Protease Inhibitors Perturb Peptide Presentation See article p. 3595
Treg Transitions Impact Tolerance See article p. 3665
Interrogating HIV Vaccine B Cell Responses See article p. 3729
Fish Virus Is Unfriendly to IFN See article p. 3744
Making Memories without IL-15 See article p. 3920
Fish Virus Is Unfriendly to IFN
The induction of IFN through intracellular viral sensors, including retinoic acid–inducible gene I (RIG-I), is a first line of defense against viral infection. However, many viruses have evolved mechanisms to evade or suppress the host IFN response, devices that have been documented many times in mammalian systems but for which there is little evidence in lower vertebrate systems. In this issue, Lu et al. (p. 3744) demonstrate that the N protein of spring viremia of carp virus (SVCV), a single-stranded RNA virus that causes significant mortality in carp, inhibits IFNφ1 production in zebrafish. It accomplishes this via the degradation of mitochondrial antiviral signaling protein (MAVS), a key component of the RIG-I intracellular nucleic acid–sensing pathway. The authors transfected a minnow epithelial cell line susceptible to SVCV, epithelioma papulosum cyprini (EPC) cells, with reporter constructs harboring the promoter region of IFNφ1, 2, 3, or 4. They found that polyinosinic-polycytidylic acid stimulation activated the reporter gene in EPC cells transfected with constructs encoding IFNφ1 or 3, but not 2 or 4, an effect that could be inhibited by SVCV infection in a dose-dependent manner. To determine the mechanism behind SVCV inhibition of IFNφ1 activation, the authors cotransfected the N-terminal domain of RIG-I, MAVS, TANK-binding kinase 1 (TBK1), or IFN regulatory factor 3 or 7 with the IFNφ1 promoter region and then infected cells with SVCV. They found that IFNφ1 promoter induction was inhibited in cells that were also transfected with a MAVS reporter construct, but not with constructs containing signaling elements up- or downstream of MAVS, suggesting that SVCV inhibits IFNφ1 production by targeting MAVS. Additional studies suggested that SVCV targeted MAVS for degradation via the ubiquitin–proteasome pathway by promoting K48-linked ubiquitination of this protein through a mechanism dependent on the N protein of SVCV. These data provide strong evidence for the use of immune evasion strategies by viruses in lower vertebrates.
Making Memories without IL-15
Memory CD8+ T cells can be divided into central memory (TCM), effector memory (TEM), and resident memory (TRM) T cells on the basis of cell surface phenotype and trafficking abilities. IL-15 regulates homeostatic proliferation (HP) and survival of TCM and TEM cells and is thought to limit the total number of memory CD8+ T cells that can survive in the host. However, heterologous prime-boost-boost (HPBB) vaccination can increase the total memory CD8+ T cell pool, suggesting that memory can be increased without eroding existing memory specificities. In this issue, Schenkel et al. (p. 3920) identified varying levels of dependence on IL-15 in the generation and survival of different types of memory CD8+ T cells. Comparison of circulating tertiary memory CD8+ T cells generated through i.v. HPBB vaccination with primary memory CD8+ T cells revealed that the tertiary TEM cells had lower levels of HP and depended less on IL-15 for their maintenance than their primary memory counterparts. HPBB vaccination also significantly increased numbers of TRM cells in the female reproductive tract and salivary glands, suggesting that there is not an IL-15–regulated cap on the numbers of these memory cells. Indeed, primary CD8+ TRM cells survived in the absence of IL-15 in several (but not all) nonlymphoid tissues tested. Further analysis of TRM cells in different tissues revealed that these cells could be divided into three subsets, including those: 1) dependent on IL-15 for HP but not survival, 2) dependent on IL-15 for both HP and survival, and 3) independent of IL-15 for both HP and survival. However, in none of the tissues examined did TRM cells require IL-15 for production of IFN-γ, IL-2, or TNF-α. Taken together, these data suggest a tissue-specific heterogeneity among TRM cells and provide encouragement for vaccine development by indicating that IL-15 does not necessarily limit memory T cell generation.
