bluuebunny/crossref_metadata_2025 huggingface dataset:
Manipulating number and function of effector CD8 T cells (IRC9P.710)
Authors: Kim Marie , Harty John
Published: January 2023

In response to infection or vaccination, CD8 T cells undergo massive proliferative expansion in numbers followed by a contraction phase where 90-95% of the expanded population is eliminated through apoptosis, with the remaining cells initiating the memory pool. Although basic CD8 T cell kinetics in response to infection and immunization have been described, the mechanisms regulating how memory CD8 T cell numbers are determined remain unknown. The current paradigm regarding memory CD8 T cell numbers states that the size of the memory pool is dictated primarily by the peak numbers achieved after infection or immunization. However, our data directly challenge this paradigm. Here, we demonstrate that DC-immunization and infection models, coupled with stimulatory IL-2/Ab complex treatment, can increase peak effector CD8 T cell numbers over 100-fold with no concurrent increase in memory CD8 T cell numbers post-contraction. Additionally, IL-2/Ab complex treatment not only increases effector CD8 T cell numbers, but also enhances and sustains expression of cytolytic molecules such as granzyme B, 4-1BB, and GITR. Thus, our data dissociate the magnitude of effector CD8 T cell expansion from memory set point, as well as highlight how IL-2/Ab complexes can enhance effector CD8 T cell function. Our findings suggest that IL-2/Ab complex therapy may be useful in short-term elevation of effector CD8 T cell numbers and function for tumor immunotherapy or during infectious disease outbreaks.
Altered CD8 T cell memory development in the presence of bystander chronic infections and inflammation (IRC9P.712)
Authors: Stelekati Erietta , Shin Haina , Doering Travis , Dolfi Douglas , Ziegler Carly , Beiting Daniel , Dawson Lucas , Liboon Jennifer , Wolski David , Ali Mohammed-Alkhatim , Katsikis Peter , Shen Hao , Roos David , Haining W. , Lauer Georg , Wherry John
Published: January 2023

Chronic infections impair immune responses to unrelated pathogens and vaccines but the underlying mechanisms are not clear. We investigated whether bystander chronic infections impact the differentiation of memory CD8 T cells. We demonstrate that diverse chronic viral and parasitic infections substantially impair both the quantity and quality of developing memory CD8 T cells, independently of initial priming. Fully-formed memory CD8 T cells were less susceptible, though not impervious, to the effects of bystander chronic infection, suggesting that the impact of inflammation on memory development is more prominent during the critical period of transitioning from effector to memory. Prolonged induction of type I IFN recapitulated the effects of chronic bystander infection on memory CD8 T cell development, though these effects were independent of IFNAR signaling on the CD8 T cells during chronic infection. However, systemic blockade of IFNAR signaling after the priming phase enhanced the survival and the numerical seeding of the developing memory CD8 T cell pool. In contrast to survival, altered CD8 T cell memory differentiation was regulated by the transcription factors Tbx21 (T-bet) and Prdm1 (Blimp-1), specifically in the post-effector phase. Thus, exposure to chronic bystander infections and inflammation impairs the transition from effector to memory. These results have relevance for immunity and vaccination during persisting infections and chronic inflammation.
Human IL-7Rα low memory CD8+ T cells are clonal anergic cells being reversed by exogenous IL-2 (IRC10P.469)
Authors: Sim Ji Hyun , Cho Bon-A , Lee Sunkyung , Ko Youngho , Seol Min A , Kim Jin-Hee , Oh Keunhee , Lee Dong-Sup , Kang Insoo , Choi Kyung-Ho , Kim Hang-Rae
Published: January 2023

We have found two subsets of CD8+ T cells expressing IL-7Rα high and IL-7Rα low with different cell survival responses to IL-7 in peripheral blood of human. Of interest, these IL-7Rα low memory CD8+ T cells that comparably express effector molecules have impaired proliferation and IL-2 production upon TCR stimulation by anti-CD3/CD28 Abs. Treating IL-7Rα low memory CD8+ T cells with anti-CD3/CD28 Abs did not produce IL-2, but comparably expressed IFN-γ and TNF-α, as shown in split anergic cells. Importantly, IL-7Rα low memory CD8+ T cells have reduced calcium flux and subsequent inhibited nuclear translocation of NF-AT1 upon in vitro TCR stimulation by anti-CD3/CD28 Abs, leading to impaired IL-2 production. Moreover, inhibition of IL-2 production and proliferation was not recovered by PMA/ionomycin treatment, suggesting that IL-7Rα low memory CD8+ T cells also have TCR-distal signaling defect of TCR signaling. Interestingly, this anergic status was reversed by exogenous IL-2 stimulation for 10 days, clearly distinct from adaptive tolerance induced by soluble peptide Ag. In conclusion, we firstly showed that specific subset of human CD8+ T cells, IL-7Rα low memory CD8+ T cells, are T-cell clonal anergy in vivo. Based on these findings, we are going to further investigate which mechanisms are in volved in anergic phenotype of human anergic IL-7Rα low memory CD8+ T cells, and eventually, what is the role of these cells in vivo using by humanized mouse.
Increased expression of costimulatory molecules on synovial monocytes affecting inflammatory T-cell responses in rheumatoid arthritis (RA) (IRC10P.470)
Authors: Lee Won-Woo , Hwang Yuri , Yoon BoRuem , Lee Hyunju
Published: January 2023

