bluuebunny/crossref_metadata_2025 huggingface dataset:
CD8 T cells prime CD8a dendritic cells for IL‐12p70 production via CD40 CD40L interactions
Authors: Wong Kok Loon
Published: June 2021

Dendritic cells prime CD8 T cells to proliferate and acquire effector function. However, how CD8 T cells could modulate DC function during this priming is less understood. Here, we show that activated CD8 T cells can prime DCs for IL‐12p70 in the presence of LPS. This ability was not seen with freshly isolated CD8 T cells or when LPS was absent. Direct contact involving MHC class I peptide and CD40‐CD40L interactions was essential for IL‐12p70 production, whereas soluble factors, including IFN‐g, TNF‐a, IL‐4 and GM‐CSF was little or no role to play in this process. CD8 T cells primed the CD8a+ but not the CD8a− DC subset for IL‐12p70 production. Hence apart from their role as killer cells, activated CD8 T cells may provide help to DCs via CD40L to pathogenically challenged CD8a+ DCs for IL‐12p70 and Th1 induction.

This work is funded by the Biomedical Research Council (Grant no. 04/1/21/19/373). Wong KL is supported by an A*STAR graduate scholarship.
Identification of a novel form of c‐Rel in a human B cell line undergoing spontaneous apoptosis
Authors: Valentin‐Acevedo Anibal J , Covey Lori R
Published: June 2021

NFκB proteins comprise a highly conserved family of eukaryotic transcription factors that are involved in many aspects of the immune response. c‐Rel is a lymphoid restricted member of this family known to affect cell proliferation differentiation and survival. Previously, our lab generated an EBV‐mediated immortalized human B cell line from a patient with non X‐linked HIGM in which the major transforming EBV protein, LMP1, is expressed under a tetracycline‐inducible promoter (pt1‐LCLtet cells). FACS analysis of these cells revealed an increased level of cell death even in the presence of LMP1 signaling; also a constitutive level of autophagy was detected as determined by LC3 expression. Western analysis of NFκB proteins revealed low levels of c‐Rel and in‐vivo labeling experiments indicate that in pt1‐LCLtet cells c‐Rel is hyperphosphorylated. Interestingly, we also detected the presence of a novel 25kD cytoplasmic form (c‐Rel25) by immunoblotting. Inhibition of caspase or proteosomal activity had no effect in the generation of c‐Rel25. However, when protein translation was inhibited we detected a gradual reduction of c‐Rel25 in a time dependent manner, suggesting that this novel form is translated from an independent mRNA. Experiments are being performed to test this hypothesis. Our working models is that c‐Rel25 is part of a novel pathway regulating B cell survival. (AJV supported by NJCCR; AI37081 to LRC)
Surface antigens are rapidly separated from bacterium‐sized microspheres in the subcapsular sinus and acquired by antigen‐specific follicular B cells
Authors: Pape Kathryn A , Catron Drew M , Jenkins Marc K
Published: June 2021

Antibody production is critical for antimicrobial immunity. The initial step in this process is the binding of antigen to the B cell receptor. Yet, the size of many microbes prevents them from entering the lymph node follicles where naïve B cells reside. Here, we show that antigen‐specific follicular B cells rapidly acquired antigen, produced peptide:MHC II complexes, and secreted antibody in a T cell‐dependent fashion following subcutaneous injection of bacterium‐sized antigen‐coupled particles even though the particles were confined to the subcapsular sinus. This form of B cell antigen acquisition did not depend on dendritic cells or subcapsular sinus macrophages. These results suggest that proteins released from the surface of microbes directly access the follicles to initiate the humoral response.
HSP90 inhibition affects MHC class II presentation of glutamic acid decarboxylase
Authors: Houlihan Josetta L. , Blum Janice S.
Published: June 2021

The central dogma of antigen processing is that MHC class I molecules present peptides derived from endogenous sources while MHC class II molecules present peptides derived from exogenous sources. Yet, certain cytoplasmic antigens have been found to be presented by MHC II molecules. The exact molecular mechanisms involved in MHC class II presentation of cytoplasmic antigens remains unknown. However, previous studies suggest that certain heat shock protein family members may play a role. Here, we implicate the heat shock protein HSP90 in the process of cytoplasmic and exogenous antigen presentation by MHC class II molecules. Our lab has generated a B cell line expressing glutamic acid decarboxylase (GAD), a key cytoplasmic autoantigen in the development of diabetes. Using HSP90 inhibitors and siRNA, we have found that HSP90 inhibition results in a decrease of endogenous and exogenous GAD antigen presentation by MHC class II molecules. HSP90 inhibition does not affect MHC class II presentation of exogenous GAD peptide or HSA (human serum albumin) antigen or peptide. These findings indicate that HSP90 plays a role in MHC class II presentation of select antigens. Research support provided by NIH RO1 AI49589
A Novel Approach for the Generation of Human Dendritic Cells from Blood Monocytes in the Absence of Exogenous Factors
Authors: Byers Anthony Michael , Schanen Brian C , Drake Donald R
Published: June 2021

Human dendritic cells (DCs) for research and clinical applications are typically derived from purified blood monocytes cultured in a mixture of cytokines for a week or more. Because it has been suggested that these cytokine‐derived DCs may be deficient in some important immunological functions attributed to antigen presenting cell (APC) populations found under normal conditions in vivo, there is an interest in developing more physiologically relevant methods of DC generation in vitro. Here, we describe a simple, reliable technique for generating large numbers of highly purified DCs based on a one‐way migration of blood monocytes through a confluent layer of human umbilical vein endothelial cells grown in the upper chamber of a Transwell device. The resultant APCs, harvested from the lower Transwell chamber, resemble other cultured DC populations in their expression of MHC and costimulatory molecules, ability to phagocytose protein antigens and capacity to elicit primary antigen‐specific T cell responses. This technique offers many advantages over the standard method of in vitro cytokine‐driven DC development: (1) DC differentiation occurs in only two days, (2) the differentiation process is more akin to the development of DCs under physiologic conditions and (3) lack of monocyte pre‐selection and recombinant cytokine addition makes the system cost‐effective.
Targeting DNA to Dendritic Cells Using Major Compatibility Complex (MHC) Class II Ligands
Authors: Kovacs Jeffrey R , Zheng Ying , Jia Liang , Gawalt Ellen S , Shen Hongmei , Meng Wilson S
Published: June 2021

Objectives:

This study was aimed at developing DNA delivery systems by exploiting MHC‐binding motifs. We tested the hypothesis that particles guided by MHC class II ligands can enhance uptake of oligodeoxynucleotides (ODN) into dendritic cells (DCs).

