Dendritic cells process synthetic long peptides better than whole protein,improving antigen presentation and T‐cell activation

The efficiency of antigen (Ag) processing by dendritic cells (DCs) is vital for the strength of the ensuing T‐cell responses. Previously, we and others have shown that in comparison to protein vaccines, vaccination with synthetic long peptides (SLPs) has shown more promising (pre‐)clinical results. Here, we studied the unknown mechanisms underlying the observed vaccine efficacy of SLPs. We report an in vitro processing analysis of SLPs for MHC class I and class II presentation by murine DCs and human monocyte‐derived DCs. Compared to protein, SLPs were rapidly and much more efficiently processed by DCs, resulting in an increased presentation to CD4⁺ and CD8⁺ T cells. The mechanism of access to MHC class I loading appeared to differ between the two forms of Ag. Whereas whole soluble protein Ag ended up largely in endolysosomes, SLPs were detected very rapidly outside the endolysosomes after internalization by DCs, followed by proteasome‐ and transporter associated with Ag processing‐dependent MHC class I presentation. Compared to the slower processing route taken by whole protein Ags, our results indicate that the efficient internalization of SLPs, accomplished by DCs but not by B or T cells and characterized by a different and faster intracellular routing, leads to enhanced CD8⁺ T‐cell activation.     

Nucleoprotein‐specific nonneutralizing antibodies speed up LCMV elimination independently of complement and FcγR

CD8⁺ T cells have an essential role in controlling lymphocytic choriomeningitis virus (LCMV) infection in mice. Here, we examined the contribution of humoral immunity, including nonneutralizing antibodies (Abs), in this infection induced by low virus inoculation doses. Mice with impaired humoral immunity readily terminated infection with the slowly replicating LCMV strain Armstrong but showed delayed virus elimination after inoculation with the faster replicating LCMV strain WE and failed to clear the rapidly replicating LCMV strain Docile, which is in contrast to the results obtained with wild‐type mice. Thus, the requirement for adaptive humoral immunity to control the infection was dependent on the replication speed of the LCMV strains used. Ab transfers further showed that LCMV‐specific IgG Abs isolated from LCMV immune serum accelerated virus elimination. These Abs were mainly directed against the viral nucleoprotein (NP) and completely lacked virus neutralizing activity. Moreover, mAbs specific for the LCMV NP were also able to decrease viral titers after transfer into infected hosts. Intriguingly, neither C3 nor Fcγ receptors were required for the antiviral activity of the transferred Abs. In conclusion, our study suggests that rapidly generated nonneutralizing Abs specific for the viral NP speed up virus elimination and thereby may counteract T‐cell exhaustion.     

CD27 costimulation contributes substantially to the expansion of functional memory CD8+ T cells after peptide immunization

Naive T cells require signals from multiple costimulatory receptors to acquire full effector function and differentiate to long‐lived memory cells. The costimulatory receptor, CD27, is essential for optimal T‐cell priming and memory differentiation in a variety of settings, although whether CD27 is similarly required during memory CD8⁺ T‐cell reactivation remains controversial. We have used OVA and anti‐CD40 to establish a memory CD8⁺ T‐cell population and report here that their secondary expansion, driven by peptide and anti‐CD40, polyI:C, or LPS, requires CD27. Furthermore, antigenic peptide and a soluble form of the CD27 ligand, CD70 (soluble recombinant CD70 (sCD70)), is sufficient for secondary memory CD8⁺ T‐cell accumulation at multiple anatomical sites, dependent on CD80/86. Prior to boost, resting effector‐ and central‐memory CD8⁺ T cells both expressed CD27 with greater expression on central memory cells. Nonetheless, both populations upregulated CD27 after TCR engagement and accumulated in proportion after boosting with Ag and sCD70. Mechanistically, sCD70 increased the frequency of divided and cytolytic memory T cells, conferred resistance to apoptosis and enabled retardation of tumor growth in vivo. These data demonstrate the central role played by CD27/70 during secondary CD8⁺ T‐cell activation to a peptide Ag, and identify sCD70 as an immunotherapeutic adjuvant for antitumor immunity.     

