bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced lung-resident T cell memory in infants following influenza infection
Authors: Zens Kyra D , Farber Donna L
Published: January 2023

Influenza is a major cause of morbidity and mortality in infants, who suffer more severe disease and are more susceptible to reinfection. In adults, influenza generates robust lung tissue-resident T cell memory (TRM) populations which are protective upon subsequent infection. Using a mouse model, we investigated whether infants also establish lung TRM after influenza infection. We found that infants and adults mount similar primary lung T cell responses to influenza. However, following recovery, infected infants generated fewer lung TRM than did animals infected as adults. Mice infected as infants also suffered greater weight loss and increased lung viral titers upon reinfection as adults in a TRM-dependent manner. Reciprocal transfer of infant or adult antigen-specific CD4 T cells to adult or infant hosts revealed that while both infant and adult cells expand in the primary response, only adult cells are efficiently maintained as TRM after resolution of infection suggesting a cell-intrinsic cause for reduced TRM in infants. RNAseq analysis of infant and adult primary effectors revealed increased T-bet and strongly decreased levels of Eomes and the survival factor Bcl-2 in infant T cells while analysis of protected lung memory from animals infected as infants or adults demonstrated lower levels of TRM-associated retention markers and decreased levels of the survival factors Bcl-2 and Bcl-xL. Further pathway analysis implicated reduced IL-7 and IL-15 signaling in infant T cells suggesting a mechanism for their reduced TRM with important implications in vaccine design and therapies for influenza in this population.
IL-15 supports the generation of lung tissue-resident memory CD4 T cells
Authors: McKinstry Karl Kai , Finn Caroline , Swain Susan L
Published: January 2023

Increasing evidence from animal models and human studies points towards a critical role for tissue-resident memory T cells (TRM) during protective recall responses. TRM able to persist at sites of infection may be especially important for clearance of pathogens such as influenza A virus (IAV) that can evade neutralizing antibody. We recently showed that while virtually all memory CD4 T cells that develop in secondary lymphoid organs following IAV priming require autocrine IL-2 signals, a population of CD69high CD127low lung CD4 TRM is generated through a unique IL-2-independent pathway. Here, using several different experimental approaches, we find that formation of this TRM subset is dependent on IL-15 signals received during the first week of IAV infection. Long-term maintenance of the TRM cells, however, does not require IL-15 or the recognition of residual IAV antigen. Finally, our results demonstrate that IL-15-dependent TRM cells can more efficiently elicit pro-inflammatory responses from dendritic cells presenting cognate peptide antigen than can conventional splenic memory CD4 T cells expressing the same T cell receptor, a function that correlates with initial control of viral titers during the first few days following IAV challenge. These studies, identifying a novel role for IL-15 in supporting the generation of a subset of lung CD4 TRM cells with specialized function following IAV priming, are highly relevant to vaccine design.
Pyruvate kinase type M2 promotes tumor cell exocytosis via phosphorylating synaptosome-associated protein-23
Authors: Liu Yuan , Wei Yao , Pan Chaoyun , Zhu Dihan , Shi Lei , Bian Zhen , Zen Ke
Published: January 2023

Tumor cells secret large amount of microvesicles (MVs) which are involved in remodeling tumor-stromal interactions to promote malignancy. The regulatory mechanisms governing the active exocytosis in tumor cells, however, remain incompletely understood. We show here that tumor cell exocytosis is tightly controlled by aerobic glycolysis (Warburg effect) and pyruvate kinase type M2 (PKM2), which is commonly upregulated and phosphorylated in tumor cells, plays critical roles in promoting exocytosis. Upon stimulation by EGF, more phosphorylated PKM2 switches to dimer form and is recruited to exosomes where it associates with synaptosome-associated protein (SNAP)-23 (SNAP-23). The association of PKM2 and SNAP-23 in secreted exosomes is confirmed by cross immunoprecipitation and Western blot analysis. Dimerized PKM2 enhances the exosome release via phosphorylating SNAP-23. Direct phosphorylation assay and mass spectrometry identifies SNAP-23 phosphorylation at Ser95by PKM2. Ectopic expression of non-phosphorylated SNAP-23 mutant (Ser95→Ala95) significantly reduces PKM2-mediated exocytosis whereas phosphomimetic SNAP-23 mutants (Ser95→Ele95) partially rescue the impaired exocytosis in tumor cells with PKM2 knockdown. Our findings reveal a previously unrecognized non-metabolic function of PKM2 in promoting tumor cell exocytosis.
PDL1 expression is elevated prior to AIDS-related Non-Hodgkin’s lymphoma diagnosis
Authors: Epeldegui Marta , Conti David , Guo Yu , Cozen Wendy , Penichet Manuel , Martinez-Maza Otoniel
Published: January 2023