Methods:

Particles fabricated from PLGA or polystyrene (PS) were coated with the peptide VHA (amino acid sequence VHASHAVHAAHAVHA) which fulfills the binding motif of the murine class II MHC molecule I‐Ad. FITC‐labeled NF‐κB decoy ODN were condensed on the surface of the particles via the cationic peptide O10H6. DCs derived from BALB/c (I‐Ad) and C57BL/6 (I‐Ab) mice were used as the surrogate target and control, respectively.

Results:

VHA‐anchored particles carrying the decoy were found to be in the nano‐sized range (120–400nm). VHA‐mediated ODN uptake was demonstrated using flow cytometry (2‐fold higher MFI in I‐Ad vs. I‐Ab DCs). The uptake in BALB/c DCs was blocked by an anti‐I‐Ad antibody. Lysotracker staining showed lysosomal escape of DNA in cells. BALB/c DCs treated with VHA‐directed ODN show decreased sensitivity to LPS as measured by CD86 expression (2‐fold, n=3 p<0.05). Differential IFN‐γ production (34%±5%, n=4, p<0.05) upon stimulation was observed in VHA‐guided ODN exposed I‐Ad DCs compared to I‐Ab DCs.

Conclusion:

The results support the concept that MHC class II ligands can be used to facilitate delivery of DNA into DCs.
Maturation pathways of dendritic cells determine TAP1 and TAP2 levels and cross‐presenting function
Authors: Albaitero Andres Lopez , Maillard Robbie B , Hackman Trevor , Filho Pedro Andrade , Wang Xinhui , Gooding William , Ferrone Soldano , Kalinski Pawel , Ferris Robert L.
Published: June 2021

Cross‐presentation of exogenous antigens in the HLA class I pathway is key to the antigen presenting function of mature tumor‐loaded dendritic cells (DC). Conditions of DC maturation have been shown to influence the ability of DCs to induce T cell effector functions. However, it remains unknown if different pathways of DC maturation are associated with modulation in the ability of DCs to cross‐present tumor‐associated antigens. We compared DC matured with three clinically relevant cytokine protocols: IL‐1β, TNF‐α, IL‐6 (DC‐0), DC‐0+PGE‐2, or IFN‐γ, IFN‐α, TNF‐α, and IL1‐β (DC‐1). We found that these DC vary in the ability to cross‐present tumor antigens to CTL, with DC‐1 protocol being significantly more effective than the other protocols. This level of TA cross presentation was correlated with the level of expression of the antigen processing machinery (APM) components, TAP1 and TAP2, and to a lesser extent, tapasin, indicating that these components could be used to standardize DC preparations for optimal function. However, upregulation of TAP1/2 was not sufficient to explain the strongest cross‐presentation ability induced by DC‐1 cells, since IFN‐γ did not generate DC as effective at cross‐presentation as the full DC‐1 cytokine cocktail. These data indicate that DC maturation pathways modulate APM component expression and that DC vary in the ability to cross‐present TA to HLA class I‐restricted CTL.
Expression and release of angiopoietin‐1 from neutrophils: intracellular mechanisms
Authors: Sirois Martin G. , Neagoe Paul‐Eduard , Brkovic Alexandre
Published: June 2021

We recently detected the expression of Tie2 receptor on neutrophils which can be activated by both angiopoietins (Ang1 and Ang2), and promoting β2‐integrin activation, platelet‐activating factor synthesis (PAF) and migration. Recruitment of neutrophils to inflammatory sites contributes to pathological angiogenesis by their capacity to produce proangiogenic cytokines, including vascular endothelial growth factor (VEGF).

Herein, we assessed if human neutrophils can produce both angiopoietins, and if so, determined their mechanisms of release.

We observed by RT‐PCR, FACScan analysis, ELISA and confocal microscopy that neutrophils express Ang1 but not Ang2. For each condition, VEGF detection was performed as positive control. Using nitrogen cavitation, we observed that Ang1 is localized in the cytosolic fraction whereas VEGF is found in the specific granules (β‐granules). By ELISA we quantified the concentration of Ang1 (2727 ± 22 pg/ml) and VEGF (609 ± 7 pg/ml) from 2.5 × 108 neutrophils. Using selective agonists (PMA, Il‐8, fMLP, TNF‐α, calcium ionophore A23187) capable to promote VEGF release by the neutrophils, we assessed their capacity to support Ang1 release. Only a treatment with PMA (50 ng/ml), and A23187 (10 μM) induced Ang1 release by up to 5.9‐ and 4‐fold respectively as compared to PBS‐treated neutrophils, and these effects were maximal within 15 minutes. Supported by the CIHR and HSFQ.
CXCL12 mediates T‐cell migration via activation of the non‐canonical Wnt signaling pathway
Authors: Ghosh Manik C. , Collins Gary D , Carter Arnell , Vandanmagsar Bolormaa , Brill Margaret , Lustig Ana , Becker Kevin G , Wood William W , Emeche Chineya D , French Amanda D , O'Connell Michael P , Dissanayake Samudra K , Weeraratne Ashani T , Taub Dennis D
Published: June 2021

