Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1998 May 18;187(10):1681-7.
doi: 10.1084/jem.187.10.1681.

Modulation of natural killer cell cytotoxicity in human cytomegalovirus infection: the role of endogenous class I major histocompatibility complex and a viral class I homolog

C C Leong  1 , T L Chapman, P J Bjorkman, D Formankova, E S Mocarski, J H Phillips, L L Lanier
Affiliations

Modulation of natural killer cell cytotoxicity in human cytomegalovirus infection: the role of endogenous class I major histocompatibility complex and a viral class I homolog

C C Leong et al. J Exp Med. .

Erratum in

  • J Exp Med 1998 Aug 3;188(3):following 614

Abstract

Natural killer (NK) cells have been implicated in early immune responses against certain viruses, including cytomegalovirus (CMV). CMV causes downregulation of class I major histocompatibility complex (MHC) expression in infected cells; however, it has been proposed that a class I MHC homolog encoded by CMV, UL18, may act as a surrogate ligand to prevent NK cell lysis of CMV-infected cells. In this study, we examined the role of UL18 in NK cell recognition and lysis using fibroblasts infected with either wild-type or UL18 knockout CMV virus, and by using cell lines transfected with the UL18 gene. In both systems, the expression of UL18 resulted in the enhanced killing of target cells. We also show that the enhanced killing is due to both UL18-dependent and -independent mechanisms, and that the killer cell inhibitory receptors (KIRs) and CD94/NKG2A inhibitory receptors for MHC class I do not play a role in affecting susceptibility of CMV-infected fibroblasts to NK cell-mediated cytotoxicity.

