bluuebunny/crossref_metadata_2025 huggingface dataset:
Pim kinases significantly impact humoral immune responses (IRM12P.648)
Authors: Willems Kristen , Schmidt Madelyn , Woodland Robert
Published: January 2023

The Provirus Integration site for Moloney murine leukemia virus (Pim) family of oncogenic serine/threonine kinases regulates a variety of metabolic functions in hematopoietic cells and lymphocytes. We examined the importance of Pim kinases in B cell activation following antigen stimulation. Humoral immune responses were significantly impaired in Pim 1 and 2 deficient mice (Pim 1-/-2-/-) T cell dependent responses were compromised while T cell independent type 1 and 2 antigen responses were virtually absent. Humoral deficiencies were owing to reduced production of antibody secreting cells due to the diminished induction of BLIMP-1 expression. The central role of Pim in these responses is consistent with our observation that Pims are induced downstream of multiple B cell activation receptors including CD40, BCR, and TLR4. We suggest that Pim 1 and 2 dependent signaling pathways are involved in B cell activation and their loss requires increased signaling through other pathways to reach an activation threshold. This hypothesis is further supported by the initial higher affinity of BCRs on Pim deficient B cells activated in a TD response and their inability to proliferate or undergo isotype switching in culture at the same rate as WT B cells when activator concentrations are decreased. Taken together, these data demonstrate a critical role for Pim kinases in affinity maturation, isotype switching, proliferation and the production of antibody secreting cells by B lymphocytes. .
IL-2 production by activated naïve and effector CD8 T cells forecasts memory formation. (LYM5P.700)
Authors: Kahan Shannon , Bakshi Rakesh , Luther Rita , Harrington Laurie , Weaver Casey , Zajac Allan
Published: January 2023

The clearance and control of many intracellular pathogens and tumors rely on the development of efficacious CD8 T cell responses. These responses are functionally diverse, however, whether and how this heterogeneity forecasts effector formation, memory potential, and protective capacity has not yet been definitively determined. To address this, we utilized IL-2 and IFN-γ double cytokine reporter mice to separate viable IL-2 and IFN-γ producing CD8 T cell subsets, and performed a series of adoptive transfer experiments to track their memory potential and fate. We discovered that within hours of activation, naïve CD8 T cells undergo a burst of cytokine synthesis and distinct populations develop that produce only IL-2, only IFN-γ, co-produce IL-2 and IFN-γ, or do not produce either cytokine. This diversity is consequential as newly activated naïve CD8 T cells that fail to produce IL-2 preferentially attain effector features. Conversely, autocrine IL-2 production by recently activated naïve CD8 T cells is associated with the formation of memory traits. Similarly, the ability of CD8 T cells to produce IL-2 during the effector phase also signified memory potential and the ability to mount vigorous secondary proliferative responses. Together these data demonstrate that functional heterogeneity influences the differentiation of CD8 T cell responses and that effector and memory formation can be predicted by the pattern of autocrine IL-2 production.
Lymphopenia-induced CD4 T cell proliferation leads to bystander activation of CD8 T cells with increased gut-homing specificity (LYM6P.722)
Authors: Kim Juhee , Surh Charles
Published: January 2023

