bluuebunny/crossref_metadata_2025 huggingface dataset:
The complement receptor CRIg dominates clearance of intravascular parasites during African trypanosome infection
Authors: Liu Gongguan , Sun Peng , Xu Jinjun , Shi Meiqing
Published: January 2023

Kupffer cells (KCs), the liver resident macrophages, play a prominent role in clearance of blood-borne pathogens. The mechanisms by which KCs engulf circulating parasites such as African trypanosomes have yet to be determined. Here we show that depletion of KCs resulted in uncontrolled parasitemia in mice infected with Trypanosoma congolense, indicating the essential role of KCs in clearance of intravascular parasites. By using intravital microscopy, we further investigated the dynamics of interactions of KCs with circulating T. congolense in real-time. The results demonstrate that KCs catch circulating parasites in the presence of specific IgM and IgG antibodies, but fail to do so in the absence of specific antibodies. Complement C3 is critically involved in the capture of the parasites mediated by both IgM and IgG. We further demonstrate that complement receptor CRIg, but not CR3, is crucial for KCs to catch the parasites. In support of the findings gained by in vivo imaging, mice deficient of C3 or CRIg, but not CR3, could not control the first wave of parasitemia, and had a dramatic reduced survival time compared to wild-type mice following infection with T. congolense (10 days vs over 100 days). Collectively, our data demonstrate that CRIg play a dominant role in the clearance of circulating parasites by KCs in an antibody dependent manner.
Brucella melitensis specifically interferes with autophagy pathway in human monocytes and enhances anti-inflammatory response.
Authors: Paliogianni Fotini , Giannopoulou Ioanna , Liopeta Kassiani , Sanoudou Despoina , Dimitrakopoulos Odyssefs
Published: January 2023

Autophagy and cytokine response are both involved in host defense against intracellular bacteria, including Brucella. However the effect of autophagy on the balance of Brucella triggered pro- and anti-inflammatory cytokine production has not been studied in the human system. In the present study, we assessed the effect of autophagy on pro- and anti-inflammatory cytokine production, using primary human monocytes infected with live clinical strain of B. melitensis in the presence or absence of specific autophagy inhibitors (Wortmannin, LY294002). Autophagy inhibition resulted in significant induction mainly of IL-1b and TNF-a release as detected at the cell culture supernatants after 16h of stimulation. The effect was less prominent on IL-6 production. Most importantly IL-10 production was drastically reduced in the presence of autophagy inhibitors. Activation of specific autophagy proteins in THP-1 cells infected with B. melitensis was detected at different time points of infection. Differentiation of THP-1 cells to macrophage phenotype induced autophagy as detected by the formation of LC3II complex. Prolonged stimulation with B. melitensis (2–24h) neither altered the formation of LC3II complex nor affected the amount of ULK1 protein. Microarray analysis of the transcriptional response of peripheral human monocytes 4h postinfection with B. melitensis, revealed a dramatic increase of a protein upstream of LC3II that has an essential role in regulation of autophagy. These results suggest that autophagy differentially regulates cytokine response against B. melitensis in humans, modulating inflammation probably by interfering in signaling pathways initiated by key autophagy proteins.
Partial role of CXCR3 in the migration of CD4 T cells into the lung of TB infected mice
Authors: Hoft Stella G , Sallin Michelle A , Sakai Shunsuke , Barber Daniel L
Published: January 2023

Mycobacterium tuberculosis (Mtb) is the leading cause of death by infectious disease worldwide. In order to develop effective vaccination strategies for Mtb, it is critical to understand the properties of host-protective CD4 T cells. We have shown that various subsets of Mtb-specific Th1 cells display dramatically different lung migratory abilities. CXCR3+ Th1 cells migrate into the lung and control Mtb infection, while CX3CR1+ Th1 cells are retained in the lung-associated blood vasculature and do not suppress bacterial growth in vivo. However, little is know about the precise role of different chemokine receptors in the entry of Mtb-specific Th1 cells into the lung. Here we quantify the individual contributions of several chemokine receptors in the pulmonary migration of Th1 cells by performing in vivo migration experiments after adoptive transfer of WT and CCR KO Mtb-specific lung effector T cells into infection matched WT recipients. Firstly, we find that Th1 cells migration into the lung is pertussis toxin sensitive, indicating that G-protein coupled receptors are required. We find that while CXCR6 is highly expressed in Th1 cells capable of migrating into the lung, it has no role in their entry. Likewise, CCR2 had no role in Th1 cell migration into Mtb infected lungs. Interestingly, CXCR3−/− CD4 T cells displayed only a ~30% reduction compared to WT cells 16 hours after transfer. Although CXCR3 and CXCR6 are defining markers expressed on all lung-homing Mtb-specific Th1 cells, our results indicate that additional chemokine receptors are involved in their migration. Identification of new mechanisms governing the migration of Mtb-specific Th1 cells into the lung may give important insight into T cell dependent immunity to Mtb infection.
TACI contributes to Plasmodium yoelii host resistance by controlling the kinetics of Tfh and GC formation and antibody development
Authors: Parra Marcela , Yang Jiyeon , Schmidt Thomas , Derrick Steven , Yang Amy , Liu Xia , Siddiqui Shafiuddin , Akkoyunlu Mustafa
Published: January 2023