Monocytes function as crucial innate effectors during inflammation. Human monocytes can be divided into three distinct subsets based on CD14 and CD16 expression. Accumulating evidences suggest that three monocyte subsets have distinct functions in inflammatory response. We investigated the characteristics of monocytes in synovial fluid (SF) of RA patients. CD16 expression on CD14++ monocytes in the SF was significantly increased compared with that in the peripheral blood (PB) of RA patients and healthy control (HC). Microarray data showed that 3,134 genes were differentially expressed in SF monocytes compared with PB monocytes. Among the genes, CD80 and CD276 expression were significantly elevated on SF monocytes, while PB monocytes of RA and HC did not express both of them without stimulation. CD80 and CD276 belong to the B7 family, which plays a checkpoint role for modulating T-cell responses. To explore how SF monocytes gain unique properties, PB monocytes were stimulated with various cytokines and TLR ligands. TGF-β was a potent inducer of CD16 expression on monocytes, whereas expressions of CD80 and CD276 were markedly elevated by IFN-γ and GM-CSF, respectively. In vitro assay, SF monocytes were found to significantly promote Th17 and Th1 responses, compared with PB monocytes of RA patients. Our findings suggest the possible role for cytokine milieu of the SF in giving unique features to SF monocytes and their crucial roles in affecting inflammatory T-cell response in RA
The B cell response to Salmonella enteric ser. Typhimurium: promiscuous B cell recruitment for early Ab followed by extrafollicular affinity maturation (MPF6P.743)
Authors: DiNiro Roberto , Lee Seung Joo , McSorley Stephen , Shlomchik Mark
Published: January 2023

We have investigated the unconventional B-cell response to Salmonella typhimurium (Stm). Stm engenders a sustained IgM and IgG extrafollicular plasmablast response, with AFC numbers increasing by >100-fold, but no notable germinal centers. To investigate the properties of this response, we screened large panels of plasmablast-derived mAbs for Stm specificity, using ELISA, flow cytometry, and antigen microarray. To our surprise, the majority of the response (>98%) appeared to be non-specific. Polyclonal B-cell activation via TLRs was an intriguing possibility but excluded based on the identical response in MyD88-deficient mice. Conversely, BCR specificity was important, as infection of BCR transgenic mice with limited repertoire led to a >10-fold loss of AFCs. We thus hypothesized that the initial response was specific, but of such low affinity that Ag-binding could not be detected. Indeed, analysis of the somatic hypermutation (SHM) pattern of specific mAb V regions suggested that SHM was necessary for detectable Ag binding. This was confirmed by reverting mAbs to germline, which caused stepwise loss of reactivity. Strikingly, laser microdissection of extrafollicular plasmablast patches demonstrated robust SHM occurring outside of GCs. These results suggest a revised vision of how clonal selection and affinity maturation operate in the response to bacteria: starting affinities are virtually undetectable, yet eventually improve by SHM occurring during the plasmablast response.
Intravital imaging shows a key role for splenic DCs in acute malaria for CD4+ T cell activation and parasite clearance (MPF6P.747)
Authors: Borges da Silva Henrique , Fonseca Raíssa , Cassado Alexandra , Salles Érika , Menezes Maria , Langhorne Jean , Marinho Claudio , Boscardin Silvia , Álvarez José , D'Império Lima Maria , Tadokoro Carlos
Published: January 2023

Heterogeneous populations of macrophages and dendritic cells (DCs) form a complex phagocyte network inside the red pulp (RP) of the spleen, which is a major site for the control of blood-borne infectious diseases, such as malaria. However, the dynamics of splenic DCs during Plasmodium infections are poorly understood, obscuring our knowledge about the protective role of these cells in malaria. To address this issue, the phagocytosis of infected red blood cells (iRBCs) by splenic DCs was quantified by confocal intravital microscopy, as well as by flow cytometry and immunofluorescence, in three distinct phases of blood-stage Plasmodium chabaudi malaria: at the first encounter, at pre-crisis concomitant with parasitemia growth and at crisis, when the parasitemia decline coincides with spleen closure. The in vivo and ex vivo imaging of the spleen during the early infection shows that DCs phagocytize iRBCs and interact with CD4+ T cells in CD4+ T cell-rich areas and the RP. This analysis also reveals that the RP DC population maintains the ability to phagocytize iRBCs during pre-crisis stages and greatly contributes to parasite control. These findings add novel information to the immunology field and demonstrate that in vivo imaging can help to unravel the protective mechanisms against malaria.
The role of plasmacytoid dendritic cells in the delay of Th17 production during Bordetella pertussis infection in mice (MPF6P.738)
Authors: Wu Victoria , You Hana , Ferguson Ryan , Niknam Shahryar , Abramson Tzvia
Published: January 2023

Whooping cough, caused by Bordetella pertussis (Bp), is a highly contagious respiratory disease. Pertussis toxin (PTX) is largely responsible for extreme leukocytosis and chronic disease. Th17 cells have been implicated in host defense and control of mucosal pathogens, including Bp. Plasmacytoid dendritic cells (pDCs) have been shown to participate in Th17 commitment through secretion of IL-6. However, stimulation of pDCs through Toll-like receptor 7 leads to the secretion of IFN-α, a cytokine documented to inhibit Th17 differentiation. pDCs are activated during lung inflammation in response to pathogens, but their role during Bp infection is understudied. Using murine infection models of Bp and BpΔPTX (Bp strain devoid of PTX), we test the hypothesis that the functions of pDCs can manipulate the rise of Th17 cells during infection. Lung Th17 cells increase significantly in Bp-infected mice only by 10 days post-infection (p.i.), while lung pDCs emerged 2-3 fold at 5 days p.i. During the late rise in Th17 cells, the frequency of pDCs secreting IFN-α drops while those secreting IL-6 increase. In addition, blocking IFN-α production in mice results in an earlier increase in Th17 cells during infection. The recruitment and cytokine secretion of lung pDCs during infection with Bp correlate with the delayed Th17 responses, suggesting a role for pDCs in Th17 differentiation that may account for a longer-lasting disease.
Protective effector memory CD4+ T cells are generated from an early effector memory precursor (MPF6P.739)
Authors: Opata Michael , Carpio Victor , Ibitokou Samad , Obiero Joshua , Dillon Brian , Langhorne Jean , Stephens Robin
Published: January 2023

