Redox modulation inhibits CD8 T cell effector function

The evolutionary preservation of reactive oxygen species in innate immunity underscores the important roles these constituents play in immune cell activity and as signaling intermediates. In an effort to exploit these pathways to achieve control of aberrant immune activation we demonstrate that modulation of redox status suppresses cell proliferation and production of IL-2, IFN-gamma, TNF-alpha, and IL-17 in two robust CD8 T-cell-dependent in vitro mouse models: (1) response to alloantigen in an mixed leukocyte reaction and (2) CD8 T cell receptor transgenic OT-1 response to cognate peptide (SIINFEKL). To correlate these findings with cytotoxic lymphocyte (CTL) function we performed cytotoxicity assays and found that redox modulation diminishes the ability of alloantigen-specific and antigen-specific OT-1 CTLs to kill their corresponding antigen-expressing target cells. To further examine the mechanisms of redox-mediated repression of CTL target cell lysis, we analyzed the expression of the effector molecules IFN-gamma, perforin, and granzyme B and the degranulation marker CD107a (LAMP-1). In both models, redox modulation reduced the expression of these effector components by at least fivefold. These results demonstrate that redox modulation quells the CD8 T cell response to alloantigen and the T cell receptor transgenic CD8 T cell response to its cognate antigen by inhibiting proliferation, proinflammatory cytokine synthesis, and CTL effector mechanisms.     

Enhancement of suboptimal CD8 cytotoxic T cell effector function in vivo using antigen-specific CD80 defective T cells

T cell upregulation of B7 molecules CD80 and CD86 limits T cell expansion in immunodeficient hosts; however, the relative roles of CD80 separate from CD86 on CD4 versus CD8 T cells in a normal immune system are not clear. To address this question, we used the parent-into-F1 (P→F1) murine model of graft-versus-host disease and transferred optimal and suboptimal doses of CD80 and/or CD86 knockout (KO) T cells into normal F1 hosts. Enhanced elimination of host B cells by KO T cells was observed only at suboptimal donor cell doses and was greatest for CD80 KO→F1 mice. Wild-type donor cells exhibited peak CD80 upregulation at day 10; CD80 KO donor cells exhibited greater peak (day 10) donor T cell proliferation and CD8 T cell effector CTL numbers versus wild-type→F1 mice. Fas or programmed cell death-1 upregulation was normal as was homeostatic contraction of CD80 KO donor cells from days 12-14. Mixing studies demonstrated that maximal host cell elimination was seen when both CD4 and CD8 T cells were CD80 deficient. These results indicate an important role for CD80 upregulation on Ag-activated CD4 and CD8 T cells in limiting expansion of CD8 CTL effectors as part of a normal immune response. Our results support further studies of therapeutic targeting of CD80 in conditions characterized by suboptimal CD8 effector responses.     

Induction of polyclonal CD8+ T cell activation and effector function by Pertussis toxin

Pertussis toxin (PTX) has pronounced adjuvant activity and strongly enhances innate and adaptive immune responses, including increased antibody production and Th1/Th2 cytokine production. Adjuvant effects of PTX on Th1 and Th2 cells are primarily mediated via CD80/86 costimulation via enhanced expression of these molecules by APCs. However, it has remained unresolved whether PTX modulates the expression of costimulatory and inhibitory molecules on CD4+ and CD8+ T cells. To address this question, we determined the expression kinetics of CD28, CTLA-4, and CD40L on spleen CD4+ and CD8+ T cells after incubation with PTX. The results show that PTX upregulated the expression of CD28 by CD8+ T cells, but not by CD4+ T cells. In contrast, the expression of CTLA-4 and CD40L was not substantially altered on CD4+ or CD8+ T cells. CD28 upregulation by CD8+ T cells was paralleled by upregulation of CD69 and the induction of IFN-γ, Granzyme B (GrB), and IL-17. CD8+ T cell activation and cytokine production could be substantially blocked with anti-CD80 and CD86 antibodies, consistent with CD28 mediated signaling. Treatment of highly purified CD8+ T cells with PTX resulted in upregulation of CD28 and CD69, and production of IFN-γ. Incubation with CD28 mAb further enhanced this effect, suggesting that PTX has direct effects on CD8+ T cells which are enhanced by CD80/86-mediated costimulation provided by APCs.     

