bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of CCL5 transfected murine mammary adenocarcinoma cells for alterations in disease progression (50.2)
Authors: Kurt Robert A , Jayasinghe Mevan , Nair Priyanka , O’Donnell Cara , Werner Michael
Published: January 2023

A previous study showed that reducing tumor-derived CCL5 expression using RNA interference did not significantly influence growth rate or metastatic ability of the 4T1 tumor. Since 4T1 is an aggressive tumor that constitutively expresses CCL5 and exhibits low immunogenicity we wanted to determine whether tumor derived CCL5 expression could influence disease progression of a less aggressive and non-metastatic tumor. For this purpose we used the 168 tumor which does not constitutively express CCL5. We cloned the full length CCL5 gene, inserted it into a eukaryotic expression vector and transfected the 168 tumor. Whereas the parental and vector transfected control 168 tumors expressed 0–30pg/ml of CCL5, transfection with the CCL5 expression vector resulted in up to 1000pg/ml of CCL5. To determine whether CCL5 expression would influence growth or metastasis of the 168 tumor we generated clones of the CCL5 transfected tumor. After 4 weeks of in vitro passaging, in the absence of G418, CCL5 expression remained elevated indicating the generation of stable lines. Analysis of tumor growth rates revealed that tumor-derived CCL5 expression alone was not sufficient to modulate growth of the 168 tumor. These clones are being used to determine whether CCL5 transgene expression is sufficient to modulate metastatic ability of the tumor.

This work was supported by the National Institutes of Health R15 CA111539-01.
TGF-beta1-induced expression of alphaE(CD103)beta7 integrin on Ag-specific tumor-infiltrating T lymphocytes promotes killing of E-cadherin-bearing tumor cells by triggering polarized cytotoxic granule exocytosis (50.22)
Authors: MAMI-CHOUAIB Fathia , JALIL Abdelali , VERGNON Isabelle , LE MAUX CHANSAC Béatrice , LAZAR Vladimir , BISMUTH Georges , CHOUAIB Salem , LEFLOC’H Audrey
Published: January 2023

Interaction of intercellular adhesion molecule (ICAM)-1 with lymphocyte function-associated antigen (LFA)-1 enhances target cell-T cell adhesion and thus promotes TCR-mediated killing. In the absence of ICAM-1, frequently lost during tumor cell dissemination, we demonstrate that the interaction of the epithelial cell marker E-cadherin with αE(CD103)/1 _7 integrin, often expressed by tumor-infiltrating T lymphocytes (TIL), plays a major role for an effective cancer cell lysis. Indeed, while tumor-specific CD103+ TIL are able to kill E-cadherin+/ICAM− autologous tumor cells, CD103− PBL-derived counterparts are inefficient. Furthermore, tumor cell killing by TIL is abrogated by anti-CD103 mAb and RNA interference targeting E-cadherin. Confocal microscopy analysis demonstrated that αEβ7 is recruited at the immunological synapse and that its interaction with E-cadherin is essential for cytolytic granule polarization and subsequent exocytosis. Interestingly, pre-incubation of PBL-derived T cells with anti-CD3 mAb and TGF-β1 induced CD103 expression resulting in a dramatic potentiation of anti-tumor effector function. Thus, CD8+/CD103+ tumor-reactive T cells infiltrating epithelial tumors most likely play a major role in anti-cancer CTL response.
Antigen priming in the presence of IL-12 selectively enhances the survival of CD62LhiCD8+ T cells and results in superior anti-tumor activity (50.23)
Authors: Diaz-Montero C. Marcela , EL-Naggar Sabry A. , Montero Alberto J. , Cole David J. , Salem Mohamed L.
Published: January 2023

It has been recently shown that early effector CD8+T cells are more efficient at killing tumors than late effector CD8+T cells. Since expression of CD62L is progressively lost as T cells differentiate, its expression on activated CD8+T cells has been associated with superior antitumor activity. In our study, we have shown that in vitro priming in the presence of IL-12 results in the accumulation of CD62LhiCD8+ T cells. The antiapoptotic effects of IL-12 were found to be selective for this population and was associated with downregulation of caspases 3 and 8, Bax, and Bad. We also found that IL-12 modulates the expression of CD62L. OT-1 cells primed with SIINFEKL with or without IL-12 were sorted into CD62Llo and CD62Lhi populations three days after priming and recultured with or without IL-12. Exposure of untreated CD62LloCD8+cells to IL-12 after sorting resulted in increased CD62L levels. CD62LloCD8+cells primed in the presence of IL-12 transiently upregulated CD62L expression. However, the levels decreased by day 7 unless cells were re-exposed to IL-12. The fact that effects were seen on sorted populations, suggests that IL-12 has a direct effect on the expression of CD62L. Finally, we found that CD8+CD62Lhi cells generated after priming with IL-12 had increased in vivo survival and enhanced recall capabilities. When compared with CD62Lhi cells primed with antigen alone, IL-12-treated- CD62Lhi cells showed enhanced anti-tumor capabilities. In conclusion, our data supports the findings that increased expression of CD62L on effector T cells results in enhanced anti-tumor activity, and suggests IL-12 treatment as an effective strategy for the generation and maintenance of such subpopulation.
NK Cell Mediated Tumor Cytotoxicity is enhanced by Mild Thermal Stress through Heat Shock Factor 1 (50.24)
Authors: Dayanc Baris Emre , Ostberg Julie R , Yuan Min , Repasky Elizabeth A.
Published: January 2023

