CD40 ligand‐expressing recombinant vaccinia virus promotes the generation of CD8+ central memory T cells

Central memory CD8⁺ T cells (T_CM) play key roles in the protective immunity against infectious agents, cancer immunotherapy, and adoptive treatments of malignant and viral diseases. CD8⁺ T_CM cells are characterized by specific phenotypes, homing, and proliferative capacities. However, CD8⁺ T_CM‐cell generation is challenging, and usually requires CD4⁺ CD40L⁺ T‐cell “help” during the priming of naïve CD8⁺ T cells. We have generated a replication incompetent CD40 ligand‐expressing recombinant vaccinia virus (rVV40L) to promote the differentiation of human naïve CD8⁺ T cells into T_CM specific for viral and tumor‐associated antigens. Soluble CD40 ligand recombinant protein (sCD40L), and vaccinia virus wild‐type (VV WT), alone or in combination, were used as controls. Here, we show that, in the absence of CD4⁺ T cells, a single “in vitro” stimulation of naïve CD8⁺ T cells by rVV40L‐infected nonprofessional CD14⁺ antigen presenting cells promotes the rapid generation of viral or tumor associated antigen‐specific CD8⁺ T cells displaying T_CM phenotypic and functional properties. These observations demonstrate the high ability of rVV40L to fine tune CD8⁺ mediated immune responses, and strongly support the use of similar reagents for clinical immunization and adoptive immunotherapy purposes.     

Enhancing immunity prevents virus‐induced T‐cell‐mediated immunopathology in B cell‐deficient mice

Hyper‐activated or deviated immune responses can result in immunopathological diseases. Paradoxically, immunodeficiency represents a frequent cause of such immune‐mediated pathologies. Immunopathological manifestations are commonly treated by immunosuppression, but in situations in which immunodeficiency is the basis of disease development, enhancing immunity may represent an alternative treatment option. Here, we tested this counterintuitive concept in a preclinical model using infection of mice with lymphocytic choriomeningitis virus (LCMV). Firstly, we demonstrate that infection of B‐cell‐deficient (B^?/?) but not of wild‐type (WT) mice with the LCMV strain Docile induced a rapid and fatal CD8⁺ T‐cell‐mediated immunopathological disease. Similar to WT mice, LCMV‐infected B^?/? mice generated a potent, functional LCMV‐specific CD8⁺ T‐cell response but exhibited prolonged viral antigen presentation and increased vascular leakage in liver and lungs. Secondly, we were able to prevent this virus‐induced immunopathology in B^?/? mice by active or passive T‐cell immunizations or by treatment with LCMV‐specific virus neutralizing or non‐neutralizing monoclonal antibodies (mAb). Thus, boosting antiviral immunity did not aggravate immunopathology in this model, but prevented it by decreasing the formation of target structures for damage‐causing CD8⁺ T cells.     

Thymus‐resident memory CD8+ T cells mediate local immunity

The thymus is a primary lymphoid organ responsible for production and selection of T cells. Nonetheless, mature T cells and in particular activated T cells can reenter the thymus. Here, we identified memory CD8⁺ T cells specific for lymphocytic choriomeningitis virus or vaccinia virus in the thymus of mice long‐time after the infection. CD8⁺ T cells were mainly located in the thymic medulla, but also in the cortical areas. Interestingly, virus‐specific memory CD8⁺ T cells in the thymus expressed the cell surface markers CD69 and CD103 that are characteristic of tissue‐resident memory T cells in a time‐dependent manner. Kinetic analyses and selective depletion of peripheral CD8⁺ T cells by antibodies further revealed that thymic virus‐specific memory CD8⁺ T cells did not belong to the circulating pool of lymphocytes. Finally, we demonstrate that these thymus‐resident virus‐specific memory CD8⁺ T cells efficiently mounted a secondary proliferative response, exhibited immediate effector functions and were able to protect the thymus from lymphocytic choriomeningitis virus reinfection. In conclusion, the present study not only describes for the first time virus‐specific memory CD8⁺ T cells with characteristics of tissue‐resident memory T (T_RM) cells in a primary lymphoid organ but also extends our knowledge about local T‐cell immunity in the thymus.     