CXCL12 acts as a strong chemoattractant during inflammation and mediates its effects by binding and signaling through its receptor, CXCR4 to regulate the trafficking of cells. To better understand the pathways by which CXCL12 mediates T‐lymphocyte migration, we performed microarray analysis on resting and migrating T‐cells exposed to CXCL12. Through these analyses, we identified a number of unique signaling molecules induced by CXCL12 treatment including the induction of several members of the non‐canonical Wnt pathway. The mRNA and protein expression of Wnt5A along with its receptors Fzd2 and Fzd3 were significantly induced upon CXCL12 treatment in a biphasic and time‐dependent manner. In addition, the transcriptional activation of the Wnt5A promoter by CXCL12 was further confirmed by electrophoretic mobility shift assay (EMSA). siRNA and shRNA knockdown of Wnt5a in primary T cells and transformed T cell lines, respectively, significantly inhibited CXCR4 signaling and function suggesting an important role for the Wnt‐Fzd pathways in chemokine‐chemokine receptor function. Moreover, chemokine‐induced Wnt and Fzd molecules appear to play a critical role in PKC and Rac activation, MMP expression and actin polymerization in response to CXCL12 stimulation. Together, these results implicate the possible involvement of the non‐canonical Wnt signaling pathway in chemokine activation and T cell trafficking
IL‐12 and IFNα/β regulate non‐redundant functions in human CD8+ T cells
Authors: Ramos Hilario J , Farrar J. David
Published: June 2021

During viral infection, dendritic cells secrete the innate cytokines IL‐12 and IFNα/β to facilitate viral clearance. These cytokines have been linked to CD8+ T cell and are widely thought to act in a redundant manner to facilitate cytokine secretion and cytolytic activity. We examined the ability of IL‐12 and IFNα/β to drive these responses in human CD8+CD45RA+ T cells. We found that IL‐12 was sufficient for the generation of CD8+ effector cell activity, marked by IFNγ and TNFα secretion and strong cytolytic activity. In contrast, IFNα/β alone did not promote IFNγ or TNFα. Although, IFNα/β markedly induced perforin and granzyme B expression as a whole, these cells displayed functionally defective lytic activity in the absence of IL‐12 signaling. Thus, IL‐12 but not IFNα/β regulates primary effector functions in human CD8+ T cells. Interestingly, IFNα/β enhanced the expression of lymph node homing receptor CCR7 on a subset of T cells. This subpopulation displayed various characteristics of a memory phenotype, such as low CXCR3 and perforin expression while retaining granzyme B expression. Further, development of this CCR7 hi sub population was regulated solely by IFNα/β and was independent of the effects of IL‐12. These results demonstrate that IL‐12 and IFNα/β are not functionally redundant and in fact, impart unique phenotypes on human CD8+ T cells.

This study is supported by a grant from the National Institutes of Health/National Institute of Allergy and Infectious Disease (AI56222) and by a predoctoral fellowship from the National Instititue of Health/Nation Institute of Allergy and Infectious Disease (AI68622).
Neonatal and aged mice are unable to respond to polysaccharide antigens due to an up regulation in p38 MAPK
Authors: Chelvarajan Lakshman , Popa Diana , Hersh Matthew , Getchell Thomas , Stromberg Arnold , Bondada Subbarao
Published: June 2021

Due to the inability of the immune systems of aged and neonatal humans and rodents to make sufficient Ab to the capsular polysaccharides (PS), they are susceptible to infections by PS encapsulated bacteria. This lack of anti‐PS Ab is due to MΦ not being able to produce B cell stimulatory cytokines. When stimulated by TLR agonists, murine neonatal and aged MΦ make less pro‐inflammatory cytokines (p‐ic) and more IL‐10 than adult MΦ. Neutralization of IL‐10 allowed them to produce levels of p‐ic comparable to that produced by young adult MΦ. A microarray analysis was performed on RNA from resting and TLR‐4‐stimulated murine splenic MΦ. Expression of genes encoding the TLR signaling pathway, cytokines, chemokines, and their receptors was decreased in neonatal and aged MΦ, but p38 MAPK was enhanced in neonatal and aged MΦ. Surprisingly p38 had a dual role in p‐ic production with excess p38 levels being inhibitory. Suboptimal levels of p38 inhibitor enhanced p‐ic production but inhibited IL‐10 production. In vivo, a partial inhibition of p38 also suppressed TLR‐4 induced IL‐10 production without affecting p‐ic production. A partial inhibition of p38, considerably enhanced production of PS specific Ab in aged mice. TLR‐4 induced IL‐10 production was similarly dependent on p38 in human cord blood monocytes. Thus, elevated p38 activity in aged and neonatal MΦ leads to diminished help for B cells to mount an Ab response against PS.
Rapid up regulation of CNS VEGF is initiated by CD8 T cells in a murine model of BBB disruption
Authors: Johnson Aaron Jon , Suidan Georgette L , Chen Yi , Pirko Istvan
Published: June 2021

The role of inflammatory cells in promoting opening of the blood brain barrier (BBB) in neurological disease remains poorly understood. We have created a novel in vivo system to study BBB disruption using the Theilers murine encephalomyelitus virus (TMEV) model of multiple sclerosis. During acute TMEV infection, 70% of CNS infiltrating CD8 T cells are specific for a TMEV peptide, VP2 121–130, presented in the context of the Db Class I molecule. Intravenous injection of the VP2 121–130 peptide during the peak of CD8 T cell expansion in the CNS results in fatal vascular permeability characterized by: 1.) Astrocyte activation as detected by GFAP expression, 2.) Alteration of BBB tight junction proteins Z0‐2, Occludin and Claudin 5, 3.) Vascular permeability as measured by quantifiable T1 weighted MRI, and 4.) Severe motor deficit. Prior to these overt changes in CNS vascular permeability we observe high levels of active VEGF protein upregulated in the CNS implying an important role for this factor in CNS vascular permeability. To our knowledge, this is the first demonstration of an antigen specific T cell response initiating the expression of VEGF in the CNS during a vascular permeability syndrome.
Regulation of Th17 differentiation by epidermal fatty acid binding protein, E‐FABP
Authors: LI BING , REYNOLDS JOSEPH M. , STOUT ROBERT D. , SUTTLES JILL
Published: June 2021

