bluuebunny/crossref_metadata_2025 huggingface dataset:
Heat shock protein gp96-Ig vaccine induces antigen specific CD8 T cell response in the placenta
Authors: Strbo Natasa , Romero Laura , Garcia Dennise
Published: January 2023

The placenta acts as a barrier against infections, due to multiple unique structural, cellular, and immune properties. The detrimental effects of congenital viruses, including Zika virus (ZIKV), on pregnancy and fetal outcomes occur in part because of impaired placental function and profound pathological changes have been observed infected placentas. Our hypothesis is that induction of appropriate vaccine-induced immune responses in the placenta are key to successful prevention of congenital viruses’ infections in developing fetus. The main goal of our study is to developed novel heat shock protein (HSP) based vaccine approach that will induce antigen-specific responses in the placenta and will lead to the prevention of the pathogen transmission to the fetus.

HSP gp96 is a biological adjuvant that activates antigen-presenting cells and simultaneously delivers antigen specific peptides to MHC I for CD8 T cell activation. B6 females were mated with B6 mice and gestation day (GD) 0.5 was determined by the presence of vaginal plug. At GD 5.5, 0.5 million of OT1-gfp cells were injected in the tail vein. Two days after, 2.5 million 3T3-gp96-OVA-Ig or 3T3-gp96-Ig cells were injected subcutaneously (SC). Five days later, pregnant uteri were collected and placental and decidual single cells suspension was obtained. Frequency of OT1-gfp cells was determent by flow cytometry.

We found that secreted gp96 vaccine delivered by SC route induces very high frequency of antigen specific CD8 T cells in the placenta (15%) and decidua (10%) without compromising placental or fetal viability.

Our findings are highly supportive of development of novel gp96-Ig vaccine as unique systemic and placenta-homing, antigen-specific CD8 CTL vaccine strategy.
IgH isotype-specific B cell receptor dosage regulates B cell fate
Authors: Wesemann Duane R , Granato Alessandra , Tong Pei , Zuo Teng , Chaudhary Neha , Han Seung Seok
Published: January 2023

Immunoglobulin heavy chain (IgH) isotypes (e.g. IgM, IgG and IgE) are generated as secreted/soluble antibodies (sIg) or as membrane-bound (mIg) B cell receptors (BCR) through alternative RNA splicing. IgH isotype dictates soluble antibody function, but how mIg isotype influences B cell behavior is unclear. We examined IgH isotype-specific BCR function by engineering polyclonal Ighγ1/γ1 and Ighɛ/ɛ mice, which initially produce IgG1 or IgE from their respective native genomic configurations. Transcripts producing IgM, IgG1, and IgE are produced in an alternative splice form bias hierarchy, where mIgμ>mIgγ1>mIgɛ—influencing respective BCR densities and fitness for populating the periphery—a pattern replicated in activated and memory B cells from wild-type mice. Restrained B cell development from Ighγ1/γ1 and Ighɛ/ɛ mice was proportional to sIg/mIg ratios, and was rescued by enforced expression of the respective mIgs. In addition, enhanced BCR signaling by way of PTEN deletion in IgE B cells generated functional IgE memory responses from IgE memory B cells themselves, which are normally absent under normal conditions, indicating that weak signal strength normally restrains IgE responses. Thus, IgH isotype-specific mIg dosage regulates B cell fate by influencing quantitative baseline BCR signal strength.
Sex-specific differences in the CD8+ T cell response to infection is linked to differences in IL-12 sensitivity
Authors: Mon Kristel Joy Yee , Goldsmith Elizabeth , Wang Jocelyn , Watson Neva B , Smith Norah , Rudd Brian D
Published: January 2023

It is well known that males and females respond differently to intracellular pathogens. In general, females mount more robust immune responses than males, which decreases susceptibility to infection but comes at the cost of increasing auto-immunopathology. However, the underlying basis for these sex-related differences is poorly understood. In this study, we investigated cell-intrinsic differences between male and female CD8+ T cells. First, we compared the ability of male and female CD8+ T cells to proliferate following in vitro stimulation. Interestingly, we found that female CD8+ T cells proliferate sooner and faster than their male counterparts. Next, we asked whether cell-intrinsic differences between male and female CD8+ T cells contribute to changes in the CD8+ T cell response to infection in vivo. We co-transferred equal numbers of male and female donor CD8+ T cells from TCR transgenic mice into the same host and compared their ability to respond to Listeria monocytogenes. The data indicated that female CD8+ T cells preferentially become short-lived effectors (SLECs), while male donor CD8+ T cells give rise to more memory precursor cells (MPECs), even when placed in the same environment. To gain insight into why female CD8+ T cells are inherently more responsive to infection, we performed a series of in vitro and in vivo experiments, whereby either antigen (signal 1) or IL-12 (signal 3) was titrated. These studies showed that male and female CD8+ T cells respond similarly to low amounts of cognate antigen, but female CD8+ T cells appear to be more sensitive to IL-12. Collectively, our data demonstrates that female CD8+ T cells are biased towards forming SLECs after infection because of their enhanced capacity to respond to IL-12.
Oxidative phosphorylation is progressively programmed during plasma cell differentiation
Authors: Price Madeline J , Barwick Benjamin G , Scharer Christopher D , Boss Jeremy M
Published: January 2023

