IL-7-primed bystander CD8 tumor-infiltrating lymphocytes optimize the antitumor efficacy of T cell engager immunotherapy.

Bispecific T cell engagers (TCEs) show promising clinical efficacy in blood tumors, but their application to solid tumors remains challenging. Here, we show that Fc-fused IL-7 (rhIL-7-hyFc) changes the intratumoral CD8 T cell landscape, enhancing the efficacy of TCE immunotherapy. rhIL-7-hyFc induces a dramatic increase in CD8 tumor-infiltrating lymphocytes (TILs) in various solid tumors, but the majority of these cells are PD-1-negative tumor non-responsive bystander T cells. However, they are non-exhausted and central memory-phenotype CD8 T cells with high T cell receptor (TCR)-recall capacity that can be triggered by tumor antigen-specific TCEs to acquire tumoricidal activity. Single-cell transcriptome analysis reveals that rhIL-7-hyFc-induced bystander CD8 TILs transform into cycling transitional T cells by TCE redirection with decreased memory markers and increased cytotoxic molecules. Notably, TCE treatment has no major effect on tumor-reactive CD8 TILs. Our results suggest that rhIL-7-hyFc treatment promotes the antitumor efficacy of TCE immunotherapy by increasing TCE-sensitive bystander CD8 TILs in solid tumors.

IL-15 induced bystander activation of CD8+ T cells may mediate endothelium injury through NKG2D in Hantaan virus infection.

Introduction: Hantaan virus (HTNV) can cause endothelium injury in hemorrhagic fever with renal syndrome (HFRS) patients. Bystander activation of CD8+ T cells by virus infection has been shown that was involved in host injury, but it is unclear during HTNV infection. This project aimed to study the effect of bystander-activated CD8+ T cell responses in HTNV infection. Methods: The in vitro infection model was established to imitate the injury of endothelium in HFRS patients. Flow cytometry was performed to detect the expression of markers of tetramer+ CD8+ T cells and human umbilical vein endothelial cells (HUVECs). The levels of interleukin-15 (IL-15) in serum and supermanant were detected using ELISA kit. The expression of MICA of HUVECs was respectively determined by flow cytometry and western blot. The cytotoxicity of CD8+ T cells was assessed through the cytotoxicity assay and antibody blocking assay. Results: EBV or CMV-specific CD8+ T cells were bystander activated after HTNV infection in HFRS patients. HTNV-infected HUVECs in vitro could produce high levels of IL-15, which was positively correlated with disease severity and the expression of NKG2D on bystander-activated CD8+ T cells. Moreover, the elevated IL-15 could induce activation of CD122 (IL-15Rß)+NKG2D+ EBV/CMV-specific CD8+ T cells. The expression of IL-15Rα and ligand for NKG2D were upregulated on HTNV-infected HUVECs. Bystander-activated CD8+ T cells could exert cytotoxicity effects against HTNV-infected HUVECs, which could be enhanced by IL-15 stimulation and blocked by NKG2D antibody. Discussion: IL-15 induced bystander activation of CD8+ T cells through NKG2D, which may mediate endothelium injury during HTNV infection in HFRS patients.

Significance of bystander T cell activation in microbial infection.

During microbial infection, pre-existing memory CD8+ T cells that are not specific for the infecting pathogens can be activated by cytokines without cognate antigens, termed bystander activation. Studies in mouse models and human patients demonstrate bystander activation of memory CD8+ T cells, which exerts either protective or detrimental effects on the host, depending on the infection model or disease. Research has elucidated mechanisms underlying the bystander activation of CD8+ T cells in terms of the responsible cytokines and the effector mechanisms of bystander-activated CD8+ T cells. In this Review, we describe the history of research on bystander CD8+ T cell activation as well as evidence of bystander activation. We also discuss the mechanisms and immunopathological roles of bystander activation in various microbial infections.

Bystander CD4 T-cell death is inhibited by broadly neutralizing anti-HIV antibodies only at levels blocking cell-to-cell viral transmission.

The progressive loss of CD4+ T cells during HIV infection of lymphoid tissues involves both the apoptotic death of activated and productively infected CD4 T cells and the pyroptotic death of large numbers of resting and abortively infected bystander CD4 T cells. HIV spreads both through cellular release of virions and cell-to-cell transmission involving the formation of virological synapses. Cell-to-cell transmission results in high-level transfer of large quantities of virions to the target cell exceeding that achieved with cell-free virions. Broadly neutralizing anti-HIV antibodies (bNAbs) binding to HIV envelope protein capably block cell-free virus spread, and when added at higher concentrations can also interdict cell-to-cell transmission. Exploiting these distinct dose-response differences, we now show that four different bNAbs block the pyroptotic death of bystander cells, but only when added at concentrations sufficient to block cell-to-cell transmission. These findings further support the conclusion that HIV killing of abortively infected bystander CD4 T cells requires cell-to-cell transfer of virions. As bNAbs attract more interest as potential therapeutics, it will be important to consider the higher concentrations of these antibodies required to block the inflammatory death of bystander CD4 T cells.