The transmembrane activator and calcium-modulator and cyclophilin ligand receptor (TACI) is involved in B-cell survival, antibody isotype switching and plasma cell generation. TACI expression is severely impaired in murine and human newborns as compared to adults. We challenged TACI knock-out (KO)-mice with Plasmodium yoelii (Py) NL in order to evaluate the role of TACI in response to malaria infection. We found that the parasitemia levels were significantly elevated in TACI KO-mice (61.8% at day 18) compared to WT-mice (11.1% at day 11), and that parasite clearance was substantially delayed in the TACI KO (27 days) mice relative to WT controls (18 days). We also determined that TACI KO-mice have a delay in anti-Py NL IgG-antibody production when compared with the WT-mice. Since the interaction of T follicular helper (Tfh) cells with antigen specific B cells in the germinal center (GC) is essential for the generation of antibody responses, we measured the formation of Tfh and GC in the spleens of Py NL infected mice. Interestingly, we determined that while Tfh cell numbers were elevated on day 10 in both the strains, WT mice Tfh cell numbers sharply declined by day 15 as the number of Tfh cells in TACI KO-mice remained high. Coinciding with parasite clearance, TACI KO Tfh cells declined to baseline values on day 25 and GC B cells remained high beyond day 15 when WT GC B cells declined. Finally, we detected elevated serum levels of the TACI ligand B cell-activating factor belonging to the TNF family (BAFF) in infected TACI KO mice as compared to the WT mice. In conclusion, susceptibility of TACI deficient mice to Py NL infection appears to be based on altered Tfh and GC kinetics, which in turn results in delayed antibody development.
The m153 gene product stabilizes expression of the inhibitory NKR-P1B ligand, Clr-b, during mouse cytomegalovirus infection
Authors: Aguilar Oscar A , Rahim Mir Munir A , Sampaio Isabella S , Samaniego Jackeline D , Popović Branka , Tilahun Mulualem E , Krmpotić Astrid , Margulies David H , Allan David S.J. , Makrigiannis Andrew , Jonjić Stipan , Carlyle James R
Published: January 2023

Natural killer (NK) cells are a subset of innate lymphoid cells (ILC) capable of recognizing stressed and infected cells through multiple germline-encoded receptor-ligand interactions. Missing-self recognition involves NK cell sensing of the loss of host-encoded inhibitory ligands on target cells, including MHC class I (MHC-I) molecules and MHC-independent ligands. Mouse cytomegalovirus (MCMV) infection has been shown to promote a rapid loss of the inhibitory NKR-P1B ligand, Clr-b, on infected cells. Here, we provide evidence that an MCMV m145 family member, m153, functions to stabilize Clr-b at the cell surface during MCMV infection. Ectopic expression of m153 in fibroblasts significantly augments Clr-b cell surface levels. Moreover, infections using m153-deficient MCMV mutants (Δm144-m158; Δm153) show an accelerated and exacerbated Clr-b downregulation. Importantly, enhanced loss of Clr-b upon infection with MCMV Δm153-mutants can be reverted to wild-type levels by exogenous m153 complementation in fibroblasts. While the effects of m153 on Clr-b levels are independent of Clec2d transcription, imaging experiments reveal that the m153 and Clr-b proteins only minimally co-localize within the same subcellular compartments, and tagged versions of the proteins were refractory to co-immunoprecipitation using gentle detergents. Indeed, a prominent intracellular vesicular localization of m153 suggests that its effects on Clr-b stabilization may be indirect. In vivo, the Δm153-mutant possesses enhanced virulence, independent of Clr-b and NKR-P1B, suggesting that m153 may modulate other Clr or activating NKR:ligand interactions.
Transgenic West Nile Virus virus-specific CD8+ T cells provide insights on activation patterns and protective role of cytotoxic T cells during infection
Authors: Aguilar-Valenzuela Renan , Seo Young-Jin , Grakoui Arash , Suthar Mehul S
Published: January 2023

West Nile Virus (WNV) is the leading cause of mosquito-borne encephalitis worldwide. Currently, there are no vaccines or therapeutics approved for use in humans. Virus-specific CD8+ T cells are essential in mediating protective immunity during WNV infection. However, the mechanisms that promote protective CD8+ T cell immunity against WNV are not well understood. In this study, we characterized a novel transgenic mouse encoding CD8+ T cells to the immunodominant epitope located within the WNV NS4b protein (WNV-I). Through adoptive transfer studies in mice, we evaluated the expansion, migration, and functional properties of WNV-I CD8+ T cells during infection. In the spleen, WNV-I CD8+ T cell numbers increased on day 7 post-infection followed by contraction in cell numbers through day 15 post-infection. We observed a robust infiltration of virus-specific CD8+ T cells to the brain on day 7 concomitant with an increase in virus replication within the CNS. Interestingly, we observed clearance of infectious virus from the brain, however, WNV-I CD8+ T cells remained elevated in the brain through day 15 post-infection. Functionally, WNV-I CD8+ T cells secrete both IFN-γ and TNF-α, with peak responses in the spleen and in the brain on days 7 and 11 post-infection, respectively. Furthermore, Rag-1−/− mice that received WNV-I CD8+ T cells had lower viral burden in spleens and serum at day 7 of infection. Thus, WNV-I cells are functionally active and can mediate protective immunity against WNV infection. Further studies are underway to better understand how CD8+ T cells mediate WNV clearance in the central nervous system.
The CD4 T cell response in acute infection with dengue virus
Authors: Weiskopf Daniela , de-Oliveira-Pinto Luzia M , Tian Yuan , de Silva Aruna D , Seumois Gregory , Vijayanand Pandurangan , Premawansa Sunil , Premawansa Gayani , Wijewickrama Ananda , Peters Bjoern , Sette Alessandro
Published: January 2023

