Channakeshava Sokke Umeshappa

CD4+ Th-APC with Acquired Peptide/MHC Class I and II Complexes Stimulate Type 1 Helper CD4+ and Central Memory CD8+ T Cell Responses

T cell-T cell Ag presentation is increasingly attracting attention. We previously showed that the in vitro OVA-pulsed dendritic cell (DCOVA)-activated CD4+ Th cells acquired OVA peptide/MHC (pMHC) class I and costimulatory molecules such as CD54 and CD80 from DCOVA and acted as CD4+ Th-APC capable of stimulating OVA-specific CD8+ CTL responses. In this study, we further applied the OVA-specific TCR-transgenic OT I and OT II mice with deficiency of various cytokines or costimulatory molecule genes useful for studying the molecular mechanisms underlying in Th-APC’s stimulatory effect. We demonstrated that DCOVA-stimulated OT II CD4+ Th-APC also acquired costimulatory molecules such as CD40, OX40L, and 4-1BBL and the functional pMHC II complexes by DCOVA activation. CD4+ Th-APC with acquired pMHC II and I were capable of stimulating CD4+ Th1 and central memory CD8+44+CD62LhighIL-7R+ T cell responses leading to antitumor immunity against OVA-expressing mouse B16 melanoma. Their stimulatory effect on CD8+ CTL responses and antitumor immunity is mediated by IL-2 secretion, CD40L, and CD80 signaling and is specifically targeted to CD8+ T cells in vivo via acquired pMHC I. In addition, CD4+ Th-APC expressing OVA-specific TCR, FasL, and perforin were able to kill DCOVA and neighboring Th-APC expressing endogenous and acquired pMHC II. Taken together, we show that CD4+ Th-APC can modulate immune responses by stimulating CD4+ Th1 and central memory CD8+ T cell responses and eliminating DCOVA and neighboring Th-APC. Therefore, our findings may have great impacts in not only the antitumor immunity, but also the regulatory T cell-dependent immune tolerance in vivo.

GP120-specific exosome-targeted T cell-based vaccine capable of stimulating DC- and CD4(+) T-independent CTL responses.

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutics. In this study, we generated ovalbumin (OVA)-pulsed and pcDNAgp120-transfected dendritic cell (DC)-released exosomes (EXOova and EXOgp120) and ConA-stimulated C57BL/6 CD8(+) T cells. OVA- and Gp120-Texo vaccines were generated from CD8(+) T cells with uptake of EXOova and EXOgp120, respectively. We demonstrate that OVA-Texo stimulates in vitro and in vivo OVA-specific CD4(+) and CD8(+) cytotoxic T lymphocyte (CTL) responses leading to long-term immunity against OVA-expressing BL6-10(OVA) melanoma. Interestingly, CD8(+) T cell responses are DC and CD4(+) T cell independent. Importantly, Gp120-Texo also stimulates Gp120-specific CTL responses and long-term immunity against Gp120-expressing B16 melanoma. Therefore, this novel HIV-1-specific EXO-targeted Gp120-Texo vaccine may be useful in induction of efficient CTL responses in AIDS patients with DC dysfunction and CD4(+) T cell deficiency.

Novel CD8+ T cell-based vaccine stimulates Gp120-specific CTL responses leading to therapeutic and long-term immunity in transgenic HLA-A2 mice

The limitations of highly active anti-retroviral therapy have necessitated the development of alternative therapeutics for human immunodeficiency virus type-1 (HIV-1)-infected patients with dysfunctional dendritic cells (DCs) and CD4(+) T cell deficiency. We previously demonstrated that HIV-1 Gp120-specific T cell-based Gp120-Texo vaccine by using ConA-stimulated C57BL/6 (B6) mouse CD8(+) T (ConA-T) cells with uptake of pcDNA(Gp120)-transfected B6 mouse DC line DC2.4 (DC2.4(Gp120))-released exosomes (EXO(Gp120)) was capable of stimulating DC and CD4(+) T cell-independent CD8(+) cytotoxic T lymphocyte (CTL) responses detected in wild-type B6 mice using non-specific PE-anti-CD44 and anti-IFN-γ antibody staining by flow cytometry. To assess effectiveness of Gp120-Texo vaccine in transgenic (Tg) HLA-A2 mice mimicking the human situation, we constructed adenoviral vector AdV(Gp120) expressing HIV-1 GP120 by recombinant DNA technology, and generated Gp120-Texo vaccine by using Tg HLA-A2 mouse CD8(+) ConA-T cells with uptake of AdV(Gp120)-transfected HLA-A2 mouse bone marrow DC (DC(Gp120))-released EXO(Gp120). We then performed animal studies to assess Gp120-Texo-induced stimulation of Gp120-specific CTL responses and antitumor immunity in Tg HLA-A2 mice. We demonstrate that Gp120-Texo vaccine stimulates Gp120-specific CTL responses detected in Tg HLA-A2 mice using Gp120-specific PE-HLA-A2/Gp120 peptide (KLTPLCVTL) tetramer staining by flow cytometry. These Gp120-specific CTLs are capable of further differentiating into functional effectors with killing activity to Gp120 peptide-pulsed splenocytes in vivo. In addition, Gp120-Texo vaccine also induces Gp120-specific preventive, therapeutic (for 6 day tumor lung metastasis) and CD4(+) T cell-independent long-term immunity against B16 melanoma BL6-10(Gp120/A2Kb) expressing both Gp120 and A2Kb (α1 and α2 domains of HLA-A2 and α3 domain of H-2K(b)) in Tg HLA-A2 mice. Taken together, the novel CD8(+) Gp120-Texo vaccine capable of stimulating efficient CD4(+) T cell-independent Gp120-specific CD8(+) CTL responses leading to therapeutic and long-term immunity in Tg HLA-A2 mice may represent a new immunotherapeutic vaccine for treatment of HIV-1 patients with CD4(+) T cell deficiency.

