The human cytomegalovirus-encoded pUS28 antagonizes CD4+ T-cell recognition by targeting CIITA

  1. Fabienne Maaßen
  2. Vu Thuy Khanh Le-Trilling
  3. Luisa Betke
  4. Thilo Bracht
  5. Corinna Schuler
  6. Malte Bayer
  7. Antonia Belter
  8. Tanja Becker
  9. Benjamin Katschinski
  10. Lori Frappier
  11. Barbara Sitek
  12. Katharina Fleischhauer
  13. Mirko Trilling

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Abstract

Human cytomegalovirus (HCMV) is a relevant pathogen especially for individuals with impaired immunity. Harnessing potent immune antagonists, HCMV circumvents sterile immunity. Given that HCMV prevents the upregulation of human leukocyte antigen (HLA)-DP and HLA-DR, we screened a library of HCMV genes by co-expression with the HLA class II (HLA-II)-inducing transcription coordinator class II transactivator (CIITA). We identified the latency regulator pUS28 as interaction factor and potent viral antagonist of CIITA-driven expression of CD74, HLA-DR, HLA-DM, HLA-DQ, and HLA-DP. Both wt-pUS28 and a mutant incapable to induce G-protein-coupled signaling (R129A), but not a mutant lacking the C-terminus, drastically reduced the CIITA protein abundance post-transcriptionally. While control CD4+ T cells from HCMV-seropositive individuals vigorously responded to CIITA-expressing cells decorated with HCMV antigens, pUS28 expression was sufficient to inhibit HLA-II induction and immune recognition by HCMV-specific CD4+ T cells. Our data uncover a mechanism employed by HCMV to evade HLA-II-mediated recognition by CD4+ T cells.

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    Referee #3

    Evidence, reproducibility and clarity

    Maaßen and colleagues followed up on their observation that infection with HCMV mutants lacking pUS2 and 3 impaired HLA-DP expression of IFN-gamma treated MRC-5 cells. This phenomenon is interesting because pUS2 and 3 are viral components that have been shown earlier to degrade HLA-DR alpha and HLA-DM alpha and thus inhibit the formation of HLA-DR alpha/beta heterodimers. These data indicated that in addition to pUS2 and 3 some other MHC II inhibitor must be encoded by HCMV. The authors tested this hypothesis by analyzing a HCMV gene expression library for the presence of a new HLA-DP antagonist. Indeed, the data revealed pUS28 as a new MHC II inhibitor that exhibited a posttranscriptional effect on CIITA, which is the key regulator of MHC II expression. In in vitro stimulation experiments, pUS28 impaired activation of antigen-specific CD4+ T cells.

    The study provides new and important information on how HCMV evades human immunity. The shown data support the main conclusions. Nevertheless, inclusion of some additional controls would facilitate understanding the overall concept.

    Minor points:

    A key element of this study is that IFN-gamma induces MHC II expression on MRC-5 fibroblasts. Nevertheless, professional antigen presenting cells such as dendritic cells express MHC II independent of any stimulation. Therefore, in Fig. 1 in addition to IFN-gamma induced DP expression on MRC-5 fibroblasts, DP and DR expression of dendritic cells should be shown. This could be easily done by analysis of dendritic cells in PBMC. Furthermore, the authors should show DP and DR expression of dendritic cells with and without IFN-gamma stimulation. Most likely, DP and DR expression of untreated dendritic cells is significantly higher than DP expression of IFN-gamma treated MRC-5 cells. It is important to include such controls to avoid the impression that IFN-gamma treated fibroblasts can have similar functions as professional antigen presenting cells.

    In Fig. 6b the proportion of CD137-positive T cells normalized to T cells activated by HeLa cells transfected with CIITA and pulsed with HCMV lysate is shown. In this kind of data presentation, the magnitude of the original effect, i.e., the percentage of CD4+ T cells that after 24 h of stimulation is CD137-positive, remains unclear. Therefore, it is recommended to first show actual data and then relative values. In the figure legend it is stated n = 4-10. Does this mean that T cells from different donors of the corresponding numbers have been tested? Or have T cells from some donors been tested more than once? More precise information should be given here.

