# CD28-deficient mice are vulnerable to mouse papillomavirus MmuPV1 infection of the skin and mucosae

## TL;DR

Mice lacking CD28 are more susceptible to mouse papillomavirus infection and experience delayed viral clearance due to impaired T-cell function.

## Contribution

This study reveals that CD28 deficiency in mice delays but does not prevent papillomavirus clearance at skin and mucosal sites.

## Key findings

- CD28-deficient mice are vulnerable to MmuPV1 infection at mucosal sites like the anogenital tract and oral cavity.
- CD28 deficiency leads to delayed viral clearance and impaired T-cell recruitment and function.
- Adoptive transfer of CD8+ T cells from B6 mice achieves more effective viral clearance than from CD28ko mice.

## Abstract

CD28 is a co-stimulatory molecule expressed on the surface of T cells. To date, three individuals with germline CD28 deficiency have been reported to develop recalcitrant, HPV-driven warts: one exhibited persistent lesions, another experienced disease resolution, and the third developed a chronic “tree-man” phenotype. In mice, we confirmed that CD28-knockout (CD28ko) animals on the C57BL/6 (B6) background are susceptible to cutaneous infection with mouse papillomavirus (MmuPV1); however, their skin warts regressed spontaneously approximately five weeks post-infection. Furthermore, we demonstrate that CD28ko mice are vulnerable to MmuPV1 infection at HPV-relevant mucosal sites, including the most HPV prevalent sites: anogenital tract and oral cavity. Virions recovered from vaginal lavage were infectious but could be neutralized by the neutralizing monoclonal antibody MPV.A4. Viral clearance at mucosal sites was delayed in CD28ko mice, persisting for up to six weeks in the lower genital tract. Blocking the CD28 ligands CD80 and CD86 in B6 mice reproduced the CD28ko phenotype following MmuPV1 infection and markedly reduced CD28 expression, implicating the CD28-CD80/CD86 axis in delayed viral clearance. Infected CD28ko mice showed a reduction in both CD4+ and CD8+ T cell population in the spleen compared to infected B6 mice, but an increase in CD11c+/F4-80+ cells, particularly the plasmacytoid dendritic cell (pDCs, SiglecH⁺) subset. Additionally, CD28ko mice exhibited delayed recruitment of activated CD4+ T cells to infected tissues. Accumulation of MmuPV1 E6/90–99-specific, tetramer-positive CD8+ cytotoxic T lymphocytes (CTLs) was slower in CD28ko than in B6 mice; these CTLs remained FoxP3 negative but displayed reduced efficacy in both in vitro killing and antiviral cytokine assays. Adoptive transfer of CTLs from either B6 or CD28ko mice into MmuPV1-infected Rag1ko mice induced viral clearance at mucosal (oral) sites, whereas B6-derived CTLs achieved more complete regression of cutaneous (tail) lesions. Collectively, these findings indicate that CD28 deficiency delays but does not prevent the clearance of papillomavirus infections at both cutaneous and mucosal sites in mice.

Human papillomaviruses (HPVs) are ubiquitous, and high-risk HPV types account for approximately 5% of all human cancers. Although prophylactic vaccines are available, no effective therapy exists for established HPV infections or their sequelae. Viral persistence is central to HPV pathogenesis, and individuals with primary or secondary immunodeficiencies exhibit elevated rates of HPV infection and dysplasia. Our previous work demonstrated that CD28-deficient patients develop recalcitrant HPV-driven lesions, including the extreme “tree-man” phenotype. In mice, CD28-knockout (CD28ko) animals develop MmuPV1-induced cutaneous warts with delayed viral clearance. Here, we investigated whether MmuPV1 can infect three HPV-relevant mucosal sites, the anal tract, genital tract and oropharynx, of CD28ko mice. All sites proved susceptible. To dissect CD28-dependent immune control, we blocked CD80/CD86-expressing cells in B6 mice; this recapitulated the CD28ko phenotype and delayed viral clearance at both skin and mucosa. Further depletion of CD4+ and CD8+ T cells in CD28ko mice prolonged viral persistence. CD28ko mice exhibited delayed expansion of CD8+ T cells and impaired recruitment of CD4+ T cells to infected tissues. MmuPV1 E6/90–99-specific CD8+ cytotoxic T lymphocytes (CTLs) from infected B6 mice displayed greater in vitro cytotoxicity and antiviral activity than those from CD28ko mice. Adoptive transfer of these CTLs from either genotype into MmuPV1-infected Rag1ko mice induced tumor regression, but B6-derived CTLs were more effective, leading to complete clearance particularly at cutaneous sites. Collectively, these findings indicate that CD28 deficiency delays but does not prevent clearance of papillomavirus at both cutaneous and mucosal sites, likely through impaired T-cell recruitment and function. This study provides new insights into the immunological mechanisms underlying HPV pathogenesis and underscores the critical role of CD28-dependent pathways in orchestrating effective antiviral immunity at both mucosal and cutaneous sites.