Manjunatha Ankathatti Munegowda

Intercellular Trogocytosis Plays an Important Role in Modulation of Immune Responses

Intercellular communication is an important means of molecular information transfer through exchange of membrane proteins from cells to cells. Advent of the latest analytical and imaging tools has allowed us to enhance our understanding of the cellular communication through the intercellular exchange of intact membrane patches, also called trogocytosis, which is a ubiquitous phenomenon. Immune responses against pathogens or any foreign antigens require fine immune regulation, where cellular communications are mediated by either soluble or cell surface molecules. It has been demonstrated that the membrane molecule transfer between immune cells such as dendritic and T cells can be derived through internalization/recycling pathway, dissociation-associated pathway, uptake of exosomes and membrane nanotube formations. Recent evidence implicates the trogocytosis as an important mechanism of the immune system to modulate immune responses. Exchange of membrane molecules/antigens between immune cells has been observed for a long time, but the mechanisms and functional consequences of these transfers remain unclear. In this review, we discuss the possible mechanisms of trogocytosis and its physiological relevance to immune system, with special reference to T cells and the stimulatory or suppressive immune responses derived from T cells with acquired dendritic cell membrane molecules.

Dendritic Cells Recruit T Cell Exosomes via Exosomal LFA-1 Leading to Inhibition of CD8+ CTL Responses through Downregulation of Peptide/MHC Class I and Fas Ligand-Mediated Cytotoxicity

Active T cells release bioactive exosomes (EXOs). However, its potential modulation in immune responses is elusive. In this study, we in vitro generated active OVA-specific CD8+ T cells by cultivation of OVA-pulsed dendritic cells (DCOVA) with naive CD8+ T cells derived from OVA-specific TCR transgenic OTI mice and purified EXOs from CD8+ T cell culture supernatant by differential ultracentrifugation. We then investigated the suppressive effect of T cell EXOs on DCOVA-mediated CD8+ CTL responses and antitumor immunity. We found that DCOVA uptake OTI T cell EXOs expressing OVA-specific TCRs and Fas ligand via peptide/MHC Ag I–TCR and CD54–LFA-1 interactions leading to downregulation of peptide/MHC Ag I expression and induction of apoptosis of DCOVA via Fas/Fas ligand pathway. We demonstrated that OVA-specific OTI T cell EXOs, but not lymphocytic choriomeningitis virus-specific TCR transgenic mouse CD8+ T cell EXOs, can inhibit DCOVA-stimulated CD8+ CTL responses and antitumor immunity against OVA-expressing B16 melanoma. In addition, these T cell EXOs can also inhibit DCOVA-mediated CD8+ CTL-induced diabetes in transgenic rat insulin promoter-mOVA mice. Interestingly, the anti–LFA-1 Ab treatment significantly reduces T cell EXO-induced inhibition of CD8+ CTL responses in both antitumor immunity and autoimmunity. EXOs released from T cell hybridoma RF3370 cells expressing OTI CD8+ TCRs have a similar inhibitory effect as T cell EXOs in DCOVA-stimulated CTL responses and antitumor immunity. Therefore, our data indicate that Ag-specific CD8+ T cells can modulate immune responses via T cell-released EXOs, and T cell EXOs may be useful for treatment of autoimmune diseases.

Direct in vivo evidence of CD4+ T cell requirement for CTL response and memory via pMHC-I targeting and CD40L signaling

CD4+ T cell help contributes critically to DC‐induced CD8+ CTL immunity. However, precisely how these three cell populations interact and how CD4+ T cell signals are delivered to CD8+ T cells in vivo have been unclear. In this study, we developed a novel, two‐step approach, wherein CD4+ T cells and antigen‐presenting DCs productively engaged one another in vivo in the absence of cognate CD8+ T cells, after which, we selectively depleted the previously engaged CD4+ T cells or DCs before allowing interactions of either population alone with naïve CD8+ T cells. This protocol thus allows us to clearly document the importance of CD4+ T‐licensed DCs and DC‐primed CD4+ T cells in CTL immunity. Here, we provide direct in vivo evidence that primed CD4+ T cells or licensed DCs can stimulate CTL response and memory, independent of DC‐CD4+ T cell clusters. Our results suggest that primed CD4+ T cells with acquired pMHC‐I from DCs represent crucial “immune intermediates” for rapid induction of CTL responses and for functional memory via CD40L signaling. Importantly, intravital, two‐photon microscopy elegantly provide unequivocal in vivo evidence for direct CD4‐CD8+ T cell interactions via pMHC‐I engagement. This study corroborates the coexistence of direct and indirect mechanisms of T cell help for a CTL response in noninflammatory situations. These data suggest a new “dynamic model of three‐cell interactions” for CTL immunity derived from stimulation by dissociated, licensed DCs, primed CD4+ T cells, and DC‐CD4+ T cell clusters and may have significant implications for autoimmunity and vaccine design.

