bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of PKA‐activating Gs‐coupled GPCR ligands in the regulation of SLE T cell responses
Authors: Khan Islam U. , Das Maitreyee , Hudgens Joseph M. , Loza Matthew J.
Published: June 2021

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by generalized T cell dysfunction. A subpopulation of SLE subjects demonstrate a T cell‐specific defect in type I PKA (PKA‐I). We hypothesize that T‐cell specific defects in PKA‐I contribute to reduced inhibition of T cell responses mediated by Treg cells and GS‐coupled GPCR ligands. Tregs (CD4+ CD25+hi) and responder T cells (CD4+ CD25−) were purified by cell sorting. The responder T cells were stimulated with CD3+CD28 mAb alone or with added Tregs, without or with added XAC (pan‐adenosine receptor antagonist) or indomethacin (pan‐cyclooxygenase inhibitor). Addition of Tregs suppressed proliferation of responder cells, with the suppression significantly greater in control subjects than in SLE subjects with a PKA‐I defect. Blocking adenosine receptors with XAC and inhibition of prostaglandin production with indomethacin significantly reversed suppression of responder cell proliferation for control but not SLE. These results demonstrate the role of GS‐coupled GPCR ligands in Treg‐mediated suppression, and provide a mechanism by which Tregs from SLE have reduced capacity to inhibit T cell responses.

Supported by NIH RO1‐AR39501.
Blockade of CD70 Exacerbates Experimental Autoimmune Myocarditis by Suppressing Regulatory T cells
Authors: Li Haiyan S. , Ligons Davinna L. , Guler Mehmet L. , Talor Monica V. , Cihakova Daniela , Yagita Hideo , Rose Noel R.
Published: June 2021

Our previous studies of genes determining susceptibility to experimental autoimmune myocarditis (EAM) revealed Eam2 locus on chr.6. In searching for differentially regulated transcripts during EAM induction in susceptible A.SW vs resistant B10.S mice by microarray analysis, CD27 was identified in Eam2. CD70/CD27 are cell surface molecules belonging to the TNF/TNF‐receptor superfamilies and provide costimulatory signals for T cell activation and survival. In this study, administration of CD70‐blocking antibody FR70 exacerbated EAM incidence, severity and mortality in A.SW but not B10.S mice, accompanied with enhanced T‐ and B‐ cell responses to cardiac myosin. We observed a significant increase in IL‐17 in the spleens of FR70‐treated group, whereas levels of IL‐2, IL‐4, IL‐10 TNFα and IFNγ were reduced. Surface staining of CD27/CD70 showed that CD27 was constitutively expressed on naïve T cells and was downregulated after activation, whereas CD70 was largely restricted to a regulatory T cell (Treg) subset before activation and was rarely upregulated in activated T, B and dendritic cells. Addition of FR70 to in vitro cultures of cardiac myosin‐ primed lymphocytes enhanced their proliferation and IL‐17 production. These results show that blockade of CD70 impairs Treg functions, thus promoting EAM. Our findings suggest a novel molecular mechanism by which Treg may exert suppressive functions.

Supported by NIH Grants R01 HL‐077611 and R01 HL‐067290
Suppression of Proteoglycan‐Induced Arthritis by Anti‐CD20 mAb Inhibits Autoantibody and T‐cell Perpetuation of Disease
Authors: Hamel Keith Michael , Doodes Paul , Cao Yanxia , Wang Yumei , Dunn Robert , Kehry Marilyn , Finnegan Alison
Published: June 2021

Recent studies demonstrate a significant improvement in clinical symptoms in rheumatoid arthritis (RA) patients treated with Rituximab, an anti‐CD20 monoclonal antibody (mAb) that depletes mature B cells but not plasma cells. To begin to understand how B cells are involved in RA, we have depleted B cells in a murine model of RA, proteoglycan (PG)‐induced arthritis (PGIA). Female BALB/c mice (≥ 3 months of age) were immunized intraperitoneally with 150 μg human PG in adjuvant [dimethy dioctadecy ammonium bromide (DDA)] twice at a 3‐week interval. B cells were depleted with a single i.v. injection of anti‐CD20 mAb (250μg/mouse) 4 days after the second PG immunization. Suppression of PGIA was observed along with reduced autoantibodies titers in mice depleted of B cells compared with controls. CD4+ T cell from B‐depleted PGIA mice exhibited reduced PG activation in primary mice as indicated by proliferation, cytokine release, and adoptive transfer experiments. Collectively, suppression of PGIA by anti‐CD20 mAb treatment provides a model for study of B cell interactions during autoimmunity after initiation phases that from these studies include autoantibody production and T cell activation. Funding was provided by NIAMS NIH grant AR47657.
Anti‐inflammatory properties of cannabinoid receptor‐2‐selective agonists in experimental autoimmune encephalomyelitis
Authors: Kong Weimin , Adhikary Sabina , Zhang Ming , Tuma Ronald , Ganea Doina
Published: June 2021

Previous studies showed that cannabinoids have anti‐inflammatory effects in experimental autoimmune encephalomyelitis (EAE), but the mechanism is elusive. Cannabinoids act through two cloned receptors, CB1 and CB2. The CB1 receptor is expressed primarily in the CNS, whereas the CB2 receptor is highly expressed in immune cells in the periphery. Previous results from our laboratories indicated that the CB2 agonist O‐1966 has both a protective and a therapeutic effect in models of chronic EAE and of remitting‐relapsing EAE. Here we investigated the effect of the CB2 agonist O‐1966 on dendritic cells and T cells. We report that the CB2 agonist inhibits antigen‐induced T cell proliferation in co‐cultures with syngeneic dendritic cells. The CB2 agonist decreased production of dendritic cell‐derived inflammatory cytokines, i.e., IL‐12p70, IL‐6 and IL‐23, and increased production of the anti‐inflammatory cytokine IL‐10. The CB2 agonist also inhibited IFNγ production by T cells. We also found that the CB2 agonist induced Foxp3 expression in T cells, as determined by quantitative RT‐PCR. We conclude that the anti‐inflammatory role of CB2 agonists could be mediated through effects on dendritic cells and subsequent T cell differentiation, and through the induction of regulatory T cells.