Fatty acid binding proteins, FABPs, have been identified as central regulators of both metabolic and inflammatory pathways. Mice deficient for expression of E‐FABP display protection from development of experimental autoimmune encephalomyelitis, exhibiting reduced expression of proinflammatory cytokines, including IFNγ and IL‐17, as compared to wild‐type mice. FABP‐deficient macrophages and dendritic cells (DC) are defective in expression of inflammatory cytokines and are inefficient in the promotion of proinflammatory T cells responses during antigen presentation. However, E‐FABP expression by T cells, themselves, was found to influence T cell cytokine expression. Purified naive CD4+ T cells from female wild‐type and E‐FABP‐deficient mice were driven towards a Th17 phenotype by culture with TGFβ and IL‐6. Flow cytometric analysis revealed that wild‐type T cell cultures contained over twice the levels of IL‐17 positive cells than did T cells isolated from E‐FABP‐deficient mice. Interestingly, when these experiments were performed using naive T cells from male mice, no differences in IL‐17 expression were observed. Overall, our studies demonstrate that E‐FABP expression by macrophages and DC promotes Th1 and Th17 differentiation. However, T cell expression of E‐FABP also plays a role in Th17 differentiation, directly, in a gender specific manner.
Oral administration of capsaicin protects mice from autoimmune diabetes by inhibition of cross‐presentation of self antigens.
Authors: Nevius Erin , Beltran Jacqueline , Basu Sreyashi
Published: June 2021

Capsaicin (CP) is the pungent component of chili peppers. We have observed previously that dendritic cells (DCs), a key cell type in immune responses, have the receptor for CP, and engagement of this receptor has powerful immune consequences. As CP can be used as a powerful adjuvant in enhancement of an immune response, we tested its immunomodulatory activity by oral administration in treatment of Type 1 diabetes (T1D) in non‐obese diabetic (NOD) mice. Here we demonstrate that oral administration of CP delays the onset of diabetes in NOD mice in a dose dependent manner and adoptive transfer of splenocytes from CP treated mice can transfer protection in recipient NOD mice. Further, CP administration attenuates OVA specific naïve T cell (OT1) proliferation in pancreatic lymph nodes (PLN) of RIP‐OVA mice. These results reflect the immunological potency of a neurological ligand in modulating cross‐presentation ability of islet DCs in the draining PLNs.
Impaired Dendritic Cell Migration and T cell Stimulation in Aging Lead to Defective Dendritic Cell‐Mediated Antitumor Immunity
Authors: Grolleau‐Julius Annabelle , Harning Erin , Yung Raymong
Published: June 2021

We recently reported that bone marrow‐derived dendritic cells (DCs) from old C57BL/6 mice are less effective than their young counterpart in inducing the regression of B16‐OVA melanomas. To examine the underlying mechanisms, we investigated the effect of aging on DC tumor antigen presentation and migration. Although aging does not affect the ability of DCs to present OVA peptide (257–264), DCs from aged (18–20 months) mice are less efficient than those from young (3–6 months) mice in stimulating MHC class I OVA‐specific T cells in vitro (p<0.0025). Phenotypic analysis revealed a selective decrease in DC‐specific/intracellular adhesion molecule type 3‐grabbing, nonintegrin (DC‐SIGN) level in aged DCs. Importantly, vaccination of young mice using old OVA‐peptide(257–264) pulsed DCs resulted in impaired activation of OVA‐specific CD8+ splenic T cells in vivo (p<0.025). These splenic T cells from aged animals were able to differentiateinto IFN‐γ‐producing and cytotoxic effectors, but to a significantly less extent than those vaccinated with young OVA PP‐DCs. A decrease influx of CD8+ T cells within the tumor was also observed seven days after old DC injection. Finally, adoptive transfer experiments showed defective in vivo DC trafficking in aging, with half as many aged DCs migrated to the regional lymph nodes (p<0.025). This correlates with reduced CCR7 gene expression, impaired in vitro migration of aged DCs in response to CCL21, and defective CCR7 signaling in aged DCs. Taken together, our findings suggest that defective DC migration and T cell stimulation contribute to the observed impaired DC tumor immunotherapeutic response in aging.

This work id supported by the NIH (1RO1AG020628‐01A2; 3RO1 AR42525) (RY), the Ann Arbor VA Health System (VA Merit Review) (RY), and by the NIH‐NIA (AG024824, University of Michigan OAIC Pepper Center) (AG).
Depletion of CD25+ T cells and IL‐7 administration enhanced anti‐tumor effects in mice with B16 melanoma
Authors: Cui Yongzhi , Mackall Crystal
Published: June 2021