The differentiation of immune cells requires a transition in metabolic programming to achieve effector function. Unlike T cells, the metabolic changes and epigenetic regulation of this process during the differentiation of antibody secreting plasma cells is not well characterized. Using in vivo and ex vivo LPS driven models of B cell differentiation, the metabolic changes and epigenetic regulation of metabolism was investigated. Naïve B cells (B220+GL7−CD138−) are metabolically quiescent, and upon activation through TLR4, B cells rapidly shift their metabolic program to use oxidative phosphorylation for their energetic demands. Activated B cells (B220+GL7+CD138−) use oxidative phosphorylation, but maintain a large spare respiratory capacity. In contrast, terminally differentiated, post-mitotic plasma cells (B220−GL7−CD138+) use oxidative phosphorylation at their maximal ability, with no spare respiratory capacity, suggesting unique metabolic changes are programmed upon differentiation. Next, an adoptive transfer model system that utilizes CFSE labeled B cells was used to distinguish molecular events during the proliferative/activation versus the differentiation phase. Analysis of epigenetic and transcriptome data in the two phases identified a subset of metabolic genes that underwent differentiation specific programming. Additionally, the role of key transcription factors was examined in terms of their control of the metabolic reprogramming steps. These results provide novel insights into how the step-wise changes in metabolism are programmed as quiescent B cells differentiate into antibody-secreting plasma cells.
The intracellular C5 system is critical to DAMP sensing and cellular responses in human monocytes
Authors: Niyonzima Nathalie , Freeley Simon , Arbore Giuseppina , Le Friec Gaelle , Lappegård Knut Tore , Mollnes Tom Eirik , Kemper Claudia , Espevik Terje
Published: January 2023

Cholesterol crystal (CC)-induced inflammasome activation is a key contributor to atherosclerosis. Phagocytosis of CC causes lysosomal damage, assembly of the NLRP3 inflammasome, activation of caspase-1 and release of mature IL-1β. We have demonstrated that complement activation by CC in serum leads to C5a-C5aR1 driven CR3 up-regulation, increased uptake and IL1B gene expression in monocytes. Intracellular complement activation now emerges as critical regulator of cellular responses for induction of Th1 cells. In this study we investigated if CC-induced NLRP3 activation and IL-1β release in monocytes involves intracellular C5 activation. We observed that resting monocytes contained stores of C5 and C5a that increased upon CC uptake. Reduction of intracellular C5 by siRNA treatment abrogated IL-1β secretion to the same extent as NLRP3 inhibition. We confirmed the importance of the intracellular C5a-C5aR1 axis using monocytes from a serum C5-deficient patient, which produced intracellular C5 and detectable C5a and mounted equally strong IL-1β response when compared to healthy donor monocytes. Monocytes expressed also active Factors B and D and contained C3 and C3 activation fragments suggesting that an intracellular C5 convertase could mediate intracellular C5 activation. This notion is supported by the finding that knockdown of Factor B and usage of a cell-permeable convertase inhibitor both reduced CC-induced IL-1β. These results indicate that intracellular C3/C5 convertase-driven C5 activation is required for DAMP sensing, such as for CC, and suggest a critical role of intracellular complement in sterile inflammation and related disease states – and, importantly, that this novel axis can be pharmacologically targeted.
Glioblastoma usurps host extracellular adenosine to circumvent host anti-tumor Responses
Authors: Bynoe Margaret S
Published: January 2023

Glioblastoma (GB) is the most aggressive, fatal, and least successfully treated of all solid brain tumors due to its high capacity to proliferate and extreme chemoresistance. Treatment for GB patients is managed with multimodal therapies including surgical resection (if possible), radiotherapy and chemotherapy with temozolomide (one of the few drugs shown to have some effect). Despite this, patient survival ranges between 6–18 months. Because GB is highly infiltrative, it often invades normal brain tissue, sometimes preventing surgical resection, which inevitably leads to recurrence. Adenosine is an immune suppressive signaling molecule that is generated by the enzymatic action of CD73. I tested the hypothesis that GB usurps host extracellular adenosine to circumvent host anti-tumor mechanisms by down regulating macrophage/microglial cell immune responses and promote M2 instead of M1 macrophage/microglial phenotype. To test this, I isolated macrophage/microglial cells from the tumor microenvironment of mice lacking or expressing CD73. Compared to WT mice, the frequency of M1 verses M2 phenotypes showed that eighty percent of the macrophage/microglial cells present in the tumor microenvironment of CD73−/− mice exhibited an M1 phenotype, while WT mice had about 30 percent M1 phenotype.
Exosomal MEK1 delays tumor growth by promoting anti-tumor immunity
Authors: Searles Stephen , Lyden David C , Bui Jack D
Published: January 2023

Exosomes mediate intercellular communication and regulation in many processes, including cancer-immune cell interactions. Exosomes are small (~100 nm) membrane vesicles that arise from the late endosomal pathway and contain bioactive molecules that are transferred to and affect the function of target cells. Tumor-derived exosomes are important regulators of anti-tumor immunity and have been shown to exert stimulatory and inhibitory effects on immune cells, depending on the experimental setting. Using a proteomic approach, we found that exosomes derived from tumors that undergo immune rejection (“regressors”) contain different proteins than exosomes from progressively growing tumors (“progressors”). Specifically, the signaling molecule MAP2K1 (“MEK1”) is highly enriched in regressor compared to progressor exosomes, leading us to hypothesize that exosomal MEK1 stimulates anti-tumor immunity and inhibits tumor progression. We found that forced expression of MEK1 in progressor exosomes delays tumor growth and increases anti-tumor immune cell infiltration, and that MEK1-hi exosomes are sufficient to delay tumor growth, but MEK1-low exosomes are not. Moreover, we found that MEK1 protein can be physically transferred from tumor cells to macrophages via exosomes, and that MEK1-hi exosomes induce pro-inflammatory cytokine production in macrophages, but MEK1-low exosomes do not. We propose that tumor-derived MEK1 is transferred to macrophages via exosomes, which enhances their anti-tumoricidal effector function and delays tumor growth. This cell extrinsic, anti-tumor activity of MEK1 contrasts with the known oncogenic properties of MEK1 and could limit the efficacy of MEK1 inhibitors currently in clinical trials.
Ectopic expression of CD127 restores the numbers of thymic and peripheral T cell populations in adenosine A2A receptor deficient mouse
Authors: Cekic Caglar , Linden Joel M , Savas Ali Can , Kayhan Merve , Koyas Altay , Akdemir Imran
Published: January 2023