This work is supported by NIH/NIAID R01AI052306.
Targeting Syk in autoimmune diabetes
Authors: Colonna Lucrezia , Ogryzlo‐Harbers Stephanie Natalia , Catalano Geoffrey , Flores Marcella , Chew Claude , Masuda Esteban , Schmitz Jochen , Thomas James , Clynes Raphael
Published: June 2021

Type I diabetes (T1D) results from autoimmune attack of the pancreatic β cell islet by inflammatory cells, autoreactive T cells being the primary effectors. However, B cells are necessary for disease in nonobese diabetic (NOD) mice, the best studied model of human T1D. B cells may participate pathogenically as antigen presenting cells and/or as producers of autoantibodies (autoAbs). We have shown in the RIP‐mOVA peripheral tolerance model (RO) that auto‐Abs induce a loss of T cell tolerance in a manner dependent on activating Fc receptors on cross‐priming dendritic cells (DCs). Therefore, we have targeted the spleen tyrosine kinase (Syk), which is required for both BCR‐ and FcR‐mediated signaling, in order to broadly inhibit B cell‐mediated as well as autoAb‐enhanced antigen presentation. Using the oral Syk inhibitor R406 (Rigel Pharmaceuticals) we show that Syk blockade abrogates both B cell‐ and DC‐mediated Ag presentation in vitro, as well as effector T cell differentiation and diabetes in RO mice (100% healthy‐treated vs. 20% healthy‐untreated mice, p=0.02). Finally, Syk inhibition decreases autoAb production, prevents diabetes (60% healthy‐treated vs 14% healthy‐untreated mice at 32 weeks, p<0.01) and prolongs survival in NOD mice (p<10−5). Thus pharmacologic blockade of Syk is a promising therapeutic strategy to inhibit the humoral component of autoimmunity and prevent T1D.

This work was supported by the training grant of the Integrated Graduate Program in Cellular, Molecular, Structural and Genetic Studies at Columbia University, NY and by the Juvenile Diabetes Research Foundation, NY.
Accumulation of nuclear [beta]‐catenin in dendritic cells from NOD mice is responsible for the pro‐inflammatory phenotype of these cells
Authors: Villard Marine , Martin Michael , Carabeo Rey A , Manirarora Jean N , Parnell Sarah A , Kosiewicz Michele M , Alard Pascale
Published: June 2021

Pro‐inflammatory cytokines appear to mediate Type I diabetes development, and suppression of pro‐inflammatory cytokines in NOD mice is associated with prevention of diabetes onset. Antigen presenting cells in NOD mice produce higher levels of the pro‐inflammatory cytokine, IL‐12, due to NFκB hyperactivity. GSK3 is a crucial modulator of pro‐versus anti‐inflammatory cytokines, since its inhibition following phosphorylation by Akt leads to the impairment in IL‐12 production and to the increase in anti‐inflammatory cytokine, IL‐10, by DC. We, therefore, examined whether the pro‐inflammatory phenotype of NOD DC was due to the alteration of GSK3 activity. We have found that the levels of GSK3 phosphorylation were identical in NOD DC versus B6 DC. However, the levels of phosphorylated β‐catenin and glycogen synthase, two GSK3 targets, were elevated. Interestingly, there was a dramatic accumulation of β‐catenin in the nucleus of NOD DC compared to B6 DC. Knocking‐down β‐catenin with β‐catenin siRNA led to the downregulation of IL‐12 and upregulation of IL‐10 production by NOD DC. Altogether, these data indicate that NOD DC exhibit a defect in the expression of β‐catenin, which upon accumulation in the nucleus appears to be responsible for the pro‐inflammatory phenotype of these cells. (‐catenin has, therefore, the potential to be a new therapeutic target for the prevention and/or treatment of type 1 diabetes.
CC chemokine receptor 1 (CCR1) is expressed by bone marrow mast cells (BMMC) and, when costimulated with FcεRI, increases cell activation and promotes reorganization of membrane rafts
Authors: Goyal Nimita F , Beer Frederick , Morohoshi Kei , Raghuwanshi Sandeep , Aye Cho Cho , Ohbayashi Masaharu , Fukuda Ken , Richardson Ricardo
Published: June 2021

We examined functions and spatial interactions of FCεRI and CCR1 during granulocyte activation, in BMMC. Using flow cytometry and mRNA analysis, we observed that BMMC endogenously express CCR1 after 6 weeks. BMMC also expressed FCεRI and c‐kit, but not CCR2, CCR3 or CCR4. Using β‐hex, we found that costimulation of FCεRI and CCR1 using antigen and MIP‐1α resulted in significantly higher BMMC degranulation compared to crosslinking‐mediated FC ε RI activation alone (90% vs. 54%, P<0.05). We observed that Ca2+ influx increased from 183 nM with FCεR activation to 223 nM with costimulation in 7‐week BMMC (P<0.05). When we examined the spatial arrangement of these receptors, we found that sensitization alone, promoted CCR1 internalization. Subsequent cross‐linking to activate FCεRI resulted in lipid mediator release and localization of all FCεRI to reorganized, GM1‐positive membrane raft clusters. FCεRI‐CCR1 costimulation resulted extensive leading edge, flattening morphology and membrane ruffles than did FCεRI stimulation alone, lipid mediator released with higher frequency and cell‐to‐cell connections in a pattern seen exclusively with costimulation. The cell networks produced by costimulation may explain the arrest of mast cell chemotaxis in late‐phase allergic reactions, and may provide a conduit for cell‐cell crosstalk molecules and signal transduction mediators during immune reactions.
Stat5 Regulates Mast Cell Activation by IgE
Authors: Barnstein Brian , Tarleton Christy , Wilson Bridget , Ryan John J
Published: June 2021