The risk of developing non-Hodgkin’s lymphoma (NHL) is greatly elevated in HIV infection, and AIDS-associated NHL (AIDS-NHL) remains a significant problem, even in those receiving anti-retroviral therapy. AIDS-NHL is thought to develop due to: 1) loss of immunoregulation of Epstein-Barr virus infected cells and/or 2) the accrual of genetic errors associated with B-cell activation driven by HIV infection. A population of B cells that express PDL1, secrete CXCL13, IL6 and IL10, and can regulate T-cell function by interacting with PD1, has been recently described. We previously showed that serum levels of CXCL13, IL-16 and IL-10 are elevated prior to, as well as after, AIDS-NHL diagnosis. Also, others have described that PDL1 and PD1 expression are elevated in HIV infection, and that this has an impact on T-cell function. PDL1 expression on B cells may contribute to the development of AIDS-NHL in a dual fashion by: 1) enhancing B-cell activation, and 2) by impairing/inhibiting T-cell function through PD1:PDL1 interactions. Hence, we hypothesized that levels of CD19+PDL1+ cells may be elevated prior to AIDS-NHL diagnosis and may play a role in lymphomagenesis. To investigate this hypothesis, we conducted a nested case-control study within the Multicenter AIDS Cohort Study, obtaining viable frozen PBMC from HIV+ subjects who later developed NHL (cases, n=31) and from HIV+ subjects who did not develop NHL (HIV+ matched controls, n=29) and performed multi-color flow cytometry. We observed that the numbers of circulating CD19+PDL1+ cells in cases were elevated prior to AIDS-NHL diagnosis, when compared to HIV+ controls (p=0.026), suggesting that CD19+PDL1+ cells may be playing an important role in the development of AIDS-NHL.
Thymic stromal lymphopoietin promotes interplay between breast tumor cells, neutrophils and Ly6Chi monocytes to regulate breast tumor progression
Authors: Kuan Emma Lo , Ziegler Steven F
Published: January 2023

Various human cancers display a T helper type 2 (Th2)-like inflammation. One possible mechanism suggested in human breast cancers is thymic stromal lymphopoietin (TSLP) secreted by breast tumor cells which can drive Th2 responses via tumor-associated dendritic cells. However, the interactions between TSLP, tumor cells, and other immune cells remain unclear.

We found in breast tumor-bearing mice TSLP can also be produced by two pro-tumor myeloid cell populations, neutrophils and Ly6Chi monocytes. This non-tumor derived TSLP is crucial for both primary breast tumor growth and its metastasis to lungs. One important mechanism is myeloid derived TSLP can directly act on tumor cells and promote survival and TSLP production of tumor cells. Mice transplanted with TSLPR deficient breast tumor cells developed smaller primary breast tumors and fewer lung metastases. Conversely, mice that constitutively express TSLP in lungs transplanted with breast tumor cells had markedly increased lung metastases. Blocking TSLP in lungs resulted in a reduction in the number of lung metastases. We further discovered that TSLP signaling in Ly6Chi monocytes is crucial for their suppressor functions and their ability to differentiate into macrophages in tumors. Transfer of TSLP receptor deficient Ly6Chi monocytes significantly reduced lung metastases in tumor-bearing mice by changing T cell populations in lungs and in tumors.

Our work is the first to show myeloid cell derived TSLP plays an important role in promoting breast tumor progression via maintaining tumor cell survival. We also provide a novel mechanism of the requirement of TSLP signaling in differentiation and suppressor functions in Ly6Chi monocytes.
Adrenergic signaling mediates immune suppression induced by cool housing temperatures for laboratory mice
Authors: Bucsek Mark J , Qiao Guanxi , Liu Haichao , Evans Lauren , MacDonald Cameron , Hylander Bonnie , Repasky Elizabeth A
Published: January 2023

Our laboratory has demonstrated that standard mildly cool housing temperatures (ST, 22°C) can significantly enhance tumor growth by suppressing anti-tumor immunity. In contrast, we have found that maintaining mice at thermoneutral temperatures (TT, 30°C) reversed immunosuppression and significantly delayed tumor growth compared to ST. Under cool conditions, mammals produce heat via adaptive thermogenesis, a process which is driven by norepinephrine (NE) acting through β-adrenergic receptors (β-ARs) on brown adipose cells and we found that NE is significantly elevated in mice at ST compared to TT. NE is known to suppress immune cell activity. We therefore hypothesized that cool housing conditions elevate NE which then suppresses anti-tumor immunity. To investigate this hypothesis, we used two syngeneic mouse tumor models: 4T1 mammary carcinoma and B16-OVA melanoma. In tumor bearing mice at ST, the β-AR antagonist propranolol significantly delayed tumor growth. β-blockade had no effect on the growth of 4T1 tumors in immunodeficient SCID mice indicating a mechanism involving the adaptive immune response. In mice bearing B16-OVA tumors, β-blockade also slowed tumor growth and this beneficial effect was negated by CD8+ T-cell depletion. In both models, reversing adrenergic stress induced immunosuppression by β-AR blockade in combination with boosting the anti-tumor T cell response by blocking programmed cell death receptor 1 (PD-1) significantly slowed tumor growth in mice housed at ST compared to either treatment alone. Therefore, combination therapy with β-receptor antagonists could improve the clinical efficacy of immunotherapy, including checkpoint inhibition.
CXCR4 is required for progression of T cell acute lymphoblastic leukemia
Authors: Chen Cynthia , Pitt Lauren , Tikhonova Anastasia , Aifantis Iannis , Schwab Susan Ruth
Published: January 2023