The role of CD4 T cell help for CD8 CTL activation

CD4 T cells play an important role in the initiation and persistence of CD8 T cells responses. In this review, we report on and evaluate the mechanisms by which CD4 T cells contribute to activation of CD8 T cells and the signal pathways of the down-streaming events after CD4 T cell help.     

Antibody-independent antiviral function of memory CD4+ T cells in vivo requires regulatory signals from CD8+ effector T cells

Previous studies have shown that vaccine-primed CD4(+) T cells can mediate accelerated clearance of respiratory virus infection. However, the relative contributions of Ab and CD8(+) T cells, and the mechanism of viral clearance, are poorly understood. Here we show that control of a Sendai virus infection by primed CD4(+) T cells is mediated through the production of IFN-gamma and does not depend on Ab. This effect is critically dependent on CD8(+) cells for the expansion of CD4(+) T cells in the lymph nodes and the recruitment of memory CD4(+) T cells to the lungs. Passive transfer of a CD8(+) T cell supernatant into CD8(+) T cell-depleted, hemagglutinin-neuraminidase (HN)(421-436)-immune muMT mice substantially restored the virus-specific memory CD4(+) response and enhanced viral control in the lung. Together, the data demonstrate for the first time that in vivo primed CD4(+) T cells have the capacity to control a respiratory virus infection in the lung by an Ab-independent mechanism, provided that CD8(+) T cell "help" in the form of soluble factor(s) is available during the virus infection. These studies highlight the importance of synergistic interactions between CD4(+) and CD8(+) T cell subsets in the generation of optimal antiviral immunity.     

Radiotherapy promotes tumor-specific effector CD8+ T cells via dendritic cell activation

Radiotherapy is an important treatment for cancer. The main mode of action is thought to be the irreversible damage to tumor cell DNA, but there is evidence that irradiation mobilizes tumor-specific immunity, and recent studies showed that the efficacy of high-dose radiotherapy depends on the presence of CD8(+) T cells. We show in this study that the efficacy of radiotherapy given as a single, high dose (10 Gy) crucially depends on dendritic cells and CD8(+) T cells, whereas CD4(+) T cells or macrophages are dispensable. We show that local high-dose irradiation results in activation of tumor-associated dendritic cells that in turn support tumor-specific effector CD8(+) T cells, thus identifying the mechanism that underlies radiotherapy-induced mobilization of tumor-specific immunity. We propose that in the absence of irradiation, the activation status of dendritic cells rather than the amount of tumor-derived Ag is the bottleneck, which precludes efficient anti-tumor immunity.     

The fate of effector T cells in vivo is determined during activation and differs for CD4+ and CD8+ cells

Effector T cells generated in the mesenteric lymph nodes (mLN) are known to accumulate in mLN and the tissue drained by them after circulating in the blood. Their accumulation is due less to preferential entry into mLN but more to preferential proliferation within mLN. The factors regulating the proliferation of effector T cells in vivo are unclear, and it is unknown whether they are different for CD4(+) and CD8(+) effector T cells. Rat T cells from mLN or peripheral lymph nodes (pLN) were stimulated polyclonally via the TCR and CD28 and injected i.v. into congenic recipients. Using three-color flow cytometry and immunohistochemistry, they were identified in mLN, pLN, and blood over time, and proliferation was determined by measuring bromodeoxyuridine incorporation. Only effector mLN T cells showed a significantly increased proliferation rate after entry into mLN compared with that in pLN (2.4 +/- 1.8% vs 0.8 +/- 0.4%). Proliferation among the injected cells was higher when they had contact with dendritic cells within mLN (9.0 +/- 4.3%) than when they did not (4.1 +/- 2.1%). Furthermore, effector mLN T cells which were observed 56 days after injection maintained the capacity for preferential proliferation within mLN. Interestingly, CD4(+) effector mLN T cells proliferated at a higher rate (4.8 +/- 0.7%), remaining in mLN, whereas CD8(+) effector mLN T cells proliferated at a lower rate (3.3 +/- 1.0%) and were able to leave the mLN into the blood. Elucidating the factors regulating the proliferation of effector T cells in vivo will help to modify their distribution for therapeutic purposes.     