Lymphopenia supports augmented expansion of adoptively transferred antigen specific T cells and enhances the effectiveness of adoptive cell therapies for cancer, which were attributed to increased availability of homeostatic cytokines (e.g. IL‐7) and diminished numbers of CD4+CD25+ T regulatory (Treg) cells. We reasoned that new approaches designed to selectively replicate these beneficial effects of “lymphocyte space” in T cell replete hosts, could be equally effective and potentially less toxic than subjecting hosts to fully lymphoablative therapies. Thus, we compared the ability for adoptively transferred tumor specific T cells to eradicate tumor in lymphopenic RAG−/− mice vs. lymphoreplete mice (euthymic and athymic) treated with Treg depletion (anti‐CD25 moAbs) and/or rhIL7 therapy. Briefly, all animals were inoculated with B16 melanoma cells SQ on day 0, and received adoptive transfer of LN cells from Pmel TCR Tg mice on Day 3 ± anti‐CD25 moAb (PC61, 3 doses with last dose on Day −10) ± rhIL‐7 3–5 doses/week x 4–6 weeks. Surprisingly, lymphoreplete hosts treated with Treg depletion ± IL‐7 demonstrated superior anti‐tumor effects following adoptive therapy compared to RAG−/− mice. The most potent effects were observed when Treg depletion was combined with rhIL‐7 therapy in athymic hosts. Thus, lymphoreplete animals treated with targeted therapies to selectively decrease Treg populations and increase availability of homeostatic cytokines (e.g. IL‐7) demonstrated superior outcomes. We conclude that the benefits of “lymphocyte space” in supporting adoptive cell therapies can be harnessed in lymphoreplete hosts with the use of targeted therapies.
Upregulation of PD‐1 expression on tumor antigen‐specific CD8+ T cells in patients with advanced melanoma is associated with reversible immune dysfunction
Authors: Fourcade Julien , Kudela Pavol , Sun Zhaojun , Shen Hongmei , Land Stephanie , Sander Cindy , Kirkwood John M. , Zarour Hassane M.
Published: June 2021

The inhibitory receptor programmed death 1 (PD‐1) is a negative regulator of activated T cells and is upregulated on the surface of exhausted virus‐specific CD8+ T cells in chronically infected mice and humans. Blockade of this pathway using antibodies against PD ligand 1 (PD‐L1) reversed CD8+ T cell dysfunction. Here, we have investigated the role of the PD‐1/PD‐L1 pathway in impairing human tumor‐induced CD8+ T cells directed against multiple tumor antigens. In sharp contrast to the Epstein Barr virus (EBV) and influenza matrix (flu), we observed that ex vivo detectable CD8+ T cells recognizing well‐defined MHC class I‐restricted tumor epitopes in patients with advanced melanoma, upregulated PD‐1. After blockade of the PD1/PD‐L1 pathway, these tumor antigen‐specific CD8+ T cells exhibited increased proliferation and production of cytokines in response to the cognate antigen. Our findings contribute to a better understanding of the intriguing coexistence of strong tumor antigen‐specific CD8+ T cell responses with clinical progression in patients with advanced melanoma. They further provide a strong rationale for the use of PD 1/PDL‐1 blockade alone or in the context of cancer vaccines to reverse CD8+ T cell dysfunction and increase the likelihood of tumor regression.

This work was supported by NIH/NCI Grants CA90360 and CA112198 (to HMZ)
Gemcitabine (gem) can enhance immunotherapy (IT) of pancreatic cancer – importance of schedule
Authors: Schultes Birgit Corinna , Daley Graham Elisabeth , Nicodemus Christopher Farley
Published: June 2021

With a 5‐year survival rate of only 4%, non‐resectable pancreatic cancer is in urgent need of novel approaches to supplement current therapies. This study assessed parameters for combining gem and IT with the anti‐MUC1 antibody, AR20.5. Upon injection, AR20.5 forms immune complexes with circulating MUC1, leading to enhanced T cell immunity. To examine the effects of gem on immune cells and antigen processing, three parallel approaches were conducted: a) an in vitro co‐culture system of immature DC, tumor and T cells; b) a time course study of gem (60 mg/kg) in Panc02 tumor bearing mice; and c) a combination chemo‐IT study in MUC1‐transgenic mice with established Panc02.MUC1 tumors. In vitro, gem did not affect T cell or DC survival at concentrations ≤5 μg/mL. Interestingly, gem enhanced maturation of DC and T cell proliferation to non‐specific stimuli (anti‐CD3/CD28) as well as to the co‐cultured tumor cells. The in vivo time course study confirmed minor effects of gem on T and B cell survival with rapid recovery. Gem increased DC maturation in the spleen and tumor as well as DC migration to the tumor by Day 5. Further, gem significantly reduced T regs in the draining LN (Day 7) and tumor (Day 10). Reduction in myeloid derived suppressor cells was transient (Day 5–7) in spleen and tumor. Tumor control studies showed synergistic effects for combination gem‐IT over single agents when IT was given 1 week delayed relative to gem.
Tolerance and immunity to tumor‐associated antigen MUC1 in MUC1.Tg mice
Authors: Ryan Sean O. , Turner Michael S. , Revers Leigh , Gariepy Jean , Finn Olivera J.
Published: June 2021

Numerous adenocarcinomas aberrantly express a hypoglycosylated form of human mucin 1 (MUC1), containing epitopes with tumor‐associated mono‐ and disaccharide moieties including Tn, sialyl‐Tn, and TF antigens along with bare protein backbone. In the MUC1 transgenic mouse the immune system maintains a level of tolerance to MUC1 peptides, most notably in decreased CD4 T cell help. We are further examining the mechanisms of tolerance to MUC1 peptide (self), as compared to responses to a tumor‐associated MUC1 glycopeptide (non‐self), which should not be subject to tolerance. We have cloned a MUC1 glycopeptide‐specific MHC‐Class II restricted TCR (RFT). that is currently being used to generate TCR transgenic mice for comparative in vivo studies with MUC1 peptide‐specific TCR transgenic mice previously generated. Preliminary adoptive transfer studies have indicated that peptide‐specific TCR transgenic T cells are able to persist in both wild type and MUC1.Tg mice, but upon MUC1 immunization, only the cells in wild type recipients appear activated. Taken together our studies explore the differences in immune responsiveness to two different tumor‐associated forms of MUC1 in a preclinical model where MUC1 is endogenously expressed and functioning and will enable us to tailor more effective and safe MUC1 vaccination strategies. (supported by NIH grants T32CA82084 and RO1CA56103; J.G. by grants from CBCRA/CCS)
Mechanism of IL‐12's suppression of VEGFR3 upregulation on tumor vessels
Authors: Sorensen Elizabeth Welty , Gerber Scott , Frelinger John , Lord Edith
Published: June 2021

IL‐12 is a powerful anticancer agent in many tumor models, but how IL‐12 exerts its anti‐tumoral effect in vivo is still largely unknown. We are exploring this using B16, a C57BL/6‐derived melanoma line transfected to express IL‐12 (B16/IL‐12), a system that provides constant, IL‐12 production within the tumor microenvironment.