OX40 controls effector CD4+ T‐cell expansion,not follicular T helper cell generation in acute Listeria infection

To investigate the importance of OX40 signals for physiological CD4⁺ T‐cell responses, an endogenous antigen‐specific population of CD4⁺ T cells that recognise the 2W1S peptide was assessed and temporal control of OX40 signals was achieved using blocking or agonistic antibodies (Abs) in vivo. Following infection with Listeria monocytogenes expressing 2W1S peptide, OX40 was briefly expressed by the responding 2W1S‐specific CD4⁺ T cells, but only on a subset that co‐expressed effector cell markers. This population was specifically expanded by Ab‐ligation of OX40 during priming, which also caused skewing of the memory response towards effector memory cells. Strikingly, this greatly enhanced effector response was accompanied by the loss of T follicular helper (TFH) cells and germinal centres. Mice deficient in OX40 and CD30 showed normal generation of TFH cells but impaired numbers of 2W1S‐specific effector cells. OX40 was not expressed by 2W1S‐specific memory cells, although it was rapidly up‐regulated upon challenge whereupon Ab‐ligation of OX40 specifically affected the effector subset. In summary, these data indicate that for CD4⁺ T cells, OX40 signals are important for generation of effector T cells rather than TFH cells in this response to acute bacterial infection.     

IL‐2 is required for the generation of viral‐specific CD4+ Th1 tissue‐resident memory cells and B cells are essential for maintenance in the lung

CD4⁺ tissue resident cells are an important first line of defense against viral infections in the lungs and are critical for promoting the localization of lung resident CD8⁺ T cells. However, relatively little is known about the signaling programs required for the development of viral‐specific CD4⁺ tissue resident cells in the lungs. Recently, it was shown that signaling through the high affinity IL‐2 receptor is required for the differentiation of lung‐resident Th2 memory (Trm) cells in a murine model of airway inflammation. We therefore tested if IL‐2 signaling is also required for the development of viral antigen‐specific CD4⁺ Th1 cells in the lung after i.n. infection with lymphocytic choriomeningitis virus. These studies demonstrate that Th1 CD4⁺ T cells also require IL‐2 for lung Trm development. Additionally, they show that B cells potently inhibit early Th1 cell lung residency, but are required for the maintenance of a long‐lived population of CD4⁺ Th1 Trm.     

CD40L expression by CD4+ but not CD8+ T cells regulates antiviral immune responses in acute LCMV infection in mice

CD40‐CD40 ligand (CD40L) signaling plays multiple indispensable roles in cellular and humoral immunity. Impaired memory T‐cell responses in the absence of CD40L have been well documented, but the requirement of this interaction for efficient priming of CD8⁺ T cells especially under inflammatory conditions has been under debate. In contrast to previous publications, we report here that virus‐specific CD8⁺ T‐cell responses as well as viral clearance are affected not only in the memory but also in the effector phase in CD40L^?/? mice infected with lymphocytic choriomeningitis virus (LCMV) Armstrong strain. Interestingly, a considerable part of the LCMV‐specific effector and memory T cells consists of CD40L⁺ CD8⁺ T cells. However, deficiency of CD40L in CD8⁺ T cells did influence neither the quantity nor the quality of primary T‐cell responses in LCMV infection. Virus‐specific CD8⁺ T cells in conditional knockout mice, with a selective deletion of the CD40L in CD8⁺ T cells, were fully functional regarding cytokine production and efficient pathogen clearance. Thus, our results unambiguously demonstrate that while CD40L is critical to generate effective primary CD8⁺ T‐cell responses also under inflammatory conditions, CD40L expression by CD8⁺ T cells themselves is dispensable in acute LCMV infection.     

Plasmodium berghei sporozoite challenge of vaccinated BALB/c mice leads to the induction of humoral immunity and improved function of CD8+ memory T cells

Protection against malaria can be achieved by induction of a strong CD8⁺ T‐cell response against the Plasmodium circumsporozoite protein (CSP), but most subunit vaccines suffer from insufficient memory responses. In the present study, we analyzed the impact of postimmunization sporozoite challenge on the development of long‐lasting immunity. BALB/c mice were immunized by a heterologous prime/boost regimen against Plasmodium berghei CSP that induces a strong CD8⁺ T‐cell response and sterile protection, which is short‐lived. Here, we show that protective immunity is prolonged by a sporozoite challenge after immunization. Repeated challenges induced sporozoite‐specific antibodies that showed protective capacity. The numbers of CSP‐specific CD8⁺ T cells were not substantially enhanced by sporozoite infections; however, CSP‐specific memory CD8⁺ T cells of challenged mice displayed a higher cytotoxic activity than memory T cells of immunized‐only mice. CD4⁺ T cells contributed to protection as well; but CD8⁺ memory T cells were found to be the central mediator of sterile protection. Based on these data, we suggest that prolonged protective immunity observed after immunization and infection is composed of different antiparasitic mechanisms including CD8⁺ effector‐memory T cells with increased cytotoxic activity as well as CD4⁺ memory T cells and neutralizing antibodies.     

