bluuebunny/crossref_metadata_2025 huggingface dataset:
CD19-dependent signaling pathways activated by IgM ligation, CD19 ligation or simultaneous IgM and CD19 ligation demonstrate differential requirements for Lyn and Syk kinase activities (34.17)
Authors: Kountikov Evgueni I , Poe Jonathan C , Tedder Thomas F
Published: January 2023

CD19 is a transmembrane protein with a cytoplasmic domain containing nine tyrosines that mediate associations with multiple signaling molecules. In response to cell surface IgM ligation, CD19 is phosphorylated by Lyn kinase and establishes a Src-family kinase amplification loop using tyrosines Y482 and Y513. These residues are also required for induction of Ca2+ response via binding of PI 3-kinase (PI3K). In this study, CD19 proteins with amino acid changes that prevent either PI3K or Lyn binding, or both, were stably expressed in CD19 deficient B cells and used to examine the role of CD19 in Ca2+ responses. Optimal IgM-induced Ca2+ responses required both Lyn and PI3K binding to phosphorylated CD19. However, CD19 ligation-induced Ca2+ responses did not require Src-family kinase amplification or PI3K binding to phosphorylated CD19. Instead, CD19 ligation induced a pool of activated Vav via a Syk kinase-dependent pathway that did not induce BLNK tyrosine phosphorylation. By contrast, IgM and CD19 co-ligation synergistically enhanced Ca2+ responses through a Syk-dependent pathway that correlated with enhanced BLNK and Vav phosphorylation. Thus, IgM or CD19 engagement alone, or co-ligation of these receptors, induced qualitatively and quantitatively unique physiological outcomes. This research was supported by NIH grant CA096547.
Reduced response to influenza vaccine associates with reduced AID in aged human B lymphocytes (34.5)
Authors: Blomberg Bonnie B. , Romero Maria , Landin Ana Marie , Diaz Alain , Riley Richard L. , Frasca Daniela
Published: January 2023

Elderly humans have compromised humoral and cellular immune responses, which lead to reduced protection to infectious agents and to vaccines. Currently, available vaccines suboptimally protect the elderly population. The capacity to class switch the Immunoglobulin (Ig) H chain is critical to the effectiveness of humoral immune responses in mice and humans. We have previously shown that elderly humans have fewer percentages of CD19+ total B cells, switch memory B cells, and increased percentages of naïve B cells. Activated CD19+ B cells also have less E47, AID and Igγ1 circle transcripts (CTs) with age. AID is critical for class switch recombination (CSR) and somatic hypermutation (SHM), both necessary for optimal function of Ig. More recently we have found the antibody hemagglutination inhibition (HI) assay response to the influenza vaccine is decreased in elderly (as others have previously shown) and this associates with the decrease in AID in the activated B cells from the same subjects. The mechanism that we have previously shown in mice and humans to regulate CSR (E47, AID, IgG CTs) in aging can now be used to evaluate the response to the influenza vaccine.

This work is supported by NIH AG-23717 and AG28586 (BBB) and AG025256 and AI064591 (RLR)
Computational studies of B cell receptor diffusion elucidate mechanisms of B cell immune synapse formation (34.8)
Authors: Raychaudhuri Subhadip , Tsourkas Philippos
Published: January 2023

B cells are shown to form immunological synapse, an ordered protein segregation structure consisting of BCR/Antigen and LFA-1/ICAM-1 molecules, at the cell-cell contact area between a B cell and an antigen-presenting cell. The formation mechanisms of the B cell immune synapse seem to be complex and a combination of different mechanisms are possibly in effect, including a directed transport of receptors to the central cell-cell contact area. We have studied receptor diffusion at the single molecule level during B cell synapse formation using a combination of theoretical and computational models. Results from detailed computer simulations show slow sub-diffusive transport of receptors during the course of synapse formation. Such a sub-diffusive transport becomes slower with increasing affinity of BCR-antigen bonds and thus directed receptor transport for high affinity antigens is needed for synapses to form in biologically relevant time-scales. Recent single molecule tracking experiments also revealed slow diffusion of receptors in regions of antigen binding. A simple theoretical model of receptor diffusion and clustering is proposed that clearly elucidates that sub-diffusive nature of receptor trafficking can arise due to receptor-ligand binding at a curved cell-cell contact zone.
Immunoglobulin A secretion is enhanced in allergic mice lacking the VPAC-2 receptor (39.15)
Authors: Samarasinghe Amali E. , Hoselton Scott A.
Published: January 2023

Chronic asthma is a TH2 mediated disease characterized by eosinophilic inflammation and elevated immunoglobulin (Ig)E. Acting through its G-protein coupled receptor VPAC-2, vasoactive intestinal peptide (VIP) supports a TH2 phenotype regulating lymphocytes and macrophages. IgA, produced at the lung mucosa, is important in host defense. VIP has been reported to enhance the number of IgA secreting precursors, and increase IgA while decreasing IgG. Since VPAC-2 transgenic mice have elevated antibodies and eosinophils, we hypothesized that VPAC-2 null mice will have decreased antibodies and eosinophils in an allergy model. C57BL/6 wild type (WT) and VPAC-2 null (KO) mice were sensitized with Aspergillus fumigatus antigen and challenged twice two weeks apart with live conidia. Cells in the bronchoalveolar lavage (BAL) were differentially stained and counted at days 3, 7, 14, 28 and 42 following allergen challenge. IgA in the BAL fluid (BALF), and IgE, IgG2a and IgA in sera were analyzed in both groups. Local IgA producing cells were identified with immunohistochemistry. Eosinophil recruitment and egress in the KO mice were delayed (peak at day 7) in comparison to the WT (peak at day 3). Decreased serum IgA and IgG2a and increased IgE was observed in the WT, while all three antibodies were elevated in the sera of KO mice. More IgA was available in the BALF of KO mice with the peak in IgA coinciding with peak eosinophils in the lumen at day 7. IgA producing cells were located around the blood vessels and airways in both groups. VPAC-2 null mice have elevated basal antibody levels compared to the WT controls which may indicate that VIP suppresses antibody production by acting through VPAC-2. Elevated IgA in the BALF may indicate enhanced mucosal defense in KO mice although the lack of VPAC-2 does not protect these mice from developing allergic asthma in our model. (2P20RR015566)
Vaginal Langerhans cells resist genomic integration of HIV-1 but transmit endocytosed virions to susceptible target cells (39.27)
Authors: Robinson Barry R , Ballweber Lamar , Kreger Allison , Lentz Gretchen M , Fialkow Michael F , McElrath M. Juliana , Hladik Florian
Published: January 2023