Inhibition of proximal TCR signals by adenosine A2A receptors (A2AR) is important for T cell development and maintaining T cell quiescence in vivo. Deletion of A2ARs causes accelerated growth of B16 and MB49 tumors, which is associated with reduced T cell accumulation in tumors and decreased CD127 expression and interleukin-7 responsiveness. Here we showed that ectopic expression of CD127 in A2AR deficient mice restores normal numbers of thymic populations and IL-7 responsiveness of T cells. Ectopic expression of CD127 in A2AR deficient mice also caused accumulation of similar numbers of T cells in the periphery as compared to A2AR proficient mice. Growth of B16 tumors in A2AR deficient mice was accelerated as compared to A2AR proficient mice. However, growth of B16 tumors was comparable between A2AR deficient or proficient mice ectopically expressing CD127. Adenosine A2A receptor deletion or ectopic CD127 expression did not change Vbeta selection suggesting changes in T cell repertoire may not be involved in increased tumor growth and reduced T cell numbers in A2AR deficient mice. Interestingly, tumor growth in A2AR and CD127 double deficient mice was slower as compared to CD127 deficient mice. Finally, adenosine preconditioning of engineered T cells significantly reduced lung colonization of B16OVA tumors and persistence of engineered T cells. Overall these results suggest that CD127 regulation by adenosine is important for T cell maintenance and functional responses in vivo and blockade of A2AR to improve anti-tumor immune responses should be further promoted by providing signals for T cell survival.
Adrenergic signaling impairs activation of CD8+ T cells by blocking metabolic reprogramming
Authors: Qiao Guanxi , Bucsek Mark J , Hylander Bonnie L , Repasky Elizabeth A
Published: January 2023

Adrenergic stress promotes tumor progression by several mechanisms. Mice housed at standard housing temperature (ST, 22°C) experience chronic adrenergic cold stress sufficient to elevate norepinephrine levels compared to mice housed at thermoneutrality (TT, 30°C) and we found that tumors grow faster at ST. We have also previously reported that the anti-tumor immune response is suppressed at ST. Tumor infiltrating CD8+ T cells from these mice have reduced expression of activation and effector function markers. These deficits are reversed by housing mice at TT which reduced adrenergic stress or by treating mice at ST with the β-adrenergic receptor antagonist propranolol. Coincident with this effect, we see increased cell surface expression of the glucose transporter-1 on CD8+ T cells, which is critical for glycolysis. Based on the fact that T cell activation and function require up-regulation of glycolysis (i.e. “metabolic reprogramming”), we hypothesize that adrenergic signaling impairs the antitumor efficacy of CD8+ T cells by impairing metabolic reprogramming. To test this hypothesis, CD8+ T cells were isolated and activated and treated +/− the β-AR agonist isoproterenol (ISO). Using Seahorse Extracellular Flux Analysis to compare the bioenergetics of control and ISO treated CD8+ T cells, we found that adrenergic signaling significantly reduced ECAR (glycolysis) and increased the ratio of oxidative phosphorylation/glycolytic rate (OCR/ECAR). These data support the idea that adrenergic signaling blocks metabolic reprogramming in CD8+ T cells thereby inhibiting T cell activation. Current work is focused on identifying the precise intracellular pathways by which adrenergic signaling and T cell metabolism intersect.
CD73 expression in cancer-associated fibroblasts exacerbates immune suppression and promotes tumor progression via augmenting adenosine accumulation in the tumor microenvironment
Authors: Yu Miao , Guo Gang , Huang Lei , Mellor Andrew , CUI YAN
Published: January 2023

CD73, an ecto-5′-nucleotidase, is the major extracellular adenosine-generating enzyme that converts AMP to immunosuppressive adenosine (ADO). Previous studies demonstrated that CD73-mediated ADO production in the tumor microenvironment (TME) is an immune evasion mechanism explored by tumors, myeloid-derived suppressor cell (MDSCs), and regulatory T cells. Targeted suppression of CD73, either via CD73 blocking antibody or inhibitors, suppresses tumor progression and metastasis. Given the complexity of the TME and crucial involvement of cancer-associated fibroblasts (CAFs) in immunosuppression and tumor progression, we explored the contribution of CD73 in CAFs to tumor progression. Using a CD73-negatvie EL4 tumor in C57BL/6 WT and CD73KO mice, we observed a high level of CD73 expression in CAFs, associated with their higher capacity of converting extracellular AMP to ADO than that of lymphocytes and myeloid cells in culture. Interestingly, as tumor progresses, CAFs in the TME of WT mice expended extensively to form an interconnected network and the percentage of CD73+ CAFs increases, while that of tumor infiltrating lymphocytes, especially CD8 T cells, decreases. However, in CD73KO mice, EL4 tumor progression was greatly delayed, associated with their significantly higher level of tumor-infiltrating CD8+ cytotoxic T cells. Functional analysis of fibroblastic stromal cells confirmed that CD73 inactivation greatly diminished their ability to generate extracellular ADO and subsequent suppression of T cell activation. Our results suggest that CD73 also represents one of the important mechanisms of CAF-mediated immunosuppression. We will discuss the potential environmental factors that enforce CD73 expression in CAFs.
Roles of SOCS1 for establishment of mycobacterial infection
Authors: Soma Shogo , Yasutomi Yasuhiro
Published: January 2023

Background

Suppressor of cytokine signaling (SOCS) 1 plays a crucial role in the negative regulation of JAK/STAT pathway. The SOCS1 is strongly upregulated by infection with several pathogens include M. bovis bacillus Calmette-Guérin (BCG). Since the function of SOCS1 induced by infection is not fully understood. In present study, we establish recombinant BCG which express a dominant-negative SOCS1 (rBCG-SOCS1DN) which acts as antagonist for SOCS1 signals.