Effector memory CD4+ T cells (Tem) are the predominant memory subset in chronic infections such as malaria and tuberculosis. Tem respond quickly by making effector cytokines. Therefore, understanding their mechanism of generation and the role of persistent antigen is essential for vaccination strategies. The current paradigm of Tem generation is that they develop progressively from mature effector T cells in the contraction phase, however this remains to be established. In order to identify a precursor of Tem we identified three effector subsets with an Early CD127-CD62Lhi CD4+ memory precursor effector T cell (MPEC) population, and short-lived CD62Llo Teff (SLEC). In adoptive transfer experiments, we show the potential of Early Teff to diverge into either terminal Teff or memory cells depending on the environment, while mature Teff lose their memory potential, in contrast to the progressive differentiation model. Furthermore, we demonstrate that resting central memory T cells can become Tem in the absence of further stimulation. All three effector subsets protect immunocompromised animals, however, only Late Effector memory (TemL) improves both infection and pathology. Based on our data we propose an early decision point for generation of protective effector memory T cells, suggesting that memory development occurs in the early phase of activation. This work also suggests that TemL represent a correlate of immunity in chronic infection and can improve vaccine design studies.
Although the induction of INF-γ+ CD8 T cells by radiation-attenuated Plasmodium berghei sporozoites is TAP-independent, the recall of protective effector CD8 T cells during infection requires TAP machinery (MPF6P.740)
Authors: Pichugin Alexander , Steers Nick , De La Vega Patricia , Krzych Urszula
Published: January 2023

Multiple exposures of humans and laboratory rodents to radiation-attenuated Plasmodium sporozoites (γ-spz) induce sterile and long-lasting protection that is thought to be directed mainly to liver-stage antigens (LS-Ags). Although protection involves B cells and CD4 T cells, MHC class I-dependent liver IFN-γ+ CD8 T cells are the key effectors against LS-Ags. Recently several LS-Ags have been shown to induce protection in murine models; nevertheless, the mechanism of processing and presentation of LS-Ag to CD8 T cells has not been investigated. We tested liver CD8 T cell responses induced by Plasmodium berghei (Pb) γ-spz in TAP-/- mice to inquire whether processing/presentation of the exogenous LS-Ags was TAP-dependent or -independent. Immunization with Pb γ-spz induced proliferation and IFN-γ production by liver CD8 T cells. Also, TAP-/- CD8 T cells eliminated hepatocytes infected with Pb sporozoites in vitro. Despite these effector activities, Pb γ-spz immunized TAP-/- mice were not protected against sporozoite infection, mainly because liver CD8 T effector cells failed to produce IFN-γ in response to infectious sporozoite challenge. Our results demonstrate that although TAP-independent vacuolar pathway operates efficiently during immunization with γ-spz, the TAP-associated phagosomal-to-cytosol antigen processing mechanism appears to be required for effective recall of effector CD8 T cells during infection. The implications of these findings will be discussed.
Neutrophils are not a primary contributor to the early chemokine and cytokine cascade during Chlamydial infection (MPF7P.748)
Authors: Agugbuem Cemeka , Yeruva Venkat , Rank Roger
Published: January 2023

Chlamydia trachomatis is a common cause of tissue scarring, pelvic inflammatory disease, ectopic pregnancy, and infertility in women. A milieu of chemokines and cytokines contribute to the resultant pathology, directing the acute inflammatory response to mitigate infection with damaging consequences for the host. Yet, there are no reports on the cellular source of this chemokine and cytokine cascade during the initial stage of the host-pathogen interaction. We utilized a mouse model to determine whether neutrophils are a primary contributor to the chemokine/cytokine response during the initial development cycle (42- 48hrs) following intracervical inoculation with Chlamydia muridarum. Antibody depletion of neutrophils significantly increased the number of chlamydial inclusion forming units at 48 hours post infection and the recruitment of macrophages and natural killer cells to the infection site. Chemokine and cytokine mRNA expression at 36 hours after inoculation showed an increase in TNF-α, CXCL1 and CXCL2 and a decrease in IFN-γ in comparison to isotype control mice. Furthermore, we observed a slight increase TGFβ2, although not significant, in the neutrophil-depleted mice but we did not see major changes in IL-1β, CXCL10, CCL2, CCL4, and CCL5. Our findings suggest neutrophils are not a primary contributor to the early chemokine and cytokine cascade during chlamydial infection and that infected epithelial cells may be the primary source of this response.
Human mesenchymal stem/stromal cells prevent experimental autoimmune uveitis by inhibiting Th17 cells and by inducing IL-10-expressing B220+CD19+ cells (MUC7P.759)
Authors: Kim Mee Kum , Oh Joo Youn , Jeong Hyun Jeong , Ryu Jin Suk , Kim Dong Hyun , Lee Hyun Ju , Kim Tae Wan , Wee Won Ryang
Published: January 2023

