Anna M. Keller

Expression of Costimulatory Ligand CD70 on Steady-State Dendritic Cells Breaks CD8+ T Cell Tolerance and Permits Effective Immunity

Steady-state dendritic cells (DCs) maintain peripheral T cell tolerance, whereas mature DCs generate immunity. CD70 is a costimulatory ligand acquired upon DC maturation. To determine its impact on T cell fate, we have generated mice that constitutively express CD70 in conventional DCs (cDCs). In these mice, naive CD4+ and CD8+ T cells spontaneously convert into effector cells. Administration of peptide without adjuvant, which is ordinarily tolerogenic, elicited tumor-eradicating CD8+ T cell responses and robust CD4+ T cell-independent memory. CD70 was also constitutively expressed in cDCs that inducibly present viral epitopes. In this case, tolerance induction was prevented as well. The antigen-presenting DCs generated protective immunity to virus infection and broke a pre-existing state of CD8+ T cell tolerance. Thus, the sole expression of CD70 by otherwise immature cDCs sufficed to convert CD8+ T cell tolerance into immunity, defining the importance of CD27-CD70 interactions at the interface between T cell and DC.

Apoptosis induction by Bid requires unconventional ubiquitination and degradation of its N-terminal fragment.

Bcl-2 family member Bid is subject to autoinhibition; in the absence of stimuli, its N-terminal region sequesters the proapoptotic Bcl-2 homology 3 (BH3) domain. Upon proteolytic cleavage in its unstructured loop, Bid is activated, although structural data reveal no apparent resulting conformational change. We found that, upon Bid cleavage, the N-terminal fragment (tBid-N) is ubiquitinated and degraded, thus freeing the BH3 domain in the C-terminal fragment (tBid-C). Ubiquitination of tBid-N is unconventional because acceptor sites are neither lysines nor the N terminus. Chemical approaches implicated thioester and hydroxyester linkage of ubiquitin and mutagenesis implicated serine and possibly threonine as acceptor residues in addition to cysteine. Acceptor sites reside predominantly but not exclusively in helix 1, which is required for ubiquitination and degradation of tBid-N. Rescue of tBid-N from degradation blocked Bids ability to induce mitochondrial outer membrane permeability but not mitochondrial translocation of the cleaved complex. We conclude that unconventional ubiquitination and proteasome-dependent degradation of tBid-N is required to unleash the proapoptotic activity of tBid-C.

Immunological and Antitumor Effects of IL-23 as a Cancer Vaccine Adjuvant

The promising, but modest, clinical results of many human cancer vaccines indicate a need for vaccine adjuvants that can increase both the quantity and the quality of vaccine-induced, tumor-specific T cells. In this study we tested the immunological and antitumor effects of the proinflammatory cytokine, IL-23, in gp100 peptide vaccine therapy of established murine melanoma. Neither systemic nor local IL-23 alone had any impact on tumor growth or tumor-specific T cell numbers. Upon specific vaccination, however, systemic IL-23 greatly increased the relative and absolute numbers of vaccine-induced CD8+ T cells and enhanced their effector function at the tumor site. Although IL-23 specifically increased IFN-γ production by tumor-specific T cells, IFN-γ itself was not a primary mediator of the vaccine adjuvant effect. The IL-23-induced antitumor effect and accompanying reversible weight loss were both partially mediated by TNF-α. In contrast, local expression of IL-23 at the tumor site maintained antitumor activity in the absence of weight loss. Under these conditions, it was also clear that enhanced effector function of vaccine-induced CD8+ T cells, rather than increased T cell number, is a primary mechanism underlying the antitumor effect of IL-23. Collectively, these results suggest that IL-23 is a potent vaccine adjuvant for the induction of therapeutic, tumor-specific CD8+ T cell responses.

CD27 instructs CD4+ T cells to provide help for the memory CD8+ T cell response after protein immunization.

For optimal quality, memory CD8+ T cells require CD4+ T cell help. We have examined whether CD4+ T cells require CD27 to deliver this help, in a model of intranasal OVA protein immunization. CD27 deficiency reduced the capacity of CD4+ T cells to support Ag-specific CD8+ T cell accumulation at the tissue site after primary and secondary immunization. CD27-dependent CD4+ T cell help for the memory CD8+ T cell response was delivered during priming. It did not detectably affect formation of CD8+ memory T cells, but promoted their secondary expansion. CD27 improved survival of primed CD4+ T cells, but its contribution to the memory CD8+ T cell response relied on altered CD4+ T cell quality rather than quantity. CD27 induced a Th1-diagnostic gene expression profile in CD4+ T cells, which included the membrane molecule MS4A4B. Accordingly, CD27 increased the frequency of IFN-γ- and IL-2-producing CD4+ T cells. It did not affect CD40L expression. Strikingly, MS4A4B was also identified as a unique marker of CD8+ memory T cells that had received CD27-proficient CD4+ T cell help during the primary response. This apparent imprinting effect suggests a role for MS4A4B as a downstream effector in CD27-dependent help for CD8+ T cell memory.

