Kamal M. Khanna

Endogenous naive CD8+ T cell precursor frequency regulates primary and memory responses to infection.

Through genetic recombination, the adaptive immune system generates a diverse T cell repertoire allowing recognition of a vast spectrum of foreign antigens. Any given CD8+ T cell specificity is thought to be rare, but none have been directly quantified. Here, major histocompatibility complex tetramer and magnetic-bead technology were coupled to quantitate naive antigen-specific CD8+ T cells and the early response to infection. Among six specificities measured, the number of naive antigen-specific precursors ranged from approximately 80 to 1200 cells/mouse. After vesicular stomatitis virus infection, the antigen-specific CD8+ T cell response occurred in discrete phases: prolonged activation of a subset of cells over the first 72 hr followed by a rapid proliferative burst. Naive precursor frequency altered response kinetics and regulated immunodominance, as well as the time required for the responding population to shift toward CD62L(hi) memory cells. Thus, initial endogenous precursor frequencies were surprisingly diverse and not only regulated initial immune response characteristics but also controlled memory CD8+ T cell lineage decisions.

Herpes Simplex Virus-Specific Memory CD8+ T Cells Are Selectively Activated and Retained in Latently Infected Sensory Ganglia

This study challenges the concept that herpes simplex virus type 1 (HSV-1) latency represents a silent infection that is ignored by the host immune system, and suggests antigen-directed retention of memory CD8(+) T cells. CD8(+) T cells specific for the immunodominant gB(498-505) HSV-1 epitope are selectively retained in the ophthalmic branch of the latently infected trigeminal ganglion, where they acquire and maintain an activation phenotype and the capacity to produce IFN-gamma. Some CD8(+) T cells showed TCR polarization to junctions with neurons. A gB(498-505) peptide-specific CD8(+) T cell clone can block HSV-1 reactivation from latency in ex vivo trigeminal ganglion cultures. We conclude that CD8(+) T cells provide active surveillance of HSV-1 gene expression in latently infected sensory neurons.

Noncytotoxic Lytic Granule–Mediated CD8+ T Cell Inhibition of HSV-1 Reactivation from Neuronal Latency

Reactivation of herpes simplex virus type 1 (HSV-1) from neuronal latency is a common and potentially devastating cause of disease worldwide. CD8+ T cells can completely inhibit HSV reactivation in mice, with interferon-γ affording a portion of this protection. We found that CD8+ T cell lytic granules are also required for the maintenance of neuronal latency both in vivo and in ex vivo ganglia cultures and that their directed release to the junction with neurons in latently infected ganglia did not induce neuronal apoptosis. Here, we describe a nonlethal mechanism of viral inactivation in which the lytic granule component, granzyme B, degrades the HSV-1 immediate early protein, ICP4, which is essential for further viral gene expression.

Gamma Interferon Can Prevent Herpes Simplex Virus Type 1 Reactivation from Latency in Sensory Neurons

ABSTRACT We recently demonstrated that CD8+ T cells could block herpes simplex virus type 1 (HSV-1) reactivation from latency in ex vivo trigeminal ganglion (TG) cultures without destroying the infected neurons. Here we establish that CD8+ T-cell prevention of HSV-1 reactivation from latency is mediated at least in part by gamma interferon (IFN-γ). We demonstrate that IFN-γ was produced in ex vivo cultures of dissociated latently infected TG by CD8+ T cells that were present in the TG at the time of excision. Depletion of CD8+ T cells or neutralization of IFN-γ significantly enhanced the rate of HSV-1 reactivation from latency in TG cultures. When TG cultures were treated with acyclovir for 4 days to insure uniform latency, supplementation with recombinant IFN-γ blocked HSV-1 reactivation in 80% of cultures when endogenous CD8+ T cells were present and significantly reduced and delayed HSV-1 reactivation when CD8+ T cells or CD45+ cells were depleted from the TG cultures. The effectiveness of recombinant IFN-γ in blocking HSV-1 reactivation was lost when its addition to TG cultures was delayed by more than 24 h after acyclovir removal. We propose that when the intrinsic ability of neurons to inhibit HSV-1 gene expression is compromised, HSV-specific CD8+ T cells are rapidly mobilized to produce IFN-γ and perhaps other antiviral cytokines that block the viral replication cycle and maintain the viral genome in a latent state.

Lung‐resident memory CD8 T cells (TRM) are indispensable for optimal cross‐protection against pulmonary virus infection

Previous studies have shown that some respiratory virus infections leave local populations of tissue TRM cells in the lungs which disappear as heterosubtypic immunity declines. The location of these TRM cells and their contribution to the protective CTL response have not been clearly defined. Here, fluorescence microscopy is used to show that some CD103+ TRM cells remain embedded in the walls of the large airways long after pulmonary immunization but are absent from systemically primed mice. Viral clearance from the lungs of the locally immunized mice precedes the development of a robust Teff response in the lungs. Whereas large numbers of virus‐specific CTLs collect around the bronchial tree during viral clearance, there is little involvement of the remaining lung tissue. Much larger numbers of TEM cells enter the lungs of the systemically immunized animals but do not prevent extensive viral replication or damage to the alveoli. Together, these experiments show that virus‐specific antibodies and TRM cells are both required for optimal heterosubtypic immunity, whereas circulating memory CD8 T cells do not substantially alter the course of disease.

