bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant human IL7 enhances naïve and T stem cell memory reconstitution and enhances repertoire diversity following cytotoxic chemotherapy (LYM5P.705)
Authors: Zhang Hua , Robins Harlan , Mackall Crystal
Published: January 2023

T cell immune reconstitution in humans is limited by poor thymic reserve. Hence, dose intensive cytotoxic chemotherapy often induces profound, prolonged lymphopenia. Lymphopenia can increase the effectively of adoptive cell therapy but also has detrimental consequences related to diminished repertoire diversity, which limits the hosts’ capacity to respond to new antigenic challenges. In this study, we analyzed circulating T cell subsets in a cohort of 50 children and young adults rendered lymphopenic via cytotoxic chemotherapy, and compared immune reconstitution ± rhIL-7. Chemotherapy induced profound T cell depletion with decreased frequencies of CD4+ and CD8+ naïve cells, but increased frequencies of CD4+ and CD8+ T stem cell memory cells. RIL-7 therapy (20 mcg/kg SQ every 2 wk) beginning 6 wk after completion of chemotherapy, increased total numbers of CD4+ and CD8+ T cells as well as CD4+ and CD8+ naïve, central memory, stem cell memory and CD4+ effector memory cells. Repertoire diversity of specific subsets was measured sequentially in three subjects from each treatment group using next generation sequencing. Chemotherapy induced substantial reductions in the number of unique TCR sequences/mcl of blood, which was dramatically reversed in patients treated with rhIL7 compared to those who were not. Thus, rhIL-7 therapy can rapidly restore T cell repertoire diversity in patients following cytotoxic chemotherapy.
Efficient culture of human naïve and memory B cells (LYM5P.711)
Authors: Su Kuei-Ying , Kuraoka Masayuki , Watanabe Akiko , Kelsoe Garnett
Published: January 2023

The ability to culture and expand B cell numbers in vitro has become a useful tool for studying human immunity. A limitation of current methods in human B cell cultures is the capacity to support continued proliferation by mature B cells. We have adapted the culture methods established by Luo et al. (Blood, 2009) to efficiently (60% cloning efficiency) activate and expand both naïve and memory human B cells. Briefly, naïve or memory B cells recovered from blood are cultured with cytokines on CD154+ feeder cells; this culture supports extensive B cell proliferation, with approximately 103 fold increases in a week; by splitting cultures onto new feeder layers, culture-derived (CD) B cells continuously proliferate, achieving 106 fold increases by day 16. The capacity for continued proliferation is stable for at least another week. In culture, a significant fraction of naïve B cells upregulate AID expression, undergo isotype switching without V(D)J hypermutation, and terminally differentiate into plasmacytes. CD B cells are readily cryopreserved and retain their ability to proliferate and differentiate. Significantly, proliferating CD B cells express high levels of MHCII, CD80, and CD86. We have examined the APC function of CD B cells and find that they present both allo- and microbial antigens to autologous T cells with comparable efficiency to PBMC. This culture method provides a platform for studying the BCR and TCR repertoires within a single individual.
Levels of the pro-apoptotic molecule Bim are determined early in the response and positively correlated with memory T cell fate (LYM5P.702)
Authors: Li Kun-Po , Sholl Allyson , Hildeman David
Published: January 2023

We and others have shown that the pro-apoptotic molecule Bim plays a critical role in controlling the contraction of T cell responses. Paradoxically, T cells destined to become memory (pre-memory) cells have higher levels of Bim than their effector counterparts. Similarly, after memory development, central memory (TCM) cells have higher levels of Bim relative to their effector memory (TEM) counterparts. However, due to the intracellular location of Bim, sorting and subsequent tracking of T cells based on Bim expression has been challenging. To circumvent this issue and determine the relationship between Bim expression and cell fate, we generated Bim-mCherry reporter mice, inserting an IRES-mCherry cassette into the 3’-UTR of the Bim gene. Similar to intracellular staining for Bim, mCherry levels were higher in pre-memory cells and TCM cells than their effector or TEM counterparts. In addition, the expression and function of Bim in T cells is not altered in Bim-mCherry reporter mice. The transfer of Bimhi or Bimlo T cells on the basis of mCherry expression favored their development into TCM or TEM cells, in recipient animals, respectively. Further, the levels of mCherry (and Bim) remained stable in the transferred mCherryhi or mCherrylo T cells across contraction of the response. These data suggest that the levels of Bim in CD8 T cells are determined at an early stage of infection and are paradoxically positively correlated with T cells having a memory fate.
Virtual memory cells: a consequence of self-affinity-linked naïve CD8 T cell heterogeneity (LYM5P.703)
Authors: White Jason , Walton Kendra , Danhorn Thomas , O'Connor Brian , Kedl Ross
Published: January 2023

Memory phenotype (MP), CD44hi CD49dlo CD8 T cells are found in nearly all mice—lymphoreplete, pathogen naïve, and even TCR transgenics. The presence of MP was long thought to be due to environmental pathogen exposure (or, in the case of transgenics, heterologous immunity to same), but studies establishing the presence of these cells in germ-free mice suggested that this was not the case. These cells, dubbed Virtual Memory (VM), are of special relevance due to their increased effector functioning, and, in contrast to other MP CD8 T cells, develop in the periphery with a dependence on IL-15. Our recent studies have determined that VM cells develop in a manner reminiscent of lymphopenic homeostatic expansion, whereby the highest self-affinity T cells (as measured by CD5) interact with available IL-15 to turn into VM cells. This indicates that the naïve CD8 T cell pool is not as homogeneous as usually assumed, and RNA sequencing has further confirmed the heterogeneity of the CD8 naïve T cell pool (separated on CD5). The sequencing has further implicated VM cells in mediating CD8 bystander protection, and ongoing studies suggest that this is indeed the case, with the VM bystander effects being IL-15 dependent.
Generation of central memory T cells by vaccination that mimics natural infection (LYM5P.704)
Authors: Dalai Sarat , Bhurani Vishakha , Mathur Aditi , Patel Hardik
Published: January 2023