B: Vaginal Langerhans cells (LCs) efficiently endocytose HIV-1 (Immunity 2007, 26:257-70), but the ability of LCs to become productively infected or to transmit HIV to mucosal CD4+ T cells is unclear.

M: Epithelial sheets isolated from healthy vaginal mucosa were challenged with HIV-1. After 3 days, isolated cells were sorted by FACS into single LCs (>99% purity) and T cell (TC)-LC conjugates. Integrated HIV-1 was tested in DNA isolated from these cell populations using real-time PCR. LCs and LC-TC conjugates from HIV-challenged tissues were also co-cultured with uninfected T cells and transmission of HIV was evaluated by HIV-1 Gag p24 content in the co-culture supernatants.

R: We detected integrated HIV-1 DNA in 100% (4/4 donors) of the LC-TC conjugates while HIV-1 genomic integration could not be detected in single LCs (0/4 donors). Nevertheless, single LCs transmitted HIV-1 to T cells, which were productively infected (2/3 donors). LC-TC conjugates transmitted HIV-1 to T cells in 3/3 donors.

C: Vaginal LCs resist genomic integration of HIV-1 and are not susceptible to productive infection. However, vaginal LCs are capable of transmitting stored virions to T cells. Thus, in contrast to reports on HIV endocytosis in skin-derived LCs, HIV-1 endocytosis into vaginal LCs is not a dead end for the virus. This infection pathway needs to be considered when designing preventive strategies. Supported by R01 NIH-HD-51455
CCR5 is not required for mucosal protection against Trypanosoma cruzi (39.28)
Authors: Sullivan Nicole L , Eickhoff Christopher S. , Giddings Olivia K , Hoft Daniel F , Lane Thomas E
Published: January 2023

Trypanosoma cruzi is an intracellular obligate parasite and the causative agent of Chagas disease. Previous work has shown that the chemokine receptor CCR5 plays a role in systemic responses against T. cruzi. We evaluated whether CCR5 plays a critical role in mucosal protection against natural oral and conjunctival challenges. Memory-immune CCR5-/- and wild-type C57BL/6 mice were generated by repeated infectious challenges with sublethal doses of insect-derived metacyclic trypomastigotes. Similar IgA, IgG and IFN-γ responses were detected in the spleen in both memory-immune and primary infected wild-type and CCR5-/- mice. More importantly, protection was evaluated in memory-immune mice via real-time PCR and limiting dilution assay for parasite outgrowth. CCR5-/- memory-immune mice developed equivalent levels of protection against both oral and conjunctival mucosal challenges. Preliminary studies from the stomachs of CCR5-/- memory-immune mice showed an increase in the transcript levels of CCL5 as compared to memory-immune wild-type mice. Overall, it appears that CCR5 does not play a critical role in the control of parasite replication and immune responses at sites of initial mucosal infection with T. cruzi. However, CCL5 signaling through CCR1 and/or CCR3 may compensate for the absence of CCR5.
Morphine impairs intestinal immunity and barrier function leading to coagulase-negative staphylococcal bacteraemia (39.30)
Authors: Wang Jinghua , Ma Jing , Barke Roderick A. , Charboneau Richard , Roy Sabita
Published: January 2023

Opiate abuse increases susceptibility to bacterial infection. However, if the intestine can be a potential source of bacterial infections in opiate abusers is not fully elucidated. Using an opiate abuse model, where mice were chronically treated with morphine or placebo pellet, we found that morphine treatment induced bacterial translocation and led to bacteraemia. After morphine exposure, group D Entercocci and coagulase negative Staphylococci were detected in the mesenteric lymph nodes and lungs. Morphine treatment enhanced bacterial growth in the intestine, and that such increased in bacteria was in direct proportion to coagulase-negative staphylococcal bacteraemia. Furthermore, our preliminary findings showed that morphine treatment modulated intestinal intraepithelial gammadelta T cells and impaired intestinal permeability. Moreover, morphine treatment induced dysfunction in intestinal intraepithelial gammadelta T cells was associated with the absence of phosphorylation of occludin and lack of claudin-3 and zona occludens-1 (ZO-1) proteins in tight junction complexes. Our studies suggest that bacterial translocation and bacteraemia occurs in opiate abusers, either on account of a breach of the mucosal barrier, impaired host immune defense, and/or bacterial overgrowth creating an ecologic imbalance in the indigenous intestinal microflora. Supported by R01 DA12104, P50 DA11806, and R03 DA023353.
Role of small intestinal intraepithelial CD11c+ CD8+ T cells in mucosal immunoregulation (39.36)
Authors: Fujiwara Daisuke , Wei Bo , Braun Jonathan
Published: January 2023