Of the most common childhood cancer, acute lymphoblastic leukemia (ALL), 15% of cases are caused by transformation of T cell progenitors (T-ALL). While survival rates have vastly improved for newly diagnosed T-ALL, substantial challenges remain in the treatment of relapsed disease and in the minimization of side-effects from central nervous system prophylaxis. Hence, the search for more effective, less toxic treatments continues. Homing receptors play multiple roles in cancer progression, yet little is known about the trafficking of T-ALL. Upon profiling homing receptor expression, we found high levels of the chemokine receptor CXCR4 on the surface of mouse and primary human T-ALL cells. Furthermore, T-ALL cells localized in close proximity to CXCL12-producing stromal cells in mouse femoral bone marrow. Deletion of Cxcl12 in vascular endothelial cells limited leukemia progression, suggesting a vascular niche for T-ALL. In parallel, we found CXCR4 expression to be essential for T-ALL maintenance and progression. Deletion of Cxcr4 in mouse T-ALL after disease onset led to disease remission and prolonged survival, and CXCR4 antagonism using the small molecule inhibitor AMD3465 suppressed human disease in a patient-derived xenograft model. Loss of CXCR4 signaling in T-ALL cells reduced Myc abundance, which was previously reported to regulate leukemia-initiating cell activity. In line with this, CXCR4-deficient T-ALL cells failed to establish disease when adoptively transferred into secondary hosts. Together, our findings highlight an important contribution of microenvironment in regulating T-ALL pathogenesis, and suggest targeting CXCL12:CXCR4 signaling as a powerful new strategy for treating this aggressive disease.
Low Dose Gemcitabine plus TH1 Dendritic Cell Vaccination Mediates Durable Cure of Orthotopic KrasG12D/p53−/− Pancreatic Ductal Adenocarcinoma via the Generation of Atypical CD3+CD8+NK1.1+ T-cells
Authors: Konduri Vanaja , Li Dali , Halpert Matthew M. , Liang Dan , Liang Zhengdong , Chen Yunyu , Fisher William E , Levitt Jonathan M , Yao Qizhi Cathy , Decker William K
Published: January 2023

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States with a five-year overall survival of only 7%. To explore the potential of immune-based treatment approaches to PDAC, we treated orthotopically-established K-rasG12D/p53−/− PDAC tumors with 40 mg/kg gemcitabine and a cell-based therapeutic vaccine previously shown to generate robust and durable cell-mediated (TH1) immunity. The results indicated that the combination of chemotherapy and a TH1 dendritic cell (DC) vaccine was effective in eliminating tumor, preventing metastasis and recurrence, and significantly enhancing OS. No animal that received the combination therapy relapsed, while 80% of mice that received gemcitabine-only relapsed and progressed. Analysis of circulating PBMC demonstrated that mice receiving the combination therapy exhibited significantly elevated levels of IFN-γ+CCR7+CD8+ T-cells with significantly reduced levels of exhausted GITR+CD8+ T-cells after the cessation of treatment. Retro-orbital tumor re-challenge of surviving animals at six months after treatment demonstrated durable antitumor immunity only among mice that had received the combination therapy. CD8+ splenocytes derived from surviving mice that had received the combination therapy were sorted into NK1.1pos and NK1.1neg populations and adoptively transferred into naïve recipients. Transfer of only 1,500 CD8+NK1.1pos T-cells was sufficient to mediate tumor rejection whereas transfer of 1,500 CD8+NK1.1neg T-cells imparted only minimal effects. The data suggest that addition of a TH1 DC vaccine regimen as an adjuvant to existing therapies can mediate eradication of tumors and offer durable protection against PDAC.
Inflammasome-associated inflammation inhibits cell-mediated immune responses to Listeria monocytogenes
Authors: Theisen Erin M , Sauer John-Demian
Published: January 2023

Listeria monocytogenes is a genetically tractable Gram-positive intracellular bacterium that activates a robust cell-mediated immune response capable of breaking self-tolerance. These properties contribute to its success as a cancer immunotherapeutic platform; however, despite these attributes, the specific mechanisms by which L. monocytogenes stimulates a robust adaptive immune response remain largely unknown. We hypothesized that cytosolic innate immune activation, specifically inflammasome activation, by L. monocytogenes would be critical for triggering a robust CD8+ T-cell response. Surprisingly, we found that inflammasome activation impairs the induction of antigen specific T-cells and ultimately the generation of protective immunity. To determine if this phenomenon is unique to inflammasome-induced pyroptosis or a general outcome of pathogen induced host cell death, we designed strains of L. monocytogenes to uniquely activate pyroptosis, necrosis, or apoptosis.

Contrary to previous studies of immunity in the context of sterile cell death, pathogen induced pyroptosis, necrosis and apoptosis each impaired the generation of cell-mediated immunity. Importantly, the mechanism of impairment was unique for each form of cell death, although in each case it was independent of dendritic cell depletion or deficits in antigen presentation. Apoptosis and necrosis impaired dendritic cell co-stimulatory molecule expression, while the pyroptotic inflammatory milieu independently impaired the generation of cell-mediated immunity. These results suggest that inhibition of inflammasome activation, or specifically certain inflammatory components, is critical for the success of L. monocytogenes immunotherapy.
Understanding heterogeneous CTL responses to the yellow fever vaccine using mathematical models.
Authors: Moore James R , Antia Rustom , Akondy Rama , Jia Jonathan , Ahmed Rafi
Published: January 2023

In an acute infection, such as those caused by attenuated vaccines, we should expect the immune response to depend in some manner upon the precursor frequency as well as the magnitude of the viral load within the patient. However, we do not have a quantitative understanding of this process. In a recent study, 80 patients received the yellow fever vaccine and their viral titer and T-cell responses were tracked for several days. It was found that there were substantial differences between individuals in peak viral load, but that the difference in CTL response was relatively small. Moreover, although the CTL response was positively correlated with viral load, the response saturated for high viral loads. Using mathematical models, we investigated which rules of T-cell proliferation could give rise to this pattern. We considered three models: antigen-dependent proliferation in which the T-cells must receive continuous antigen stimulation to continue to divide, programmed proliferation in which T-cells commit to a fixed number of division upon recruitment and a hybrid model in which T-cells commit to a variable number of division based on viral load. We find that this latter model is the most plausible description of the data. We also considered whether negative feedback in the form of T-cell competition may play a role in determining CTL response, however we found no evidence for any such mechanism.
Coincident Type 2 diabetes mellitus modulates the systemic and antigen – specific induction of Type 1, Type 17, and other cytokines in both active and latent tuberculosis
Authors: Babu Subash , Kumar Nathella Pavan , Nutman Thomas B
Published: January 2023

