CD70 and IFN‐1 selectively induce eomesodermin or T‐bet and synergize to promote CD8+ T‐cell responses

CD70‐mediated stimulation of CD27 is an important cofactor of CD4⁺ T‐cell licensed dendritic cells (DCs). However, it is unclear how CD70‐mediated stimulation of T cells is integrated with signals that emanate from signal 3 pathways, such as type‐1 interferon (IFN‐1) and IL‐12. We find that while stimulation of CD27 in isolation drives weak Eomesodermin^hiT‐bet^lo CD8⁺ T‐cell responses to OVA immunization, profound synergistic expansion is achieved by cotargeting TLR. This cooperativity can substantially boost antiviral CD8⁺ T‐cell responses during acute infection. Concomitant stimulation of TLR significantly increases per cell IFN‐γ production and the proportion of the population with characteristics of short‐lived effector cells, yet also promotes the ability to form long‐lived memory. Notably, while IFN‐1 contributes to the expression of CD70 on DCs, the synergy between CD27 and TLR stimulation is dependent upon IFN‐1's effect directly on CD8⁺ T cells, and is associated with the increased expression of T‐bet in T cells. Surprisingly, we find that IL‐12 fails to synergize with CD27 stimulation to promote CD8⁺ T‐cell expansion, despite its capacity to drive effector CD8⁺ T‐cell differentiation. Together, these data identify complex interactions between signal 3 and costimulatory pathways, and identify opportunities to influence the differentiation of CD8⁺ T‐cell responses.     

Developing and maintaining protective CD8+ memory T cells

Summary: A critical aim of vaccine‐related research is to identify the mechanisms by which memory T cells are formed and maintained over long periods of time. In recent years, we have designed experiments aimed at addressing two key questions: (i) what are the factors that maintain functionally responsive CD8⁺ memory cells over long periods of time, and (ii) what are the signals during the early stages of infection that drive the differentiation of long‐lived CD8⁺ memory T cells? We have identified a role for CD4⁺ T cells in the generation of CD8⁺ T‐cell‐mediated protection from secondary challenge. While CD4⁺ T cells appear to play a role in the programme of CD8 memory, we find that they are also required for the long‐term maintenance of CD8⁺ memory T‐cell numbers and function. This property is independent of CD40–CD40L interactions, and we propose a role for CD4⁺ T cells in maintaining the ability of CD8⁺ memory T cells to respond to interleukin‐7 (IL‐7) and IL‐15. By manipulating both the time course of infection and the timing of antigen presentation to newly recruited CD8⁺ T cells, we also demonstrate that the programming of effector and memory potential are at least partially distinct processes.     

ICAM‐1 and B7‐1 provide similar but distinct costimulation for CD8+ T cells,while CD4+ T cells are poorly costimulated by ICAM‐1

The function of purified ICAM‐1 in costimulating CD4⁺ and CD8⁺ T cell responses has been directly compared to that of B7‐1 in a model system that minimizes contributions of other receptor‐ligand interactions. While B7‐1 costimulates both subsets of T cells, ICAM‐1 is much more effective in the costimulation of CD8⁺ cells. ICAM‐1 also synergizes with B7‐1 for the induction of IL‐2 production in CD8⁺ but not CD4⁺ T cells. These differences are not explained by differences in LFA‐1 receptor expression on the two subsets of T cells. The CD8⁺ T cell response to ICAM‐1 costimulation is associated with increased proliferation and IL‐2 production at levels similar to those seen with B7‐1 costimulation, but clonal expansion in response to ICAM‐1 is not as great due to decreased cell survival. ICAM‐1‐mediated costimulation is effective for both naive and memory CT8⁺ T cells, is independent of CD28 engagement, and does not appear to be due solely to effects on adhesion. These results suggest that ICAM‐1‐dependent, B7‐independent costimulation may be important in initiating a CTL response to class I antigen presented by cells that are not professional APC.     