Optimal TLR9 signal converts tolerogenic CD4–8– DCs into immunogenic ones capable of stimulating antitumor immunity via activating CD4+ Th1/Th17 and NK cell responses

Abstract

A Distinct Role of CD4 + Th17- and Th17-Stimulated CD8 + CTL in the Pathogenesis of Type 1 Diabetes and Experimental Autoimmune Encephalomyelitis

Both CD4+ Th17-cells and CD8+ cytotoxic T lymphocytes (CTLs) are involved in type 1 diabetes and experimental autoimmune encephalomyelitis (EAE). However, their relationship in pathogenesis of these autoimmune diseases is still elusive. We generated ovalbumin (OVA)- or myelin oligodendrocyte glycoprotein (MOG)-specific Th17 cells expressing RORγt and IL-17 by in vitro co-culturing OVA-pulsed and MOG35-55 peptide-pulsed dendritic cells (DCOVA and DCMOG) with CD4+ T cells derived from transgenic OTII and MOG-T cell receptor mice, respectively. We found that these Th17 cells when transferred into C57BL/6 mice stimulated OVA- and MOG-specific CTL responses, respectively. To assess the above question, we adoptively transferred OVA-specific Th17 cells into transgenic rat insulin promoter (RIP)-mOVA mice or RIP-mOVA mice treated with anti-CD8 antibody to deplete Th17-stimulated CD8+ T cells. We demonstrated that OVA-specific Th17-stimulated CTLs, but not Th17 cells themselves, induced diabetes in RIP-mOVA. We also transferred MOG-specific Th17 cells into C57BL/6 mice and H-2Kb−/− mice lacking of the ability to generate Th17-stimulated CTLs. We further found that MOG-specific Th17 cells, but not Th17-activated CTLs induced EAE in C57BL/6 mice. Taken together, our data indicate a distinct role of Th17 cells and Th17-stimulated CTLs in the pathogenesis of TID and EAE, which may have great impact on the overall understanding of Th17 cells in the pathogenesis of autoimmune diseases.

Antigen Specificity Acquisition of Adoptive CD4+ Regulatory T Cells via Acquired Peptide-MHC Class I Complexes

The Ag-specific CD4+ regulatory T (Tr) cells play an important role in immune suppression in autoimmune diseases and antitumor immunity. However, the molecular mechanism for Ag-specificity acquisition of adoptive CD4+ Tr cells is unclear. In this study, we generated IL-10- and IFN-γ-expressing type 1 CD4+ Tr (Tr1) cells by stimulation of transgenic OT II mouse-derived naive CD4+ T cells with IL-10-expressing adenovirus (AdVIL-10)-transfected and OVA-pulsed dendritic cells (DCOVA/IL-10). We demonstrated that both in vitro and in vivo DCOVA/IL-10-stimulated CD4+ Tr1 cells acquired OVA peptide MHC class (pMHC) I which targets CD4+ Tr1 cells suppressive effect via an IL-10-mediated mechanism onto CD8+ T cells, leading to an enhanced suppression of DCOVA-induced CD8+ T cell responses and antitumor immunity against OVA-expressing murine B16 melanoma cells by ≈700% relative to analogous CD4+ Tr1 cells without acquired pMHC I. Interestingly, the nonspecific CD4+25+ Tr cells can also become OVA Ag specific and more immunosuppressive in inhibition of OVA-specific CD8+ T cell responses and antitumor immunity after uptake of DCOVA-released exosomal pMHC I complexes. Taken together, the Ag-specificity acquisition of CD4+ Tr cells via acquiring DC’s pMHC I may be an important mean in augmenting CD4+ Tr cell suppression.

CD4+ Th2 cells function alike effector Tr1 and Th1 cells through the deletion of a single cytokine IL-6 and IL-10 gene.