Natural killer (NK) cells directly kill tumor cells and regulate downstream adaptive immune responses. Previous studies in our lab suggest that mild thermal stress (39.5 C) significantly enhances the cytotoxicity of human peripheral blood NK cells compared to normothermic conditions (37 C) against human colon tumor cells. This enhancement was not seen when syngeneic human PBMC were used as targets, nor when the IL-2 dependent human NK cell lines NKL or NK92MI, or IL-2 activated primary NK cells were used as effectors. Thus, we hypothesized that resting NK cell cytotoxic activity is regulated by mild thermal stress. Blocking of the activating receptor NKG2D and its stress-induced ligand MICA with blocking antibodies suggested the involvement of these molecules in the thermal regulation of NK cytotoxicity. We found that inhibitory receptor KIR2DL1 levels on NK cells were not changed with thermal stress. Increased clustering of NKG2D on NK cells was observed with thermal stress, suggesting the potential for enhanced efficiency of activating signals. However prevention of lipid raft clustering with methyl B-cyclodextrin on NK cells shows no significant affect on thermally enhanced cytotoxicity. On target cells, MICA expression appeared to be upregulated by mild thermal stress in a manner that is dependent on the transcription factor HSF-1. Overall, these data suggest that thermal therapy might be clinically utilized to help enhance NK mediated tumor cell killing.

Supported by NIH P01 CA94045, R01 CA71599, Komen Foundation DISS0402487 and Fulbright Program.
OX40-ligand inhibits function of IL-10-producing intratumoral CD4+CD25+ regulatory T cells in human follicular B-cell lymphoma (50.25)
Authors: Voo Kui Shin , Bover Laura del Carmen , Neelapu Sattva S , Kwak Larry W , Liu Yong-Jun
Published: January 2023

Recent studies showed that infiltrating T regulatory cells inhibit effector T-cell function in B-cell non-Hodgkin lymphomas. Therefore, it was suggested that strategies that inactivate or remove regulatory T-cell populations should be done in conjunction with established clinical protocols in B-cell lymphomas bearing patients. In the present study, we purified CD4+CD25−, CD4+CD25highICOS+ and CD4+CD25highICOS- cell subsets by flow cytometry from a follicular lymphoma patient. We examined their intracellular FoxP3 expression and determined their IL-10 secretion in cell supernatants under different conditions. The cell subsets were stimulated with anti-CD3 and anti-ICOS antibodies in the presence of IL-2 on parental L cells or OX40L-L cells. Cytokine production in supernatants was measured by ELISA. Both CD4+CD25highICOS+ and CD4+CD25highICOS- cells showed FoxP3+ expression by FACS analysis, while it was negative in CD4+CD25−. After 3 or 6 days of culture, only ICOS+ cells produced large amounts of IL-10 (470 and 3,250 pg/ml respectively) and OX40L dramatically inhibited this production (87 and 94% of inhibition). Immunohistochemistry staining demonstrated simultaneous expression of FoxP3 and ICOS on T-cells infiltrating B-cell lymphoma tissues. These results suggest that OX40L could be potentially used as a therapeutic agent to modulate T regulatory cells function in B-cell lymphoma patients.
Depletion of CD4+CCR4+ cells enhances T cell-mediated responses in vitro and vaccine-induced anti-tumor immune responses in mice. (50.27)
Authors: Biragyn Arya , Olkhanud Purevdorj B , Sumitomo Kenya , Baatar Dolgor
Published: January 2023

CCR4 is a chemokine receptor which is preferentially expressed on T helper 2 (Th2) CD4+ cells and CD25+CD4+ T regulatory cells (Tregs). Tregs and Th2-type cytokines (IL-10 and IL-4) were implicated in suppression of anti-tumor immune responses. We have hypothesized that depletion of CCR4+CD4+ cells, particularly Tregs and Th2 cells, may improve anti-tumor T cell immune responses. To test this, we generated a recombinant protein consisting of TARC (CCL17, ligand for CCR4) fused with 38kD truncated form of Pseudomonas exotoxin (PE38), designated TARC-PE38. TARC-PE38 efficiently and specifically killed CCR4+ cells, including sorted human peripheral blood CCR4+CD4+ and CCR4+ Tregs. We demonstrate that, while CCR4+non-Tregs mostly suppress Th1-type polarization without affecting T cell proliferation, CCR4+Tregs primarily regulate T cell proliferation via contact-mediated suppressive pathways. As a result, depletion of CCR4+ cells enhance proliferation of T cells and promote the Th1-type polarized responses. Moreover, the depletion also significantly enhanced tumor antigen-specific T cell responses in vitro. Importantly, in vivo depletion of CCR4+ cells augmented tumor - specific CD8+ CTL responses improving survival of tumor bearing mice, when combined with cancer DNA vaccines. Taken together, our data suggest that poor immune responses to tumors can be reversed by transiently depleting suppressive CCR4+ cells.

This work was supported by the Intramural Research Program of the National Institute on Aging, NIH.
Aberrantly expressed neutrophil elastase (ELA2) in the nucleus and cytoplasm of acute lymphocytic leukemia (ALL) cells cleaves cyclin E (CCNE) into low-molecular-weight forms (LMWFs) yielding novel HLA-A2 restricted determinants (50.28)
Authors: Ishiyama Ken , Kondo Yukio , Wieder Eric , Lu Sijie , Molldrem Jeffrey
Published: January 2023