Mesenchymal stem/stromal cells (MSCs) have the remarkable ability to protect tissues by modulating immune responses in various autoimmune diseases. We here investigated that systemic administration of MSCs might prevent development of experimental autoimmune uveitis (EAU) in mice. The study was conducted in conformance with the FASEB Statement of Principles for the use of animals in research and education. Analysis of the retina during the efferent phase revealed that a single intraperitoneal injection of human bone marrow-derived MSCs (hMSCs) at the time of immunization markedly protected the retina from inflammation-mediated damage and suppressed levels of innate and adaptive pro-inflammatory cytokines including IL-1β, IL-6, TNF-α, IFN-γ, IL-17A, and IL-2 in the eye. Analysis of draining lymph nodes (DLNs) during the afferent phase showed that hMSCs significantly decreased the proportions of IL-17- or IFN-γ-expressing CD4+ T cells as well as levels of their cytokines, IL-17A and IFN-γ. The levels of IL-1β, IL-6, IL-12, and IL-23 were not decreased by hMSCs. However, hMSCs significantly increased the level of immunoregulatory cytokine IL-10 and markedly induced the population of IL-10+B220+CD19+ cells in DLNs. Together, data demonstrate that systemic administration of hMSCs attenuates EAU by inducing IL-10-secreting B220+CD19+ cells and by suppressing the Th1/Th17 immune responses.
Trojan T cell horse: TLR5 ligand-secreting T cells reshape the tumor microenvironment and exhibit enhanced antitumor activity (TUM2P.909)
Authors: Kaczanowska Sabina , Geng Degui , Tsai Alexander , Ochoa Augusto , Johnson Laura , Davila Eduardo
Published: January 2023

T cell responses are downregulated by the immunosuppressive tumor microenvironment. On the basis of previous studies demonstrating that Toll-like receptor (TLR) engagement on T cells augments their responses, we hypothesized that T cells engineered to secrete the TLR5 ligand flagellin would enhance T cell costimulation in the tumor environment and augment antitumor activity. Human T cells engineered to express the MART-1 TCR and secrete TLR5-ligand (TLR5-L) responded to lower tumor antigen levels resulting in increased proliferation, cytolytic activity and reversed tumor growth associated with prolonged mouse survival in a xenogeneic model. Treatment with TLR5-L-secreting T cells eradicated tumor in 40% of mice in a syngeneic model. Intratumoral secretion of TLR5-L by T cells reshaped the tumor microenvironment in part by inducing of a distinct chemokine/cytokine profile rendering it more immunogenic and permissive to T cell infiltration. Furthermore, tumor lysis by TLR5-L-producing T cells triggered the priming of endogenous tumor-reactive CD8 and CD4 T cells. Characterization of the cellular composition of the tumor and spleen revealed a reduction in MDSCs in the spleen and increased expression of MHC I in the tumor. This study demonstrates for the first time the ability to use T cells to deliver a TLR agonist to the tumor site and that intratumoral TLR5-L contributes to various processes involved in tumor regression.
Alterations in Kv1.3 channels in tumor infiltrated lymphocytes of patients with head and neck tumors. (TUM2P.913)
Authors: Chimote Ameet , Hajdu Peter , Filipovich Alexandra , Casper Keith , Conforti Laura
Published: January 2023

The progression of solid tumors, such as head and neck squamous cell carcinoma (HNSCC), is strongly dependent on the host immune system. Tumor infiltrating lymphocytes (TIL) are potent mediators of an anti-tumor response; however their function is attenuated in solid tumors. T cell effector functions like cytokine production and cytotoxicity are dependent on cytoplasmic Ca2+ which is regulated by ion channels. In human T cells, Kv1.3 channels are responsible for membrane hyperpolarization that allows the sustained Ca2+ influx necessary for effector functions, but their role in TILs is not understood. We hypothesize that Kv1.3 activity is decreased in TILs thereby contributing to their decreased functionality. We obtained tumor samples and matched peripheral blood (PB) from 6 patients with HNSCC. CD3+ TILs were separated by mechanical dissociation of tumors and negative selection. TILs were 99% viable and comprised of 57% CD4+ (85% TEM and 10% Treg) and 36% CD8+ cells. Electrophysiology revealed a 70% reduction in functional Kv1.3 channels in TILs as compared to PB CD3+ cells from paired patients. The Kv1.3 currents in the PBTs of cancer patients were similar to those of healthy donors. Furthermore, we observed a 40% reduction in the ion channel-dependent component of Ca2+ influx (measured bypassing the T cell receptor) in TILs. Overall, these data indicate that defective Kv1.3 and Ca2+fluxes in TILs could contribute to reduced immune surveillance in cancer.
Tim-3 is expressed by a majority of NK cells at periphery but not at tumor site in patients with advanced melanoma (TUM2P.919)
Authors: Sun Zhaojun , Fourcade Julien , Pagliano Ornella , Chauvin Joe-Marc , Sander Cindy , Kirkwood John , Kuchroo Vijay , Zarour Hassane
Published: January 2023