The chronic intestinal inflammation tends to occur in the large intestine (LI) rather than in the small intestine (SI) partly due to the relative abundant microbial colonization in LI. However, intestinal intraepithelial T lymphocytes (IEL) in the large intestine are also distinguished from these in the small intestine. In this study, we investigated the possibility that the phenotypic difference of immune populations in two immune compartments might represent their differed immunoregulatory functions for regional control of mucosal inflammation. We observed that in total IELs population, about 36-40% cells in SI and 12-15% cells in LI are CD11c+ and CD3+ double positive cells. The majority of these CD11c+ CD3+T cells were CD8 positive expressing either TCRαβ or γδ. Their T cell lineage was defined by TCR expression and generation of IEL CD11c+ T cells after transfer of CD45.1 splenic T cell into RAG2-/- mice. Compared with CD11c- CD3+ T cells, CD11c+ CD3+ T cells produced significantly lower levels of pro-inflammatory cytokines including TNF-α?and IFN-γ. In mouse model of Gαi2 KO T cells induced colitis, 2.5×105 of SI CD11c+ CD3+ T cells suppressed pro-inflammatory cytokine-producing CD4 T cells at the mucosal sites and protected mice from colitis development. In contrast, LI CD11c+ CD3+ T cells or SI CD11c- CD3+ T cells were ineffective in colitis protection. Taken together, these findings indicate that SI CD11c+ CD8+ T cells might be a functional T cell population that plays an immunoregulatory role in control of pro-inflammatory CD4+ T cells and maintenance of mucosal homeostasis in the intestine.
Antibody-mediated neutralization of RANKL prevents differentiation of M cells in the follicle-associated epithelium of Peyer’s patches (39.4)
Authors: Knoop Kathryn A. , Kumar Nachiket , Butler Betsy R. , Sakthivel Senthil , Taylor Rebekah T. , Nochi Tomonori , Akiba Hisaya , Yagita Hideo , Kiyono Hiroshi , Williams Ifor R.
Published: January 2023

Mucosal surfaces are bombarded with commensal bacteria and foreign antigens. Intestinal Peyer's patches (PP) are inductive sites where secretory IgA production and T cell tolerance are initiated. The follicle-associated epithelium (FAE) over PP includes M cells that transcytose particulate antigens. We have shown that RANKL, a TNF-related cytokine expressed by PP stromal cells, is required for M cell development. UEA-I+ M cells were reduced by >98% in RANKL-/- mice, a defect corrected by s.c. injection of RANKL. We investigated whether acute depletion of RANKL by neutralizing monoclonal antibody (mAb) in BALB/c mice also led to M cell loss. Loss of 70% of UEA-I+ M cells was detected 4 days after every other day injections of 250 μg of rat anti-mouse RANKL mAb (IK22-5). Further loss of M cells occurred after longer treatment, with loss of 92% of M cells after 8 days. Anti-RANKL treatment also led to functional loss of M cell antigen sampling as measured by uptake of 200 nm diameter fluorescent beads. We conclude that continuous RANKL expression on PP stromal cells is essential for the normal differentiation of M cells from epithelial stem cells in crypts adjacent to PP domes.
Phospholipase A2 alters membrane lipid composition during ischemia/reperfusion (39.55)
Authors: Sparkes Byron Lloyd , Woods Keith , Roth Mary , Welti Ruth , Fleming Sherry D
Published: January 2023

Ischemia/reperfusion (IR) injury results in significant tissue damage. Naturally occurring anti-phospholipid antibodies (αPL Ab) induce intestinal prostaglandin E2 (PGE2) and damage when administered to IR resistant Rag-1-/- mice, suggesting involvement of a lipid antigen. Phospholipase A2 (PLA2) hydrolyzes phospholipids releasing lysophospholipid and arachadonic acid, a precursor to PGE2 production. We hypothesized that IR induced PLA2 expression, results in a lipid antigen recognized by αPL Ab. At multiple time points after Sham or IR treatment, lipids extracted from mouse intestinal sections were analyzed by mass spectrometry to determine concentrations of lipid species. Western blot analysis of additional intestinal samples detected specific PLA2 isoforms. Preliminary data indicate that lipid composition is conserved between wildtype and Rag-1-/- mice. However, within 15 min of reperfusion, IR induces significantly more lysophosphatidylcholine and lysophosphatidylethanolamine than Sham treatment. These results correlated with increased PLA2 expression. These data suggest that activation of PLA2 may create a phospholipid neoantigen which is recognized by αPL Ab and lead to tissue damage.
A single pair of CD4+ and CD8+ T cell epitopes can induce protective T. cruzi immunity (39.56)
Authors: Zhang Xiuli , Eickhoff Chris S. , Sullivan Nicole L. , Hoft Daniel F.
Published: January 2023

Both CD4+ and CD8+ T cells are required for immunity protective against the protozoan parasite, Trypanosoma cruzi. The unique T. cruzi trans-sialidase (TS) antigen can induce both mucosal and systemic protection. BALB/c mice develop CD8 responses directed against the H2-Kd-restricted TS epitope IYNVGQVSI (aa 359-367). We have used overlapping peptides (18mers with 15 aa overlaps) spanning the TS amino-terminus (aa 33-275) to identify an H2-IAd-restricted CD4 epitope that induces potent TS-specific Th1 responses (KVTERVVHSFRLPALVNV; aa 58-75). Here we demonstrate that dendritic cells (DC) pulsed only with KVTERVVHSFRLPALVNV and IYNVGQVSI can induce potent protective immunity. DC generated in vivo with flt3-ligand were pulsed with 50 ug/ml of each peptide and injected intravenously into BALB/c mice (1 million/mouse) 3 times at 2 week intervals. One month later these mice were challenged with 5000 Tulahuén blood form trypomastigotes. Controls included unvaccinated mice and mice given DC only. Both parasitemia and mortality were significantly reduced (p<0.05) in mice receiving DC pulsed with the TS peptides. These results indicate that monospecific CD4+ and CD8+ T cells can protect against a complex parasitic pathogen. We are preparing transgenic mice expressing TCR specific for these two epitopes with the goal of assessing in detail the mechanisms involved in the development of protective T. cruzi immunity.

