Memory CD8+ T cells in the circulation rapidly infiltrate non-lymphoid tissues following infection and provide protective immunity in an antigen-specific manner. However, the subsequent fate of memory CD8+ T cells after entering non-lymphoid tissues such as the skin during a secondary infection is largely unknown. Furthermore, because expression of CD62L is often used to identify the central memory (TCM) CD8+ T cell subset, uncoupling the physical requirement for CD62L-mediated lymph node homing versus other functional attributes of TCM CD8+ T cells remains unresolved. Here, we show that in contrast to naïve CD8+ T cells, memory CD8+ T cells traffic into the skin independent of CD62L-mediated lymph node re-activation and provide robust protective immunity against Vaccinia virus (VacV) infection. TCM, but not effector memory (TEM), CD8+ T cells differentiated into functional CD69+/CD103- tissue residents following viral clearance, which was also dependent on local recognition of antigen in the skin microenvironment. Finally, we found that memory CD8+ T cells expressed granzyme B after trafficking into the skin and utilized cytolysis to provide protective immunity against VacV infection. Collectively, these findings demonstrate that TCM CD8+ T cells become cytolytic following rapid infiltration of the skin to protect against viral infection and subsequently differentiate into functional CD69+ tissue-residents.

CD4+ helper T cells play important roles in providing help to B cells, macrophages, and cytotoxic CD8+ T cells, but also exhibit direct effector functions against a variety of different pathogens. In contrast to CD8+ T cells, CD4+ T cells typically exhibit broader specificities and undergo less clonal expansion during many types of viral infections, which often makes the identification of virus-specific CD4+ T cells technically challenging. In this study, we have generated recombinant vaccinia virus (VacV) vectors that target I-Ab–restricted peptides for MHC class II (MHC-II) presentation to activate CD4+ T cells in mice. Conjugating the lymphocytic choriomeningitis virus immunodominant epitope GP61–80 to either LAMP1 to facilitate lysosomal targeting or to the MHC-II invariant chain (Ii) significantly increased the activation of Ag-specific CD4+ T cells in vivo. Immunization with VacV-Ii-GP61–80 activated endogenous Ag-specific CD4+ T cells that formed memory and rapidly re-expanded following heterologous challenge. Notably, immunization of mice with VacV expressing an MHC-II–restricted peptide from Leishmania species (PEPCK335–351) conjugated to either LAMP1 or Ii also generated Ag-specific memory CD4+ T cells that underwent robust secondary expansion following a visceral leishmaniasis infection, suggesting this approach could be used to generate Ag-specific memory CD4+ T cells against a variety of different pathogens. Overall, our data show that VacV vectors targeting peptides for MHC-II presentation is an effective strategy to activate Ag-specific CD4+ T cells in vivo and could be used to study Ag-specific effector and memory CD4+ T cell responses against a variety of viral, bacterial, or parasitic infections.

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The COVID-19 pandemic is a global health emergency, and the development of a successful vaccine will ultimately be required to prevent the continued spread and seasonal recurrence of this disease within the human population. However, very little is known about either the quality of the adaptive immune response or the viral Ag targets that will be necessary to prevent the spread of the infection. In this study, we generated recombinant Vaccinia virus expressing the full-length spike protein from SARS-CoV-2 (VacV-S) to evaluate the cellular and humoral immune response mounted against this viral Ag in mice. Both CD8+ and CD4+ T cells specific to the SARS-CoV-2 spike protein underwent robust expansion, contraction, and persisted for at least 40 d following a single immunization with VacV-S. Vaccination also caused the rapid emergence of spike-specific IgG-neutralizing Abs. Interestingly, both the cellular and humoral immune responses strongly targeted the S1 domain of spike following VacV-S immunization. Notably, immunization with VacV-expressing spike conjugated to the MHC class II invariant chain, a strategy previously reported by us and others to enhance the immunogenicity of antigenic peptides, did not promote stronger spike-specific T cell or Ab responses in vivo. Overall, these findings demonstrate that an immunization approach using VacV or attenuated versions of VacV expressing the native, full-length SARS-CoV-2 spike protein could be used for further vaccine development to prevent the spread of COVID-19.

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