Tim-3 is a type I glycoprotein expressed by innate and adaptive immune cells. Tim-3 is an inhibitory receptor expressed by dysfunctional tumor antigen-specific CD8+ T cells of patients with advanced melanoma at periphery and in the tumor microenvironment. Tim-3 is also constitutively expressed by functional and mature NK cells in healthy donors (HD). The expression of Tim-3 on NK cells isolated from melanoma patients (MP) and its role in regulating NK functions have not been evaluated yet. To this end, we have investigated Tim-3 expression by NK cells in PBMCs isolated from HD and MP. In sharp contrast with HD, Tim-3 is upregulated by CD56bright as compared to CD56dim NK cells in PBMCs of MP. Upon stimulation with IL-12 and IL-18, NK cells in HD and MP upregulated Tim-3 expression and Tim-3high NK cells produced more IFN-γ than Tim-3neg, suggesting they are more functional. IFN-γ production increased upon NK stimulation with cytokines and Tim-3 ligand Galectin-9 and did not decrease upon Tim-3 blockade. In contrast to circulating NK cells, tumor-infiltrating NK cells (TINKs) are CD56dim in their large majority (> 98%), and exhibit lower levels of Tim-3 expression. Collectively, our data show that Tim-3 is expressed by a majority of functional circulating NK cells in HD and MP but only a minority of TINK cells, suggesting that TINK may be less functional than their peripheral counterpart.
High density of cytotoxic and regulatory T lymphocytes in gastric cancers with microsatellite instability (TUM2P.920)
Authors: Kim Hyoung-Il , Park Chung-Gyu , Hyung Woo Jin
Published: January 2023

Background: the association between cancer phenotype and host immune response has rarely been evaluated in gastric cancer. We hypothesized that microsatellite instability (MSI) is associated with pattern of tumor infiltrating lymphocytes (TILs). Methods: we evaluated the status of MSI and subset of TILs in gastric cancer patients who received curative resection. The number of MSI-High and non-MSI-High were matched 1:3. The immunohistochemical staining with anti-CD3, CD4, CD8, Foxp3, and granzyme B were used to quantify the subset of TILs. Results: of the 98 carcinomas studied, 27 were MSI-High and 71 were non-MSI-High. Clinicopathologic profiles were comparable for both groups. The absolute number of CD3+ T-cells was not significantly different. MSI-High tumors carried significantly higher numbers of CD8+ T-cells than non-MSI-High tumors, suggesting favorable prognosis. Paradoxically, MSI-High tumors also contained higher numbers Foxp3+ T-cells and higher ratio of Foxp3+/CD4+ T-cells suggesting unfavorable prognosis. In subgroup analysis using T classification, distribution of TILs according to MSI type changed in CD8 and CD4 subsets. Conclusions: Microsatellite instable gastric cancers harbor distinctive distribution of subset of tumor infiltrating lymphocytes according to tumor progression.
PD-1 expression on peripheral blood cells increases with stage in renal cell carcinoma patients and is rapidly reduced after surgical tumor resection (TUM2P.881)
Authors: MacFarlane Alexander , Jillab Mowafaq , Plimack Elizabeth , Hudes Gary , Uzzo Robert , Litwin Samuel , Dulaimi Essel , Al-Saleem Tahseen , Campbell Kerry
Published: January 2023

In this study, we analyzed expression of numerous immune cell markers on fresh PBMC from 90 RCC patients preoperatively and 25 age-matched healthy controls by 10-color flow cytometry. Postoperative blood samples were also analyzed from 23 members of the RCC patient cohort. The most striking phenotypic immune biomarker in RCC patients was a significant increase in PD-1 expression on certain PBMC in a subset of patients. Increased PD-1 expression on CD14bright myelomonocytic cells, effector T cells, and NK cells correlated to disease stage. Importantly, we also observed that expression was significantly reduced on all cell types in virtually all patients within 2 weeks after surgical resection of the primary tumor. Based upon our results, we conclude that PD-1 expression on fresh peripheral blood leukocytes may provide a useful indicator of RCC disease progression. Furthermore, measuring PD-1 levels in peripheral blood may assist in identifying patients likely to respond to PD-1 blocking antibodies, and these therapies may be most effective before and immediately after surgical resection of the primary tumor, when PD-1 expression is most prominent. Finally, we conclude that long-term PD-1 blocking therapy after nephrectomy may only be effective in stage 3 and stage 4 patients that retain PD-1 expression, presumably maintained by the presence of residual metastatic tumor.
Alpha fetoprotein hypersensitizes human natural killer cells to interleukin-2-mediated activation (TUM2P.885)
Authors: Vujanovic Lazar , Stahl Elizabeth , Butterfield Lisa
Published: January 2023

Alpha fetoprotein (AFP) is an oncofetal antigen commonly produced by hepatocellular carcinomas (HCC). Multiple studies have shown that tumor-derived AFP (tAFP) has an immunosuppressive role on T, B and dendritic cells which may play a role in HCC pathogenesis. However, a direct tAFP effect on natural killer (NK) cells remains unclear. We examined the ability of cord blood-derived AFP (nAFP) and tAFP to modulate human NK cell activity in vitro. We show that tAFP and, in particular, nAFP hypersensitize healthy donor NK cells to interleukin-2 (IL-2)-mediated activation. Individual nAFP and tAFP treatments specifically induce IL-1β and IL-6 secretion by NK cells, but do not enhance their overall activation as measured by CD69 upregulation, type-1 cytokine secretion, tumor cell killing and proliferation. Interestingly, by the same assessments, NK cells simultaneously stimulated with AFP and IL-2 have a higher degree of activation than those activated with only IL-2. The observed synergism between AFP and IL-2 may be mediated by the ability of AFP to modulate IL-2 receptor signaling, as shown by the ability of AFP to upregulate CD25, and downregulate CD122 and CD132 on NK cells. Overall, these data show that nAFP and tAFP hypersensitize NK cells to IL-2-mediated activation. Defining the impact of circulating AFP on NK cells may be crucial to understand the NK cell functional deficits described in HCC patients, and for the development of an effective HCC-targeting immunotherapy.
The effects of key pro-inflammatory cytokines on influenza vaccine induced memory CD8+ T cell homeostasis (VAC2P.938)
Authors: Zhou Xin , Wang Chongkai , Swain Susan , Haynes Laura , Kuchel George , McElhaney Janet
Published: January 2023