This work was funded by NIH RO1 #AI040196 awarded to DFH.
Anti- and pro-tumor activities of the TLR2 ligand FSL-1 (40.2)
Authors: Shibata Ken-ichiro , Kiura Kazuto , Hasebe Akira , Iyori Mitsuhiro , Haque Mohammad Shamsul , Ohtani Makoto
Published: January 2023

The TLR2 ligand FSL-1, a mycoplasmal diacyllipopeptide, possessed TLR2-mediated anti- and pro-tumor activities in vivo when immunized with and without tumor-associated antigens (TAAs), respectively. CpG DNA possessed the anti-tumor activity as described previously, but did not possess the pro-tumor activity when immunized with CpG DNA alone. The anti-tumor activity of FSL-1 was explained by induction of TAAs-specific CTLs, ADCC of NK cells and reduction of the number of CD4+ CD25+ FOXP3+ regulatory T cells (Treg) cells in the draining lymph node. CpG DNA also induced production of TAAs-specific CTLs and reduction of the number of Treg cells when immunized with TAAs, but did not induce ADCC of NK cells. The pro-tumor activity of FSL-1 was explained by enhancing the immunosuppressive activity of Treg cells in the microenvironment of tumor, because the immunization with FSL-1 alone, but not CpG alone, increased significantly the number of Treg cells in the draining lymph node of mice when compared with that of control mice.
Anti-tumor activity of vitamin C through the maintenance of IFN-producing dendritic cell (IKDC) population. (40.20)
Authors: Kong Joo Myoung , Bae Se Yeon , Kang Jae Seung , Lee Na Eun , Maeng Hyung Gun , Kim Hye Min , Hwang Young Il , Lee Wang Jae
Published: January 2023

Natural killer (NK) cells play an important role in elimination of altered self cell, such as tumor cells. Recently, vitamin C is an essential component for augmentation of NK cell activity, especially in cancer patients.

In this study, the effects of vitamin C on the anti-tumor activity of NKDC (CD49b+CD11c+NK1.1+) were investigated in Gulo(-/-) mice, which can not biosynthesize vitamin C like human beings. Body weight and the size of spleen of vitamin C depleted Gulo(-/-) mice was significantly decreased at 2 weeks after vitamin C depletion, comparing to those of wild type or vitamin C-supplemented Gulo(-/-) mice. To investigate the function of NKDC, CD49b+cells from splenocytes were isolated by MACS and then analyzed by flow cytometry. As a result, the number of NKDC population was completely reduced in vitamin C depleted Gulo(-/-) mice, which showed decrease of a secretion of IFN-gamma ƒ×. In addition, Fms-like tyrosine kinase 3 ligand (Flt3L), which stimulates the expansion and differentiation of IKDCs, was significantly reduced in vitamin C depleted Gulo(-/-) mice serum. Finally, the anti-tumor function of splenocytes from vitamin C depleted Gulo(-/-) mice was decreased, compared with those of wild type or vitamin C supplemented Gulo(-/-) mice. Taken together, it suggests that vitamin C shows their anti-tumor activity through the maintenance of Flt3L and the number of IKDC population.
Cell cycle arrest and apoptosis induced by engagement of B7-H4 on EBV-positive B lymphoma cells (40.21)
Authors: Hur Dae Young , Park Ga Bin , Song Hyunkeun , Kim Yeong-Seok , Sung Minjung , Lee Hyun-Kyung , Lee Wang Jae
Published: January 2023

B7-H4 is a recently discovered B7 family member that has inhibitory effect on T cell immunity. However, the signaling mechanism of the B7-H4-expressing cells remains unclear. Previous work has shown that B7-H4 expression was enhanced on B cells by infection of Epstein-Barr virus (EBV), and engagement of induced B7-H4 exerted cell death of EBV-transformed B cells. Here we found that B7-H4 was constitutively expressed on EBV-positive Burkitt¡ s lymphoma cells and that engagement of B7-H4 significantly reduced cell viability of Raji cell. Engagement of B7-H4 resulted cell cycle arrest at G0-G1 phase in a dose- and time-dependent manner. To clarify the mechanism of B7-H4 triggering induced cell cycle arrest, cell cycle regulatory factor was examined by RT-PCR and immunoblotting. B7-H4 triggering down-regulated CDK4/6 and up-regulated p21 expression at both protein and RNA level. Furthermore, CDK2 and cyclineE/D expression was down-regulated by B7-H4 triggering. Additionally, the down-regulation of phospho-AKT and phospho-cyclinE was clearly detected in B7-H4 triggering Raji cells, but the phosphorylation of p53 was constitutively maintained. These results represent that B7-H4-mediated signaling on EBV-positive B cells modulates cell cycle through down-regulation of AKT pathway. We conclude that B7-H4 may have the potency for use in EBV-associated cancer therapy.
Characterization of T cell responses specific for a self-antigen expressed in the mouse prostate (40.23)
Authors: Chou Cassie K , Teague Ryan M , Tan Xiaoxia , Ratliff Timothy L , Greenberg Norman M , Greenberg Philip D
Published: January 2023

OBJECTIVE: Examine if functional prostate-specific T cells exist in mice and determine if such T cells might be harnessed for prostate cancer therapy.

METHODS: Studies were performed in transgenic mice (POET) expressing ovalbumin (OVA) under control of a prostate-specific promoter and double transgenic POETxOTI and POETxOTII mice, which contain OVA-specific CD8 or CD4 T cells, respectively.

RESULTS: Double transgenic mice revealed incomplete central deletion of OTI and OTII cells, with functional prostate-specific T cells still present in the periphery. To model the fate of prostate-specific T cells in which the self-protein is not expressed during development, we transferred OTI or OTII cells into POET mice. While most transferred OTI cells were deleted, the persisting OTI cells were antigen experienced and capable of expanding and inducing prostate inflammation. Transferred OTII cells by contrast maintained a naïve and responsive phenotype.

CONCLUSIONS: Antigen experienced prostate-specific CD8 T cells persist, can be induced to expand and infiltrate the normal prostate, and thus may prove useful for cancer therapy. CD4 T cells specific for the same prostate antigen remain naïve, suggesting limited presentation by APC. We are generating TRAMPxPOET mice, which develop OVA-expressing prostate cancer, to study targeting self-antigens for tumor therapy.