    Considering the higher MHC II levels expressed by dendritic cells, it would be interesting to see to which extent pUS28 expression reduces MHC II expression of dendritic cells. Such experiments can be performed by lentiviral pUS28 expression for example in monocyte-derived dendritic cells.

    The conclusion in the last paragraph of the discussion that NKG2C+ memory NK cells might have activated antigen-specific CD4+ T cells is confusing. In the end, professional antigen presenting cells that have taken up viral proteins most probably stimulated antigen-specific CD4+ T cells. And since antigen presentation on MHC II is independent of infection of the antigen presenting cell, it is difficult to understand why under such conditions a red-queen race should have been taken place.

    Significance

    This is a highly relevant study. It adds important new information about pUS28 and how different viral components interact to evade human immunology. My expertise is on HCMV infection of human dendritic cells and the impact on antigen presentation.

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    Referee #2

    Evidence, reproducibility and clarity

    In this study, Maaßen and colleagues investigate how HCMV US28 functions to antagonize the class II Transactivator (CIITA) transcription factor and subsequent HLA class II expression. Through physical interaction with CIITA, US28 triggered a post-transcriptional decline in CIITA protein, leading to reduced cell surface expression of HLA class II molecules, including HLA-DR, HLA-DQ, HLA-DM, CD74, and HLA-DP. Moreover, the authors show that US28-mediated degradation of CIITA hindered the activation of HCMV-specific CD4+ T cells.

    Strengths of this article include the rigorous methodologies and analysis, clear and concise writing, and relevance within a clinical context. Despite the strengths, a few deficiencies were identified. Many figures lack statistical analysis to support the claims made by the authors. Where applicable, the authors should include these or explicitly state that only significant comparisons are shown. There is a lack of information regarding how the expression library screen was performed and how hits were determined/chosen for further analysis. While the observation that US28 antagonizes CIITA is well supported, the mechanism behind the antagonism is somewhat lacking.

    These findings have major implications for understanding the immune response to HCMV, particularly in immunocompromised patients where impaired HLA-II presentation poses clinical risks. The authors suggest that a comprehensive understanding of the molecular mechanisms governing HCMV immune evasion could guide the development of tailored protocols for risk protection, such as vaccination, cellular therapies, or drugs targeting US28-mediated CIITA degradation.

    Significance

    Major Comments:

    • Figure 1: Lacks statistical analysis and the number of replicate experiments that were performed.
    • Figure 2: Lacks information regarding how hits from the expression screen were determined. It would be helpful to understand the selection criteria.
    • Figure 3: While semi-quantitative RT-PCR is a useful method for determining the levels of mRNA, it would be beneficial to conduct RT-qPCR experiments to support the claim that US28 does not affect CIITA mRNA levels.
    • Figure 3: While the blots here support the authors claim that US28 antagonize CIITA at the post transcriptional level, it would be beneficial to make these observations within the context of viral infection.
    • Figure 5B: Labeling of this panel is confusing. The authors should attempt to relabel, or perform the experiment again and run samples on one gel.
    • Figure 5: data for the claim that US28-mediated antagonism of CIITA is independent of neddylation, proteasomal degradation, etc. should be shown if the authors wish to make this claim.

    Minor Comments:

    • Labeling for immunoblots should be clearer regarding transfection conditions. The terms "empty" and "vector" is ambiguous and is not descriptive enough for the conclusions the authors are drawing.
    • Many figure legends lack the information regarding the number of replicate experiments that were performed.
    • Many figures, lack statistical analysis supporting the claims being made. If observations do not reach statistical significance, these should be explicitly stated within the legends. (i.e. comparisons are shown where statically significant).
    • The authors should move away from making conclusions without showing any data substantiating their claims.
    • Minor grammatical and citation errors were identified throughout the manuscript. The authors should carefully read and fix any errors prior to publication.
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    Referee #1

