Stephen P. Schoenberger

T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions.

Although in vivo priming of CD8+ cytotoxic T lymphocytes (CTLs) generally requires the participation of CD4+ T-helper lymphocytes,, the nature of the ‘help’ provided to CTLs is unknown. One widely held view is that help for CTLs is mediated by cytokines produced by T-helper cells activated in proximity to the CTL precursor at the surface of an antigen-presenting cell (APC). An alternative theory is that, rather than being directly supplied to the CTL by the helper cell, help is delivered through activation of the APC, which can then prime the CTL directly. CD40 and its ligand, CD40L, may activate the APC to allow CTL priming. CD40L is expressed on the surface of activated CD4+ T-helper cells and is involved in their activation and in the development of their effector functions,. Ligation of CD40 on the surface of APCs such as dendritic cells, macrophages and B cells greatly increases their antigen-presentation and co-stimulatory capacity. Here we report that signalling through CD40 can replace CD4+ T-helper cells in priming of helper-dependent CD8+ CTL responses. Blockade of CD40L inhibits CTL priming; this inhibition is overcome by signalling through CD40. CD40–CD40L interactions are therefore vital in the delivery of T-cell help for CTL priming.

CD4 + T cells are required for secondary expansion and memory in CD8 + T lymphocytes

A long-standing paradox in cellular immunology concerns the conditional requirement for CD4+ T-helper (TH) cells in the priming of cytotoxic CD8+ T lymphocyte (CTL) responses in vivo. Whereas CTL responses against certain viruses can be primed in the absence of CD4+ T cells, others, such as those mediated through ‘cross-priming’ by host antigen-presenting cells, are dependent on TH cells. A clearer understanding of the contribution of TH cells to CTL development has been hampered by the fact that most TH-independent responses have been demonstrated ex vivo as primary cytotoxic effectors, whereas TH-dependent responses generally require secondary in vitro re-stimulation for their detection. Here, we have monitored the primary and secondary responses of TH-dependent and TH-independent CTLs and find in both cases that CD4+ T cells are dispensable for primary expansion of CD8+ T cells and their differentiation into cytotoxic effectors. However, secondary CTL expansion (that is, a secondary response upon re-encounter with antigen) is wholly dependent on the presence of TH cells during, but not after, priming. Our results demonstrate that T-cell help is ‘programmed’ into CD8+ T cells during priming, conferring on these cells a hallmark of immune response memory: the capacity for functional expansion on re-encounter with antigen.

Na|[iuml]|ve CTLs require a single brief period of antigenic stimulation for clonal expansion and differentiation

In defense of the host, the immune system must often raise an effective cytotoxic T lymphocyte (CTL) response from a small number of clonal precursors. The degree to which activation stimuli regulate the expansion and differentiation of naïve CTLs, however, remains unknown. Using an engineered antigen-presenting cell (APC) system that allows control over antigenic stimulation, we studied the signaling duration requirements for priming and clonal expansion of naïve CTLs. We found that naïve CTLs become committed after as little as 2 h of exposure to APCs and that their subsequent division and differentiation can occur without the need for further antigenic stimulation of the daughter cells, whether priming is in vitro or in vivo. These data show that after a brief interaction with stimulatory APCs, naïve CTLs initiate a program for their autonomous clonal expansion and development into functional effectors.

CD4+ T-cell help controls CD8+ T-cell memory via TRAIL-mediated activation-induced cell death.

The ‘help’ provided by CD4+ T lymphocytes during the priming of CD8+ T lymphocytes confers a key feature of immune memory: the capacity for autonomous secondary expansion following re-encounter with antigen. Once primed in the presence of CD4+ T cells, ‘helped’ CD8+ T cells acquire the ability to undergo a second round of clonal expansion upon restimulation in the absence of T-cell help. ‘Helpless’ CD8+ T cells that are primed in the absence of CD4+ T cells, in contrast, can mediate effector functions such as cytotoxicity and cytokine secretion upon restimulation, but do not undergo a second round of clonal expansion. These disparate responses have features of being ‘programmed’, that is, guided by signals that are transmitted to naive CD8+ T cells during priming, which encode specific fates for their clonal progeny. Here we explore the instructional programme that governs the secondary response of CD8+ T cells and find that helpless cells undergo death by activation-induced cell death upon secondary stimulation. This death is mediated by tumour-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Regulation of Trail expression can therefore account for the role of CD4+ T cells in the generation of CD8+ T cell memory and represents a novel mechanism for controlling adaptive immune responses.