In the presence of high levels of certain cytokines, such as IL-2 and/or IL-15, and self-peptide-TCR interaction, CD8 T cells undergo strong proliferation, referred to as “bystander proliferation”. Up to now, bystander activated CD8 T cells have not been investigated on a role in normal condition. In order to define a role of bystander activated CD8 T cells, we co-transferred OT-I T cells with naïve polyclonal CD4 T cells into conventional immunodeficient Rag-/- mice to make CD4 T cells be activated in response to commensal microbiota and secrete large amount of IL-2. The bystander activated OT-I T cells which were driven by IL-2 from activated CD4 T cells underwent massive proliferation, acquired effector functions, up-regulated a4b7 and migrated into mostly gut. Moreover, we induced OT-I T cells to undergo bystander proliferation in normal conventional B6 hosts by co-injection of smarta CD4 T cells, a strong inducer of IL-2, after LCMV injection and by depletion of host T cells to increase consumption of IL-2 for donor OT-I T cells. Interestingly, bystander activated OT-I T cells driven by IL-2 from activated smarta CD4 T cells in normal conventional B6 hosts also up-regulated a4b7 and migrated into gut. Based on the finding, we will further investigate on the role of bystander activated CD8 T cells to protect against mucosal infection, such as Listeria monocytogenes-OVA (LM-OVA) and the factor for up-regulating a4b7 on bystander activated CD8 T cells.
Long-lived IgM plasma cells confer host protection against viral challenge (LYM6P.717)
Authors: Bohannon Caitlin , Powers Ryan , Satyabhama Lakshmipriyadarshini , Cui Ang , Tipton Christopher , Michaeli Miri , Mehr Ramit , Mittler Robert , Kleinstein Steven , Sanz Ignacio , Jacob Joshy
Published: January 2023

Long-lived plasma cells are critical to immune memory, as they continuously secrete antigen-specific antibodies for up to a lifetime. Antigen-stimulated B cells, with T cell help, undergo affinity maturation within germinal centers and home to the bone marrow microenvironment where they persist as long-lived IgG plasma cells. Here we show that antigen-specific IgM plasma cells can also persist in the spleen for a lifetime. These IgM plasma cells can develop independent of the germinal center. Interestingly, despite being germinal center-independent, these cells exhibited somatic mutations in their expressed IgV heavy chain loci. Finally, we present data that antibodies from these IgM plasma cells can confer protective immunity to the host.
Low surrogate light chain in pro-B cells from old mice suppresses apoptosis and alters the idiotypes and proportions of phosphorylcholine reactive B cells (LYM6P.718)
Authors: Riley Richard , McAvoy Kelly , Ratliff Michelle , Alter Sarah , Frasca Daniela , Khan Wasif , Blomberg Bonnie
Published: January 2023

The generation of new B cells is substantially decreased in old mice (>2 years). Early B cell precursors (pro-B cells) are reduced in old mice and exhibit a reduction in the surrogate light chain (SLC) protein λ5. We hypothesize that this alters the expression and function of the pre-B cell receptor and influences the “read-out” of antibody specificities in the B cell repertoire. In old mice, the remaining pro-B cells, low in SLC, are resistant to apoptotic stimuli (TNFα), in contrast to young adult pro-B cells. Apoptotic sensitivity reflects levels of the SLC, since young pro-B cells partially or completely deficient in λ5 are also resistant to apoptosis. Age-associated B cells (ABC) represent a B cell subset which expands in old mice; ABC are pro-inflammatory and secrete TNFα. Adoptive transfer of ABC into young RAG-2 KO recipients results in partial loss of pro-B cells and, in particular, loss of pro-B cells with higher levels of SLC λ5. Anti-phosphorylcholine (PC) reactive B cells, especially those of the T15 idiotype, are important in defense against pneumococci. In old age, the proportion of PC reactive B cells, particularly with less protective T15 negative idiotypes, increases. Low SLC promotes both increased PC reactivity and T15 negative idiotypes. These studies suggest that ABC promote an “SLClow” B cell pathway that alters antibody specificities in old age.
TNFα-induced epigenetic modifications support a DC1 program in dendritic cells during protective immunity to cryptococcal infection (MPF4P.732)
Authors: Eastman Alison , Potchen Nicole , Carolan Jacob , Malachowski Antoni , Kryczek Ilona , Kunkel Steven , Osterholzer John , Olszewski Michal
Published: January 2023