    Evidence, reproducibility and clarity

    In this study entitled "The human cytomegalovirus-encoded pUS28 antagonizes CD4+ T-cell recognition by targeting CIITA", Maassen et al. tried to identify viral genes/proteins downregulating the HLA class II molecule HLA-DP. In a transfection-based screening assay, they identified US28 as a viral gene downregulating CIITA-dependent HLA-DP expression in transfected HeLa cells. Other HLA class II molecules and other proteins expressed in a CIITA-dependent manner were downregulated as well. The authors went on to show that CIITA transcripts were not reduced in the presence of pUS28, but CIITA protein levels were massively reduced, suggesting that US28 downregulates CIITA on the post-transcriptional level. A signaling-deficient US28 mutant, but not a mutant lacking the cytoplasmic carboxy terminus, was capable of reducing CIITA levels. In a final set of T cell activation experiments, the authors showed that US28-expression in HeLa cells reduced HLA-II-dependent restimulation of CD4+ T cells.

    The data presented in the paper are generally very clean and convincing. However, not all conclusions are sufficiently supported by the data. A major weakness of the study is the fact that most experiments were done with transfected HeLa cells. Whether the proposed US28-mediated HLA-II downregulation occurs in HCMV-infected cells remains unclear. Moreover, the proposed pUS28-CIITA interaction was demonstrated in a single co-IP experiment, and the mechanism of CIITA downregulation remains obscure. Hence, the conclusion that they have identified "a mechanism employed by HCMV to evade HLA-II-mediated recognition by CD4+ T cells" (abstract) is not justified.

    Major comments:

    1. The mechanism of US28-dependent CIITA downregulation remains unresolved. The authors have made several attempts to clarify the mechanism, but these experiments have not met with success. Therefore, conclusions on the underlying mechanism should be toned down or removed.
    2. The claim that US28-dependent CIITA downregulation occurs by pUS28 interacting with CIITA is based on a single co-IP experiment. The result would be more convincing if the authors could show the same interaction in a reverse IP, and ideally also in HCMV-infected cells. Is the US28 C-terminus required for this interaction?
    3. The authors could not demonstrate US28-dependent HLA-II downregulation in HCMV-infected cells. Hence, they cannot conclude that HCMV employs this mechanism. Transfected HeLa cells are a somewhat artificial system. This does not invalidate the data, but one has to be careful when interpreting the data. Others have shown that HCMV downregulates CIITA transcript levels in myeloid cells (PMID 21458073 and 31915281). This apparent discrepancy could either be explained by several redundant mechanisms (as proposed by the authors of this manuscript) or by differences and limitations of the respective experimental systems.
    4. The possibility that US28 might downregulate CIITA in latently infected cells is intriguing. Have the authors tested this in an HCMV latency system, e.g. in infected THP-1 or Kasumi-3 cells? I acknowledge that such experiments are not trivial and may be beyond the scope of the present study. However, as latently infected cells express US28 but not the other viral genes previously shown to affect HLA-II expression (US2, US3, IE1 and 2), the latency model might a way to demonstrate biological significance in virus-infected cells.

    Minor comments:

    1. Figure 2. Why was US29 used as a control and not US27 as in other experiments. The authors pointed out themselves that US27 is probably an ideal control for US28 as both genes encode related GPCRs.
    2. Figure 2. The use of "empty" for mock-transfected cells is confusing, particularly as empty vector-transfected cells are labeled "vector".

    Significance

    Recognition of virus-infected cells by CD4+ T cells is an important immune defense mechanism. Viruses like HCMV have evolved numerous immune evasion mechanisms. Previous studies have identified HCMV proteins targeting HLA-II for degradation (US2, US3) or downregulating CIITA transcription (probably IE1+IE2). The findings of the present manuscript now demonstrate that US28 is capable of contributing to HLA-II downregulation. This is potentially of great significance as US28 is expressed in latently infected cells. However, the significance of the present study is limited by the fact that the studies were done in transfected HeLa cells, not in HCMV-infected cells, and that the mechanism of CIITA post-transcriptional downregulation remains unknown.

    In its present form, the study should be of interest for virologists and immunologists interested in new viral immune evasion strategies. The significance and appeal to a wider audience would be massively increased if the authors could clarify the mechanism or show its importance in virus-infected cells (or both).

  5. Version published to 10.1101/2023.10.17.562683v1 on bioRxiv