bluuebunny/crossref_metadata_2025 huggingface dataset:
Genes encoding proteins of immunological interest in the Oryctolagus cuniculus (rabbit) whole genome sequences and ENCODE (81.19)
Authors: Mage Rose G.
Published: January 2023

Rabbits are a valuable resource for diagnostic and therapeutic antibodies, are becoming increasingly important for vaccine development and as animal models of human diseases. They remain a major source of polyclonal and monoclonal antibodies because unique characteristics of their immune system result in antibodies of high affinity and specificity. The whole genome shotgun (2x WGS) "unfinished Oryctolagus cuniculus database" had serious gaps, but assemblies of the 2x WGS sequence were useful for designing primers for PCR by predicting mRNA sequences and exon boundaries. Deeper 7x WGS coverage was completed in 2008 and assembly of a high quality draft rabbit genome sequence is nearing completion. The "Thorbecke Inbred Rabbit" chosen for sequencing at Broad Institute was less heterozygous than outbred NZW but the Trace Archive of Oryctolagus cuniculus (WGS) reveals heterozygosity at the immunoglobulin heavy chain locus. The ENCODE Project with ~ 1% of rabbit genomic sequence from a different animal has rabbit genes of immunological interest including a gene cluster syntenic with human and mouse containing IL13, IL3, IL4, IL5 and IRF1. Rabbit genome sequences can contribute comparative genomic data, reveal variants of genes of immunological interest, and genetic contributions to animal and human disease susceptibilities. This research was supported by the Intramural Research Program of the NIH, NIAID.
Cloning and functional characterization of a rainbow trout CD80/CD86 ortholog (81.22)
Authors: Zhang Yong-An , Hikima Jun-ichi , Li Jun , Sunyer J. Oriol
Published: January 2023

In mammals, interaction of CD28 with CD80 or CD86 molecules provides costimulatory signals for T cell activation. Such signals lead to increased gene and protein expression of IL-2 by activated T-cells. CD80 and CD86 have only been cloned in mammals and birds thus far, and their functional characterization is basically limited to their study in mammals. Thus, in the present study we show for the first time the cloning of a rainbow trout molecule (rtCD80/86) that shows a high degree of homology to CD80 and CD86 molecules. In addition, we have identified and cloned trout IL-2 and we have shown that recombinantly produced trout CD80/86 upregulates the expression of IL-2 in trout blood leukocytes. This finding indicates that this capacity to modulate IL-2 expression is a primordial function that has been conserved both in fish and mammalian CD80/CD86 molecules throughout evolutionary time.
Non-apoptotic caspase-8 activation balances T lymphocyte autophagy (82.14)
Authors: Bell Bryan D , Leverrier Sabrina , Weist Brian M , Walsh Craig M
Published: January 2023

Apoptosis and autophagy are critical mediators of a functional immune response. FADD and caspase-8 (casp8) were originally discovered to transduce apoptotic signals delivered through TNF family members. Paradoxically, lymphocytes lacking FADD or casp8 function have impaired clonal expansion following antigen receptor crosslinking and succumb to a caspase-independent cell death. We show that T cells lacking FADD or casp8 are subject to hyperactive autophagy. T cell autophagy induces casp8 activation through its interaction with FADD:Atg5:Atg12 complexes. Blockade of autophagic signaling with shRNA knockdown of the autophagy-dependent protein Atg7 leads to a rescue of T cells lacking FADD and casp8. Similarly, inhibition of RIPK1 kinase activity with necrostatin-1 completely restored T cell cycle and survival. We hypothesize that the non-apoptotic casp8 activity serves to dampen the autophagic response of proliferating T cells through direct cleavage of RIPK1 or the casp8 homolog, c-FLIP, which may secondarily inhibit RIPK1-dependent authophagic signaling. While rapidly dividing T cells require autophagy, this negative feedback loop can prevent hyperactive autophagy. Linking apoptotic proteins to this signaling paradigm may serve to prevent an inflammatory response driven by autophagic cell death in lymphocytes during an immune response. (Funded by NIH T32CA09054 to B.D.B. and R01AI63419 to C.M.W.)
Naturally occurring memory (CD44hi) CD4 T cell responses are differentially regulated by the expression of programmed death-1 receptor (82.4)
Authors: Suvas Susmit , Channappanavar Rudragouda , Twardy Brandon
Published: January 2023

CD4 T cells in unprimed mice can acquire CD44hi memory phenotype (MP) and the MP CD4 T cell subset become progressively more prominent with age. Although it is not clearly evident if MP CD4 T cells arise due to lymphopenia-induced proliferation or environmental antigen exposure, studies carried out in mice model suggest that naturally occurring MP CD4 T cells are hyporesponsive to both polyclonal and TCR mediated stimulus. In this report we determined that a large proportion of naturally occurring MP CD4 T cell pool in unprimed mice expressed multiple inhibitory receptor PD-1, ICOS and CTLA-4. Furthermore, inhibitory receptor expressing MP CD4 T cells become more prominent with advancing age. Our results showed that PD-1hi MP CD4 T cells isolated from aged mice were more susceptible to spontaneous apoptosis when compared with PD-1lo MP CD4 T cells. We also determined the differential expression of IFN-γ, IL-2 and IL-17 cytokines in PD-1+ versus PD-1- MP CD4 T cells isolated from unprimed young and old mice. Lastly, MP CD4 T cells purified from aged mice proliferated differentially to Con A stimulus depending on the expression of PD-1 receptor. Thus, we conclude that responses of naturally occurring MP CD4 T cells are differentially regulated by the expression of inhibitory PD-1 receptor.