Transient loss of MHC class I tetramer binding after CD8+ T cell activation reflects altered T cell effector function

Engagement of the Ag receptor on naive CD8+ T cells by specific peptide-MHC complex triggers their activation/expansion/differentiation into effector CTL. The frequency of Ag-specific CD8+ T cells can normally be determined by the binding of specific peptide-MHC tetramer complexes to TCR. In this study we demonstrate that, shortly after Ag activation, CD8+ T cells transiently lose the capacity to efficiently bind peptide-MHC tetramer complexes. This transient loss of tetramer binding, which occurs in response to naturally processed viral peptide during infection in vitro and in vivo, is associated with reduced signaling through the TCR and altered/diminished effector activity. This change in tetramer binding/effector response is likewise associated with a change in cell surface TCR organization. These and related results suggest that early during CD8+ T cell activation, there is a temporary alteration in both cell surface Ag receptor display and functional activity that is associated with a transient loss of cognate tetramer binding.     

Medroxyprogesterone acetate inhibits CD8+ T cell viral-specific effector function and induces herpes simplex virus type 1 reactivation

Clinical research suggests hormonal contraceptive use is associated with increased frequencies of HSV reactivation and shedding. We examined the effects of medroxyprogesterone acetate (MPA), the compound most commonly used for injectable hormonal contraception, on HSV type 1 (HSV-1) reactivation and CD8(+) T cell function in murine trigeminal ganglia (TG). In ex vivo TG cultures, MPA dramatically inhibited canonical CD8(+) T cell effector functions, including IFN-gamma production and lytic granule release, and increased HSV-1 reactivation from latency. In vivo, MPA treatment of latently infected ovariectomized mice inhibited IFN-gamma production and lytic granule release by TG resident CD8(+) T cells stimulated directly ex vivo. RNA specific for the essential immediate early viral gene ICP4 as well as viral genome DNA copy number were increased in mice that received MPA during latency, suggesting that treatment increased in vivo reactivation. The increase in HSV-1 copy number appeared to be the result of a two-tine effect, as MPA induced higher reactivation frequencies from latently infected explanted TG neurons in the presence or absence of CD45(+) cells. Our data suggest hormonal contraceptives that contain MPA may promote increased frequency of HSV reactivation from latency through the combinatory effects of inhibiting protective CD8(+) T cell responses and by a leukocyte-independent effect on infected neurons.     

Regulation of effector and memory CD8+ T cell function by inflammatory cytokines

Cells communicate with each other through the production and secretion of cytokines, which are integral to the host response to infection. Once recognized by specific cytokine receptors expressed on the cell surface, these exogenous signals direct the biological function of a cell in order to adapt to their microenvironment. CD8⁺ T cells are critical immune cells that play an important role in the control and elimination of intracellular pathogens. Current findings have demonstrated that cytokines influence all aspects of the CD8⁺ T cell response to infection or immunization. The cytokine milieu induced at the time of activation impacts the overall magnitude and function of the effector CD8⁺ T cell response and the generation of functional memory CD8⁺ T cells. This review will focus on the impact of inflammatory cytokines on different aspects of CD8⁺ T cell biology.     