Progesterone promotes maternal–fetal tolerance by reducing human maternal T‐cell polyfunctionality and inducing a specific cytokine profile

Progesterone is a steroid hormone essential for the maintenance of human pregnancy, and its actions are thought to include promoting maternal immune tolerance of the semiallogenic fetus. We report that exposure of maternal T cells to progesterone at physiological doses induced a unique skewing of the cytokine production profile of CD4⁺ and CD8⁺ T cells, with reductions not only in potentially deleterious IFN‐γ and TNF‐α production but also in IL‐10 and IL‐5. Conversely, production of IL‐4 was increased. Maternal T cells also became less polyfunctional, focussing cytokine production toward profiles including IL‐4. This was accompanied by reduced T‐cell proliferation. Using fetal and viral antigen‐specific CD8⁺ T‐cell clones, we confirmed that this as a direct, nonantigen‐specific effect. Yet human T cells lacked conventional nuclear progesterone receptors, implicating a membrane progesterone receptor. CD4⁺ and CD8⁺ T cells responded to progesterone in a dose‐dependent manner, with subtle effects at concentrations comparable to those in maternal blood, but profound effects at concentrations similar to those at the maternal–fetal interface. This characterization of how progesterone modulates T‐cell function is important in understanding the normal biology of pregnancy and informing the rational use of progesterone therapy in pregnancies at risk of fetal loss.     

Stimulation through very late antigen‐4 and ‐5 improves the multifunctionality and memory formation of CD8+ T cells

T cells express multiple integrin molecules. The significance of signaling through these molecules on acquisition of T‐cell effector functions and memory formation capacity remains largely unknown. Moreover, the impact of stimulation through these signals on the generation of T cells for adoptive immunotherapy has not been elucidated. In this study, using a recombinant fragment of fibronectin, CH‐296, we demonstrated that stimulation via very late Ag (VLA)‐4 and VLA‐5 in human and BALB/c mouse CD8⁺ T cells, in combination with TCR stimulation, enhances effector multifunctionality and in vivo memory formation. Using TCR‐transgenic mouse‐derived CD8⁺ T cells expressing TCR specific for the syngeneic CMS5 fibrosarcoma‐derived tumor Ag, we showed that stimulation by CH‐296 improved the ability of tumor‐specific CD8⁺ T cells to inhibit CMS5 tumor growth when adoptively transferred into hosts with progressing tumors. Improved antitumor effects were associated with decreased infiltration of Foxp3⁺CD4⁺ Treg cells in tumors. These results suggest that stimulation via VLA‐4 and VLA‐5 modulates the qualities of effector T cells and could potentially increase the efficacy of adoptive therapy against cancer.     

Central tolerance spares the private high‐avidity CD4+ T‐cell repertoire specific for an islet antigen in NOD mice

Although central tolerance induces the deletion of most autoreactive T cells, some autoreactive T cells escape thymic censorship. Whether potentially harmful autoreactive T cells present distinct TCRαβ features remains unclear. Here, we analyzed the TCRαβ repertoire of CD4⁺ T cells specific for the S100β protein, an islet antigen associated with type 1 diabetes. We found that diabetes‐resistant NOD mice deficient for thymus specific serine protease (TSSP), a protease that impairs class II antigen presentation by thymic stromal cells, were hyporesponsive to the immunodominant S100β_1‐15 epitope, as compared to wild‐type NOD mice, due to intrathymic negative selection. In both TSSP‐deficient and wild‐type NOD mice, the TCRαβ repertoire of S100β‐specific CD4⁺ T cells though diverse showed a specific bias for dominant TCRα rearrangements with limited CDR3α diversity. These dominant TCRα chains were public since they were found in all mice. They were of intermediate‐ to low‐avidity. In contrast, high‐avidity T cells expressed unique TCRs specific to each individual (private TCRs) and were only found in wild‐type NOD mice. Hence, in NOD mice, the autoreactive CD4⁺ T‐cell compartment has two major components, a dominant and public low‐avidity TCRα repertoire and a private high‐avidity CD4⁺ T‐cell repertoire; the latter is deleted by re‐enforced negative selection.     