Supported by the funds derived from American Federation for Aging Research (AFAR).
A high level of isoform V in the CD40 receptor composition leads to survival/proliferation signals in autoimmune Th40 cells (82.6)
Authors: Vaitaitis Gisela M , Wagner, Jr David H
Published: January 2023

CD40 - CD154 interactions are critical in autoimmunity. CD4loCD40+ T cells (Th40) are expanded in autoimmunity and are necessary and sufficient in transferring disease. The expansion, as well as disease onset, is prevented by blocking CD154. Autoimmune Th40 constantly recruit high levels of CD40 and TRAF2 to the raft microdomain for signaling. CD40 stimulation further increases those levels and induces high levels of Bcl-XL and cFLIPp43 leading to survival and proliferation. In non-autoimmune Th40, only low levels of CD40 and TRAF2 are recruited to the raft even upon CD40 stimulation and this only induces Bcl-XL without an effect on survival and proliferation. There are five CD40 isoforms that multimerize to form a functional receptor in mice. We speculated that differing CD40 receptor composition could be responsible for the signaling differences. Here we show that autoimmune Th40 have a high ratio of isoform V to isoform I while it is drastically lower in non-autoimmune Th40. Th40 from autoimmune mice that had been treated to block CD154 demonstrated a ratio similar to that of non-autoimmune Th40 suggesting that the CD40 receptor composition depends on the availability of CD154 and that a receptor predominantly composed of isoform V is directly related to the ability to induce proliferation. Interestingly, when non-autoimmune mice were injected with agonistic CD40 antibodies the Th40 population expanded and the CD40 isoform ratio changed to look like those in autoimmune animals. Therefore it is important to understand what conditions lead to the expression of one CD40 isoform over another and whether those conditions can be altered in autoimmunity.
MS4a4B: a novel regulator in T cell proliferation (82.9)
Authors: Xu Hui , Yan Yaping , Williams Mark S , Fowler Daniel H , Carey Gregory B , Zhang Guang-Xian , Rostami Abdolmohamad
Published: January 2023

MS4a4B is a novel member of the recently discovered MS4A gene family (Membrane-spanning 4-domain family, subfamily A). The most well-known members of MS4A gene family include CD20, FcεRIβ and HTm4. An increasing body of evidence has demonstrated roles of the MS4A proteins in cell function, particularly for CD20 and HTm4. CD20 is involved in cell cycle regulation and apoptosis in B cells. HTm4 has been found to regulate the cell cycle through the KAP-CDK2 cascade. To date, the function of MS4a4B is unknown. In a screen for genes potentially involved in T cell development we cloned MS4a4B. Our data show that MS4a4B was expressed in primary T cells. The expression of MS4a4B in T cells was enhanced upon T cell activation. Interestingly, we observed a loss of MS4a4B expression in all the malignant T cells we have tested to date, including thymoma cells and T hybridoma. To determine the role of MS4a4B, we expressed MS4a4B in T cells by MS4a4B-expressing-vectors. Over-expression of MS4a4B reduced proliferation of T cells. Similarly, forced-expression of MS4a4B in EL4 cells, a thymoma cell line, by viral vector also inhibited the proliferation of the thymoma cells. Our results suggest that MS4a4B plays a regulatory role in T cell proliferation.

Supported by American Heart Association grant AHA-0830072N (to H.X)
Differential localization of effector and memory T cell subsets (83.16)
Authors: Jung Yong Woo , Kaech Susan M
Published: January 2023

During viral infections, it is not well defined where within tissues effector and memory CD8 T cells form and persist. Our recent findings show that two subsets of effector CD8 T cells that have distinct cell fates are formed during acute infection - the KLRG1hi IL-7Rlo short-lived effector cells (SLECs) and the KLRG1lo IL-7Rhi memory precursor effector cells (MPECs). Most of the SLECs undergo apoptosis after infection whereas most MPECs survive and become long-lived memory T cells. Here, we show that MPECs and SLECs are differentially localized within the spleen during LCMV infection. Using immunofluorescence microscopy, we demonstrate that the majority of MPECs were found in the T cell zone, while SLECs were exclusively localized in the red pulp during the course of an immune response to LCMV infection. At memory time points, only T cells with an MPEC phenotype were found in the T cell zone. MPECs homed to the T cell zone using pertussis toxin-sensitive chemokine receptors, and appeared to contact with gp38+ stromal cells, which produce chemokines CCL19 and CCL21 and the T cell survival cytokine IL-7. Employing a transwell migration assay, we found that MPECs showed higher chemotactic ability toward CCL19 than SLECs. Together, our data suggest that the preferential survival of the MPECs and the memory T cells may be tightly linked to their localization in the T cell zone and the interaction with IL-7-producing stromal cells.
Triglyceride lipids can enhance the cytotoxicity of induced cytotoxic T lymphocytes (CTLs) (83.8)
Authors: Hudig Dorothy , Alves Bryce N , Marshall Kristen , Tamang David L , Leong Jeffrey , Redelman Doug , Elliott Viki , Lowe Mark E
Published: January 2023