Conserved epitopes dominate cross‐CD8+ T‐cell responses against influenza A H1N1 virus among Asian populations

Novel strains of influenza A viruses (IAVs) have emerged with high infectivity and/or pathogenicity in recent years, causing worldwide concern. T cells are correlated with protection in humans through cross‐reactive immunity against heterosubtypes of IAV. However, the different hierarchical roles of IAV‐derived epitopes with distinct levels of polymorphism in the cross‐reactive T‐cell responses against IAV remain elusive. In this study, immunodominant epitopes scattered throughout the entire proteome of 2009 pandemic influenza A H1N1 virus and seasonal IAVs were synthesized and divided into different pools depending on their conservation. The overall profile of the IAV‐specific CD8⁺ T‐cell immunity was detected by utilizing these peptide pools and also individual peptides. A dominant role of the conserved peptide‐specific T‐cell immunity was illuminated within the anti‐IAV responses, while the CD8⁺ T‐cell responses against the variable epitopes were lower than the conserved peptides. As previously demonstrated within a Caucasian population, we determined that GL9‐specific T cells, which also utilize Vβ 17 TCR (BV19), play a pivotal role in IAV‐specific T‐cell immunity within an HLA‐A2⁺ Asian population. Our study objectively reveals the different predominant roles of T‐cell epitopes among IAV‐specific cross‐reactive cellular immunity. This may guide the development of vaccines with cross‐T‐cell immunogenicity against heterosubtypes of IAV.     

AMPK: A metabolic switch for CD8+ T‐cell memory

Adenosine monophosphate‐activated protein kinase (AMPK) is a serine/threonine kinase and is crucial for cellular energy homeostasis. The exact role of AMPK during memory CD8⁺ T‐cell differentiation, a process that changes from the metabolically active state of effector T cells to one of quiescence in memory cells is not well understood; however, a report by Cantrell and colleagues [Eur. J. Immunol. 2013. 43: 889‐896] in this issue of the European Journal of Immunology shows that AMPK, by sensing glucose stress, is an important upstream molecule of mammalian target of rapamycin (mTOR) complex 1 for memory CD8⁺ T‐cell differentiation. This study provides new insights into how AMPK monitors energy stress to control effector and memory CD8⁺ T‐cell formation as discussed in this Commentary.     

Identification of novel markers for mouse CD4+ T follicular helper cells

CD4⁺ T follicular helper (T_FH) cells are central for generation of long‐term B‐cell immunity. A defining phenotypic attribute of T_FH cells is the expression of the chemokine R CXCR5, and T_FH cells are typically identified by co‐expression of CXCR5 together with other markers such as PD‐1, ICOS, and Bcl‐6. Herein, we report high‐level expression of the nutrient transporter folate R 4 (FR4) on T_FH cells in acute viral infection. Distinct from the expression profile of conventional T_FH markers, FR4 was highly expressed by naive CD4⁺ T cells, was downregulated after activation and subsequently re‐expressed on T_FH cells. Furthermore, FR4 expression was maintained, albeit at lower levels, on memory T_FH cells. Comparative gene expression profiling of FR4^hi versus FR4^lo Ag‐specific CD4⁺ effector T cells revealed a molecular signature consistent with T_FH and T_H1 subsets, respectively. Interestingly, genes involved in the purine metabolic pathway, including the ecto‐enzyme CD73, were enriched in T_FH cells compared with T_H1 cells, and phenotypic analysis confirmed expression of CD73 on T_FH cells. As there is now considerable interest in developing vaccines that would induce optimal T_FH cell responses, the identification of two novel cell surface markers should be useful in characterization and identification of T_FH cells following vaccination and infection.     