Supported by the NCI & a CRI Predoctoral Fellowship
HSP90 inhibitor 17-DMAG enhances EphA2+ tumor cell recognition by specific CD8+ T cells (40.27)
Authors: Kawabe Mayumi , Mandic Maja , Taylor Jennifer , Vasquez Cecilia , Wesa Amy , Neckers Leonard , Storkus Walter
Published: January 2023

EphA2, a member of receptor tyrosine kinases (RTK), is commonly expressed by a broad range of cancer types, where its level of (over)expression correlates with poor clinical outcome. Since tumor cell expressed EphA2 is a non-mutated "self" protein, specific CD8+ T cells are subject to self-tolerance mechanisms and typically exhibit only moderate-to-low functional avidity, rendering them marginally competent to recognize EphA2+ tumor cells in vitro or in vivo. We have recently reported that the ability of specific CD8+ T cells to recognize EphA2+ tumor cells can be augmented after the cancer cells are pretreated with EphA2 agonists that promote proteasomal degradation (Wesa et al., J. Immunol., in press, 2008). In the current study we show that treatment of EphA2+ tumor cells with the HSP90 inhibitor, 17-DMAG, similarly enhances EphA2 degradation by the proteasome, minimally impairs MHC class I antigen presentign machinery, and increases tumor cell recognition by specific CD8+ T cell lines and clones in vitro. These studies suggest that EphA2 represents a novel HSP90 client protein and that the treatment of cancer patients with 17-DMAG-based "pulse" therapy may improve the anti-tumor efficacy of CD8+ T effector cells reactive against EphA2-derived epitopes.
KIR receptors implicated in NK cell activation provide a risk of developing breast cancer (40.29)
Authors: Ashouri Elham , Farjadian Shirin , Ghaderi Abbas , Rajalingam Raja
Published: January 2023

The polymorphic killer-cell immunoglobulin-like receptors (KIR) are the key receptors of human NK cells that trigger early immune response against infection and tumors. To investigate if certain KIRs are associated with the development of breast cancer, we determined the KIR gene content in 153 women with breast cancer and 248 healthy controls. Frequency of group-B haplotypes that possess 2-6 activating KIR genes was predominant in patients compared to controls (60.8% vs. 42.3%, P = 4.9x10-7; 95% CI, 0.35-0.63, OR = 0.47). Particularly 3 activating KIRs that are associated with group-B haplotypes, 3DS1 (47.0% vs. 33.0%, P = 0.008; 95% CI, 0.37-0.85, OR = 0.57), 2DS1 (52.0% vs. 35.0%, P = 0.001; 95% CI, 0.34-0.77, OR = 0.51) and 2DS5 (43.0% vs. 25.4%, P = 0.0003; 95% CI, 0.29-0.69, OR = 0.45) were significantly increased in patients compared to controls. Moreover, 34% of the patients carried all these three KIRs compared to only 21.4% controls (P = 0.007; 95% CI, 0.33-0.83, OR = 0.52). We further observed a similar trend in patients with lung cancer, suggesting an universal tumor-susceptibility function for 3DS1- 2DS1-2DS5. Our data suggests that NK cells expressing activating receptors 3DS1, 2DS1 and/or 2DS5 may trigger an inappropriate localized hyperresponsiveness exacerbating the risk of cancer growth. Functional studies are underway to test if NK cells expressing KIR repertoire with and without these activating KIRs mount varied response against tumor lines.
Lipopolysaccharide treated murine mammary carcinomas secrete proinflammatory chemokines and exhibit reduced growth rates in vivo, but not in vitro (40.3)
Authors: Kurt Robert A , Nair Priyanka , O'Donnell Cara , Janasek Karolina , Sajduk Monika , Smith Emily , Golden Jacqueline , Vasta Casey , Huggins Alison
Published: January 2023

Toll-like receptors (TLR) are pattern recognition receptors that play a pivotal role in the initiation of immune responses. Ligation of TLR on antigen presenting cells enhances immunological responses through multiple mechanisms including secretion of proinflammatory mediators. Here we report that the murine mammary carcinoma 4T1 constitutively expressed genes encoding TLR2, 3, 4 and 5. Moreover, treatment of 4T1 with peptidoglycan (PGN), polyinosinic-polycytidylic acid (Poly(I:C)) or lipopolysaccharide (LPS), agonists for TLR2, 3 or 4 respectively, induced nuclear translocation of NFkB and secretion of CCL2, CCL5 and CXCL1 in a dose dependent manner. Although treating the tumor cells with the TLR agonists did not modulate growth or viability of the tumor cells in vitro, 4T1 exhibited a decreased growth rate in vivo following treatment with LPS that was dependent upon the presence of CD8+ T cells. Analysis of three additional murine mammary carcinomas revealed that they also secreted CCL2, CCL5 and CXCL1 in response to TLR agonist treatment, and LPS treated 168 and SM1 tumors exhibited decreased growth rates in vivo, but not in vitro. These data indicated that four out of four murine mammary carcinomas secreted proinflammatory chemokines following treatment with TLR agonists, and three out of four of the mammary carcinomas responded to LPS treatment in a manner that decreased tumor growth in vivo.
Role of uridine kinase-like 1 (UCKL-1) in NK-mediated tumor cytotoxicity (40.9)
Authors: Ambrose Elise C , Kornbluth Jacki
Published: January 2023

NK lytic associated molecule (NKLAM), which is upregulated in NK cells by activation, has been shown to play a critical role in NK-mediated tumor cytotoxicity. We have found that NKLAM interacts with uridine kinase-like 1 protein (UCKL-1) and causes its ubiquitination and subsequent degradation. This suggests that NKLAM may play a role in tumor killing by decreasing UCKL-1 expression in target cells. In support of this is the finding that UCKL-1 mRNA is most abundant in tumor cell lines, indicating that it may be important in tumor cell growth and/or survival. In order to determine the function of UCKL-1 in tumor cell killing, K562 target cells were treated with UCKL-1 RNAi or control RNAi. Selective knockdown of UCKL-1 resulted in increased apoptosis and decreased proliferation of K562. In addition, decreased UCKL-1 expression increased the susceptibility of K562 to the apoptosis inducers staurosporine and etoposide. This finding indicates that UCKL-1 plays a role in the proliferation of K562 cells and protects them from apoptosis. By ubiquitinating and degrading UCKL-1 in target cells, NKLAM released from NK cells would therefore enhance tumor killing.