Reprogramming of microRNA expression via E2F1 downregulation promotes Salmonella infection both in infected and bystander cells.

Cells infected with pathogens can contribute to clearing infections by releasing signals that instruct neighbouring cells to mount a pro-inflammatory cytokine response, or by other mechanisms that reduce bystander cells' susceptibility to infection. Here, we show the opposite effect: epithelial cells infected with Salmonella Typhimurium secrete host factors that facilitate the infection of bystander cells. We find that the endoplasmic reticulum stress response is activated in both infected and bystander cells, and this leads to activation of JNK pathway, downregulation of transcription factor E2F1, and consequent reprogramming of microRNA expression in a time-dependent manner. These changes are not elicited by infection with other bacterial pathogens, such as Shigella flexneri or Listeria monocytogenes. Remarkably, the protein HMGB1 present in the secretome of Salmonella-infected cells is responsible for the activation of the IRE1 branch of the endoplasmic reticulum stress response in non-infected, neighbouring cells. Furthermore, E2F1 downregulation and the associated microRNA alterations promote Salmonella replication within infected cells and prime bystander cells for more efficient infection.

Quantitative Evaluation of the Effect of Antigen Expression Level on Antibody-Drug Conjugate Exposure in Solid Tumor.

Antibody-drug conjugates (ADCs) rely on high expression of target antigens on cancer cells to effectively enter the cell and release a cytotoxic payload. Previous studies have shown that ADC efficacy is not always tied to antigen expression. However, our recent in vitro study suggests a linear relationship between antigen expression and the intracellular levels of the ADC payload. In this study, we have explored the relationship between antigen expression and intratumoral ADC exposure in vivo. Using trastuzumab-vc-MMAE (T-vc-MMAE) and four cell lines with varying expression of human epithelial growth factor receptor 2 (HER2), the pharmacokinetics of total trastuzumab, released ("free") MMAE, and total MMAE were evaluated in a tumor xenograft model. Nude mice were implanted with tumors originating from BT-474, MDA-MB-453, MCF-7, and MDA-MB-468 cell lines and dosed with 10 mg/kg or 1 mg/kg of ADC. Observed data were mathematically characterized using a mechanism-based PK model. A strong positive correlation was observed between antigen expression levels and free/total MMAE exposure (R2 ≥ 0.91) (total MMAE being the sum of released and conjugated MMAE) within the tumor, but not for total trastuzumab exposure. The PK model was able to recapitulate plasma PK through simulation; however, the tumor PK was overpredicted or underpredicted in some cases potentially due to differences in tumor vasculature or extracellular matrix conditions. Our results indicate a linear relationship between antigen expression and tumor exposure of free/total ADC payload in vivo, validating our previous finding in vitro, while also revealing the need to understand complex physiology of the tumor to predict tumor PK of ADC and its components. Our findings also support the concept of antigen expression screening in patients for targeted therapies like ADCs to achieve the maximum therapeutic benefit of the treatment.

Viral infection modulates Qa-1b in infected and bystander cells to properly direct NK cell killing.

Natural killer (NK) cell activation depends on the signaling balance of activating and inhibitory receptors. CD94 forms inhibitory receptors with NKG2A and activating receptors with NKG2E or NKG2C. We previously demonstrated that CD94-NKG2 on NK cells and its ligand Qa-1b are important for the resistance of C57BL/6 mice to lethal ectromelia virus (ECTV) infection. We now show that NKG2C or NKG2E deficiency does not increase susceptibility to lethal ECTV infection, but overexpression of Qa-1b in infected cells does. We also demonstrate that Qa-1b is down-regulated in infected and up-regulated in bystander inflammatory monocytes and B cells. Moreover, NK cells activated by ECTV infection kill Qa-1b-deficient cells in vitro and in vivo. Thus, during viral infection, recognition of Qa-1b by activating CD94/NKG2 receptors is not critical. Instead, the levels of Qa-1b expression are down-regulated in infected cells but increased in some bystander immune cells to respectively promote or inhibit their killing by activated NK cells.

The Ugly Duckling Turned to Swan: A Change in Perception of Bystander-Activated Memory CD8 T Cells.