We have recently found that the transcription factor Stat5 is rapidly activated by IgE crosslinkage, and that its expression is critical to IgE‐mediated degranulation, LTB4 production, cytokine secretion, and survival signals. Stat5KO mast cells induced cytokine mRNAs normally following IgE crosslinkage, but these mRNAs were not sustained over time, and were degraded at twice the rate observed in wild type cells. Interestingly, the RNA destabilizing protein tristetraprolin (TTP) was induced following IgE crosslinkage in Stat5KO but not wild type cells. Moreover, reducing TTP expression via shRNA transfection significantly increased IL‐13 production in Stat5KO mast cells. We find that Stat5 is associated with the IgE receptor after antigen‐mediated activation. Moreover, cholesterol depletion or Fyn kinase deletion prevents Stat5 tyrosine phosphorylation in response to IgE crosslinkage. These data suggest that Stat5 is activated through a lipid raft‐ and Fyn‐dependent pathway. Our work demonstrates that Stat5 is a critical factor in IgE‐induced mast cell activation that could provide a new clinical target for mast cell‐associated inflammatory diseases. Supported by NIH grant 1R01 AI59638.
Inducing an Anergic State in Human Mast Cells (MC) and Basophils (HB) without Secretion
Authors: MacGlashan Donald W , Undem Bradley J
Published: June 2021

IgE‐mediated secretion from MC or HB depends on the activity of both syk and PI3K but several specific down‐regulatory pathways (e.g., loss of syk expression) do not. We tested whether stimulation with antigen, in the presence of a syk inhibitor (SI = NVP‐QAB205) would ablate secretion while simultaneously allowing anergy. The anergic state was assessed after a relatively short period of time (45–60 min.) by removing the SI and re‐stimulating the cells. HB, cultured‐derived MC and in situ stimulated airway MC (human bronchi) were examined. Antigen caused 35 ±65% and 62 ±10% histamine release from HB and MC, respectively; and it caused an 87±5% histamine/LTD4‐dependent contraction of bronchi. All of these responses were blocked > 95% by the SI. Re‐challenging the preparations with antigen, after first washing out the SI and antigen, revealed that near complete anergy (92–100%) occurred in each case. In control experiments we demonstrated that the SI was readily reversible and easily washed out of the cells and tissues. Thus, even though the SI nearly abolished the antigen‐induced secretion from MC and HB, it had little effect on the pathways involved in anergy. A similar result was found when using a PI3K inhibitor, LY294002. These results suggest that syk and PI3K are not involved in down‐regulation leading to anergy and that it would be possible to induce anergy in vivo without a significant induction of MC/HB secretion.
Tumor progression despite the presence of functional tumor‐infiltrating CD8+ T cells
Authors: Singh Vinod , Feigenbaum Lionel , Hurwitz Arthur
Published: June 2021

Many of the antigens recognized by tumor‐reactive cytotoxic CD8+ T cells are self‐antigens. Tyrosinase‐related protein 2 (TRP‐2) is a melanosomal enzyme expressed by both normal melanocytes and melanomas that is believed to be a candidate melanoma rejection antigen. To study the role of self‐reactive CD8+ T cells in tumor immunity and autoimmunity, we generated T cell receptor transgenic (TCR Tg) mice which bear a TCR transgene specific for TRP‐2 (180–188). TRP‐2 TCR Tg mice did not develop depigmentation despite systemic expression of TRP‐2 in the skin and their T cells exhibit a naïve phenotype: CD44low, CD62hi and CD69 and CD25 negative. Furthermore, endogenous TRP‐2 expression has no effect on the TRP‐2 specific CD8+ T cells development and antigen responsiveness. Using a system of Tg T cell adoptive transfer into wild‐type C57BL/6 mice, we have observed that in vitro‐activated TRP‐2‐specific CD8+ T cells reduced B16/BL6 pulmonary nodules. However, provision of a TRP‐2‐pulsed dendritic cell vaccine induced proliferation of the Tg T cells resulting in migration to the subcutaneous tumor. These tumor‐infiltrating Tg T cells remained reactive to TRP‐2 for at least 15 days after vaccination but did not reduce tumor growth. Thus, these findings demonstrate that tumors can evade functional anti‐tumor immune responses. Our on‐going studies will test whether priming these tumor‐reactive T cells in the presence of various cytokine regimens will sufficiently enhance their cytotoxic capacity to induce more potent tumor immunity.
Tumor specific CD4 T cells promote the accumulation of low avidity tumor specific CD8 T cells within the tumor milieu
Authors: Bos Rinke , Wong Justin , Marquardt Kristi , Biggs Judy , Sherman Linda
Published: June 2021

Using a murine model of spontaneous tumor formation in the pancreatic islets, RIP‐Tag2HA, in which tumors express the hemagglutinin (HA) protein of influenza as a surrogate tumor antigen, we previously reported that tumor ablation by low‐avidity Clone 1 CD8 cells was enhanced greatly by the presence of CD4 help. This was true despite the fact that the mice received influenza virus to stimulate the CD8 cells. This enhancement of tumor ablation was associated with increased infiltration of CD8+ T cells into the tumor within the first 4 days after vaccination, but it wasn't clear whether this was due to an effect of CD4+ T cells on the priming of CD8+ T cells or was attributable to the activity of CD4+ T cells at the site of the tumor. We have shown now that only CD4+ T cells that recognize antigen within the tumor environment are adequate to facilitate tumor infiltration by tumor specific CD8+ T cells. This is not only true for high affinity CD4+ T cells specific for HA, but also for lower affinity BDC2.5 CD4+ T cells that recognize an auto‐antigen in the islets. To determine whether CD4 cells were also required to sustain tumor eradication, tumor specific CD4+ T cells were depleted at different time points from RIP‐Tag2HA mice that received tumor specific CD8 cells, CD4 cells and vaccine. This revealed that the presence of CD4+ T cells during the first 6 days after immunization is very effective in the induction of good anti‐tumor immunity. We are now further elucidating the mechanisms of CD8+ T cell recruitment into the tumor and the effect of CD4+ T cells on the long‐term anti‐tumor response.
A Chimeric 4‐1BBL as a Potent Adjuvant for Therapeutic Cancer Vaccines
Authors: Sharma Rajesh Kumar , Elpek Kutlu G , Yolcu Esma S , Schabowsky Richard H , Zhao Hong , Bandura Laura L , Shirwan Haval
Published: June 2021

Objective: Inasmuch as adaptive immune responses are critical to immune surveillance and such responses are regulated by CD28 and TNFR family costimulatory molecules, we herein tested whether 4‐1BBL can serve as an effective adjuvant for therapeutic cancer vaccines. The choice of 4‐1BBL is due to its pleiotropic effect on the cells of innate, adaptive, and regulatory immunity. Inasmuch as 4‐1BBL has no function in soluble form, we generated a chimeric molecule consisting extracellular portions of 4‐1BBL fused C‐terminus to core streptavidin.

