Early effector maturation of naïve human CD8+ T cells requires mitochondrial biogenesis

The role of mitochondrial biogenesis during naïve to effector differentiation of CD8⁺ T cells remains ill explored. In this study, we describe a critical role for early mitochondrial biogenesis in supporting cytokine production of nascent activated human naïve CD8⁺ T cells. Specifically, we found that prior to the first round of cell division activated naïve CD8⁺ T cells rapidly increase mitochondrial mass, mitochondrial respiration, and mitochondrial reactive oxygen species (mROS) generation, which were all inter‐linked and important for CD8⁺ T cell effector maturation. Inhibition of early mitochondrial biogenesis diminished mROS dependent IL‐2 production – as well as subsequent IL‐2 dependent TNF, IFN‐γ, perforin, and granzyme B production. Together, these findings point to the importance of mitochondrial biogenesis during early effector maturation of 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.     

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.     

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.     

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.     

CD28 and CD57 define four populations with distinct phenotypic properties within human CD8+ T cells

After repeated antigen exposure, both memory and terminally differentiated cells can be generated within CD8⁺ T cells. Although, during their differentiation, activated CD8⁺ T cells may first lose CD28, and CD28⁻ cells may eventually express CD57 as a subsequent step, a population of CD28⁺CD57⁺(DP) CD8⁺ T cells can be identified in the peripheral blood. How this population is distinct from CD28⁻CD57⁻(DN) CD8⁺ T cells, and from the better characterized non-activated/early-activated CD28⁺CD57⁻ and senescent-like CD28⁻CD57⁺ CD8⁺ T cell subsets is currently unknown. Here, RNA expression of the four CD8⁺ T cell subsets isolated from human PBMCs was analyzed using microarrays. DN cells were more similar to “early” highly differentiated cells, with decreased TNF and IFN-γ production, impaired DNA damage response and apoptosis. Conversely, increased apoptosis and expression of cytokines, co-inhibitory, and chemokine receptors were found in DP cells. Higher levels of DP CD8⁺ T cells were observed 7 days after Hepatitis B vaccination, and decreased levels of DP cells were found in rheumatoid arthritis patients. More DP and DN CD8⁺ T cells were present in the bone marrow, in comparison with PBMCs. In summary, our results indicate that DP and DN cells are distinct CD8⁺ T cell subsets displaying defined properties.     

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.     

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.     

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.     

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.     

Proteomic definition of human mucosal‐associated invariant T cells determines their unique molecular effector phenotype

Mucosal‐associated invariant T cells (MAIT) constitute the most abundant anti‐bacterial CD8⁺ T‐cell population in humans. MR1/TCR‐activated MAIT cells were reported to organize cytotoxic and innate‐like responses but knowledge about their molecular effector phenotype is still fragmentary. Here, we have examined the functional inventory of human MAIT cells (CD3⁺Vα7.2⁺CD161⁺) in comparison with those from conventional non‐MAIT CD8⁺ T cells (cCD8⁺) and NK cells. Quantitative mass spectrometry characterized 5500 proteins of primary MAIT cells and identified 160 and 135 proteins that discriminate them from cCD8⁺ T cells and NK cells donor‐independently. Most notably, MAIT cells showed a unique exocytosis machinery in parallel to a proinflammatory granzyme profile with high levels of the granzymes A, K, and M. Furthermore, 24 proteins were identified with highest abundances in MAIT cells, including CD26, CD98, and L‐amino‐oxidase (LAAO). Among those, expression of granzyme K and CD98 were validated as MAIT‐specific with respect to non‐MAIT CD8⁺ effector subsets and LAAO was found to be recruited together with granzymes, perforin, and CD107a at the immunological synapse of activated MAIT cells. In conclusion, this study complements knowledge on the molecular effector phenotype of MAIT cells and suggest novel immune regulatory functions as part of their cytotoxic responses.     

T cells compete by cleaving cell surface CD27 and blocking access to CD70‐bearing APCs

T cells compete against each other for access to molecules on APCs in addition to peptide/MHC complexes. However, the identity of cell surface molecules that influence T‐cell competition, other than peptide/MHC, have yet to be defined. Here, we identify CD70, a TNF ligand expressed on activated APCs, as an important mediator of T‐cell competition for APCs. Upon engagement of CD27 by CD70, CD27 is proteolytically cleaved from the surface of the interacting CD8⁺ T cell and captured by CD70 expressing dendritic cells. The capture of CD27 effectively masks CD70 on APCs, disallowing the interaction with CD27 on other competing T cells. Collectively, our data indicate that T cells compete against each other for access to the TNF‐ligand CD70, an interaction that affects the duration and potency of T cell/DC interactions, thus influencing the repertoire of responding CD8⁺ T cells to self or foreign antigens.     

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.     

CD39 is involved in mediating suppression by Mycobacterium bovis BCG‐activated human CD8+CD39+ regulatory T cells

Regulatory T (Treg) cells can balance normal tissue homeostasis by limiting inflammatory tissue damage, e.g. during pathogen infection, but on the other hand can also limit protective immunity induced during natural infection or following vaccination. Because most studies have focused on the role of CD4⁺ Treg cells, relatively little is known about the phenotype and function of CD8⁺ Treg cells, particularly in infectious diseases. Here, we describe for the first time the expression of CD39 (E‐NTPDase1) on Mycobacterium‐activated human CD8⁺ T cells. These CD8⁺CD39⁺ T cells significantly co‐expressed the Treg markers CD25, Foxp3, lymphocyte activation gene‐3 (LAG‐3), and CC chemokine ligand 4 (CCL4), and suppressed the proliferative response of antigen‐specific CD4⁺ T helper‐1 (Th1) cells. Pharmacological or antibody mediated blocking of CD39 function resulted in partial reversal of suppression. These data identify CD39 as a novel marker of human regulatory CD8⁺ T cells and indicate that CD39 is functionally involved in suppression by CD8⁺ Treg cells.