We reported that CCNE-specific CTL elicited from healthy donors (HDs) kill ALL and CML cells, which aberrantly express CCNE. Further, 5 LMWFs of CCNE1 are present only in malignant cells and constitutively active to promote cell division. In addition, we showed ELA2-specific CTL also kill AML and CML, and ELA2 is normally expressed only in myeloid cells. To determine whether ELA2 might be aberrantly expressed in ALL and whether ELA2 cleaves CCNE to LMWF’s, we first found CCNE of LMWF to be overexpressed by western blot of cell lysates from ALL, but not from ALL in remission or HD B-cells. ELA2 added to ALL lysates increased LMWF’s and was inhibited by the ELA2 inhibitor elafin. Subcellular fractionation and co-immunoprecipitation showed ELA2 to be aberrantly associated with CCNE in nucleus, cytoplasm, and membrane-bound organelles in ALL, but not normal bone marrow cells. Because determinants recognized by CCNE-CTL are also found in the LMWFs and lysis of leukemia correlates with target protein overexpression, we studied the effect of induced CCNE expression on target susceptibility to cytolysis. While normal PBMC were not killed, PHA or α-CD3/28 stimulated PBMC were killed by CCNE-CTL, although non-specifically. We conclude that in ALL, aberrant expression of the myeloid protein ELA2 results in mistrafficking of ELA2 to cytoplasm and nucleus, thus mediating CCNE cleavage to LMWF’s and increasing susceptibility to CCNE-CTL killing. This shows novel tumor antigen interaction that may cooperatively reverse CTL tolerance.
TNF-related apoptosis inducing ligand (TRAIL) enhances TCR-mediated renal cancer recognition by a novel CD4+ T cell clone (50.29)
Authors: Wang Qiong , Hanada Ken-ichi , Yang James C
Published: January 2023

A human CD4+ T cell clone (HC/2G-1) was established by stimulating peripheral blood T cells with dendritic cells pre-incubated with the autologous apoptotic renal tumor line in the presence of IFN-α. It recognizes multiple MHC-mismatched RCCs by IFN-γ release (10 of 11 lines) and lysis (9 of 10 lines), but does not recognize multiple EBV-B cells or fibroblasts. It shows little or no recognition of a panel of melanomas, breast cancer and non-small cell lung cancers. Phenotypically, HC/2G-1 is CD3+CD4+TCRα/β+, but CD161−CD16−NKG2D−. HC/2G-1 does not utilize Vα or Vβ TCRs described for classical NKT cells, but rather Vα14, Vβ2.1. Tumor recognition was TCR α/β mediated as demonstrated by conferring reactivity by transducing third party T-cells with the cloned TCR from HC/2G-1. Tumor recognition by clone HC/2G-1 was not blocked by antibodies to HLA-class I or class II, but was significantly reduced by both anti-TCRα/β and anti-TRAIL Ab. HC/2G-1 expresses low level of TRAIL, and can be upregulated with type I IFNs. Although most RCC tumor lines were resistant to TRAIL induced apoptosis, treatment of soluble TRAIL trimer enhances tumor recognition such as IFN-γ production by HC/2G-1 as well as by HC/2G-1 TCR-transduced T cells. Furthermore, tumor recognition by HC/2G-1 TCR-transduced T cells was blocked by anti-TRAIL Ab. This suggests that TRAIL may be inducing the antigen, or a restriction element on tumor cells. Attempts to understand the molecular mechanism of tumor recognition by HC/2G-1 are underway.
Host-derived CCL5 is critical for mammary tumor growth and metastasis (50.30)
Authors: Liu Jianguo , Ma Xiaojing
Published: January 2023

CCL5 is a chemokine produced by a variety of human cells under stress. It plays an important role in inflammation by recruiting T cells, macrophages and eosinophils to inflammatory sites. It has been shown in animal studies that tumor-derived CCL5 is able to help tumors to form new blood vessels (angiogenesis), thus fostering their growth. Tumor-derived CCL5 can also inhibit the T cell response and enhance the growth of breast cancer. Human studies have shown high incidence of CCL5 expression in tissue sections of breast carcinomas. Furthermore, in stage II breast cancer patients, the expression of CCL5 increased significantly the risk for disease progression. However, the mechanisms of CCL5 promote the tumor growth and metastasis is still unknown.

Using a syngeneic 4T1 mammary tumor model that closely resembles human breast cancer in primary tumor growth characteristics and metastatic properties, we have observed that, compared to control wild type mice of identical background, mice that are genetically deficient in CCL5 (CCL5-ko) are highly resistant to 4T1 tumor growth and to tumor-induced death. In addition, the remaining tumors in CCL5-ko mice developed little lung metastasis after 60 days. The CCL5-ko mice that regressed the tumor promptly and strongly rejected rechallenge with 4T1 tumor, indicating establishment of protective immunity. Furthermore, mAb-mediated neutralization of CCL5 in vivo prevented 4T1 tumor growth and lung metastasis. Our data also indicate that 4T1 tumor cells spontaneously produce high levels of CCL5 driven by highly active NF-kB and other yet to be characterized transcription factors. In conclusion, CCL5 plays a critical role in the growth and metastasis of mammary tumors, and that CCL5 represents a desirable and accessible target in breast cancer therapy.
Substance P modulation of anti-tumor responses: enhanced tumor resistance by adoptive transfer of SP-treated T cells and increased NK cell activity (50.5)
Authors: Manske Jill , Brand Lucas , Kastelic Amanda , Westin Angelene
Published: January 2023

Substance P (SP) is a neuropeptide that has been shown to have immunoregulatory properties including effects on mediators involved in anti-tumor immunity. In previous studies we have shown that treatment of mice with SP provides protection against tumor growth. This protection requires both T cells and NK cells, and adoptive transfer of cells from SP-treated animals can transfer tumor protection. These studies suggest a model in which SP treatment prior to tumor challenge primes immune mediators to prevent or delay tumor establishment. In this study we examined the role of NK cells and T cells in tumor protection. The cytotoxic activity of NK cells against Yac-1 targets was examined by labeling cells with annexin-V and measuring apoptotic cell death by flow cytometry. NK cells from mice treated with SP had significantly higher cytotoxic activity compared to NK cells from controls. To examine whether SP-primed T cells could increase tumor resistance, purified T cells from SP-treated donor mice were adoptively transferred into animals prior to tumor challenge. Mice adoptively transferred with T cells from SP-treated donors developed significantly fewer tumors than did controls. These results suggest that SP mediates anti-tumor immunity in part by increasing NK cytotoxic activity. Since SP-primed T cells can transfer tumor protection, these cells may play a role in this increase in NK activity.
Targeting Activation Induced Cytidine Deaminase Promotes Rejection of Large Established Tumors by Cytotoxic T Lymphocytes in a Mouse Myeloma Model (50.8)
Authors: Bai Xue-Feng , Liu Jin-Qing , Joshi Pramod S. , Liu Chang-Gong , Wu Lai-Chu , Liu Yang
Published: January 2023