Methods

C57BL/6, Rag1−/−, Nos2−/− and Rag1−/−Nos2−/− (DKO) mice were inoculated intratracheally with rBCGSOCS1DN or vector control rBCG (rBCG-pSO). Lungs and bronchoalveolar lavage fluids were harvested and analyzed. The mRNA and supernatants from bone marrow-derived myeloid dendritic cells (BMmDCs) infected with rBCG were harvested and nos2 gene expression and the production of nitric oxcide (NO) were measured. These cells were analized by electron microscopy.

Results

The amount of rBCG-SOCS1DN was significantly reduced by not only C57BL/6 but also Rag1−/− mice comparison with those of rBCG-pSO. The cytokine/chemokine profiles were not significant difference between rBCG-SOCS1DN and rBCG-pSO; however, nos2 gene expression and NO release from BMmDCs infected with rBCG-SOCS1DN were significantly higher than BMmDCs infected with rBCG-pSO. In Nos2−/− and DKO mice, there were no significant difference of bacterial burdens between rBCG-SOCS1DN and rBCG-pSO. Furthermore, the BMmDCs infected rBCG-SOCS1DN were necrotic damaged less than BMmDCs infected with rBCG-pSO.

Conclusions

These data suggest that BCG induce SOCS1 activation for evasion of microbicidal mechanism by nitric oxide from the innate immune responses.
The non-classical CD8+ T cell response to murine cytomegalovirus
Authors: Anderson Courtney K , Reilly Emma C , Lee Angus Y , Vance Russell E , Brossay Laurent
Published: January 2023

There is a growing interest in the role of MHC class Ib-restricted CD8+ T cells, referred to as non-classical T cells, such as MAIT cells and NKT cells. Non-classical CD8+ T cells have been shown to participate during the immune response towards a number of bacterial and viral pathogens. However, the overall role of these cells remains largely undetermined. We sought to investigate their contribution during murine cytomegalovirus (MCMV) infection, a well-established model for human cytomegalovirus. To circumvent the known role of conventional CD8+ T cells we utilize KbDb−/− mice, which lack MHC class Ia molecules. Following MCMV challenge, a subset of non-classical CD8+ T cells undergoes robust expansion, gains effector functions, and subsequently contracts. Interestingly, there is a prolonged inflammatory phenotype not observed in B6 or β2m−/− control animals. Non-classical CD8+ T cells from long-term infected KbDb−/− mice are also capable of robust proliferation in response to secondary MCMV infection, indicating that these T cells exhibit a memory phenotype. Using RAGKbDb−/− animals, we determined that adoptively transferred non-classical CD8+ T cells are sufficient to protect against MCMV-induced lethality. The population we describe is more phenotypically similar to conventional CD8+ T cells than innate non-classical T cells. Altogether, our data indicate that MHC class Ib-restricted CD8+ T cells may substitute for conventional CD8+ T cells when their functions are compromised during CMV immunoevasion.
Cytokine induced SH2-containing (CIS) protein mediated intrinsic and extrinsic CD8+ T cell regulation
Authors: Dlugolenski Daniel Aaron , Ward Alister , Stambas John
Published: January 2023

Small nuclear polymorphisms in CIS exhibit strong correlation with enhanced disease susceptibility in humans. CIS is a member of the SOCS family of proteins, and SOCS proteins are intracellular negative regulators of JAK/STAT signalling. The SH2 domain of CIS inhibits STAT5 interaction with JAK1 and JAK3. STAT5 is critical in downstream signalling of IL-2 and IL-15 in CD4+ and CD8+ T cells. IL-2 and IL-15 signalling are important in the regulation of critical T cell checkpoints including activation, proliferation, survival and differentiation. The regulatory role of CIS in immune cell development and function is relatively undefined, and recent studies have provided brief insights on the role of CIS in CD8+ T cell biology and its overall contribution to infection and immunity. Using CIS knock-out mice, we show that CIS plays a significant role in intrinsic and extrinsic regulation of CD8+ T cell responses to influenza A virus infection. Infection in the absence of CIS results in reduced morbidity and improved recovery compared with wildtype mice associated with enhanced recruitment of influenza A virus specific CD8+ T cells in the lungs of knockout mice. The observed changes in influenza A virus specific CD8+ T cells is supported by intrinsic modification of the proliferative capacity of CD8+ T cells lacking CIS. We also observe significant changes in DC subsets in the lung and mediastinal lymph nodes altering the extrinsic regulation of influenza specific CD8+ T cells. This work highlights a comprehensive understanding of the role of CIS in viral immunity and CD8+ T cell biology.
Efficient activation of latent HIV reservoir in human memory T cells by depletion of regulatory T cells in humanized mice in vivo
Authors: Li Guangming , Nunoya Jun-ichi , Reszka-Blanco Natalia , Cheng Liang , Tsao Li-Chung , Su Lishan
Published: January 2023