Type 2 diabetes mellitus (DM) is known to be a major risk factor for the development of active tuberculosis, although its influence on active and latent Mycobacterium tuberculosis infection and disease remains poorly characterized. To identify the influence of coincident DM on cytokine levels in active pulmonary tuberculosis and latent tuberculosis, we examined circulating levels of a panel of cytokines in the plasma of individuals with PTB-DM or PTB alone and LTB-DM or LTB alone. PTB-DM is characterized by elevated circulating levels of type 1 (IFNγ, TNFα, and IL-2), type 2 (IL-5), type 17 (IL-17A) but decreased circulating levels of IL-10 family of cytokines (IL-19, IL-20, IL-22 and IL-24). This was associated with increased systemic levels of other pro-inflammatory cytokines (IL-1β, IL-6, and IL-18) and an anti-inflammatory cytokine (IL-10) but not type 1 IFNs. In contrast, LTB-DM is characterized by diminished circulating levels of type 1 (IFNγ, IL-2, and TNFα), type 17 (IL-17F), IL-10 family (IL-10, IL-19, IL-20 and IL-24) and other pro-inflammatory (IL-1β and IL-18) cytokines. Moreover, PTB-DM showed increased levels of pro-inflammatory cytokines upon tuberculosis antigen stimulation whereas LTB-DM showed diminished pro-inflammatory cytokines upon tuberculosis antigen stimulation. Our data reveal that PTB-DM is characterized by heightened cytokine responsiveness, indicating that chronic inflammation underlying DM potentially contributes to increased immune pathology. On the other hand, LTB-DM is characterized by diminished production of cytokines, implicated in the control of M. tuberculosis, allowing for a potential immunological mechanism that could account for the increased risk of active tuberculosis.
The immunogenic HSP receptor CD91 is an essential mediator of tumor immunosurveillance
Authors: Sedlacek Abigail L , Zhou Yu Jerry , Binder Robert J
Published: January 2023

Immunogenic Heat Shock Proteins (HSPs) are capable of eliciting potent immune responses against their chaperoned antigens and are being investigated as a potential cancer immunotherapy. HSPs signal through the receptor CD91 on the surface of antigen presenting cells and result in the development of a Th1 immune response. We have previously shown that when antigen load is low, HSPs and CD91 are required to prophylactically immunize against tumor development. Since antigen load is also low during nascent tumor development, we hypothesized that CD91 is critical during tumor immunosurveillance. We induced tumors using methylcholanthrene in CD91fl/flx CD11cCremice as well as CD91fl/fllittermates. Tumors developed more rapidly and at a higher incidence in the CD91fl/flx CD11cCremice as compared to the littermate controls. Additionally, tumors in these animals grew more progressively. We are currently immune-phenotyping the tumors that develop in each cohort at multiple time points to determine how the lack of CD91 on CD11c+ cells influences the immune cell infiltrate. Collectively, our data demonstrate that CD91 is a critical mediator of tumor immuneosurveillance and influences the immune infiltration of developing tumors.
Autoreactive CD8+ T-cells are highly dependent on CD8 for activation and as such targeting CD8 is an effective way of blocking autoreactive CD8+ T-cell activation
Authors: Wooldridge Linda , Morgan David , Pearson James Alexander , Gras Stephanie , van den Berg Hugo , Lissina Anya , Llewellyn-Lacey Sian , Willis Mark , Dockree Tamsin , McLaren James E , Ekeruche-Makinde Julia , Gostick Emma , Robertson Neil , Rossjohn Jamie , Burrows Scott R , Price David , Wong Susan , Peakman Mark , Skowera Ania , Clement Matthew
Published: January 2023

Autoimmune diseases such as type I diabetes (TID) and multiple sclerosis (MS) are chronic conditions that have a significant impact on quality of life. CD8+ T-cells play an important role in the pathogenesis of these diseases. However, drugs that target the entire CD8+ T-cell population are not desirable because this population provides protection against infection. Accordingly, there is an urgent requirement to develop novel treatment strategies that exclusively target the autoreactive CD8+ T-cell population. CD8+ T-cells express a molecule called CD8 at their cell surface which assists with activation. We have demonstrated that autoreactive CD8+ T-cells are entirely dependent on CD8 for activation. In stark contrast, pathogen specific CD8+ T-cells are characterized by higher functional sensitivity and are relatively CD8 independent. This represents an intrinsic difference that can be exploited for therapeutic benefit. Our data suggests that “blocking anti-CD8 antibodies” can be used to block autoreactive CD8+ T-cell attack without affecting pathogen specific immunity, a key goal in the treatment of autoimmunity. The generation of “blocking anti-CD8 antibodies” offers an opportunity to develop a novel therapeutic approach that can be used to treat CD8+ T-cell mediated autoimmunity. In order to conduct a further assessment, we are currently developing a strategy for in vivo validation of the approach.
Ras-mediated missing-self recognition via NKR-P1B:Clr-b interactions facilitates detection of oncogenic transformation and cancer immunosurveillance
Authors: Tanaka Miho , Fine Jason H. , Kirkham Christina L. , Aguilar Oscar A. , Ketela Troy , Moffat Jason , Allan David S.J. , Carlyle James R.
Published: January 2023