Activation induced cytidine deaminase (AID) is an essential enzyme that controls the generation of antibody diversity in B cells and is considered a general gene mutator. Using transgenic T cells specific to P1A (P1CTL) to treat mice with large established plasmacytoma J558 tumors, we frequently observed mutations in P1A antigenic epitope, which lead to cancer cell evasion of destruction by P1CTL. To determine whether AID plays a role in CTL-mediated rejection of plasmacytoma, we generated AID-silenced plasmacytoma J558 cells and controls and tested whether large established AID-silenced J558 tumors could be rejected by P1CTL. Complete rejection of large established tumors by P1CTL was rare (0–10%) in mice with AID+ J558 tumors while the rejection rates were 30–90% in mice with AID-silenced J558 tumors. AID knock down did not reduce mutation rate of P1A gene, but rather caused more genetic instability and altered gene expression profile. Notably, we observed strong up-regulation of OX-2 (CD200) gene and down-regulation of CaMKII-δ gene in AID-silenced J558 cells, whose roles may be related to enhanced immune rejection of AID-silenced J558 tumors. Our results suggest that simultaneously targeting AID in CTL-targeted therapy of myeloma or other AID+ tumors may be of therapeutic value.
Agent based model of the TH1/TH2 differentiation decision (51.3)
Authors: Lawrence Michael Brandeau , Schmidt Brian J. , Chavali Arvind K. , Glaw Lydia
Published: January 2023

Adaptive immune responses tend to polarize to predominantly TH1 or TH2-directed, and this decision is critical for the clinical outcome of many diseases. For example, Leishmania species that elicit a TH2 response, such as donavani, can lead to visceral leishmaniasis, which is fatal if not treated. In contrast, the body is able to combat leishmaniasis and recover from species that tend to elicit a TH1 response, such as L. major. The type of response the immune system mounts is governed by a multifactorial process involving the nature of the antigen and co-stimulatory molecules expressed on antigen presenting cells (APCs). We developed an agent-based computational model using knowledge of immunological interactions recently elucidated by in vivo two-photon microscopy studies to investigate whether APCs could be engineered to elicit a TH1 response after a TH2 response was previously established by a pathogen. The model supports the stochastic repertoire scanning model and further suggests that this mechanism may help T-cells coordinate their response by sampling the cytokine cues left by other differentiating T-cells. Additional cell types, such as natural killer and memory cells, may be important for establishing the dynamics of the response and the ratio of TH1 to TH2 cells. Our model additionally suggests that engineered APCs have the potential to be a viable treatment method for diseases such as visceral leishmaniasis.

Supported by NIH R01 HL54614.
A soluble Dectin-1 Fc fusion protein enhances innate immunity against invasive pulmonary aspergillosis (51.8)
Authors: Mattila Polly E , Metz Allison E , Repaka Rekha R , Steele Chad
Published: January 2023

The Dectin-1 beta-glucan receptor is essential for alveolar macrophage (AM)-mediated defense against Aspergillus fumigatus. Increased incidence of invasive pulmonary aspergillosis (IPA) over the last several decades has necessitated the development of novel vaccines and immunotherapies to augment host defense against A. fumigatus in susceptible individuals. In our studies, the extracellular carbohydrate binding domain of Dectin-1 was fused with murine IgG1 Fc (sDect-Fc) by PCR and cloned into a mammalian expression vector. sDect-Fc was also cloned into a non-replicating adenovirus for in vivo expression. The viability index (VI) in AM cultures containing sDect-Fc was 80% lower than untreated AM cultures, indicating sDect-Fc significantly augments AM-mediated killing of swollen conidia. sDect-Fc also minimized the AM inflammatory response to A. fumigatus. IV administration of AdsDect-Fc, but not AdLuc, to naive mice resulted in sDect-Fc protein in the lungs. In preliminary studies, mice receiving AdLuc had 66% mortality within 16 days after A. fumigatus challenge whereas mice that received AdsDect-Fc had no mortality within a 21 day period. These results support the use of sDect-Fc as an immunotherapeutic strategy to augment host defense against IPA. Grant HL080317 from the NIH and grant CI-21330-N from the ALA.
Cyclophosphamide effects on avian macrophages in vitro (52.10)
Authors: Kolawole Abimbola Olayinka , McCorkle Fred Miller
Published: January 2023

Cyclophosphamide is an alkylating agent used in the treatment of a variety of cancers. It has been shown to cause immunodepression by reducing T cell, B cell and antibody production, but little is known about the effects of the drug on macrophages. Avian macrophages are good models for human macrophages because they have the same functions in maintaining immunity. This study examined the cytotoxic effect of cyclophosphamide on the activated avian macrophage cell line MQ-NCSU, as well as the effects of the drug on attachment to a plastic substrate, phagocytosis, nitrite production and RNA synthesis for macrophage chemoattractant protein-1 (MCP-1) and nitrous oxide synthetase-1 (NOS-1). Avian macrophages were exposed to doses ranging from 101 to 10−7 mg/mL for a period of 30 min. Cyclophosphamide was not cytotoxic to the avian macrophages and did not affect the ability to phagocytize sheep red blood cells and E. coli. Macrophage attachment was reduced by 21% after treatment at concentrations of at least 10−4 mg/mL. At 10 mg/mL, cyclophosphamide reduced nitrite production by 67% and was associated with a qualitative reduction in the amount of nos-1 RNA extracted from macrophages. The total cellular RNA extracted from and the amount of mcp-1 RNA in macrophages was not affected. This study shows that the clinical doses of cyclophosphamide used for cancer patients are not detrimental to avian macrophage functions.