There are accumulated evidences show that many age related immune defects are attributed to declined T lymphocyte helper and effector function involved CD4+ and CD8+ T cells. The lower efficacy of current influenza vaccine in aged people and other high risk population such as hospitalized patients cause need to develop more effective vaccine adjuvants which can significantly enhance vaccine mediated T cell immune response in response to influenza virus. We investigated the influenza live virus and vaccine induced pre-inflammatory cytokine secretion profile in human PBMCs, evaluated the effects of several key cytokines on vaccine induced T cell homeostasis. Our data show that IL-2, IL-6 and IL-17A can affect the flu vaccine induced human T cell homeostasis by enhancing the naïve T cell proliferation and differentiation as well as driving memory into effector CD8+ T cells through classic cytokine signaling transduction pathway. Our results will be able to help evaluate the efficacy of future possible vaccine adjuvant for developing better influenza vaccine compounds.
Induction of polyfunctional human memory T cells by Wnt pathway activation (VAC3P.949)
Authors: Chiu Yen-Ling , Sung Boyi , Joy Jaimy , Blankson Joel , Oelke Mathias , Schneck Jonathan
Published: January 2023

Polyfunctionality is a hallmark of protective immunity against pathogens and cancer. However, the molecular mechanisms governing the induction of polyfunctional T cells are not completely understood. We found that during antigen-driven expansion of human virus-specific T cells, Wnt pathway activation enhances the CD62L+, CD28+, and KLRG1- central memory T cell phenotype and promoted the generation of highly polyfunctional virus-specific T cells. Such effects are not only seen in influenza-specific responses but also in terminally differentiated CMV- and HIV-specific T cells. Mechanistically induction of polyfunctionality was independent of arresting antigen-specific cell expansion and is T cell-intrinsic. These findings provide the first evidence that Wnt pathway activation leads to polyfunctional antigen-specific memory human T cells responses and therefore have implications for treatment of chronic viral infections and cancer.
Defining the role of the alarmin IL-33 during the CD8 T cell effector response (VAC3P.958)
Authors: St. Rose Marie-Clare , Tsurutani Naomi , Adler Adam , Vella Anthony
Published: January 2023

ST2, the receptor for the alarmin IL-33, is expressed on CD8 T cells and both IL-33 and ST2 can optimize anti-viral CD8 T cell immune responses. Further, signaling through ST2 in combination with innate-derived cytokines such as IL-12 can induce TCR-independent production of IFN-gamma. We sought to understand the role of the IL-33/ST2 pathway during the effector CD8 T cell response elicited after immunization with CD134 and CD137 costimulatory agonists. ST2 is rapidly up-regulated on CD27+ effector CD8 T cells during the earliest phase of T cell re-activation, even before the up-regulation of other well-known T cell activation markers. Also, CD27+ST2+ CD8 T cells expressed increased cytokines compared to CD27-veST2+ CD8 T cells, and blockade of ST2 impeded the expression of those cytokines. Moreover, CD27+ST2+ CD8 T cells can kill EL4 tumor targets. Thus, ST2 seems to be marking effector CD8 T cells with the best capacity to exert effector functions. Upon TCR engagement, viable effector CD8 T cells rapidly up-regulate ST2, which facilitates the delivery of additional activation signals through IL-33 that further augment T cell activation. Based on these findings, we hypothesize that IL-33 may be one of the first signals that link T cell activation with the local stromal environment. Thus, direct targeting of the more highly functional ST2+ CD8 T cells may improve the outcome of T cell-based cancer immunotherapies.
Cytokine-based cancer immunotherapy impairs primary T cell immunity (VAC3P.942)
Authors: Sckisel Gail , Bouchlaka Myriam , Mirsoian Annie , Sungur Can , Soulika Athena , Wiltrout Robert , Blazar Bruce , Murphy William
Published: January 2023

Cancer immunotherapy has made great strides towards becoming a viable alternative to traditional cancer treatment options. Under the premise of specificity and long lasting memory, immunotherapy is hypothesized to provide what traditional cancer therapies lack. However, despite success in tumor efficacy, the smoking gun of tumor-specific immunity has yet to be uncovered. Few studies to date have found correlations between tumor specific T cells and outcomes, particularly with stimulatory regimens. Here we show evidence that following cytokine based immunotherapy regimens, primary CD4 T cell responses towards mitogenic or cognate antigenic stimulation were greatly hampered. This lack of CD4 immunity was further exacerbated by acute thymic involution. Interestingly, the paralysis was confined to the CD4 T cell subset and was transient, resolving within 2 weeks of cessation of therapy. Paralyzed T cells significantly upregulated SOCS3 and failed to undergo proper STAT phosphorylation and CD25 upregulation following TCR engagement. The consequences of this paralysis were impaired vaccination and viral clearance, and also suggest impairment in the development of subsequent primary anti-tumor T cell responses. Taken together, these data suggest that cytokine-based immunotherapies may actually impair the development of immunity towards novel tumor antigens and may put patients at risk for opportunistic infections.
Denileukin diftitox depletes regulatory T cells without clinical benefit in advanced stage epithelial ovarian carcinoma (VAC3P.945)
Authors: Curiel Tyler , Thibodeaux Suzanne , Wall Shawna , Pandeswara Sri Lakshmi , Daniel Benjamin , Drerup Justin , Murthy Kruthi , Kryczek Ilona , Zou Weiping , Barnett Brian
Published: January 2023