Dengue virus (DENV) is the etiologic agent of dengue fever (DF), the most significant mosquito-borne viral disease in humans. DENV transmission occurs in more than 100 countries and is an increasing public health problem in tropical and sub-tropical regions. Up to 400 million DENV infections occur every year and severity can range from asymptomatic to an acute self-limiting febrile illness (DF) or the more severe hemorrhagic fever (DHF) characterized by severe vascular leakage, thrombocytopenia, and hemorrhagic manifestations. In this study we have investigated the acute CD4+ T cell response in hospitalized patients with different severity of disease manifestations. Samples from patients either diagnosed with DF or DHF have been collected in the acute phase and in the convalescent phase after resolving the infection. Virus specific CD4 T cells were detected based on their ability to produce cytokines in response to stimulation with DENV specific peptide pools in an intracellular cytokine assay. All donors elicited IFNγ in the convalescent phase of infection, regardless of the severity of disease. Interestingly, in the acute phase DF patients elicited IFNγ in response to the peptide pool, while the response in DHF patients was dominated by IL-10. Further characterization revealed that these responding IFNγ and IL 10 producing CD4 T cells differ in memory phenotype as well as activation markers. We are currently establishing specific gene expression signatures, associated with these characteristic phenotypes. Detailed knowledge of phenotype and function of DENV specific T cells will aid in the establishment of correlates of protection against severe disease.
Immune signatures of CD4 T cell subsets in healthy and dengue virus-infected individuals
Authors: Tian Yuan , Babor Mariana , Seumois Gregory , Vijayanand Pandurangan , Weiskopf Daniela , Peters Bjoern , Sette Alessandro
Published: January 2023

Dengue virus (DENV) has spread through most tropical and subtropical areas of the world and represents a serious public health problem. The control of DENV infections has been hindered by lack of effective therapeutics or vaccines. Nevertheless, a better understanding of the immune system both at steady state and following DENV infections may reveal new strategies for eliciting and improving anti-DENV immunity. T cells provide protective immunity against various viral infections, and recent findings demonstrate that a subset of CD4 T cells, designated effector memory re-expressing CD45RA T (Temra) cells, expand following DENV infections and may play an important role in protecting the host from severe dengue diseases. In this study, we report that CD4 Temra cells display unique gene expression profiles characterized by the upregulation of cytotoxic molecules such as perforin, granzyme B and GPR56, as well as transcription factors including T-bet, Eomes, Runx3 and Hobit, which direct T cell differentiation, function and tissue residency. Furthermore, our results indicate that CD4 Temra cells are heterogeneous and can be largely divided into two sub-populations, which exhibited differential expression of various effector molecules and transcriptional regulators. Finally, using high-dimensional mass cytometry, we further dissected the heterogeneity of DENV-specific CD4 Temra cells. Over 30 molecules were analyzed simultaneously, resulting in the identification of novel subsets within the CD4 Temra population. Overall, our findings reveal immune signatures of CD4 Temra cells in healthy and DENV-infected individuals and provide new insights into the antiviral immunity against DENV and potentially other emerging pathogens.
T cell receptor repertoire in Kenyan HIV exposed but uninfected infants at birth: evidence of in utero clonal expansions
Authors: Gabriel Benjamin , Bosire Rose , Mbori-Ngacha Dorothy , Farquhar Carey , John-Stewart Grace , Overbaugh Julie , Richardson Barbra , Payne Barbara Lohman
Published: January 2023

With the expansion of global strategies to prevent mother to child transmission of HIV, the number of perinatal HIV infections had dropped dramatically. This public health achievement has created a population of HIV exposed but uninfected (HEU) infants with substantially more morbidity and mortality than their HIV unexposed (HU) peers. We hypothesize that in utero exposure to maternal HIV induces a premature activation and expansion of fetal T cells, potentially leading to infant immune dysregulation (Th1 polarization/immune exhaustion) upon exposure to pathogens after birth.

We selected pre-ARV era cord blood samples from 17 HEU infants born to viremic Kenyan women and 17 cord blood samples from HIV unexposed infants born to Kenyan women with similar sociodemographics. Next generation technology including addition of unique molecular identifiers was used to generate amplicon libraries for sequencing (MiSeq, Illumina). We calculated the Simpson Diversity Index for the 100 most frequent clones per TRB family. The median Ds for HU cord blood samples was 0.1, representing near maximal diversity of the TCR repertoire. Overall, HEU samples displayed a 30% reduction in diversity compared to the samples from HU infants, indicative of fetal T cell clonal expansion. Of note, the median diversity of Kenyan HEU samples located closer to the diversity of American adult peripheral blood than to the diversity of American cord blood.

Although the antigen specificity of these cells is unknown, these results support the hypothesis that HIV exposure in utero stimulates fetal immune responses and point towards the existence of specific features of the immune system of newborn HEU infants which might be linked to their poor morbidity and mortality rates.
The contribution of CD8 T cells targeting Cryptic Epitopes on viral evolution during acute HIV-1 infection.
Authors: Peng Binghao J , Carlson Jonathan M , Hunter Eric , Bansal Anju , Goepfert Paul A
Published: January 2023

Alternative reading frames (ARF) of HIV, in both sense and antisense directions, have been shown to produce polypeptides of unknown functions. These peptides, known as cryptic epitopes (CE), commonly induce CD8 T cell responses in HIV; however, their role in controlling HIV-1 infection remains unclear.

We hypothesized that CD8 T-cells target CE shortly following acute HIV infection and force viral escape. Such a finding would indicate that CE may serve as viable immunogens for inclusion in preventative HIV-1 vaccines. Using an HLA-I binding prediction algorithm applied to fixed mutations in ARF during the first two years of infection in a Zambian patient cohort, we predicted 66 CD8 T-cell escape mutations within gag, pol, and nef, which we further tested for immunogenicity.