IL-4 induces pancreatic lipase related protein 2 (PLRP2) in cytotoxic T lymphocytes (CTLs). Because PLRP2 in semen mediates lipid-dependent toxicity to sperm, we questioned whether CTL-derived PLRP2 could support similar cytotoxicity towards tumor cells. First we tested recombinant PLRP2. Microgram/ml concentrations were toxic to P815 cells in 48 hr assays when lipid and co-lipase were present. Next we found that CTLs secrete triglyceride lipase but that most IL-2 or IL-4 induced CTLs were unable to mediate lipid-dependent cytotoxicity. A single, exceptional lot of IL-4 reproducibly induced lipid-dependent cytotoxicity that was also perforin-independent. In efforts to induce the lipid-dependent CTL killing again, attempts were made to block type 1-polarizing cytokines, including biasing the T cells cultures with more IL-4 or with IL-12 blocking antibodies, and using T cells without IFN-ã receptors. These attempts were unsuccessful at inducing triglyceride-enhanced cytotoxicity. The CTLs induced with IL-4 consistently had mRNA for PLRP2 but lacked mRNA for co-lipase. Therefore, we added exogenous co-lipase to the cytotoxicity assays. Cytotoxicity was unaltered. We conclude (1) that lipid-dependent cytotoxicity by PLRP needs co-lipase and (2) that more than PLRP2 is required for lipid-dependent cytotoxicity by CTLs. Supported by NIH R01CA38942 and T32CA09563.
The C3d/Epstein-Barr virus receptor (gp140, CR2, CD21, EBV/C3dR) triggers distinct signaling pathways, independently of CD19 or BCR, in human B lymphocytes. (84.1)
Authors: FRADE Raymond
Published: January 2023

We originally isolated gp140, the receptor of human C3 (the third component of complement) from Raji, a human B lymphoma cell line (1981). gp140 is the C3d receptor (1983, 1985a) and also the Epstein-Barr virus receptor (1985b). Activated EBV/C3dR regulates B lymphocyte proliferation (1985c). EBV interaction with EBV/C3dR is the first step of B lymphocyte transformation (1985b). Activated EBV/C3dR interacts through its intracytoplasmic domain, with the p53 oncoprotein (1989, 1992, 1995) or a p68 Cabp (1991, 1992, 1995). We identified the two first signaling pathways specifically triggered by activated EBV/C3dR. The first pathway, independent of CD19 and BCR activation, is characterized by: 1) activation of pp60src kinase to tyrosine phosphorylate nucleolin; 2) tyrosine phosphorylated nucleolin interacts with the SH2 domains of the p85 subunit of PI-3 kinase, activating this latter; 3) activated PI 3-kinase recrutes the AKT-GSK3 pathway (1999, 2001, 2003). The second pathway is characterized by: 1) Cbl phosphorylation; 2) interactions of phosphorylated Cbl with Crk-L, Syk and the p85 subunit of PI-3-kinase; 3) dissociation of phosphorylated Cbl from Vav (2006). This dissociation, opposed to the Cbl-Vav association triggered by CD19 or BCR, emphasizes the specificity of EBV/C3dR signaling pathways. We herein identified the functional relationship between these two pathways.
Cyclooxygenase-2 is Essential for the Optimal Differentiation of Human Antibody Secreting Cells: Implications for Vaccination (84.11)
Authors: Phipps Richard P , Bernard Matthew P
Published: January 2023

Generation of optimal humoral immunity in response to vaccination is essential in order to protect the public from potentially devastating infectious agents. Cyclooxygenase inhibitors are amongst the most widely used and commonly prescribed medications. However, relatively little is understood about their influence on human immune responses. Herein, we report that non-steroidal anti-inflammatory drugs (NSAIDs) and small molecule Cox-2 selective inhibitors attenuated human B cell immunoglobulin production in a Cox-2-dependent manner. We further demonstrated that the Cox-2 selective inhibitors SC-58125 and NS-398 non-selectively blunt the production of human antibody isotypes including IgM, IgG1, IgG2, IgG3 and IgG4. This correlated with a significant reduction in the generation of CD38+ IgM+ and CD38+IgG+ antibody secreting plasma cell precursors. Interestingly, we discovered that inhibition of Cox-2 activity with SC-58125 severely reduced the mRNA and protein levels of the essential plasma cell transcription factors, Blimp-1 and Xbp-1 in normal human B cells. These new results demonstrate an essential role for Cox-2 in the terminal differentiation of human B lymphocytes to antibody secreting cells. Our findings suggest that the use of drugs that attenuate Cox-2 activity can reduce the generation of plasma cells and ultimately optimal antibody responses to vaccines and pathogenic infections.

This research was supported by DE011390, ES01247, AI071064 and T32-DE007202.
Molecular mechanism of allelic exclusion of TCR alpha chains (84.2)
Authors: Rybakin Vasily , Gascoigne Nicholas R.J.
Published: January 2023

Allelic exclusion is a mechanism whereby a product of single allele is selected for expression. In case of the T cell receptor, allelic exclusion takes place differently for TCRa and TCRb chains. For the TCRb chain, a successfully completed in-frame rearrangement of one locus effectively inhibits the rearrangement of the second locus. This inhibition is achieved as a result of signaling originated from a successfully formed dimer of the b chain with pre-Ta.

In contrast, both TCRa alleles are rearranged, often multiple times, until the positive selection stage, when a single a chain in conjunction with a fixed b chain allows the thymocyte to differentiate. Majority of immature thymocytes express more than one a chain on the cell surface, while after positive selection only one chain is expressed.