The goal of vaccination is to generate long-lived antigen specific memory T & B cells. Often we notice that the immune responses generated by the vaccines fail to achieve the desired goal, particularly the T cell responses. In many infection models it is well established that long-lived memory T cells could be generated in hosts exposed to pathogens through natural infection. Here we are adopting the strategies that would mimic natural infection with the hope to induce immune responses in a manner similar to that triggered during natural infection. In our study we have mimicked the natural infection to vaccinate the host by (i) providing the danger signals to the immune system before or during encounter of Ag to mimic the course of infection, (ii) combining non-target Ag(s) with target Ag, while priming the host in the presence of danger signals.We have used OVA as the model antigen to immunize C57BL/6 mice, and Poly I:C/R848 as danger signals to represent viral infection. For non-target antigens, we have used immunogenic (HEL) as well as non-immunogenic (MBP) antigens. We found that vaccination through mimicking course of infection favors generation of Central MemoryT cells whereas conventional vaccination favors effector memory T cells. When we mimicked natural infection to vaccinate the host by including poorly immunogenic non-target antigen with the vaccine target we found it to favor generation of central memory T cells compared to that of immunogenic non- target.
In situ hematopoiesis-derived mast cells promote protective immunity to Trichinella spiralis (MPF4P.738)
Authors: Siracusa Mark , Henry Everett , Sy Chandler
Published: January 2023

In situ hematopoiesis (ISH) refers to the differentiation of hematopoietic stem/progenitor cells (HSPCs) into effector cells that occurs at active sites of inflammation. Previous studies have demonstrated that ISH contributes to the population expansion of basophil and mast cell populations in the context of multiple helminth infections. However, whether ISH-derived effector cell populations are critical regulators of TH2 cytokine-mediated inflammation and protective immunity to helminth parasites remain unknown. Here, we demonstrate a previously unrecognized role for ISH in promoting the population expansion of intestinal mast cell populations, TH2 cytokine responses and protective immunity to Trichinella spiralis. Further, discovery based-studies suggest that helminth-induced HSPCs exhibit distinct transcriptional profiles compared to their bone marrow-resident counterparts. These data demonstrate that ISH contributes to the development of protective immunity to helminth parasites and that helminth-induced HSPCs are phenotypically distinct cells that may represent viable therapeutic targets.
Activation of inflammasome by rickettsiae in human and murine macrophages (MPF4P.727)
Authors: Smalley Claire , Bechelli Jeremy , Zhao Xuemei , Walker David , Fang Rong
Published: January 2023

Rickettsiae are Gram-negative, obligately intracellular bacteria that infect macrophages. However, the molecular mechanisms involved in interaction of rickettsiae with macrophages remain poorly understood. In this study, we investigated the inflammasome activation by rickettsiae in human and mouse macrophages. R. australis activated the inflammasome in mouse bone-marrow derived macrophages (BMDM), marked by caspase (casp)-1/11-dependent secretion of IL-1β and IL-18, and induction of pyroptosis. Kinetic studies revealed that low dose (MOI 2) R. australis activated the inflammasome in mouse macrophages at 12 h post infection (p.i.), while high dose (MOI 6) activated at 8 h p.i., with no significant difference in activation between doses at 24 h p.i.. In contrast, R. australis activated the inflammasome in human THP-1 derived macrophages at as early as 3 h p.i., resulting in production of IL-1β, IL-18, induction of pyroptosis, and activation of casp-1. Studies using siRNA and inhibitors specific for casp-4 suggest that rickettsiae activated non-canonical inflammasome pathway in human macrophages. Taken together, our data, for the first time, illustrated the activation of inflammasome by rickettsiae in both mouse and human macrophages, although with different kinetic mechanisms. This study provides invaluable information for developing novel therapeutic interventions for rickettsioses by employing current knowledge derived from mouse infection models.
A subset of protective γ9δ2 T cells is activated by novel mycobacterial antigens (MPF5P.740)
Authors: Xia Mei , Hesser Danny , Sakala Isaac , Spencer Charles , Abate Getahun , Chatterjee Delphi , Dobos Karen , Hoft Daniel
Published: January 2023

γ9δ2 T cells provide a natural bridge between innate and adaptive immunity and are prominently induced by both tuberculosis (TB) infection and bacillus Calmette Guérin (BCG) vaccination. Mycobacteria-expanded γ9δ2 T cells represent only a subset of the simple phosphoantigen (IPP and HMBPP)-responsive γ9δ2 T cells, this subset expresses a more oligoclonal set of TCR sequences, and only this subset efficiently recognizes and inhibits intracellular BCG/Mtb. We have been searching for new mycobacterial antigens capable of inducing the unique subset of γ9δ2 T cells that can recognize mycobacteria-infected macrophages and mediate protective effects inhibiting intracellular mycobacterial growth. We first ruled out protein, nucleic acid and apolar lipids with basic separations and enzymatic digestions. Acid hydrolysis, which digests complex carbohydrate structures, had the largest effect on eliminating Mtb fraction specific activity. Organic extractions resulted in sub-fractions with >100 fold enriched specific activity. A combination of MS, NMR, TLC indicate that methyl-glucose lipopolysaccharides (mGLP) are predominant components of the most active fractions. We are currently attempting to use synthetic techniques to confirm the specific activity of mGLP for induction of protective γ9δ2 T cells. These results have important implications for the development of new immunotherapeutic approaches for prevention/treatment of TB.
Inhibition of Interleukin 10 receptor signaling induces microbiota-dependent chronic colitis in Apolipoprotein E deficient mice (MUC5P.754)
Authors: Yeoh Beng San , Kumar Manish , Singh Vishal , Kennett Mary , Vijay-Kumar Matam
Published: January 2023