8/32 chronic patient samples responded to at least one predicted CE as shown by IFNγ ELISpot. Furthermore, all ten immunogenic CE identified in the study were derived from a single antisense reading frame within the coding region of pol. Longitudinal studies failed to show any evidence of early viral escape to CE, as evaluation of these CE-specific CD8 T-cells following acute infection was not temporally associated with the occurrence of potential escape mutations. Our data suggest that although T-cell responses to CE can be detected during early HIV infections, they rarely establish HIV-1 escape mutations. As such their contribution to viral control is likely limited. Potential reasons for their limitations in forcing escape mutations include their infrequent expression and the weak magnitude of T-cell responses generated. Nevertheless, these data indicate that cryptic epitopes are not optimal targets for a preventative HIV vaccine.
Activation of intrahepatic mucosal associated invariant T cells resolves with antiviral therapy for hepatitis C
Authors: Bolte Fabian J , O’Keefe Ashley C. , Webb Lauren M , Serti Elisavet , Rivera Elenita , Liang Jake , Ghany Marc , Rehermann Barbara
Published: January 2023

BACKGROUND

Chronic hepatitis profoundly affects the phenotype of innate and adaptive immune cells. Mucosal associated invariant T (MAIT) cells are innate-like T cells that are enriched in the intestine and the liver. They can be activated by cytokines and by bacteria translocated from the gut. The relative roles of cytokines and bacteria in MAIT cell activation and function are unknown in chronic hepatitis.

METHODS

MAIT cells and monocytes were studied in liver and blood of 37 patients with chronic hepatitis C prior to and after Sofosbuvir/Velpatasvir-induced viral clearance.

RESULTS

MAIT cells were more frequent and showed a more activated and cytotoxic phenotype in the liver than in the blood. The frequency of intrahepatic MAIT cells correlated inversely with liver inflammation. The amelioration of liver inflammation by week 4 of antiviral therapy was associated with a significant decrease in MAIT cell activation and cytotoxicity. MAIT cell activation correlated with the frequency of intermediate/pro-inflammatory monocytes and the quality of MAIT cell effector responses was dependent on monocyte-derived IL-18. MAIT cell responses to T cell receptor-dependent (E. coli) and -independent (IL-12/IL-18) stimulation were not different before and after antiviral therapy and were not impaired compared to uninfected controls.

CONCLUSIONS

MAIT cells are activated by monocyte-derived cytokines in chronic HCV infection. They are reduced in number but maintain their response to cytokines and bacteria. MAIT cell activation rapidly declines during antiviral therapy and their frequency increases in the liver.
Neonatal mouse model to study Zika virus pathogenesis: Host immune response determines ZIKV tropism and outcome of disease.
Authors: Manangeeswaran Mohanraj , Ireland Derek D.C. , Sykes Jacob , McWilliams Ian , Verthelyi Daniela
Published: January 2023

Zika virus (ZIKV) is a mosquito-borne flavivirus that has spread to more than 70 countries and has infected more than 2 million people worldwide. The recent ZIKV outbreak has resulted in CNS complications including microcephaly, Guillain Barre and polyneuropathy. Current ZIKV infection models use interferon (IFN) deficient mice that develop high viral loads systemically and die within a week. Establishing an Immunocompetent animal model that replicates ZIKV neurotropism is critical to understand ZIKV pathology and testing of immunotherapies. We developed an immunocompetent neonatal mouse model in wild type C57BL/6 mice by challenging 1 day old mice with contemporary ZIKV isolate PRVABC59. Infected mice developed unsteady gait, loss of balance, kinetic tremors, severe ataxia and seizures beginning 13 days post infection (dpi) that resolved by 28 dpi. Following challenge the mice developed a systemic infection with high virus titers in spleen, liver and kidney that was cleared by 10 dpi. In the Central Nervous System (CNS) and the eye, the virus was detected starting 6 dpi, peaked at 9–15 dpi, and then decreased over 6 weeks. Unlike IFN deficient that have a significant increase in mRNA expression of complement (C3), Cox2, Il1a, Il1b, and Il6, the CNS of infected B6 mice showed increased expression of genes associated with T cell responses including CD3, CD4, CD8, MHC-II, CD80, IFNg, perforin and granzyme B., Despite the absence of clinical disease in convalescent B6wt mice, there remain increased expression of inflammatory and T cell associated genes that suggest that the virus could have long-term effects on the CNS. Further investigation including behavioral studies is needed to explore possible long term CNS sequelae of ZIKV infection.
T cell infiltration associated with neurodegeneration and Zika virus control in the CNS of immunocompetent, neonatal mice.
Authors: Ireland Derek D.C. , Manangeeswaran Mohanraj , Miller-Novak Leia , Sykes Jacob , Verthelyi Daniela
Published: January 2023

The recent spread of Zika virus (ZIKV) and its association with neurological disorders as well as congenital defects has created an urgent need to develop animal models that allow the elucidation of pathogenesis following ZIKV infection and to develop and test therapeutic agents that will prevent or eliminate CNS disease. We have developed a novel model of ZIKV infection in mice, using B6 WT, neonatal mice that are susceptible to contemporary ZIKV (PRVABC59) CNS infection following peripheral, subcutaneous inoculation at P1. Immunocompetent B6 WT mice develop a discrete neurological disease that includes: unsteady gait, kinetic tremors, severe ataxia and seizures by 12–15 dpi that gradually resolve. Gene expression and immunohistochemistry demonstrate inflammation in the CNS of ZIKV infected B6 WT mice starting after 9 dpi and infiltration of immune cells into the parenchyma of the CNS beginning concomitantly. At the peak of disease (15 dpi), immune cell infiltrates in the CNS consisted primarily of T cells, particularly CD8+ T cells. Gene expression analysis indicated an upregulation of genes consistent with this infiltration. Immunohistochemistry of the CNS indicates a concomitant increase in apoptosis and neurodegeneration after 9 dpi in the CNS particularly in the granular and Purkinje cell layers of the cerebellum. The role of T cells in the control of ZIKV infection in the CNS and the periphery will be explored.
Differential susceptibility to granzymes drives delayed apoptosis of HIV-infected macrophages and promotes CTL pro-inflammatory cytokine release
Authors: Clayton Kiera , Collins David , Dotiwala Farokh , Lieberman Judy , Walker Bruce D
Published: January 2023