Acknowledgements: Dr. Jennifer Schisa, Graduate student research and creative endeavors grant, Central Michigan University.
Structural and functional characterization of fowlicidin-1, a cationic host defense peptide with potent antibacterial and LPS-neutralizing activities (52.13)
Authors: Alexander Justin R , Bommineni Yugendar , Xiao Yanjing , Dai Huaien , Prakash Om , Zhang Guolong
Published: January 2023

Cathelicidins are a group of cationic host defense peptides with pluripotent immunomodulatory activities. We recently identified fowlicidin-1, a chicken cathelicidin possessing broad-spectrum antibacterial and LPS-neutralizing activities but with considerable toxicity toward mammalian cells. NMR spectroscopy revealed that fowlicidin-1 mainly adopts an α-helical conformation with glycine-induced kink in the center and a short flexible unstructured region at the N-terminal end. To reveal functional significance of different structural regions and identify peptide analog(s) with enhanced therapeutic potential, a series of deletion and substitution analogs of fowlicidin -1 were synthesized. Eight amino acids (residues 16–23) at the C-terminal end were found to be critically involved in bacterial killing, LPS binding and cytotoxicity. We also revealed that the N-terminal unstructured segment (residues 1–5) plays a minimum role in antibacterial and LPS-neutralizing activities. Removal of valine at position 5 reduced the cytotoxicity by 4-fold while retaining a similar antibacterial potency to the parent peptide. However, omission of tryptophan at position 6 resulted in at least 4-fold reduction in bacteria-killing efficiency, indicating the critical involvement of tryptophan. Taken together, fowlicidin-1(6–26) represents a short peptide analog with excellent potential to be explored as a novel antimicrobial.

This project was supported by Oklahoma Center for the Advancement of Science and Technology, Oklahoma Agricultural Experiment Station and HHMI-Undergraduate Research Scholarship.
Impaired expression of Toll-like receptors in peripheral blood monocytes and granulocytes in type 2 diabetic subjects (52.15)
Authors: MORENO-FIERROS LETICIA , Martinez-Barrera Luis Enrique
Published: January 2023

Type 2 diabetes represents a prevalent serious health problem worldwide. Type 2 diabetes (non-insulin-dependent) is characterized by chronic hyperglycemia and disturbances of carbohydrate, fat and protein metabolism. Complications among diabetic patients include increased recurrence and severity to many infections. Low-grade inflammation also seems to be involved in the pathogenesis of diabetes. However the underlying mechanisms of the immunological impairment in diabetes are unclear. Toll-like receptors (TLRs) play a key role in pathogen recognition by the innate immunity, interestingly altered TLR expression has been recently reported in diabetic rodent models but it has never been studied in humans. Therefore using flow cytometry we tested if, under basal conditions, TLR expression was altered in peripheral blood monocytes and granulocytes from Type 2 diabetic Mexican subjects. We found a decreased frequency of monocytes and granulocyte expressing TLR3 and TLR4 in diabetic subjects, nevertheless TLR2 and TLR9 expression was not significantly different between diabetic and healthy groups. Whereas in hyperglycemic conditions lower frequencies of TLR2, TLR3 and TLR4 expression were found, TLR9 expression was significantly higher. Our findings support that the increased incidence of infections occurring in diabetes maybe related an impaired TLR expression.
4-1BB stimulation induces pregnancy failure that is mediated by CD8+ T cells (52.16)
Authors: Kim Kwang H , Kim Young H , Lee Myeong J , Choi Beom K , Kim Jung D , Kwon Byoung S
Published: January 2023

4-1BB (CD137) regarded as a powerful T-cell costimulatory molecule. However, recent studies indicate that in vivo 4-1BB stimulation is able to actively suppress the antigen-specific immune response by inducing indoleamine 2,3-dioxygenase (IDO) in antigen-presenting cells. Since we found that both 4-1BB and IDO were induced in the placenta during pregnancy, we questioned whether 4-1BB signaling would be negative or positive on the pregnancy. By treating agonistic anti-4-1BB mAb or blocking anti-4-1BBL mAb to the pregnant mice, we tested the role of 4-1BB. Treatment of agonistic anti-4-1BB mAb markedly increased the abortion of allogeneic fetus, which was primarily dependent on the CD8+ T cells. Since anti-4-1BB treatment induced the accumulation of C3 component in the placenta, which was reversed by treating depleting anti-CD8 mAb, C3 complement seemed to be functioned as an effector molecule in the placenta. However, blockade of 4-1BB / 4-1BBL interaction or 4-1BB stimulation to the syngeneic fetus showed a marginal effect on the pregnancy. These results indicate that 4-1BB signal may be involved in the regulation of immune responses between maternal T cells and fetal tissues.