Methods: Mice were injected with 1×105 TC‐1 cells expressing HPV E7 oncoprotein in the right flank. Once tumor was established, animals were vaccinated s.c. once with 50 μg of an E7 peptide serving as the dominant epitope for CD8+ T cells and 25 μg of SA‐4‐1BBL. The efficacy of vaccine was assed by monitoring animals for tumor growth, in vivo killing response, and intracellular cytokine staining.

Results: Vaccination resulted in the eradication of existing tumors as well as prevention of recurrences after surgical removal of the tumors. The efficacy of immunotherapy was correlated with higher CD8+ T cell infiltration into tumors, the generation of robust T cell killing response, higher frequency of IFN‐γ expressing CD8+ T cells, and long‐term memory.

Conclusion: This novel form of 4‐1BBL has the potential to serve as an effective adjuvant for the development of therapeutic cancer vaccines because of its role on adaptive, innate, and regulatory immunity.

Funded in parts by 1R41AI071618, R43CA109866, R41 CA121665, and JBCC Fellowship
Activation of tumor‐specific CD8+ T cells after intratumoral Ad5‐TRAIL/CpG combination therapy
Authors: VanOosten Rebecca L. , Griffith Thomas S.
Published: June 2021

CD8+ T cell activation via cross‐presentation of antigens from apoptotic tumor cells is controversial. Dendritic cells (DC) capture shed tumor antigens and cross‐present them to CD8+ T cells; yet, the frequency of activated CD8+ T cells is often too low to mount an effective antitumor response. By increasing the amount of antigen for presentation, a larger T cell response can be theoretically elicited. We used a recombinant adenovirus encoding TRAIL (Ad5‐mTRAIL) to induce tumor cell apoptosis, and when given intratumorally to mice bearing experimental renal cell carcinoma (Renca) tumors, Ad5‐mTRAIL only minimally prolonged survival and induced low CTL activity. To enhance DC efficiency, CpG was combined with Ad5‐mTRAIL, which significantly augmented in vivo antigen‐specific T cell proliferation and CTL activity, and prolonged the survival of Renca tumor‐bearing mice. Interestingly, depletion of CD4+ or CD25+ cells prior to therapy further enhanced survival and in vivo CTL activity. In addition, tumor‐free mice depleted of CD4+ cells were able to reject a subsequent challenge of Renca cells, but not MHC‐matched RM‐11 prostate tumor cells, demonstrating the existence of immunologic memory. These results collectively show that local treatment with Ad5‐mTRAIL and CpG can augment tumor antigen cross‐presentation resulting in T cell proliferation, enhanced CTL activity, and increased animal survival.
Overcoming brain immune privilege with Flt3L and TK treatment in a recurrent rat glioma model
Authors: King Gwendalyn D. , Muhammad Ghulam , Larocque Daniel , Candolfi Marianela , Bergeron Josee , Liu Chunyan , Kroeger Kurt , Van Rooijen Nico , Lowenstein Pedro R. , Castro Maria G.
Published: June 2021

GBM is an invasive primary brain cancer which invariably recurs. Intratumoral treatment of a large syngeneic rat model of GBM with AdFlt3L and AdTK induce ~70% long term survival. Here we evaluated Flt3L and TK treatment in a model of recurrence. Long term GBM survivors post‐therapy were rechallenged with GBM in the contralateral striatum. 100% of animals survived to ~200 days post‐GBM rechallenge. In vitro elispot assays detected tumor specific circulating T cells; in vivo DTH was detected in all long term survivors. Depletion of CD8+ cells but not CD4+ or macrophages abrogated survival of GBM rechallenged animals. To model neo‐antigen expression as a means of tumor escape, we generated CNS‐1 cells stably expressing hemagglutinin CNS1‐HA cells grew similarly in vitro and in vivo and responded to Ad‐Flt3L/Ad‐TK treatment in an indistinguishable fashion from CNS1 tumors. When long term survivors were rechallenged with CNS‐1HA cells, INF γ secreting T‐cells were detected against HA. We conclude that Ad‐Flt3L/Ad‐TK treatment induces immunological memory that protects against recurrence and tumor escape by overcoming brain immune privilege.

Funding: NIH/NINDS Grant 1R01 NS44556.01; 1R21‐NSO54143.01; 1UO1 NS052465.01; 1RO1 NS 054193.01; RO1 NS 42893.01, U54 NS045309‐01 and 1R21 NS047298‐01 to P.R.L. GDK and MC are supported by NIH/NINDS 1F32 N50503034‐01 and 1F32 NS058156.01 respectively.
Gadd45β Is Important for CD8+T Cell Mediated Tumor Surveillance
Authors: Ju Songguang , Li Changyou , Zhao Yani , He Yukai , Gu Qin , Liu Lin , Zhang Xueguang , Lu Binfeng
Published: June 2021

Recent studies have shown that growth arrest and DNA damage‐inducible protein 45β (Gadd45β) plays an important role in Th1 responses in the acute infectious model and promotes Th1‐mediated autoimmune diseases in experimental allergic encephalomyelitis (EAE). However, the function of Gadd45β in CD8+T cells is unclear. In this study, it is shown that Gadd45β deletion results in decreased IFNγ production and reduced levels of activated p38 MAP kinase in CD8+T cells upon TCR or IL12/IL18 stimulation. Gadd45β−/− mice are more susceptible to tumor development. The compromised CD8 T cell function caused by Gadd45b deletion is mainly responsible for the failure of tumor immunosurveillance. Taken together, our study demonstrated that Gadd45β plays an important role in tumor surveillance by promoting t tumor suppression by CD8+T cells.