Regulatory T (Treg) cells play an important role in modulating immune activation and human immunodeficiency virus type 1 (HIV-1) infection. However, the role of Treg cells in HIV-1 latency establishment or maintenance in vivo remains unknown because of the lack of a robust model. We have established a humanized mouse model that supports long-term stable HIV-1 replication. When treated with combination antiretroviral therapy (cART), HIV-1 infection was stably suppressed to undetectable levels. Upon cART interruption, HIV-1 replication rebounded rapidly to pre-treatment levels. Interestingly, HIV-1 viral replication was significantly increased upon Treg cell depletion under effective cART. The HIV-1 reactivation during cART was mainly through increased gene expression from T cells. The rebound viruses during cART did not acquire drug resistance mutations. We found that most activated HIV-1 reservoir target cells were CD4 memory T cells in all lymphoid organs. These findings demonstrate that Treg cells suppress HIV-1 replication and its reactivation from latently infected T cells in vivo. Treg cell depletion provides an efficient way to reverse latency and reactivate HIV-1 reservoirs in vivo.
The CD38+ HLA-DR+ PD-1+ CD4 T cell population represents a short-lived part of the viral reservoir and predicts poor immunologic recovery upon initiation of ART
Authors: Eller Michael A. , Creegan Matthew , Hong Ting , Nau Marty , Slike Bonnie M. , Krebs Shelly J. , McElrath M. Juliana , Katabira Elly T , Michael Nelson L. , Robb Merlin L. , Tovanabutra Sodsai , Baeten Jared M. , Sandberg Johan K.
Published: January 2023

The activated PD-1+ CD4 T cell population is associated with disease progression in untreated HIV-1 infection, and with residual viral replication in individuals on ART. Few studies have examined cell associated viral load (CAVL) in phenotypically different activated CD4 T cell populations to define the HIV reservoir. We followed 32 HIV-1 chronically infected individuals, from Kampala, Uganda, and determined their CD4 T cell counts, and viral load at baseline, 6, and 12 months after the initiation of ART. Peripheral blood T cell populations were sorted based on activation profiles and gag DNA was measured to determine CAVL within these populations. Soluble inflammatory factors were measured in plasma using a multiplexed platform. The median HIV-1 viral load declined from 95,315 copies/ml at baseline to below detection for all patients after 12 months of ART, whereas CD4 T cells counts averaged 204 cells/μl at baseline and increased to 373 cells/μl by month 12. Median levels of the activated PD-1+ CD4 T cell population contracted from 4.5% of CD4 T cells at baseline to 2.3% of CD4 T cells after 12 months of ART. Baseline levels of activated PD-1+ CD4 T cells positively correlated with plasma levels of IP-10 (R2=0.362, P<0.001) and TNFRII (R2=0.237, P=0.006). Higher baseline level of activated PD-1 positive CD4 T cells was associated with poorer CD4 T cell recovery after 12 months of ART (R2=0.240, P=0.039). CAVL was similar within the activated PD-1+ CD4 T cell population as other CD4 populations at baseline, but significantly lower after 12 months of ART compared to baseline. Taken together, the activated PD-1+ CD4 T cell population is a short-lived component of the viral reservoir and high levels of these cells predict poor immunologic recovery on ART.
HTLV-1 infection and neuropathogenesis in the context of Rag1−/−γc−/− (RAG1-hu) and BLT mice
Authors: Madagula Kiran Kumar , Ginwala Rashida , Caruso Breanna , Khan Zafar K , Chew Glen M , Pattekar Ajinkya , Corley Michael J. , Loonawat Ronak , Jacobson Steven , Sreedhar Sreesha , Ndhlovu Lishomwa C , Jain Pooja
Published: January 2023

To date, the lack of a suitable small animal model has hindered our quest to understand the immuno- and neuropathogenesis of HTLV-1, the causative agent of chronic disabling neuroinflammatory disease HAM/TSP. Host immune response that plays a critical role in the outcome of HTLV-1 infection could be better tested in the context of humanized (hu) mice. Thus, we infected neonatal and adult Balb/c-Rag1−/−γc−/− (Rag1) as well as Bone marrow-Liver-Thymic (BLT) mice with HTLV-1. Proviral load (PVL) was determined in the peripheral blood, spleen, and other organs by droplet digital PCR. Within blood, PVL and viral protein Tax was detected as early as 2 wks post-infection (wpi). Tax showed peak expression at 14 wpi in Rag1 mice with continued expression until 16 weeks. Both PVL and Tax expression was considerably higher in the adult Rag1 mice as compared to the neonates with the latter showing less than 20% PVL in the peripheral blood, brain, and liver. Moreover, signs of lymphocytic infiltration with concomitant Tax expression and resulting myelin disruption were observed in the spinal cord and brain of infected mice. Thus far, several members of the CD28:B7 family of co-signaling molecules have been associated with T-cell dysfunctions in HTLV-infected patients. We found increased expression of PD-1, TIGIT and TIM-3 on CD8+ T cells in the CNS of infected hu-mice. This represents the first attempt to establish HTLV-1 neuropathogenesis in the context of Rag-1 and BLT hu-mice suggesting the possibility of developing a small animal model of HAM/TSP for testing groundbreaking therapies.
MAIT cells (CD8+CD161++TCR7.2++) are functionally impaired during chronic SHIV infection.
Authors: Murugesan Amudhan , Ibegbu Chris , Styles Tiffinay , Hicks Sakeenah , Sabula Micheal , Reddy Pradeep Babu Jagadeesh , Jones Andrew , Shankar Esaki Muthu , Amara Rama Rao , Velu Vijayakumar
Published: January 2023