We have shown that the presence of intratumoral IL‐12 delays tumor growth, and causes phenotypic changes in the blood vasculature. The vessels within B16/IL‐12 tumors have a more normal morphology and fail to upregulate expression of the angiogenic vessel marker, vascular endothelial growth factor receptor 3 (VEGFR3). Studies performed in transgenic mice have shown that the failure to upregulate VEGFR3 is mediated by IFN‐γ and that T cells are required for this effect. We hypothesize that IFN‐γ is acting directly on the endothelial cells to prevent upregulation of VEGFR3. This is being tested with IFN‐γ receptor knock out mice in a bone marrow chimera system. IL‐12 also stimulates the immune system resulting in a marked increase in the number of tumor infiltrating lymphocytes (TILs) and more of these are producing IFN‐γ. These studies suggest that the vascular effects of immunotherapy may be as important to the beneficial effects of immunotherapy on tumor growth as the effects on the immune cells themselves.

Supported by NIH R01‐CA28332
Effect of tumor burden on myeloid derived suppressor cells and cytokine expression
Authors: Talmadge James E. , Dafferner Alicia , Donkor Moses , Westphal Sherry , Younos Ibrahim , Verma Vivek , Hoke Traci , Scholar Eric
Published: June 2021

Tumors growth has been suggested to suppress host immunity, including tumor infiltration by histocytes and T cells. We report herein that the frequency of myeloid derived suppressor cells (MDSCs) are significantly increased in the tumors, spleens, lungs and livers of mice bearing orthotopic clone‐66, mammary tumors in a tumor burden and duration dependant manner, while T cell numbers are suppressed. The increases in tumor burden and MDSC number are associated with significantly increased levels of VEGF‐a transcripts from tumor infiltrating myeloid cells (TIMCs), but not spleen cells or tumor parenchyma. Tumor burden and MDSC numbers are also directly correlated with G‐ and GM‐CSF transcript levels from the tumor parenchyma. In contrast, T cell numbers are inversely correlated with NOS‐2, ARG‐1 and COX‐2 transcript levels in the TIMCs, but not spleen cells, while MDSC numbers in the spleen and TIMCs are directly correlated with these enzyme transcript levels. These results suggest that the tumor associated increase in MDSC numbers was associated with G‐ and GM‐CSF and VEGF‐a secretion, while the activation of the MDSCs and secretion of ARG‐1 and NOS‐2 was associated with increased COX‐2 activity.
Myeloid‐derived suppressor cells (MDSCs) and tumor‐associated macrophages (TAMs) produce CCL22 which selectively recruits regulatory T‐cells (Tregs) to the tumor microenvironment
Authors: Mailloux Adam William , Young M Rita I
Published: June 2021

Tumor‐induced immune suppression involves the accumulation of immune suppressive infiltrates in the microenvironment. We demonstrated an increased number of Tregs in Lewis Lung Carcinoma (LLC). High levels of CCR4 on Tregs lead us to hypothesize that CCR4‐chemokines are secreted by LLC and selectively recruit Tregs. ELISA shows a large increase in CCL22 secretion by LLC bearing tissue. Lack of CCL22 in primary cultures suggests that CCL22 is NOT produced by the tumor, but rather by some other component of the microenvironment. Migration assays showed that normal, and to a greater extent, LLC‐bearing tissue selectively recruits Tregs. Neutralization of CCL22 significantly reduced the selective Treg migration. Fractionated normal and LLC bearing tissue shows that while NK, CD31+, and CD11c+ cells all produce CCL22, only the CD11b+ population secretes higher levels in LLC bearing tissue. This is magnified by the increased number of CD11b+ cells found in LLC bearing tissue. Of the CD11b+ cells, two populations emerge. One population is absent in normal lung tissue and expresses F4/80, suggesting that these are TAMs. The other population expresses Gr‐1, which is consistent with MDSCs. Together, our data suggests that other immune suppressive phenotypes such as TAMS and MDSCs play an active role in Treg recruitment to the microenvironment through the production of CCL22.

Supported by the Dept. of Veteran's Affairs & NIH
CD8+ T cell Recognition of Polymorphic Wild Type Sequence p5365–73 Peptides in Squamous Cell Carcinoma of the Head and Neck
Authors: Filho Pedro Alcantara Andrade , Ito Daisuke , Whiteside Theresa L , DeLeo Albert , Ferris Robert L
Published: June 2021

The HLA‐A*0201‐restricted wt peptide, p5365–73, encompasses a well‐studied polymorphism in TP53, at codon 72 (R/P), which is homozygous (R/R) in over half of the world population. In vitro stimulation of PBMC from healthy donors led to the generation of wt p5365–73‐specific CD8+ T cells in a substantial number of individuals. Generation of CTL recognizing either p5365–73P or p5365–73R, after IVS of PBMC from homozygous or heterozygous donors was successful, and recognition of either allelic peptide was observed, regardless of the genotype of the donor T cells or the peptide used as a target. HLA A*0201+ tumor cell lines which are either homozygous (R/R or P/P) or heterozygous (R/P) at codon 72, demonstrated cross‐recognition by a panel of p5365–73‐specific CTL. More efficient HLA‐A*0201 binding was observed with the p5365–73P versus the p5365–73R allelic peptide, indicating that p5365–73P variant peptide could be useful as an enhanced epitope for vaccine application. However, we found preferential recognition of p5365–73R expressing target cells, regardless of the allelic peptide used to generate in IVS cultures. In addition, since p53 homozygosity (R/R) is widely prevalent, this enables vaccination with the nonself p5365–73P, peptide avoiding self tolerance and future thymic deletion and may permit the utilization of p5365–73P to most efficiently target this epitope in wt p53‐based vaccines.
Relative contribution of membrane‐bound complement regulatory proteins CD55, CD59, CD46, and soluble factor H to resistance of human B lymphoma cells to complement
Authors: Ferreira Viviana P , Pangburn Michael K.
Published: June 2021