Natural killer (NK) cells are involved in tumor immune surveillance, immunity to infection, and various pathological conditions. One mode of NK cell recognition, ‘missing-self’ recognition, is triggered by downregulation of inhibitory self ligands on target cells, thereby decreasing the net signaling threshold to promote NK cell activation. Clr-b, ligand for the NKR-P1B inhibitory NK receptor, is downregulated during cellular transformation, viral infection, and genotoxic stress, in turn promoting MHC-independent missing-self recognition. However, the mechanisms underlying the loss of Clr-b during oncogenic transformation remain unknown. Here, we demonstrate that proto-oncogene, Ras, is directly involved in Clr-b downregulation on mouse fibroblasts via the Raf/MEK/ERK and PI3K pathways. We show that c-myc overexpression also promotes Clr-b downregulation. To examine a link between the loss of Clr-b and tumour progression using a spontaneous B cell lymphoma model, we backcrossed the Emu-c-myc transgene onto a Clr-b−/− background, then assessed the survival rates of transgenic littermates. Preliminary results reveal a trend towards accelerated tumor progression of Clr-b+/+ littermates versus Clr-b+/− littermates, suggesting a potential protective role for Clr-b loss in tumour immune surveillance, one revealed under loss-of-heterozygosity conditions. Notably, previous results using Nkrp1b−/− mice also suggest that NKR-P1B-mediated inhibition may also facilitate deleterious tumour immune escape. Collectively, our data suggest that Clr-b downregulation during oncogenic transformation plays a role in the detection of oncogenic transformation and cancer immunosurveillance by NK cells.
Natural Killer cells from primary human head and neck squamous cell carcinomas upregulate NKG2A
Authors: Korrer Michael J , Kim Young
Published: January 2023

Immunotherapy has revolutionized cancer therapy primarily by targeting checkpoint inhibitors found on endogenous immune cells. We analyzed and compared NK cells from the blood and tumor of HNSCC patients by flow cytometry for the expression of NK cell activating and inhibitory receptors. We found that NK cells from the primary tumor, but not the blood of HNSCC patients, significantly downregulated activating receptors NKG2D, DNAM-1, NKp30, CD16 and 2B4. Surprisingly, the majority of inhibitory KIR receptors on tumor-infiltrating NK cells were decreased compared to NK cells from the blood. However, the inhibitory receptors NKG2A and PD-1 were found to be highly overexpressed in tumor-associated NK cells. We believe that this study provides the first in-depth look at NK cells from primary HNSCC tumors. We found that tumor-associated NK cells express a profoundly different phenotype than NK cells from the blood of the same patients, with multiple activating receptors downregulated and the inhibitory receptor NKG2A over-expressed. These results support the therapeutic strategy of targeting NKG2A.
Toll-like receptors 2 and 4 are necessary to generate a fully functional commensal-specific ocular immune response.
Authors: Almaghrabi Fatimah , St Leger Anthony , Caspi Rachel R
Published: January 2023

Conjunctiva-associated lymphoid tissue (CALT) resembles a conventional mucosal immune tissue, which harbors various immune cells and is exposed to a wide array of microorganisms at the ocular surface. In a recent study, we demonstrated that colonization of the ocular mucosa with the commensal bacterium, Corynebacterium mastitidis (C. mast), results in increased resistance of the ocular surface to infectious fungal and bacterial pathogens. Further, we showed that this effect is dependent on IL-17 produced by gd T cells that respond to C. mast. In this study, we used mice deficient in the toll-like receptors, TLR2 and TLR4, to examine the role of these receptors in generating a gdT-17 immune response at the ocular surface. In vitro, we show that these receptors are necessary on dendritic cells (DCs) to effectively induce IL-17 production and proliferation of Vg4+ T cells. In vivo, we show that TLR2 KO mice and TLR4 KO mice fail to recruit Vg4+ T cells and neutrophils to the conjunctiva. These data suggest that both TLR2 and TLR4 are required to generate a fully functional mucosal IL-17 response in the conjunctiva and its draining lymph nodes. Our data highlight the importance of innate receptors in the generation of the ocular mucosal immune response and maintenance of gd T cell responses in vivo.
SMN deficiency alters the CNS and the peripheral immune system in a mouse model of spinal muscular atrophy (SMA)
Authors: Khairallah Marie-Therese , Franklin Craig , Simon Madeline , O’Day Siri
Published: January 2023

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss or mutation of the SMN1 gene. SMA rises from the insufficient levels of the ubiquitously expressed survival motor neuron (SMN) protein and is characterized by progressive α-lower motor neuron (LMN) degeneration and muscle atrophy. SMN is vital to all tissues, and the specific vulnerability of the LMN is still puzzling. However, currently there is a growing list of tissues affected in SMA. Recently, we have analyzed the development and the immune components of SMA spleen and found a selective reduction of the red pulp (RD) and RP macrophages in SMA mice, while white pulp and lymphocyte populations were largely preserved. Furthermore, previous studies suggested a role of glia cells in LMN pathology. These findings prompt us to investigate the role of the immune system in SMA pathogenesis. Looking into the cytokines production in the widely used “SMNΔ7” mouse model of SMA, we found that at steady state there is a higher production of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in SMA central nervous system (CNS) that starts at an early symptomatic age and escalates at the late symptomatic time. However, the anti-inflammatory cytokine TGF-β had lower expression. Interestingly, after in vivo LPS stimulation, SMA astrocytes and splenocytes exhibit less potency in increasing the pro-inflammatory cytokines except for IL-1β that had significantly higher levels in SMA at all points. These findings suggest that there is a role of SMN in the homeostasis and function of resident macrophages in the CNS and the spleen, and denote a yet undiscovered immune component of SMA where targeting cytokines would be a potential new supportive therapy for SMA patients.
The inflamed phenotype in PD-L1 negative non-small cell lung cancer (NSCLC) and response to checkpoint inhibitors
Authors: Pabla Sarabjot , Dy Grace , Nesline Mary , Gandhi Shipra , Pandey Manu , Ernstoff Marc C. , Conroy Jeffrey M. , Glenn Sean , Burgher Blake , Andreas Jonathan , Giamo Vincent , Papanicolau-Sengos Antonios , Galluzzi Lorenzo , He Ji , Gardner Mark , Morrison Carl
Published: January 2023

Background

NSCLC with a PD-L1 tumor proportion score (TPS) by immunohistochemistry (IHC) of greater than 50% has a positive predictive value (PPV) of 42% for response to pembrolizumab. Across all tumor types and different checkpoint inhibitors (CPIs), the evidence supports the inflamed phenotype associated with responders. Currently very little is known about predicting response to CPIs in PD-L1 negative NSCLC. Additionally, the association of the PD-L1 negative NSCLC with the inflamed phenotype has not been well described.