PubMed Disclaimer

Figures

Figure 1
Figure 1
(A) Downregulation of class I MHC in hCMV-infected HFFs. AD169 or mock-infected HFFs were stained with anti–class I (DX17) or control Ig (cIg), followed by PE-conjugated goat anti–mouse Ig. HFFs were infected with AD169 at an MOI of 3, and cells were stained at 24 h after infection. (B) NK cell cytotoxicity assay against hCMV or mock-infected HFF cells. hCMV or mock-infected HFFs were used as targets in a 4-h 51Cr–release assay. HFFs were infected at an MOI of 3 and used at 48 h after infection. Mock-infected, white squares; AD169, black diamonds. (C) Blocking class I MHC, KIR, or CD94 does not induce NK cell killing of uninfected HFFs. Normal HFFs were incubated in the presence of NK cells and with cIg or a mixture of mAb against class I MHC (DX15, DX16, and DX17 at 20 μg/ml) or a mixture of anti-KIR mAbs (DX27, DX30, DX31, and HP-3E4) and anti-CD94 mAb (DX22), each at 20 μg/ml. cIg, black bars; anti-KIR/CD94, gray bars; anti–class I, hatched bars. (D) AD169 or mock-infected HFFs were incubated in the presence of NK cells with cIg or mAb directed at class I MHC (DX15, DX16, and DX17 at 20 μg/ml) or a cocktail of mAbs against KIR and CD94. Cytotoxicity assays were performed at E/T ratios of 5:1. As controls, 721.221 class I HLA transfectants expressing HLA-B*0702 or HLA-Cw*0702 were analyzed, using NK clones expressing the relevant CD94/NKG2A or KIR, respectively. cIg, white bars; anti-KIR/CD94, black bars; anti–class I, hatched bars.
Figure 1
Figure 1
(A) Downregulation of class I MHC in hCMV-infected HFFs. AD169 or mock-infected HFFs were stained with anti–class I (DX17) or control Ig (cIg), followed by PE-conjugated goat anti–mouse Ig. HFFs were infected with AD169 at an MOI of 3, and cells were stained at 24 h after infection. (B) NK cell cytotoxicity assay against hCMV or mock-infected HFF cells. hCMV or mock-infected HFFs were used as targets in a 4-h 51Cr–release assay. HFFs were infected at an MOI of 3 and used at 48 h after infection. Mock-infected, white squares; AD169, black diamonds. (C) Blocking class I MHC, KIR, or CD94 does not induce NK cell killing of uninfected HFFs. Normal HFFs were incubated in the presence of NK cells and with cIg or a mixture of mAb against class I MHC (DX15, DX16, and DX17 at 20 μg/ml) or a mixture of anti-KIR mAbs (DX27, DX30, DX31, and HP-3E4) and anti-CD94 mAb (DX22), each at 20 μg/ml. cIg, black bars; anti-KIR/CD94, gray bars; anti–class I, hatched bars. (D) AD169 or mock-infected HFFs were incubated in the presence of NK cells with cIg or mAb directed at class I MHC (DX15, DX16, and DX17 at 20 μg/ml) or a cocktail of mAbs against KIR and CD94. Cytotoxicity assays were performed at E/T ratios of 5:1. As controls, 721.221 class I HLA transfectants expressing HLA-B*0702 or HLA-Cw*0702 were analyzed, using NK clones expressing the relevant CD94/NKG2A or KIR, respectively. cIg, white bars; anti-KIR/CD94, black bars; anti–class I, hatched bars.
Figure 1
Figure 1
(A) Downregulation of class I MHC in hCMV-infected HFFs. AD169 or mock-infected HFFs were stained with anti–class I (DX17) or control Ig (cIg), followed by PE-conjugated goat anti–mouse Ig. HFFs were infected with AD169 at an MOI of 3, and cells were stained at 24 h after infection. (B) NK cell cytotoxicity assay against hCMV or mock-infected HFF cells. hCMV or mock-infected HFFs were used as targets in a 4-h 51Cr–release assay. HFFs were infected at an MOI of 3 and used at 48 h after infection. Mock-infected, white squares; AD169, black diamonds. (C) Blocking class I MHC, KIR, or CD94 does not induce NK cell killing of uninfected HFFs. Normal HFFs were incubated in the presence of NK cells and with cIg or a mixture of mAb against class I MHC (DX15, DX16, and DX17 at 20 μg/ml) or a mixture of anti-KIR mAbs (DX27, DX30, DX31, and HP-3E4) and anti-CD94 mAb (DX22), each at 20 μg/ml. cIg, black bars; anti-KIR/CD94, gray bars; anti–class I, hatched bars. (D) AD169 or mock-infected HFFs were incubated in the presence of NK cells with cIg or mAb directed at class I MHC (DX15, DX16, and DX17 at 20 μg/ml) or a cocktail of mAbs against KIR and CD94. Cytotoxicity assays were performed at E/T ratios of 5:1. As controls, 721.221 class I HLA transfectants expressing HLA-B*0702 or HLA-Cw*0702 were analyzed, using NK clones expressing the relevant CD94/NKG2A or KIR, respectively. cIg, white bars; anti-KIR/CD94, black bars; anti–class I, hatched bars.