Mucosa-associated invariant T-cells (MAIT) are abundant in humans and recognize bacterial ligands. Here, we have studied the distribution and function of MAIT cells during chronic SHIV infection using rhesus macaques (RM). Two groups of RM, healthy and SHIV infected were studied. lymphocytes from various tissues were analysed for MAIT (TCR7.2++ CD161++) cells using multicolor flow cytometry and characterized for their distribution, phenotype and function during chronic SHIV infection. Similar to human, we found significant fraction of (~2%) RM CD8 T cells co-express MAIT cell markers TCR7.2, CD161, IL-18R, CCR6 and display central memory (CCR7+CD45RA−) phenotype. The frequency of MAIT cells were decreased during chronic SHIV infection and tissue analysis showed a significant enrichment of MAIT cells in liver (~8%) and BAL (3%) than in blood (1%), spleen(0.5%), lymph node(0.13%) and gut (0.2%). Importantly, during chronic SHIV infection more than 80% of MAIT cells expressed T cell exhaustion marker PD-1 in blood and the level of expression was higher (>95%) in tissue resident CD69+MAIT cells. These cells lack proliferating capacity (Ki-67) and cytokine production (IFN- g and IL-17) in the blood, suggesting their functional impairment. The preserved blood and gut MAIT cells correlate inversely with plasma viral RNA levels and associate directly with gut CD4 T cell levels, suggesting that CD4 help maintains MAIT cell frequencies in the gut. These data demonstrate that MAIT cells are decreased in blood and are functionally exhausted during chronic SHIV infection. Future studies focused on blocking the PD-1 pathway to retrive MAIT cell exhaustion during chronic SHIV/HIV infection may yield therapeutic benefit to HIV/SHIV infection.
Characterising ZBTB20 as a novel regulator of effector CD8 T cell longevity and differentiation
Authors: Sun Yanbo , Usherwood Edward J
Published: January 2023

During pathogen infection a T cell response is induced in order to control the infection. T cell differentiation can follow a variety of paths, including terminal differentiation to effector cells or differentiation to memory cells with stem cell-like self-renewal capacity. This process is known to be controlled in part by several transcription factors such as TCF-1, T-bet, eomesodermin, Blimp-1 and Bcl-6. Zbtb20, also known as HOF or DPZF, belongs to an evolutionarily conserved transcription factor family named BTB-ZF family. BTB-ZF proteins, including BCL-6, PLZF, BAZF and ZBTB20, play critical roles in a wide range of biological process including developmental events, cell cycle progression in normal and oncogenic tissues and maintenance of the stem cell pool. In the past decade, an emerging volume of literature has demonstrated that many BTB-ZF proteins are also key players in the development and function of lymphocytes and myeloid cells. Here we sought to examine if ZBTB20 affects T cell differentiation or function in response to infection by viruses or intracellular bacteria. We present data using both conditional deletion and overexpression strategies testing how zbtb20 affects the size, quality and memory potential of the pathogen-specific T cell response. This work has important implications for strategies to understand and enhance the T cell response to pathogens.
Crucial role of FOXO1 in inflationary CD8+ T-cell responses during persistent viral infection
Authors: Delpoux Arnaud , Michelini Rodrigo Hess , Verma Shilpi , Lai Chenyen , Omilusik Kyla D , Benedict Chris A , Hedrick Stephen M
Published: January 2023

Cytomegalovirus (CMV) establish persistent infection that induce the accumulation of virus-specific T-cells over time in a process called memory inflation. Although persistence of antigen (Ag) is considered essential, the molecular pathway for the inflationary T-cell development is poorly understood. Mainly, inflationary CD8 T-cells are KLRG1hi CD27−, but a low percentage of them are KLRG1lo CD27+, which are considered as their memory precursors. To study the role of FOXO1 in CD8 T cells during MCMV infection, we analyzed mouse bone marrow chimeras in which FOXO1 was specifically deleted in CD8 T cells. These mice were infected with MCMV-Δm157 strain, and the expansion and phenotype of inflationary was examined. The results revealed that the IE3 and M38 CD8 T-cell responses do not inflate in absence of FOXO1. Furthermore, with an absence of FOXO1, there were fewer Ag-specific CD8 T cells that produced both IFNγ and TNFα. Consistent with this lack of CD8 effector cells the mice were less able to control the virus in the spleen and the liver at day 6 after infection. Moreover, the FOXO1 KO Ag-specific CD8 T cells fail to up-regulate the memory associated transcription factors, TCF-1 and EOMES, correlating with a lower percentage of memory precursors. Then, we found that FOXO1 KO Ag-specific CD8 T cells display an anergic state and are prone to apoptosis. Finally, we delete FOXO1 at day 40 after infection, we showed that the FOXO1 KO Ag-specific CD8 T cells survive less efficiently and lose their memory characteristics as well as their recall capacities. Collectively, these results demonstrate an intrinsic role for FOXO1 in establishing and maintaining the inflationary memory program that is essential to forming long-lived effector and memory cells.
FCRL3 modulates the function of human thymic regulatory T cells
Authors: Kraus Zachary J , Agarwal Stuti , Dement-Brown Jessica , Tolnay Mate
Published: January 2023