Apolipoprotein E (ApoE), a secreted protein, plays major role in cholesterol metabolism. ApoE is also known to exhibit anti-inflammatory properties. However, its role in the intestine, specifically during inflammation is not very clear. First, we verified upregulation of colonic ApoE transcripts in various models of murine acute and chronic colitis models. Age and gender matched ApoEKO mice and their WT littermates (n=5) were given either one or 4 weekly injections of IL-10 receptor (IL-10R) neutralizing mAb intraperitoneally and colitis development was monitored. ApoEKO mice given single injection of IL-10R mAb displayed acute colitis. Four weekly injections IL-10R mAb resulted in the development of chronic colitis as measured by colomegaly, splenomegaly and elevated colonic pro-inflammatory gene expression (TNFα, IFNγ) and histological score when compared to WT littermates. IL-10R neutralization also induced substantial levels of lipocalin 2, a general inflammatory marker in the feces, colon, and circulation in ApoEKO mice. Ablation of gut microbiota by broad-spectrum antibiotics substantially protected ApoEKO mice against IL-10R neutralization induced chronic colitis. In addition, cohousing ApoEKO mice with IL-10KO mice exaggerated spontaneous colitis (rectal prolapse) in IL-10KO mice when compared to WT suggesting ApoEKO harbor colitogenic microbiota. Collectively, our results demonstrate that ApoE plays a critical role in protecting the gut against inflammation.
Autoantigen-targeted therapy reinforces impaired central tolerance in autoimmune mice (THER5P.916)
Authors: Bonami Rachel , Rachakonda Amita , Hulbert Chrys , Thomas James
Published: January 2023

Immune tolerance breakdown precedes autoimmune disease. Receptor editing is an immune tolerance mechanism that alters BCR specificity to combat frequent autoreactivity in the developing B cell repertoire. Much of what is known about receptor editing derives from multivalent antigens, or soluble antigens which interact with the BCR with supra-physiologic affinity (1010 M-1). A system was therefore developed in which the BCR interacts with insulin autoantigen with a more physiologic affinity (107 M-1) to better reflect B cells that arise in the developing bone marrow repertoire. Surprisingly, circulating insulin was found to elicit receptor editing in non-autoimmune mice. This process occurred less efficiently in the type 1 diabetes-prone NOD strain, consistent with enhanced anti-insulin B cell escape into the periphery from the bone marrow observed in this strain. Receptor editing efficiency was enhanced by treatment with an insulin-targeting mAb that selectively eliminates anti-insulin B lymphocytes and prevents type 1 diabetes. A F(ab’)2 of this antibody also depleted developing anti-insulin B lymphocytes, confirming receptor editing as a central tolerance mechanism targeted by this approach. These studies show that a small protein hormone of modest affinity elicits receptor editing, but with less efficiency in the context of autoimmunity. This defect can be overcome with autoantigen-targeted therapy, which offers promise for the treatment of autoimmune disease.
Molecular mechanism underlying immune modulation during stem cell therapy of overt type 1 diabetes (THER5P.917)
Authors: Chen Weirong , Ukah Tobechukwu , Miller Mindy , Ellis Jason , Barik Subhasis , Zaghouani Habib
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disorder in which insulin-producing pancreatic beta-cells are selectively destroyed by self-reactive T cells. In previous studies, we incorporated glutamic acid decarboxylase 206-220 peptide into an Ig molecule and the resulting Ig-GAD2 chimera was shown to target diabetogenic T cells and prevent disease progression. However, despite its efficacy, Ig-GAD2 was not able to reverse overt T1D. In contrast, transfer of bone marrow (BM) cells alongside Ig-GAD2 induces recovery from overt T1D. This was due to effective immune modulation that stems from diminished function of the mammalian target of rapamycin (mTOR) complex 1. In addition, there was generation of BM-derived endothelial cells that contributed to the repair of islet vascular network which facilitated endogenous β-cell formation and restoration of β-cell mass. These findings suggest that targeting the function of mTOR synergizes with stem cell transfer to control overt T1D.
Inhibition of hyaluronan synthesis restores immune tolerance during autoimmune insulitis (THER5P.919)
Authors: Bollyky Paul , Kaber Gernot , Johnson Pamela , Gebe John , Preisinger Anton , Falk Ben , Gooden Michel , Vernon Robert , Bogdani Marika , Kuipers Hedwich , Campbell Daniel , Wight Thomas , Nagy Nadine
Published: January 2023

