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.     

Residual LCMV antigen in transiently CD4+ T cell‐depleted mice induces high levels of virus‐specific antibodies but only limited B‐cell memory

Infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong (Arm) induces an acute infection with rapid virus clearance by CD8⁺ T cells independently of CD4⁺ T cell help. Residual viral antigen may, however, persist for a prolonged time. Here, we demonstrate that mice that had been transiently depleted of CD4⁺ T cells during acute LCMV Arm infection generated high levels of virus‐specific IgG antibodies (Ab) after viral clearance. Robust induction of LCMV‐specific IgG after transient CD4⁺ T cell depletion was dependent on Fcγ receptors but not on the complement receptors CD21/CD35. In contrast to the potent production of LCMV‐specific IgG, the generation of LCMV‐specific isotype‐switched memory B cells after transient CD4⁺ T cell depletion was considerably reduced. Moreover, mice depleted of CD4⁺ T cells during acute infection were strongly impaired in generating a secondary LCMV‐specific B cell response upon LCMV rechallenge. In conclusion, our data indicate that LCMV antigen depots after viral clearance were capable of inducing high levels of virus‐specific IgG. They failed, however, to induce robust virus‐specific B cell memory revealing a previously unappreciated dichotomy of specific Ab production and memory cell formation after priming with residual antigen.     

The immunoproteasome subunit LMP7 is required in the murine thymus for filling up a hole in the T cell repertoire

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine‐inducible variant of the proteasome which has been implicated in regulating inflammatory responses and antigen presentation. In the thymus, medullary thymic epithelial cells (mTECs) and cortical thymic epithelial cells (cTECs) do express different proteasome subunits exerting chymotrypsin‐like activities suggesting distinct functions in thymic T cell selection. Employing the lymphocytic choriomeningitis virus (LCMV) infection model, we could show that the immunoproteasome subunit LMP7 was absolutely required for the generation of LCMV GP_118‐125‐specific T cells although the class I mediated presentation of GP_118‐125 was not dependent on LMP7. Using bone marrow chimeras and adoptive transfer of LMP7‐deficient CD8⁺ T cells into RAG1‐deficient mice we show that LMP7‐deficient mice lacked GP_118‐125‐specific T cell precursors and that LMP7 was required in radioresistant cells – most likely thymic epithelial cells ‐ to enable their selection. Since LMP7 is strongly expressed in negatively selecting mTECs but barely in positively selecting cTECs our data suggest that LMP7 was required to avoid excessive negative selection of GP_118‐125‐specific T cell precursors. Taken together, this study demonstrates that the immunoproteasome is a crucial factor for filling up holes within the cytotoxic T cell repertoire.     

Tissue‐resident memory T cells: Local guards of the thymus

T‐cell surveillance of nonlymphoid tissues has traditionally been ascribed to recirculating memory T cells that continuously patrol the body. Extending this concept, recent evidence suggests that T cells also exist as nonmigratory memory cells that provide local immune protection in a broad range of peripheral tissues, including barrier locations such as skin and mucosa. In this issue of the European Journal of Immunology, Pircher and colleagues [Eur. J. Immunol. 2013. 43: 2295–2304] demonstrate, for the first time, the existence of such permanently tissue‐resident CD8⁺ memory T (T_RM) cells in a primary lymphoid organ, the thymus. T_RM cells in this location provide potent local immunity, which may help to preserve thymic integrity and normal T‐cell development in the face of infection with thymus‐invading pathogens.     