CD8+ T‐cell memory in tumor immunology and immunotherapy

Summary: The cellular and molecular mechanisms underlying the formation of distinct central, effector, and exhausted CD8⁺ T‐cell memory subsets were first described in the setting of acute and chronic viral diseases. The role of these T‐cell memory subsets are now being illuminated as relevant to the tumor‐bearing state. The generation and persistence of productive CD8⁺ T‐cell memory subsets is determined, in part, by antigen clearance, costimulation, responsiveness to homeostatic cytokines, and CD4⁺ T‐helper cells. By contrast, chronic exposure to antigen, negative costimulation, and immunomodulation by CD4⁺ T regulatory cells corrupt productive CD8⁺ T memory formation. It has become clear from human and mouse studies that the mere generation of CD8⁺ T‐cell memory is not a ‘surrogate marker’ for cancer vaccine efficacy. Some current cancer vaccine strategies may fail because they amplify, rather than correct or reset, the corrupted CD8⁺ memory population. Thus, much of the present effort in the development of vaccines for cancer and chronic infectious diseases is aimed at creating effective memory responses. Therapeutic vaccines for cancer and chronic infectious diseases may achieve consistent efficacy by ablation of the dysfunctional immune state and the provision of newly generated, non‐corrupted memory cells by adoptive cell transfer.     

Splenic CD8α⁺ dendritic cells undergo rapid programming by cytosolic bacteria and inflammation to induce protective CD8⁺ T-cell memory

Memory CD8⁺ T lymphocytes are critical effector cells of the adaptive immune system mediating long‐lived pathogen‐specific protective immunity. Three signals – antigen, costimulation and inflammation – orchestrate optimal CD8⁺ T‐cell priming and differentiation into effector and memory cells and shape T‐cell functional fate and ability to protect against challenge infections. While among the conventional spleen DCs (cDCs), the CD8α⁺ but not the CD8α^? cDCs most efficiently mediate CD8⁺ T‐cell priming, it is unclear which subset, irrespective of their capacity to process MHC class I‐associated antigens, is most efficient at inducing naïve CD8⁺ T‐cell differentiation into pathogen‐specific protective memory cells in vivo. Moreover, the origin of the required signals is still unclear. Using mice infected with the intracellular bacterium Listeria monocytogenes, we show that splenic CD8α⁺ cDCs become endowed with all functional features to optimally prime protective memory CD8⁺ T cells in vivo within only a few hours post‐immunization. Such programming requires both cytosolic signals resulting from bacterial invasion of the host cells and extracellular inflammatory mediators. Thus, these data designate these cells as the best candidates to facilitate the development of cell‐based vaccine therapy.     

IL‐27 enhances the survival of tumor antigen‐specific CD8+ T cells and programs them into IL‐10‐producing,memory precursor‐like effector cells

IL‐27 is a member of the IL‐12 family of cytokines that is comprised of an IL‐12 p40‐related protein subunit, EBV‐induced gene 3, and a p35‐related subunit, p28. IL‐27 functions through IL‐27R and has been shown to have potent antitumor activity via activation of a variety of cellular components, including antitumor CD8⁺ T‐cell responses. However, the exact mechanisms of how IL‐27 enhances antitumor CD8⁺ T‐cell responses remain unclear. Here we show that IL‐27 significantly enhances the survival of activated tumor antigen‐specific CD8⁺ T cells in vitro and in vivo, and programs tumor antigen‐specific CD8⁺ T cells into memory precursor‐like effector cells, characterized by upregulation of Bcl‐6, SOCS3, Sca‐1, and IL‐10. While STAT3 activation and the CTL survival‐enhancing effects can be independent of CTL IL‐10 production, we show here that IL‐27‐induced CTL IL‐10 production contributes to memory precursor cell phenotype induction, CTL memory, and tumor rejection. Thus, IL‐27 enhances antitumor CTL responses via programming tumor antigen‐specific CD8⁺ T cells into a unique memory precursor type of effector cells characterized by a greater survival advantage. Our results have important implications for designing immunotherapy against human cancer.     

Programming, demarcating, and manipulating CD8+ T-cell memory

Summary: In response to infection, antigen‐specific CD8⁺ T cells undergo massive expansion in numbers, acquire effector mechanisms, and disseminate throughout the body. The expansion phase is followed by a contraction (death) phase, where 90–95% of antigen‐specific CD8⁺ T cells are eliminated. The remaining antigen‐specific CD8⁺ T cells form the initial memory pool, which can be stably maintained for life. In this review, we discuss evidence that early events after infection ‘program’ CD8⁺ T cells to expand, contract, and generate memory in a fashion that is largely insensitive to the duration of infection or antigen display. Recent data demonstrate, despite numerical stability, that memory CD8⁺ T‐cell populations undergo phenotypic and functional changes with time after immunization. However, the early suggestion that specific markers can be used to identify memory CD8⁺ T cells has not been supported by recent studies. Thus, we argue that specific functional characteristics, such as the ability to persist and undergo vigorous secondary expansion leading to elevated memory cell numbers, remain the best markers of ‘good’ memory cells. Finally, we discuss experimental approaches to manipulate and accelerate generation of CD8⁺ T cells with memory characteristics, and how these systems can inform both basic and applied immunology.     