TNFα is required for protective Th1 immunity to Cryptococcus neoformans (Cneo) and these effects are linked to the stable, early classical activation of dendritic cells (DC1), preventing alternative (DC2) activation. We hypothesized that TNFα stabilizes DC1 DC by inducing epigenetic modification of key DC1 genes. We modeled TNFα-stabilized DC (TSDC) in vitro by treating DC with DC1-skewing IFNγ +/- TNFα followed by a switch to DC2-skewing IL-4, and we assessed transcriptional and epigenetic signatures. Epigenetic studies focused on histone 3 methylation at lysines 4, 9 and 27 (H3K4, 9 or 27, respectively). H3K4 methylation is associated with activation of proximal genes, but H3K9 and 27 methylations are associated with repression. TSDC showed resistance to induction of DC2 genes and increased transcript levels of the key histone methyltransferases G9a (H3K9), EZH2 (H3K27) and MLL1 (H3K4). Further, we performed ChIP on TSDC and found that IL12b and IFNγ promoters are associated with the activating H3K4 methylation while non-stabilized DC1 and DC2 were not. We next assessed EZH2, G9a, and MLL1 transcript levels and the global methylation state of H3K4 in Cneo-infected lungs and found that both mRNA transcript levels and H3K4 methylation increased significantly in control but not TNFα-depleted mice. We conclude that histone modifications in DC are significantly altered by TNFα, and that this correlates with increased DC1 stability during protective responses to Cneo infection.
Large-scale sequencing of antibody responses to Borrelia burgdorferi infection (MPF5P.743)
Authors: Blum Lisa , Lahey Lauren , Kongpachith Sarah , Ju Chia-Hsin , Aucott John , Soloski Mark , Robinson William
Published: January 2023

Patients with Lyme disease present with protean manifestations and kinetics of disease, and these differences cannot be solely attributed to pathogen load or strain. Better understanding of antibody-mediated immunity in the human host is crucial for developing improved diagnostics and could provide insights into immune targeting of B. burgdorferi antigens associated with successful clearance of infection. We have developed an “antibody repertoire capture” (ARC) strategy that utilizes DNA barcodes to enable large-scale sequence analysis of the paired heavy-chain (HC) and light-chain (LC) immunoglobulin (Ig) genes expressed by individual Ig-expressing cells. This method allows us to generate evolutionary trees of the paired (HC+LC) repertoires and thus identify and recombinantly express the affinity-matured antibodies that are likely to drive immune protection and pathogenicity. We have applied ARC to peripheral blood plasmablasts from patients with early (weeks-long) and late (months-long) untreated B. burgdorferi infections. The size and number of clonally expanded Ig families was significantly greater in infections that went untreated for many months. Recombinant mAbs derived from these sequences bound to both host- and pathogen-associated antigens. These human plasmablast-derived mAbs provide us with a powerful tool to interrogate the functions of host antibodies in vitro and in animal models of infection, and may prove to have diagnostic or therapeutic potential.
mTOR mediates IL-23 induction of neutrophil IL-17 and IL-22 production (MUC5P.745)
Authors: Chen Feidi
Published: January 2023

It has been shown recently that neutrophils are able to produce IL-22 and IL-17, which differentially regulate the pathogenesis of inflammatory bowel disease. However, it is still largely unknown how the neutrophil production of IL-22 and IL-17 is regulated, and what the receptors are required. We demonstrated that, in a microbiota antigen specific T-cell mediated colitis model, neutrophils accumulated in the intestines prior to intestinal inflammation. Depletion of neutrophils resulted in a significant increase of colitis development, indicating a protective role of neutrophils in colitis. We found that neutrophils expressed both rorc and ahr. IL-23 promoted neutrophil expression of rorc and ahr, and stimulated production of IL-17 and IL-22. RORgt and AHR differentially regulated IL-23 induction of neutrophil IL-17 and IL-22, in that blockade of RORgt inhibited both IL-17 and IL-22 while AHR inhibitor only inhibited IL-22 but not IL-17 production. IL-23 induced mTOR activation in neutrophils. Blockade of mTOR pathway decreased expression of rorc and ahr, and inhibited IL-23-induced IL-17 and IL-22 production. Collectively, our data indicates that neutrophils negatively regulate microbiota antigen specific T cell induction of colitis, and IL-23 induces neutrophil production of IL-22 and IL-17 through induction of rorc and ahr, which is mediated by mTOR.
The NLRP1 inflammasome attenuates colitis and colitis-associated tumorigenesis (MUC5P.750)
Authors: Allen Irving , Williams Tere , Leeth Rachel , Rothschild Daniel , Coutermarsh-Ott Sheryl , McDaniel Dylan , Simmons Alysha , Heid Bettina , Cecere Thomas
Published: January 2023