α4β1 integrin promotes accumulation of tissue‐resident memory CD8+ T cells in salivary glands

The salivary glands (SGs) of virus‐immune mice contain substantial numbers of tissue‐resident memory CD8⁺ T cells (T_RM cells) that can provide immunity to local infections. Integrins regulate entry of activated T cells into nonlymphoid tissues but the molecules that mediate migration of virus‐specific CD8⁺ T cells to the SGs have not yet been defined. Here, we found that polyinosinic‐polycytidylic acid (poly(I:C)) strongly promoted the accumulation of P14 TCR‐transgenic CD8⁺ T_RM cells in SGs in an α₄β₁ integrin‐dependent manner. After infection with lymphocytic choriomeningitis virus, accumulation of P14 T_RM cells in SGs and intestine but not in kidney was also α₄ integrin dependent. Blockade of α₄β₇ by monoclonal antibodies (mAbs) inhibited lymphocytic choriomeningitis virus‐induced accumulation of P14 T_RM cells in the intestine but not in SGs. In conclusion, our data reveal that α₄β₁ integrin mediates CD8⁺ T_RM accumulation in SGs and that poly(I:C) can be used to direct activated CD8⁺ T cells to this organ.     

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.     

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.     

Brain‐resident memory CD8+ T cells induced by congenital CMV infection prevent brain pathology and virus reactivation

Congenital HCMV infection is a leading infectious cause of long‐term neurodevelopmental sequelae. Infection of newborn mice with mouse cytomegalovirus (MCMV) intraperitoneally is a well‐established model of congenital human cytomegalovirus infection, which best recapitulates the hematogenous route of virus spread to brain and subsequent pathology. Here, we used this model to investigate the role, dynamics, and phenotype of CD8⁺ T cells in the brain following infection of newborn mice. We show that CD8⁺ T cells infiltrate the brain and form a pool of tissue‐resident memory T cells (T_RM cells) that persist for lifetime. Adoptively transferred virus‐specific CD8⁺ T cells provide protection against primary MCMV infection in newborn mice, reduce brain pathology, and remain in the brain as T_RM cells. Brain CD8⁺ T_RM cells were long‐lived, slowly proliferating cells able to respond to local challenge infection. Importantly, brain CD8⁺ T_RM cells controlled latent MCMV and their depletion resulted in virus reactivation and enhanced inflammation in brain.     

Antigens expressed by myelinating glia cells induce peripheral cross‐tolerance of endogenous CD8+ T cells

Auto‐reactivity of T cells is largely prevented by central and peripheral tolerance. Nevertheless, immunization with certain self‐antigens emulsified in CFA induces autoimmunity in rodents, suggesting that tolerance to some self‐antigens is not robust. To investigate the fate of nervous system‐specific CD8⁺ T cells, which only recently came up as being important contributors for MS pathogenesis, we developed a mouse model that allows inducible expression of lymphocytic choriomeningitis virus‐derived CD8⁺ T‐cell epitopes specifically in oligodendrocytes and Schwann cells, the myelinating glia of the nervous system. These transgenic CD8⁺ T‐cell epitopes induced robust tolerance of endogenous auto‐reactive T cells, which proved thymus‐independent and was mediated by cross‐presenting bone‐marrow‐derived cells. Immunohistological staining of secondary lymphoid organs demonstrated the presence of glia‐derived antigens in DC, suggesting that peripheral tolerance of CD8⁺ T cells results from uptake and presentation by steady state DC.     

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.     

NK1.1+ innate lymphoid cells in salivary glands inhibit establishment of tissue-resident memory CD8+ T cells in mice

NK1.1⁺ cells found in salivary glands (SG) represent a unique cell population of innate lymphoid cells (ILC) with characteristics of both conventional NK cells and ILC1. Here, we demonstrate that these NK1.1⁺ cells limit the accumulation and differentiation of virus-specific tissue-resident memory CD8⁺ T cells (T_RM cells) in SG of mice infected with lymphocytic choriomeningitis virus (LCMV). The negative regulation of LCMV-specific CD8⁺ T_RM cells by NK1.1⁺ cells in SG is independent of NKG2D, NKp46, TRAIL, and perforin. Moreover, analysis of NKp46^iCre+Eomes^fl/fl mice revealed that Eomes-dependent conventional NK cells are dispensable for negative regulation. Since the SG are prone to autoimmune reactions, regulation of T_RM cells by tissue-resident ILC may be particularly important to prevent immunopathology in this organ.     

