Reduced Effector Function Research Articles

Out-of-sequence signal 3 as a mechanism for virus-induced immune suppression of CD8 T cell responses.

Virus infections are known to induce a transient state of immune suppression often associated with an inhibition of T cell proliferation in response to mitogen or cognate-antigen stimulation. Recently, virus-induced immune suppression has been linked to responses to type 1 interferon (IFN), a signal 3 cytokine that normally can augment the proliferation and differentiation of T cells exposed to antigen (signal 1) and co-stimulation (signal 2). However, pre-exposure of CD8 T cells to IFN-inducers such as viruses or poly(I∶C) prior to antigen signaling is inhibitory, indicating that the timing of IFN exposure is of essence. We show here that CD8 T cells pretreated with poly(I∶C) down-regulated the IFN receptor, up-regulated suppressor of cytokine signaling 1 (SOCS1), and were refractory to IFNβ-induced signal transducers and activators of transcription (STAT) phosphorylation. When exposed to a viral infection, these CD8 T cells behaved more like 2-signal than 3-signal T cells, showing defects in short lived effector cell differentiation, reduced effector function, delayed cell division, and reduced levels of survival proteins. This suggests that IFN-pretreated CD8 T cells are unable to receive the positive effects that type 1 IFN provides as a signal 3 cytokine when delivered later in the signaling process. This desensitization mechanism may partially explain why vaccines function poorly in virus-infected individuals.

Out-of-sequence signal 3 as a mechanism for virus-induced immune suppression of CD8 T cell proliferation (VIR1P.960)

Abstract Virus infections are known to induce a transient state of immune suppression often associated with an inhibition of T cell proliferation in response to mitogen or cognate-antigen stimulation. Recently, virus-induced immune suppression has been linked to responses to type 1 IFN, a signal 3 cytokine that normally can augment the proliferation and differentiation of T cells exposed to antigen (signal 1) and co-stimulation (signal 2). However, pre-exposure of CD8 T cells to IFN-inducers such as viruses or poly(I:C) prior to antigen signaling is inhibitory, indicating that the timing of IFN exposure is of essence. Studies with T cells lacking IFN receptors implicated a direct role for type 1 IFN in this process. CD8 T cells pretreated with poly(I:C) down-regulated the IFN receptor, up-regulated SOCS1, and were refractory to IFNβ-induced STAT phosphorylation. When exposed to a viral infection these CD8 T cells behaved more like 2-signal than 3-signal T cells, showing defects in short lived effector cell differentiation, reduced effector function, delayed cell division, and reduced levels of survival proteins. This suggests that IFN-pretreated CD8 T cells are unable to receive the positive effects that type 1 IFN provides as a signal 3 cytokine when delivered later in the signaling process. This desensitization mechanism may partially explain why vaccines function poorly in virus-infected individuals.

Temporally-defined inflammatory pathways modulate distinct mechanisms of effector and regulatory CD4 T cell responses. (P1189)

Abstract Potentially autoreactive T cells are controlled by both cell-intrinsic and dominant trans-suppressive mechanisms. One of the major mediators of dominant suppression of immune responses are regulatory CD4+ T cells that express the transcription factor Foxp3. These cells can arise during thymic T cell development or be induced in the periphery from naïve CD4+ T cells. The factors that drive development of induced regulatory T cells can represent a critical choice for health or autoimmunity in the case of potentially self-reactive CD4 T cells. Numerous studies have implicated a role for enhanced effector differentiation and suppressed induction of regulatory T cells in response to antigen in the context of inflammatory signals. Here we show that inflammatory stimuli can drive expansion of functional Foxp3+ regulatory T cells in vivo, largely driven by type I interferon signals. Furthermore, naïve CD4+ T cells that are bystander-activated by interferon in the absence of cognate antigen exhibit tolerance, reduced effector function, and enhanced induction of Foxp3 upon subsequent antigen encounter. These data suggest that transient bystander activation imposes a dynamic state of responsiveness in naïve CD4+ T cells, in which the nature of subsequent differentiation is shaped by temporal coordination of antigen/TCR and inflammatory signals. This work provides an interesting new framework for potential antigen-specific therapeutic immunization strategies.