We recently reported that abundant deposits of the extracellular matrix polysaccharide hyaluronan (HA) characterize autoimmune insulitis in human type 1 diabetes (T1D) but the significance of these deposits was unclear. Here, we demonstrate that HA is critical for the pathogenesis of autoimmune diabetes. Using the DO11.10xRIPmOVA (DORmO) mouse model of T1D, we show that HA deposits are temporally and anatomically associated with the development of insulitis. Moreover, treatment with an inhibitor of HA synthesis, 4‑methylumbelliferone (4-MU), halted progression to diabetes even after the onset of insulitis. 4-MU reduced HA accumulation, constrained effector T-cells to non-destructive insulitis, and increased numbers of intra-islet Foxp3+ regulatory T-cells (Treg). Consistent with this, Treg differentiation was inhibited by HA and anti-CD44 antibodies and rescued by 4-MU in an ERK1/2-dependent manner. These data may explain how peripheral immune tolerance is impaired in tissues under autoimmune attack, including islets in T1D. We propose that 4-MU, already an approved drug used to treat biliary spasm, could be repurposed to prevent, and possibly treat, T1D in at-risk individuals.
Selective ROCK2 inhibitor reverses experimental chronic graft-versus-host disease via concurrent regulation of STAT3/STAT5 phosphorylation (TRAN1P.933)
Authors: Zanin-Zhorov Alexandra , Flynn Ryan , Luznik Leo , Nyuydzefe melanie , Panoskaltsis-Mortari Angela , Du Jing , Goodman Katelyn , Weiss Jonathan , Chen Wei , Serody Jonathan , MacDonald Kelli , Hill Geoffrey , O'Shea John , Waksal Samuel , Blazar Bruce
Published: January 2023

Rho-associated kinase 2 (ROCK2) was recently shown to be implicated in regulation of IL-21 and IL-17 secretion in mice and humans. Here, we report that the potent and highly selective ROCK2 inhibitor KD025 effectively reverses an established chronic graft-versus-host (cGVHD) murine model driven by IL-21 and characterized by an aggressive multiorgan system fibrotic disease. KD025 treatment leads to a dose dependent decrease in the development of pathogenic pulmonary function which correlates with a marked reduction of antibody and collagen deposition in the lungs of treated mice to levels comparable to non-cGVHD controls. The spleens of mice treated with KD025 had a decrease in the frequency of germinal centers associated with a reduction in STAT3 and concurrent increase in STAT5 phosphorylation, supporting the molecular mechanism of targeted ROCK2 inhibition in humans. The critical role of STAT3 activation in this cGVHD model was further confirmed by data showing that mice transplanted with inducible STAT3 deficient T cells or BM cells had pulmonary function comparable to the healthy negative controls. Finally, KD025 effectively blocked the progression of sclerodermatous cGVHD model characterized by improvements in weight loss, posture and skin integrity of treated animals. Together, these data demonstrate the importance of ROCK2 signaling in STAT3-dependent regulation of cytokine secretion and highlight the potential of targeted ROCK2 inhibition for cGVHD therapy.
Immunization with donor splenocytes in testis of recipient mice promotes induction of long-term survival of islet allografts transplanted in a conventional site (TRAN1P.920)
Authors: Dai Zhenhua , Xie Qingfeng , Zhang Shuzi
Published: January 2023

Immune responses are subdued in immune privileged sites, which provide a unique opportunity to prolong cellular allograft survival. We and others have previously shown that inducing tolerance of islet allografts transplanted in murine testes is easier than in conventional sites. However, it is not practical to transplant islets in human testes. Here we found that immunization of B6 recipients with irradiated Balb/C splenocytes through testicular injection, 7 and 14 days before islet transplantation under the kidney capsule, promotes long-term allograft survival induced by low doses of anti-CD40L (MR1) or CTLA4-Ig (0.1mg/day, days 0,2,4,6), with 75% islet allografts achieving long-term survival (>120 days). Same Ab treatment without the immunization failed to do so. Immunization without MR1 or CTLA4-Ig did not prolong allograft survival. We also found that islet transplantation under renal capsule of previously immunized recipients generates much less CD44+CD8+ memory T cells but induces more CD4+CD25+ Tregs than under renal capsule of unimmunized recipients. These Tregs exhibited donor-specific suppression of alloimmune responses in vitro. These findings reveal new mechanisms of immune privilege and may provide novel approaches to inducing long-term allograft survival or transplant tolerance in both immunologically privileged and unprivileged sites.
Molecular identification of endothelial cell surface antigens using proteomic approach to the molecules recognized by antibodies in transplant recipients (TRAN1P.939)
Authors: Zou Yizhou , Hu Juan , Luo Weiguang , Li Tingting
Published: January 2023

It is known that transplant recipients may have antibodies to endothelial cells (EC) but not to lymphocytes, by which the rapid and irreversible destruction of allograft is induced. We characterize EC surface antigens using well-identified recipient sera with strong antibodies against EC. 9 serum samples from 7 patients with kidney allograft rejection and antibodies against freshly isolated human umbilical vein endothelial cells (HUVECs) were selected. Target antigen proteins of EC were immunoprecipited by serum antibodies and separated by SDS/PAGE, followed by protein identification with mass spectrometry. Monoclonal antibodies against HLA and MICA were used as the procedure control in our experiment. We successfully captured the HLA and MICA molecules from ECs by these mAbs respectively. Using patient sera, three proteins were identified in these experiments: 1) human MICA glycoproteins, already known surface molecules expressed on ECs and associated with antibody-mediated rejection; 2) human keratin 1, a protein known to be expressed on the surface of endothelial cells; 3) 70KDA (local name) molecule, a protein underwent investigation. A variety of antigens appears to be harbored on the surface of endothelial cells and can elicit immune responses in transplant recipients. Our results indicated that MICA, keratin 1 and 70KDA proteins might be important antigens in organ transplant rejection. The role of these molecules on transplant rejection requires further investigation.
IL-33/ST2 activation of IL-9-secreting T cells alters the balance of fatal immunity and tumor immunity (TRAN1P.926)
Authors: Ramadan Abdulraouf , Zhang Jilu , Griesenauer Brad , Kapur Reuben , Hanenberg Helmut , Sun Jie , Kaplan Mark , Paczesny Sophie
Published: January 2023

