Generation of highly effective and stable murine alloreactive Treg cells by combined anti‐CD4 mAb,TGF‐β, and RA treatment

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long‐term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti‐CD4 antibody (aCD4). Here, we investigated whether adding TGF‐β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg‐cell generation and function. Murine CD4⁺ T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF‐β+RA or aCD4+Rapa. Addition of TGF‐β+RA or Rapa resulted in an increase of CD25⁺Foxp3⁺‐expressing T cells. Expression of CD40L and production of IFN‐γ and IL‐17 was abolished in aCD4+TGF‐β+RA aTreg cells. Additionally, aCD4+TGF‐β+RA aTreg cells showed the highest level of Helios and Neuropilin‐1 co‐expression. Although CD25⁺Foxp3⁺ cells from all culture conditions displayed complete demethylation of the Treg‐specific demethylated region, aCD4+TGF‐β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF‐β+RA aTreg cells suppressed effector T‐cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF‐β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.     

Tumor‐induced myeloid‐derived suppressor cell subsets exert either inhibitory or stimulatory effects on distinct CD8+ T‐cell activation events

Tumor growth coincides with an accumulation of myeloid‐derived suppressor cells (MDSCs), which exert immune suppression and which consist of two main subpopulations, known as monocytic (MO) CD11b⁺CD115⁺Ly6G^?Ly6C^high MDSCs and granulocytic CD11b⁺CD115^?Ly6G⁺Ly6C^int polymorphonuclear (PMN)‐MDSCs. However, whether these distinct MDSC subsets hamper all aspects of early CD8⁺ T‐cell activation — including cytokine production, surface marker expression, survival, and cytotoxicity — is currently unclear. Here, employing an in vitro coculture system, we demonstrate that splenic MDSC subsets suppress antigen‐driven CD8⁺ T‐cell proliferation, but differ in their dependency on IFN‐γ, STAT‐1, IRF‐1, and NO to do so. Moreover, MO‐MDSC and PMN‐MDSCs diminish IL‐2 levels, but only MO‐MDSCs affect IL‐2Rα (CD25) expression and STAT‐5 signaling. Unexpectedly, however, both MDSC populations stimulate IFN‐γ production by CD8⁺ T cells on a per cell basis, illustrating that some T‐cell activation characteristics are actually stimulated by MDSCs. Conversely, MO‐MDSCs counteract the activation‐induced change in CD44, CD62L, CD162, and granzyme B expression, while promoting CD69 and Fas upregulation. Together, these effects result in an altered CD8⁺ T‐cell adhesiveness to the extracellular matrix and selectins, sensitivity to FasL‐mediated apoptosis, and cytotoxicity. Hence, MDSCs intricately influence different CD8⁺ T‐cell activation events in vitro, whereby some parameters are suppressed while others are stimulated.     

Mixed functional characteristics correlating with TCR‐ligand koff‐rate of MHC‐tetramer reactive T cells within the naive T‐cell repertoire

The low frequency of antigen‐specific naïve T cells has challenged numerous laboratories to develop various techniques to study the naïve T‐cell repertoire. Here, we combine the generation of naïve repertoire‐derived antigen‐specific T‐cell lines based on MHC‐tetramer staining and magnetic‐bead enrichment with in‐depth functional assessment of the isolated T cells. Cytomegalovirus (CMV) specific T‐cell lines were generated from seronegative individuals. Generated T‐cell lines consisted of a variety of immunodominant CMV‐epitope‐specific oligoclonal T‐cell populations restricted to various HLA‐molecules (HLA‐A1, A2, B7, B8, and B40), and the functional and structural avidity of the CMV‐specific T cells was studied. Although all CMV‐specific T cells were isolated based on their reactivity toward a specific peptide‐MHC complex, we observed a large variation in the functional avidity of the MHC‐tetramer positive T‐cell populations, which correlated with the structural avidity measured by the recently developed Streptamer k_off‐rate assay. Our data demonstrate that MHC‐tetramer staining is not always predictive for specific T‐cell reactivity, and challenge the sole use of MHC‐tetramers as an indication of the peripheral T‐cell repertoire, independent of the analysis of functional activity or structural avidity parameters.     

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.