in patients with congenital infections and immunosuppressed patients. Thus, it is important to understand the modulation of the immune response by HCMV, which is understudied in the context of endogenous MHC class II regulation. Using Kasumi-3 cells as a myeloid progenitor cell model endogenously expressing MHC class II (HLA-DR), this study shows that HCMV decreases the expression of HLA-DR in infected cells by reducing the transcription of HLA-DR transcripts early during infection independently of the expression of previously implicated genes. This is an important finding, as it highlights a mechanism of immune evasion utilized by HCMV to decrease the expression of MHC class II in a relevant cell system that endogenously expresses the MHC class II complex.

Keywords: Kasumi-3; MHC class II; cytomegalovirus; myeloid.

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Figures

FIG 1
FIG 1
HCMV downregulates surface expression of MHC class II in a myeloid progenitor cell line. (A) Flow cytometry analysis of unstained Kasumi-3 cells or cells stained for HLA-DR, HLA-DQ, and HLA-DP. (B) Flow cytometry scatter plot of HCMV-infected (72 hpi) and uninfected cells showing the relationship between mCherry (a marker of HCMV infection) and HLA-DR. (C) Histograms of HCMV-infected (mCherry) and uninfected Kasumi-3 cells stained for surface HLA-DR at 72 and 120 h postinfection. (D) Bar graph of the geometric mean (GM) fluorescence values for the samples used in the assay whose results are presented in panel C, displayed as a percentage of the value for the uninfected sample. (E) Geometric mean fluorescence values from uninfected or HCMV-infected CD14+ human peripheral blood monocytes (HPBM) at 72 hpi. Values are averages from a minimum of three independent experiments. *, P < 0.05.
FIG 2
FIG 2
Total MHC class II is reduced during HCMV infection. (A) Bar graph of the geometric mean fluorescence values of total HLA-DR protein in uninfected and infected samples (72 and 120 hpi). Values are a percentage of the value for the uninfected sample and are averages from at least three independent experiments. *, P < 0.05. (B) Western blot analysis of uninfected and HCMV-infected Kasumi-3 cells at 24 and 72 hpi to detect HLA-DR, IE2, and p115 (loading control) protein levels. (C) Immunofluorescence of total HLA-DR protein (green) in uninfected and HCMV-infected Kasumi-3 cells at 72 and 120 hpi. Infected cells expressed mCherry (red) from the genome as marker for infection. (D) Immunofluorescence of total HLA-DR (pink) and LAMP1 (green) in HCMV-infected (red) Kasumi-3 cells at 72 and 120 hpi. Nuclei stained with DAPI. Bars (C and D), 1 μm.
FIG 3
FIG 3
Kasumi-3 cells do not regulate MHC class II using viral proteins previously reported to decrease MHC class II expression and localization. (A) RT-PCR analysis of US2, US3, IE1, and GAPDH transcripts from uninfected fibroblasts or fibroblasts infected with the AD169 or TB40/E strains of HCMV (96 hpi). (B) (Left) Histograms of surface HLA-DR protein in Kasumi-3 cells 48 h after electroporation with either the GFP control or GFP-pp65. (Right) Quantification of the geometric mean fluorescence as a percentage of the value for the GFP control sample. (C) Infectious titers at 120 hpi of wild-type TB40/E (wild type [WT]) and the UL111A-STOP and UL78-galK viruses following infection of fibroblasts at an MOI of 3. (D) (Left) Histograms of surface HLA-DR protein in Kasumi-3 cells infected with mCherry expressing wild-type TB40/E or a virus lacking UL111A (UL111A-STOP) expression at 72 hpi. (Middle) Western blot analysis confirms the absence of the UL111A protein. (Right) Quantification of the geometric mean fluorescence as a percentage of the value for the uninfected sample. (E) (Left) Histograms of surface HLA-DR protein in Kasumi-3 cells infected with mCherry expressing wild-type TB40/E or a virus lacking UL78 expression (UL78-galK) at 72 hpi. (Right) Quantification of the geometric mean fluorescence of TB40/E or a virus lacking UL78 (UL78-galK) as a percentage of the value for the uninfected sample. (F) Graph representing the geometric mean fluorescence of surface HLA-DR protein in Kasumi-3 cells at 48 h postelectroporation with either the vector control or a plasmid expressing UL78. The histograms in panels B, D, and E are representative images from one of at least three independent experiments. The values in the bar graphs in panels B to F are percentages of the geometric mean fluorescence for uninfected or control transfected cells and are averages from at least three independent experiments.