Allogeneic hematopoietic cell transplantation (HCT) is the most potent curative therapy for cancers of the bone marrow; however, graft-versus-host disease (GVHD) continues to be the major limitation to successful HCT, with mortality rates around 50%. Current strategies to suppress GVHD also compromise the beneficial graft-versus-tumor (GVT) activity. We have shown that elevated IL-33-sequestering soluble ST2 in the plasma is a risk factor for severe GVHD. In addition, the IL-9-secreting Th9 and Tc9 cell subsets have higher antitumor activity than Th1 and Tc1. We hypothesized that induction of the ST2/IL-33 pathway will both alleviate GVHD and increase GVT activity. Differentiation of Th9 with IL-33 increased expression of membrane ST2 (ST2L) and PU.1, a transcription factor that promotes IL-9 production. Adoptive transfer of IL-33-treated Th9 cells with bone marrow cells in HCT murine models showed less severe GVHD compared to Th9 without IL-33, or Th2 cells that are protective of GVHD. Polarization of total T cells with IL-33 to IL-9 secreting cells showed that IL-9 was also increased in Tc9 cells via Th9 help. Transcriptome analysis of IL-33-T9 cells showed increased expression of genes with anti-tumor activity. Higher GVT activity with IL-33-T9 cells was demonstrated in models of GFP+MLL-AF9 acute myeloid leukemia. Human IL-33-T9 cells also showed a strong anti-leukemia cytolytic activity. Thus, IL-33-T9 cells represent a promising curative cellular therapy following HCT.
Semaphorin7A Inhibition interrupts hypoxia induction of angiogenic factors in murine monocytes (TUM6P.963)
Authors: Garcia-Areas Ramon , Libreros Stephania , Keating Patricia , Karram Joseph , Castro Camilla , Iragavarapu-Charyulu Vijaya
Published: January 2023

We discovered that mammary tumor cells express high levels of the protein Semaphorin7A (SEMA7A) and that tumor-derived SEMA7A induces monocytes to secrete pro-angiogenic chemokines to enhance tumor growth. Decreased SEMA7A limits tumor growth and production of angiogenic CXCL-2/MIP-2 and VEGF-A by monocytes. The mechanisms controlling SEMA7A expression during tumor progression are largely unknown. Recent reports describe a hypoxia responsive element in the promoter of SEMA7A. It is well established that hypoxia enhances tumor growth by promoting angiogenesis and alternatively activating monocytes. We hypothesize that SEMA7A may be a hypoxia-inducible factor and its inhibition may deter production of monocyte-derived angiogenic proteins. Monocytes from wild-type (WT) and SEMA7A null BALB/c mice were exposed to CoCl2-induced hypoxia. CoCl2 induced expression of SEMA7A in both mammary cells and WT monocytes. Following hypoxic stimuli, WT monocytes increased production of CXCL2/MIP-2, VEGF-A and Chitinase-3-like protein1 (CHI3L1). However, SEMA7A null monocytes failed to respond to hypoxia, which may be attributed to an impaired MAPK signaling response. Thus, inhibiting SEMA7A may abrogate the detrimental effects of tumor induced hypoxia on monocytes. We are further delineating how the lack of host SEMA7A can impact mammary tumor growth in vivo. Understanding the role of SEMA7A may unravel important tumor-host immune interactions in breast cancer.
Defined combinations of cytokines plus survival factors drive the differentiation of human monocytic myeloid derived suppressor cells into M1- or M2-like macrophages (TUM6P.969)
Authors: Bayik Defne , Wang Jing , Gursel Ihsan , Klinman Dennis
Published: January 2023

Myeloid derived suppressor cells (MDSCs) support tumor progression by inhibiting the activity of tumor-infiltrating natural killer cells and T cells. The subpopulation represented by monocytic MDSCs (mMDSCs) is the most immunosuppressive on a per cell basis. We studied mMDSCs FACS purified from the peripheral blood of 20 healthy donors. Results showed that specific TLR agonists (targeting TLRs 4, 7, 8 and 9) induced these cells to differentiate into either M1- or M2-like macrophages. We then defined the soluble factors responsible for this maturation. Differentiation was optimized by the inclusion of maturation factors such as GM-CSF or PGE2 during culture. GM-CSF alone induced the maturation of only a minority of mMDSCs, but nearly all of these became M2-like, suggesting that that M2 generation represents the default differentiation pathway of mMDSCs. Additional study showed that optimal mMDSC differentiation required the addition of IL-6 (and that blocking IL-6 activity prevented differentiation). The type of macrophage generated was influenced by other environmental cytokines, including IL-12, IL-4, TNFa, IL-10 and IL-13. Thus, specific combinations of cytokines plus survival factors can be harnessed to generate M1- or M2-like macrophages, raising the possibility of selectively producing such cells for the treatment of malignancies or autoimmune diseases, respectively.
Tumor-associated macrophages and heparanase in lung cancer microenvironment. (TUM6P.970)
Authors: Castelo-Branco Morgana , Souza Heitor , Paschoal Marcos , Pavão Mauro , Rumjanek Vivian
Published: January 2023