This study was supported by the Science Research Center Fund from KOSEF and the Korean Ministry of Science and Technology.
ASP-C5L2 neutralizing antibodies alter triglyceride metabolism in vitro and in vivo (53.10)
Authors: Cianflone Katherine , Cui Wei , Paglialunga Sabina , Kalant David , Lu HuiLing , Roy Christian
Published: January 2023

There are growing data that show the linkage between immune and metabolic systems. C3a is an anaphylatoxin. Its inactive product, C3a desArg, known as Acylation Stimulating Protein (ASP), is well documented as a lipogenic factor stimulating triglyceride synthesis (TGS) and glucose transport (GT). Recently, C5L2 was identified as a functional receptor of ASP. Interestingly, C5a also binds C5L2, but binding has no inflammatory response, suggesting C5L2 is a C5a decoy receptor. The aim of this study is to determine in vitro and in vivo effects of blocking ASP-C5L2 interaction using antiASP and antiC5L2 IgG. In C5L2 transfected HEK cells, antiASP or antiC5L2 competes for ASP binding (IC50: ASP 93±27 nM; antiC5L2, 199±19 nM;) and inhibit TGS and GT. In mice, antibody injection had no effect on body and tissue weight, food intake, and plasma levels of insulin, leptin, or adiponectin, but caused delayed TG (AUC: control 2.0±0.3 mM*5 h; antiASP 11.8±1.5 mM*5h p<0.001; antiC5L2 10.4±0.9 mM*5h, p<0.001) and non-esterified fatty acid (AUC: control 1.3±0.3 mM*5h; antiASP 7.2±0.5 mM*5h p<0.001; antiC5L2 6.9±0.3 mM*5h p<0.001) clearance. TG content decreased in liver (antiASP -28.0% p<0.05; anti-C5L2 −40.9% p<0.01), but increased in muscle (62.0%, antiASP, p<0.01; 81.4%, antiC5L2, p<0.001). AntiASP and antiC5L2 block ASP-C5L2 interaction, demonstrating ASP functions through C5L2 receptor.

Funding: CIHR
Complement-mediated hemolytic anemia and platelet activation in mCd59a and mCd59b double knockout mice (53.4)
Authors: Qin Xuebin , Hu Weiguo , Song Wenping , Grubissich Luciano , Song Yanli , Wu Gongxiong , Hu Xuemei , Ferris Sean , Dobarro Martin , Bauer Selena , Feelisch Martin , Leopold Jane A. , Loscalzo Joseph , Halperin Jose A
Published: January 2023

The complement regulatory protein CD59 inhibits formation of the complement membrane attack complex (MAC). Humans express only one while mice express two Cd59 genes; each mCd59 gene contains two promoters that control its expression in a tissue specific manner. Thus, total deficiency of mCd59 activity is required to study unambiguously the role of MAC and CD59 in mouse models of human diseases including paroxysmal nocturnal hemoglobinuria (PNH), a complement mediated hemolytic anemia with thrombosis due to the lack of GPI-linked membrane proteins, especially CD59. Here we report the targeted deletion of both mCd59 genes in mCd59ab−/− mice, their PNH-like phenotype, and the use of these mice in complement sufficient and complement (C3) deficient background to establish the role of complement in the activation of PNH-like platelets in vivo. Low NO bioavailability in the vasculature due to increased NO-scavenging free hemoglobin in their blood has long been suspected to contribute to the thrombotic diathesis and smooth muscle dystonias of PNH-patients. Using an NO-synthase inhibitor in vivo and an NO donor in vitro, we established a novel balancing effect of nitric oxide (NO) in the complement-mediated activation of mCd59ab−/− (CD59 deficient, PNH-like). These results unmask a hitherto unrecognized role for NO in balancing complement-mediated platelet activation.
C-Reactive Protein Stimulated Spleen Cells Transfer Suppression of Experimental Immune Thrombocytopenia (53.9)
Authors: Marjon Kristopher David , Clos Terry W Du , Marnell Lorraine L , Verbeek J. Sjef , Mold Carolyn
Published: January 2023

C-reactive protein (CRP) is an innate recognition molecule, which regulates immune responses by interaction with FcγR. CRP suppresses inflammation and autoimmunity in diverse experimental models. Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease in which platelets are cleared by splenic macrophages through FcγR. IVIG is used to treat human ITP. IVIG induces dendritic cells to transfer suppression of ITP (Siragam et al., 2006, Nat Med). In this study, splenocytes were treated with 200 μg/ml CRP or BSA or 1.8 mg/ml IVIG for 30 min, washed and transferred to naïve recipients. After 24 h, ITP was induced by injection of anti-CD41 mAb. Platelets were counted 24 h later. Anti-CD41 decreased platelet counts from 945±29 to 312±61 x 103/mm3. Platelet counts in recipients of 106 treated spleen cells were: BSA, 225±35; CRP, 672±25; and IVIG 590±35. Spleen cells from FcR γ-chain−/− or FcγRI−/− mice treated with CRP or IVIG were not protective and no protection was observed in FcγRIIb−/− mice. Macrophage depletion in vivo by Clodronate liposome treatment of the donor eliminated protection by CRP (363±9, p<0.001 vs CRP), but not IVIG (560±38). Thus, CRP treatment of spleen cells in vitro generates a cell that transfers suppression of ITP. Generation of this cell by CRP is dependent on FcγRI and macrophages. The findings allow for the characterization of the cells involved in the immunoregulatory properties of CRP.
RNA-Binding Protein Pum2 Promotes Self-renewal and Suppresses Differentiation of Multipotent Hematopoietic Cells by Maintaining Them in Inactive CD34− State (81.3)
Authors: Zayas Jennifer , Nachtman Ronald G , George John , Jurecic Roland
Published: January 2023

Over-expression of RNA-binding protein Pum2 supports maintenance and suppresses mutilineage differentiation of murine HSC-like cell line EML. Analysis of HSC markers on wild type EML cells and EML cells over-expressing Pum2 (Pum2-EML cells) has revealed that almost all wt EML cells are Sca-1+c-kit+ Flk2−, but exhibit heterogeneous expression of CD34, with ~20% of cells being CD34−, and ~80% being CD34+. Similarly, almost all Pum2-EML cells are Sca-1+c-kit+Flk2−, but the vast majority of Pum2-EML cells are CD34− (80–95%). Functional analysis has revealed that purified CD34+ subpopulation of wt EML and Pum2-EML cells repeatedly contained almost all of the cells capable of mutilineage differentiation in response to cytokines. In contrast, the CD34− subpopulations of wt EML and Pum2-EML cells differentiated poorly or not at all. Previous studies have shown that mouse HSC can alternate between “activated” CD34+ and CD34− “inactive” state. Thus, we testedwhether CD34− EML cells are more primitive and give rise to CD34+ cells, orwhether EML cells alternate between CD34− inactive (differentiation inhibited) and CD34+ active (differentiation ready) state.

