Khawaja Ashfaque Ahmed

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.

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.

Differential expression of mannose‐6‐phosphate receptor regulates T cell contraction

CD8+ T cells provide protection against pathogens and cancer. After encountering a pathogenic antigen, CD8+ T cells undergo a triphasic program of rapid proliferation, contraction, and memory formation. Most (∼90–95%) CD8+ T cells die after vigorous proliferation in the T cell contraction phase, yet the mechanism that triggers apoptotic T cell death remains elusive. This study tested the hypothesis that differential cell‐surface expression of M6PR, a multifunctional receptor that regulates lysozyme biogenesis, but also uptakes apoptosis‐inducing serine‐protease Gzm‐B, critically determines life vs. death decisions in T cells. We demonstrate that M6PR‐expression on CD8+ T cell surfaces is dynamically regulated during LmOVA bacterial infection. Notably, time‐lapse, confocal microscopy and flow cytometry confirms that M6PRlow effectors, but not M6PRhigh effectors, escape Gzm‐B lethal‐hit derived from CD4+25+ Treg cells. Adoptive cotransfer of M6PRlow effectors and M6PRhigh effectors sorted from LmOVA‐infected, congenic mice at the peak of CD8+ T cell response, reveals that M6PRlow effectors with the CD8+ T cell memory precursor phenotype preferentially survive the CD8+ T cell contraction and differentiate into functional, long‐lasting memory CD8+ T cells. Taken together, our data provide the first evidence, to our knowledge, that selective M6PR down‐regulation has a critical role in CD8+ T cell survival, and our findings have implications for efficient vaccine design and immunotherapy.

Transgene IL-6 enhances DC-stimulated CTL responses by counteracting CD4+25+Foxp3+ regulatory T cell suppression via IL-6-induced Foxp3 downregulation.

Dendritic cells (DCs), the most potent antigen-presenting cells have been extensively applied in clinical trials for evaluation of antitumor immunity. However, the efficacy of DC-mediated cancer vaccines is still limited as they are unable to sufficiently break the immune tolerance. In this study, we constructed a recombinant adenoviral vector (AdVIL-6) expressing IL-6, and generated IL-6 transgene-engineered DC vaccine (DCOVA/IL-6) by transfection of murine bone marrow-derived ovalbumin (OVA)-pulsed DCs (DCOVA) with AdVIL-6. We then assessed DCOVA/IL-6-stimulated cytotoxic T-lymphocyte (CTL) responses and antitumor immunity in OVA-specific animal tumor model. We demonstrate that DCOVA/IL-6 vaccine up-regulates expression of DC maturation markers, secretes transgene-encoded IL-6, and more efficiently stimulates OVA-specific CTL responses and therapeutic immunity against OVA-expressing B16 melanoma BL6-10OVA in vivo than the control DCOVA/Null vaccine. Moreover, DCOVA/IL-6-stimulated CTL responses were relatively maintained in mice with transfer of CD4+25+Foxp3+ Tr-cells, but significantly reduced when treated with anti-IL-6 antibody. In addition, we demonstrate that IL-6 down-regulates Foxp3-expression of CD4+25+Foxp3+ Tr-cells in vitro. Taken together, our results demonstrate that AdV-mediated IL-6 transgene-engineered DC vaccine stimulates potent CTL responses and antitumor immunity by counteracting CD4+25+ Tr immunosuppression via IL-6-induced Foxp3 down-regulation. Thus, IL-6 may be a good candidate for engineering DCs for cancer immunotherapy.

Acquired pMHC I Complexes Greatly Enhance CD4+ Th Cell's Stimulatory Effect on CD8+ T Cell-Mediated Diabetes in Transgenic RIP-mOVA Mice