Although CD4+ T cells represent the main target cell for HIV/SIV infection, macrophages also contribute to viral reservoirs. Given CD8+ cytotoxic T lymphocytes (CTL) are essential for controlling viral replication, characterizing how they interact with HIV-infected targets will provide insights into mechanisms of reservoir persistence and cure strategies. CTL killing of SIV-infected macrophages is less efficient than CD4+ T cells, but the mechanism of this differential killing and whether this applies to HIV is not known. Here, we demonstrate that in contrast to infected CD4+ T cells, HIV-infected macrophages are intrinsically resistant to ex vivo CTL-mediated killing, resulting in inefficient suppression of viral infection. However, when macrophages are killed, apoptosis is delayed and caspase-3-dependent versus CD4+ T cell apoptosis, which is rapid and caspase-independent. Induction of these separate apoptosis pathways is a result of differential sensitivity to granzymes, specifically granzyme B, which is poorly expressed by ex vivo HIV-specific CTLs. Differential killing is not a consequence of poor recognition. On the contrary, CTL interaction with antigen-loaded macrophages is associated with longer effector-target contact time and increased CTL production of IFN-γ. This excess IFN-γ induces production of macrophage pro-inflammatory chemokines, CXCL9 and CXCL10, which recruit activated CD4+ T cell targets. These results suggest that inefficient CTL killing of HIV-infected macrophages may contribute to reservoir persistence, chronic inflammation, and recruitment of CD4+ T cells to sites of infection.
Systematic analysis of T cell responses specific to the Epstein-Barr virus proteome using ATLAS™
Authors: Kaufmann Johanna K , Liu Biao , Jacques Judy , Wu Ning , Yan Zheng , Alami Aula , Kelley Christine , O’Keeffe Michael S , Hoover Maegan E , McManus Margaret M , Henderson Jennifer , Ghamsari Lila , Olivieri Kevin C , Flechtner Jessica B , Broom Wendy , Flano Emilio , Luzuriaga Katherine
Published: January 2023

Epstein-Barr virus (EBV) is a human herpesvirus infecting 95% of the world population. Primary infection can cause acute infectious mononucleosis (AIM), mainly when EBV is acquired later in life. In addition, EBV establishes latency and has been associated with several cancers. To date, efforts to develop vaccines for prevention of infection or disease by induction of gp350-specific neutralizing antibody responses have failed. To improve antigen selection for future vaccines, we utilized ATLAS™, a screening platform that can characterize the specificity of CD4+ and CD8+ T cell recall responses to the entire EBV proteome without bias of immunodominance.

Firstly, we measured antigen-specific T cell activation in 15 AIM patients longitudinally during and 6 weeks after the symptomatic disease phase. For both T cell subsets, the breadth of response significantly decreased with resolution of disease, likely in parallel to the contraction of the CD8+ T cell compartment associated with AIM clearance. Notably, we detected a substantial overlap in antigen specificity between T cell responses in samples collected during AIM and in convalescence.

Secondly, we identified EBV antigens that elicit T cell responses in both seropositive and seronegative but possibly exposed adolescents. Interim analysis revealed that ATLAS™ not only confirmed several previously described CD4+ and CD8+ T cell targets in latently infected individuals, but also identified novel T cell antigens independently of serostatus. Ongoing comparative analyses will focus on differences in the memory T cell repertoire between cohorts and on antigens overrepresented in exposed seronegative individuals that may be protective and therefore of value for vaccine development.
Cognate epitope is dispensable in maintenance of PD-1 expression on tissue-resident CD8+ cells
Authors: Shwetank  , Frost Elizabeth L. , Abdelsamed Hossam Aly , Mockus Taryn , Maru Saumya , Youngblood Ben A , Lukacher Aron
Published: January 2023

Persistent CNS infection by mouse polyomavirus (MuPyV) establishes a tissue-resident memory (TRM) CD8+ T cell population possessing higher TCR affinity than the memory cells in spleen. This necessitates a mechanism to regulate the functions of a non-replenished TRM population. Here, we report that virus-specific CD8+ T cells express PD-1 during acute infection and remain PD-1hion brain-infiltrating T cells. In contrast, antiviral memory CD8+ T cells in the spleen downregulate PD-1 expression during persistent infection, despite having similar viral load as the brain. The Pdcd1 promoter was found to be partially remethylated following its downregulation in the splenic population while the locus remained unmethylated in the brain-resident cells. Antiviral TRM cells purified from infected brain when transferred into a naïve mouse via intracerebral route, remained PD-1hi. Thus, epitope as well as inflammation are dispensable for maintenance of PD-1 expression. Absence of PD-1 signaling in an in vitro peptide stimulation assay resulted in augmented IFN-γ production and in vivo absence of PD-1 or PD-L1 resulted in increased IFN-γ-dependent microglial activation, indicating that PD-1 operates to limit CD8+ T cell-dependent neuroinflammation. PD-L1 was found to be expressed on infiltrating myeloid cells and astrocytes; astrocytes were also found to support productive MuPyV infection. PD-L1−/− mice had fewer anti-MuPyV brain-TRM cells, but increased proportion of the cells that were CD103hi, an αE integrin expressed by TRM cells. In summary, our data shows that PD-1 plays an important role in maintenance of antiviral CD8+ TRM cells in the brain and its expression is independent of the cognate epitope.
CD11c+ MHCIIint bone marrow-derived dendritic cells as adjuvants for neoepitope – based cancer immunotherapy
Authors: Ebrahimi-Nik Hakimeh , Corwin William L. , Yamamoto Ryo , Srivastava Pramod K
Published: January 2023