Peripheral tolerance is achieved as the result of a delicate balance between inflammatory and tolerogenic signals. Regulatory T cells (Treg) are at the forefront in maintaining this balance, and external factors capable of modifying their suppressive function can be a powerful tool in future therapies for cancer and autoimmune disease. FCRL3 is a member of a novel family of antibody binding receptors expressed by specific human B cell and thymic Treg (tTreg) subsets that are reported to have altered suppressive function. Most importantly, overexpression of FCRL3 is strongly associated various autoimmune diseases. In particular, FCRL3 overexpression on Tregs is associated with more severe cases of rheumatoid arthritis. FCRL3 in B cells was shown to have dual signaling capacity; however no study addressed the functional role of FCRL3 in Treg. We used tTregs purified from human blood and pre-stimulated through FCRL3 to directly test the role of FCRL3 on Treg function using in vitro Treg suppression assays. We observed that engagement of FCRL3 increased Treg proliferation while reduced their suppressive activity, suggesting for the first time a direct link between FCRL3 and Treg function. TLR2 signaling has also been shown to repress the suppressive effect of human Tregs, therefore we tested possible integration with FCRL3 signaling. When TLR2 agonists were used together with FCRL3 pre-stimulation, we found indications of signal integration by tTreg downstream of FCRL3 and microbial sensing. We propose that FCRL3 is a novel regulator of Treg functionality, suggesting a mechanism for its association with autoimmune diseases.
Microbubbles: A high-throughput platform for the sustained culture, characterization, and isolation of single cells based on morphology, cytokine production, and antibody secretion.
Authors: Kobie James , DeLouise Lisa , Scheinman Andrew
Published: January 2023

Analysis of lymphocytes in bulk masks the heterogeneity of cellular processes. Through advancements in single cell technologies finer resolution and the true complexities of cell populations can be revealed. A major limitation of single cell technologies, especially those that enable high-throughput screening is the poor survival and proliferation of individual cells when binned separately in vitro. We have developed a novel platform technology, termed microbubbles which are scalable arrays of individual nanoliter size wells of unique spherical architecture and gas exchange properties that enable single cells to rapidly condition their microenvironment and enable long-term culture. Additionally, morphological heterogeneity is maintained in the microbubbles, unlike in conventional tissue culture wells. Microbubble arrays are compatible with conventional fluorescent microscopes and morphology and cell surface protein expression can be easily monitored longitudinally. Secreted cellular products such as antibodies and cytokines accumulate and are readily detected from cells in real-time using fluorescent probes. Arrays with 100,000 microbubbles, contained within a footprint of a standard microscope slide, can be screened using custom software in brightfield and three fluorescent channels within 10 minutes, and rare cells of interest identified based on morphological and/or functional properties are easily recovered for downstream analysis. Applications of microbubbles developed thus far include identification and isolation of rare antibody secreting cells based on antibody-specificity for monoclonal antibody development, monitoring T cell cytokine production, and characterizing cancer stem cells.
Poly Lactic-co-glycolic acid nanoparticles encapsulation enhance the effectiveness of curcumin for rescuing trafficking-defective gp91phox in chronic granulomatous disease leukocytes.
Authors: Yen Chia Liang , Liao Chia Li , Shieh Dar Bin , Wu Ping Ching , Yeh Chen Sheng , Chang Chuan Fa , Jiang Si Tse , Shieh Chi Chang
Published: January 2023

Mutations in leukocyte NADPH oxidase genes lead to defective reactive oxygen species (ROS) production and cause chronic granulomatous disease (CGD) in humans. Previously we have shown that the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump inhibitor, thapsigargin, rescues the endoplasmic reticulum (ER) retention of the mutant protein in a CGD patient with a gp91phox (H338Y) mutation. Curcumin, a natural constituent of Curcuma longa, is also a SERCA pump inhibitor. However, the clinical effectiveness of curcumin is not satisfactory due to its poor in vivo bioavailability and potential cytotoxicity. In this study, we investigated the effects of organelle-targeting nanoparticles on the delivery of curcumin to leukocytes for the treatment of CGD. First, we investigated the cytotoxicity of curcumin with poly (lactic-co-glycolic acid) (PLGA) nanoparticles encapsulation. Next, we investigated the therapeutic potential of PLGA encapsulated curcumin in mice expressing gp91phox (H338Y) mutant protein. Our results showed that (1) ER-targeting peptide decoration enhanced the localization of PLGA encapsulated curcumin in the ER region; (2) PLGA encapsulated curcumin induced ER calcium release, but the following extracellular calcium entry and ER stress-associated cell death was inhibited; (3) The in vivo test showed that PLGA encapsulated curcumin with ER-targeting peptide decoration enhanced leukocytes gp91phox (H338Y) mutant protein processing and the ROS production. This study showed the therapeutic potential of PLGA encapsulated curcumin for diseases with protein-trafficking defective mutations. Efficient organelle-targeting may promote the efficiency of nanoparticle-based curcumin delivery system.
Functional stability of ACD anticoagulated blood samples stored for 72 hours at room temperature.
Authors: Kunz Hawley E , Quiriarte Heather , Nelman-Gonzalez Mayra , Chew Darilyn , Crucian Brian
Published: January 2023

Characterization of the immune system in remote locations is challenging, requiring technical expertise and specific analytical equipment. Immune functional assessments require live cells, and the window of viability following blood donation is limited. To test the ability of ACD blood collection tubes to expand this window of viability, cellular function was analyzed over 4 consecutive days, beginning on the date of blood collection. T-cell activation, characterized by CD69 and CD25 expression, was measured following a 24-hour stimulation with soluble anti-CD3 and anti-CD28 antibodies (CD3/CD28) or with staphylococcus enterotoxins A and B (SEA+SEB). Mitogen stimulated cytokine profiles were determined following a 48-hour stimulation with CD3/CD28, phytohemagglutinin and ionomycin (PMA+I), or lipopolysaccharide (LPS). T-cell activation following stimulation with SEA+SEB was not altered by the processing delay; however, following stimulation with CD3/CD28, T-cell activation increased over the 72 hour period. The processing delay did not affect cytokine production following CD3/CD28 stimulation. Following stimulation with LPS and PMA+I, results were varied. While some cytokine responses were unaltered following the processing delay, others were impaired. These findings confirm the general viability of whole blood stored at room temperature (68 to 72°F) for 48 to 72 hours in ACD tubes and suggest many phenotypic and functional assays are feasible on blood samples aged as described. Investigators using this protocol should confirm their specific culture technique and measured output. This protocol may assist studies of subjects in remote locations with no immediate access to laboratory instrumentation.
Application of super-resolution microscopy to the study of B and T lymphocyte mitochondria morphology and metabolism
Authors: Roesler Alexander Scott , Miozzo Pietro , Akkaya Billur , Smelkinson Margery , Brzostowski Joseph , Kabat Juraj , Traba Javier , Dorward David W , Pierce Susan K , Akkaya Munir
Published: January 2023