With(out) a little help from my friends: An IL‐12/CD40L‐mediated feed‐forward loop between CD8+ T cells and DCs

CD40–CD40L interactions are important for both antigen‐dependent B‐cell differentiation and effector and memory T‐cell formation. The prevailing view is that CD40L is expressed on activated CD4⁺ T cells, which enables them to provide help to high‐affinity B cells in GCs and to license DCs for efficient induction of CD8⁺ T‐cell responses. Interestingly, CD8⁺ T cells themselves can also express CD40L and, in this issue of the European Journal of Immunology, Thiel and colleagues [Eur. J. Immunol. 2013. 43: 1511‐1517] show that CD40L expression on these cells can be part of a self‐sustaining feed‐forward loop, in which expression of CD40L is induced by IL‐12 and TCR signaling. This provides a paradigm shift in our thinking about the requirements of effector CD8⁺ T‐cell development and the role herein of CD4⁺ T cells to provide help in this process.     

Tolerogenic dendritic cells impede priming of naïve CD8+ T cells and deplete memory CD8+ T cells

Type 1 diabetes is a T‐cell‐mediated autoimmune disease in which autoreactive CD8⁺ T cells destroy the insulin‐producing pancreatic beta cells. Vitamin D3 and dexamethasone‐modulated dendritic cells (Combi‐DCs) loaded with islet antigens inducing islet‐specific regulatory CD4⁺ T cells may offer a tissue‐specific intervention therapy. The effect of Combi‐DCs on CD8⁺ T cells, however, remains unknown. To investigate the interaction of CD8⁺ T cells with Combi‐DCs presenting epitopes on HLA class I, naive, and memory CD8⁺ T cells were co‐cultured with DCs and proliferation and function of peptide‐specific T cells were analyzed. Antigen‐loaded Combi‐DCs were unable to prime naïve CD8⁺ T cells to proliferate, although a proportion of T cells converted to a memory phenotype. Moreover, expansion of CD8⁺ T cells that had been primed by mature monocyte‐derived DCs (moDCs) was curtailed by Combi‐DCs in co‐cultures. Combi‐DCs expanded memory T cells once, but CD8⁺ T‐cell numbers collapsed by subsequent re‐stimulation with Combi‐DCs. Our data point that (re)activation of CD8⁺ T cells by antigen‐pulsed Combi‐DCs does not promote, but rather deteriorates, CD8⁺ T‐cell immunity. Yet, Combi‐DCs pulsed with CD8⁺ T‐cell epitopes also act as targets of cytotoxicity, which is undesirable for survival of Combi‐DCs infused into patients in therapeutic immune intervention strategies.     

Inflammation enhances the vaccination potential of CD40‐activated B cells in mice

Vaccination with antigen‐pulsed CD40‐activated B (CD40‐B) cells can efficiently lead to the in vivo differentiation of naive CD8⁺ T cells into fully functional effectors. In contrast to bone marrow‐derived dendritic cell (BMDC) vaccination, CD40‐B cell priming does not allow for memory CD8⁺ T‐cell generation but the reason for this deficiency is unknown. Here, we show that compared to BMDCs, murine CD40‐B cells induce lower expression of several genes regulated by T‐cell receptor signaling, costimulation, and inflammation (signals 1–3) in mouse T cells. The reduced provision of signals 1 and 2 by CD40‐B cells can be explained by a reduction in the quality and duration of the interactions with naive CD8⁺ T cells as compared to BMDCs. Furthermore, CD40‐B cells produce less inflammatory mediators, such as IL‐12 and type I interferon, and increasing inflammation by coadministration of polyriboinosinic‐polyribocytidylic acid with CD40‐B‐cell immunization allowed for the generation of long‐lived and functional CD8⁺ memory T cells. In conclusion, it is possible to manipulate CD40‐B‐cell vaccination to promote the formation of long‐lived functional CD8⁺ memory T cells, a key step before translating the use of CD40‐B cells for therapeutic vaccination.     