Total splenocytes, macrophage and bone-marrow-derived dendritic cells (BMDCs) were compared for adjuvanticity in a tumor rejection assay, where a neoepitope of the BALB/c Meth-fibrosarcoma, was the immunogen. BMDCs showed the highest activity, providing 100% tumor protection. BMDCs were sorted into various sub-populations, each of which was tested individually in parallel in the same assay. The CD11c+, MCHIIint, CD11bhi, CD86−, CD40−, CD24− BMDCs provided the highest tumor protection. The high adjuvanticity of BMDCs was not MHC I-dependent, as the neoepitope-pulsed BMDCs from C57BL/6 or BALB/c mice provided roughly equivalent protection. These data show that as adjuvants, BMDCs function as antigen reservoirs, facilitating cross-presentation by endogenous antigen presenting cells. These results have obvious implications for neoepitope-based human cancer immunotherapy.
Tumor Lysate Loaded Dendritic Cells Induce a T cell Specific Antitumor Response Against Gallbladder Cancer
Authors: Rojas-Sepúlveda Daniel Alberto , Gleisner Mª Alejandra , Pereda Cristian , López Mercedes , Salazar-Onfray Flavio
Published: January 2023

Introduction

Although infrequent, Gallbladder cancer (GBC) has high incidence rates in South America. In fact, Chile has the highest GBC incidence and mortality worldwide. Currently, surgery is the only effective treatment, and the five-year survival rate is less than 10%. Previously, we demonstrated that allogeneic melanoma cell lysates-loaded dendritic cells (DCs) provide a standardized, applicable tumor-specific vaccine; capable to induce a cell mediated immune response in melanoma patients, improving survival. Here, we investigated the effect of tumor lysates derived from gallbladder cancer cell (GCC) lines on immature DCs and their capacity to induce a T cell mediated anti GBC responses in vitro.

Material and Methods

DCs were stimulated with GCC lysates and analyzed by flow cytometry for a panel of surface markers. Autologous CD3+ T cells were co-cultured with these DCs for 14 days. CD4+ and CD8+ T cells were analyzed by flow cytometry for activation and chemokine receptor expression. CD8+ T cells were isolated and co-cultured with GCC lines GBd1, TGBC-2TKB and melanoma cell line Mel1 for 16hrs. IFN-γ secretion was measured by ELISPOT.

Results

All GCC lysates induced DCs maturation, increasing surface expression of MHC-I, MHC-II, CD80, CD83, CD86, CCR7. DCs maturated with GCC lysate induced CD3+ T cells activation, increasing expression of CD25, CD69, CXCR3, CXCR4 in both CD4+ and CD8+ T cells compared with non-stimulated lymphocytes. Additionally, CD8+ T cells co-cultured with GBC-loaded DCs, released higher amounts of IFN-γ than controls.

Discussion

Based on these results, DCs maturated with GCC lysates are capable to induce specific T cells activation against GBC and can be considered for future immunotherapy approaches.
Activation of the TLR2-MyD88 pathway is required for in-vivo efficacy of Lipid Nanoparticle based vaccine formulation
Authors: Swaminathan Gokul , Lin Shu-An , Patel ManishKumar , DiFelice Katie , Smith Jeffrey , Gindy Marian , Mahne Ashley , Cua Daniel , Casimiro Danilo , Espeseth Amy , Bett Andrew
Published: January 2023

Emerging evidence suggests that bioengineered nanoparticles can be used as immunomodulatory agents. Using murine and non-human primate vaccination models, we have identified novel cationic lipid nanoparticles (LNPs) that significantly enhance antigen-specific B-cell, CD4+T cell, and most notably, CD8+ T-cell responses. Importantly, we found that LNP-containing vaccine formulations resulted in protective efficacy in a virus challenge model, as well as, in syngeneic murine tumor models. In-vivo imaging and immunophenotyping studies showed that LNPs prolong antigen retention at the injection site and enhance antigen migration to the lymph nodes. Unbiased screening of LNPs ability to activate innate immune sensors showed that LNPs selectively activate Toll-Like Receptor-2 (TLR2). Using genetic ablation, and antibody mediated blockade, we uncovered that TLR2-MyD88 pathway is required for LNPs ability to boost immune responses in-vivo. Ex-vivo and in-vitro studies using dendritic cells (DC) showed that neutralization of TLR2 does not impact the LNP mediated increase in antigen uptake. However, LNP dependent enhancement of DC maturation, antigen processing & presentation, and chemokine production, were significantly reduced when TLR2 was neutralized. Pharmacological inhibition TIRAP, and CD14-MD2, further confirmed the need for the activation of TLR2-MyD88 pathway in LNP’s functionality. Future studies are aimed at utilizing chosen Merck chemical inhibitors of innate immune kinases to further interrogate the TLR2-MyD88 pathway’s involvement in LNP functionality. Such detailed knowledge of LNP’s mode of action will significantly influence the future design of LNPs for vaccines and immune oncology applications.
Targeting adenosine A2A receptors to improve vaccine efficacy
Authors: Cekic Caglar , Savas Ali Can , Kayhan Merve , Koyas Altay , Akdemir Imran
Published: January 2023