CD154 and IL-2 signaling of CD4+ T cells play a critical role in multiple phases of CD8+ CTL responses following adenovirus vaccination.

Adenoviral (AdV) vectors represent most commonly utilized viral vaccines in clinical studies. While the role of CD8+ cytotoxic T lymphocyte (CTL) responses in mediating AdV-induced protection is well understood, the involvement of CD4+ T cell-provided signals in the development of functional CD8+ CTL responses remain unclear. To explore CD4+ T helper signals required for AdVova-stimulated CTL responses, we established an adoptive transfer system by transferring CD4+ T cells derived from various knock out and transgenic mice into wild-type and/or CD4-deficient animals, followed by immunizing with recombinant ovalbumin (OVA)-expressing AdVova vector. Without CD4+ T help, both primary and memory CTL responses were greatly reduced in this model, and were associated with increased PD-1 expression. The provision of OVA-specific CD4+ T help in CD4+ T cell-deficient mice restored AdVova-induced primary CTL responses, and supported survival and recall responses of AdVova-stimulated memory CTLs. These effects were specifically mediated by CD4+ T cell-produced IL-2 and CD154 signals. Adoptive transfer of “helped” or “unhelped” effector and memory CTLs into naïve CD4+ T cell-deficient or -sufficient mice also revealed an additional role for polyclonal CD4+ T cell environment in the survival of AdVova-stimulated CTLs, partially explaining the extension of CTL contraction phase. Finally, during recall responses, CD4+ T cell environment, particularly involving memory CD4+ T cells, greatly enhanced expansion of memory CTLs. Collectively, our data strongly suggest a critical role for CD4+ T help in multiple phases of AdV-stimulated CTL responses, and could partially explain certain failures in AdV-based immunization trials targeting malignant tumors and chronic diseases that are often associated with compromised CD4+ T cell population and function.

Simulated Microgravity Promotes Cell Apoptosis Through Suppressing Uev1A/TICAM/TRAF/NF-κB-Regulated Anti-Apoptosis and p53/PCNA- and ATM/ATR-Chk1/2-Controlled DNA-Damage Response Pathways.

Microgravity has been known to induce cell death. However, its underlying mechanism is less studied. In this study, BL6‐10 melanoma cells were cultured in flasks under simulated microgravity (SMG). We examined cell apoptosis, and assessed expression of genes associated with apoptosis and genes regulating apoptosis in cells under SMG. We demonstrate that SMG induces cell morphological changes and microtubule alterations by confocal microscopy, and enhances apoptosis by flow cytometry, which was associated with up‐ and down‐regulation of pro‐apoptotic and anti‐apoptotic genes, respectively. Moreover, up‐ and down‐regulation of pro‐apoptotic (Caspases 3, 7, 8) and anti‐apoptotic (Bcl2 and Bnip3) molecules was confirmed by Western blotting analysis. Western blot analysis also indicates that SMG causes inhibition of an apoptosis suppressor, pNF‐κB‐p65, which is complemented by the predominant localization of NF‐κB‐p65 in the cytoplasm. SMG also reduces expression of molecules regulating the NF‐κB pathway including Uev1A, TICAM, TRAF2, and TRAF6. Interestingly, 10 DNA repair genes are down‐regulated in cells exposed to SMG, among which down‐regulation of Parp, Ercc8, Rad23, Rad51, and Ku70 was confirmed by Western blotting analysis. In addition, we demonstrate a significant inhibition of molecules involved in the DNA‐damage response, such as p53, PCNA, ATM/ATR, and Chk1/2. Taken together, our work reveals that SMG promotes the apoptotic response through a combined modulation of the Uev1A/TICAM/TRAF/NF‐κB‐regulated apoptosis and the p53/PCNA‐ and ATM/ATR‐Chk1/2‐controlled DNA‐damage response pathways. Thus, our investigation provides novel information, which may help us to determine the cause of negative alterations in human physiology occurring at spaceflight environment. J. Cell. Biochem. 117: 2138–2148, 2016.