Mycobacterium tuberculosis PstS1 amplifies IFN‐γ and induces IL‐17/IL‐22 responses by unrelated memory CD4+ T cells via dendritic cell activation

The immunological mechanisms that modulate protection during Mycobacterium tuberculosis (Mtb) infection or vaccination are not fully understood. Secretion of IFN‐γ and, to a lesser extent, of IL‐17 by CD4⁺ T cells plays a major role both in protection and immunopathology. Few Mtb Ags interacting with DCs affect priming, activation, and regulation of Ag‐unrelated CD4⁺ T‐cell responses. Here we demonstrate that PstS1, a 38 kDa‐lipoprotein of Mtb, promotes Ag‐independent activation of memory T lymphocytes specific for Ag85B or Ag85A, two immunodominant protective Ags of Mtb. PstS1 expands CD4⁺ and CD8⁺ memory T cells, amplifies secretion of IFN‐γ and IL‐22 and induces IL‐17 production by effector memory cells in an Ag‐unrelated manner in vitro and in vivo. These effects were mediated through the stimulation of DCs, particularly of the CD8α^? subtype, which respond to PstS1 by undergoing phenotypic maturation and by secreting IL‐6, IL‐1β and, to a lower extent, IL‐23. IL‐6 secretion by PstS1‐stimulated DCs was required for IFN‐γ, and to a lesser extent for IL‐22 responses by Ag85B‐specific memory T cells. These results may open new perspectives for immunotherapeutic strategies to control Th1/Th17 immune responses in Mtb infections and in vaccinations against tuberculosis.     

Effector T‐cell differentiation during viral and bacterial infections: Role of direct IL‐12 signals for cell fate decision of CD8+ T cells

To study the role of IL‐12 as a third signal for T‐cell activation and differentiation in vivo, direct IL‐12 signaling to CD8⁺ T cells was analyzed in bacterial and viral infections using the P14 T‐cell adoptive transfer model with CD8⁺ T cells that lack the IL‐12 receptor. Results indicate that CD8⁺ T cells deficient in IL‐12 signaling were impaired in clonal expansion after Listeria monocytogenes infection but not after infection with lymphocytic choriomeningitis virus, vaccinia virus or vesicular stomatitis virus. Although limited in clonal expansion after Listeria infection, CD8⁺ T cells deficient in IL‐12 signaling exhibited normal degranulation activity, cytolytic functions, and secretion of IFN‐γ and TNF‐α. However, CD8⁺ T cells lacking IL‐12 signaling failed to up‐regulate KLRG1 and to down‐regulate CD127 in the context of Listeria but not viral infections. Thus, direct IL‐12 signaling to CD8⁺ T cells determines the cell fate decision between short‐lived effector cells and memory precursor effector cells, which is dependent on pathogen‐induced local cytokine milieu.     

Human virus-specific CD8+ T cells: diversity specialists

Summary: Human viruses elicit functionally highly diverse CD8⁺ T‐cell responses. This variation, which manifests in the memory or latency stage, includes differences in expansion requirements, migratory properties, homeostatic maintenance mechanisms, and constitutive effector properties, and it may reflect specific adaptations of the human immune system to challenges posed by individual pathogens. Longitudinal follow‐up of individuals after primary virus infections has revealed how divergent virus‐specific CD8⁺ T cells may develop from the initially expanded virus‐specific T‐cell effector pool. Recent findings have shown that CD8⁺ T cells reactive toward latent viruses may depend on other mechanisms for their homeostatic maintenance than T cells specific for cleared viruses. Whereas the latter can respond to the homeostatic cytokine interleukin‐7 (IL‐7), many persistent virus‐specific T cells, recognizing latent herpesviruses such as Epstein–Barr virus and cytomegalovirus (CMV), lack IL‐7 receptor α (IL‐7Rα) and depend on viral antigens to persist. Finally, CMV is unique in that it generates a vast pool of resting virus‐specific T cells with constitutive cytolytic effector function. The putative role of functionally diverse CD8⁺ T cells in protective immunity to persistent viruses is discussed in this review.     