Reversal of chronic to resolved infection by IL‐10 blockade is LCMV strain dependent

Chronic viral infections lead to CD8⁺ T‐cell exhaustion, characterized by impaired cytokine secretion. The immune‐regulatory cytokine IL‐10 promotes chronicity of infection with lymphocytic choriomeningitis virus (LCMV) Clone 13, as absence of IL‐10 or blocking of IL‐10R during early LCMV Clone 13 infection results in viral clearance. Thus, treatment of humans suffering from chronic viral infections with IL‐10 neutralizing or IL‐10R blocking antibodies was proposed to boost virus‐specific T‐cell responses to enhance control or even clear the viral infection. Here we demonstrate that although CD4⁺ and CD8⁺ T cells can produce elevated levels of cytokines in IL‐10^?/? mice early after infection compared with WT mice, IL‐10^?/? mice cannot clear an infection with the quicker replicating LCMV strain Docile, eventually resulting in T‐cell exhaustion. These data suggest that the success of IL‐10 blockade to control chronic viral infections may critically depend on the virulence of the infecting strain.     

Inhibition of the Fibrinogen‐Like Protein 2:FcγRIIB/RIII immunosuppressive pathway enhances antiviral T‐cell and B‐cell responses leading to clearance of lymphocytic choriomeningitis virus clone 13

Persistent viruses evade immune detection by interfering with virus‐specific innate and adaptive antiviral immune responses. Fibrinogen‐like protein‐2 (FGL2) is a potent effector molecule of CD4⁺ CD25⁺ FoxP3⁺ regulatory T cells and exerts its immunosuppressive activity following ligation to its cognate receptor, FcγRIIB/RIII. The role of FGL2 in the pathogenesis of chronic viral infection caused by lymphocytic choriomeningitis virus clone‐13 (LCMV cl‐13) was assessed in this study. Chronically infected fgl2^+/+ mice had increased plasma levels of FGL2, with reduced expression of the maturation markers, CD80, CD86 and MHC‐II on macrophages and dendritic cells and impaired production of neutralizing antibody. In contrast, fgl2^?/? mice or fgl2^+/+ mice that had been pre‐treated with antibodies to FGL2 and FcγRIIB/RIII and then infected with LCMV cl‐13 developed a robust CD4⁺ and CD8⁺ antiviral T‐cell response, produced high titred neutralizing antibody to LCMV and cleared LCMV. Treatment of mice with established chronic infection with antibodies to FGL2 and FcγRIIB/RIII was shown to rescue the number and functionality of virus‐specific CD4⁺ and CD8⁺ T cells with reduced total and virus‐specific T‐cell expression of programmed cell death protein 1 leading to viral clearance. These results demonstrate an important role for FGL2 in viral immune evasion and provide a rationale to target FGL2 to treat patients with chronic viral infection.     

Myelin oligodendrocyte glycoprotein induces incomplete tolerance of CD4+ T cells specific for both a myelin and a neuronal self‐antigen in mice

T‐cell polyspecificity, predicting that individual T cells recognize a continuum of related ligands, implies that multiple antigens can tolerize T cells specific for a given self‐antigen. We previously showed in C57BL/6 mice that part of the CD4⁺ T‐cell repertoire specific for myelin oligodendrocyte glycoprotein (MOG) 35–55 also recognizes the neuronal antigen neurofilament medium (NF‐M) 15–35. Such bi‐specific CD4⁺ T cells are frequent and produce inflammatory cytokines after stimulation. Since T cells recognizing two self‐antigens would be expected to be tolerized more efficiently, this finding prompted us to study how polyspecificity impacts tolerance. We found that similar to MOG, NF‐M is expressed in the thymus by medullary thymic epithelial cells, a tolerogenic population. Nevertheless, the frequency, phenotype, and capacity to transfer experimental autoimmune encephalomyelitis (EAE) of MOG_35‐55‐reactive CD4⁺ T cells were increased in MOG‐deficient but not in NF‐M‐deficient mice. We found that presentation of NF‐M_15‐35 by I‐A^b on dendritic cells is of short duration, suggesting unstable MHC class II binding. Consistently, introducing an MHC‐anchoring residue into NF‐M_15‐35 (NF‐M_15‐35T20Y) increased its immunogenicity, activating a repertoire able to induce EAE. Our results show that in C57BL/6 mice bi‐specific encephalitogenic T cells manage to escape tolerization due to inefficient exposure to two self‐antigens.     