T cell TRAIL promotes murine lupus by sustaining effector CD4 Th cell numbers and by inhibiting CD8 CTL activity

T cells play an essential role in driving humoral autoimmunity in lupus. Molecules such as TRAIL exhibit strong T cell modulatory effects and are up-regulated in lupus, raising the possibility that they may influence disease severity. To address this possibility, we examined the role of TRAIL expression on pathogenic T cells in an induced model of murine lupus, the parent-into-F(1) (P-->F(1)) model of chronic graft-vs-host disease (GVHD), using wild-type or TRAIL-deficient donor T cells. Results were compared with mice undergoing suppressive acute GVHD. Although chronic GVHD mice exhibited less donor T cell TRAIL up-regulation and IFN-alpha-inducible gene expression than acute GVHD mice, donor CD4(+) T cell TRAIL expression in chronic GVHD was essential for sustaining effector CD4(+) Th cell numbers, for sustaining help to B cells, and for more severe lupus-like renal disease development. Conversely, TRAIL expression on donor CD8(+) T cells had a milder, but significant down-regulatory effect on CTL effector function, affecting the perforin/granzyme pathway and not the Fas ligand pathway. These results indicate that, in this model, T cell-expressed TRAIL exacerbates lupus by the following: 1) positively regulating CD4(+) Th cell numbers, thereby sustaining T cell help for B cells, and 2) to a lesser degree by negatively regulating perforin-mediated CD8(+) CTL killing that could potentially eliminate activated autoreactive B cells.     

CD80 binding polyproline helical peptide inhibits T cell activation

The critical role played by the CD28/CD152-CD80/CD86 costimulatory molecules in mediating T cell activation and suppression provides attractive targets for therapeutic strategies. CD28 and CD152 share a conserved polyproline motif in the ligand-binding region. Similar proline-rich regions in globular domains preferentially adopt a polyproline type II (PP) helical conformation and are involved in transient (II)protein-protein interactions. Interestingly, in the human CD80-CD152 complex, Pro(102) of CD152 restricts the preceding proline to PP(II) helix in the binding orientation in relation to the shallow binding pocket of CD80. Peptide agents derived from binding sites of receptors that mimic the bioactive conformation have been shown to block receptor-ligand interactions. Contact preferences of the interface amino acids at the protein-protein interaction sites and the propensity of each residue to form PP(II) helix were integrated in the design of novel peptide agents referred to as CD80 competitive antagonist peptides. Structural and functional studies suggest potential therapeutic value for select CD80 competitive antagonist peptides.     

Cutting edge: rapid in vivo CTL activity by polyoma virus-specific effector and memory CD8+ T cells

For viruses that establish persistent infection, continuous immunosurveillance by effector-competent antiviral CD8(+) T cells is likely essential for limiting viral replication. Although it is well documented that virus-specific memory CD8(+) T cells synthesize cytokines after short term in vitro stimulation, there is limited evidence that these T cells exhibit cytotoxicity, the dominant antiviral effector function. Here, we show that antiviral CD8(+) T cells in mice acutely infected by polyoma virus, a persistent mouse pathogen, specifically eliminate viral peptide-pulsed donor spleen cells within minutes after adoptive transfer and do so via a perforin-dependent mechanism. Antiviral memory CD8(+) T cells were similarly capable of rapidly mobilizing potent Ag-specific cytotoxic activity in vivo. These findings strongly support the concept that a cytotoxic effector-memory CD8(+) T cell population operates in vivo to control this persistent viral infection.     

A new dynamic model of CD8+ T effector cell responses via CD4+ T helper-antigen-presenting cells