IL‐10 promotes homeostatic proliferation of human CD8+ memory T cells and,when produced by CD1c+ DCs,shapes naive CD8+ T‐cell priming

IL‐10 is an anti‐inflammatory cytokine that inhibits maturation and cytokine production of dendritic cells (DCs). Although mature DCs have the unique capacity to prime CD8⁺ CTL, IL‐10 can promote CTL responses. To understand these paradoxic findings, we analyzed the role of IL‐10 produced by human APC subsets in T‐cell responses. IL‐10 production was restricted to CD1c⁺ DCs and CD14⁺ monocytes. Interestingly, it was differentially regulated, since R848 induced IL‐10 in DCs, but inhibited IL‐10 in monocytes. Autocrine IL‐10 had only a weak inhibitory effect on DC maturation, cytokine production, and CTL priming with high‐affinity peptides. Nevertheless, it completely blocked cross‐priming and priming with low‐affinity peptides of a self/tumor‐antigen. IL‐10 also inhibited CD1c⁺ DC‐induced CD4⁺ T‐cell priming and enhanced Foxp3 induction, but was insufficient to induce T‐cell IL‐10 production. CD1c⁺ DC‐derived IL‐10 had also no effect on DC‐induced secondary expansions of memory CTL. However, IL‐15‐driven, TCR‐independent proliferation of memory CTL was enhanced by IL‐10. We conclude that DC‐derived IL‐10 selects high‐affinity CTL upon priming. Moreover, IL‐10 preserves established CTL memory by enhancing IL‐15‐dependent homeostatic proliferation. These combined effects on CTL priming and memory maintenance provide a plausible mechanism how IL‐10 promotes CTL responses in humans.     

TLR2 engagement on memory CD8+ T cells improves their cytokine‐mediated proliferation and IFN‐γ secretion in the absence of Ag

Persistence of memory CD8⁺ T cells is known to be largely controlled by common gamma chain cytokines, such as IL‐2, IL‐7 and IL‐15. However, other molecules may be involved in this phenomenon. We show here that TLR2^?/? mice have a decreased frequency of memory phenotype CD8⁺ T cells when compared with WT mice. This prompted us to investigate the role of TLR2 in the homeostasis of memory CD8⁺ T cells. We describe here a new TLR2‐dependent mechanism which, in the absence of specific antigen, directly controls memory CD8⁺ T‐cell proliferation and IFN‐γ secretion. We demonstrate that TLR2 engagement on memory CD8⁺ T cells increases their proliferation and expansion induced by IL‐7 both in vitro and in vivo. We also show that TLR2 ligands act in synergy with IL‐2 to induce IFN‐γ secretion in vitro. Both conclusions are obtained with spontaneously arising memory phenotype and antigen‐specific memory CD8⁺ T cells. Altogether, our data support the idea that continuous TLR2 signaling in response to microbial stimuli or endogenous danger signals might directly contribute to the maintenance of the diversity memory CD8⁺ T cells in the organism.     

Dendritic cells activated by an anti‐inflammatory agent induce CD4+ T helper type 2 responses without impairing CD8+ memory and effector cytotoxic T‐lymphocyte responses

Prevalence of pro‐inflammatory diseases is rising in developed country populations. The increase in these diseases has fuelled the search for new, immune suppressive, anti‐inflammatory therapies, which do not impact, or minimally impact, CD4⁺ and/or CD8⁺ T‐cell‐mediated immunity. The goal of this study was to determine if antigen‐presenting cells (APCs) activated by the anti‐inflammatory oligosaccharide, lacto‐N‐fucopentaose III (LNFPIII), would have an impaired ability to drive CD4⁺ T helper (Th) or CD8⁺ memory and effector T‐cell responses. To investigate this we activated splenic dendritic cells (SDCs) with LNFPIII and examined their ability to drive antigen‐specific CD4⁺ Th, and CD8⁺ memory and cytotoxic T‐cell (CTL) responses compared with lipopolysaccharide (LPS) ‐stimulated SDCs. The LNFPIII‐activated SDCs had altered co‐stimulatory molecule expression compared with LPS‐stimulated SDCs, while the levels of SDC chemokines following activation by either compound were similar. LNFPIII‐activated SDCs produced significantly lower levels of interleukin‐12 but surprisingly higher levels of interleukin‐6 than LPS‐activated SDCs. Similar to previous studies using bone‐marrow‐derived DCs, LNFPIII‐activated SDCs induced strong Th2 responses in vivo and ex vivo. LNFPIII activation of APCs was independent of the Toll‐interleukin‐1 receptor adaptor myeloid differentiating factor 88. Importantly, LNFPIII‐matured DCs induced CD8⁺ memory and effector CTL responses similar to those driven by LPS‐matured DCs, including the frequency of interferon‐γ‐producing CD8⁺ T cells and induction of CTL effectors. Treatment of APCs by the anti‐inflammatory glycan LNFPIII did not impair their ability to drive CD8⁺ effector and memory cell‐mediated immunity.     