We developed and experimental system allowing to directly assess the fate of each of two specific a chains by flow cytometry and microscopy. Our studies are aimed at the better characterization of posttranslational modifications and signaling events leading to the selection of a single TCRa chain expressed at the cell surface.
Stim1 sets a threshold for activation of a pro-apoptotic Erk pathway in B cells (84.21)
Authors: Limnander Andre , Weiss Arthur
Published: January 2023

Developing B cells differ from mature B cells in signaling downstream of the B cell receptor (BCR), and these differences play a crucial role in repertoire selection. While BCR crosslinking in mature B cells leads to proliferation and activation, in immature B cells it triggers apoptosis. BCR-induced Ca2+ entry is higher and prolonged in immature B cells with respect to mature B cells. While it is known that differences in Ca2+ signals play a role in the differing BCR responses during B cell development, the exact signaling pathways involved in the apoptosis vs. proliferation decision remain elusive. Stim1 is a Ca2+ sensor that is required for gating of the CRAC channels, and its expression is transiently increased during B cell development. We demonstrate that increasing Stim1 expression is sufficient to augment the amplitude and duration of Ca2+ entry in B cells, and consequently sensitizes B cells to antigen-induced apoptosis. Increased intracellular Ca2+ activates a pro-apoptotic, Ca2+-dependent pathway to Erk, and the biochemical requirements for Ca2+-dependent Erk activation differ from those of diacyl glycerol (DAG)-mediated Erk activation. In immature B cells, increases in Ca2+ entry that outlast increases in DAG levels may lead to activation of pro-apoptotic, Ca2+-dependent ERK signals that override DAG-mediated proliferative signals, thereby triggering negative selection.
The class II Histone deacetylase 7 controls Interleukin 2 receptor expression and proliferation of primary cytotoxic T cells (84.8)
Authors: Navarro Maria N , Goebel Jurgen , Feijoo-Carnero Carmen , Cantrell Doreen A
Published: January 2023

Regulation of chromatin accessibility via class II histone deacetylases is a critical mechanism controlling gene expression. The paradigm for class II HDAC regulation is that in quiescent lymphocytes these molecules localise to the nucleus and form repressive complexes on chromatin. Antigen receptor activation of Protein Kinase D induces phosphorylation and nuclear export of HDACs thereby relieving their repressive actions on chromatin and gene expression. In this context, the present study has examined the regulation and function the class II histone deacetylase 7 in effector cytotoxic T cells (CTLs). This HDAC has been shown previously to function as a signal dependent repressor of gene transcription during T cell development in the thymus. We now show that HDAC7 is a critical regulator of Interleukin 2 receptor expression and proliferation in primary CTL. Strikingly, there is constitutive and sustained phosphorylation and export of HDAC7 from the nucleus to the cytosol in CTLs. The cytosolic localisation of HDAC7 is determined by the phosphorylation of key serine residues in the molecule S156, S322, S457 and by a balance of src kinase and diacylglycerol signalling pathways that increase its nuclear export and block its nuclear import. An HDAC7 mutant (S156A, S322A, S457A) that cannot be phosphorylated is thus maintained in the nucleus and prevents expression of the Interleukin2 (IL-2) receptor and accordingly inhibits CTL responses to IL-2. HDAC7 thus acts as a molecular switch to mediate IL-2 receptor expression and cellular responsiveness to IL-2 in primary T cells.
IL-12 enhances generation of CD25+ cells from common lymphoid progenitors in OP9-DL1 co-cultures. (85.14)
Authors: Van De Wiele C. Justin , Tan Chibing , Davis Brenda K. , Teague T. Kent
Published: January 2023

Efficient in vitro production of T cells from bone marrow precursors represents a major technical hurdle that must be overcome in order to fully realize the potential benefits of future tailored immunotherapies. We have utilized the OP9-DL1 system to determine whether interleukin-12 (IL-12) influences development of common lymphoid progenitors (CLPs) towards the T cell fate. Inclusion of 5 ng/ml recombinant mouse IL-12 in addition to standard levels of IL-7 (5 ng/ml) and Flt3L (5 ng/ml) doubled the number of recoverable viable cells after one week of culture in comparison to control cultures. As the cells differentiated to a DN3a-like stage (Lin-CD4-CD8-CD25+CD44-CD117-CD28lo)during the following two weeks of culture, the effectiveness of IL-12 gradually diminished. This was not completely unexpected as IL-12 response assays on developing thymocytes demonstrated induction of STAT4 phosphorylation only within the very earliest DN1cKit+ and DN2 thymic subsets. The effectiveness of IL-12 in promoting production of bona fide T cell precursors was demonstrated by the observation of enhanced cell yields from IL-12 containing DN2/OP9-DL1 co-cultures. The IL-12 effect was titerable and was abrogated by genetic absence of the IL-12Rβ2, indicating a direct effect of IL-12 on the developing lymphoid cells.
Bcl11b is a positive regulator of T-lineage specification (85.19)
Authors: Li Long , Rothenberg Ellen V.
Published: January 2023

Hematopoietic stem cells (HSC) commit to the T-lineage by gradually losing developmental plasticity and gaining T cell identity. A recently discovered transcription factor Bcl11b is expected to be a key regulator in this process. It is known Bcl11b is crucial for alpha/beta T cell development and for survival of double positive cells specifically. However, little is known about how Bcl11b works in the very early stages of T cell development. We have cloned cDNA of four transcripts of Bcl11b, and over-expressed these Bcl11b isoforms in fetal liver HSCs. Our data so far has shown that certain Bcl11b isoforms positively regulate T-lineage specification. While full-length Bcl11b and Bcl11b 1-2-4 are growth inhibitory, full-length Bcl11b stimulates emerging of Thy1+ cells from HSCs under a condition that does not normaly favor T cell development. In another experiment, fetal liver HSCs transduced with Bcl11b 1-3-4 and 1-4 were co-cultured with OP9-DL1 cells or OP9 control cells. The data showed that after 4 days of co-culturing HSCs with OP9 control cells, empty vector infected HSCs showed no developmental progression: almost all cells were c-kit+ CD25-; however, some Bcl11b transfected cells became c-kit+, Thy1+ and CD25+ DN2-like cells, normally seen only on OP9-DL1 cells. Our data revealed that certain isoforms of Bcl11b may stimulate T cell development and may even have a novel relationship to Notch signaling.
Foxn1 is required to maintain Notch ligand expression on TECs (86.11)
Authors: Xiao Shiyun , Manley Nancy
Published: January 2023