Activated CD8+ T cells exhibit blunted effector functions in early HIV-1 disease (P3035)

Abstract Elevated, persistent CD8+ T cell activation is a hallmark of HIV-1 infection. CD8+ T cell activation is measured by expression of CD38 and HLA-DR antigens on CD8+ T cells from the blood. How CD8+ T cell activation state may define impairments to CD8+ T cell function remains unclear. ERK1/2 signaling is critical for multiple T cell functions, and interruption of this pathway due to immune activation and inflammation may blunt CD8+ T cell function. We previously demonstrated that a major proportion of activated (CD38+HLA-DR+) CD8+ T cells from recently HIV-1-infected antiretroviral-treatment-naïve adults are refractory to phosphorylation of the ERK1/2 kinases (p-ERK1/2-refractory). Here we hypothesized that p-ERK1/2-refractory CD8+ T cells would exhibit reduced effector function compared to CD8+ p-ERK1/2-responsive cells. We combined single-cell phospho-kinase flow cytometry with intracellular cytokine staining into a single assay (PFICS), to examine IFN-γ and perforin responses in CD8+ T cells by ERK1/2 signaling ability. On a per cell basis, p-ERK1/2-refractory cells produced less IFN-γ in response to polyclonal or HIV-1 Gag peptide stimulation (% IFN-γ+ cells was 5.5-fold lower, p=0.006 for polyclonal and 1.5-fold lower, p=0.048 for Gag) and expressed reduced levels of perforin (2.1 and 1.6-fold lower perforin MFI). Blunted CD8+ T effector functions, secondary to ERK1/2 signaling deficits concentrated within activated CD8+ T cells, may contribute to HIV immunodeficiency.

Tregs suppress CD8 T cell effector function in inflamed tissue in a mouse model of autoimmune diabetes (P1018)

Abstract The aim of this study was to determine the means by which Tregs control disease in the NOD mouse model of autoimmune diabetes. Treatment of pre-diabetic mice with ongoing islet destruction with islet antigen-specific Tregs expanded from BDC2.5 TCR transgenic mice led to a rapid restoration of insulin production and a concomitant selective downregulation of a cytotoxic T lymphocyte signature, which included CD8α, IFNγ, granzymes A and B, and Cxcl9. This downregulation could be partially attributed to a decrease in intra-islet CD8 T cell numbers due to impaired recruitment of CD8 T cells to the islets. In addition, Treg treatment also reduced effector function of persisting CD8 T cells. Two-photon imaging showed that Tregs did not significantly disrupt interactions between islet CD8 T cells and dendritic cells, but abrogated IFNγ production by CD8 T cells. This suppression appeared to take place at multiple stages of CD8 T cell differentiation. Tregs inhibited differentiation of newly recruited CD8 T cells to IFNγ producers completely, but prevented IFNγ protein expression without affecting IFNγ message in pre-activated CD8 T cells. Altogether, these results indicate that CD8 T cells are the primary early targets of Treg control of tissue inflammation and rapid disease resolution by Tregs can be attributed to efficient control of CD8 T cell infiltration, reactivation in the tissue, and their expression of effector functions.

An Effector-Reduced Anti- -Amyloid (A ) Antibody with Unique A Binding Properties Promotes Neuroprotection and Glial Engulfment of A

Passive immunization against β-amyloid (Aβ) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aβ monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aβ, protected against Aβ1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aβ oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aβ, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aβ antibody that takes advantage of a unique Aβ binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.

ATROSAB, a humanized antagonistic anti-tumor necrosis factor receptor one-specific antibody

Tumor necrosis factor (TNF) signals through two membrane receptors, TNFR1 and TNFR2, and TNFR1 is known to be the major pathogenic mediator of chronic and acute inflammatory diseases. Present clinical intervention is based on neutralization of the ligand TNF. Selective inhibition of TNF receptor 1 (TNFR1) provides an alternative opportunity to neutralize the pro-inflammatory activity of TNF while maintaining the advantageous immunological responses mediated by TNFR2, including immune regulation, tissue homeostasis and neuroprotection. We recently humanized a mouse anti-human TNFR1 monoclonal antibody exhibiting TNFR1-neutralizing activity. This humanized antibody has been converted into an IgG1 molecule (ATROSAB) containing a modified Fc region previously demonstrated to have greatly reduced effector functions. Purified ATROSAB, produced in CHO cells, showed strong binding to human and rhesus TNFR1-Fc fusion protein and mouse embryonic fibroblasts transfected with a recombinant TNFR1 fusion protein with an affinity identical to the parental mouse antibody H398. Using chimeric human/mouse TNFR1 molecules, the epitope of ATROSAB was mapped to the N-terminal region (amino acid residues 1-70) comprising the first cysteine-rich domain (CRD1) and the A1 sub-domain of CRD2. In vitro, ATROSAB inhibited typical TNF-mediated responses like apoptosis induction and activation of NFκB-dependent gene expression such as IL-6 and IL-8 production. These findings open the way to further analyze the therapeutic activity of ATROSAB in relevant disease models in non-human primates.