Memory T cells (Tmem) rapidly mount Ag-specific responses during pathogen reencounter. However, Tmem also respond to inflammatory cues in the absence of an activating TCR signal, a phenomenon termed bystander activation. Although bystander activation was first described over 20 years ago, the physiological relevance and the consequences of T cell bystander activation have only become more evident in recent years. In this review, we discuss the scenarios that trigger CD8 Tmem bystander activation including acute and chronic infections that are either systemic or localized, as well as evidence for bystander CD8 Tmem within tumors and following vaccination. We summarize the possible consequences of bystander activation for the T cell itself, the subsequent immune response, and the host. We highlight when T cell bystander activation appears to benefit or harm the host and briefly discuss our current knowledge gaps regarding regulatory signals that can control bystander activation.

A noninflammatory mRNA vaccine for treatment of experimental autoimmune encephalomyelitis.

The ability to control autoreactive T cells without inducing systemic immune suppression is the major goal for treatment of autoimmune diseases. The key challenge is the safe and efficient delivery of pharmaceutically well-defined antigens in a noninflammatory context. Here, we show that systemic delivery of nanoparticle-formulated 1 methylpseudouridine-modified messenger RNA (m1Ψ mRNA) coding for disease-related autoantigens results in antigen presentation on splenic CD11c+ antigen-presenting cells in the absence of costimulatory signals. In several mouse models of multiple sclerosis, the disease is suppressed by treatment with such m1Ψ mRNA. The treatment effect is associated with a reduction of effector T cells and the development of regulatory T cell (Treg cell) populations. Notably, these Treg cells execute strong bystander immunosuppression and thus improve disease induced by cognate and noncognate autoantigens.

A Critical Role for Fas-Mediated Off-Target Tumor Killing in T-cell Immunotherapy.

T cell-based therapies have induced cancer remissions, though most tumors ultimately progress, reflecting inherent or acquired resistance including antigen escape. Better understanding of how T cells eliminate tumors will help decipher resistance mechanisms. We used a CRISPR/Cas9 screen and identified a necessary role for Fas-FasL in antigen-specific T-cell killing. We also found that Fas-FasL mediated off-target "bystander" killing of antigen-negative tumor cells. This localized bystander cytotoxicity enhanced clearance of antigen-heterogeneous tumors in vivo, a finding that has not been shown previously. Fas-mediated on-target and bystander killing was reproduced in chimeric antigen receptor (CAR-T) and bispecific antibody T-cell models and was augmented by inhibiting regulators of Fas signaling. Tumoral FAS expression alone predicted survival of CAR-T-treated patients in a large clinical trial (NCT02348216). These data suggest strategies to prevent immune escape by targeting both the antigen expression of most tumor cells and the geography of antigen-loss variants. SIGNIFICANCE: This study demonstrates the first report of in vivo Fas-dependent bystander killing of antigen-negative tumors by T cells, a phenomenon that may be contributing to the high response rates of antigen-directed immunotherapies despite tumoral heterogeneity. Small molecules that target the Fas pathway may potentiate this mechanism to prevent cancer relapse.This article is highlighted in the In This Issue feature, p. 521.

Tolerogenic nanoparticles suppress central nervous system inflammation.

Therapeutic approaches for the induction of immune tolerance remain an unmet clinical need for the treatment of autoimmune diseases, including multiple sclerosis (MS). Based on its role in the control of the immune response, the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) is a candidate target for novel immunotherapies. Here, we report the development of AhR-activating nanoliposomes (NLPs) to induce antigen-specific tolerance. NLPs loaded with the AhR agonist ITE and a T cell epitope from myelin oligodendrocyte glycoprotein (MOG)35-55 induced tolerogenic dendritic cells and suppressed the development of experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS, in preventive and therapeutic setups. EAE suppression was associated with the expansion of MOG35-55-specific FoxP3+ regulatory T cells (Treg cells) and type 1 regulatory T cells (Tr1 cells), concomitant with a reduction in central nervous system-infiltrating effector T cells (Teff cells). Notably, NLPs induced bystander suppression in the EAE model established in C57BL/6 × SJL F1 mice. Moreover, NLPs ameliorated chronic progressive EAE in nonobese diabetic mice, a model which resembles some aspects of secondary progressive MS. In summary, these studies describe a platform for the therapeutic induction of antigen-specific tolerance in autoimmune diseases.

Detecting Tumor Antigen-Specific T Cells via Interaction-Dependent Fucosyl-Biotinylation.