Regulators of T-cell memory generation: TCR signals versus CD4 + help?

In the event of pathogen entry, antigen (Ag)-specific naive CD8+ T cells undergo activation and rapid clonal expansion that results in the generation of millions of effector CD8+ cytotoxic T lymphocytes (CTLs), and subsequently, a small cohort of memory cells. This dynamic event is largely controlled by signaling provided by the immunological synapse, proinflammatory cytokines and CD4+ T cells.1,2 However, how these signals contribute to the generation of heterogeneous populations of effector and memory cells from a relatively homogeneous and rare naive CD8+ T-cell population is still not clearly understood. Two recent reports in Blood from Smith-Garvin et al.3 and Wiehagen et al.4 now show that altered T-cell receptor (TCR) signals can affect differentiation, heterogeneity, and the functions of effector and memory cells, supporting growing evidence that the strength of TCR signals, at least in part, determines the fate of CD8+ T-cell lineage choices. To verify whether altered TCR signals impact effector and memory CD8+ T-cell differentiation fates, Smith-Garvin et al. use genomic knock-in mice that express tyrosine to phenylalanine mutations in SH2 domaincontaining leukocyte phosphorylation of 76 kDa (SLP-76), and a well-defined infectious model, Armstrong strain of lymphocytic choriomeningitis virus (LCMV). On the other hand, Wiehagen et al.4 used conditional knockout mice where they ablated the SLP-76 gene by administering estrogen analog, tamoxifen. As SLP-76 mediates initial TCRinduced phosphorylation signals to various downstream effector molecules, mutation in its tyrosine residues or its conditional deletion results in defective phosphorylation of critical molecules that propagate TCR signals.3,4 Thus, this elegant approach allowed the study of how TCR signal strengths determine memory differentiation fates. On the basis of temporal expression of CD62L, a central memory (Tcm) marker, Smith-Garvin et al.3 showed an increased rate of effector memory (Tem) to Tcm conversion in LCMVinfected SLP-76-knock-in mice. Similarly, when SLP-76 expression was abolished during the contraction or memory phase, Wiehagen et al.4 also observed an increased rate of Tem to Tcm conversion. In line with these results, Sarkar et al.5 showed that this rate of conversion is largely influenced by priming signals, such as Ag strength (strong versus subdominant epitopes), and/or duration of infection. Fousteri et al.2 showed that, during later stage of viral infection, naive CD8+ T cells receiving weaker TCR signals due to reduced Ag concentration convert into memory cells efficiently. Similarly, the preferential development of Tcm from latecomer CD8+ T cells that received weaker stimuli following infection by intracellular pathogens was also reported.6,7 Notably, inducing short-term transgene expression following adenovirus serotype-5 or plasmid DNA immunization also produced higher levels of Tcm and a more robust secondary response compared with more sustained expression, in which persistence and predominance of effector CTLs and Tem are observed.8,9 Smith-Garvin et al.3 and Wiehagen et al.4 attempt to address the functional characteristics of memory cells by re-challenging the mice with potent immunostimulants. Interestingly, although Tcm exhibited a mature memory phenotype expressing CXCR3, CD27, CD44, CD127, CD122 or CD62L, they observed partial or complete loss of interferon-g-secreting and proliferative responses to re-challenge by LCMV-specific peptide(s) and Listeria monocytogenes3 or LCMV clone-13,4 respectively (Figure 1a). However, stimulation with phorbol myristate acetate/ ionomycin, which circumvents proximal TCR signaling, showed that these Tcm can secrete cytokines similar to wild-type cells, suggesting they are not terminally differentiated following TCR stimulation. As phorbol myristate acetate/ionomycin provides very potent, nonspecific stimulation, its signaling is possibly strong enough to drive cytokine secretion from these Tcm even if they are poorly functional. Thus, although their studies provide compelling evidence of a role for TCR signals in Tcm generation, they are unable to assess the requirements for fully functional Tcm during secondary responses. One possible explanation for the poorly functional Tcm described in these two papers is that the strength of recall stimulus might determine memory response10 (Figure 1a). Alternatively, it is possible that defective Tcm generation occurs because of the reduced or lack of TCR signals during priming or contraction. Supporting this notion, Teixeiro et al.,1 by directly introducing point mutation into the TCR-b-transmembrane domain, showed that reduced TCR signals leading to poor TCR polarization and a severe decrease in nuclear translocation and DNA binding of nuclear factor-kB, resulted in defective memory, but normal primary, responses. Furthermore, a study by Gett et al.11 suggested that stronger TCR signals increase the fitness of T cells by enhancing survival and cytokine responsiveness, whereas weaker signals result in T-cell death because of the poor responsiveness to cytokines, CS Umeshappa is at the Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada or Dr J Xiang and Channakeshava Sokke Umeshappa are at the Cancer Research Unit, Saskatchewan Cancer Agency, 20 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 4H4. E-mail: jim.xiang@saskcancer.ca Immunology and Cell Biology (2011) 89, 578–580 & 2011 Australasian Society for Immunology Inc. All rights reserved 0818-9641/11

