Research identifies Th1 memory CD4+ T cells as key to protect against emerging poxvirus skin infections

By Neha MathurReviewed by Lily Ramsey, LLMApr 27 2023

In a recent article published in the Cell Reports Journal, researchers used a preclinical mouse model to demonstrate that T helper 1 (Th1) effector memory cluster of differentiation 4 (CD4)⁺ T cells provide complete immunity against poxviruses-triggered skin infections.

Study: T helper 1 effector memory CD4⁺ T cells protect the skin from poxvirus infection. Image Credit: MarinaDemidiuk/Shutterstock.com

Introduction

Pox viruses are an emerging public health concern after the World Health Organization (WHO) declared the mpox virus a public health emergency of international concern (PHEIC) in 2022.

During the 2022 mpox outbreak, this deadly virus spread in >100 countries, causing more than 80,000 cases amid the pandemic of the ongoing coronavirus disease 2019 (COVID-19).

Background

Within 200 years of its introduction, the vaccinia virus (VACV)-based vaccine eradicated smallpox, a deadly communicable disease caused by Variola virus.

Globally, immunizations by VacV ended by the 1980s; thus, most of the world population is no longer immune to zoonotic poxviruses, such as mpox virus and cowpox virus (CPXV) of genus Orthopoxvirus, which spread primarily by direct contact with infected skin lesions.

Countries dealing with mpox outbreaks used a modified VacV Ankara (MVA) vaccine to combat mpox infections. However, it remains largely unknown whether the MVA vaccine protects against poxvirus skin infections and elicits adequate immunological memory to prevent the dissemination of zoonotic poxviruses.

Mpox and CPXV express genes that inhibit antigen presentation by major histocompatibility complex (MHC) class I molecules, thereby evading the anti-viral activity of CD8⁺ cytotoxic T lymphocytes (CTLs), raising the possibility that Th1 CD4⁺ T cells might be the key players in combating poxvirus skin infections.

Skin, the largest human organ system, is a physical barrier between the “self” and the environment.

Keratinocytes, cells that detect pathogen invasion and initiate inflammatory responses by secreting different cytokines, constitute the outermost layer of skin called the epidermis.

Although there is experimental data on the role of keratinocytes in harmonizing immune responses within the skin, whether they are the targets of anti-viral adaptive immunity combating viral skin infections is not fully known.

About the study

In the present study, researchers vaccinated mice with tk⁻ VacV by skin scarification 30 days before infecting their distal skin with CPXV. This was to determine whether VacV elicited adequate memory CD8⁺ or CD4⁺ T cells to protect the skin from zoonotic poxvirus infections.

Further, they used MHC class I and class II epitopes highly conserved in the genus Orthopoxvirus to stimulate mice splenocytes and then analyzed whether T cells secreted interferon-gamma (IFNγ)/tumor necrosis factor-alpha (TNF-α).

Furthermore, they tested whether inhibition of MHC class I expression allowed CPXV to evade memory CD8⁺ T cells.

The team transferred SMARTA T cell receptor (TCR)-transgenic CD4+ T cells into B6 mice. Next, they infected test animals with lymphocytic choriomeningitis virus (LCMV) to generate a readily trackable population of CD4+ T cells.

These T cells were specific to an immunodominant epitope from the LCMV glycoprotein, amino acids 61–80 (GP61). Finally, they infected LCMV-immune mice with VacV and VacV-GP61 on the right and left ear skin, respectively.

Study findings

VacV vaccination generated memory CD8⁺ and CD4⁺ T cells; however, the relative contributions of memory T cells and neutralizing antibodies in mediating adaptive immunity against poxvirus skin infections remain incomprehensible.

Antigen-specific memory CD8⁺ and CD4⁺ T cells underwent secondary expansion following CPXV infection of the skin. Compared to naive animals, VacV-immune mice did not develop necrotic skin lesions.

The team infected VacV-vaccinated mice and controlled animals with wild-type (wt) CPXV or Δ203CPXV. Due to the depletion of CD8+ T cells before infection, test animals had no immunity against Δ203CPXV, demonstrating that memory CD8+ T cells recognized and eliminated the virus.

However, “bystander” activation of memory CD8+ T cells did not limit wt VacV Western Reserve (VacV-WR) replication in the skin. Conversely, loss of antigen-specific memory CD4+ T cells significantly reduced the magnitude of protection as these cells alone eliminated the virus.

Depletion of CD8+ and CD4+ T cell populations before infection with VacV-WR increased viral loads by ∼1,000–5,000 times compared with control-treated animals, demonstrating that only memory CD4⁺ T cells protected the skin from CPXV infection.

Memory SMARTA CD4⁺ T cells infiltrated towards VacV- and VacV-GP61-infected skin equally in an antigen-independent manner. GP61-specific memory CD4⁺ T cells conferred complete protection against the VacV-GP61 infection but not against the VacV infection.

Accordingly, LCMV-immune mice remained protected from VacV-GP61 skin infection, and within seven days of infection, they cleared the virus.

Regardless of antigen and tissue-specificity, virus-specific memory CD4⁺ T cells conferred adequate immunity against poxvirus infection in the skin.

Memory CD4⁺ T cells infiltrating the skin following VacV infection were predominately Ly6C⁺ effector memory (T_EM) cells, which expressed core two O-glycans, identified via the monoclonal antibody, 1B11.

It demonstrated that the Th1 memory CD4⁺ T cells trafficking into the skin were Ly6C⁺ T_EM and synthesized core two O-glycans. More importantly, these cells exhibited robust anti-viral activity within the skin.

Conclusions

Several earlier studies demonstrated that CD4+ T cells orchestrate adaptive immune responses by providing “assistance” to B cells. For example, Th1 CD4⁺ T cells activated by IFNγ increase the expression of nitric oxide (NO) and reactive oxygen species (ROS) to mediate the killing of bacterial pathogens, e.g., Salmonella enterica.

In many instances, antigen-specific memory CD4⁺ T cells also become pathogenic rather than protective during viral infections.

This study highlighted some previously underappreciated functions of T cells that protect against viral skin infections. First, antigen-specific Th1 T_EM CD4+ T cells provided protection and limited pathology during poxvirus infection of the skin. Second, CD4+ and CD8+ memory T cells used distinct but synergistic mechanisms to combat viral skin infections.

Third, the researchers observed that keratinocytes were highly responsive to IFNγ produced in response to local activation of antigen-specific memory CD4+ T cells during a viral skin infection.

However, keratinocytes activated different signaling pathways in response to IFNγ during commensal bacterial colonization than poxvirus infection of the epidermis.

Rapid delivery of IFNγ to keratinocytes prompted them to express interferon-stimulating genes and increase antigen presentation.

The study demonstrated that virus-specific memory CD4+ T cells traffic into skin keratinocytes to limit viral spread and replication within the host.

Given the critical function of memory CD4+ T cells in providing immunity against poxvirus infections of the skin, studies should investigate whether the MVA vaccine elicits poxvirus-specific Th1 effector memory CD4+ T cells.