Cellular‐FLIP,Raji isoform (c‐FLIPR) modulates cell death induction upon T‐cell activation and infection

Dysregulation of apoptosis caused by an imbalance of pro‐ and anti‐apoptotic protein expression can lead to cancer, neurodegenerative, and autoimmune diseases. Cellular‐FLIP (c‐FLIP) proteins inhibit apoptosis directly at the death‐inducing signaling complex of death receptors, such as CD95, and have been linked to apoptosis regulation during immune responses. While the isoforms c‐FLIP_L and c‐FLIP_S are well characterized, the function of c‐FLIP_R remains poorly understood. Here, we demonstrate the induction of endogenous murine c‐FLIP_R in activated lymphocytes for the first time. To analyze c‐FLIP_R function in vivo, we generated transgenic mice expressing murine c‐FLIP_R specifically in hematopoietic cells. As expected, lymphocytes from c‐FLIP_R transgenic mice were protected against CD95‐induced apoptosis in vitro. In the steady state, transgenic mice had normal cell numbers and unaltered frequencies of B cells and T‐cell subsets in lymphoid organs. However, when challenged with Listeria monocytogenes, c‐FLIP_R transgenic mice showed less liver necrosis and better bacterial clearance compared with infected wild‐type mice. We conclude that c‐FLIP_R expression in hematopoietic cells supports an efficient immune response against bacterial infections.     

Peripheral CD4+ T‐cell tolerance is induced in vivo by rare antigen‐bearing B cells in follicular,marginal zone,and B‐1 subsets

B cells are efficient APCs when they internalize antigen via BCR‐mediated uptake. Adoptively transferred antigen‐presenting B cells can induce T‐cell tolerance to foreign and self antigens; however, it is unknown whether endogenous B cells presenting self‐peptides interact with naïve T cells and contribute to peripheral T‐cell self‐tolerance. Moreover, the relative abilities of mature B‐cell subsets to induce T‐cell tolerance have not been examined. To address these questions, we created a new mouse model wherein a very small fraction of B cells expresses an antigen transgene that cannot be transferred to other APCs. We limited antigen expression to follicular, marginal zone, or B‐1 B‐cell subsets and found that small numbers of each subset interacted with naïve antigen‐specific T cells. Although antigen expressed by B‐1 B cells induced the most T‐cell division, divided T cells subsequently disappeared from secondary lymphoid tissues. Independent of which B‐cell subset presented antigen, the remaining T cells were rendered hypo‐responsive, and this effect was not associated with Foxp3 expression. Our data show that physiologically relevant proportions of B cells can mediate peripheral T‐cell tolerance, and suggest that the mechanisms of tolerance induction might differ among follicular, marginal zone, and B‐1 B‐cell subsets.     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.     

Competition between T cells maintains clonal dominance during memory inflation induced by MCMV

Both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) establish persistent infections that induce the accumulation of virus‐specific T cells over time in a process called memory inflation. It has been proposed that T cells expressing T‐cell receptors (TCRs) with high affinity for HCMV‐derived peptides are preferentially selected after acute HCMV infection. To test this in the murine model, small numbers of OT‐I transgenic T cells, which express a TCR with high affinity for the SIINFEKL peptide, were transferred into congenic mice and recipients were challenged with recombinant MCMV expressing SIINFEKL. OT‐I T cells were selectively enriched during the first 3 weeks of infection. Similarly, in the absence of OT‐I T cells, the functional avidity of SIINFEKL‐specific T cells increased from early to late times postinfection. However, even when exceedingly small numbers of OT‐I T cells were transferred, their inflation limited the inflation of host‐derived T cells specific for SIINFEKL. Importantly, subtle minor histocompatibility differences led to late rejection of the transferred OT‐I T cells in some mice, which allowed host‐derived T cells to inflate substantially. Thus, T cells with a high functional avidity are selected shortly after MCMV infection and continuously sustain their clonal dominance in a competitive manner.