Methods

PD-L1 (22C3) IHC and a custom NGS cancer immune gene expression assay were used to interrogate 50 NSCLC samples of which 23 were treated with one or more CPIs. PD-L1 TPS >50% was considered positive for IHC. Select over expression of 43 genes related to T-cell activation and 11 genes associated with tumor infiltrating lymphocytes were considered positive for the inflamed phenotype. RECIST v1.1 was used to assess patient response.

Results

9 of the 50 samples were PD-L1 IHC positive and showed an inflamed phenotype. An additional 7 samples exhibited an inflamed phenotype that were PD-L1 IHC negative. Of the 23 patients that could be evaluated for response to CPIs, 9 showed an inflamed phenotype while 14 were non-inflamed.

Conclusion

A subset of PD-L1 IHC negative NSCLC patients with an inflamed phenotype show a favorable response rate to CPIs. No complete or partial responses were identified in patients that were PD-L1 IHC negative with a non-inflamed phenotype.
IFN-ɛ regulated innate immune responses in the female reproductive tract during Chlamydia infection
Authors: Horvat Jay C , Mayall Jemma R , Mangan Niamh E , Brown Alexandra C , Chevalier Anne , Starkey Malcolm R , Kim Richard Y , Hertzog Paul J , Hansbro Philip M
Published: January 2023

The immune processes involved in the clearance and immunopathology of Chlamydia infection in the female reproductive tract (FRT) are not well understood. In previous ground-breaking studies we showed that IFN-ɛ, a novel type I IFN that is exclusively and constitutively expressed in the FRT, plays an important role in protecting against Chlamydia infections. Here, we examined the effects of IFN-ɛ on innate immune processes in the FRT in order to elucidate the mechanisms by which IFN-ɛ protects against Chlamydia infections. Female WT and IFN-ɛ−/− C57BL/6 mice were pre-treated with progesterone and infected intra-vaginally with Chlamydia muridarum or sham-infected. Uterine horns were harvested and the effects of IFN-ɛ deficiency on immune factor expression and cellular infiltration were assessed using microarray/bioinformatics analyses, real-time qPCR and flow cytometry. IFN-ɛ uniquely regulated the expression of 744 genes at baseline and 802 genes during Chlamydia infection, and universally regulated the transcription of 61 genes, regardless of infection status. Of note, pathways associated with protective innate responses, such as IFN regulatory factor activation and pattern recognition receptor signalling were down-regulated in Chlamydia-infected IFN-ɛ−/− mice compared to WT controls. These changes correlate with a decrease in the expression of many important IFN-γ signalling molecules and number of IFN-γ-producing NK cells in the FRT during infection. These findings suggest that IFN-ɛ may protect against Chlamydia FRT infections by potentiating innate immune processes important for the clearance of infection, particularly pathways associated with IFN-γ signalling and the activation of NK cell responses.
Inhibition of Glycogen Synthase Kinase 3 beta Provides Adjuvant-like Effect on Antigen-specific CAR-T cells
Authors: Sengupta Sadhak , Sengupta Sudarshana , Sampath Prakash
Published: January 2023

Although strong CAR T cell mediated immune response in hematological tumors has been credited for its success in cancer remission, unfortunately the same cannot be replicated in solid tumors. Weak exposure to antigens in solid tumors results in less than optimal CAR T cell activation. This leads to anemic expansion of activated CAR T cells and early clonal contraction, which ultimately fails to induce a proper anti-tumor immune response. Addition of CD28 and other costimulatory signaling moieties, along with the use of supplementary IL2 therapy has been shown to be not sufficient to overcome this impediment and have its own cytotoxic limitations. Glycogen-synthase kinase 3 beta (GSK3β), a constitutively active signaling molecule in naïve T cells, is transiently inactivated during TCR-mediated T cell activation and has been attributed to rapid T cell proliferation. Pharmacological inhibition of GSK3β promotes antigen-specific CAR T cell proliferation that is supported by both increases in cell division as well as long-term survival of these T cells. GSK3β inhibition further promotes development of very specific CD8+ effector CAR T memory phenotype that can be modulated with variability in antigen exposure. Tumor-rechallenge experiments with these GSK3β-inhibited antigen- experienced CAR T cells resulted in 100% tumor elimination and progress-free survival in experimental animals. We predict that this adjuvant-like effect of GSK3β inhibition on activated CAR T cells will be the portent to successful implementation of CAR T immunotherapy against solid tumors.
IL-11 induces encephalitogenic Th17 cells
Authors: Kiapour Nazanin , Zhang Xin , Khan Tabish Hasan , Thamilarasan Madhan , Kapoor Sahil , Markovic-Plese Silva
Published: January 2023

Objective

To examine the capacity of IL11 to induce encephalitogenic Th17 cells.