High-resolution microscopy techniques have advanced our understanding of lymphocyte biology. An emerging focus in the field of immunometabolism on activation-induced metabolic reprogramming and its effects on immune cell function will require in-depth analyses of mitochondria. However, even with newer imaging technologies, obtaining super-resolution images of lymphocyte mitochondria remains challenging due to their small cytoplasmic sizes. Here, we compared standard confocal laser microscopy with three commonly used super-resolution methods to analyze activation-induced mitochondrial changes in B and T cells. Naïve B cells were stimulated in vitro via Toll-like receptor 9 and/or the B cell receptor, and naïve T cells were differentiated in vitro into activated or T regulatory phenotypes. The cells were imaged using standard laser confocal microscopy, AiryScan, stimulated emission depletion (STED) microscopy, and computerized tomography electron microscopy (TEM). Mitochondrial morphology was best resolved by staining both the mitochondrial membrane and matrix simultaneously. In preliminary analyses, we observed activation-induced changes in the distribution of lymphocyte mitochondria. Of the imaging methods used, only TEM allowed for 3D reconstructions of the mitochondria’s internal structure, which revealed swelling of the inner matrix and significant loss of cristae in B cells, but not in T cells. In conclusion, the super-resolution light microscopy techniques we developed appear to be powerful tools for immunometabolism studies of lymphocyte mitochondria, having demonstrated clear visualization even at the molecular level, while TEM answered key questions about intra-mitochondrial morphology.
Dynamic characterization of the phagocytic process utilizing an automated microfluidic platform
Authors: Chen Cindy , Yeh Victor , Booth Ross , Helton James , cappione amedeo
Published: January 2023

Phagocytosis is a complex pathway that involves removal of pathogens, foreign particles, or cell debris through engulfment by unique cell types. Such innate responses provide the first line of immune defense with evidence showing their critical involvement in prevention of chronic inflammation or autoimmune disease, tissue remodeling and removal of apoptotic tumor cells following therapeutic intervention. Traditional tools evaluate cell behavior in bulk context thus masking inherent cellular heterogeneity or employ end-point assays thus losing temporal process dynamics. Moreover, non-adherent immune cells are mobile complicating single cell analysis. Characterizing functional changes with single cell resolution requires precise spatial cell positioning, controlled micro-environment and high-throughput multiplexed measurement. We developed a novel microfluidic approach to trap single non-adherent immune cells in a microfluidic platform capable of perfusion-based media supply with gas and temperature control. The device is comprised of a controller system and optically transparent microfluidic plates, each with four culture chambers. Each chamber [dimension: 3mm × 3mm × 40μm (L × W × H)] contains a trap array tailored to the dimensions of the cells of interest. Each unit can be addressed by programmable manipulation of up to six reagents, enabling uninterrupted real-time live cell imaging assays in response to changing conditions. To demonstrate utility, the platform was used in conjunction with fluorescent markers to monitor the phagocytic process in real-time via, including temporal changes in morphology and the rate of phagosome formation/inhibition in the presence of different stimuli and concentration.
Treatment for severe combined immunodeficiency (SCID) in the era of newborn screening (NBS)
Authors: Wahlstrom Justin , Nguyen Alan , Dvorak Christopher C. , Puck Jennifer M , Cowan Morton J
Published: January 2023

SCID is associated with excellent survival when treated with allogeneic hematopoietic cell transplantation (HCT), gene therapy (GT), or enzyme replacement therapy (ERT) within 3–4 months of birth or prior to infection. NBS for SCID results in the diagnosis by 2–3 weeks of age. This retrospective analysis describes therapy and immune reconstitution of 28 infants diagnosed by NBS in California since 2010 and treated at the University of California, San Francisco at a median of 56 (24–217) days of age. HCT with alkylating chemotherapy (N=6), serotherapy alone (N=6), or no conditioning (N=12) was used in 24 infants, and ERT followed by definitive GT at University of California, Los Angeles, was used in 4 ADA-deficient infants. Overall survival at >2 years was 93% with 1 death from HHV6 and 1 from CMV. The median time to CD4 T cell recovery (>200 T cells/uL) was 73 (10–96) days for ERT patients prior to GT, and 138 days (14–220) for HCT patients. Time from first appearance of donor chimerism to CD4 recovery was evaluable for 14 HCT survivors who lacked pre-HCT maternal chimerism or whose donors were not their mothers. After donor chimerism appeared in peripheral blood at a median of 21.5 (14–169) days, appearance of T cells took 32 (0–84) more days. Second transplant or stem cell boost was required for 10 of 24 patients at a median of 77 (35–382) days following first HCT; for patients eligible for analysis (N=15), need for 2nd therapy was significantly higher in those without detectable donor chimerism by 28 days following therapy (0% vs 83%, p=0.04). NBS for SCID allowed rapid institution of anti-infective and definitive treatment to optimize outcomes, and early detection of donor chimerism was associated with successful T cell recovery.