Re-activation and clonal expansion of tumor-specific antigen (TSA)-reactive T cells are critical to the success of checkpoint blockade and adoptive transfer of tumor-infiltrating lymphocyte (TIL)-based therapies. There are no reliable markers to specifically identify the repertoire of TSA-reactive T cells due to their heterogeneous composition. We introduce FucoID as a general platform to detect endogenous antigen-specific T cells for studying their biology. Through this interaction-dependent labeling approach, intratumoral TSA-reactive CD4+, CD8+ T cells, and TSA-suppressive CD4+ T cells can be detected and separated from bystander T cells based on their cell-surface enzymatic fucosyl-biotinylation. Compared to bystander TILs, TSA-reactive TILs possess a distinct T cell receptor (TCR) repertoire and unique gene features. Although exhibiting a dysfunctional phenotype, TSA-reactive CD8+ TILs possess substantial capabilities of proliferation and tumor-specific killing. Featuring genetic manipulation-free procedures and a quick turnover cycle, FucoID should have the potential of accelerating the pace of personalized cancer treatment.

The nanomaterial-induced bystander effects reprogrammed macrophage immune function and metabolic profile.

Bystander effects in biological systems are the responses shown by nontargeted neighboring cells, and critical to the bio-nano interface interactions. In addition to direct effects, bystander effects also determine the design, applications and safety of nanomaterials, although the related information of nanomaterial-induced bystander effects remain largely unknown. A coculture system of A549 and THP-1 was established to mimic the lung microenvironment to study the bystander effects of WS2 nanosheets (representative transition-metal dichalcogenide nanosheets) on microenvironment macrophages during the inhalation exposure or the nanomaterial biomedical application in the lung. Lung cells exposed to WS2 nanosheet resulted in an increase in reactive oxygen species and the depolarization of mitochondrial membrane potential in neighboring macrophages. Bystander exposure also induced macrophage polarization toward the anti-inflammatory M2 phenotype, which is adverse to disease therapy. Metabolomics showed that WS2 nanosheets disturbed the energy metabolism and amino acid metabolism of macrophages, consistent with the metabolic characteristics of M2 macrophages. Nitric oxide-transforming growth factor-ß1 played an important mediator in the bystander effects. Importantly, WS2 nanosheet bystander exposure affected macrophage phagocytosis and migration and altered the macrophage immune response to endotoxin. This study improves the current understanding of bio-nano interactions and highlights the importance of neighboring cell responses, allowing us to use the maximum benefits of nanomaterials while limiting their adverse bystander effects.

Bystander CD4+ T cells: crossroads between innate and adaptive immunity.

T cells are the central mediators of both humoral and cellular adaptive immune responses. Highly specific receptor-mediated clonal selection and expansion of T cells assure antigen-specific immunity. In addition, encounters with cognate antigens generate immunological memory, the capacity for long-term, antigen-specific immunity against previously encountered pathogens. However, T-cell receptor (TCR)-independent activation, termed "bystander activation", has also been found. Bystander-activated T cells can respond rapidly and secrete effector cytokines even in the absence of antigen stimulation. Recent studies have rehighlighted the importance of antigen-independent bystander activation of CD4+ T cells in infection clearance and autoimmune pathogenesis, suggesting the existence of a distinct innate-like immunological function performed by conventional T cells. In this review, we discuss the inflammatory mediators that activate bystander CD4+ T cells and the potential physiological roles of these cells during infection, autoimmunity, and cancer.

Irradiated tumor cell-derived microparticles mediate tumor eradication via cell killing and immune reprogramming.

Radiotherapy (RT) is routinely used in cancer treatment, but expansion of its clinical indications remains challenging. The mechanism underlying the radiation-induced bystander effect (RIBE) is not understood and not therapeutically exploited. We suggest that the RIBE is predominantly mediated by irradiated tumor cell-released microparticles (RT-MPs), which induce broad antitumor effects and cause immunogenic death mainly through ferroptosis. Using a mouse model of malignant pleural effusion (MPE), we demonstrated that RT-MPs polarized microenvironmental M2 tumor-associated macrophages (M2-TAMs) to M1-TAMs and modulated antitumor interactions between TAMs and tumor cells. Following internalization of RT-MPs, TAMs displayed increased programmed cell death ligand 1 (PD-L1) expression, enhancing follow-up combined anti-PD-1 therapy that confers an ablative effect against MPE and cisplatin-resistant MPE mouse models. Immunological memory effects were induced.

The future of radiation-induced abscopal response: beyond conventional radiotherapy approaches.