Culture and flow cytometry analysis of purified CD34− and CD34+ wt EML cells has revealed that both CD34− and CD34+ EML cells reproducibly generate each other, and that the “secondary” CD34− and CD34+ EML cells exhibit the same properties. Identical experiments have revealed that Pum2-EML cells are maintained predominantly in an inactive CD34-state. These results support the notion that Pum2 protein supports proliferation and suppresses differentiation (two critical components of self-renewal) of multipotent hematopoietic cells by maintaining them in inactive CD34− state.
Multiple prethymic defects underlie age-related loss of T progenitor competence (81.6)
Authors: Zediak Valerie P. , Maillard Ivan , Bhandoola Avinash
Published: January 2023

Aging in mice and humans is characterized by declining T lymphocyte production in the thymus, yet it is unclear whether aging impacts the T lineage potential of hematopoietic progenitors. Although alterations in the lymphoid progenitor content of aged mouse bone marrow have recently been described, irradiation- reconstitution experiments have failed to reveal defects in T lineage potential of bone marrow (BM) progenitors or purified hematopoietic stem cells (HSC) from aged mice. Here, we assessed T progenitor potential in young recipient mice without conditioning irradiation. T potential was reduced in aged BM compared to young BM and this reduction was apparent at the earliest stages of intrathymic differentiation. Further, purified populations of aged HSC or multipotent progenitors (MPP) also gave rise to fewer T lineage cells than their young counterparts. Limiting dilution experiments in vitro showed that the T precursor frequency within the MPP pool was unchanged. However, there was a fourfold reduction in T precursor frequency within the aged HSC pool, indicating that there is an accumulation of T-incompetent cells in this population. Additionally, among the T-competent HSC clones, there were fewer highly proliferative clones in the aged HSC pool than in the young HSC pool. These results define the nature and cellular sites of prethymic age-related defects in T lineage differentiation potential.
The Jackson Laboratory repository: model selection for immunology research (82.10)
Authors: Lake Jeffrey , Donahue Leah Rae , Lutz Cathy , Rockwood Stephan , Sasner Michael
Published: January 2023

The mouse continues as one of the most important model organisms. The Jackson Laboratory Repository serves as a centralized facility for developing, rederiving, cryopreserving and distributing mouse models. The Repository contains a large repertoire of mouse strains related to immunology research that have applications in studies of basic immunology, innate immunity, cancer research, autoimmunity, allergic diseases, and infectious diseases.

Selecting appropriate mutant mice is often difficult. For example, mice devoid of CD4+ T cells are found in 3 different strains including a Cd4 −/− strain (B6.129S2-Cd4tm1Mak/J), a strain where MHC class II genes were deleted (B6.129-H2dlAb1-Ea/J), or a targeted mutation in the class II transactivator (B6.129S2-C2tatm1Ccum/J). A wide variety of cre-expressing strains, targeted mutants with loxP-flanked genes and tetracycline regulated transactivator-expressing mice are available that require careful selection. Other factors for model selection include mutation characteristics related to mechanism of action, cell types affected, effects of the mutation on breeding and lifespan, and other associated phenotypes. The strain background including breeding performance, MHC haplotype, disease resistance/susceptibilility and known mutations, such as hemolytic complement deficiency found in a subset of inbred strains, could confound results.

Supported by NCRR (RR09781, RR11083, RR16049), NIA, HHMI, and donations from several private foundations
Development of all CD4 T lineages requires nuclear factor TOX (82.2)
Authors: Aliahmad Parinaz , Kaye Jonathan
Published: January 2023

We have previously identified a nuclear factor member of the HMG-box superfamily, designated TOX, which is transiently expressed in the thymus during both positive selection and β-selection. We had shown that Tox is upregulated by calcineurin-dependent signaling and transgenic mice that express TOX generate CD8 single positive thymocytes (CD8SP), even in the absence of TCR signaling. We now report that mice deficient in TOX (TOX−/−) are outwardly normal but lack all CD4 lineage cells, namely CD4 αβ T, CD4 regulatory T and NK-T cells, due to a block in positive selection at the transitional CD4loCD8lo stage. In contrast, some CD8SP thymocytes develop in TOX−/− mice. These CD8SP can colonize the periphery and respond to TCR triggering. The block in CD4 T but not CD8 T cell development in TOX−/− mice is also observed in TCR transgenic animals. These results demonstrate differential requirements for the CD4 and CD8 lineages, where CD4-destined cells require induction of TOX for their development.
Negative selection of Th1-skewed NKT cells by the thymic epithelial CD1d (82.8)
Authors: Lee Hyunji , Hong Changwan , Park Se-Ho
Published: January 2023

