Rescue Of CD8 Research Articles

CD40 ligand stimulation affects the number and memory phenotypes of human peripheral CD8+ T cells

BackgroundCD40L is primarily expressed on activated CD4+ T cells and binds to CD40 which is expressed by various cells including dendritic cells, macrophages and B lymphocytes. While CD40-CD40L interaction is known to be direct between B cells and CD4+ T cells which results in proliferation and immunoglobulin isotype switching, antigen presenting cells (APCs) were thought to be involved in the delivery of CD4+ help to CD8+ T cells by cross-talk between CD4+ and CD8+ T cells and APCs. However, subsequent study demonstrated that CD40L signal can be directly delivered to CD8+ T cells by CD40 expression on CD8+ T cells. Since most studies have been carried out in murine models, we aimed to investigate the direct effect of CD40L on human peripheral CD8+ T cells.ResultsHuman peripheral CD8+ T cells were isolated to exclude the indirect effect of B cells or dendritic cells. Upon activation, CD40 expression on CD8+ T cells was transiently induced and stimulation with artificial APCs expressing CD40L (aAPC-CD40L) increased the number of total and central memory CD8+ T cells and also pp65 specific CD8+ T cells. Stimulation with aAPC-CD40L also resulted in higher proportion of central memory CD8+ T cells.ConclusionsOur study suggests that CD40L has an effect on the increased number of CD8+ T cells through CD40 expressed on activated CD8+ T cells and has influence on memory CD8+ T cell generation. Our results may provide a new perspective of the effect of CD40L on human peripheral CD8+ T cells, which differ according to the memory differentiation status of CD8+ T cells.

CD8+ CD226high T cells in liver metastases dictate the prognosis of colorectal cancer patients treated with chemotherapy and radical surgery

CD226 has been reported to participate in the rescue of CD8+ T cell dysfunction. In this study, we aimed to assess the prognostic value of CD226 in tumor-infiltrating lymphocytes (TILs) derived from colorectal cancer (CRC) liver metastases treated with chemotherapy and radical surgery. TILs from 43 metastases were isolated and analyzed ex vivo using flow cytometry. CD155 and CD3 levels in the tumor microenvironment were assessed by immunohistochemistry. Exploration and validation of biological processes highlighted in this study were performed by bioinformatics analysis of bulk RNA-seq results for 28 CRC liver metastases pretreated with chemotherapy as well as public gene expression datasets. CD226 expression contributes to the definition of the immune context in CRC liver metastases and primary tumors. CD226 on CD8+ T cells was not specifically coexpressed with other immune checkpoints, such as PD1, TIGIT, and TIM3, in liver metastases. Multivariate Cox regression analysis revealed CD226 expression on CD8+ T cells to be an independent prognostic factor (p = 0.003), along with CD3 density at invasion margins (p = 0.003) and TIGIT expression on CD4+ T cells (p = 0.019). CD155 was not associated with the prognostic value of CD226. Gene expression analysis in a validation dataset confirmed the prognostic value of CD226 in CRC liver metastases but not in primary tumors. Downregulation of CD226 on CD8+ TILs in the liver microenvironment was restored by IL15 treatment. Overall, CD226 expression on liver metastasis-infiltrating CD8+ T cells selectively contributes to immune surveillance of CRC liver metastases and has prognostic value for patients undergoing radical surgery.

Effector function prior to establishment of the phagosomal pathogen niche is required for protective CD4+ T cell-mediated immunity against Leishmania

Abstract Leishmania is an appealing model organism to study CD4+ T cell-mediated protective immunity against phagosomal pathogens which features localized primary and secondary infection sites with defined innate and adaptive responses. Upon secondary challenge of chronic L. major-infected C57Bl/6 mice, rapid CD4+T effector (TEFF) function via IFN-g-mediated activation of infected monocytes is associated with optimal immunity. However, the requirement for immediate effector function has yet to be demonstrated. Thus, we isolated time as a variable in the delivery of CD4+ TEFF effector function. We adoptively transferred (AT) chronic mouse-derived Ly6C+CD4+TEFFs into naïve recipients immediately (D0) or 4 days (D4) post-L. major challenge. In this time window Leishmania establishes an intracellular niche but does not proliferate. At day 21 post-challenge, D4 AT resulted in a total loss of the parasite control mediated by D0 transfer. To address whether parasite niche establishment modulated Th1 TEFF cell recruitment, we employed intravital imaging. Ag-sp T cells were present in the infected dermis at significantly lower numbers following D4 vs D0 transfer at 4 days post-T cell transfer, indicating a recruitment deficit. Rapid CD4+ Th1 effector function was required for circulating Th1 TEFF cells to capitalize on an early recruitment window associated with cxcl9 and cxcl10 expression and to prevent parasite niche establishment through altered cell recruitment, gene expression, and functional capacity of both innate and adaptive immune cells. Thus, acute TEFF cell availability is required to prevent host immunomodulation by Leishmania and is a key consideration for vaccination against phagosomal pathogens reliant on Th1 immunity.