Background

IL11 is the most up-regulated cytokine in the serum and CSF samples from patients with clinically isolated syndrome (CIS) suggestive of multiple sclerosis (MS). Studies of relapsing remitting experimental autoimmune encephalomyelitis (RREAE), confirmed a causative role of IL11 in worsening clinical severity of the disease and inducing increased numbers of IL17+CD4+ cells within the spinal cord inflammatory infiltrates. Passive transfer RREAE is used to directly examine the role of IL11 in the induction of the encephalitogenic Th17 cells.

Methods

SJL Mice were immunized with PLP139–151, sacrificed at day 11 at the onset of clinical EAE, and LN cells were re-stimulated with: 1) PLP, 2) PLP+IL11, and 3) PLP+IL23 for 7 days. Clinical scores and weight were measured following passive transfer of CD4+ cells daily in three groups for 70 days.

Results

Antigen recall experiments on LN cells revealed that invitro restimulation with PLP 139–151 in the presence of IL11 induces an increased percentage of IL17A+CD4+ cells (17.80%, p<0.001), similar to IL23 (18.33%, p<0.001). Upon passive transfer of CD4+ cells, mice that received IL-11-stimulated cells had an earlier onset of the disease (day 9 vs. 14) and increased average scores compared to mice that received cells restimulated with antigen only (ANOVA, p<0.001). There was no statistically significant difference between the severity of the disease induced by IL11 and IL23-polarized cells.

Conclusion

Our study demonstrated that IL11 induced encephalitogenic Th17 cells, similar to the prototypical Th17 polarizing cytokine IL23.

The study was supported by R03 AI111592-01 grant from NIH/NINDS.
Novel approaches to assess therapeutic efficacy in the MRL/lpr model of lupus
Authors: Schaevitz Laura , Lim Maria , Defensor Erwin
Published: January 2023

Spontaneous genetic models of systemic lupus erythematosus (SLE), such as MRL/lpr and NZB/W F1 mice, are commonly used to assess therapeutic efficacy. Therapies are evaluated against several reliable standard measures, including proteinuria, anti-dsDNA, skin lesions, splenomegaly, nephritis, and survival. We hypothesize that continuous monitoring of behavioral and physiologic parameters will provide additional meaningful data to assess disease and efficacy. Here, we treated MRL/lpr mice with two reference compounds, cyclophosphamide (CP: 25mg/kg, IP, weekly) and dexamethasone (DEX: 2mg/kg, IP, 3× week). Throughout the experiment, mice were housed in Vium’s Digital Vivarium, which provides continuous monitoring and assessment of behavioral and physiologic measures using a network of cameras and sensors. Digital metrics and standard measures were displayed and analyzed in near real-time, through an online interface. Group disease profiles were compared using automatically generated breathing rate and motion metrics, in addition to standard measures. Proteinuria, body weight, and spleen size showed disease establishment in MRL/lpr mice as early as 16 weeks of age, which was ameliorated by CP or DEX (p’s < 0.03). Breathing rate significantly increased in MRL/lpr vehicle-treated mice as early as 13 weeks of age, while nighttime motion significantly decreased in MRL/lpr vehicle-treated mice by 14 weeks. Changes in breathing rate and motion detected in MRL/lpr mice were rescued by administration of CP or DEX (p’s < 0.0001). Our results demonstrate that continuous measurement of breathing rate and motion are useful additional measures for evaluating disease severity and therapeutic efficacy in the MRL/lpr murine model of SLE.
A Novel Clone Ranking and Selection Assay that Combines all the Relevant Insights Needed to Choose the Most Optimal Clones in a Single Experiment
Authors: Duensing Thomas , Liu Zhaoping , Hauser Caroline
Published: January 2023

When developing cell lines for the production of antibodies, it is essential to choose healthy clones with high levels of IgG per cell. This process typically requires several assays on multiple platforms to get enough information to select optimal clones. Due to the expense and effort involved, this limits the number of clones that can be evaluated. We have developed the Cy-Clone™ PLUS Kit, a rapid, no-wash screening assay that simultaneously provides IgG concentration, IgG concentration per cell and per viable cell, cell viability, and cell density in every well of a 384-well plate.

CHO cells transfected with human IgG genes were inoculated, plated by limiting dilution, and cultured for 20 days. Cells were mixed with the reagents from the Cy-Clone PLUS kit and then read on the iQue® Screener PLUS platform in ~22 minutes. Using ForeCyt® Software, readouts for each well were visualized using heat maps and Profile Map to identify wells meeting multiple IgG production criteria.

The Cy-Clone PLUS kit with the iQue Screener platform provides all the relevant data needed to select and rank the best clones in a single well without screen-size limitations. The Cy-Clone PLUS kit enables you to make more informed decisions earlier, shorten development time, and decrease costs.
Central immune tolerance establishment under thymic involution (Supported by NIH/NIAID 1R01AI121147 to D-M Su)
Authors: Su Dongming , Oh Jiyoung , Wang Weikan , Thomas Rachel
Published: January 2023

The establishment of central immune tolerance in the thymus is occurs via thymocyte negative selection to deplete self-reactive T clones and thymic CD4+ regulatory T cell (tTreg) generation. Due to limited published data, it is largely unclear whether these two mechanisms are both defective in the involuted thymus, as a result of aging or other insults. We used mouse models with thymic epithelial cell (TEC) conditional defect (FoxN1 conditional knockout) and naturally aged defect to study mechanistic changes in central tolerance establishment. We identified a defect in thymocyte negative selection but not in tTreg generation. In comparison to tTreg with thymic conventional CD4+ T cells (tTcon) in the same thymus, the ratios were increased with severity of thymic involution, suggesting that activity of tTreg generation is increased in the involuted condition. We found that these changes were due to alterations in signaling strength received by T cell receptors, characterized by a decline of Nur77 and CD5 in CD4+ single positive thymocytes and an enhancement of Zap-70 in newly-generated tTregs. This is due to weakened signaling provided by MHC/self-peptide complex ligand from defective TECs, but not due to changes in tTreg development-required factors (TGFb, IL-2, IL-15, and NF-kB). Our results suggest that the involuted thymus attempts to balance the defective negative selection by enhancing tTreg generation to maintain central immune tolerance. If this balance is broken, age-related inflammatory diseases could take place.
CD46 activation on monocyte-derived dendritic cells
Authors: Biltoft Mette , Hansen Aida S. , Höllsberg Per
Published: January 2023