NLR proteins are a diverse family of pattern recognition receptors that modulate immune system homeostasis in the gastrointestinal system. Recent studies have identified a sub-group of inflammasome forming NLRs that modulate the mucosal immune response during inflammatory bowel disease (IBD) and colitis associated tumorigenesis. To better elucidate the contribution of NLR family members in IBD and cancer, we conducted a retrospective analysis of gene expression metadata from human patients. These data revealed that NLRP1, an inflammasome forming NLR, was significantly dysregulated in IBD and colon cancer. To better characterize the function of NLRP1 in disease pathogenesis, we utilized Nlrp1b-/- mice in experimental colitis and colitis associated cancer models. Nlrp1b-/- mice demonstrated significant increases in inflammation and tumorigenesis compared to wild type mice. The increased inflammation and tumor burden was correlated with attenuated levels of IL-1β and IL-18. Further mechanistic studies revealed the increased disease pathogenesis in the Nlrp1b-/- mice was associated with non-hematopoietic derived cells and suggests that NLRP1 functions in the colon epithelial cells to attenuate tumorigenesis. Together, these data identify NLRP1 as an essential mediator of the host immune response during IBD and cancer. These findings are consistent with a model whereby multiple NLR inflammasomes attenuate disease pathobiology through modulating IL-1β and IL-18 levels in the colon.
Treatment with a selective histone deacetylase 6 inhibitor decreases lupus nephritis in NZB/W mice (THER5P.910)
Authors: Vieson Miranda , Barrett Sarah , Gojmerac Alex , Reilly Christopher
Published: January 2023

To date, there are 17 histone deacetylase (HDAC) enzymes, divided into 4 classes, which alter protein function by removing acetyl groups from lysine residues. Prior studies report that non-selective HDAC inhibitors decrease disease in various lupus mouse models. Selective HDAC-6 inhibition acts on cytosolic proteins to decrease B cell proliferation and differentiation by inhibiting α-tubulin function. Since B cells play a critical role in the initiation and propagation of systemic lupus erythematosus, we hypothesized that a selective HDAC-6 inhibitor (HDAC6i) will alleviate disease in a mouse model. Intraperitoneal injections of HDAC6i (0.3 mg/kg, 1 mg/kg, or 3 mg/kg), vehicle control, or dexamethasone were administered to 21-week-old, female NZB/W mice, 5 days a week, for 13 weeks. Disease was evaluated by body weight, proteinuria, serum levels of anti-dsDNA antibody, cytokines and immunoglobulins, and post mortem evaluation of nephritis and B cell populations in the bone marrow and spleen. Treatment with HDAC6i decreased glomerular scores, spleen weights, and urine protein scores when compared to vehicle-treated mice. No differences in B cell development and differentiation in the bone marrow, and B cell activation in the spleen were noted. We conclude that HDAC-6 inhibitors are effective at decreasing lupus nephritis and disease in NZB/W mice, however further studies are warranted to investigate the underlying mechanism, particularly in regards to B cell pathophysiology.
Gut bacteria modulation of antigen presenting cells in a mouse model of autoimmune diabetes. (THER5P.915)
Authors: Sidi Mohamed Zaynab , Larkin Joseph
Published: January 2023