This research is supported by a merit review award from the Department of Veterans Affairs.
Analysis of in vivo homing of cytokine-induced killer cells into lymph nodes (41.17)
Authors: Han Sang-Bae , Kim Yeon Jin , Kim Jee Youn , Ryu Hwa Sun , Kim Hyung Suk , Yun Jun Ho , Kim Hwan Mook , Kang Jong Soon , Song Sukgil , Hong Jin Tae , Kim Youngsoo
Published: January 2023

The goal of immune cell-based cancer therapy is to eliminate cancer cells through the transfer of ex vivo expanded and activated immune cells. Cytokine-induced killer (CIK) cells are ex vivo expanded T cell mixture with proven anticancer activity in vitro and in vivo. In the presence of immobilized anti-CD3 antibody and IL-2 for 14 days, mouse splenocytes changed to heterogeneous CIK cell population, which comprised 97% CD3+, 86% CD8+, 7% CD4+, 25% CD3+NK1.1+, and 30% CD8+NK1.1+. Although CD3+NK1.1+ cells were rare in fresh human peripheral blood mononuclear cells, they could expand more than 1,000-fold on day 14. Ex vivo proliferation of splenocytes was critically dependent on IL-2 concentration and cell density; in that IL-2 was required at least 750 U/ml and cell number was maintained at one or two million cells per milliliter during the cultivation. CIK cells strongly expressed IFN-gamma and IL-2, moderately IL-4 and TNF-alpha, but not IL-1beta, IL-5, and IL-10. Compared with purified T cells, CIK cells also expressed lower levels of adhesion molecules, such as L-selectin and CD49d, and chemokine receptors, such as CCR7 and CXCR4. It was resulted in less trafficking of CIK cells to lymph nodes than purified T cells did, which was determined by confocal microscopic and flow cytometric analysis. The data shown here demonstrated that inappropriate trafficking of CIK cells to lymph nodes might be one of the hurdles for in vivo application of CIK cell therapy, and suggest that L-selectin, CD49d, CCR7 and CXCR4 might be good targets to increase CIK cell trafficking to lymph nodes.
Increase of antigen expression and activation of APCs by targeting adenovirus to CD40 significantly enhanced therapeutic effect of antigen presenting cell-based anti-tumor vaccine (41.18)
Authors: Kim Yun-Sun , Han Seung-Hee , Kim Yeon-Jeong , Ko Hyun-Jeong , Park Hae-Jung , Pereboev Alexander V. , Nguyen Huan H. , Kang Chang-Yuil
Published: January 2023

The efficacy of antigen presenting cell (APC)-based vaccines generated in vitro by genetic transduction critically depends on the expression level of target antigen and the activation status of APCs. Although APCs such as DCs and B cells transduced by recombinant adenovirus (AdV) readily induced antigen specific immune responses, they are relative resistant to adenoviral transduction due to the lack of coxsackievirus-adenovirus receptor (CAR), which mediates adenovirus entry. In this study, we used recombinant adapter molecule, CFm40L, which consists of CAR genetically fused to CD40 ligand via a trimerization motif, to target Her-2/neu or HPV16 E6/E7-encoding AdV to DCs and B cells that express CD40. Such transduced DCs and B cells enhanced substantially immunity against Her-2/neu or HPV16 E6/E7-expressing tumor resulted in significant inhibition of tumor growth. Especially, the anti-tumor effect of B cell-based vaccines transduced with CD40-targeted adenoviral vector is comparable to that of DC-based vaccines, indicating the feasibility of B cell-based vaccine for anti-cancer therapy. Our results suggest that targeting AdV to APC via CD40 using CFm40L offers a novel approach in anti-tumor therapy
Ex vivo induction and expansion of Natural Killer T cells by CD1d1-Ig coated artificial antigen presenting cells (41.22)
Authors: Webb Tonya J , Bieler Joan G , Schneck Jonathan P , Oelke Mathias
Published: January 2023

Natural killer T (NKT) cells play a pivotal role in maintaining immune homeostasis. Many studies examining patients with cancer have shown that there is a reduction in both NKT cell number and function. Due to the complexities of manipulating NKT cells in vivo, ex vivo expanded effector NKT cells would be an excellent therapeutic modality. To date, immunotherapy utilizing the NKT/CD1d system has been dependent on the use of autologous antigen presenting cells pulsed with α-galactocylceramide (α-GalCer). In this study, we have generated CD1d- expressing artificial antigen presenting cells (aAPC) to examine co-stimulatory requirements for NKT cells and to expand NKT cells from the peripheral blood of healthy controls and cancer patients. We tracked the level of NKT cell expansion and the expanded population was characterized both phenotypically and functionally. We found that CD1d-based aAPC can effectively propagate both canonical (iNKT cells) and noncanonical NKT cells. Thus, CD1d-expressing aAPC will enhance our knowledge of NKT cell biology and could potentially be used as a novel tool in adoptive immunotherapeutic strategies.
Chimeric Antigen Receptors for Stem Cell Based Immunotherapy (41.23)
Authors: Badowski Michael S , Tsang Tom C , Zhang Tong , Harris David T
Published: January 2023