Evidence that a significant number of naive T cells enter non-lymphoid organs as part of a normal migratory pathway.

Only activated and effector memory T cells are thought to access non‐lymphoid tissues. In contrast, naive T cells are thought to circulate only between the blood, lymph and secondary lymphoid organs. We examined the phenotype of endogenous T cells in various non‐lymphoid organs and showed that a subset of cells exhibited an apparently naive phenotype and were functionally inactive. FTY720 treatment selectively depleted this population from the non‐lymphoid tissues. In addition, RAG‐deficient TCR transgenic CD4 and CD8 T cells were present in non‐lymphoid tissues in bone marrow chimeric mice and in situ imaging analysis revealed their location in the parenchymal tissues. Moreover, migration of TCR transgenic T cells to non‐lymphoid tissues after adoptive transfer was pertussis‐toxin resistant. Overall, the results suggest that naive T cells may circulate through non‐lymphoid tissues as part of their normal migratory pathway.

Cell-surface residence of sphingosine 1-phosphate receptor 1 on lymphocytes determines lymphocyte egress kinetics

The sphingosine 1-phosphate receptor 1 (S1P1) promotes lymphocyte egress from lymphoid organs. Previous work showed that agonist-induced internalization of this G protein–coupled receptor correlates with inhibition of lymphocyte egress and results in lymphopenia. However, it is unclear if S1P1 internalization is necessary for this effect. We characterize a knockin mouse (S1p1rS5A/S5A) in which the C-terminal serine-rich S1P1 motif, which is important for S1P1 internalization but dispensable for S1P1 signaling, is mutated. T cells expressing the mutant S1P1 showed delayed S1P1 internalization and defective desensitization after agonist stimulation. Mutant mice exhibited significantly delayed lymphopenia after S1P1 agonist administration or disruption of the vascular S1P gradient. Adoptive transfer experiments demonstrated that mutant S1P1 expression in lymphocytes, rather than endothelial cells, facilitated this delay in lymphopenia. Thus, cell-surface residency of S1P1 on T cells is a primary determinant of lymphocyte egress kinetics in vivo.

Persistent Antigen Presentation after Acute Vesicular Stomatitis Virus Infection

ABSTRACT Long-term antigen expression is believed to play an important role in modulation of T-cell responses to chronic virus infections. However, recent studies suggest that immune responses may occur late after apparently acute infections. We have now analyzed the CD8 T-cell response to vesicular stomatitis virus (VSV), which is thought to cause to an infection characterized by rapid virus clearance by innate and adaptive immune system components. Unexpectedly, virus-encoded antigen was detectable more than 6 weeks after intranasal VSV infection in both draining and nondraining lymph nodes by adoptively transferred CD8 T cells. Infection with Listeria monocytogenes expressing the same antigen did not result in prolonged antigen presentation. Weeks after VSV infection, discrete T-cell clustering with dendritic cells within the lymph node was observed after transfer of antigen-specific CD8 T cells. Moreover, memory CD8 T cells as defined by phenotype and function were generated from naïve CD8 T cells entering the response late after infection. These findings suggested that protracted antigen presentation after an apparently acute virus infection may contribute to an ongoing antiviral immune response.

Latent Virus Influences the Generation and Maintenance of CD8+ T Cell Memory

The influence of latent virus on CD8+ T cell memory is poorly understood. HSV type 1 specifically establishes latency in trigeminal ganglia (TG) after corneal infection of mice. In latently infected TG, IL-15 deprivation reduced the following: 1) accumulation of HSV-specific CD8+ effector T cells (HSV-CD8eff), 2) accumulation of CD127+ putative HSV-CD8 memory precursors, and 3) the size and functionality of the memory (HSV-CD8mem) population. Although compromised in IL-15−/− mice, the HSV-CD8mem pool persisted in latently infected tissue, but not in noninfected tissue of the same mice. Anti-IL-2 treatment also dramatically reduced the size of the HSV-CD8eff population in the TG, but did not influence the concomitant generation of the CD127+ putative HSV-CD8mem precursor population or the size or functionality of the HSV-CD8mem pool. Thus, the size of the memory pool appears to be determined by the size of the CD127+ CD8mem precursor population and not by the size of the overall CD8eff pool. HSV-CD8mem showed a higher basal rate of proliferation in latently infected than noninfected tissue, which was associated with a reduced population of CD4+FoxP3+ regulatory T cells. Thus, the generation, maintenance, and function of memory CD8+ T cells is markedly influenced by latent virus.

In situ imaging reveals different responses by naïve and memory CD8 T cells to late antigen presentation by lymph node DC after influenza virus infection

Pulmonary influenza infection causes prolonged lymph node hypertrophy while processed viral antigens continue to be presented to virus‐specific CD8 T cells. We show that naïve, but not central/memory, nucleoprotein (NP)‐specific CD8 T cells recognized antigen‐bearing CD11b+ DC in the draining lymph nodes more than 30 days after infection. After these late transfers, the naïve CD8 T cells underwent an abortive proliferative response in the mediastinal lymph node (MLN), where large clusters of partially activated cells remained in the paracortex until at least a week after transfer. A majority of the endogenous NP‐specific CD8 T cells that were in the MLN between 30 and 50 days after infection also showed signs of a continuing response to antigen stimulation. A high frequency of endogenous NP‐specific CD8 T cells in the MLN indicates that late antigen presentation may help shape the epitope dominance hierarchy during reinfection.