A long-standing paradox in cellular immunology has been the conditional requirement for CD4(+) Th cells in priming of CD8(+) CTL responses. We propose a new dynamic model of CD4(+) Th cells in priming of Th-dependent CD8(+) CTL responses. We demonstrate that OT II CD4(+) T cells activated by OVA-pulsed dendritic cells (DC(OVA)) are Th1 phenotype. They acquire the immune synapse-composed MHC II/OVAII peptide complexes and costimulatory molecules (CD54 and CD80) as well as the bystander MHC class I/OVAI peptide complexes from the DC(OVA) by DC(OVA) stimulation and thus also the potential to act themselves as APCs. These CD4(+) Th-APCs stimulate naive OT I CD8(+) T cell proliferation through signal 1 (MHC I/OVAI/TCR) and signal 2 (e.g., CD54/LFA-1 and CD80/CD28) interactions and IL-2 help. In vivo, they stimulate CD8(+) T cell proliferation and differentiation into CTLs and induce effective OVA-specific antitumor immunity. Taken together, this study demonstrates that CD4(+) Th cells carrying acquired DC Ag-presenting machinery can, by themselves, efficiently stimulate CTL responses. These results have substantial implications for research in antitumor and other aspects of immunity.     

Plasmacytoid dendritic cells take up opsonized antigen leading to CD4+ and CD8+ T cell activation in vivo

Plasmacytoid dendritic cells (pDC) are the body's main source of IFN-alpha, but, unlike classical myeloid DC (myDC), they lack phagocytic activity and are generally perceived as playing only a minor role in Ag processing and presentation. We show that murine pDC, as well as myDC, express Fcgamma receptors (CD16/CD32) and can use these receptors to acquire Ag from immune complexes (IC), resulting in the induction of robust Ag-specific CD4(+) and CD8(+) T cell responses. IC-loaded pDC stimulate CD4(+) T cells to proliferate and secrete a mixture of IL-4 and IFN-gamma, and they induce CD8(+) T cells to secrete IL-10 as well as IFN-gamma. In contrast, IC-loaded myDC induce both CD4(+) and CD8(+) T cells to secrete mainly IFN-gamma. These results indicate that pDC can shape an immune response by acquiring and processing opsonized Ag, leading to a predominantly Th2 response.     

Disparate primary and secondary allospecific CD8+ T cell cytolytic effector function in the presence or absence of host CD4+ T cells

The role of CD4+ T cells in promoting CD8+ T cell effector activity in response to transplant Ags in vivo has not been reported. We used a hepatocellular allograft model known to initiate both CD4-dependent and CD4-independent rejection responses to investigate the contribution of CD4+ T cells to the development, function, and persistence of allospecific CD8+ T cell effectors in vivo. Complete MHC-mismatched hepatocellular allografts were transplanted into C57BL/6 (CD4-sufficient) or CD4 knockout (CD4-deficient) hosts. The development of in vivo allospecific cytotoxicity was determined by clearance of CFSE-labeled target cells. CD8+ T cell cytotoxic effector activity was enhanced in response to allogeneic hepatocellular grafts with a greater magnitude of allocytotoxicity and a prolonged persistence of CTL effector activity in CD4-sufficient hosts compared with CD4-deficient hosts. Cytolytic activity was mediated by CD8+ T cells in both recipient groups. In response to a second hepatocyte transplant, rejection kinetics were enhanced in both CD4-sufficient and CD4-deficient hepatocyte recipients. However, only CD4-sufficient hosts developed recall CTL responses with an augmented magnitude and persistence of allocytotoxicity in comparison with primary CTL responses. These studies show important functional differences between alloreactive CD8+ T cell cytolytic effectors that mature in vivo in the presence or absence of CD4+ T cells.     

In vivo ligation of CD40 enhances priming against the endogenous tumor antigen and promotes CD8+ T cell effector function in SV40 T antigen transgenic mice