Activation of immune cells is associated with increased expression of adenosine receptors. Extracellular adenosine is present in primary and secondary lymphoid organs and limit the amplitude of immune responses suggesting that antagonists for adenosine receptors can be targeted to improve the efficacy of vaccines. Here we showed that vaccine adjuvant Monophosphoryl Lipid A (MPLA) strongly increased adenosine A2A and adenosine A2B receptor expression in primary macrophages and dendritic cells. Adenosine A2A but not adenosine A2B receptor blockade significantly increased both numbers and percentages of antigen specific endogenous T cells in both spleen and draining lymph nodes after primary immunizations with MPLA and ovalbumin. We observed a similar increase in both endogenous or adoptively transferred T cells in mice with myeloid-specific deletion of A2A receptors after primary immunization. Adenosine receptor blockade significantly increased the total IgG titers and IgG2c/IgG1 ratio after rechallenge with ovalbumin. These results suggest that adenosine A2A receptor blockade potentially improve vaccine efficacy by promoting both cellular and humoral immune responses and promoting Th1 type immunity. Our results also suggest that A2ARs on antigen presenting cells are important targets to improve vaccine efficacy by adenosine A2A receptor blockade. These findings have important implications for the design of novel and more efficacious vaccine formulations.
Blocking CD13 in combination with a toll like receptor agonist increases the efficacy of cancer vaccine adjuvants
Authors: Qendro Veneta , Ghosh Mallika , Shapiro Linda
Published: January 2023

Vaccines have been an extraordinary tool in the fight against human disease. Recent cancer research has revealed promising results leading to approved cervical and prostate cancer vaccines. Despite the success, a major downfall of these vaccines remains poor immunogenicity.

MPLA (monophosphorylated lipid A) is a synthetic analog of LPS that binds to TLR4 leading to activation of the adaptive immune response without triggering deleterious inflammatory consequences. As such, MPLA has recently been FDA approved as an adjuvant of the prophylactic vaccine against HPV-driven (Human Papilloma Virus) cervical cancer. However, this vaccine is limited to non-preexisting HPV cases while protecting against only two out of the dozen HPV strains that can potentially lead to cervical cancer.

Pertinent to this subject, we have found that the absence of CD13 in antigen presenting cells (APCs), leads to increased TLR4 endocytosis and bias toward the same endocytic-signaling pathway triggered by MPLA. CD13 is a large, multifunctional cell surface peptidase constitutively expressed on all lineages of myeloid cells. We have previously shown that lack of CD13 increases tumor antigen uptake and presentation, resulting in enhanced activation of tumor-specific cytotoxic T cells.

The combination of these facts has led to the intriguing possibility that blocking CD13 may amplify the efficacy of MPL as an adjuvant and ameliorate the efficacy of prophylactic and therapeutic cancer vaccines. Therefore, we hypothesize that CD13-blocking agents will act as dual-action adjuvants by enhancing MPL-TLR4 endocytosis, and as a delivery system through increased endocytic antigen uptake and subsequent cytotoxic T cell activation.
Combination of Peptide-based Vaccine and Chinese Herbal Medicine as a Strategy to Enhance Immune responses in Virus-associated Cancer
Authors: Song Ying-Chyi , Yen Hung-Rong
Published: January 2023

Virus infections may account for the development of several cancers, such as HPV16/18 are known to cause around 70% of cervical cancer cases. Until now, several immunotherapeutic approaches for virus-induced cancer are under development. Peptide-based vaccines have several advantages over conventional whole-protein vaccines in terms of purity, lot-to-lot consistency, production costs, and the high antigenic specificity. However, the use of peptide antigens in vaccine development has been hampered by problems, such as weak immunogenicity coupled with a paucity of potent adjuvants. Specific T cell immune response is crucial for anti-tumor immunity. Manipulating specific T cell immunity by Chinese herbal medicine (CHM) is a promising field to explore. Therefore, we aimed to investigate whether CHM can modulate specific T cell responses to apply for the development of cancer vaccine. We have identified a CHM extract that could increase expression of maturation cytokines and activation markers of murine bone marrow-derived dendritic cells. Furthermore, in a murine TC-1 tumor-bearing model, we found that the CHM extract could act as an adjuvant to induce cellular immune responses and anti-tumor effect in peptide vaccine strategy. We suggested that immune-stimulator CHM combined with cancer vaccine endows them with increased immunologic activity, which may be used to bypass the requirement for the conditional adjuvant. Further delineation of the mechanism may provide new clues for vaccination strategy.
Dendritic cell CD40 targeting vaccines elicit CD8+ T cell-mediated therapeutic immunity against cancers
Authors: Yin Wenjie , Duluc Dorothée , Joo HyeMee , Xue Yaming , Gu Chao , Wang Zhiqing , Wang Lei , Oxford Lance , Clark Amelia , Parikh Falguni , Thompson-Snipes LuAnn , Woo Jung-Hee , Zurawski Sandra , Sikora Andrew G , Zurawski Gerard , Oh SangKon
Published: January 2023