Tumor-associated macrophages promote aspects of tumor initiation, growth, and progression. Tumor growth takes place in a complex microenvironment of stromal cells and extracellular matrix. Heparanase is the only known mammalian glycosidase capable of cleaving heparan sulfate. Its expression has been associated with tumor development. Immunohistochemistry and immunofluorescence were performed in samples of 37 lung cancer patients to assess heparanase, macrophages and lymphocytes presence and localization. All types of tumors expressed heparanase within the cytoplasm and nucleus. Adenocarcinoma showed the highest intensity and widespread distribution. The other types had similar intensity with a less widespread distribution. Tumor surroundings also presented heparanase co-expression with fibroblast, macrophages and epithelial cells. Heparanase expression correlates to TNM staging. The heparanase possesses a different distribution according to the type of lung tumor. Heparanase from fibroblasts might regulate the tumor microenvironment through HS cleavage, by enabling invasion and release of growth factors from extracellular matrix. We showed lung cancer TAMs expressing heparanase and described heparanase localization in the tumor microenvironment providing knowledge concerning lung cancer. Therefore, we suggest that heparanase can constitute a possible therapeutic target for lung carcinoma and that macrophages are attractive targets as part of combination therapy in cancer.
Lgr4 deficiency attenuates tumor formation by reprograming macrophage polarization in lung cancer (TUM6P.973)
Authors: Du Bing , Tan Binhe , Wei Yinglei , Qin Juliang , Qian Min , Liu Mingyao
Published: January 2023

Macrophage polarization is increasingly recognized as an important pathogenetic factor in tumor formation. Our previous study has shown that Lgr4 negatively regulates TLR2/4 associated pattern recognition in innate immune responses. However, the exact roles and mechanisms of Lgr4 in macrophage polarization remain largely unknown. Here, we found that patients with Lgr4-high lung cancers displayed much poorer survival than the rest of the cohorts. Meanwhile, Lgr4 is also aberrantly expressed at high levels in macrophages from clinical lung adenocarcinomas (ADs), implying the potential role of Lgr4 in tumor-associated macrophages (TAMs). In addition, the expression of Lgr4 in bone marrow derived macrophages (BMMs) was reduced by IFN-γ and increased by IL-4 significantly. Interestingly, Lgr4 deficiency BMMs showed a obviously increased M1 phenotypes and reduced M2 phenotypes. Accordingly, host deficiency in Lgr4 resulted in defective and reduced growth of subcutaneously implanted Lewis lung carcinoma (LLC). Consistent with the defective growth, LLC cells implanted into Lgr4 conditional knockout mice also displayed reduced TAMs and increased M1 like macrophages in tumor tissues, suggesting impaired TAMs polarization. These findings suggest that deletion of Lgr4 may inhibits tumor growth via reprograming TAMs polarization. Thus, Lgr4 expressed in TAMs may potentially become a novel target for cancer therapy.
IRF8 expressed in T cells regulates GM-CSF expression to control myeloid derived suppressor cell differentiation (TUM6P.955)
Authors: Paschall Amy , Zhang Rui-hua , Qi Chen-Feng , Bardhan Kankana , Peng Liang , Lu Geming , Yang Jianjun , Merad Miriam , McGaha Tracy , Zhou Gang , Mellor Andrew , Abrams Scott , Morse Herbert , Ozato Keiko , Xiong Huabao , Liu Kebin
Published: January 2023

During hematopoiesis, hematopoietic stem cells differentiate into granulocytes and macrophages via a distinct differentiation program that is controlled by myeloid lineage-specific transcription factors. Mice with a null mutation of IFN Regulatory Factor 8 (IRF8) accumulate CD11b+Gr1+ myeloid cells that phenotypically and functionally resemble tumor-induced myeloid-derived suppressor cells (MDSCs), indicating an essential role of IRF8 in myeloid cell lineage differentiation. However, whether IRF8 functions intrinsically or extrinsically in regulation of myeloid cell differentiation is not fully understood. Here we report an intriguing finding that mice with IRF8 deficiency only in myeloid cells exhibit no abnormal myeloid cell lineage differentiation. Instead, mice with IRF8 deficiency only in T cells exhibited MDSC accumulation. We further demonstrated that IRF8-deficient T cells exhibit elevated GM-CSF expression and secretion. Treatment of mice with GM-CSF increased MDSC accumulation, and adoptive transfer of IRF8- deficient T cells, but not GM-CSF-deficient T cells, increased MDSC accumulation in the recipient mice. Overexpression of IRF8 decreased GM-CSF in T cells. Our data determine that in addition to its intrinsic role as an apoptosis regulator in myeloid cells, IRF8 also acts extrinsically to repress GM-CSF expression in T cells to control myeloid cell lineage differentiation, revealing a novel mechanism of adaptive immune cell regulation of myelopoiesis in vivo.
EPOR expression by peritoneal macrophages and B1b cells (TUM6P.1010)
Authors: Riggs James , Goldman Naomi , Orlowski Michelle
Published: January 2023