T1D is an autoimmune disease whereby the beta cells of the pancreas are destroyed, resulting in the inability to self-regulate blood glucose. While genetics play a role, the contribution of environmental factors such as gut bacteria have also been implicated. Notably, it was shown that the oral administration of L. johnsonii N6.2 (LjN6.2) conveyed diabetes resistance to diabetes prone rodents while a second strain, Lactobacillus reuteri TD1, had no effect. However, the exact mechanisms of microbiota action on diabetes prevention remain poorly understood. As it has been shown that defective antigen presentation by dendritic cells (DC) contributes to T1D progression, we tested the hypothesis that gut flora can serve to modulate DC maturation. We observed that the administration of LjN6.2 to NOD leukocytes in vitro mediated increases in CD11c+MHCII+ DC. Moreover, LjN6.2 facilitated enhanced myeloid dendritic cell maturation denoted by the increase in number of CD8alo DEC205lo that were comparable to levels present in NOR mice. In addition, LjN6.2 also increased IL6 production. This study highlights the role of gut bacteria in autoimmunity as a critical component for T1D therapeutic strategies and might be used to extrapolate treatment approaches in humans in the future.
Sex-dependent response to treatment for arthritis in humanized mice (THER5P.903)
Authors: Taneja Veena
Published: January 2023

Rheumatoid arthritis (RA) occurs twice more often in women than men. B cell depletion has been shown to be efficacious in treating RA though it is unknown if efficacy is dependent on the sex of the patient. Our previous studies suggested that antigen presentation via B cells results in a sex-specific immune response and cytokines in humanized mice. Now we show that the efficacy of B cell depletion in arthritic DQ8 mice is sex-dependent. Arthritic female DQ8 mice treated with anti-CD20 antibody in therapeutic protocols show milder disease severity associated with decreased antibodies to citrullinated proteins along with increased numbers of T regulatory as compared to male DQ8 mice. The milder disease severity in CD20-depleted females was associated with reduced levels of IL-23 and CCL5 with increased IL-13 as compared to males. Treated male mice showed higher numbers of subsets of dendritic cells, known producers of pro-inflammatory cytokines, as compared to females. A window where males can benefit from B cell depletion needs to be defined. Our observations suggest that in therapeutic strategies that target B cells during established disease may benefit females while DCs might be relatively more important for men than women.
TREM1 intervention does not affect renal pathology upon ischemia/reperfusion injury (TRAN1P.929)
Authors: Tammaro Alessandra , Emal Diba , stroo Ingrid , Teske Gwendolin , Butter Loes , Claessen Nike , florquin sandrine , Leemans Jacklien , Dessing Mark
Published: January 2023

Rationale:Following transplantation, renal ischemia is a major cause of Acute Kidney injury(AKI). TREM1(Triggering receptor expressed on myeloid cells 1) is an innate immune receptor expressed on neutrophils and monocytes/macrophages, the most common cells infiltrating the kidney upon IR. TREM1 enhances Toll-like receptor (TLR)-induced inflammatory response in several models of infection and sterile inflammation.TREM1 inhibition reduced disease severity in mesenteric ischemia-reperfusion induced injury (IRI).We hypothesized that TREM1 intervention will also reduce renal IRI. Methods:Mice were subjected to renal IRI induced by occlusion of the renal artery for 25’ and reperfused for 24h. Mice were treated i.p. with different doses of LP17, LR12, TREM1 fusion protein and control peptides.Renal TREM1 protein expression and plasma sTREM1 were measured. TREM1 expression on circulating granulocyte and monocyte, renal function (creatinine, urea), damage (PASD) and inflammation (KC and LY6G-positive cell infiltration) were evaluated. Results:IRI was associated with increased renal and circulating TREM1 protein levels. TREM1 levels were similar on circulating granulocyte but decrease on monocyte upon IR. TREM1 intervention with LP17, LR12 and TREM1 fusion protein did not reduce renal dysfunction, injury or inflammation following IRI. Conclusion: The modulation of the TREM-1 pathway by means of synthetic peptides and fusion protein does not represent a useful tool to prevent renal IRI.
The microbiota accelerates skin graft rejection through enhanced T cell priming (TRAN1P.953)
Authors: Lei Yuk , Chen Luqiu , Wang Ying , Nair Lekha , Molinero Luciana , Theriault Betty , Bartman Caroline , Chong Anita , Alegre Maria-Luisa
Published: January 2023