Depending on polarizing cytokine signals during activation by antigen, naïve CD4+ T cells can be stimulated and differentiated into distinct functional CD4+ T cell subsets such as Th1, Th2 and Tr1 cells. Among them, Th2 cells are pathogenic in allergic diseases such as asthma, which are characterized by transcription factor GATA3 expression and IL-4, IL-5, IL-6, and IL-10 cytokine secretion. The overlapping expression of some signature cytokines by Th2 and other subsets of CD4+ T cells may not only indicate the plasticity of CD4+ T cells, but could also suggest the possibility of the deletion of a single signature cytokine gene leading to the functional differentiation of naïve CD4+ T cells into effector Th1 or Tr1 cells under Th2 differentiation conditions. In this work, we stimulated naïve CD4+ T cells derived from OT II mice or OT II mice that were deficient in individual cytokines (IL-4, IL-5, IL-6 and IL-10) with OVA-pulsed dendritic cells (DC(OVA)) in the presence of IL-4 and anti-IFN-γ, to generate OVA-specific wild-type (WT) Th2, and Th2(IL-4 KO), or Th2(IL-5 KO), or Th2(IL-6 KO), or Th2(IL-10 KO) cells, and to assess their capacity in modulating DC(OVA)-induced CD8+ cytotoxic T lymphocyte (CTL) responses, and antitumor immunity in WT C57BL/6 mice. We conclusively demonstrate that GATA-3-expressing Th2 cells enhance DC(OVA)-induced CTL responses via IL-6 secretion. We also show that IL-6 and IL-10 gene deficient Th2(IL-6 KO) and Th2(IL-10 KO) cells, but not IL-4 and IL-5 gene deficient Th2(IL-4 KO) and Th2(IL-5 KO) cells, behave like functional Tr1 and Th1 cells by inhibiting and enhancing DC(OVA)-induced OVA-specific CD8+ CTL responses and antitumor immunity, respectively. We further elucidate that inhibition and enhancement of DC(OVA)-induced OVA-specific CTL responses by Th2(IL-6 KO) and Th2(IL-10 KO) cells are mediated by their immune suppressive IL-10 and pro-inflammatory IL-6 secretion, respectively. Taken together, our study suggests that deletion of a single cytokine gene IL-6 and IL-10 makes CD4+ Th2 cells become effector CD4+ Tr1- and Th1-like cells, respectively. Our data thus not only provide new evidence for another type of CD4+ T cell plasticity, but also have a potential to impact the development of a new direction in immunotherapy of allergic diseases.

T cell precursor frequency differentially affects CTL responses under different immune conditions.

Generation of effective CTL responses is the goal of many vaccination protocols. However, to what extant T cell precursor frequencies will generate a CD8(+) CTL response has not been elucidated properly. In this study, we employed a model system, in which naive CD4(+) and CD8(+) T cells derived from ovalbumin (OVA)-specific TCR transgenic OT II and OT I mice were used for adoptive transfer into wild-type, Ia(b-/-) gene knockout and transgenic RIP-mOVA mice, and assessed OVA-pulsed DC (DC(OVA))-stimulated CD8(+) CTL responses in these mice. We demonstrated that (i) a critical threshold exists above which T cells precursor frequency cannot enhance the CTL responses in wild-type C57BL/6 mice, (ii) increasing CD8(+) T cell precursors is required to generate CTL responses but with functional memory defect in absence of CD4(+) T cell help, and (iii) increasing CD4(+) and CD8(+) T cell precursors overcomes immune suppression to DC(OVA)-stimulated CD8(+) CTL responses in transgenic RIP-mOVA mice with OVA-specific self immune tolerance. Taken together, these findings may have important implications for optimizing immunotherapy against cancer.

Transgene expression of |[alpha]| tumor necrosis factor with mutations D142N and A144R under control of human telomerase reverse transcriptase promoter eradicates well-established tumors and induces long-term antitumor immunity

Recombinant adenoviral vectors (AdVTNF-α) expressing α tumor necrosis factor (TNF-α) under control of cytomegalovirus (CMV) promoter have been used in cancer gene therapy. To reduce its cytotoxicity, we constructed a recombinant AdVTERTmTNF-α expressing a mutant TNF-α (mTNF-α) with mutations at D142N and A144R under control of human telomerase reverse transcriptase (hTERT) promoter for treatment of well-established ovalbumin (OVA)-expressing murine B16 melanoma (BL6-10OVA) (6 mm in diameter). We demonstrated that the mTNF-α with mutations at D142N and A144R has less in vitro cytotoxicity, but maintains its functional effect in the stimulation of T-cell proliferation. The in vitro and in vivo transgene expressions under control of hTERT promoter are highly restricted in tumor cells compared with those under the control of the CMV promoter. AdVTERTmTNF-α gene therapy by intratumoral injection of AdVTERTmTNF-α vector (2 × 109 PFU) expressing the mutant mTNF-α under control of hTERT promoter reduces its in vivo toxicity, eradicates well-established BL6-10OVA tumors in 4/10 tumor-bearing mice, and induces OVA-specific CD8+ T-cell-mediated long-term antitumor immunity. Therefore, AdVTERTmTNF-α gene therapy may be very useful in the immunotherapy of cancer.