Erythropoietin (EPO) is used in the treatment of chemotherapy-related anemia. A higher mortality rate among cancer patients treated with EPO has been documented. Research in this area has focused upon EPOR expression by cancer cells, either cell lines or ex vivo tumor tissue. We find that addition of exogenous EPO suppresses the activation of peritoneal cavity (PerC) T cells. Via FACs analysis of cultured C57BL/6J (wildtype,WT) PerC cells, we detected the EPO receptor (EPOR) on F4/80+, CD11b+ macrophages (Mfs). The specificity of the anti-EPOR pAb was validated by inhibition of staining with soluble EPOR. The percentage of Mfs expressing the EPOR increased with culture duration. EPOR+ Mfs also increased expression of the immunoinhibitory molecule PDL1/B7H1. Interestingly, EPOR expression also appeared on cultured B1b (IgMhi, CD11blo) cells. Although less evident in WT mice assessed ex vivo, EPOR+ PerC Mfs were found in IL10KO mice. Considerably more EPOR+ B1b cells were observed in the inflammatory ascites of mice with the ovarian cancer that develops following injection of the ID8 mouse epithelial carcinoma cell line. We are currently investigating how EPO contributes to T cell suppression in vitro and impacts Mf and B1b cell function and phenotype. Our data support a link between inflammation and EPOR expression by PerC Mfs and B1b cells.
Increasing Th1 phenotype in Th17 cells improves anti-tumor T cells function (VAC7P.1032)
Authors: Chatterjee Shilpak , Kesarwani Pravin , Soloshchenko Myroslawa , Fu Jianing , Paulos Chrystal , Yu Xue-Zhong , Mehrotra Shikhar
Published: January 2023

Immunotherapy of cancer is a promising approach for successful eradication of large and established tumor. While cytolytic CD8+ T cells have been extensively investigated and used for adoptive cell therapy (ACT), accumulating evidence indicates that CD4+ T helper (Th) cells including Th1 and Th17 subsets are also effective in tumor immunotherapy. Although Th1 cells possess key anti-tumor effector functions as IFNγhi, CD107ahi, T-bethi, Granzyme Bhi, but their poor persistence due to lack of stem-cell like characteristic effect long-term tumor control and reduce their potential in ACT. However, Th17 cells programmed ex vivo in TGFβ exhibit increased stem-cell like features and reduced effector function as compared to Th1 cells, which corelates to their increased persistence but reduced long-term tumor control. Herein we identified the ex vivo programming conditions that combine potent Th1 effector conditions with key stemness feature of Th17 to generate hybrid Th17/Th1 cells that exhibit improved control of murine melanoma (B16-F10) and human melanoma (624-MEL), as compared to Th1 or Th17 cells alone. The hybrid Th1/Th17 cells were less susceptible to activation induced cell death, exhibited increased persistence along with potent effector function, and have potential in future translational studies.
Improving anti-tumor immunity by engineering T cell receptors to prevent mispairing (VAC7P.1033)
Authors: Bethune Michael , Gee Marvin , Lee Mark , Kuhns Michael , Baltimore David
Published: January 2023

T cells transduced with the genes encoding the α and β chains of a tumor-specific T cell receptor (TCR) can mediate anti-tumor immunity in patients. Such TCR-transduced T cells co-express both the therapeutic and an endogenous TCR. As each receptor is an αβ heterodimer, the endogenous α chain can mispair with the therapeutic β chain and vice versa. Such mispairing - which is observed in transduced human T cells - reduces export of the therapeutic TCR and produces potentially self-reactive TCRs. The latter can precipitate graft-vs-host disease, a lethal complication reported in mice following TCR gene transfer. We have developed a general strategy for preventing TCR mispairing. Select constant domains are swapped between the α and β chains of a tumor-specific T cell receptor, creating a domain-swapped (ds)TCR. When correctly paired, these dsTCR chains retain all domains necessary to recruit CD3 proteins, to express on the T cell surface, and to mediate functional T cell responses upon engaging a target antigen. By contrast, mispaired TCRs containing one dsTCR chain and one wild-type TCR chain lack key domains necessary for CD3 recruitment, export, and signaling, and thus are unable to mediate deleterious autoimmunity. We validate the potential of this approach in cell-based assays, as well as in a mouse model of TCR gene transfer-induced graft-vs-host disease. Thus, this work will enable the design of safer, more effective TCR gene therapy candidates for cancer immunotherapy.
Preservation of Raboral V-RG® viability by vitrification (VAC9P.1100)
Authors: Tran Yen-Nhi , Kemp Jeffrey , Nash Paul
Published: January 2023

Raboral V-RG® is a recombinant vaccinia virus used to orally immunize wildlife such as raccoons and coyotes against rabies. Since it is a live virus vaccine that expresses the rabies virus glycoprotein, stabilizing the virus and allowing for more methods of incorporation into bait can both be advantageous. This may also be true for other live viruses or cells. Two methods of preserving the virus were tested: vitrification and centrifugal evaporation. Vitrification in a sugar or polymer matrix provides protection for the virus through the transition of the matrix into a glass state and eliminates the need for containment of liquid virus suspension. Centrifugal evaporation in a sugar matrix provides protection for the virus through drying out the sample and is an alternative method of eliminating the need for containment of liquid virus suspension. Vitrification in several different matrices provided the virus with a significant level of protection. Using vaccinia virus as a surrogate for Raboral V-RG, we also examined whether simple drying might also be effective.
Novel approach to simultaneously monitor local and systemic in vivo effects of varying doses of intradermally injected LPS (VET1P.1123)
Authors: Byrne Kristen , Erf Gisela , Falcon Daniel
Published: January 2023