Chimeric antigen receptors (CARs) can provide anti-tumor specificity, and adoptive transfer of CAR-modified stem cells offers a method to introduce and expand tumor specific T-cells. This adoptive immunotherapy can eliminate malignant cells or supplement traditional therapies. The use of a single chain TCR (scTCR) in which the TCR Vα and Vβ segments are joined by a flexible linker provides a better alternative to introduction of full length TCR genes. We have developed a HER2 specific scTCR as well as a single chain antibody based receptor (scFv) to increase avidity to the tumor antigen and avoid the potential limitation of MHC restriction. Our lab has previously developed a signaling cassette based on the CD28 and p56Lck proteins which are prominent in the T-cell signaling pathway. The single chain specificities are linked to the signaling cassette that we have shown to function in T-cells. With specificity and signaling coupled, the chimeric antigen receptor can be transduced into hematopoietic stem cells (HSC). Engraftment of the transduced HSC shows multiple cell types expressing our transgene reporter. Anti-tumor activity of transduced cells is measured by cytokine release upon stimulation.
Targeting Lymphoma with Bispecific Antibody-Armed T Cells and their Potential for use in an Allogeneic Setting (41.31)
Authors: Cousens Leslie  P. , Davol Pamela A.
Published: January 2023

Patients with high-risk refractory or relapsed lymphomas face a poor prognosis and limited therapeutic options. Current treatment regimens of chemotherapy with either rituximab (anti-CD20 mAb) or with allogeneic stem cell transplant (alloSCT) yield serious toxicities and high relapse rates. In particular, benefits of alloSCT are grossly limited by life-threatening graft vs host disease (GvHD). New approaches to enhance graft vs lymphoma (GvL) effects without increased GvHD are needed. Bispecific antibodies (BiAbs) are produced by linking anti-CD3 for T cell binding to an antibody specific for a tumor antigen. Anti-CD3 activated T cells (ATC) "armed" with BiAb express tumor-specific CTL function independent of further activation, proliferation, antigen presentation or costimulation. Studies here examined allo-reactivity of BiAb-armed ATC for potential application in an allogeneic setting. Data show that ATC armed with anti-CD3 x anti-CD20 BiAb specifically target and kill B cell lymphomas. However, BiAb-armed ATC are remarkably poor stimulators of, or responders to, allo-stimlulation. These results provide important evidence of a functional separation between the GvL and GvH effects of BiAb-armed ATC.
Differential expression of CD4+ TReg cell subpopulations among ovarian cancer patients following autologous MUC1-specific Th1 effector cell immunotherapy (41.38)
Authors: Dobrzanski Mark J , Rewers-Felkins Kathleen , Samad K.A. , Robinson William , Wright Stephen E
Published: January 2023

Adoptive T cell immunotherapy is a viable treatment for cancer patients and optimally requires participation of Ag specific CD4 T cells. Here, we assessed the therapeutic effects of adoptively transferred autologous MUC1 peptide stimulated CD4+ effector cells in patients with ovarian cancer. Using MUC1 peptide and IL-2 for ex vivo CD4+/Th1 effector cell generation, we show that 3 monthly treatment cycles of T cell restimulation and intraperitoneal re-infusion modulated T cell mediated immune responses that correlated with diminished serum CA125 tumor marker levels and enhanced patient survival. One patient survived long term (>5 yrs), another patient survived long term with recurrent disease (>5 yrs) and two patients expired within 100 days following final infusion. Long term survivors showed elevated levels of systemic memory (CD45RO) and naïve (CD45RA) CD3/CD4/CD25+ T cells. Such cells co-expressed different levels of Foxp3 and CTLA-4 that resulted in lower systemic Foxp3/CTLA-4 memory T cell ratios. Lastly, these patients showed elevated levels of MUC1 specific T cells expressing the CCR5 and CCR1 chemokine receptors associated with T cell differentiation and select migration. This data suggests that effective immunotherapy with autologous MUC1 peptide-stimulated CD4+ effector cells induce differential levels of systemic TReg cell subpopulations that influence long term tumor immunity in ovarian cancer patients following adoptive T cell transfer.
A novel form of 4-1BB ligand has better efficacy than agonistic 4-1BB antibody as well as TLR agonists in a peptide based cancer vaccine (41.5)
Authors: Sharma Rajesh K. , Elpek Kutlu G. , Yolcu Esma S. , Schabowsky Rich-Henry , Zhao Hong , Bandura-Morgan Laura L. , Shirwan Haval
Published: January 2023

Toll like receptor (TLR) agonists, MPL and CpG-ODN, have been the subject of intense preclinical and clinical investigations as most promising vaccine adjuvants. Given the paramount importance of costimulation in the modulation of immune responses, we hypothesized that agonistic costimulatory ligands may serve effective alternatives to TLR agonists for vaccine development. Unlike TLR agonists, costimulatory ligands primarily function as cell membrane-bound proteins and lack activity in soluble form. We, therefore, used core streptavidin as a chaperon to generate costimulatory ligands that function in soluble forms. We compared the activity of a novel form of 4-1BBL composed of the extracellular domain of murine 4-1BBL fused to the C-terminus of core streptavidin (SA-4-1BBL) with an agonistic Abs to 4-1BB and TLR agonists as components of peptide based vaccines in a cervical cancer mouse model. Vaccination with SA-4-1BBL resulted in better efficacy than agonistic Ab as well as MPL/LPS/CpG in the eradication of established tumors. Therapeutic efficacy of SA-4-1BBL was associated with enhanced tumor specific primary and memory responses and reversal of T cell anergy. Importantly, therapeutic efficacy was achieved in the absence of sever toxicity associated with the agonistic Ab. Taken together, these data demonstrate the feasibility of using SA-4-1BBL as an effective alternative to TLR agonists or agonistic Abs to 4-1BB as immunomodulatory component of therapeutic cancer vaccines.