Costimulatory ligand CD70 allows induction of CD8+ T-cell immunity by immature dendritic cells in a vaccination setting

The use of dendritic cells (DCs) as anticancer vaccines holds promise for therapy but requires optimization. We have explored the potential of costimulatory ligand CD70 to boost the capacity of DCs to evoke effective CD8(+) T-cell immunity. We show that immature conventional DCs, when endowed with CD70 expression by transgenesis, are converted from a tolerogenic state into an immunogenic state. Adoptively transferred CD70-expressing immature DCs could prime CD8(+) T cells, by CD27, to become tumor-eradicating cytolytic effectors and memory cells with a capacity for robust secondary expansion. The CD8(+) T-cell response, including memory programming, was independent of CD4(+) T-cell help, because the transferred immature DCs were loaded with major histocompatibility complex class I-restricted peptide only. Without CD70 expression, the DCs generated abortive clonal expansion, dysfunctional antitumor responses, and no CD8(+) T-cell memory. CD70-expressing CD8(+) DCs were the primary subset responsible for CD8(+) T-cell priming and performed comparably to fully matured DCs. These data highlight the importance of CD27/CD70 interactions at the T-cell/DC interface and indicate that CD70 should be considered in the design of DC vaccination strategies.

Costimulatory ligand CD70 is delivered to the immunological synapse by shared intracellular trafficking with MHC class II molecules

TNF family member CD70 is the ligand of CD27, a costimulatory receptor that shapes effector and memory T cell pools. Tight control of CD70 expression is required to prevent lethal immunodeficiency. By selective transcription, CD70 is largely confined to activated lymphocytes and dendritic cells (DC). We show here that, in addition, specific intracellular routing controls its plasma membrane deposition. In professional antigen-presenting cells, such as DC, CD70 is sorted to late endocytic vesicles, defined as MHC class II compartments (MIIC). In cells lacking the machinery for antigen presentation by MHC class II, CD70 travels by default to the plasma membrane. Introduction of class II transactivator sufficed to reroute CD70 to MIIC. Vesicular trafficking of CD70 and MHC class II is coordinately regulated by the microtubule-associated dynein motor complex. We show that when maturing DC make contact with T cells in a cognate fashion, newly synthesized CD70 is specifically delivered via MIIC to the immunological synapse. Therefore, we propose that routing of CD70 to MIIC serves to coordinate delivery of the T cell costimulatory signal in time and space with antigen recognition.

The Pim Kinase Pathway Contributes to Survival Signaling in Primed CD8+ T Cells upon CD27 Costimulation

Stimulation of the costimulatory receptor CD27 by its ligand CD70 has proved important for the generation of primary and memory CD8+ T cell responses in various models of antigenic challenge. CD27/CD70-mediated costimulation promotes the survival of primed T cells and thereby increases the size of effector and memory populations. In this paper, we reveal molecular mechanisms underlying the prosurvival effect of CD27. CD27 signaling upregulated expression of the antiapoptotic Bcl-2 family member Bcl-xL. However, genetic reconstitution of Cd27−/− CD8+ T cells with Bcl-xL alone or in combination with the related protein Mcl-1 did not compensate for CD27 deficiency in the response to influenza virus infection. This suggested that CD27 supports generation of the CD8+ effector T cell pool not only by counteracting apoptosis via Bcl-2 family members. Genome-wide mRNA expression profiling indicated that CD27 directs expression of the Pim1 gene. Pim-1 is a serine/threonine kinase that sustains survival of rapidly proliferating cells by antiapoptotic and prometabolic effects that are independent of the mammalian target of rapamycin (mTOR) pathway. In TCR-primed CD8+ T cells, CD27 could increment Pim-1 protein expression and promote cell survival throughout clonal expansion independent of the mTOR and IL-2R pathways. In addition, introduction of the Pim1 gene in Cd27−/− CD8+ T cells partially corrected their defect in clonal expansion and formation of an effector pool. We conclude that CD27 may contribute to the survival of primed CD8+ T cells by the upregulation of antiapoptotic Bcl-2 family members but also calls the Pim-1 kinase survival pathway into action.

The invariant chain transports TNF family member CD70 to MHC class II compartments in dendritic cells

CD70 is a TNF-related transmembrane molecule expressed by mature dendritic cells (DCs), which present antigens to T cells via major histocompatibility complex (MHC) molecules. In DCs, CD70 localizes with MHC class II molecules in late endosomal vesicles, known as MHC class II compartments (MIICs). MIICs are transported to the immune synapse when a DC contacts an antigen-specific CD4+ T cell. Consequently, MHC class II and CD70 are simultaneously exposed to the T cell. Thereby, T-cell activation via the antigen receptor and CD70-mediated co-stimulation are synchronized, apparently to optimize the proliferative response. We report here that the invariant chain (Ii), a chaperone known to transport MHC class II to MIICs, performs a similar function for CD70. CD70 was found to travel by default to the plasma membrane, whereas Ii coexpression directed it to late endosomes and/or lysosomes. In cells containing the MHC class II presentation pathway, CD70 localized to MIICs. This localization relied on Ii, since transport of CD70 from the Golgi to MIICs was impeded in Ii-deficient DCs. Biophysical and biochemical studies revealed that CD70 and Ii participate in an MHC-class-II-independent complex. Thus, Ii supports transport of both MHC class II and CD70 to MIICs and thereby coordinates their delivery to CD4+ T cells.