Abstract 1104: Targeting the stress response kinase GCN2 to restore immunity in the tumor microenvironment

Abstract The tumor microenvironment (TME) is characterized by deficiencies in oxygen and key nutrients, such as glucose and amino acids, resulting in an overall immune-suppressive environment. Key suppressive cell types in the TME include tumor, stromal and myeloid-derived suppressor cells (MDSC) which create a nutrient-poor environment that supports tumor growth and limits immune surveillance. General control nonderepressible 2 (GCN2), a stress response kinase, plays a key role in sensing and modulating the cellular response to amino acid deprivation. GCN2 activation in T cells triggers the integrated stress response pathway and promotes T cell anergy and apoptosis. We have developed small molecule GCN2 inhibitors (GCN2i) that are highly potent and selective in vitro. Culturing primary mouse or human immune cells under low nutrient conditions activates the GCN2 pathway limiting T cell proliferation and function. Treatment of these nutrient-deprived T cells with GCN2i resulted in rescue of CD8+ T cell proliferation and effector functions. In addition, GCN2 inhibition in MDSC alone fully reversed CD33+MDSC-induced T cell suppression and effector functions. Our GCN2 inhibitors are orally bioavailable with drug like in vivo ADME properties. Our GCN2i is currently being evaluated in vivo, in murine syngeneic tumor models. Our results demonstrate that inhibition of GCN2 is an attractive approach for relieving T cell suppression and promoting effector function, demonstrating GCN2 as a promising therapeutic target for the treatment of cancer. Note: This abstract was not presented at the meeting. Citation Format: Buvana Ravishankar, Lavanya Adusumilli, Deepa Pookot Pookot, Emily Huang, Raashi Sreenivasan, Lisa Marshall, Deepika Kaveri, Oezcan Talay, Silpa Suthram, Svetlana Miakicheva, Abood Okal, Mikhail Zibinsky, Jeffrey Jackson, Grant Shibuya, Paul Leger, Parcharee Tivitmahaisoon, Scott Jacobson, Steve Wong, Angela Wadsworth, Jerick Sanchez, Martin Brovarney, David Chian, Sachie Marubayashi, Aparna Jorapur, Delia Bradford, Christophe Colas, Gene Cutler, Jacob Schwartz, David Wustrow, Paul Kassner, Dirk Brockstedt. Targeting the stress response kinase GCN2 to restore immunity in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1104.

Abstract 4701: Verteporfin inhibits surface PD-L1 expression in triple-negative breast cancer (TNBC) cells

Abstract Background: Programmed death ligand 1 (PDL1) is commonly expressed on the surface of many tumor cells, including breast cancer. The activity of tumor-infiltrating lymphocytes (TIL) is inhibited by PDL1. High PD-L1 basal cells (particularly basal B) overexpress genes involved in invasion, motility, and chemoresistance (Soliman et al., 2014). Targeting and blocking PD-L1 may enhance eradication of aggressive breast cancer cells by the immune system. Verteporfin (VP), a photosensitizer used to treat macular degeneration, was found to inhibit PD-L1 expression in a high-throughput screen. Studies demonstrated VP inhibits YAP activation by disrupting YAP-TEAD interactions and preventing YAP induced oncogenic growth (Zhang et al., 2016). Here we demonstrate the inhibitory effect of VP on PD-L1 expression in TNBC cell lines. Methods: MDA-MB-231 cells co-cultured with human PBMCs were treated with VP (Sigma) in presence of concanavalin A (ConA) and analyzed using flow cytometry to study levels of CD8+IFNg+ cells. TNBC cell lines (MDA-MB-231, BT-20, HCC-1143, Hs-578T) were treated with VP at doses ranging from 1µM-10µM for 24 hrs. The cells were processed for flow cytometry, Western blot and RT-PCR to check PD-L1 in mechanistic studies looking at manipulation of YAP pathway genes. Results: When MDA-MB-231 cells were co-cultured with normal human PBMCs in the presence of ConA, the CD8+IFNg+ stained cells were reduced compared to PBMC + ConA alone. Interestingly, in the group treated with VP, rescue of CD8+IFNg+ cells was observed. Moreover, MDA-MB-231, BT-20, HCC-1143 and Hs-578T TNBC cells treated with VP showed a significant dose-dependent inhibition of PD-L1 expression by flow cytometry. Western blot analysis also showed complete clearance of PD-L1 protein band with the lowest dose (1µM) used. However, RT-PCR analysis did not show a significant fold change in mRNA levels of PD-L1 in MDA-MB-231 treated cells. Surprisingly, mechanistic studies performed by silencing YAP1, E2F1, and TBK1 in MDA-MB-231, BT-20 and HS-578 T showed a decline in PD-L1 in E2F1 silenced cells, highlighting a plausible role of E2F1-PDL1 signaling axis. However, no change in PD-L1 expression was seen in cells silenced with YAP1 and TBK1. Further, chromatin-immunoprecipitation assay demonstrated E2F1 binding to PD-L1 promoter. Conclusion: Our data so far demonstrate that verteporfin treatment leads to inhibition of PD-L1 in TNBC cell lines and improvement in CD8+IFNg+ cells, indicating that VP might have potential for treatment approaches. Our study warrants further attention towards understanding the mechanism of action of VP in inhibiting PD-L1 and the role of E2F1 in the process. Citation Format: Fatema Khambati, Neha Jaiswal, Srikumar Chellappan, Hatem Soliman. Verteporfin inhibits surface PD-L1 expression in triple-negative breast cancer (TNBC) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4701.