Grant support: Cancer Research Institute Investigator award to Binfeng Lu; Pittsburgh Cancer and Aging Program (NIH grant #P20 CA103730) to Binfeng Lu. National Natural Science Foundation of China (No. 30400394) to Songguang Ju.
NKG2D dependent killing of Adenovirus serotype 5 E1A expressing tumor cells by bone marrow derived murine macrophages
Authors: Mishra Manoj K , Ryan Sharon , Lanier Lewis L , Routes John M
Published: June 2021

Prior studies indicate that the expression of the adenovirus (Ad) E1A oncoprotein in human fibrosarcoma (H4) or mouse methylchoranthrene‐induced (MCA‐205) tumor cells up‐regulates NKG2D ligands thereby sensitizing these tumor cells to lysis by NK cells. This study examined whether the ability of macrophages to lyse tumor cells that express E1A is also dependent on the expression of the activating NKG2D receptor on the surface of activated macrophages. By FACS and western analysis, we demonstrate that activation of macrophages with LPS and IFN? upregulates the expression of NKG2D. The monoclonal antibody, CX5, which blocks the interaction of NKG2D with NKG2D ligands, abrogated the ability of macrophages to kill H4 or MCA‐205 cells that express E1A. Furthermore, transfection of MCA‐205 cells with the NKG2D ligand, Rae‐1, renders these tumor cells to lysis by macrophages. These data demonstrate that macrophages express NKG2D and that the lysis of tumor cells that express E1A is NKG2D‐NKG2D ligand dependent.
The effects of B‐cell depletion therapies on humoral immune responses in normal mice
Authors: Dunn Robert Joseph , Ma Gina , Tamraz Susan , Huang Flora , Cao Xianjun , Mertsching Elisabeth , Peach Robert James , Kehry Marilyn Rose
Published: June 2021

Anti‐CD20 mediated B cell depletion is a therapy for treating rheumatoid arthritis and B‐cell malignancies. To examine how B cell depletion affects an individual's capacity to generate antibody responses we assessed in a mouse model primary and secondary antibody responses after treatment with anti‐mouse CD20 and/or BAFFR‐Ig. BALB/c mice dosed with anti‐CD20 one week prior to primary TNP‐KLH immunization had a 93% decrease in anti‐TNP IgG titers at day 10 compared to controls. Mice treated with anti‐CD20 one week prior to immunization with TNP‐Ficoll had a ~50% increase in TNP‐specific IgG and IgM titers. The secondary antibody response to TNP‐KLH was not significantly affected by anti‐CD20 treatment one week prior to immunization. BAFFR‐Ig treatment reduced the secondary response to TNP‐KLH by 57%. Treatment of mice with the combination of anti‐CD20 and BAFFR‐Ig dramatically reduced secondary antibody responses to either antigen. After B‐cell repopulation all treatment groups mounted a strong response to a tertiary challenge. Therefore, B cell depletion with anti‐CD20 results in a strong reduction in the primary antibody response to a neoantigen, however memory B cells appear resistant to anti‐CD20 treatment. Simultaneous anti‐CD20 and BAFFR‐Ig treatment results in elimination of the secondary response by blocking memory B cell activation but does not deplete memory B cells.
A Bax‐dependent apoptosis pathway of CD4+CD25highFoxp3+ regulatory T cells is a new therapeutic target in regulation of immune responses
Authors: Yang Xiao‐Feng , Yan Yan , Xiong Zeyu , Lachman Lawrence , Wang Hong
Published: June 2021

The question of whether T cell responses to more than 2000 serologically‐defined tumor antigens are under regulation of naturally occurring CD4+CD25highFoxp3+ regulatory T cells (nTreg cells) has not been answered. To address this issue, we first identified a HLA‐A2.1‐restricted T cell antigen epitope of self‐tumor antigen CML66L, 66Pa. CML66L was identified in chronic myelogenous leukemia (CML) (Yang et al, PNAS USA, 98:7492, 2001). We found that the HLA‐A2.1/66Pa peptide complex in vitro stimulated the in vivo‐primed T cells, which suggests that CML66L elicits T cell immune responses. We also developed a novel internal reference epitope for identification of T cell epitopes by construction of chimeric CML66L containing myeloid antigen proteinase 3 epitope Pr1 as a control. In addition, previous reports showed that homeostasis of nTreg cells require CD28 T cell co‐stimulation and interleukin‐2 (IL‐2)/IL‐2 receptor signaling. We demonstrated that anti‐apoptotic proteins, such as Bcl‐x isoforms and their interaction protein translationally controlled tumor protein (TCTP), play a critical role in CD28‐ and IL‐2‐signaled T cell survival pathways (Yang et al, Oncogene 24, 4778, 2005). Thus, we wanted to examine our hypothesis that Bcl‐xL‐interacting, pro‐apoptotic protein Bax mediates the apoptotic pathway in nTreg cells. We found that nTreg cells regulates CD8+ T cell responses to 66Pa, and that depletion of nTreg cells via a proapoptotic protein Bax‐dependent apoptotic pathway enhances CD8+ T cell responses to 66Pa. These findings suggest new therapeutic targets in enhancing anti‐tumor immunotherapy by modulation of survival and homeostasis of nTreg cells.
Tumor‐associated MHC II‐restricted phosphopeptides: New targets for immune recognition
Authors: Depontieu Florence , Qian Jie , Zarling Angela L. , McMiller Tracee L. , Shabanowitz Jeffrey , Hunt Donald F. , Engelhard Victor H. , Topalian Suzanne L.
Published: June 2021