We recently established the dermis (pulp) of growing feathers (GF) as a minimally invasive test-site to monitor in vivo tissue effects of test substances. The objective of this study was to use the GF test-system to simultaneously monitor local and systemic effects of varying doses of endotoxin (LPS) injected into GF on leukocyte profiles and activities. Growing feathers of 16 chickens were injected with either 10 µL of endotoxin-free PBS or 10, 100, or 1000 ng of LPS per feather (18 GF/chicken). Blood and three GF were collected at each time point: 0 (before injection), 2, 4, 8, and 24 h post-injection. The concentration of leukocytes in the blood and proportions of leukocytes within the feather pulp were determined for each time point. Both LPS 1000 and 100 exhibited similar patterns of leukocyte recruitment in the blood and GF. Specifically, GF injection of LPS resulted in an initial drop (2 h) in blood leukocytes followed by increasing levels at 4 and 8 h and returning to near control levels by 24 h. The drop in blood leukocytes at 2 h was not observed with the LPS 10 treatment. At the site of injection (GF), increased levels of heterophils were already observed at 2 h for LPS 10 and 100, whereas this increase was delayed for LPS 1000. At 8 h post LPS injection, leukocyte levels were greatly elevated for all LPS treatments. Overall, based on leukocyte recruitment patterns in blood and tissue, the lowest dose of LPS may serve as a model for the local inflammatory response.
Understanding manatee immunogenetics and the implications for conservation (VET1P.1121)
Authors: Breaux Breanna , Heard-Ganir Ashley , Sena Leo , Hunter Margaret , Bonde Robert , Criscitiello Michael
Published: January 2023

The completion of the genome draft of the Florida manatee (Trichechus manatus latirostris) in 2012 made immunogenetic exploration of the Sirenia order more feasible. The endangered manatees are sentinel species for coastal ecosystems and their evolutionary origins are complex. It is believed that they evolved from a wading terrestrial mammal that became fully aquatic, which suggests distinctive adaptations for transitioning to a new environment. Discovering the genetic details of their immune system will help understand manatee evolution and allow species-specific diagnostic tools to be developed. Due to bottleneck signatures at other loci, the genetic diversity of the major histocompatibility complex (MHC) is expected to be low. This could pose problems for conservation efforts because low diversity can increase the chance of extirpation. The primary focus of this project is to determine the immune robustness of manatees by looking at their antibody repertoire and MHC polymorphism. Two species will be compared: the West Indian manatee (T. manatus) and the Amazonian manatee (T. inunguis). Using next generation sequencing, we will robustly sample the IgH and IgL rearrangements contributing to the antibody repertoire, and identify dominant MHC alleles and estimate their frequencies. The allelic distribution between the species could reveal clues to their evolutionary relationship and aid conservation efforts to help this species become more stable in their environment.
IL-15 drives CD8 T cell cycling and differentiation in chronic HIV-1 infection (VIR6P.1162)
Authors: younes souheil , shive carey , Reynaldi Arnold , Freeman Michael , Panigrahi Soumya , Estes Jacob , Anderson Jodi , Schacker Timothy , Davenport Miles , McCune Joseph , Hunt Peter , Lee Sulggi , Serrano-Villar Sergio , Canaday David , Cameron Cheryl , Sekaly Rafick-Pierre , Rodriguez Benigno , Sieg Scott , Lederman Michael
Published: January 2023

HIV-1 infection is characterized by sustained CD8 T cell expansion that is linked to morbid outcomes even after HIV replication is controlled with antiretroviral therapy (ART). The drivers of this persistent CD8 expansion in HIV-1 infection are not well understood. Here we show that cycling frequency is increased in memory CD8 T cells in untreated HIV-1 infected patients and cycling CD8 T cells have a broad T cell receptor repertoire tightly linked to the repertoire of non-cycling cells suggesting that they are largely driven into cycle by bystander mechanisms. In HIV-1 infection, cycling and granzyme B expression were increased among CD8 T cells binding viral peptide/HLA tetramers and this enriched cycling/cytolytic phenotype among virus-reactive CD8 T cells could be generated in vitro by exposure to interleukin (IL)-15. Lymph nodes of HIV-1 infected untreated patients were enriched for interleukin-15 expression that correlated with circulating CD8 T cell counts and normalized with drug-induced control of HIV-1 replication. The determinants of persistent CD8 T cell expansion and the mechanisms whereby this expansion is linked to increased morbidity merit further examination.
Sensor function for butyrophilin 3A1 in prenyl pyrophosphate stimulation of human Vγ2Vδ2 T cells (APP5P.100)
Authors: Morita Craig , Wang Hong
Published: January 2023

Vγ2Vδ2 T cells play important roles in human immunity to pathogens and in cancer immunotherapy by responding to isoprenoid metabolites such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and isopentenyl pyrophosphate. The immunoglobulin superfamily protein, butyrophilin 3A1 (BTN3A1), has been shown to be required for prenyl pyrophosphate stimulation. We proposed that the intracellular B30.2 domain of BTN3A1 binds prenyl pyrophosphates resulting in a change in the extracellular BTN3A1 dimer that is detected by Vγ2Vδ2 TCRs. Such B30.2 binding has been recently demonstrated. However, others have reported that the extracellular BTN3A1 IgV domain binds prenyl pyrophosphates leading to the proposal that the Vγ2Vδ2 TCR recognizes the complex. To distinguish between these mechanisms, we mutagenized residues in the binding sites to alanine and tested the mutant BTN3A1 proteins for their ability to mediate prenyl pyrophosphate stimulation of Vγ2Vδ2 T cells to proliferate and secrete TNF-α. Mutagenesis of residues in the IgV site had no effect whereas mutagenesis of residues within the basic pocket and surrounding V regions of the B30.2 domain abrogated prenyl pyrophosphate-induced proliferation and reduced or abolished TNF-α production. Some mutations further from the B30.2 site also diminished stimulation suggesting that the B30.2 domain may interact with a second protein. These findings support intracellular sensing of prenyl pyrophosphates rather than extracellular presentation.