CD4+ T Cell Help Selectively Enhances High-Avidity Tumor Antigen-Specific CD8+ T Cells.

Maintaining antitumor immunity remains a persistent impediment to cancer immunotherapy. We and others have previously reported that high-avidity CD8(+) T cells are more susceptible to tolerance induction in the tumor microenvironment. In the present study, we used a novel model where T cells derived from two independent TCR transgenic mouse lines recognize the same melanoma antigenic epitope but differ in their avidity. We tested whether providing CD4(+) T cell help would improve T cell responsiveness as a function of effector T cell avidity. Interestingly, delivery of CD4(+) T cell help during in vitro priming of CD8(+) T cells improved cytokine secretion and lytic capacity of high-avidity T cells, but not low-avidity T cells. Consistent with this observation, copriming with CD4(+) T cells improved antitumor immunity mediated by higher avidity, melanoma-specific CD8(+) T cells, but not T cells with similar specificity but lower avidity. Enhanced tumor immunity was associated with improved CD8(+) T cell expansion and reduced tolerization, and it was dependent on presentation of both CD4(+) and CD8(+) T cell epitopes by the same dendritic cell population. Our findings demonstrate that CD4(+) T cell help preferentially augments high-avidity CD8(+) T cells and provide important insight for understanding the requirements to elicit and maintain durable tumor immunity.

Spatiotemporally Distinct Interactions with Dendritic Cell Subsets Facilitates CD4+ and CD8+ T Cell Activation to Localized Viral Infection

The dynamics of when and where CD4(+) Tcells provide help for CD8(+) Tcell priming and which dendritic cells (DCs) activate CD4(+) Tcells invivo after localized infection are poorly understood. By using a cutaneous herpes simplex virus infection model combined with intravital 2-photon imaging of the draining lymph node (LN) to concurrently visualize pathogen-specific CD4(+) and CD8(+) Tcells, we found that early priming of CD4(+) Tcells involved clustering with migratory skin DCs. CD8(+) Tcells did not interact with migratory DCs and their activation was delayed, requiring later clustering interactions with LN-resident XCR1(+) DCs. CD4(+) Tcells interacted with these late CD8(+) Tcell clusters on resident XCR1(+) DCs. Together, these data reveal asynchronous Tcell activation by distinct DC subsets and highlight the key role of XCR1(+) DCs as the central platform for cytotoxic T lymphocyte activation and the delivery of CD4(+) Tcell help.

The role of CD4 T cell help in restoring function of exhausted CD8 T cells during chronic infection

Exhaustion of CD8 T cell function is a hallmark of chronic viral infections. Although much advance has been made in understanding the mechanisms mediating this dysfunction, little is known about the functional role of CD4 T cell help in this process. In the current study we investigated the effect newly primed antigen specific CD4 T cells may have on CD8 T cell exhaustion and disease outcome in the murine model of chronic viral infection LCMV Clone‐13. We show that adoptive transfer of monoclonal CD4 T cells during the late stages of an ongoing chronic infection can potentially restore function of virus specific CD8 T cells and lead to reduced viral levels. Furthermore, we demonstrate that blocking the PD‐1 (Programmed Death Recptor‐1) inhibitory pathway following the CD4 T cell transfer results in a synergistic effect in terms of both functional rescue of CD8 T cells and viral control. Our data provides new evidence that delivery of CD4 T cell help during ongoing chronic infection may lead to restoration of CD8 T cell function and allow favorable disease outcome.Funding for this work is through NIH grants to R. Ahmed.

