American Society for Investigative Pathology Awards & Honors
1999 Predoctoral Merit Award

      CMV is a pernicious cause of morbidity and mortality in immunocompromised populations, e.g., AIDS patients and transplant recipients. The reactivation of latent or persistent virus which was acquired prior to immunosuppression is responsible for a sizable proportion of these serious cases. Determining the factors which promote CMV persistence is thus a critical step in understanding and preventing CMV disease.

      Cell mediated immunity, consisting primarily of NK and CD8+ T cells, is widely documented as the primary means of controlling CMV and other viral infections. A more complex story is unfolding, however, as the role of CD4+ T cells in controlling and clearing CMV infection is elucidated. Mice depleted of CD8+ T cells are able to halt disseminated CMV disease with similar kinetics as non-depleted controls (1). In addition, the clearance of CMV from select organs is completely dependent on CD4+ T cells (2, 3). Finally, cytolysis and antiviral cytokine production by CMV-specific class II restricted CD4+ cells also play a role in controlling CMV infection (2, 4, 5, 6, 7).

      CD4+ dependent anti-CMV immunity relies on the presentation of viral antigen by MHC class II. Many viruses employ immunoevasive strategies which inhibit class II expression induced by interferons. Our laboratory has demonstrated that CMV disrupts this pathway through inhibition of Jak/Stat signaling (8). The next critical question is whether CMV is able to evade the immune system at the level of inhibiting constitutive class II expression. Monocytes and macrophages which constitutively express class II are natural sites of infection by CMV. CMV infection of monocyte-derived macrophages has been shown to decrease surface HLA-DR levels late in infection (9). Decreasing the constitutive class II expression of these cells could contribute to viral persistence, preventing its detection by the immune system.

      To determine the mechanisms responsible for a CMV-mediated decrease in constitutive class II expression, we generated a constitutive class II model system by transfecting U373 cells with a plasmid containing the class II transactivator (CIITA) gene or with the vector alone pcDNA3.1 (generous gifts from Jeremy Boss). CIITA is an important transcription factor which induces expression of MHC class II and coordinately expressed genes such as the invariant chain (Ii) (10). Stable transfection was confirmed with RT-PCR and by verifying consistent HLA-DR expression by flow cytometry after several cell passages. CIITA transfectants (CIITAt) were infected with the Towne strain of CMV (MOI = 6) and analyzed for surface HLA-DR expression by flow cytometry. HLA-DR levels were decreased by 2 days post-inoculation (d.p.i.) and were maximally decreased by 3 d.p.i., yielding approximately a 50% reduction in class II staining. As an internal control for infection, we monitored levels of MHC class I in these cell populations since the CMV-mediated decrease in MHC class I is a well-documented effect of infection. We confirmed the progressive decrease in class I surface levels in each infected cell group.

      Three independent Northern blot experiments demonstrate that HLA-DR mRNA levels in CIITAt at 0, 1, and 3 d.p.i. do not decrease relative to GAPDH levels despite the significant decrease in surface HLA-DR expression. CIITA regulates class II expression at the level of transcription (11). These Northern blot results suggested that the decrease in class II is not due to altered class II transcription, and it excluded the possibility that the decrease in class II was mediated by viral effects on the CIITA promoter.

      Supernatant transfer experiments were used to determine if a soluble factor decreased class II. Supernatants from CMV-infected and uninfected CIITAt were centrifuged to remove virions and placed on uninfected CIITAt for 24 hours. Flow cytometry for HLA-DR indicated that class II levels were not significantly decreased by the supernatants of infected cells. Western blot analysis of total cell levels of HLA-DRa and -DRb at 3 d.p.i. in CIITAt revealed that HLA-DRa and -DRb were not decreased by CMV infection, and DRb levels were increased. (-DRa and -DRb were detected by mAb TAL.1B5 and DK22 respectively). Similar results were observed with invariant chain levels as detected by mAb LN-2 (anti c-terminal). In conclusion, CMV disrupts constitutive MHC class II surface expression in this CIITA transfectant model system. This effect is not mediated by a decrease in class II mRNA levels, a soluble mediator, or a decrease in the class II whole cell protein levels. Together these results support that CMV disrupts the trafficking of MHC class II to the cell surface and that the class II is retained intracellularly. A similar pattern of class II retention has been demonstrated in splenocytes from invariant chain knock-out mice and in the developmental regulation of class II expression in dendritic cells (12, 13, 14). We are currently employing confocal microscopy colocalization studies to confirm and characterize the existence of aberrant class II trafficking.

References:

1. Jonjic et al. 1990. J. Virol. 64:5457-5464
2. Lucin et al. 1992. J. Virol. 66:1977-1984
3. Jonjic et al. 1989. J. Exp. Med. 169:1199-1212
4. Davignon et al. 1996. J. Virol. 70:2162-2169
5. Hengel et al. 1994. J. Virol. 68:289-297
6. Muller et al. 1992. Science. 255:1576-1578
7. Lindsley et al. 1986. J. Immunol. 136: 3045-3051
8. Miller et al. 1998. J. Exp. Med. 187(5):675-683
9. Fish et al. 1996. J. Virol. 70(3):1855-1862
10. Chang and Flavell. 1995. J. Exp. Med. 181:765
11. Boss. 1997. Curr. Opin. Immunol. 9:107-113
12. Viville et al. 1993. Cell. 72:635-648
13. Elliott et al. 1994. J. Exp. Med. 179:681-694
14. Pierre et al. 1997. Nature. 388:787-792

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