Searching for more optimal targets for CD4+ T cell mediated anti‐tumor immunity, we have investigated phosphopeptides, a new category of tumor‐derived epitopes. It is unknown whether human MHC II molecules can present phosphopeptides for specific CD4+ T cell recognition, although MHC I molecules can display phosphopeptides recognized by CD8+ T cells. Using biochemical enrichment, mass spectrometric sequencing and comparative analysis, 79 HLA‐DR‐associated phosphopeptides from 42 proteins were isolated from an autologous pair of melanoma and EBV‐transformed B lymphoblast lines. Three sequences were common to both cell types, but others were unique to melanoma or B cells. Many sequences occurred as nested sets, typical of MHC II‐associated peptides. Importantly, many of the parent proteins have defined roles in cancer development, growth and metastasis. In parallel, we raised human CD4+ T cells against a candidate melanoma‐associated phospho‐B‐Raf V600E peptide, BRAFmutpT599, which could specifically recognize the phosphopeptide versus its non‐phosphorylated counterpart. This is the first demonstration, to our knowledge, of human MHC II‐associated phosphopeptides and of phosphopeptide‐specific reactivity in human CD4+ T cells, providing potential new targets for cancer immunotherapy. Funded by Dept. of Surgery and Kimmel Cancer Center at Johns Hopkins, and by USPHS grants to DFH and VHE.
CM1 ligation induces the apoptosis of human lung cancer cells via Fas/FasL interaction
Authors: Lee Hyun‐Kyung , Kim Yang‐Gun , Park Ga Bin , Kim Yeong‐Seok , Song Hyunkeun , Lee Wang Jae , Hur. Dae Young
Published: June 2021

CM1 (centrocyte/‐blast marker I) defined by a monoclonal antibody developed against concanavalin‐A activated PBMC (peripheral blood mononuclear cell),is expressed specifically on a subpopulation of centroblasts and centrocytes of human germinal center (GC) B cells. We observed the existence of CM1 in human lung cancer tissue. So we investigated the function of CM1 in lung cancer cell lines (A549). We observed that CM1 ligation with anti‐CM1 mAb enhanced the apoptosis of A549 cells. And CM1 ligation induced the expression of both FasL and Fas in theses cells. Furthermore, both anti‐Fas blocking antibody, ZB4, and anti‐Fas ligand antibody NOK‐1 blocked CM1‐mediated apoptosis effectively. Caspase‐8 inhibitor, z‐IETD effectively blocked CM1‐induced cell death. Western blot analysis showed down‐regulated expression of procaspase‐8 and PARP. The release of cytochrome c and translocation to nucleus of endoG and AIF were showed by confocal micoscopy. The findings suggested that Fas/FasL pathway was activated by CM1 ligation on human lung cancer cell line.
Anti apoptotic role of CCR9 in prostate cancer
Authors: Sharma Praveen Kumar , Singh Rajesh , Lillard James W. , Chung Leland W.K. , Grizzle William E. , Singh Shailesh
Published: June 2021

Limited treatment choices are available for the PCa patients with hormone refractory and metastatic disease. One of the better choices for treating hormone refractory metastatic disease is chemotherapy. Unfortunately better efficacy of these therapeutic modalities cannot be achieved due to the indolent nature of PCa with a very low rate of apoptosis and proliferation. We have recently shown that PCa cells (PC3, LNCaP and C4‐2b) express CCR9 and play significant role in cancer cell migration and invasion in vitro. Here we show that, CCR9‐mediated activation of PI3K/AKT pathway, which inhibits the activation of caspase‐3 and ‐9. Apoptosis induced by Etoposide or Doxorubicin in PCa cells was significantly inhibited after CCL25 treatment and further achieved after blocking CCR9‐CCL25 interaction with anti‐CCR9 monoclonal antibody. These results suggest that CCR9‐mediated cell signals provide protection against drug‐induced apoptosis. Therefore, inhibition of this axis will increase the efficacy of therapeutics for prostate cancer.
Immune cells facilitate lung metastasis of breast cancer cells
Authors: Olkhanud Purevdorj B. , Baatar Dolgor , Biragyn Arya
Published: June 2021

The Paget's ‘seed and soil’ metastasis theory is often simplistically assumed to consist of two components: cancer cells and the organ destination site that enables attachment and growth of recruited cancer cells. To analyse this theory, we have asked following questions: (1) Although chemokine receptors are often not detectable on cancer cells using conventional surface staining techniques, what is their role in metastasis; and (2) whether the recruitment/attachment of cancer cells alone at their metastatic site is sufficient to promote cancer growth. To study this, we have generated “natural” variants of highly metastatic murine breast cancer 4T1 cells using manipulations that do not introduce artificial features. The cells were depleted of chemokine receptor‐expressing subsets using chemokines fused with toxins (chemotoxins). We demonstrate that metastasis is mediated by a small subset of 4T1 cells (seed) that express a specific chemokine receptor. However, chemokine receptors alone and as well as the “soil” (the destination site) alone were not sufficient to promote tumor growth. Instead, it required the active participation of immune cells, indicating that the metastasis also requires a third player – “sun”. This work was supported by the Intramural Research Program of the National Institute on Aging, NIH.
MCF7 Side Population (SP) cells with characteristics of cancer stem/progenitor cells express the tumor antigen MUC1
Authors: Engelmann Katja , Shen Hongmei , Finn Olivera J
Published: June 2021

Cancer stem and progenitor cells, which are resistant to standard cancer therapy, are suspected to initiate tumor growth and cause tumor relapse. Our study aimed to characterize expression of MUC1 tumor antigen at various stages during tumor progression and to validate MUC1 as immunotherapy target on cancer stem/progenitor cells.

We isolated a population of stem/progenitor cells, the so‐called Side Population (SP), from MCF7 cell line, assessed expression of proposed cancer stem cell markers and studied MUC1 expression and in vivo tumorigenicity.

About 80% of MCF7 SP cells (7.5% ±1.19 SD) showed a CD44+/CD24−/low phenotype. Microarray analysis confirmed expression of stem/progenitor genes in SP cells; cell morphology and growth supported their stem/progenitor cell phenotype. For the first time we show expression of MUC1 on SP cells (77.8% ±6.9 SD) that was similar to that on mature tumor cells. The minor MUC1− subpopulation accumulated MUC1 protein intracellularly. In vivo injected MUC1+ and MUC1− MCF7 SP cells gave rise to MUC1+ tumors that maintained MUC1+ SP.