Role of Naturally Occurring CD4 + CD25 + Regulatory T Cells in Experimental Atherosclerosis

Naturally occurring CD4+ CD25+ regulatory T cells (Tregs) exert suppressive effects on effector CD4 cells and downregulate experimental autoimmune disorders. We investigated the importance and potential role of Tregs in murine atherogenesis. Tregs were investigated comparatively between aged and young apolipoprotein E-knockout (ApoE-KO) mice and age-matched C57BL/6 littermates. The effect of oxidized LDL (oxLDL) was tested on the functional suppressive properties of Tregs from ApoE-KO and C57BL/6 mice. Tregs, CD4+ CD25- cells, and saline were infused into ApoE-KO mice to study their effects on atherogenesis. Treg numbers were reduced in atherosclerotic compared with nonatherosclerotic ApoE-KO mice. The functional suppressive properties of Tregs from ApoE-KO mice were compromised in comparison with those from their C57BL/6 littermates. Thus, oxLDL attenuated the suppressive properties of Tregs from C57BL/6 mice and more so in ApoE-KO mice. Transfer of Tregs from age-matched ApoE-KO mice resulted in significant attenuation of atherosclerosis compared with that after delivery of CD4+ CD25+/- T cells or phosphate-buffered saline. CD4+ CD25+ Tregs may play a protective role in the progression of atherosclerosis and could be considered a therapeutic tool if results from human studies can solidify observations in murine models.

Rescue of CD8 T cell–mediated antimicrobial immunity with a nonspecific inflammatory stimulus

Reconstitution of protective immunity by adoptive transfer of pathogen-specific T cells has been successful in patients with compromised cellular immunity. The in vivo effectiveness of in vitro–expanded CD8 CTLs is variable, however. For example, adoptively transferred Listeria monocytogenes–specific CD8 CTLs only confer protective immunity if challenge infection occurs within 48 hours of T cell infusion. Herein we show that transferred CTLs persist in lymphoid compartments for many weeks, but that their response to bacterial challenge decreases during the first week following transfer. While T cells transferred less than 48 hours before infection proliferate, those transferred 7 days before infection die. Remarkably, treatment of mice with anti-CD40 at the time of T cell infusion reprograms transferred T cells, allowing them to proliferate and confer protective immunity upon bacterial challenge 7 days later. Our study demonstrates, for the first time to our knowledge that CD40-mediated stimuli can influence CD8 T cell activation independent of concurrent antigen exposure. The ability to modulate long-term responsiveness of CD8 T cells with a transient, nonspecific inflammatory stimulus has importation implications for adoptive immunotherapy.

Rescue of CD8 T cell-mediated antimicrobial immunity with a nonspecific inflammatory stimulus.

Reconstitution of protective immunity by adoptive transfer of pathogen-specific T cells has been successful in patients with compromised cellular immunity. The in vivo effectiveness of in vitro-expanded CD8 CTLs is variable, however. For example, adoptively transferred Listeria monocytogenes-specific CD8 CTLs only confer protective immunity if challenge infection occurs within 48 hours of T cell infusion. Herein we show that transferred CTLs persist in lymphoid compartments for many weeks, but that their response to bacterial challenge decreases during the first week following transfer. While T cells transferred less than 48 hours before infection proliferate, those transferred 7 days before infection die. Remarkably, treatment of mice with anti-CD40 at the time of T cell infusion reprograms transferred T cells, allowing them to proliferate and confer protective immunity upon bacterial challenge 7 days later. Our study demonstrates, for the first time to our knowledge that CD40-mediated stimuli can influence CD8 T cell activation independent of concurrent antigen exposure. The ability to modulate long-term responsiveness of CD8 T cells with a transient, nonspecific inflammatory stimulus has importation implications for adoptive immunotherapy.

Rescue of CD8 T cell–mediated antimicrobial immunity with a nonspecific inflammatory stimulus

Rescue of CD8 T cell–mediated antimicrobial immunity with a nonspecific inflammatory stimulus

CD40 stimulation accelerates deletion of tumor-specific CD8(+) T cells in the absence of tumor-antigen vaccination.

Previous work has established a role for CD40-mediated signals in eliciting helper-dependent CD8(+) T cell responses. Here we investigated the effects of in vivo CD40 stimulation on the survival and function of tumor-specific CD8(+) T cells in a mouse melanoma model system. We found that agonistic anti-CD40 antibody treatment alone of tumor-bearing mice accelerated the deletion of tumor-antigen-specific T cells. However, long-term survival and function of tumor-antigen-specific T cells could be achieved when viral immunization with tumor antigen and anti-CD40 treatment were combined. This rescue of CD8(+) T cells could not be easily replicated by inflammatory or antigen-specific stimuli alone, demonstrating the specificity of signals that regulate the deletion or survival of tumor-specific T cells. These results demonstrate that opposing effects can be elicited by CD40 stimulation in vivo and suggest the need for caution in using this treatment for cancer patients.