Thymic epithelial cells (TECs) are required to promote T cell development inside the thymus. We recently generated a novel Foxn1lacZ allele, in which Foxn1 expression is reduced below 50% of normal at about 7 days postnatal, resulting in rapid thymic degeneration. Using this allele, we showed that Foxn1 is required to maintain TEC function and T cell production in the postnatal thymus. Reduced Foxn1 results in reduced TEC proliferation and MHCIIhi TEC numbers. Total T cell and CD4 SP cells were also reduced, but CD4-8- DN cells were significantly increased. Compared to control mice, we found that the percent of CD44+25- DN1 cells gradually increased from day 14, reaching a peak of 60-80% of all DN cells at day 28. These DN1 cells were predominantly CD19+HSAhi immature B cells, which first increased between day 9 and 12. The ratio of B cells to total thymocytes was 20 fold higher at day 28 in Foxn1lacZ thymus compared to controls, and B cell number also increased. Kithi DN1a and DN1b cells, which have been identified as the progenitors of TCRαβ T cells, were significantly reduced starting at day 9. The ratio of DN1a/b cells to total thymocytes was 15 fold lower at day 9 in Foxn1lacZ thymus than in control thymus. A significant reduction of DN1a/b subsets also appeared in control mice at about 2 months, associated with a significant reduction of total T cell number and down-regulation of Foxn1 expression in TEC. Further, we show that the mRNA level of Notch ligands Dll1 and Dll4 were down regulated in Foxn1lacZ TSCs. These results suggest that Foxn1 may be required to maintain Notch ligand expression in TECs; therefore, down regulation of Foxn1 expression in Foxn1 lacZ mice changes the thymic environment to be favorable to produce B cells instead of T cells.
Tbx1 antagonizes thymus organogenesis (86.4)
Authors: Cardenas Kim T. , Liu Zhijie , Laurent Micheline , Carter Carla , Manley Nancy , Richie Ellen
Published: January 2023

The thymus and parathyroids originate from organ-specific domains in endoderm of the 3rd pharyngeal pouch (PP), identified by Foxn1 and Gcm2 expression respectively at embryonic day 11 (E11). The molecular mechanisms regulating fate determination in the 3rd PP are not clear. Our studies show that neural crest cells (NCCs) play a role in this process (Griffin et al, in press). We also showed that lack of Sonic hedgehog (Shh) results in absence of the parathyroid domain and expansion of the thymus domain (Moore-Scott et al, 2005). It has been proposed that the Tbx1 transcription factor is also required for thymus development and is regulated by Shh. However, Tbx1 is expressed in the Gcm2, but not the Foxn1 domain of 3rd PP at E10.5.

We propose a model of thymus organogenesis in which 3rd PP endoderm assumes a thymus fate unless Shh plus NCC-derived signals specify a parathyroid fate. We further propose that Tbx1 is required to establish parathyroid fate and is non-permissive for thymus fate. To test the model, we generated knock-in mice containing a Cre-inducible allele that allows temporal and spatial control of Tbx1 expression. Ectopic Tbx1 expression using Foxn1Cre resulted in markedly hypoplastic thymi, supporting the notion that Tbx1 antagonizes thymus differentiation. Current studies focus on determining the molecular basis for this phenotype and testing the effects of activating Tbx1 expression at earlier stages. Supported by NIH HD056315 to ER and HD035920 to NM.
Forced-expression of Foxn1 in stromal cells maintains thymic architecture and thymopoiesis in aged mice (86.5)
Authors: Zook Erin C , Zhang Shubin , Witte Pamela L , Le Phong T
Published: January 2023

The thymus is the primary site for thymopoiesis and production of naïve T cells; however, thymic function declines with age. Thymopoiesis requires continuous thymic seeding by early T cell progenitors (ETP), a progeny of the bone marrow-derived lineage marker negative, Sca1pos, c-kitpos (LSK) progenitors that express Flt3 (LSK Flt3pos). A decrease in the frequency of ETP, and in the number of LSK Flt3pos with age has been implicated as a mechanism for the decline in thymopoiesis. We have shown that a decrease in thymocyte numbers occurs as early as 3m of age and correlates with a decrease in Foxn1 expression; however, a role for Foxn1 in thymopoiesis remains unknown. Here, we show that thymi of aged Foxn1 transgenic mice (Tg) have a higher number of thymocytes and ETP compared to aged wild type mice. Forced expression of Foxn1 also rescues aged-associated alterations of thymic architecture. Unexpectedly, we found that Foxn1 is over expressed in the bone marrow (BM). While the LSK and LSK Flt3pos are reduced in aged wild type mice, these populations remain unchanged in aged Foxn1Tg. Furthermore, the aged Foxn1 Tg also have a higher number of the lineage marker negative CD90.2pos CD2pos, a population absent in the Foxn1 mutated nude mice. Thus, our data suggest that expression of Foxn1 is critical for functional thymic and BM stromal cell niches required for the survival of T cell progenitors (NIA AG023809).
Haploinsufficiency of Flt3-ligand limits RAG1 locus activation (87.2)
Authors: Dolence Joseph J , Gwin Kimberly , Medina Kay L
Published: January 2023