Figure 1
Figure 1
(A) Downregulation of class I MHC in hCMV-infected HFFs. AD169 or mock-infected HFFs were stained with anti–class I (DX17) or control Ig (cIg), followed by PE-conjugated goat anti–mouse Ig. HFFs were infected with AD169 at an MOI of 3, and cells were stained at 24 h after infection. (B) NK cell cytotoxicity assay against hCMV or mock-infected HFF cells. hCMV or mock-infected HFFs were used as targets in a 4-h 51Cr–release assay. HFFs were infected at an MOI of 3 and used at 48 h after infection. Mock-infected, white squares; AD169, black diamonds. (C) Blocking class I MHC, KIR, or CD94 does not induce NK cell killing of uninfected HFFs. Normal HFFs were incubated in the presence of NK cells and with cIg or a mixture of mAb against class I MHC (DX15, DX16, and DX17 at 20 μg/ml) or a mixture of anti-KIR mAbs (DX27, DX30, DX31, and HP-3E4) and anti-CD94 mAb (DX22), each at 20 μg/ml. cIg, black bars; anti-KIR/CD94, gray bars; anti–class I, hatched bars. (D) AD169 or mock-infected HFFs were incubated in the presence of NK cells with cIg or mAb directed at class I MHC (DX15, DX16, and DX17 at 20 μg/ml) or a cocktail of mAbs against KIR and CD94. Cytotoxicity assays were performed at E/T ratios of 5:1. As controls, 721.221 class I HLA transfectants expressing HLA-B*0702 or HLA-Cw*0702 were analyzed, using NK clones expressing the relevant CD94/NKG2A or KIR, respectively. cIg, white bars; anti-KIR/CD94, black bars; anti–class I, hatched bars.
Figure 2
Figure 2
(A) Downregulation of class I MHC after Δ18 and AD169 hCMV infection of HFFs. HFFs were infected with Δ18 or AD169 at an MOI of 5. At 48 h after infection cells were stained with anti–class I MHC or cIg, followed by PE-conjugated goat anti–mouse second step. (B) Expression of UL18 in AD169 but not Δ18 cell lysates. Lysates were prepared from HFF-infected with Δ18 or AD169 at 48 h after infection and blotted with anti-UL18 or anti-hCMV IE mAb, followed by horseradish peroxidase–conjugated second step. (C) AD169-infected HFFs were lysed more efficiently than were Δ18-infected HFFs. HFFs were infected with AD169 or Δ18 at an MOI of 5, and used as targets at 48 h after infection in 4-h 51Cr–release assays. Assays were performed at an E/T ratio of 20:1, 10:1, and 1:1. Standard deviation between triplicates was <10%. Δ18, black circles; AD169, black diamonds; mock-infected, black squares. (D) Surface UL18 expression on 293EBV transfectants. 293EBV cells were transfected with pREP10 UL18. Cells were cultured for 48 h before use in cytotoxicity assays. Histograms of sorted UL18-positive cells. Cells were stained with anti-UL18 mAb 10C7 followed by PE-conjugated goat anti–mouse Ig. (E) 293EBV transfectant expressing UL18 were lysed at an enhanced level compared with parental controls. UL18-transfected 293EBV cells were used as targets in NK cell cytotoxicity assays. Experiments were performed at an E:T ratio of 5:1. All transfectants other than vector only were positively sorted using mAb directed against the protein encoded by the transfected cDNA. Cells were cultured for 48 h and used in 4-h cytotoxicity assays as described above.
Figure 2
Figure 2