NKT cells are a specialized subset of T cells mainly utilizing invariant Vα14-Jα18 chain for their T cell receptor. Unlike conventional T cells which are dependent on MHC class I or II, NKT cells are dependent on a MHC class I-like molecule, CD1d, for their development. Unlike MHC class I and II molecules, which support both of the positive and negative selection of conventional T cells, it was not clear whether CD1d also participate in negative selection of NKT cells. By comparing the NKT cell population and their characteristics in CD1d-deficient Vα14 transgenic mouse and CD1d-deficient Vα14/Eα-CD1d double transgenic mouse, we found that a subset of NKT cells with the phenotype of NK1.1+, CD44high, CD69high and showing Th1-skewed cytokine production was subjected to the negative selection by the CD1d expressed in thymic epithelial tissue. These results suggest that CD1d is critical not only for the positive selection of NKT cells, but also for the negative selection of NKT cells which have potentially harmful excessive self reactivity.
Thymic selection in the absence of TCR/coreceptor agreement (82.9)
Authors: Riddle David S , Frelinger Jeffrey , Collins Edward
Published: January 2023

The contribution of TCR coreceptor to T cell lineage commitment has not been firmly established. Models proposed to explain lineage commitment require that the MHC specificity of TCR and coreceptor agree in order for thymocytes to develop successfully. We have generated a mouse model, MHC II°/CD8°, in which TCR/coreceptor agreement cannot occur. This mouse has between one half and one third of the number of peripheral T cells compared to wild type mice, depending on age. Nonetheless, the T cells utilize an array of TCR variable genes similar to wild type, suggesting that the reduced number of peripheral T cells is not due to a bottleneck in the population of TCRs that can be positively selected in such a restrictive environment. Both CD4+ and coreceptor negative T cells produce IL-2 and IFNg in response to antigen-independent stimulation. Furthermore, despite the lack of CD8, peripheral coreceptor negative T cells display cytolytic function against allogeneic MHC, and against syngeneic MHC following LCMV infection. These results indicate that TCR/coreceptor agreement during thymic selection is not necessary for development of a heterogeneous population of functional T cells, and thus have implications for current models of lineage commitment.
Impaired pre-B cell development in the alpha 1,6-fucosyltransferase deficient mice (83.13)
Authors: Kondo Akihiro , Li Wenzhe , Ishihara Katsuhiko , Yokota Takafumi , Nakagawa Takatoshi , Miyoshi Eiji , Koyama Nobuto , Taniguchi Naoyuki
Published: January 2023

The alpha 1,6-fucosyltransferase (FUT8) is responsible for the core-fucosylation of N-glycans and core fucose of N-glycans could modify function of glycoproteins on the cell surface. Mice with a targeted gene disruption of Fut8 (Fut8−/−) had impaired B cell development at the pre-B cell stage, reduced numbers of immature B cells, reduced B cell proliferative responses and antibody production. Our finding for the first time showed impaired early B cells development owing to the low binding affinity of VLA-4 to VCAM-1 in Fut8-KD-pre-B cells. Consistently, reintroduction of Fut8 partly restored the binding affinity by an increase in the percentage of binding cells from 29% to 44%, indicating that core fucosylation is required for VLA-4/VCAM-1 interaction during B cell development. Loss of early B cell lineage was associated with down-regulation of several gene expressions, such as CD79a, CD79b, Ebf1, Tcfe2a in Fut8−/− CD45R+IgM− cells. Indeed, the ratio of pre-BCR+CD79b+ cells in gated Fut8−/− pre-B cells was much lower than in Fut8+/+ pre-B cells, indicating a differentiation defect in the transition from pro-B cell to pre-B cell. These results provide evidence that Fut8 is functionally linked to the VLA-4/VCAM-1 interaction in mediating B cell tethering to stromal cells and is pivotal in B cell development and activation.
Plasma cell differentiation and lipid metabolism (83.18)
Authors: Bommiasamy Hemamalini , Sriburi Rungtawan , Jackowski Suzanne , Brewer Joseph W.
Published: January 2023

Plasma cells are terminally differentiated B cells that produce and secrete profuse amounts of antibodies. Plasma cell differentiation involves an increase in exocytic pathway components and expansion of the endoplasmic reticulum (ER) in order to accommodate high-rate immunoglobulin synthesis. ER expansion requires membrane biogenesis, a process that requires phospholipids. Increased phospholipid synthesis is dependent upon a sufficient supply of fatty acids. We have found that de novo fatty acid biosynthesis increases in CH12 B cells differentiating in response to lipopolysaccharide (LPS). Increased fatty acid biosynthesis correlates with upregulation of acetyl CoA carboxylase (ACC) and fatty acid synthase (FAS), enzymes that catalyze the formation of fatty acids. Further investigation will shed light into mechanisms that regulate lipid synthesis and ER expansion in differentiating plasma cells.

Supported by NIH-GM61970 and NIH-T32 A1007508
The beta-catenin pathway is involved in normal B cell development and function (83.3)
Authors: Ko Alex Y , Zierold-Fairman Claudia , Ranheim Erik A
Published: January 2023

Wnt/Fzd signaling plays a key role in development, stem cell maintenance, and tumorigenesis, particularly via stabilization of ß-catenin protein levels. TCF-1 and LEF-1 knockout mice suggested a role for this pathway in lymphoid development as well. We have previously shown that Fzd9−/− mice exhibit a decrease in pre-B cells at a stage when self-renewing division is occurring in preference over further differentiation, prior to light chain Ig recombination (Hardy C-D). We now report further evidence for a role of ß-catenin in normal B cell function and development. By flow cytometric analysis, we find that cellular ß-catenin levels rise at precisely the stage of B cell development that shows diminished numbers in Fzd9−/− mice. Expression of LEF-1, the transcriptional partner of ß-catenin, also is tightly regulated during this developmental sequence by differential transcription of full length and dominant negative forms, further suggesting that Wnt/Fzd signals impact on B-cell lymphopoiesis. In mature B-cells, activation through BCR, Toll-like receptors, and CD40 all induce an increase in beta-catenin protein levels, while changes in the expression of genes involved in both positive and negative regulation of canonical Wnt signaling are differentially regulated by these stimuli, highlighting a potential novel role for this pathway in B-lymphocyte proliferation, differentiation, and survival.