Impact of Molecular Processing in the Hinge Region of Therapeutic IgG4 Antibodies on Disposition Profiles in Cynomolgus Monkeys

The IgG4 isotype antibody is a potential candidate for immunotherapy when reduced effector functions are desirable. However, antigen binding fragment (Fab) arm exchange leads to functional monovalency with potentially reduced therapeutic efficacy. Mutagenesis studies suggested that the CH3 domain and not the core hinge is dominantly involved in in vivo molecular processing. This work investigated whether stabilization of the core hinge of a therapeutic IgG4 antibody by mutation of Ser228 to Pro (S228P) would be sufficient to prevent in vivo Fab arm exchange. In vitro experiments evaluated the influence of different levels of oxidation-reduction conditions in buffer and serum on Fab arm exchange (swapping) of wild-type (WT) IgG4 and IgG1 and of IgG4 S228P, which included a sterically neutral second mutation (Leu235 replaced by Glu). The objective of single-dose pharmacokinetic experiments in cynomolgus monkeys was to determine whether the mutation reduced IgG4 swapping in vivo. The results indicated that S228P mutation did not completely prevent Fab arm exchange in vitro in buffer under reducing conditions relative to IgG4 WT. The immunoassay findings were confirmed by mass spectrometry measurements. Results of the in vivo studies suggested that the therapeutic IgG4 WT antibody exchanged Fab arms with endogenous cynomolgus monkey IgG4, resulting in bispecific IgG4 antibodies with monovalency for the therapeutic target. In contrast, serum from cynomolgus monkeys dosed with the IgG4 mutant was virtually free of swapped IgG4. In conclusion, the results indicated that IgG4 swapping in vivo was markedly attenuated by S228P mutation.

The Lytic Potential of Human Liver NK Cells Is Restricted by Their Limited Expression of Inhibitory Killer Ig-Like Receptors

The human liver is enriched in NK cells which are potent effectors of the innate immune system. We have determined that liver NK cells freshly isolated from surgical specimens from patients with hepatic malignancy have less cytolytic activity than autologous blood NK cells. This difference was due to a higher proportion of CD16(-) NK cells in the liver and reduced cytotoxicity by CD16(+) liver NK cells compared with their blood counterparts. CD16(+) liver NK cells had similar expression of activating NK receptors and had similar intracellular granzyme B and perforin content compared with CD16(+) blood NK cells. CD16(+) liver NK cells contained a reduced fraction of cells with inhibitory killer Ig-like receptors specific for self-MHC class I (self-killer Ig-related receptor (KIR)) and an increased fraction of self-KIR(neg)NKG2A(pos) and self-KIR(neg)NKG2A(neg) cells. Using single-cell analysis of intracellular IFN-gamma production and cytotoxicity assays, we determined that CD16(+) liver NK cells expressing self-KIR were more responsive to target cells than those cells that did not express self-KIR molecules. CD16(+) liver NK cells gained cytolytic function when stimulated with IL-2 or cultured with LPS or poly(I:C)-activated autologous liver Kupffer cells. Thus, the human liver contains NK cell subsets which have reduced effector function, but under appropriate inflammatory conditions become potent killers.

IL‐15 is critical for the maintenance and innate functions of self‐specific CD8+ T cells

IL-15 is a pleiotropic cytokine involved in host defense as well as autoimmunity. IL-15-deficient mice show a decrease of memory phenotype (MP) CD8(+) T cells, which develop naturally in naïve mice and whose origin is unclear. It has been shown that self-specific CD8(+) T cells developed in male H-Y antigen-specific TCR transgenic mice share many similarities with naturally occurring MP CD8(+) T cells in normal mice. In this study, we found that H-Y antigen-specific CD8(+) T cells in male but not female mice decreased when they were crossed with IL-15-deficient mice, mainly due to impaired peripheral maintenance. The self-specific TCR transgenic CD8(+) T cells developed in IL-15-deficient mice showed altered surface phenotypes and reduced effector functions ex vivo. Bystander activation of the self-specific CD8(+) T cells was induced in vivo during infection with Listeria monocytogenes, in which proliferation but not IFN-gamma production was IL-15-dependent. These results indicated important roles for IL-15 in the maintenance and functions of self-specific CD8(+) T cells, which may be included in the naturally occurring MP CD8(+) T-cell population in naïve normal mice and participate in innate host defense responses.