TGF‐β induces the differentiation of human CXCL13‐producing CD4+ T cells

In the ectopic lymphoid‐like structures present in chronic inflammatory conditions such as rheumatoid arthritis, a subset of human effector memory CD4⁺ T cells that lacks features of follicular helper T (Tfh) cells produces CXCL13. Here, we report that TGF‐β induces the differentiation of human CXCL13‐producing CD4⁺ T cells from naïve CD4⁺ T cells. The TGF‐β‐induced CXCL13‐producing CD4⁺ T cells do not express CXCR5, B‐cell lymphoma 6 (BCL6), and other Tfh‐cell markers. Furthermore, expression levels of CD25 (IL‐2Rα) in CXCL13‐producing CD4⁺ T cells are significantly lower than those in FoxP3⁺ in vitro induced Treg cells. Consistent with this, neutralization of IL‐2 and knockdown of STAT5 clearly upregulate CXCL13 production by CD4⁺ T cells, while downregulating the expression of FoxP3. Furthermore, overexpression of FoxP3 in naïve CD4⁺ T cells downregulates CXCL13 production, and knockdown of FoxP3 fails to inhibit the differentiation of CXCL13‐producing CD4⁺ T cells. As reported in rheumatoid arthritis, proinflammatory cytokines enhance secondary CXCL13 production from reactivated CXCL13‐producing CD4⁺ T cells. Our findings demonstrate that CXCL13‐producing CD4⁺ T cells lacking Tfh‐cell features differentiate via TGF‐β signaling but not via FoxP3, and exert their function in IL‐2‐limited but TGF‐β‐rich and proinflammatory cytokine‐rich inflammatory conditions.     

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.     

Spontaneous CD4+ and CD8+ T‐cell responses directed against cancer testis antigens are present in the peripheral blood of testicular cancer patients

Cancer/testis antigen (CTAg) expression is restricted to spermatogenic cells in an immune‐privileged site within the testis. However, these proteins are expressed aberrantly by malignant cells and T‐cell responses against CTAgs develop in many cancer patients. We investigated the prevalence, magnitude and phenotype of CTAg‐specific T cells in the blood of patients with testicular germ cell tumors (TGCTs). CD8⁺ and CD4⁺ T‐cell responses against MAGE‐A family antigens were present in 44% (20/45) of patients’ samples assayed by ex vivo IFN‐γ ELISPOT. The presence of MAGE‐specific CD8⁺ T cells was further determined following short‐term in vitro expansion through the use of pMHC‐I multimers containing known immunogenic peptides. Longitudinal analysis revealed that the frequency of MAGE‐specific T cells decreased by 89% following orchidectomy suggesting that persistence of tumor antigen is required to sustain CTAg‐specific T‐cell immunity. Notably, this decrease correlated with a decline in the global effector/memory T‐cell pool following treatment. Spontaneous T‐cell immunity against CTAg proteins therefore develops in many patients with testicular cancer and may play an important role in the excellent clinical outcome of patients with this tumor subtype.     

Non‐neutralizing antibodies protect from chronic LCMV infection independently of activating FcγR or complement

Chronic viral infections lead to CD8⁺ T cell exhaustion, characterized by impaired cytokine secretion. The presence of the immune‐regulatory cytokine IL‐10 promotes chronic lymphocytic choriomeningitis virus (LCMV) Clone 13 infection in mice, whereas the absence of IL‐10/IL‐10R signaling early during infection results in viral clearance and higher percentages and numbers of antiviral, cytokine‐producing T cells. However, it is currently unclear which cell populations and effector molecules are crucial to protect against chronic infection. In this study, we demonstrate that antiviral, LCMV‐binding, non‐neutralizing antibodies are needed, in addition to CD4⁺ and CD8⁺ T cells, to clear a high‐dose LCMV infection in mice, in the absence of IL‐10. The interaction between CD4⁺ T cells and B cells in B‐cell follicles via CD40/CD40L, in addition to class switch and/or somatic hypermutation, is crucial for viral control in the absence of IL‐10. Interestingly, transfer of LCMV‐binding non‐neutralizing antibodies protected recipients from chronic infection. In addition, viral clearance in the absence of IL‐10R signaling was independent of activating Fcγ receptors and complement. These data highlight that non‐neutralizing antibodies effectively contribute to the control of LCMV infection when present prior to infection, suggesting that the induction of neutralizing antibodies is not implicitly necessary for the generation of successful vaccines.