Funded in parts by NIH R43 CA 109866 (HS), R43 AI071618 (HS, ESY)

R41 CA121665 (HS), KLCRP (HS), KSTC-145-402-22 (HS).
Amplifying TLR2 signals in Tumor-specific T cells Lowers the Activation Threshold to a Weakly Immunogenic Tumor Antigen and Enhances Antitumor Activity (41.50)
Authors: Davila Eduardo , Zheng Liquin , Geng Degui , Asprodites Nicole
Published: January 2023

Although toll-like receptor (TLR) agonists are primarily believed to potentiate T cell responses by activating antigen-presenting cells, we recently demonstrated that TLR2 engagement on CD8 T cells enhanced proliferation and increased the production of several effector molecules. Here, we examined the antitumor activity of TLR2-stimulated pmel CD8 T cells, which recognize the weakly immunogenic melanoma tumor antigen gp100. TLR2 engagement on pmel T cells but not TLR2-/-pmel or MyD88-/-pmel cells reduced the activation threshold up to 100-fold as measured by IFN-γ, granzyme B, perforin and IL-2 production and cytolytic activity. Adoptive T cell transfer of pmel T cells in wild type or MyD88 knockout mice and injection with TLR2 ligand improved the antitumor activity against an established melanoma tumor as compared with mice treated with TLR2-/-pmel or MyD88-/-pmel T cells and TLR2 ligand. Furthermore, TLR2-ligated T cells persisted longer than did control T cells. Overexpressing TLR2 in pmel T cells improved effector responses above that of T cells expressing wild type levels of TLR2. These data highlight an important and physiologically relevant role for activating TLR2-MyD88 signals within T cells and could enhance immunotherapeutic approaches to treat cancer patients by augmenting T cell responses to weakly immunogenic TAs. Supported by a NIH (1P20 RR021970) and the Louisiana Cancer Research Consortium.
Continuous Intra-Lymphatic Dendritic Cell Infusion in Patients with Advanced Melanoma (41.51)
Authors: Kalinski Pawel , Edington Howard , Brown Charles , Whiteside Theresa , Butterfield Lisa , Bartlett David , Kirkwood John
Published: January 2023

Continuous intralymphatic delivery of dendritic cells (DCs), using implantable lymphatic cannulas, allows to efficiently deliver "non-exhausted" DCs to draining lymph nodes without the disruption of nodal structures, and to recreate the physiologic kinetics of DC accumulation observed during physiologic immune responses.

We have completed initial feasibility and safety evaluation of semi-continuous intralymphatic infusions of antigen-loaded DCs (a total of 300,000 DCs over 4 days) in six patients with stage IIIb-IV melanoma in clinical trial UPCI 03-118. Four of the initial six patients received two four-day-long courses of the intralymphatic DC infusions, completing the protocol. Two patients did not complete the treatment for reasons unrelated to the protocol.

Already at this ultra-low dose of DCs, we have observed evidence of clinical efficacy of DC infusions in 3 of 4 patients with stage IV melanoma who completed both courses of treatment. We observed one near-complete response (currently >20 months), and two disease stabilizations (10 months; and > 7 months), with only one patient progressing. We have recently initiated the evaluation of "high-dose" intralymphatic infusions (total of 3 million DCs over 4 days).

Our data demonstrate the feasibility of prolonged intralymphatic delivery of biotherapeutic agents and provide preliminary indication that DCs can be clinically effective in patients with advanced cancer at the doses 100-fold lower than previously considered to be necessary.
Dissecting the integration of Natural Killer cell receptor signaling (41.52)
Authors: Xu Bin , Kaiser Brett , Strong Roland K.
Published: January 2023

Natural Killer cell is a major component of the innate immune system. It plays important roles in rejection of cancerous and viral-infected cells. NK cells express numerous surface receptors that can be activating or inhibitory. To investigate how activating and inhibitory NK receptors integrate signals in the decision to activate or to be tolerant, we studied the activating/inhibitory effects of NKG2D-MIC and NKG2A/CD94-peptide-HLA-E pathways, both alone or in combination using human NKL leukemia cell line and in vitro purified protein ligands. Our results demonstrate NKG2D receptors have antibody-induced cell activation effect. MIC ligand induces NKG2D receptor down-regulation on cell surface but alone is not sufficient to induce receptor activation. Surprisingly, NKG2A/CD94 ligand HLA-E/peptide potentiates NKG2D receptor activation. Possible molecular signaling mechanisms will be discussed. Supported by NIH grant AI48675.
Photodynamic Therapy Elicited Adaptive Immunity is Critically Dependent on IL-17 Bioactivity (41.56)
Authors: Brackett Craig , Maier Patricia , Ramsey Kimberley , Gollnick Sandra
Published: January 2023

Photodynamic therapy (PDT) is a FDA-approved local cancer treatment of early stages of cancer and palliation of advanced disease. A hallmark of PDT is the induction of acute local inflammation characterized by increased infiltration of innate immune cells into the treated tumor bed, which is dominated by CD11b+Ly-6GhiF4/80- leukocytes that morphologically resemble neutrophils. We have previously shown that enhanced anti-tumor CD8+ T cell activation following PDT is dependent upon the presence of CD11b+Ly-6GhiF4/80- leukocytes in the tumor and tumor draining lymph nodes (TDLNs). We now show that PDT promotes the differential expression of IL-23 and IL-17 within the treated tumor bed. Blocking the action of IL-17 using IL-17RA-/- mice does not diminish bone marrow release of CD11b+Ly-6GhiF4/80- leukocytes or their migration to the tumor post PDT but does abrogate accumulation of these cells in the TDLN. Furthermore, blocking IL-17 inhibits post-PDT activation of tumor specific CD8+ T cells. These findings suggest that IL-17 differentially regulates CD11b+Ly-6GhiF4/80- leukocyte migration and indicates that CD11b+Ly-6GhiF4/80- leukocyte accumulation in the TDLN is critical for induction of T cell activation. Neutrophils are capable of informing and shaping the immune response by influencing DC, monocyte, and lymphocyte activation. Understanding this behavior of neutrophils holds promise for the rationale design of PDT regimens to optimally enhance anti-tumor immunity in a clinical setting.