Evidence is accumulating that the molecular circuitry initiating lymphoid lineage specification correlates with expression of the receptor tyrosine kinase Flt3. Recombination activating gene-1 (RAG1) locus activation is a hallmark of lymphoid lineage specification. Previous studies established that expression of a RAG1-GFP/+ reporter begins in a subset of lineage negative (Lin-) cells that express high levels of c-kit and Sca-1 (LSK+). To investigate a regulatory connection between Flt3 signaling and RAG1 locus activation from this stage, RAG1-GFP/+ knockin mice expressing wildtype, heterozygote, and knockout Flt3-ligand (FL) alleles were generated. Mono-allelic expression of the FL gene reduced FL transcripts by half, providing an in vivo model to evaluate threshold requirements for FL in lymphocyte development. Percentages and numbers of LSK+ cells were similar in FL+/+ and FL+/- mice, but significantly decreased in FL-/- animals. Analysis of GFP expression within the LSK+ compartment revealed a FL dose dependent reduction in frequencies and numbers of GFP+ cells. The requirement for threshold levels of FL in regulation of GFP expression extended into the B220- c-kitlo IL-7R+ lymphoid progenitor and CD43+ B220+ Pro-B compartments, but not in later stage B cell precursors. These findings establish a critical requirement for FL in regulating one event in lymphoid specification, RAG1 locus activation.

These studies were supported by startup funds to K.L.M. from Mayo Clinic.
A comparison of “steady-state” and “emergency” granulopoiesis: evidence of a single pathway for neutrophil production (87.5)
Authors: Cain Derek W , Ueda Yoshihiro , Holl Thomas Matt , Snowden Pilar B , Kondo Motonari , Kelsoe Garnett
Published: January 2023

Normally, neutrophil pools are maintained by steady-state granulopoiesis in bone marrow (BM). Inflammation, however, elicits neutrophilias that outstrip the capacity of steady-state granulopoiesis, and are thought to rely on a distinct program of accelerated production known as "emergency" granulopoiesis. In mice immunized with alum adjuvant, IL-1RI expression by radioresistant cells is required for both the mobilization of neutrophils from BM and increased proliferation by hematopoietic progenitors. Thus, an IL-1RI dependent trans-acting factor(s) mediates the accelerated neutrophil output needed for alum-induced neutrophilias, consistent with models of emergency granulopoiesis where inflammation elicits growth factors, i.e., G-CSF, GM-CSF, IL-3, and IL-6. These models do not, however, explain the emergency-like responses that follow non-inflammatory neutrophil depletion. Similarly, progenitor compartments are constitutively expanded in mice rendered neutropenic by the conditional loss of Mcl-1. Our observations suggest that neutrophilias elicited by inflammation are not the result of a distinct pathway of granulopoiesis, but represent the stabilizing effects of feedback that correct for the loss of BM neutrophils. We propose that inflammatory signals decrease the number of BM neutrophils via mobilization to increase progenitor proliferation through a homeostatic mechanism.

Research support: NIH grants AI24335 and AI56363 (to G.K.)
Estrogen receptor expression promotes the differentiation and activation of splenic dendritic cells and macrophages during inflammation in vivo (87.6)
Authors: Agrawal Hemant , Carreras Esther , Bajana Sandra , Kovats Susan
Published: January 2023

De novo differentiation of inflammatory dendritic cells (DC) and macrophages (MΦ) occurs in autoimmune diseases that are strongly female biased in incidence. We previously showed that estrogen receptor alpha (ERα) signaling promotes DC differentiation mediated by GM-CSF, an inflammatory pathway. To compare the efficiency of myeloid cell differentiation from ERα+ and ERα-/- bone marrow (BM) cells in vivo, we generated mixed competitive ERα+/ERα-/- BM chimeric mice. In the radiation-induced inflammatory environment 3 weeks post reconstitution, DC and MΦ in spleen and BM were derived preferentially from ERα+ BM cells, and ERα+ DC displayed higher levels of CD86. These data show that during inflammation ERα signaling promotes the new differentiation and activation of myeloid cells. In contrast, in the homeostatic environment 3 months post reconstitution, DC, MΦ and lymphocytes were predominantly derived from ERα-/- BM cells, consistent with reports that ectopic estradiol depletes hematopoietic progenitors. In chimeric mice, ER signaling also increased splenic DC activation induced by in vivo exposure to TLR9 ligands: ERα+ cDC displayed significantly more CD86 than ERα-/- DC. Thus, during inflammation in vivo, ERα signaling in response to physiological levels of endogenous estrogens promotes DC and MΦ development and activation. Supported by OCAST HR06-157 & NIH AI079616, RR020143.
Unexpected expression of peptidoglycan recognition protein 2 in lymphocytes and bone marrow hematopoietic stem cells (87.8)
Authors: Wang Zhien , Xu Min , Locksley Richard M
Published: January 2023

Peptidoglycan recognition factors are highly conserved molecules involved in recognition of fragments of bacterial peptidoglycans in innate immune responses. Peptidoglycan recognition protein 2 (PGLYRP2) is a serum N-acetylmuramoyl-alanine amidase secreted by the liver. We used a green fluorescent reporter allele engineered into the gene to label cells in the mouse that endogenously express PGLYRP2. Unexpectedly, PGLYRP2 was expressed constitutively in resting CD4 and CD8 lymphocytes and in NK cells. Reconstitution of PGLYRP2-deficient mice with PGRLYRP2-sufficient T cells led to the accumulation of PGLYRP2 in serum. In assessing bone marrow populations in these mice, we identified constitutive PGLYRP2 expression in a subset of lineage-negative, c-kit+Sca1+ cells that had the properties of hemopoietic stem cells, including the capacity to establish hematopoietic lineages and for self renewal when transferred into irradiate recipient mice. Expression of PGLYRP2 was also confirmed within the conventionally identified hematopoietic stem cell compartment in wildtype mice, confirming the unexpected expression of this enzyme in bone marrow stem cells.
CM1 ligation induces the apoptosis of cervical cancer Hela 229 cells via Fas/FasL interaction (88.11)
Authors: Hur Dae Young , Park Ga-Bin , Kim Yeong-Seok , Song Hyunkeun , Sung Minjung , Lee Hyun-Kyung , Lee Wang Jae
Published: January 2023