Denileukin diftitox (DT) depletes regulatory T cells (Treg) that correlates with immune and clinical benefits in metastatic human melanoma and improved clinical outcomes in a renal cancer vaccine trial. We tested immune and clinical effects of Treg depletion using DT in a phase 0/I cancer trial and a phase II ovarian cancer trial. In our phase 0/I trial, we noted reductions in blood Treg prevalence and concentration (median ~18% and ~50%, respectively) 3-7 days after one intravenous DT infusion at 9 or 12 μg/kg, in 6 of 7 evaluable patients with breast, lung, and ovarian cancers, and melanoma, and increased blood IFN-γ+ and Ki-67+ T cells. Weekly DT significantly reduced metastatic tumors in one ovarian cancer patient prompting a small phase II trial in epithelial ovarian cancers. 28 patients received DT once every 3-4 weeks which significantly depleted functional Tregs from blood and the tumor microenvironment, but with variable immune outcomes and no significant clinical efficacy. Weekly DT eventually reduced effector T cells. In mouse ovarian cancer models we found that: i) DT efficacy depended on adaptive immunity, ii) its IL-2 moiety did not mediate clinical effects, and iii) its treatment mechanism appeared distinct from anti-CD25 antibody, which depleted Tregs in a human breast cancer trial. DT depletes Tregs in various carcinomas but requires more dosing and schedule studies. Treg-specific agents and combination treatments could also improve Treg depletion efficacy.
Mathematical modeling of the human immune response to vaccination (VIR1P.969)
Authors: Ganusov Vitaly , Le Dustin
Published: January 2023

With major advances in experimental techniques to track antigen-specific immune responses many basic questions on the kinetics of virus-specific immunity in humans remain unanswered. To gain insights into kinetics of T and B cell responses in human volunteers we combine mathematical models and experimental data from recent studies employing vaccines against yellow fever and smallpox. We found that the peak of the yellow fever-specific CD8 T cell response is determined by the rate of T cell proliferation and not by the precursor frequency of antigen-specific cells as has been suggested in several studies in mice. We also found that the rise in the virus-specific T cell numbers accurately explains clearance of yellow fever virus in the blood. Our additional mathematical model describes well the kinetics of virus-specific plasma cell and antibody response to vaccinia virus in vaccinated individuals. The model suggests that to explain the data either the rate of antibody production by plasma cells has to increase with time since immunization, or production of the majority of antibodies comes from cells located in tissues and not in the blood. Taken together, our analysis provides novel insights into mechanisms by which live vaccines induce highly efficient immunity to viral infections.
Critical roles for interferon-gamma producing immune cells during neonatal central nervous system infections (VIR1P.1005)
Authors: Ganesan Priya , O'Donnell Lauren
Published: January 2023

Viral infections in the central nervous system (CNS) are associated with devastating neurological consequences, particularly in newborns. Despite mounting innate and adaptive immune responses, neonates are often unable to control viruses in the brain and suffer extensive neuronal loss, potentially due to deficits in anti-viral cytokines. To study the response of the neonatal immune system to viral CNS infections, our laboratory uses a transgenic mouse model (NSE-CD46) of neuron-restricted measles virus (MV) infection. NSE-CD46 mice express the human isoform of CD46, a MV receptor, under the control of the neuron specific enolase (NSE) promoter, allowing for infection only in CNS neurons. Adult NSE-CD46 mice clear MV from the CNS in an interferon gamma (IFNγ) and T-cell dependent manner. In contrast, neonatal NSE-CD46 mice succumb, with 100% mortality at 15 days post infection (dpi). Neonatal mice lacking IFNγ (CD46+/IFNγ-KO) succumb more rapidly than NSE-CD46+ neonates (100% mortality, 10 dpi) despite higher T-cell infiltration in the CNS. CD46+/RAG-2-KO neonates, which lack T- and B-cells, show reduced mortality in comparison to other CD46+ genotypes and have lower MV RNA. We hypothesize that MV control in CD46+/RAG2-KO neonates could be provided by IFNγ-producing natural killer (NK) cells. Current experiments aim to define the mechanism of viral control in the CD46+/RAG2-KO neonates. This work will answer critical questions about viral control in the neonatal CNS.
Out-of-sequence signal 3 as a mechanism for virus-induced immune suppression of CD8 T cell proliferation (VIR1P.960)
Authors: Urban Stina , Welsh Raymond
Published: January 2023

Virus infections are known to induce a transient state of immune suppression often associated with an inhibition of T cell proliferation in response to mitogen or cognate-antigen stimulation. Recently, virus-induced immune suppression has been linked to responses to type 1 IFN, a signal 3 cytokine that normally can augment the proliferation and differentiation of T cells exposed to antigen (signal 1) and co-stimulation (signal 2). However, pre-exposure of CD8 T cells to IFN-inducers such as viruses or poly(I:C) prior to antigen signaling is inhibitory, indicating that the timing of IFN exposure is of essence. Studies with T cells lacking IFN receptors implicated a direct role for type 1 IFN in this process. CD8 T cells pretreated with poly(I:C) down-regulated the IFN receptor, up-regulated SOCS1, and were refractory to IFNβ-induced STAT phosphorylation. When exposed to a viral infection these CD8 T cells behaved more like 2-signal than 3-signal T cells, showing defects in short lived effector cell differentiation, reduced effector function, delayed cell division, and reduced levels of survival proteins. This suggests that IFN-pretreated CD8 T cells are unable to receive the positive effects that type 1 IFN provides as a signal 3 cytokine when delivered later in the signaling process. This desensitization mechanism may partially explain why vaccines function poorly in virus-infected individuals.
Anti-viral immune response in the central nervous system induces glial commitment in neural stem/progenitor cells (VIR1P.1010)
Authors: Kulkarni Apurva , O'Donnell Lauren
Published: January 2023