(A) Downregulation of class I MHC after Δ18 and AD169 hCMV infection of HFFs. HFFs were infected with Δ18 or AD169 at an MOI of 5. At 48 h after infection cells were stained with anti–class I MHC or cIg, followed by PE-conjugated goat anti–mouse second step. (B) Expression of UL18 in AD169 but not Δ18 cell lysates. Lysates were prepared from HFF-infected with Δ18 or AD169 at 48 h after infection and blotted with anti-UL18 or anti-hCMV IE mAb, followed by horseradish peroxidase–conjugated second step. (C) AD169-infected HFFs were lysed more efficiently than were Δ18-infected HFFs. HFFs were infected with AD169 or Δ18 at an MOI of 5, and used as targets at 48 h after infection in 4-h 51Cr–release assays. Assays were performed at an E/T ratio of 20:1, 10:1, and 1:1. Standard deviation between triplicates was <10%. Δ18, black circles; AD169, black diamonds; mock-infected, black squares. (D) Surface UL18 expression on 293EBV transfectants. 293EBV cells were transfected with pREP10 UL18. Cells were cultured for 48 h before use in cytotoxicity assays. Histograms of sorted UL18-positive cells. Cells were stained with anti-UL18 mAb 10C7 followed by PE-conjugated goat anti–mouse Ig. (E) 293EBV transfectant expressing UL18 were lysed at an enhanced level compared with parental controls. UL18-transfected 293EBV cells were used as targets in NK cell cytotoxicity assays. Experiments were performed at an E:T ratio of 5:1. All transfectants other than vector only were positively sorted using mAb directed against the protein encoded by the transfected cDNA. Cells were cultured for 48 h and used in 4-h cytotoxicity assays as described above.
Figure 2
Figure 2
(A) Downregulation of class I MHC after Δ18 and AD169 hCMV infection of HFFs. HFFs were infected with Δ18 or AD169 at an MOI of 5. At 48 h after infection cells were stained with anti–class I MHC or cIg, followed by PE-conjugated goat anti–mouse second step. (B) Expression of UL18 in AD169 but not Δ18 cell lysates. Lysates were prepared from HFF-infected with Δ18 or AD169 at 48 h after infection and blotted with anti-UL18 or anti-hCMV IE mAb, followed by horseradish peroxidase–conjugated second step. (C) AD169-infected HFFs were lysed more efficiently than were Δ18-infected HFFs. HFFs were infected with AD169 or Δ18 at an MOI of 5, and used as targets at 48 h after infection in 4-h 51Cr–release assays. Assays were performed at an E/T ratio of 20:1, 10:1, and 1:1. Standard deviation between triplicates was <10%. Δ18, black circles; AD169, black diamonds; mock-infected, black squares. (D) Surface UL18 expression on 293EBV transfectants. 293EBV cells were transfected with pREP10 UL18. Cells were cultured for 48 h before use in cytotoxicity assays. Histograms of sorted UL18-positive cells. Cells were stained with anti-UL18 mAb 10C7 followed by PE-conjugated goat anti–mouse Ig. (E) 293EBV transfectant expressing UL18 were lysed at an enhanced level compared with parental controls. UL18-transfected 293EBV cells were used as targets in NK cell cytotoxicity assays. Experiments were performed at an E:T ratio of 5:1. All transfectants other than vector only were positively sorted using mAb directed against the protein encoded by the transfected cDNA. Cells were cultured for 48 h and used in 4-h cytotoxicity assays as described above.
Figure 2
Figure 2
(A) Downregulation of class I MHC after Δ18 and AD169 hCMV infection of HFFs. HFFs were infected with Δ18 or AD169 at an MOI of 5. At 48 h after infection cells were stained with anti–class I MHC or cIg, followed by PE-conjugated goat anti–mouse second step. (B) Expression of UL18 in AD169 but not Δ18 cell lysates. Lysates were prepared from HFF-infected with Δ18 or AD169 at 48 h after infection and blotted with anti-UL18 or anti-hCMV IE mAb, followed by horseradish peroxidase–conjugated second step. (C) AD169-infected HFFs were lysed more efficiently than were Δ18-infected HFFs. HFFs were infected with AD169 or Δ18 at an MOI of 5, and used as targets at 48 h after infection in 4-h 51Cr–release assays. Assays were performed at an E/T ratio of 20:1, 10:1, and 1:1. Standard deviation between triplicates was <10%. Δ18, black circles; AD169, black diamonds; mock-infected, black squares. (D) Surface UL18 expression on 293EBV transfectants. 293EBV cells were transfected with pREP10 UL18. Cells were cultured for 48 h before use in cytotoxicity assays. Histograms of sorted UL18-positive cells. Cells were stained with anti-UL18 mAb 10C7 followed by PE-conjugated goat anti–mouse Ig. (E) 293EBV transfectant expressing UL18 were lysed at an enhanced level compared with parental controls. UL18-transfected 293EBV cells were used as targets in NK cell cytotoxicity assays. Experiments were performed at an E:T ratio of 5:1. All transfectants other than vector only were positively sorted using mAb directed against the protein encoded by the transfected cDNA. Cells were cultured for 48 h and used in 4-h cytotoxicity assays as described above.