We conclude that MUC1 is aberrantly expressed not only on mature cancer cells, but also on cancer stem/progenitor cells and suggest that epithelial cancer stem/progenitor cells could be targets of MUC1‐specific tumor immunotherapy.

Support: Human Immunology Grant Irvington Institute/Dana Foundation, NIH grants 5P01 CA73743‐07 and 2R01 CA56103‐13.
Anti‐TNF therapy inhibits pancreatic tumor growth and metastasis
Authors: Kalthoff Holger , Egberts Jan Hendrik , Wajant Harald , Trauzold Anna
Published: June 2021

Chronic inflammation has been implicated in the pathogenesis of many severe autoimmune disorders, as well as in diabetes, pulmonary diseases and cancer. Inflammation accompanies most solid cancers including pancreatic ductal adenocarcinoma (PDAC), one of the most fatal cancers with surgery being the only curative therapeutic approach currently available. In the present work we investigated the role of the major proinflammatory cytokine TNFα for the malignancy of PDAC cells in vitro and in vivo. In vitro, TNFα strongly increased invasiveness of Colo357‐, BxPc3‐ and PancTuI‐cells and showed only moderate anti‐proliferative effect. TNFα treatment of mice bearing orthotopically growing PDAC tumors led to dramatically enhanced tumor growth and metastasis. Notably, we found that PDAC cells themselves secrete TNFα. While inhibition of TNFα with infliximab or etanercept only marginally affected proliferation and invasiveness of PDAC cells in vitro, both reagents exerted strong anti‐tumoral effects in vivo. In SCID mice with orthotopically growing Colo357‐, BxPc3‐ or PancTuI‐tumors, infliximab reduced tumor growth and metastasis by 30% and 50%, respectively. Importantly, in a PDAC resection model performed with PancTuI cells, we found a significant therapeutic effect for both anti‐ TNF compounds. Infliximab‐treatment reduced the volume of recurrent tumor by 70% and etanercept by almost 60%. Furthermore, both drugs strongly reduced the number of liver metastases. Thus, tumor cell‐derived TNFα plays a profound role in malignancy of PDAC and inhibition of TNFα represents a promising therapeutic option particularly in adjuvant therapy after subtotal pancreatectomy.
Sometimes a picture is worth a thousand words – use of old‐fashioned microscopy to gain a new appreciation for the cells found in human milk
Authors: Collier Natalie , McGuire Michelle K , Beerman Kathy A , Williams Janet , Shafii Bahman , McGuire Mark A
Published: June 2021

The visualization of exfoliated human milk cells for breast cancer detection was described over 50 years ago. However, microscopic examination of these cells is now rare. We are interested in using these cells to study nutrigenomics in mammary epithelium and nutrient‐host‐pathogen interactions during breast infections. As such, we have “redeveloped” the methods required to visualize these cells and have now used these methods to visualize and characterize ~80 such samples. Milk was collected from healthy, lactating women via complete breast expression. Samples were centrifuged, the milk fat removed, supernatant aspirated, and the remaining cell pellet resuspended. After recentrifugation, the supernatant was aspirated, and the remaining cell pellet rewashed. Slides were produced with 50 μL of the resuspended cell pellet, and stained with Wright‐Giemsa. Overall, 47, 32, 15, 2, and 2% of the cells were identified as macrophage/epithelial, neutrophils, lymphocytes, eosinophils, and squamous epithelial, respectively. Subjective analysis indicates a high degree of intrasubject consistency. Interestingly, many samples were laden with neutrophils even when no signs or symptoms of breast infection were present. These observations will be systematically illustrated through a series of images. Funded by the United Dairymen of Idaho and NIH‐NRRI grant P20 RR15587.
Regulation of Δ9‐desaturase activity by dietary fatty acids in lactating dairy cows
Authors: Mosley Erin E , Shafii Bahman , McGuire Mark A
Published: June 2021

The desaturation of lauric (LA), myristic (MA), palmitic (PA), stearic (SA), and vaccenic (VA) acids by the lactating dairy cow fed a saturated or unsaturated lipid was investigated. Multiparous ruminally cannulated Holstein cows (n = 3) were continuously abomasally infused with 400 g/d of coconut oil or corn oil for 7 d in a cross over design with 3 d between each period. On d 5, a bolus of [1–13C] labeled fatty acids (7 g LA, 7.5 g MA, 50 g PA, 65 g SA, and 2 g VA) was abomasally infused to determine desaturase activity. Milk yield was greater (P < 0.05) with corn oil compared to coconut oil (32.9 vs 34.8 kg/d). Delivering coconut or corn oil post‐ruminally resulted in increased LA (9.1 vs. 3.0%; P < 0.1) or linoleic acid (3.7 vs. 15.8%; P < 0.05) concentrations in milk fat, respectively. Nonlinear regression techniques were used to model enrichment of 13C which was detected in all 9‐desaturase substrate:product pairs in milk fat. Lipid treatment did not alter the percent of product originating from substrate or percent of substrate desaturated. Supported by the NRI Competitive Grant nos. 2003‐35206‐13669 and 2006‐35206‐16819 from the USDA CSREES, and NIH‐NRRI grant P20 RR15587.
The effects of exercise on bone density and hormones in lactating women
Authors: Colleran Heather L , Bopp Melanie , Mackie Heather , Wideman Laurie , Lovelady Cheryl
Published: June 2021