The complement system uses a complex set of cell‐bound and plasma regulators to protect host cells from complement‐mediated lysis. Although membrane‐bound complement regulatory proteins (mCRPs) are known to protect cancer cell surfaces, thus interfering with anti‐cancer immunotherapy, the role of soluble factor H (fH) in their protection remains less clear. While large variations in expression of mCRPs (DAF, CD46, CD59, CR1) have been observed among tumors, fH, a key regulator of fluid phase and cell surface complement activity, is present at 500 μg/ml in blood. We have shown that recombinant C‐terminal domains 19–20 of fH (rH19–20) specifically block fH cell surface complement regulatory functions. In this study, rH19–20 + anti‐DAF and anti‐CD59 + anti‐DAF treatments similarly increased lysis of Raji lymphoma cells, compared to anti‐DAF alone. rH19–20 + anti‐DAF + Rituximab (an antibody treatment for B cell lymphoma) also increased lysis of cells vs anti‐DAF + Rituximab. Interestingly, blocking fH‐mediated cell surface protection and DAF resulted in the greatest increase in C3b and C9 deposition vs inhibiting any other combination of two regulators. The results highlight the importance of fH‐mediated cell surface protection, compared to mCRPs, and indicate that fH may be as important as mCRPs in its potential for posing an obstacle to antibody based cancer therapy. Support: NIH DK35081 and AHA 0735101N.
DIFFERENT PROCESSING SITES IN THE GENERATION OF TUMOR ANTIGEN EPITOPES AND VIRAL EPITOPES SUGGEST NEW DIRECTION FOR ANTI‐TUMOR IMMUNOTHERAPY
Authors: Yang Xiao‐Feng , Zhang Sheng , Mirkovic Dragan , Zhang Q.E. , Wang Hong
Published: June 2021

In order to improve the processing efficiency of T cell tumor antigen epitopes, this bioinformatic study compared proteolytic sites in the generation of 47 experimentally identified HLA‐A2.1‐restricted immunodominant tumor antigen epitopes to those of 52 documented HLA‐A2.1‐restricted immunodominant viral antigen epitopes. Our results showed that the amino acid frequencies in the C‐terminal cleavage sites of the tumor antigen epitopes, as well as several positions within the 10 amino acid (aa) flanking regions, were significantly different from those of the viral antigen epitopes. In the 9 amino acid epitope region, frequencies differed somewhat in the secondary‐anchored amino acid residues on E3 (the third aa of the epitope), E4, E6, E7, and E8; however, frequencies in the primary‐anchored positions, on E2 and E9, for binding in the HLA‐A2.1 groove remained nearly identical. The most frequently occurring amino acid pairs in both N‐terminal and C‐terminal cleavage sites in the generation of tumor antigen epitopes were different from those of the viral antigen epitopes. Our findings demonstrate, for the first time, that these two groups of epitopes may be cleaved by distinct sets of proteasomes and peptidases or similar enzymes with lower efficiencies for tumor epitopes. In the future, in order to more effectively generate tumor antigen epitopes, targeted activation of the immunoproteasomes and peptidases could be achieved that mediate the cleavage of viral epitopes; thus, enhancing our potential for antigen‐specific tumor immunotherapy.
Identification for human TCRgamma/delta‐recognized epitopes/proteins via CDR3delta peptide‐based immuno‐biochemical strategy
Authors: He Wei , Chen Hui , He Xiaojuan , Wang Zhun , Wu Di , Cui Lianxian
Published: June 2021

To identify antigenic epitopes/proteins recognized by TCR gamma/delta, we established a new immuno‐biochemical strategy, which is mainly based on binding activity of complementarity determining region 3 in TCR delta (CDR3delta). CDR3delta peptides were firstly synthesized. After validations for their binding specificity to target cells/tissues, the CDR3¦Ä peptides were employed as probes to pan putative epitopes in a 12‐mer random peptide phage‐displayed library, or to identify putative protein ligands within ovarian tumor protein extracts by CDR3¦Ä peptide‐mediated affinity chromatography and the analysis of LC ESI‐MS/MS. We identified nine peptides and two proteins for TCR gamma/delta, including human mutS homolog 2 (hMSH2) and heat shock protein 60. All nine tested epitope peptides could not only bind to gamma/deltaT cells, but also functionally activate gamma/deltaT cells in vitro. The hMSH2 on ovarian tumor cells was recognized by Vdelta2 T cells, suggesting that hMSH2 might be a new protein ligand for TCRdelta. Taken together, our findings provide a novel and facilitative immuno‐biochemical technical strategy to find epitopes/proteins recognized by gamma/deltaT cells.
Baseline plasma C‐reactive protein level affects oxidative stress response to a diet rich in macadamia nuts
Authors: Cao Yumei , Bagshaw Deborah M. , Cifelli Amy M. , Griel Amy E. , Kris‐Etherton Penny M.
Published: June 2021