The microbiota is known to influence local and systemic immune responses. To test whether the microbiota can modulate the strength of an alloresponse, we used a minor mismatched (H-Y) skin transplant mouse model in both germ-free (GF) and antibiotic-pre-treated (Abx) mice. Abx mice showed a marked reduction in bacterial diversity of their intestinal and skin flora. Skin allograft survival more than doubled in either GF or Abx mice compared to controls and colonization with a complex microbial community from conventional mice was sufficient to restore faster rejection kinetics in GF mice. Mechanistically, CFSE-labeled CD4+ anti-H-Y Marilyn T cells displayed a decrease in proliferation following in vivo adoptive transfer into Abx hosts as well as after in vitro culture with antigen presenting cells from Abx mice. Together, these data indicate decreased priming of allogeneic T cells when the diversity of resident bacterial communities is diminished. This effect is not due to a reduction in bacterial load but rather a loss of taxon-specific host-microbe interactions. This study illustrates the profound systemic effects of the microbial environment in transplant recipients and points to the microbiota as a potential therapeutic target for enhancing graft acceptance.
The expansion and activity of myeloid-derived suppressor cells in nasopharyngeal carcinoma mediated by up-regulating COX-2 (TUM6P.964)
Authors: Li Jiang
Published: January 2023

The expansion of myeloid-derived suppressor cells (MDSCs) is linked to tumor metastasis, but the molecular mechanism related to this link remains unclear. Here, we investigated the interactions between MDSCs and tumor cells in patients with nasopharyngeal carcinoma (NPC) and demonstrated that NPC cells induced the expansion of tumor-induced MDSCs (T-MDSCs); in turn T-MDSCs enhanced NPC cell migration and metastasis. Up-regulation of tumor cyclooxygenase-2 (COX-2) expression positively correlated with the expansion of MDSCs in NPC patients and both of COX-2 and MDSCs were poor predictors for disease-free survival. In vitro, knockdown of COX-2 expression in TW03 cells hampered T-MDSC induction by blocking IL-6. In addition, T-MDSCs promoted NPC cell proliferation, invasion and migration by triggering the epithelial-mesenchymal transition (EMT) on cell-to-cell contact; furthermore, T-MDSCs enhanced tumor lung metastasis in nude mice. Interestingly, T-MDSCs-mediated EMT was through enhancing COX-2 expression and activating the b-catenin/TCF4 pathway in NPC cells; Blockage of TGFb and iNOS significantly abolished the T-MDSC-mediated up-regulation of COX-2 and EMT in NPC cells, whereas the administration of TGFb and L-arginine supplements up-regulated COX2 expression and the b-catenin/TCF4 pathway in NPC cells. Taken together, these findings unveil a pivotal role for COX-2 in the development and cancerous contributions of MDSCs in NPC.
Isolation and characterization of human MDSC from peripheral blood of patients with various malignancies (TUM6P.971)
Authors: Wyczechowska Dorota , Hernandez Claudia , Zheng Liqin , Rodriguez Paulo , Ochoa Augusto
Published: January 2023

Myeloid-derived suppressor cells are a heterogeneous population of early myeloid progenitors that possess a strong immunosuppressive capacity and accumulate in multiple diseases characterized by chronic inflammation. In tumors, MDSC play a major role in the down-modulation of anti-tumor immune responses mediated by T cells and natural killer cells. Murine MDSC are defined as CD11b+/GR1+ and specific phenotypic markers have been identified for the distinction of monocytic MDSC and granulocytic MDSC. However, there is no consensus on how human subsets of MDSC should be phenotypically defined. Using peripheral blood mononuclear cells from patients with various malignancies, we determined by flow cytometry specific panels for the distinction of M-MDSC and G-MDSC. M-MDSC’s were identified as CD11b+ CD14+ CD33+high HLA-DRneg/low and CD66bneg, while G-MDSC’s are identified as CD11b+ CD14neg CD33+low HLA-DRneg CD66b+. Additional experiments established the expression of specific markers associated with energy metabolism in MDSC subsets. Therefore, our results characterize a panel for the distinction of MDSC subsets in patients with cancer and suggest potential metabolic differences between MDSC subpopulations in humans.
Mycobacterium bovis Bacillus Calmette-Guérin (BCG) reprograms M2 macrophages to promote antitumor T cell responses (TUM6P.1007)
Authors: Lardone Ricardo , Ramos Romela , Navarrete Marian , Torisu-Itakura Hitoe , Faries Mark , Sieling Peter , Lee Delphine
Published: January 2023