Dendritic cells expand antigen-specific Foxp3+CD25+CD4+ regulatory T cells including suppressors of alloreactivity

Summary: Thymic derived naturally occurring CD25⁺CD4⁺ T regulatory cells (Tregs) suppress immune responses, including transplantation. Here we discuss the capacity of dendritic cells (DCs) to expand antigen‐specific Tregs, particularly polyclonal Tregs directed to alloantigens. Initial studies have shown that mature DCs are specialized antigen‐presenting cells (APCs) for expanding antigen‐specific CD25⁺ CD4⁺ Tregs from TCR transgenic mice. When triggered by specific antigen, these Tregs act back on immature DCs to block the upregulation of CD80 and CD86 costimulatory molecules. More recently, DCs have been used to expand alloantigen‐specific CD25⁺CD4⁺ Tregs from the polyclonal repertoire in the presence of interleukin‐2 (IL‐2). Allogeneic DCs are much more effective than allogeneic spleen cells for expanding CD25⁺CD4⁺ Tregs. The DC‐expanded Tregs continue to express high levels of Foxp3, even without supplemental IL‐2, whereas spleen cells poorly sustain Foxp3 expression. When suppressive activity is tested, relatively small numbers of DC‐expanded CD25⁺CD4⁺ Tregs exert antigen‐specific suppression in the mixed leukocyte reaction (MLR), blocking immune responses to the original stimulating strain 10 times more effectively than to third party stimulating cells. DC‐expanded Tregs also retard graft versus host disease (GVHD) across full major histocompatibility complex (MHC) barriers. In vitro and in vivo, the alloantigen‐specific CD25⁺CD4⁺ Tregs are much more effective suppressors of transplantation reactions than polyclonal populations. We suggest that the expansion of Tregs from a polyclonal repertoire via antigen‐presenting DCs will provide a means for antigen‐specific control of unwanted immune reactions.     

Pathways of memory CD8+ T‐cell development

CD8⁺ T‐cell responses must have at least two components, a replicative cell type that proliferates in the secondary lymphoid tissue and that is responsible for clonal expansion, and cytotoxic cells with effector functions that mediate the resolution of the infection in the peripheral tissues. To confer memory, the response must also generate replication‐competent T cells that persist in the absence of antigen after the primary infection is cleared. The current models of memory differentiation differ in regards to whether or not memory CD8⁺ T cells acquire effector functions during their development. In this review we discuss the existing models for memory development and the consequences that the recent finding that memory CD8⁺ T cells may express granzyme B during their development has for them. We propose that memory CD8⁺ T cells represent a self‐renewing population of T cells that may acquire effector functions but that do not lose the naïve‐like attributes of lymphoid homing, antigen‐independent persistence or the capacity for self‐renewal.     

Thymic but not splenic CD8⁺ DCs can efficiently cross-prime T cells in the absence of licensing factors

Cross‐presentation is an important mechanism to elicit both immune defenses and tolerance. Although only a few DC subsets possess the machinery required for cross‐presentation, little is known about differences in cross‐presenting capabilities of DCs belonging to the same subpopulation but localized in different lymphoid organs. In this study, we demonstrate that steady‐state thymic CD8⁺ DCs can efficiently cross‐prime naïve CD8⁺ T cells in the absence of costimulation. Surprisingly, cross‐priming by splenic CD8⁺ DCs was dependent on licensing factors such as GM‐CSF. In the absence of GM‐CSF, antigen–MHC‐class‐I complexes were detected on thymic but not on splenic CD8⁺ DCs, indicating that the cross‐presentation capacity of the thymic subpopulation was higher. The observed cross‐priming differences between thymic and splenic CD8⁺ DCs did not correlate with differential antigen capture or costimulatory molecules found on the surface of DCs. Moreover, we did not detect overall impairment of antigen presentation, as peptide‐loaded splenic CD8⁺ DCs were able to induce CD8⁺ T‐cell proliferation. The observation that thymic CD8⁺ DCs are more efficient than splenic CD8⁺ DCs in T‐cell cross‐priming in the absence of licensing factors indicates that the requirements for efficient antigen presentation differ between these cells.