Tumor-derived HLA-G1 acquisition by monocytes through trogocytosis: possible functional consequences

We read the article recently published in the April 2010 issue by HoWangYin et al. [1] with an interest in understanding the functional consequences of tumor-derived HLA-G1 on monocytes. The authors demonstrated that HLA-G1 in tumors is rapidly acquired by monocytes through trogocytosis as acquired by antigen-presenting cells (APC), NK cells and T cells. Surprisingly, although trogocytosis is commonly observed in activated immune cells, the authors demonstrated this phenomenon in both resting and activated monocytes. Furthermore, they showed that HLA-G1 acquisition is transient and quickly disappears in LPS-activated monocytes, but found no evidence of the functional roles of these molecules. Intercellular communication through the transfer of cellsurface membrane proteins is a widespread phenomenon in multicellular organisms. It can occur through various mechanisms (reviewed in references [2, 3]), including removal of part of the membrane of the target cell by strong ligand-receptor affinity, enzymatic cleavage and transfer of the cell-surface protein ectodomains, transfer in the form of exosomes and membrane nanotubules, and transfer after formation of membrane bridges, such as T cell receptor-mediated acquisition of APC’s antigenpresenting machineries by T lymphocytes following immunological synapse formation via the internalization and recycling pathway [4]. The latter mechanism, popularly known as ‘trogocytosis’, is most commonly observed in various immune cells and their target cells. In the immune system, the functional consequences of trogocytosis vary with the type of antigens involved, differences in the nature of the immune cells, and the microenvironment in which the immune interaction occurs, which in turn direct the developmental path of the different immune cell subsets with distinct functions [5–7]. In addition to producing various tolerance-inducing factors such as IL-10 and TGF-b, and downregulating MHC-class I molecules, it is increasingly evident that tumors transfer cell-surface, membrane-immunoregulatory proteins, such as HLA-G1, to immune cells through secretion of exosomes, and/or trogocytosis, facilitating the escape of tumors from immunosurveillance mechanisms. HLA-G1 is a nonclassical HLA class I molecule, expressed pathologically in various cancers, and infectious and autoimmune diseases [7]. Its expression endows tumor cells with a multitude of functions (reviewed in references [2, 3, 8, 9]), including inhibition of proliferative, cytokine secreting and cytolytic properties of innate (NK cells) and adaptive immune cells [CD8 cytotoxic T cells and CD4 Th cells], induction of regulatory cells and drug resistance, and inhibition of dendritic cell (DC) maturation. As APCs form bridge between innate and adaptive immune responses, tolerization of APCs could profoundly affect both innate and adaptive antitumor responses. It has been shown that tumorinfiltrating, splenic, bone marrow-derived and circulating APCs from cancer patients express HLA-G1 and exhibit Comments on: HoWangYin KY, Alegre E, Daouya M, Favier B, Carosella ED, LeMaoult J (2010) Different functional outcomes of intercellular membrane transfers to monocytes and T cells. Cell Mol Life Sci 67(7):1133–45.

Differential requirements of CD4+ T‐cell signals for effector cytotoxic T‐lymphocyte (CTL) priming and functional memory CTL development at higher CD8+ T‐cell precursor frequency

Increased CD8+ T‐cell precursor frequency (PF) precludes the requirement of CD4+ helper T (Th) cells for primary CD8+ cytotoxic T‐lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) ‐pulsed dendritic cells (DCOVA) derived from C57BL/6, CD40 knockout (CD40−/−) or CD40 ligand knockout (CD40L−/−) mice were used to immunize C57BL/6, Iab−/−, CD40−/− or CD40L−/− mice, whose PF was previously increased with transfer of 1 × 106 CD8+ T cells derived from OVA‐specific T‐cell receptor (TCR) transgenic OTI, OTI(CD40−/−) or OTI(CD40L−/−) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4+ T‐cell help and Th‐provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA‐expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4+ T‐cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4+ T‐cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4+ T‐cell functions.