Advances in the immunological pharmaceuticals, such as checkpoint inhibitors and agonists, have positive implications for the future of the radiotherapy abscopal response. A once rare phenomenon, whereby distant nonirradiated tumor sites regressed after radiotherapy alone, may become more common when combined with the immune modulating agents. Radiotherapy can increase neoantigen expression, increased tumor PD-L1 expression, increase MHC class I expression, reverse exhausted CD8 T cells and increase tumor-infiltrating tumors within the tumor microenvironment. These changes in the tumor and the tumor microenvironment after radiotherapy could potentiate responses to anti-CTL-4, anti-PD-L1/PD-1 and other immunotherapy agents. Thus, advances in checkpoint inhibitors have increased interest in re-evaluation of the role of conventional radiotherapy approaches on the immune system. We reviewed newer nonconventional approaches such as SBRT-PATHY, GRID, FLASH, carbon ion and proton therapy and their role in eliciting immune responses. We believe that combining these novel radiation methods may enhance the outcome with the newly US FDA approved immune modulating agents.

Malaria exposure drives both cognate and bystander human B cells to adopt an atypical phenotype.

Atypical memory B cells (aMBCs) are found in elevated numbers in individuals exposed to malaria. A key question is whether malaria induces aMBCs as a result of exposure to Ag, or non-Ag-specific mechanisms. We identified Plasmodium and bystander tetanus toxoid (TT) specific B cells in individuals from areas of previous and persistent exposure to malaria using tetramers. Malaria-specific B cells were more likely to be aMBCs than TT-specific B cells. However, TT-specific B cells from individuals with continuous exposure to malaria were more likely to be aMBCs than TT-specific B cells in individuals from areas where transmission has ceased. Finally, sequences of BCRs specific for a blood stage malaria-Ag were more highly mutated than sequences from TT-specific BCRs and under strong negative selection, indicative of ongoing antigenic pressure. Our data suggest both persistent Ag exposure and the inflammatory environment shape the B-cell response to malaria and bystander Ags.

Memory B Cell Activation, Broad Anti-influenza Antibodies, and Bystander Activation Revealed by Single-Cell Transcriptomics.

Antibody memory protects humans from many diseases. Protective antibody memory responses require activation of transcriptional programs, cell proliferation, and production of antigen-specific antibodies, but how these aspects of the response are coordinated is poorly understood. We profile the molecular and cellular features of the antibody response to influenza vaccination by integrating single-cell transcriptomics, longitudinal antibody repertoire sequencing, and antibody binding measurements. Single-cell transcriptional profiling reveals a program of memory B cell activation characterized by CD11c and T-bet expression associated with clonal expansion and differentiation toward effector function. Vaccination elicits an antibody clone, which rapidly acquired broad high-affinity hemagglutinin binding during affinity maturation. Unexpectedly, many antibody clones elicited by vaccination do not bind vaccine, demonstrating non-specific activation of bystander antibodies by influenza vaccination. These results offer insight into how molecular recognition, transcriptional programs, and clonal proliferation are coordinated in the human B cell repertoire during memory recall.

A Matter of Life or Death: Productively Infected and Bystander CD4 T Cells in Early HIV Infection.

CD4 T cell death or survival following initial HIV infection is crucial for the development of viral reservoirs and latent infection, making its evaluation critical in devising strategies for HIV cure. Here we infected primary CD4 T cells with a wild-type HIV-1 and investigated the death and survival mechanisms in productively infected and bystander cells during early HIV infection. We found that HIV-infected cells exhibited increased programmed cell death, such as apoptosis, pyroptosis, and ferroptosis, than uninfected cells. However, productively infected (p24+) cells and bystander (p24-) cells displayed different patterns of cell death due to differential expression of pro-/anti-apoptotic proteins and signaling molecules. Cell death was triggered by an aberrant DNA damage response (DDR), as evidenced by increases in γH2AX levels, which inversely correlated with telomere length and telomerase levels during HIV infection. Mechanistically, HIV-infected cells exhibited a gradual shortening of telomeres following infection. Notably, p24+ cells had longer telomeres compared to p24- cells, and telomere length positively correlated with the telomerase, pAKT, and pATM expressions in HIV-infected CD4 T cells. Importantly, blockade of viral entry attenuated the HIV-induced inhibition of telomerase, pAKT, and pATM as well as the associated telomere erosion and cell death. Moreover, ATM inhibition promoted survival of HIV-infected CD4 T cells, especially p24+ cells, and rescued telomerase and AKT activities by inhibiting cell activation, HIV infection, and DDR. These results indicate that productively infected and bystander CD4 T cells employ different mechanisms for their survival and death, suggesting a possible pro-survival, pro-reservoir mechanism during early HIV infection.