CM1(centrocyte/-blast marker I) defined by a mAb developed against concanavalin-A activated PBMC, is expressed specifically on a subpopulation of centroblasts and centrocytes of human germinal center(GC) B cells. Interestingly cisplatin or PMA induced CM1 expression on cervical cancer cell (Hela 229). In addition, we observed the existence of CM1 in cervical cancer patients' tissues. In this study, we observed the function of CM1 induced by cisplatin on cervical cancer cells. So we designed to investigate the enhancement of the chemoresponse to cisplatin by anti-CM1 mAb treatment in human cervical cancer Hela 229 cells. We observed that CM1 ligation with anti-CM1 mAb enhanced the apoptosis of Hela cells pre-treated with cisplatin and induced the expression of both FasL and Fas in theses cells. Furthermore, anti-Fas blocking antibody, ZB4, blocked CM1-mediated apoptosis effectively in Hela cells. Caspase-8 inhibitor, z-IETD effectively blocked CM1-induced cell death. And CM1 ligation down-regulated expression of procaspase-8 and PARP. RT-PCR results showed up-regulated the expression of Bax and Bad and down-regulated the expression of Bcl-2. The release of cytochrome c and the translocation to nucleus of endoG and AIF were showed by confocal micoscopy. Based on these findings, it can be concluded that CM1 might act as an effective apoptosis inducer in chemotherapy of cervical cancer by regulating the expression of various apoptosis-related genes via Fas/FasL pathway.
Tumor Associated Ascites Inhibits CD1d-Mediated NKT cell Activation (88.18)
Authors: Webb Tonya J , Rogers Ophelia , Giuntoli, II Robert L , Schneck Jonathan P , Oelke Mathias
Published: January 2023

Natural killer T (NKT) cells recognize lipid antigen presented by CD1d molecules. It has been shown that NKT cells can both directly and indirectly mediate anti-tumor immunity. However, it is thought that the anti-tumor effects mediated by these cells are being hampered because the number and function of NKT cells are reduced in cancer patients. In addition, it has been shown that tumor associated glycolipids are frequently shed into the tumor microenvironment, which can mediate immunosuppressive activity. In these studies we examined antigen presentation by CD1d following treatment with ascites from ovarian cancer patients. It was found that pretreatment with ascites inhibited the ability of CD1d expressing cells to activate NKT cells in a dose-dependent manner. We found that this inhibition does not require antigen processing, as canonical (Vα14+) and noncanonical NKT cells, which do not require trafficking through endocytic compartments, were both inhibited. Also, ascites treatment of artificial Antigen Presenting Cells (aAPC) preloaded with α-GalCer, abrogated their ability to present antigen to NKT cells, which further suggests that the inhibition is antigen processing independent. In addition, our data demonstrate that treatment antigen presenting cells with ascites does not inhibit activation of classical CD8+ T cells. Together, these data suggest that glycolipid shedding in ascites may be a mechanism of immune evasion specifically utilized used by cancers to inhibit CD1d- mediated activation of NKT cells.
New biomolecules for cancer therapy (88.2)
Authors: Campigna Emmanuelle , Morisseau Sebastien , Garambois Veronique , Germain Claire , Larbouret Christel , Mach Jean-Pierre , Pèlegrin André
Published: January 2023

It has been previously demonstrated that tumor cells expressing MICA are no more able to grow in vivo due to the killing of tumor cells by both NK and CD8+ T cells.

In order to restore the presence of the MICA ligand at the surface of tumor cells, we designed biomolecules composed of MICA and an scFv (single chain fragment variable) that can recognise tumor associated antigens (TAA). The biomolecules should target tumor cells and re-direct the specific lysis of tumor cells by NK cells after interaction between NKG2D and exogenous MICA at the surface of tumor cells.

In vitro cytometry experiments showed that the biomolecules specifically bind different tumor cells expressing TAA. Using 51Cr release assay, we demonstrated that the biomolecules induce an efficient and specific killing of tumor cells mediated by NK cells or human PMBCs. In vivo, biodistribution studies showed that the biomolecules could target xenografted tumor when injected intravenously. Consequently, a protocol is investigated for therapy.
MicroRNAs regulate NKG2D ligands on tumor cells (88.21)
Authors: Yadav Deepak , Ngolab Jennifer , Lim Rod S , Krishnamurthy Siddharth , Bui Jack D
Published: January 2023

NKG2D is a receptor used by NK cells to detect virally infected and transformed cells. It recognizes ligands that are expressed constitutively on primary tumors and tumor cell lines. We have identified four microRNAs (miRNAs) that each was sufficient to reduce the expression of the NKG2D ligand MHC class I-related chain A (MICA). These include miR-106b, -302b, -372, and -520b. Interestingly, mir-372 was also found to reduce the expression of another NKG2D ligand, UL-16 binding protein 2 (ULBP2). One of these miRNAs (miR-520b) was induced by IFN-gamma, leading to a reduction in MICA surface protein levels. miR-520b acted on both the MICA 3'-untranslated region and the promoter region and caused a decrease in the levels of MICA transcript. In contrast, an antisense oligonucleotide inhibitor of miR-520b increased the expression of a reporter construct containing the MICA 3'-untranslated region but not the MICA promoter region. These findings demonstrate the novel regulation of NKG2D ligands by endogenous microRNAs.
B cells are required for T cell activation during tumor immunity (88.27)
Authors: DiLillo David J. , Tedder Thomas F.
Published: January 2023