Interrogating HIV Vaccine B Cell Responses
The highly mutable and variable nature of HIV envelope glycoproteins (Env) has hindered the production of a vaccine capable of eliciting a long-term broadly neutralizing Ab response. As a result, researchers have focused their vaccine efforts on targeting the few stable conserved Env sites, including the docking site for HIV on CD4+ T cells (the CD4 binding site [CD4bs]). In this issue, Wang et al. (p. 3729) conduct a thorough analysis of the genetics, functionality, and durability of Env-elicited Ab responses to determine how B cell immunity evolves over time upon repeated challenge with the first generation trimeric HIV Env target YU2 gp140-F in adjuvant. The authors immunized two rhesus macaques five times at monthly intervals and collected PBMCs 1–3 wk after each vaccination for single-cell memory B cell analysis. They found that Env-specific B cells were highly diversified, whereas the CD4bs-specific memory B cell lineages had less diversity and exhibited unique genetic and functional properties, including BCRs with longer CDR3 lengths, increased hydrophobicity, altered electrostatic properties, and decreased thermostability. These unique features may have facilitated access to the CD4 binding loop epitope on gp120. A sizeable proportion of Env-specific and CD4bs-specific clones persisted throughout the immunization schedule and accumulated progressively more extensive somatic hypermutation (SHM) after each challenge. Memory B cells with a higher degree of SHM collected after later stage immunizations exhibited increased Env-binding affinity and more potent virus neutralization abilities, indicating that improved functionality paralleled the observed increase in SHM. These data provide crucial information about the dynamic evolution of HIV Env-specific B cell responses that may inform the development of future HIV vaccines.
Protease Inhibitors Perturb Peptide Presentation
Protease inhibitors (PIs) are an important component of highly active antiretroviral therapy, which has shifted HIV infection from a death sentence to a chronic illness. In addition to inhibiting the HIV-1 protease, PIs can also affect proteasomes and aminopeptidases of host cells and thus alter Ag processing and presentation. Kourjian et al. (p. 3595) have now assessed the ability of 5 HIV PIs to also alter cross-presentation by analyzing their effects on cathepsins, proteases that cleave Ags in endosomes and phagosomes and are activated by acidification of these organelles. Different PIs could directly augment or reduce the activity of different cathepsins, and the extent of these effects varied among immune cell types. Cathepsin activity is affected by pH changes, and two of the PIs, saquinavir (SQV) and nelfinavir (NFV), could reduce the phagosomal pH in dendritic cells and the endolysosomal pH in CD4+ T cells. Phagosomal pH reduction appeared to occur via SQV- and NFV-mediated inhibition of kinases responsible for NADPH oxidase 2 activity and subsequent reactive oxygen species production, suggesting an indirect route by which these PIs might enhance cathepsin activity. As a result of their effects on cathepsin activity, PIs could alter the degradation of peptides from HIV-1 Gag p24 and change the representation of epitopes presented on MHC class I and MHC class II molecules. These alterations were linked to PI-induced changes in CTL responses to cross-presented HIV-1 Gag p24 epitopes. The PIs under study could also affect the degradation of Ags from Mycobacterium tuberculosis and hepatitis C virus, which frequently coinfect HIV-infected individuals, and SQV treatment altered the self-derived MHC-bound peptidome in uninfected PBMCs, suggesting PI treatment could also modulate autoimmunity. This study has important implications for understanding how PI treatment can alter cathepsin activity both directly and indirectly and thereby broadly affect host immune responses.
Treg Transitions Impact Tolerance
A diverse pool of regulatory T cells (Tregs) is necessary to dampen immune responses and establish and maintain self-tolerance. Within this pool, phenotypically distinct subsets of quiescent central Tregs (cTregs) residing in secondary lymphoid tissues are thought to give rise to activated effector Tregs (eTregs) with enhanced suppressive capacity that localize to sites of inflammation in the tissues. In this issue, Toomer et al. (p. 3665) used immune profiling of mRNA transcripts and deep sequencing of TCR-β V regions to help define the genetic heterogeneity of and functional association between eTreg and cTreg subsets. They found that cTregs have heterogeneous Ly-6C expression, with Ly-6C+ cTregs exhibiting lower affinity TCRs, less suppressive capacity, and a diminished propensity to transition into eTregs than their Ly-6C− counterparts. Ly-6C+ cTregs shared fewer TCR-β V region sequences with eTregs than did Ly-6C− cTregs, which exhibited sequences in common with both Ly-6C+ cTregs and eTregs. This sequence sharing suggests that Ly-6C− cTregs represent a transitional phenotype between eTregs and cTregs. eTregs were divided into four CD62Llo subsets based on CD69 and CD103 expression that had a high degree of similarity in their TCR-β repertoire but high variability in their transcriptional immune profiles. CD103− eTreg population transcriptional immune profiles shared similarities with those of cTregs, suggestive of a transitional population between cTregs and eTregs. cTregs adoptively transferred into autoimmune-prone recipients were able to transition into the eTreg phenotype, but these cells were less capable of suppressing autoimmunity than were cells that were eTregs at the time of adoptive transfer. Together, these results suggest that some, but not all, cTregs are capable of transitioning into eTregs, but it is likely that environmental programming of this more flexible Treg subset is necessary for the development of optimized suppression of autoimmunity.