Figure 2
Figure 2
(A) Downregulation of class I MHC after Δ18 and AD169 hCMV infection of HFFs. HFFs were infected with Δ18 or AD169 at an MOI of 5. At 48 h after infection cells were stained with anti–class I MHC or cIg, followed by PE-conjugated goat anti–mouse second step. (B) Expression of UL18 in AD169 but not Δ18 cell lysates. Lysates were prepared from HFF-infected with Δ18 or AD169 at 48 h after infection and blotted with anti-UL18 or anti-hCMV IE mAb, followed by horseradish peroxidase–conjugated second step. (C) AD169-infected HFFs were lysed more efficiently than were Δ18-infected HFFs. HFFs were infected with AD169 or Δ18 at an MOI of 5, and used as targets at 48 h after infection in 4-h 51Cr–release assays. Assays were performed at an E/T ratio of 20:1, 10:1, and 1:1. Standard deviation between triplicates was <10%. Δ18, black circles; AD169, black diamonds; mock-infected, black squares. (D) Surface UL18 expression on 293EBV transfectants. 293EBV cells were transfected with pREP10 UL18. Cells were cultured for 48 h before use in cytotoxicity assays. Histograms of sorted UL18-positive cells. Cells were stained with anti-UL18 mAb 10C7 followed by PE-conjugated goat anti–mouse Ig. (E) 293EBV transfectant expressing UL18 were lysed at an enhanced level compared with parental controls. UL18-transfected 293EBV cells were used as targets in NK cell cytotoxicity assays. Experiments were performed at an E:T ratio of 5:1. All transfectants other than vector only were positively sorted using mAb directed against the protein encoded by the transfected cDNA. Cells were cultured for 48 h and used in 4-h cytotoxicity assays as described above.
Figure 3
Figure 3
(A) Upregulation of ICAM-1 on hCMV-infected cells. AD169 or mock-infected HFFs were stained (24 h after infection) with FITC-conjugated anti–ICAM-1 (LB2 mAb) or FITC-conjugated cIg. Mean fluorescence intensity of ICAM-1 increased from 351 to 705. (B) Enhanced cytotoxicity against hCMV-infected HFFs was reversed with anti–LFA-1β (CD18). hCMV or mock-infected HFFs were incubated with cIg or anti– LFA-1β (20 μg/ml). 4-h 51Cr– release assays were performed at an E/T ratio of 5:1.
Figure 3
Figure 3
(A) Upregulation of ICAM-1 on hCMV-infected cells. AD169 or mock-infected HFFs were stained (24 h after infection) with FITC-conjugated anti–ICAM-1 (LB2 mAb) or FITC-conjugated cIg. Mean fluorescence intensity of ICAM-1 increased from 351 to 705. (B) Enhanced cytotoxicity against hCMV-infected HFFs was reversed with anti–LFA-1β (CD18). hCMV or mock-infected HFFs were incubated with cIg or anti– LFA-1β (20 μg/ml). 4-h 51Cr– release assays were performed at an E/T ratio of 5:1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ho, M. 1982. Epidemiology of cytomegalovirus in man. In Cytomegalovirus, Biology and Infection: Current Topics in Infectious Disease. W.B. Greenough III and T.C. Merigan, editors. Plenum Medical Book Co., New York. 77–104.
    1. Quinnan GV, Kirmani N, Rook AH, Manishewitz JF, Jackson L, Moreschi G, Santos GW, Saral R, Burns WH. Cytotoxic T cells in cytomegalovirus infection. N Engl J Med. 1982;307:6–13. - PubMed
    1. Riddell SR, Watanabe KS, Goodrich JM, Li CR, Agha ME, Greenberg PD. Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science. 1992;257:238–241. - PubMed
    1. Biron CA, Byron KS, Sullivan JL. Severe herpesvirus infections in an adolescent without natural killer cells. N Engl J Med. 1989;320:1731–1735. - PubMed
    1. Bukowski J, Warner J, Dennert G, Welsh R. Adoptive transfer studies demonstrating the anti-viral effect of natural killer cells in vivo. J Exp Med. 1985;161:40–52. - PMC - PubMed

Publication types

MeSH terms

Substances