The ability to initiate and sustain CD8(+) T cell responses to tumors in vivo is hindered by the development of peripheral T cell tolerance against tumor-associated Ags. Approaches that counter the onset of T cell tolerance may preserve a pool of potentially tumor-reactive CD8(+) T cells. Administration of agonist Ab to the CD40 molecule, expressed on APCs, can enhance immunization approaches targeting T lymphocytes in an otherwise tolerance-prone environment. In this report, the effects of anti-CD40 administration on priming of naive CD8(+) T cells against an endogenous tumor Ag were investigated. Line 501 mice express the SV40 large T Ag oncoprotein as a transgene from the alpha-amylase promoter, resulting in the development of peripheral CD8(+) T cell tolerance to the H-2-D(b)-restricted immunodominant epitope I of T Ag by 6 mo of age, before the appearance of osteosarcomas. We demonstrate that naive epitope I-specific TCR transgenic (TCR-I) T cells undergo peripheral tolerance following adoptive transfer into 6-mo-old 501 mice. In contrast, administration of agonistic anti-CD40 Ab led to increased expansion of TCR-I T cells in 501 mice, the acquisition of effector function by TCR-I T cells and the establishment of T cell memory. Importantly, this enhanced priming effect of anti-CD40 administration did not require immunization and was effective even if administered after naive TCR-I T cells had encountered the endogenous T Ag. Thus, anti-CD40 administration can block the onset of peripheral tolerance and enhance the recruitment of functionally competent effector T cells toward an endogenous tumor Ag.     

EBV-specific CD8+ T cell memory: relationships between epitope specificity, cell phenotype, and immediate effector function

EBV infection in humans induces CD8+ T cell memory to viral epitopes derived from both lytic and latent cycle Ags. We have analyzed the relationship between the phenotype and function of the memory pool of T cells specific for these Ags. Lytic epitope-specific populations were heterogeneous in terms of CD45RO/RA and CD28 expression, whereas latent epitope-specific populations were uniformly CD45RO+ and CD28+, consistent with the higher antigenic challenge from lytic epitopes driving some memory cells toward a CD45RA+, CD28- phenotype. However, both types of memory population showed immediate epitope-specific cytotoxicity and type 1 cytokine production in ex vivo assays. Cytotoxic function was not associated with preactivated T cells, as EBV-specific populations were negative for activation markers such as CD69 or CD38, nor could cytotoxic function be ascribed to CD27- or CD56+ subsets, as such cells were not detected in EBV-specific memory. Furthermore, cytotoxicity was not limited to CD45RA+ and/or CD28- fractions, but also was observed in CD45RO+, CD28+ populations in lytic and latent epitope-specific memory. Cytokine (IFN-gamma, TNF-alpha) responses, measured by intracytoplasmic staining after peptide stimulation, also were detectable in CD45RO+ and RA+ subsets as well as CD28+ and CD28- subsets. Of other markers that were heterogeneous in both lytic and latent epitope populations, CCR7 gave the best discrimination of functionality; thus, CCR7+ cells consistently failed to give an IFN-gamma or TNF-alpha response, whereas many CCR7- cells were responsive. Our data are consistent with effector functions having a broad distribution among phenotypically distinct subsets of "effector memory" cells that have lost the CCR7 marker.     

Novel exosome-targeted CD4+ T cell vaccine counteracting CD4+25+ regulatory T cell-mediated immune suppression and stimulating efficient central memory CD8+ CTL responses

T cell-to-T cell Ag presentation is increasingly attracting attention. In this study, we demonstrated that active CD4+ T (aT) cells with uptake of OVA-pulsed dendritic cell-derived exosome (EXO(OVA)) express exosomal peptide/MHC class I and costimulatory molecules. These EXO(OVA)-uptaken (targeted) CD4+ aT cells can stimulate CD8+ T cell proliferation and differentiation into central memory CD8+ CTLs and induce more efficient in vivo antitumor immunity and long-term CD8+ T cell memory responses than OVA-pulsed dendritic cells. They can also counteract CD4+25+ regulatory T cell-mediated suppression of in vitro CD8+ T cell proliferation and in vivo CD8+ CTL responses and antitumor immunity. We further elucidate that the EXO(OVA)-uptaken (targeted)CD4+ aT cell's stimulatory effect is mediated via its IL-2 secretion and acquired exosomal CD80 costimulation and is specifically delivered to CD8+ T cells in vivo via acquired exosomal peptide/MHC class I complexes. Therefore, EXO-targeted active CD4+ T cell vaccine may represent a novel and highly effective vaccine strategy for inducing immune responses against not only tumors, but also other infectious diseases.