The objective of this study was to determine the effects of exercise on bone‐related hormones in lactating women. At 4wks postpartum (PP) women were randomized to either exercise group (EX, n=10) [weight bearing aerobic exercise (3d/wk, 45 min/d) and 3d/wk of resistance exercise] or control group (C, no exercise, n=10) for 16 wks with a 1‐yr PP follow‐up. Lumbar spine (LS) and hip bone mineral densities (BMD) were measured by DXA. Prolactin (Prl), estradiol (E2), growth hormone (GH), insulin‐like growth factor (IGF‐1), and its associated binding protein‐3 (IGFBP‐3) were analyzed using ELISA. RMANOVA was used to determine differences between groups. During the intervention, C lost significantly more LS BMD then EX (−7 ± 2% vs −5 ± 1%, p < 0.01), but not hip BMD (−2 ± 3% vs −3 ± 2%). GH levels decreased in the C (2.2 ± 2.7 to 1.0 ± 1.6 μg/L) and increased in the EX (2.0 ± 2.7 to 4.0 ± 4.7 μg/L), p=0.1, with no change in IGF‐1 and IGFBP‐3. Prl levels declined in both groups during 1‐yr PP, whereas E2, GH, IGF‐1 and IGFBP‐3 levels remained stable in both groups. At 1‐yr PP, controlling for Prl levels, LS and hip BMD were within baseline values for EX (−1% and +0.4%, respectively) vs C (−4% and −2%), p=0.1. These results suggest exercise during lactation affects GH levels and given the role of GH in bone metabolism, may ultimately influence BMD.

Supported by NC Agricultural Research Services
Complementary feeding practices in contemporary Guatemalan infants: zinc (Zn) and iron (Fe) intakes
Authors: Mazariegos Manolo , Westcott Jamie , Krebs Nancy F. , Goco Norman , Gonzalez Michelle , Kraushaar Hatai , Wright Linda , Solomons Noel W , Hambidge K Michael
Published: June 2021

Background:

The lack of adequate complementary feeding (CF) practices in the infants of developing countries is a major contributor to morbidity in young children. Objective: In the context of a community nutrition trial in the Western Highlands of Guatemala, the nutrient content of Fe and Zn in diets of older infants was assessed. Methods: Cross‐sectional 24h‐interactive dietary recalls were obtained at 6 (n=78), 9 (130) and 12 (110) mo. Nutrient values were estimated by WFS (Berkeley, CA) and INCAP food composition tables. Results: Breastfeeding was reported in 100% of infants, with 90% having >6 feeds/d at each timepoint. Mean ± SD intakes/d at 6, 9 and 12 mo were: energy (kcal) 146±139 (min‐max, 0–848), 344±209 (29–1102), 359±198 (52–1130); protein (g) 4.2±5.0, 11.8±9.1, 12.0±7.8; animal protein (% of total) 35±34, 36±26, 34±25; Fe (mg) 1.1±1.6, 2.7±2.8, 3.3±3.5; Zn (mg) 0.7±1.0, 1.5±1.2, 1.5±0.9; animal source Zn (%) 26±37, 28±27, 24±24; phytate (mg) 119±172, 338±252, 413±283; phytate:Zn molar ratios 26±26, 24±14, 28±12. Conclusion: Infants’ plant‐based CF diets are low in Zn and Fe and have a high content of an absorption inhibitor. This suggests an increased risk of mineral deficiencies in this population.

Supported by Global Network for Women's and Children's Health Research, Bill and Melinda Gates Foundation, and NIH Office of Dietary Supplements.
Childhood Overweight and Obesity in China
Authors: Lum Jessica May , Wang May Choo
Published: June 2021

PURPOSE: This study examines the published literature on trends, risk factors, and emerging directions for intervention regarding childhood overweight and obesity in China.

METHODS: Literature searches were conducted via Pub Med with the key words “childhood overweight China” and “risk factors obesity China.” Only articles featuring ethnically Chinese children, ages 0.1–18 years, in China were used for analysis. Approximately 40 articles were reviewed in this study.

RESULTS: The prevalence of overweight has increased significantly in China since the late 1980s. Studies consistently report higher prevalence rates in urban versus rural areas. For example, in the capital city of Beijing, the overall prevalence of obesity in children ages 6–12 years increased from 10.9% in 1990 to 17.2% in 1993. Multiple studies also indicate that that there are higher rates in boys than in girls. Additionally, several studies report that household socioeconomic status (SES) is positively associated with BMI. Possible etiological factors that have been identified include low physical activity, parental overweight, and increasing consumption of high‐fat, Westernized diets.

CONCLUSIONS: Further research is needed on the effects of the nutrition transition on children's health status in developing countries. With China's distinct patterns of higher rates of obesity in boys and in urban areas, cultural and environmental factors must be considered a part of the etiology. As diets become westernized, the values and norms of local communities are also influenced. These relations must be analyzed to yield the most effective interventions to counter childhood obesity.
Prevalence of The Metabolic Syndrome in the United Arab Emirates
Authors: Al‐Sarraj Taif , Saadi Hussain , Volek Jeff S , Fernandez Maria Luz
Published: June 2021

The prevalence of type 2 diabetes mellitus in the United Arab Emirates (UAE) is one of the highest in the world. This has been attributed to a sedentary lifestyle related to recent affluence leading to obesity and insulin resistance. We evaluated a population‐based sample of Emirati subjects to determine the presence of the metabolic syndrome and the risk for cardiovascular disease in this population. Anthropometrics, fasting venous glucose and lipids, and blood pressure were measured in 309 subjects who were not known to have diabetes mellitus (98 men, 215 women; mean age 38.3 ± 14.6 yrs). Mean (SD) fasting levels (mmol/L) were 5.5 ± 1.0 for glucose, 3.5 ± 1.0 for LDL cholesterol, 1.1 ± 0.3 for HDL cholesterol, and 1.0 ± 0.6 for triglycerides. Mean systolic and diastolic blood pressure values were 119 ± 16 and 71 ± 10 mm of Hg, respectively. Mean waist circumference was 87 ± 14 cm. The prevalence of the metabolic syndrome based on the modified ATP III criteria was 25.2%. Most subjects led a sedentary lifestyle and only a minority (34%) reported that they had performed any form of exercise at least once in the previous 2 weeks. Diet analysis indicated that subjects take a diet high in carbohydrate with rice and dates being major food components. These results indicate that this population is at high risk for cardiovascular disease and other complications associated with diabetes. Results from this baseline evaluation suggest that subjects from this population would benefit from a dietary intervention including carbohydrate restriction.