Viral infections in the central nervous system (CNS) are characterized by infiltration of lymphocytes into the brain parenchyma and release of pro-inflammatory cytokines. The effects of cytokines on neural cell function and survival varies between cell types, and may depend upon the expression of endogenous signaling molecules. Both inflammatory and developmentally-regulated cytokines alter the activity of neural stem/ progenitor cells (NSPCs), which are critical for CNS development and repair. Moreover, the proliferation and fate of NSPCs are affected during viral CNS infections. Interferon-gamma (IFNγ) is a cytokine that is released in response to a viral infection in the brain and is required for clearance of certain viruses from the CNS. IFNγ signals via the Janus kinase/Signal transducer and activator of transcription (Jak/STAT) signaling pathway, which is also activated by developmental cytokines during cell fate decisions. The JAK/STAT pathway leads to transcription of genes (p21, p27) that affect proliferation. Therefore, we will test the hypothesis that the IFNγ influences NSPC proliferation and differentiation through Jak/STAT signaling. In preliminary studies, we found that IFNγ activates STAT1 and STAT3 proteins and decreases NCPS proliferation in vitro. Through these studies, we will define the role of the inflammatory response in modulating NSPC activity during viral infections.
Interleukin-1α released from HSV-1 infected skin keratinocytes initiates protective immunity against systemic infection (VIR1P.964)
Authors: Jensen Liselotte , Milora Katelynn , Miller Samantha , Sanmiguel Julio
Published: January 2023

Herpes simplex virus-1 (HSV-1) is a common human pathogen that typically causes self-limiting disease. However, neonates and patients with compromised immune systems are at risk of developing potentially fatal systemic disease. Previously we reported that IL-1α is released by keratinocytes treated with poly(I:C), a double stranded RNA analogue often utilized to simulate virus infections. The objective of this study was to examine whether IL-1 signaling plays a role in initiating protective immunity against HSV-1 infections. Extracellular release of IL-1α protein could be detected 6-24 hours after HSV-1 infection of primary human keratinocytes (4-5-fold increase, p<0.01). This release was not associated with a correlating change in mRNA levels suggesting that the cells released a pre-formed pool of protein. HSV-1 triggered activation of inflammatory caspases-1/4 as determined using fluorescence labeled peptides (6-fold, p<0.001); however, the cellular release of IL-1α was independent of caspases-1/4. IL-1R knockout (KO) mice exhibited an increased mortality following cutaneous HSV-1 infection (60 versus 25%, p<0.01). Caspase-1/4 deficiency did not affect survival rates; hence, the protective function of IL-1R must be exclusively due to IL-1α signaling. Recruitment of leukocytes to the infected epidermis was delayed in IL-1R KO mice. Hence, HSV-1 triggered IL-1α release from infected cells appears important for initiating protective immunity against systemic HSV-1 infections.
Cell death in pandemic and seasonal influenza viruses (VIR2P.1027)
Authors: Hartmann Boris , Albrecht Randy , Marjanovic Nada , Patil Soanli , Fribourg Miguel , Sealfon Stuart
Published: January 2023

Pandemic influenza virus strains are exceptionally transmissible or virulent, leading to their potential for increased morbidity and mortality. Seasonal influenza strains are relatively predictable causing ~ 40,000 deaths, whereas the influenza pandemic of 1918 caused up to 100 million deaths. Little is known about the virus host interactions that contribute to pandemic and seasonal infections. To gain insight into these differences, we compared temporal gene programs and cell viability in human monocyte-derived dendritic cells (DCs) infected with pandemic (Cal/09, Brevig/1918) or seasonal (Tex/91, NC/99) strains. Influenza infection is associated with cell death at late timepoints related to virion release. Strikingly, as early as 4 hrs after infection, the seasonal strains caused a degree of widespread mRNA degradation and cell death not seen with the pandemic viruses. In contrast, in epithelial cells, only the late occurance (24 hrs) of cell death was observed. These studies identify a dramatic difference in the virus-host interaction between seasonal and pandemic influenza viruses in a crucial cell type of the innate and adaptive immune system.
TNF-α processing is required to limit the CD8+ T-cell response and the extent of lung injury during influenza infection (VIR2P.1015)
Authors: DeBerge Matthew , Ely Kenneth , Enelow Richard
Published: January 2023

Influenza infection in humans evokes a potent CD8+ T-cell response, which is important for clearance of virus but may also exacerbate lung injury. TNF-α is a pleotropic cytokine that is important in regulating T-cell responses to disease. We sought to characterize the role of TNF-α during influenza infection by utilizing genetically modified mice that lack TNF-α or only express transmembrane TNF-α. We found that while TNF-α was dispensable for influenza virus clearance, soluble (s) TNF-α expression was required to limit the extent of injury. In the absence of sTNF-α, there was a significant increase in the virus-specific CD8+ T-cell response, which was due in part to increased resistance to activation-induced cell death. We found that sTNF-α mediated T-cell responses primarily through TNFR1, since mice lacking TNFR1 exhibited dysregulated immune responses and exacerbated injury similar to that observed in mice lacking sTNF-α. Interestingly, mice lacking TNFR2 had an impaired CD8+ T-cell response with attenuated injury. We also found that antibody neutralization of TNF-α early during infection enhanced the magnitude of the CD8+ T-cell response indicating that TNF-α expression has a dominant effect during T-cell priming to limit the size of the effector response later during infection. Taken together, these findings suggest that proteolytic processing of TNF-α is a critical event regulating the CD8+ T-cell response and the extent of immunopathology during influenza infection.