Objective

CD46 is an important immune modulatory receptor that influences the expression of certain cytokines in different cell types. This function has been proposed to depend on expression of specific isoforms of CD46. The common isoforms are named BC1, BC2, C1 and C2, and activation of T cells induces a switch towards dominance of BC2 and C2. However, the effect of CD46 activation on the cytokine production in dendritic cells (DCs) has so far been inconclusive and the relative distribution of CD46 isoforms in monocytes and DCs is unknown.

Methods

Monocytes were isolated from peripheral mononuclear blood cells and differentiated to dendritic cells (moDC). The maturation of moDCs was confirmed by flow cytometry using a panel of maturation markers. Monocytes and moDCs were stimulated with LPS in presence or absence of CD46 activation and the production of various cytokines were analyzed by real-time PCR. The monocytes and moDCs were furthermore analyzed for their expression of different CD46 isoforms by an isoform-specific real-time PCR assay.

Results

Upregulation of CD40, CD80, and CD83 on the surface of moDCs demonstrated the maturation of monocytes to moDCs. Activation of CD46 in moDCs during LPS stimulation was found to reduce the mRNA expression of IL12p40 and CXCL10 while no effect of CD46 activation on these cytokines was observed in monocytes. Examination of CD46 isoforms revealed a dominant expression of the isoforms BC2/C2 in moDCs upon differentiation from monocytes.

Conclusion

The results presented here suggest that CD46 activation reduces the pro-inflammatory potential of LPS stimulation in moDCs but not in monocytes. These functional changes are associated with a switch towards dominant expression of BC2/C2 isoforms.
Using molecular chemokine adjuvants to target the germinal center reaction and enhance HIV-specific humoral responses to DNA immunogens in the mucosa or the periphery
Authors: Gary Ebony Nicole , Bernui Mariana , Weiner David , Kutzler Michele A.
Published: January 2023

The germinal center (GC) is a dynamic cellular substructure found in lymphoid organs where follicular helper T cells (TFH) provide the necessary proliferation stimuli and cytokine signaling that promote antibody classs-witching and affinity maturation of B cell receptors. DNA vaccines have demonstrated great potential as vaccine immunogens and in vivo electroporation (EP) has enhanced this immunogenicity. The ability to deliver plasmid encoded molecular adjuvants to direct immune responses is an advantage of the DNA platform. Mucosa-associated epithelial chemokine (MEC, or CCL28) is secreted by epithelial cells that line mucosal surfaces, it signals through CC-chemokine receptors 10 and 3 (CCR10 and CCR3) which are expressed on IgA+ plasma cells, TH17 cells, and mucosa-resident antigen presenting cells. IL-12 is a T cell stimulating cytokine that promotes a helper-T cell phenotype. Mice were vaccinated twice intramuscularly with either consensus HIV-1 env DNA alone or DNA plus molecular MEC or IL-12 delivered by in vivo EP. Fourteen days post vaccination, animals were sacrificed and spleens and draining lymph nodes were harvested for flow cytometric analysis. Both MEC and IL-12 significantly increased antigen-specific GC B cells (B220+CD138−Igm−IgD−PNA+CXCR5+gp120+) (11.1% and 8.91% respectively, compared to 6% with DNA alone). Antigen specific isotype- ELISA confirmed that MEC increases IgA in serum (900ng/ml compare to 600ng/ml with DNA alone) and fecal extracts (27ng/ml compared to 22ng/ml), while IL-12 increases IgG in serum alone (2200ng/ml compared to 1300ng/ml with DNA alone). These data indicate that molecular adjuvants can be used to specifically direct immune responses to the mucosa or the periphery.
Dengue NS1-specific IgY antibodies neutralizes dengue infection without inducing antibody dependent enhancement.
Authors: O’Donnell Kyle Lee , Fink Ashley , Nilles Matthew L. , Bradley David S
Published: January 2023

Dengue virus (DENV) is the most prevalent arbovirus with worldwide cases increasing from 2.2 to 3.2 million in the last 5 years. Symptoms of DENV infection range from a mild fever to extensive vasculature permeability resulting in hemorrhagic fever and shock. Passive and active vaccines have been unsuccessful in protecting from or treatment for DENV infection because of antibody dependent enhancement (ADE), increased viral load due to increased monocyte opsonization by non-neutralizing Abs from a different DENV serotype. We have previously demonstrated that polyclonal avian IgY generated against whole, killed DENV-2 ameliorates DENV infection in mice without ADE. The lack of ADE with therapeutic IgY supported our hypothesis that IgY may provide a viable treatment for DENV infection as mammalian Fc receptors are unable to bind to IgY. Here we isolated DENV epitope-specific IgY and test these purified IgY pools for their ability to neutralize DENV in vitro. Avian IgY specific for Non-Structural-1 (NS1) demonstrated neutralization capacity in vitro equal to the whole polyclonal DENV IgY preparation. DENV-NS3 specific IgY, on the other hand, did not demonstrate any neutralization of DENV in vitro. We propose that NS1-specific IgY may provide a viable oligoclonal treatment for DENV infection in humans without inducing ADE.