Oxidation is important in early development of atherosclerotic plaques. High‐MUFA diets reduce susceptibility of LDL to oxidative modification. Twenty‐five mildly hypercholesterolemic subjects were recruited to a 2‐period randomized, crossover, controlled feeding study to compare the effects of a macadamia nut‐rich (MAC) diet (33% total fat, 7% SFA, 18% MUFA, 5% PUFA; ~1.5 ounces/day of macadamia nuts/2000 kcal/day) on CRP and oxidative stress, to an average American (AAD) diet (33% total fat, 13% SFA, 11% MUFA, 5% PUFA). There was no main effect of diet on CRP. Lipid hydroperoxide (LPO) decreased after both AAD and MAC diets vs. BL (baseline) (p<0.05); LPO was similar on both diets. A subgroup analysis of subjects with CRP < or ≥1 mg/L was conducted to evaluate effects of BL inflammatory status on oxidative stress response of the test diets. LPO was decreased after AAD (−0.6±0.2 μM; p<0.01) and MAC diets (−0.4±0.2 μM; p<0.06) vs. BL in the low CRP group while it did not differ from BL in the high CRP group. In conclusion, BL CRP levels may modify oxidative stress responses to a cholesterol‐lowering diet. Funded by the Hershey Company; Partial support provided by the PSU GCRC (NIH grant M01RR10732).
Evaluation of the hypocholesterolemic effect of Lactobacillus acidophilus fermented maize meal (ogi) in rats
Authors: Adebawo Olugbenga Obajimi , Banjoko Yemi , Osilesi Tola
Published: June 2021

Lactic acid bacteria (LAB) consist of micro‐organisms important in food fermentation. In Nigeria, LAB is involved in the fermentation of two main substrates: cassava and cereals. Cereals are fermented into ogi, burukutu and pito (indigenous beer) and kunu (a beverage). However, Lactobacillus acidophilus, a gut inhabiting LAB has not been isolated as natural micro flora in any of the food items. The importance of Lact acidophilus as probiotic has been well documented. Thus in the present work, we have tested strains of Lact acidophilus in the fermentation of ogi, a popular fermented maize meal eaten as breakfast cereal in Nigeria to serve as a medium of delivery of Lact acidophilus. Results showed that there was no significant difference (P < 0.05) in the pH, total titratable acidity, total reducing sugars, color, flavor, taste and overall acceptability of the Lact acidophilus fermented ogi compared to the traditional ogi. The Lact. acidophilus fermented maize mash (ogi) as well as the traditional ogi were used in compounding high cholesterol diets and tested for hypocholesterolemic effect in rats against a control diet containing unfermented maize meal. The results revealed significant differences (P < 0.05) in total cholesterol values, 3.56 ± 0.32mmolL−1, 5.32 ± 0.21mmolL−1 and 6.30 ± 0.32mmolL−1 for the diets containing Lact acidophilus fermented ogi, the traditional ogi and unfermented maize meal respectively. We concluded that Lact acidophilus was suitable as starter culture for the production of indigenous maize based fermented food products which might serve as medium for the delivery of Lact. acidophilus as probiotic for hypocholesterolemic effect.
Anthocyanin‐rich extract mitigates skin photoaging in ultraviolet‐B irradiation‐induced human dermal fibroblasts
Authors: Bae Ji‐Young , Kang Young‐Hee
Published: June 2021

Bog blueberries are rich in anthocyanins, phytochemicals that belong to the flavonoid family and provide pigmentation to fruits and vegetables. Anthocyanins have received attention as agents that may have potential in preventing chronic diseases such as cardiovascular diseases and certain cancers. The present study investigated the capacity of an anthocyanin(ATH)‐rich extract from bog blueberry to inhibit degradation of collagen, secretion of collagenolytic MMP enzymes and production of inflammatory cytokines in ultraviolet (UV)‐B irradiated human dermal fibroblasts. The ATH‐rich extract attenuated the UV‐B‐induced cytotoxicity accompanying a blockade of ROS production. In addition, the ATH‐rich extract markedly suppressed collagen degradation in UV‐B‐exposed dermal fibroblasts by blunting production of these MMP enzymes. We next attempted to explore whether the ATH‐rich extract down‐regulated MMP production via interfering with the MAPK‐dependent and/or NF‐κB‐responsive pathways. This extract mitigated activation of MAPK, which resulted in a consequent decrease in MMP‐1 secretion. Additionally, the ATH‐rich extract reduced release of multiple inflammatory cytokines such as IL‐1β, IL‐6, IL‐8 and TNF‐α. These results demonstrate that the ATH‐rich extract dampens UV‐B‐triggered collagen destruction and inflammatory responses through modulating MAPK‐ and NF‐κB‐dependent pathways. Therefore, the ATH‐rich extract from bog blueberries may be a protective agent against UV‐induced skin photoaging.

Grant Funding Source: Korea Research Foundation (KRF‐2006‐521‐F00072) and Brain Korea 21
Study of a new biomarker of bone collagen as estimator of calcium adequacy in healthy young school children
Authors: Esther Rio Naria , C Dupraz Hernan , B Zago Liliana , N Zeni Susana
Published: June 2021

The search of an adequate method to assess calcium (Ca) nutritional status in children is a pending subject of nutritional biochemistry. In this work, preliminary results of an assay addressed to add a new biomarker to the available battery is presented. Bone growth is a process with high probability of being affected by body Ca status. For that, a degradation product of collagen type I, measurable by CrossLaps (CTX) immunoassays in urine samples, was selected: the α‐CTX biomarker that arises from newly synthesized bone collagen and could be associated with growth at childhood. For that purpose, basal void samples were obtained from 173 healthy scholars attending public schools of Vicente Lopez. Children, aged 6.2 to 9.2 years, presented normal BMI. Alfa‐CTX (ELISA, Nordic Bioscience Diag); Ca (EAA) and creatinine (WienerLab®.Argentina) were performed on those samples to calculate the respective α‐CTX and Ca/Creatinine ratios. The obtained equation for α‐CTX vs Ca/C was: 1,0256x + 0,7706; R= 0,52; p<0,01. Results show a quite good correlation between both variables. Standardization procedures are in progress. UBCyT 753. Part of the H Dupraz PhD Thesis.