CD4+ helper T (Th) cells play pivotal roles in induction of CD8+ CTL immunity. However, the mechanism of CD4+ T cell help delivery to CD8+ T cells in vivo is still elusive. In this study, we used ovalbumin (OVA)-pulsed dendritic cells (DCOVA) to activate OT-II mouse CD4+ T cells, and then studied the help effect of these CD4+ T cells on CD8+ cytotoxic T lymphocyte (CTL) responses. We also examined CTL mediated islet β cell destruction which led to diabetes in wild-type C57BL/6 mice and transgenic rat insulin promoter (RIP)-mOVA mice expressing β cell antigen OVA with self OVA-specific tolerance, respectively. In adoptive transfer experiments, we demonstrated that help, in the form of peptide/major histocompatibility complex (pMHC) I acquired from DCOVA by DCOVA activation, was required for induction of OVA-specific CTL responses in C57BL/6 mice. However, in combination with TCR transgenic OT-I mouse CD8+ T cells, the tolerogenic dosage of CD4+ Th cells with acquired pMHC I, but not CD4+ (Kb−/−) Th cells without acquired pMHC I were able to cause diabetes in 8/10 (80%) RIP-mOVA mice. This study thus expands the current knowledge in T cell-mediated autoimmunity and provides insight into the nature of CD4+ T cell-mediated help in CD8+ CTL induction.

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.

mTORC1 regulates mannose-6-phosphate receptor transport and T-cell vulnerability to regulatory T cells by controlling kinesin KIF13A

Mannose-6-phosphate receptor (M6PR) that facilitates cellular uptake of M6P-bearing proteins, including serine-protease granzyme-B (Gzm-B) has an important role in T-cell activation, migration and contraction. However, molecular mechanisms controlling M6PR expression in T cells remain poorly understood. Here, we show that M6PR expression on T cells is distinctively controlled by two common γ-chain cytokines interleukin-2 (IL-2) and IL-7, and the differential M6PR expression is not caused by an altered synthesis of M6PR protein, but is a result of distinct regulation of kinesin-3 motor-protein KIF13A that transport M6PR onto cell surfaces. Using signaling pathway-specific inhibitors, we determine that IL-2 and IL-7 distinctly regulate KIF13A and β1-adaptin and cell-surface M6PR by controlling a kinase mammalian target of rapamycin complex-1 (mTORC1). Inflammatory cytokine IL-2 and prosurvival cytokine IL-7 induce strong and weak activation of mTORC1, leading to up- and downregulation of motor-protein KIF13A and KIF13A-motorized M6PR on T cells, and formation of IL-2 and IL-7 effectors with M6PRhigh and M6PRlow cell-surface expression, respectively. Inhibition of mTORC1 by rapamycin reduces T-cell expression of KIF13A and cell-surface M6PR, and increases T-cell survival in Listeria monocytogenes-infected mice. Using regulatory T (Treg)-cell-enriched mouse tumor model, we determine that M6PRhigh IL-2 effectors but not M6PRlow IL-7 effectors adoptively transferred into tumors are vulnerable to Treg Gzm-B-mediated cell apoptosis. Inhibition of mTORC1 or small interfering RNA-mediated knockdown of KIF13A or M6PR renders IL-2 effectors refractory to Treg Gzm-B lethal hit. Overall, our data offer novel mechanistic insights into T-cell M6PR regulation, and Treg-resistant/Treg-susceptible phenomenon. Furthermore, regulation of T-cell fate vis-à-vis Treg suppression via the mTORC1-KIF13A-M6PR axis provides a proof of concept for therapeutic strategies to target cancer, infectious and autoimmune diseases.

A new dynamic model of three cell interactions for CTL responses.

The exact mechanisms of CD4 help in the generation of memory cytotoxic T lymphocytes (CTLs) remain largely illusive. We propose that dendritic cells (DCs) first interact with CD4+ T cells, resulting in DC licensing and CD4+ T-cell priming. Thereafter, CD8+ T cells can receive stimulatory signals from DC-CD4+ T-cell clusters and as well as individually from licensed DCs and primed CD4+ T cells.

Multiple effects of CD40–CD40L axis in immunity against infection and cancer

CD8+ cytotoxic T lymphocyte (CTL) protects against infection and cancer cells. Understanding the mechanisms involved in generation and maintenance of effective CTL responses is essential for improving disease therapy and vaccine protocols. During CTL responses, immune cells encounter several tightly regulated signaling pathways; therefore, in such a dynamic process, proper integration of critical signals is necessary to orchestrate an effective immune response. In this review, we have focused on CD40–CD40L interactions (a key signal) in the regulation of dendritic cell (DC)–T cell (CD4+ T and CD8+ T) cross-talk, rescuing CTL exhaustion, and converting DC tolerization. We have also highlighted the knowledge gap and future directions to design immunotherapies.