Requirement for CD45 in fine-tuning mast cell responses mediated by different ligand–receptor systems

The receptor-like protein tyrosine phosphatase CD45, the most abundant cell surface phosphatase on all nucleated hemopoietic cells, is a critical regulator of the activation status of Src family kinases (SFKs). To study the impact of CD45 on mast cell function, we compare bone marrow-derived mast cells (BMMCs) from CD45-deficient mice and from mice expressing an activating point mutation (E613R) in the juxtamembrane wedge of CD45. In response to Ag-triggered FcεR1-mediated activation, CD45-deficient BMMCs exhibit increased inhibitory Lyn phosphorylation and drastically reduced effector functions (degranulation and cytokine secretion). In contrast, CD45 E613R BMMCs show stronger effector functions after Ag-triggering than wild-type (WT) BMMCs. Despite these dichotomous phenotypes, phosphorylation of the inhibitory tyrosine in the SFK Lyn of CD45 E613R BMMCs is comparable to CD45-deficient BMMCs. This unexpected phenotype most likely is due to attenuated interaction between CD45 E613R and Lyn and a hyper-activation of the Fyn-regulated phosphatidylinositol-3-kinase pathway. Interestingly, depending on the receptor system addressed, CD45-deficient and CD45 E613R BMMCs show uniform phenotypes as well. Proliferation of both cell types in response to IL-3 and/or SF is enhanced compared to WT BMMCs. Together, the data indicate that CD45 plays a complex and essential role in fine-tuning mast cell responses mediated by different ligand–receptor systems.

Effect of phosphodiesterase 7 inhibitor ASB16165 on development and function of cytotoxic T lymphocyte

In the present study, possible role of phosphodiesterase 7 (PDE7) in development and function of cytotoxic T lymphocyte (CTL) was examined using ASB16165, a specific inhibitor for PDE7. ASB16165 inhibited generation of CTL activity in mixed lymphocyte reaction (MLR), in which splenocytes from C57BL/6N mice were stimulated with those from BALB/c mice. Flow cytometric analysis revealed that ASB16165 suppressed induction of activated CD4+ as well as CD8+ T cells in MLR. In cell division analyses using 5-carboxyfluorescein diacetate succinimide ester (CFSE), ASB16165 was shown to markedly inhibit proliferation of CD4+ and CD8+ T cells. In addition, ASB16165 reduced effector function of CTL, while the effect was less than that observed in CTL induction in MLR. Forskolin and dibutyryl cAMP also inhibited both the induction and effector function of CTL. PDE4 inhibitor rolipram showed similar but weaker inhibition for the development and proliferation of CD8+ T cells compared with ASB16165, and failed to impair effector function of CTL. These findings suggest that PDE7 but not PDE4 has the major role in induction and function of CTL in mice, and that the effect might be mediated by elevation of intracellular cAMP level. ASB16165 may be useful for treatment of the diseases in which CTL has a pathogenic role ( e.g. autoimmune diseases).

Intracranial administration of deglycosylated C-terminal-specific anti-Aβ antibody efficiently clears amyloid plaques without activating microglia in amyloid-depositing transgenic mice

BackgroundAntibodies against the Aß peptide clear Aß deposits when injected intracranially. Deglycosylated antibodies have reduced effector functions compared to their intact counterparts, potentially avoiding immune activation.MethodsDeglycosylated or intact C-terminal specific high affinity anti-Aβ antibody (2H6) were intracranially injected into the right frontal cortex and hippocampus of amyloid precursor protein (APP) transgenic mice. The untreated left hemisphere was used to normalize for the extent of amyloid deposition present in each mouse. Control transgenic mice were injected with an antibody against a drosophila-specific protein (amnesiac). Tissues were examined for brain amyloid deposition and microglial responses 3 days after the injection.ResultsThe deglycosylated 2H6 antibody had lower affinity for several murine Fcγ receptors and human complement than intact 2H6 without a change in affinity for Aß. Immunohistochemistry for Aβ and thioflavine-S staining revealed that both diffuse and compact deposits were reduced by both antibodies. In animals treated with the intact 2H6 antibody, a significant increase in Fcγ-receptor II/III immunostaining was observed compared to animals treated with the control IgG antibody. No increase in Fcγ-receptor II/III was found with the deglycosylated 2H6 antibody. Immunostaining for the microglial activation marker CD45 demonstrated a similar trend.ConclusionThese findings suggest that the deglycosylated 2H6 is capable of removing both compact and diffuse plaques without activating microglia. Thus, antibodies with reduced effector functions may clear amyloid without concomitant immune activation when tested as immunotherapy for Alzheimer's disease.