Conversion of tumor-specific CD4+ T-cell tolerance to T-cell priming through in vivo ligation of CD40.

Tumor antigen-specific T-cell tolerance limits the efficacy of therapeutic cancer vaccines. Antigen-presenting cells mediate the induction of T-cell tolerance to self-antigens. We therefore assessed the fate of tumor-specific CD4+ T cells in tumor-bearing recipients after in vivo activation of antigen-presenting cells with antibodies against CD40. Such treatment not only preserved the responsiveness of this population, but resulted in their endogenous activation. Established tumors regressed in vaccinated mice treated with antibody against CD40 at a time when no response was achieved with vaccination alone. These results indicate that modulation of antigen-presenting cells may be a useful strategy for enhancing responsiveness to immunization.

Dynamic programming of CD8+ T lymphocyte responses.

The initial encounter with an antigen-presenting cell (APC) is the primary force behind the expansion, differentiation and survival of naive T cells. Using an APC that permits temporal control of priming, we examined whether the duration of antigenic stimulation can influence the functional development of CD8+ cytotoxic T lymphocytes (CTLs) in vivo. Whereas CTLs given a 4-h stimulus underwent an abortive clonal expansion with transient surface CD25 expression, those given a 20-h stimulus sustained CD25 up-regulation, proliferated extensively, and efficiently mediated destruction of peripheral target tissues. Our results show that an instructional program preceding the first cell division integrates differences in signal strength into the decision to activate versus tolerize specific CTL clones.

Effectors and memories: Bcl-6 and Blimp-1 in T and B lymphocyte differentiation

Bcl-6 and Blimp-1 have recently been identified as key transcriptional regulators of effector and memory differentiation in CD4+ T cells and CD8+ T cells. Bcl-6 and Blimp-1 were previously known to be critical regulators of effector and memory differentiation of B lymphocytes. The new findings unexpectedly point to the Bcl-6 and Blimp-1 regulatory axis as a ubiquitous mechanism for controlling effector and memory lymphocyte differentiation and function. Bcl-6 and Blimp-1 are antagonistic transcription factors and can function as a self-reinforcing genetic switch for cell-fate decisions. However, their influences in different lymphocytes are complex. Here we review and examine the commonalities and differences in the functions of these transcription factors in CD4+ follicular helper TFH lymphocytes, effector CD8+ T lymphocytes and B lymphocytes.

A Critical Role for Stat3 Signaling in Immune Tolerance

Antigen-presenting cells (APCs) can induce T cell activation as well as T cell tolerance. The molecular mechanisms by which APCs regulate this critical decision of the immune system are not well understood. Here we show that Stat3 signaling plays a critical role in the induction of antigen-specific T cell tolerance. Targeted disruption of Stat3 signaling in APCs resulted in priming of antigen-specific CD4(+) T cells in response to an otherwise tolerogenic stimulus in vivo. Furthermore, APCs devoid of Stat3 effectively break antigen-specific T cell anergy in vitro. Conversely, increased Stat3 activity in APCs led to impaired antigen-specific T cell responses. Stat3 signaling provides, therefore, a novel molecular target for manipulation of immune activation/tolerance, a central decision with profound implications in autoimmunity, transplantation, and cancer immunotherapy.

Mutant MHC class II epitopes drive therapeutic immune responses to cancer

Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient’s tumour possesses a unique set of mutations (‘the mutanome’) that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient’s individual tumour-specific mutations. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4+ T cells. Vaccination with such CD4+ immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4+ T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope target repertoire of cancers, enabling the effective targeting of every patient’s tumour with vaccines produced ‘just in time’.

A Subset of Toll-Like Receptor Ligands Induces Cross-presentation by Bone Marrow-Derived Dendritic Cells

Dendritic cells (DCs) are capable of cross-presenting exogenous Ag to CD8+ CTLs. Detection of microbial products by Toll-like receptors (TLRs) leads to activation of DCs and subsequent orchestration of an adaptive immune response. We hypothesized that microbial TLR ligands could activate DCs to cross-present Ag to CTLs. Using DCs and CTLs in an in vitro cross-presentation system, we show that a subset of microbial TLR ligands, namely ligands of TLR3 (poly(inosinic-cytidylic) acid) and TLR9 (immunostimulatory CpG DNA), induces cross-presentation. In contrast to presentation of Ag to CD4+ T cells by immature DCs, TLR-induced cross-presentation is mediated by mature DCs, is independent of endosomal acidification, and relies on cytosolic Ag processing machinery.