Dendritic cells (DCs) are major antigen-presenting cells that can efficiently cross-prime antigen-specific cytotoxic T lymphocytes (CTLs). Antigen targeting to DCs through surface receptors is therefore a promising strategy to mount CD8+ CTL-mediated immunity against viral infections and cancers. We have previously reported that viral (Influenza HA and NP) and tumor antigen (MART-1) targeting to human DCs via CD40 resulted in greater antigen-specific functional CD8+ CTL responses in vitro. In this study, we have tested CD40 targeting prototype vaccines for HPV16-associated malignancies (anti-CD40-HPV16.E6/7) and prostate cancer (anti-CD40-PSA). Our data show that both anti-CD40-HPV16.E6/7 and anti-CD40-PSA prototype vaccines are able to activate antigen-specific CD8+ T cells in the blood of head-and-neck and prostate cancer patients, respectively. We further demonstrate that the in vivo administration of anti-CD40-HPV16.E6/7 or anti-CD40-PSA plus poly(I:C) can mount tumor-specific CTL-mediated therapeutic and preventive immunity against the respective tumors in human CD40 transgenic mice. Data from this study support the clinical development of CD40 targeting vaccines against cancers and viral infections.
MHC class II-peptide complexes displayed on activated T cells guide Treg suppression
Authors: Germana Sharon K , Fryer Madeline L. , Junius Steffie , Turka Laurence A , LeGuern Christian A
Published: January 2023

Regulatory T cell (Treg) therapy is a promising strategy for extending allograft survival. Treg suppression requires initial T cell receptor (TCR)/peptide-MHC-II (pMHC-II) contacts, the nature of which remains elusive. As inflammation facilitates transfer of pMHC-II from antigen presenting cells (APCs) to activated T cells, we hypothesize that during transplantation associated inflammation, pMHC-II relocate on alloreactive T effector cells and serve as Treg activation signals. To test this, suppression assays were performed using murine CD4+ CD25− (Teff), CD90 depleted APCs, and CD4+ CD25+ Treg cells of different MHC haplotypes. We also used a transplantation model of skin allografts in RAG1 deficient mice injected with Teff and Treg cells from various MHC backgrounds. Data from more than 150 co-culture experiments show that Treg suppression occurs only when Treg and Teff share the same MHC-II, but independently of Treg matching with APC MHC-II. Further experiments demonstrate that pMHC-II complexes, displayed on activated Teff cells, are crucial to the induction of Treg suppression. In vivo results recapitulate the in vitro data: Treg suppression, leading to graft survival, occurs only when Teff and Treg cells are MHC-II matched. Collectively, these findings suggest that suppression of alloreactivity is directed by Treg TCR recognition of pMHC-II complexes exposed on Teff cells. The nature, origin (host vs. donor), and significance of MHC-II loaded peptides have yet to be established. Future studies to improve targeted Treg therapies for transplantation tolerance will focus on mechanisms coupling Treg function with Teff cell activation.
Imaging flow cytometry enhances a new model of dynamic FOXO1 nuclear localization in human CD4 T cells
Authors: Golding Amit , Hritzo Molly
Published: January 2023

The forkhead box O (FOXO) family contains transcription factors critical to differentiation and homeostasis in T cells. FOXO1, acts as a key integrator of proliferation and differentiation signals in both effector T cells (Teff) and regulatory T cells (Treg). In Teff, when the phosphoinositide 3-kinase (PI3K)/Akt pathway is activated downstream of T cell receptor (TCR) signaling and co-stimulation, Akt phosphorylates FOXO1 and excludes it from the nucleus. This promotes T cell proliferation and effector functions. In contrast, nuclear FOXO1 is essential for Treg thymic development and peripheral stability. Multiple studies have examined the effects of FOXO1 deficiency or overexpression in T cells, but to date, few have been able to directly examine the dynamics of the native FOXO1 signaling in human T cells. The accepted method of examining FOXO1 signaling dynamics is nuclear/cytoplasmic protein extraction and subsequent western blotting (WB). In order to assess native FOXO1 signaling dynamics, we obtained the human CD4+ T cell line, HuT102. Using the classic WB approach, we demonstrate that HuT102 cells have more nuclear FOXO1 compared to a similar CD4+ T cell line, HuT78. However, WB does not allow us to examine FOXO1 signaling dynamics in subpopulations. To answer this question, we developed a staining protocol to assess FOXO1 nuclear translocation in HuT102 cells using imaging flow cytometry on an Amnis Imagestream MKII. We show that addition of PI3K and Akt inhibitors increases nuclear FOXO1, while TCR signaling mimics such as PMA/ionomycin and anti-CD3/CD28 decrease nuclear FOXO1 kinetically. Using this protocol, we are now able to directly examine native FOXO1 signaling dynamics in human CD4+ T cells.
Myeloid derived suppressor cells are controlled by regulatory T cells via TGF-b during murine colitis
Authors: Park Sung-Gyoo , Lee Cho Rong
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells composed of immature myeloid cells including precursors of macrophages, granulocytes, dendritic cells, and myeloid cells at early stages of differentiation. In mice, MDSCs are defined as CD11b+Gr1+ cells and are normally present in low numbers in the bone marrow and spleen of healthy mice. However, in response to inflammatory diseases, MDSCs accumulate in greater numbers in the spleen, blood, and lymph nodes where they suppress immune responses. Moreover, MDSCs are well-known regulators of regulatory T-cells (Treg cells); however, the direct regulation of MDSCs by Treg cells, has not been well characterized. Interestingly, we found that colitis caused by functional deficiency of Treg cells leads to altered expansion and reduced function of MDSCs. In addition, during differentiation of MDSCs in vitro from bone marrow cells, Treg cells enhanced MDSC function and controlled their differentiation in a mechanism involving TGF-β. Moreover, TGF-β-deficient Treg cells were not able to regulate MDSC function in an experimentally-induced model of colitis. Finally, we evaluated the therapeutic effect of TGFb-mediated in vitro differentiated MDSCs on colitis. Adoptive transfer of MDSCs that differentiated with TGF-β was more capable of preventing colitis than the transfer of those MDSCs that differentiated without TGF-β. Our results demonstrate an interaction between Treg cells and MDSCs that contributes to regulation of MDSC proliferation and acquisition of immunosuppressive functions.