CD 8 1 T lymphocyte mobilization to virus - infected tissue requires CD 4 1 T - cell help

CD4 T helper cells are well known for their role in providing critical signals during priming of cytotoxic CD8 T lymphocyte (CTL) responses in vivo. T-cell help is required for the generation of primary CTL responses as well as in promoting protective CD8 memory T-cell development. However, the role of CD4 help in the control of CTL responses at the effector stage is unknown. Here we show that fully helped effector CTLs are themselves not selfsufficient for entry into the infected tissue, but rely on the CD4 T cells to provide the necessary cue. CD4 T helper cells control the migration of CTL indirectly through the secretion of IFN-c and induction of local chemokine secretion in the infected tissue. Our results reveal a previously unappreciated role of CD4 help in mobilizing effector CTL to the peripheral sites of infection where they help to eliminate infected cells. Elimination of invading pathogens often requires coordinated effort by effector lymphocytes for containment and clearance. Successful defence against intracellular pathogens requires neutralizing antibodies and CTL responses, both of which largely depend on CD4 T-cell help. Whereas the generation of primary CD8 T-cell responses to noninflammatory antigens and certain virus infections, such as herpes simplex virus (HSV), require CD4 T-cell help, primary CTL responses to acute infection with Listeria monocytogenes and lymphocytic choriomeningitis virus can occur in the absence of CD4 T cells. Instead, the latter type of infections requires CD4 help in promoting memory CTL development. The critical role of CD4 help in the priming and maintenance of CTL responses is well characterized; however, whether CD4 T cells help at the stages after CTL differentiation has not been described and is at present unknown. To explore the role of CD4 help in effector CTL responses, we used a physiological model of local virus infection that enables tracking of antigen-specific CD8 T cells. HSV-2 infects humans through sexual contact and causes genital herpes. When inoculated into the vaginal cavity, HSV-2 replicates predominantly in the mucosal epithelial cells and establishes latency in the innervating neurons. Because the viral infection is localized, the genital herpes model enabled us to dissect the role of CD4 help in CTL migration to the site of infection. To avoid neurovirulence associated with wild-type HSV-2, without compromising the ability to prime robust innate and adaptive immunity, we used thymidine-kinase (TK)-defective HSV-2 (TK HSV-2). After TK HSV-2 infection, both CD4 and CD8 T cells are primed in the local draining lymph nodes, and both total (Supplementary Fig. 1a) and virus-specific (Supplementary Fig. 1b, c) effector T cells migrate into the vaginal mucosa starting with CD4 T cells around day 3–4, followed by CD8 T cells on day 4–5. Notably, migration of virus-specific CD8 T cells to the infection site was highly dependent on the presence of CD4 T cells, evidenced by the failure of CD8 T cells to migrate to the local tissue in mice that were either CD4-deficient, or depleted of CD4 T cells (Supplementary Fig. 2a). However, because primary CTL expansion after HSV-1 infection has been reported to depend on CD4 T cells through their ability to license dendritic cells, we examined the total number of congenically marked (CD45.1) HSV-gB-specific T-cell receptor (TCR) transgenic T cells (gBT-I) generated in Cd4 and CD4-depeleted mice. Consistent with previous reports, gBT-I responses in various tissues after local HSV-2 infection also depended largely on the presence of CD4 T cells (Supplementary Fig. 2b–d). To determine the mechanism by which CD4 T cells license CTL migration, fully ‘helped’ CD8 effector T cells were first generated in wild-type hosts (Fig. 1a). A physiological number (23 10 cells per mouse) of gBT-I cells were transferred into naive wild-type mice. Subsequently, these mice were infected with TK HSV-2, and effector CD8 T cells were isolated (Supplementary Fig. 3a, b) and transferred into recipient mice that had been infected with TK HSV-2 3.5 days earlier. It is well known that effector CTLs migrate to various lymphoid and non-lymphoid organs including the lung, liver and intestine. Accordingly, effector CD8 T cells were found in lymphoid and peripheral organs irrespective of the infection status of the host (Fig. 1b–d). This homeostatic distribution pattern did not depend on the presence of CD4 T cells. In stark contrast, although the fully helped effector CTLs migrated into the infected vaginal tissue in the wild-type hosts, their ability to do so was significantly impaired in the absence of CD4 T cells (Fig. 1e and Supplementary Fig. 3c). Similar results were obtained using a different time course (Supplementary Fig. 4). In contrast, adoptively transferred fully helped gBT-I T cells were able to migrate to the HSV-infected vagina in CD8-deficient hosts (Fig. 1f), indicating that CD4, but not CD8, T cells are required for licensing CTL entry into the vaginal mucosa. T regulatory (Treg) cells have been shown to facilitate early protective responses to local HSV-2 infection by allowing a timely entry of immune cells into infected tissue. To examine whether effector or Foxp3 CD4 T cells account for the mobilization of CTL into the infected vaginal mucosa, either total or Foxp3 HSV-primed CD4 T cells were adoptively transferred into HSV-infected Cd4 hosts. The analysis of migration of helped gBT-I cells in such animals showed that effector CD4 T cells were equally capable of CTL mobilization into the infected tissue as compared to total CD4 T cells (including effectors and Treg cells) (Supplementary Fig. 5). These data indicated that although Treg cells are capable of facilitating effector lymphocyte entry, effector CD4 T cells alone mediate CTL recruitment and can override the requirement for Treg cells. Collectively, our results showed that, although homeostatic migration of effector CTLs occurs independently of CD4 T-cell help, fully differentiated CTLs are not self-sufficient for accelerated recruitment to the infected tissue during a viral infection. This situation is reminiscent of the requirement for ‘pioneering’ CD4 T cells for entry by pathogenic CD4 T cells in the central nervous system.