T cells are considered the major contributors to tumor immunity. Because B cells can regulate T cell-mediated immune responses, the role of B cells in tumor immunity was assessed. B cells were depleted in mice with otherwise intact immune systems using CD20-specific mAbs before tumor administration. Subcutaneous B16 melanoma tumors grew to be twice as large in mice lacking B cells compared to control mice. Similarly, the number of visible melanoma metastasis foci on the surface of the lungs was doubled in B cell deficient mice. The numbers of CD4+ and CD8+ effector-memory T cells generated during the anti-tumor response were reduced by 48% and 33%, respectively, in the draining lymph nodes of B cell deficient mice compared to control mice. The induction of IFNγ-secreting CD4+ and CD8+ T cells was impaired by 42% and 34%, respectively, in mice that lacked B cells. Likewise, the induction of TNFα-secreting CD4+ and CD8+ T cells was impaired by 47% and 50%, respectively, in B cell deficient mice. Finally, the proliferation of adoptively-transferred tumor antigen-specific T cells was reduced by 30% in mice lacking B cells. Thereby, tumor growth and metastasis were dramatically enhanced in B cell deficient mice due to a significant reduction of the tumor-specific T cell response. Thus, B cells are required for optimal T cell activation during tumor immunity.

Supported by NIH Funding (CA105001 and AI056363)
Anti-MUC1 antibody responses in endometriosis and ovarian cancer in humans and preclinical MUC1 transgenic models (88.29)
Authors: Budiu Raluca Andreia , Edwards Robert P , Vlad Anda M
Published: January 2023

Ovarian cancer is the deadliest gynecologic malignancy and endometriosis is the only currently described precursor lesion. MUC1 mucin is an ovarian epithelial tumor-associated antigen and a vaccine candidate for ovarian cancer immunotherapy. Natural and vaccine-induced MUC1 immunity, often studied in ovarian cancer is much less understood in endometriosis. Human and murine mucin 1 share little homology in the extracellular region and transgenic mouse models expressing the human antigen are needed. We generated two novel preclinical models for MUC1 positive endometriosis and MUC1-expressing ovarian tumors, respectively. The MUC1KrasG12D mice develop MUC1-positive ovarian endometriosis after AdCre injection under the ovarian bursa. MUC1KrasG12D transgenic mice injected with MUC1-expressing immortalized ovarian surface epithelial cells (OSE) develop tumors throughout the peritoneal cavity and serve as a transplantable in vivo tumor model. Our results show that MUC1 is immunogenic in humans and mice with either malignant or premalignant lesions and triggers MUC1-specific IgM and Th2-driven IgG antibodies. Furthermore, the presence of IgG in cancer patients correlates inversely with survival. In additions, mice and humans show several differences in immune regulation by other CD4 T cell subsets (Th17, Treg) with consequences for MUC1 vaccination in preclinical models. PA Dept of Health, CMRF.
ADAM33 regulates IL-18-secreted process in gastric cancer cells (88.4)
Authors: Kim Kyung-Eun , Cho Dae-Ho
Published: January 2023

Interleukin (IL)-18 has recently been reported to play a critical role in tumor migration, invasion, and metastasis. Because IL-18 has various biological activities after its secretion as an 18 kDa mature form, the regulation of IL-18-secretion process is an important step in tumor progression. This study investigated the implication of IL-18 in vascular endothelial growth factor (VEGF)-D-regulated migration, along with the role of IL-18-secretion process. VEGF-D enhanced cell migration, which was then blocked by inhibiting IL-18. VEGF-D increased IL-18 expression and secretion, suggesting that IL-18 is a critical mediator for VEGF-D-enhanced migration. VEGF-D induced a disintegrin and metalloprotease 33 (ADAM33) expression, which has a metalloproteinase domain. VEGF-D-enhanced IL-18 secretion and cell migration were inhibited by ADAM33 knock-down. Moreover, cell proliferation was considerably reduced in ADAM33 siRNA transfectants. In conclusion, ADAM33 has a key role in gastric cancer pathogenesis by up-regulating IL-18 secretion process, resulting in increased cell migration and proliferation.
Targeted knockdown of the B lineage transcriptional activator E2A in B cell chronic lymphocytic leukemia (CLL) enhances lenalidomide sensitivity and regulates cellular proliferation (88.7)
Authors: Borghesi Lisa , Kardava Lela , Yang Qi , Foon Kenneth , Lentzsch Suzanne , Vallejo Abbe , Milcarek Christine
Published: January 2023

Chronic lymphocytic leukemia (CLL) is a common malignancy characterized by the accumulation of B lymphocytes with an antigen-experienced, activated CD19+CD5+ clonal phenotype. Clinically, approximately 50% of cases will behave more aggressively. Here we used siRNA knockdown to investigate the contribution of E2A, a key transcriptional regulator of B cell activation, to CLL survival and proliferation. E2A silencing in primary CLL cells leads to a modest increase in spontaneous apoptosis in both CD38+ (aggressive) and CD38- (indolent) cases. E2A is known to restrain lymphocyte proliferation, and E2A disruption promotes cell cycle progression of aggressive but not indolent CLL. Lenalidomide, an immunomodulatory (IMiD) anticancer drug with pro-apoptotic activity in some B lineage cancers, shows synergistic effects with E2A knockdown. While lenalidomide alone fails to induce detectable apoptosis in CLL, the combined treatment of lenalidomide plus E2A silencing dramatically increases apoptosis beyond either treatment alone. Thus, disruption of a transcription factor associated with the B cell activation profile enhances the anti-tumor potential of lenalidomide, providing the opportunity for improved drug efficacy.