FIG 4
FIG 4
The unique short region is not required for the downregulation of surface MHC class II molecules in Kasumi-3 cells. (A) Schematic of the US region of the HCMV genome showing the strategy for segmentally knocking out proteins expressed from the US region. Black arrows indicate primer sets either flanking the galK insertion region or binding within galK and a neighboring flanking region. (B) PCR analysis showing replacement of the indicated segment of the US region with galK. The expected values for wild-type and galK-containing bands are indicated below the images. (C) Infectious titers at 120 hpi of wild-type TB40/E (WT) and the US deletion mutants (indicated by Δ7-12, Δ13-18, Δ19-24, Δ26-30, and Δ31-34A) following the infection of fibroblasts at an MOI of 3. (D) Bar graph generated from the geometric mean fluorescence values of surface HLA-DR staining of Kasumi-3 cells infected with wild-type TB40/E or viruses lacking segments of the US region gene (the ΔUS7-US12, ΔUS13-US18, ΔUS19-US24, ΔUS26-US30, and ΔUS31-US34A viruses) at 72 hpi. NS, not significant. (E) Histograms from one representative experiment for the samples for which the results are shown in panel D. The values in panels C and D are averages from three independent experiments.
FIG 5
FIG 5
Reduced MHC class II protein requires early viral gene synthesis. (A) Western blot analysis of uninfected (UI) or HCMV-infected Kasumi-3 cells at 24, 72, and 120 hpi, showing the results for two immediate early proteins (IE1 and IE2), two delayed early proteins (UL71 and pp150), two late proteins (pp71 and pp28), and a loading control (p115). (B) (Left) Histograms of one representative experiment of surface HLA-DR staining of uninfected Kasumi-3 cells or cells infected with wild-type TB40/E and treated with DMSO or acyclovir. (Right) The bar graph shows the geometric mean fluorescence values for infected samples at 72 hpi. (C) Histograms of one representative experiment of surface HLA-DR staining at 72 hpi of Kasumi-3 cells infected with UV-inactivated virus. (D) Infectious titers at 120 hpi of wild-type TB40/E (WT) and the ΔUL20 viruses following infection of fibroblasts at an MOI of 3. (E) (Left) Histograms of one representative experiment of surface HLA-DR staining at 72 hpi of Kasumi-3 cells infected with wild-type TB40/E or a virus expressing GFP in place of UL20 (ΔUL20). (Right) Bar graph generated from geometric mean fluorescence values. The values graphed in panels A, D, and E are averages from a minimum of three independent experiments.
FIG 6
FIG 6
HCMV does not alter the rate of MHC class II internalization in Kasumi-3 cells. (A) Schematic showing the strategy for measuring the rate of MHC class II internalization. (B) Graph plotting the remaining surface HLA-DR levels in uninfected or infected samples at 0, 4, 8, 12, 24, and 36 h after the addition of primary antibody. Values are the means from three independent experiments.
FIG 7
FIG 7
HCMV reduces MHC class II in Kasumi-3 cells by downregulating the expression of CIITA. (A and B) Quantitative PCR analysis of HLA-DRα (A) or CIITA (B) transcript levels in uninfected (UI) and HCMV-infected samples at 24 and 72 hpi. The values shown are relative to those for uninfected samples after normalization to the value for GAPDH. (C) Quantitative PCR analysis of CIITA transcripts derived from promoter I, III, or IV (PI, PIII, or PIV, respectively) in uninfected and HCMV-infected samples at 24 and 72 hpi. Values are the absolute numbers of transcripts per 105 copies of GAPDH. ND, not detected. (D) Quantification of the geometric mean fluorescence for surface HLA-DR in Kasumi-3 cells electroporated with a DNA control (Vector) or pSVH. (E) Quantitative PCR analysis of CIITA transcripts following electroporation of a DNA control (Vector) or pSVH into Kasumi-3 cells. (F) (Left) Quantification of the geometric mean fluorescence for surface HLA-DR in Kasumi-3 cells electroporated with the pCDH vector, pCDH-IE1, or pCDH-IE2. (Right) Western blots of the samples for which the results are shown in panels D and F, showing the expression of IE1 and IE2 after transfection of the pSVH, pCDH-IE1, and pCDH-IE2 plasmids. All values in panels A to F are averages from at least three independent experiments. *, P < 0.05; ns, not statistically significant.
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