CD69+ memory T lymphocytes of the bone marrow and spleen express the signature transcripts of tissue‐resident memory T lymphocytes

It is a matter of current debate whether the bone marrow is a hub for circulating memory T lymphocytes and/or the home of resident memory T lymphocytes. Here we demonstrate for CD69⁺ murine CD8⁺, and CD69⁺ murine and human CD4⁺ memory T lymphocytes of the bone marrow, making up between 30 and 60% of bone marrow memory T lymphocytes, that they express the gene expression signature of tissue‐resident memory T lymphocytes. This suggests that a substantial proportion of bone marrow memory T lymphocytes are resident. It adds to previous evidence that bone marrow memory T cells are resting in terms of mobility and proliferation, and maintain exclusive long‐term memory to distinct, systemic antigens.     

Epitope specificity of memory CD8+ T cells dictates vaccination-induced mortality in LCMV-infected perforin-deficient mice

Perforin‐deficient (PKO) mice serve as models for familial hemophagocytic lympho‐histiocytosis, a uniformly fatal disease associated with viral infection of perforin‐deficient humans. Naïve perforin‐deficient BALB/c mice survive while vaccinated PKO mice containing virus‐specific memory CD8 ⁺ T cells rapidly succumb to lymphocytic choriomeningitis virus (LCMV) infection. Thus, vaccination converts a nonlethal persistent infection into a fatal disease mediated by virus‐specific memory CD8 ⁺ T cells. Here, we determine the extent to which vaccination‐induced mortality in PKO mice following LCMV challenge is due to differences in vaccine modalities, the quantity or epitope specificity of memory CD8⁺ T cells. We show that LCMV‐induced mortality in immune PKO mice is independent of vaccine modalities and that the starting number of memory CD8 ⁺ T cells specific to the immunodominant epitope NP_118‐126 dictates the magnitude of secondary CD8 ⁺ T‐cell expansion, the inability to regulate production of CD8⁺ T‐cell‐derived IFN‐γ, and mortality in the vaccinated PKO mice. Importantly, mortality is determined by the epitope specificity of memory CD8 ⁺ T cells and the associated degree of functional exhaustion and cytokine dysregulation but not the absolute magnitude of CD8 ⁺ T‐cell expansion. These data suggest that deeper understanding of the parameters that influence the outcome of vaccine‐induced diseases would aid rational vaccine design to minimize adverse outcomes after infection.     

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.     

c‐IAP ubiquitin protein ligase activity is required for 4‐1BB signaling and CD8+ memory T‐cell survival

Cellular inhibitor of apoptosis proteins (c‐IAP) 1 and 2 are widely expressed ubiquitin protein ligases that regulate a variety of cellular functions, including the sensitivity of T cells to costimulation. 4‐1BB is a TNF receptor family member that signals via a complex that includes TRAF family members and the c‐IAPs to upregulate NF‐κB and ERK, and has been implicated in memory T‐cell survival. Here, we show that effector and memory T cells from mice expressing a dominant negative E3‐inactive c‐IAP2 (c‐IAP2^H570A) have impaired signaling downstream of 4‐1BB. When infected with lymphocytic choriomeningitis virus, unlike mice in which c‐IAPs were acutely downregulated by c‐IAP antagonists, the primary response of c‐IAP2^H570A mice was normal. However, the number of antigen‐specific CD8⁺ but not CD4⁺ T cells declined more rapidly and to a greater extent in c‐IAP2^H570A mice than in WT controls. Studies with T‐cell adoptive transfer demonstrated that the enhanced decay of memory cells was T‐cell intrinsic. Thus, c‐IAP E3 activity is required for 4‐1BB coreceptor signaling and maintenance of CD8⁺ T‐cell memory.