M2 Macrophages (MΦs), also known as tumor-associated MΦs, infiltrate solid tumors and impair antitumor immunity. M. bovis Bacille Calmette-Guérin (BCG) is an intralesional (IL) therapy for cutaneous metastatic melanoma (NCCN Guidelines), and induces regression of injected, but also non-injected lesions. Since MΦs play a pivotal role in tumors and mycobacterial infections, we hypothesized that IL-BCG affects tumor regression by altering M2 MΦs. Thus, we investigated the effects of BCG on in vitro polarized M2 MΦs. BCG induced dramatic transcriptional changes in M2 MΦs on genes involving inflammation, immune cell recruitment, cross-talk and activation. To determine whether BCG could alter the classical M2 phenotype, we compared BCG vs mock-treated M2-MΦ cell surface phenotype and response to LPS. BCG-treated M2-MΦ expressed lower CD163 (p<0.01) and secreted less IL-10 (p<0.05), with no effect on IL-12p40, vs mock-treated M2-MΦ. We also investigated the ability of BCG-treated M2-MΦ to activate T cells, and found an increased frequency of IFN-γ producing CD4+ T cells responding to BCG vs mock-treated M2-MΦ (p<0.05), suggesting an adjuvant effect of BCG. Moreover, supernatant from BCG-treated M2-MΦ increased the frequency of granzyme B-producing CD8+ T cells responding to autologous melanoma cell lines (p<0.01). Together, our data indicates IL-BCG therapy could contribute to a tumor-rejecting environment by inducing a reprogramming of M2 MΦs towards a proinflammatory phenotype.
PD-1 blockade reverts motility arrest of transferred tumor-infiltrating CD8+ T cells in adoptive cell transfer immunotherapy (TUM7P.1014)
Authors: Mehta Gautam , Sukumar Madhuhasan , McGavern Dorian , Restifo Nicholas
Published: January 2023

Adoptive cell transfer (ACT) immunotherapy can result in complete responses in the treatment of metastatic solid cancers, however the majority of patients eventually progress. Although many immune regulatory programs are context-dependent, the in vivo behavior of adoptively-transferred CD8+ T cells over time remains unknown. Using the Pmel-1 model, we performed intravital microscopy of subcutaneous tumors in mice to visualize the in vivo motility of transferred tumor-specific CD8+ T cells. This revealed that CD8+ T cell motility arrest increased over time after adoptive transfer. By ex vivo analyses, behavioral change coincided with a reduction in IFN-γ production and glucose uptake. As the transferred cells strongly expressed PD-1, we treated mice with both ACT and anti-PD-1 blocking antibody to determine the effect of immune checkpoint blockade on cellular motility. In mice treated with anti-PD-1 blocking antibody, transferred intratumoral CD8+ T cell motility arrest diminished, as compared with controls. This corresponded with an increase in CD8+ T cell glucose uptake. These data demonstrate that intratumoral CD8+ T cell behavior in ACT is dynamic and that greater motility is associated with increased functionality and bioenergetic demand. Furthermore, concurrent PD-1 blockade exerts a cell-intrinsic effect on transferred CD8+ T cells suggesting synergy between ACT and anti-PD-1 immunotherapy and the potential for their combinatorial use in the treatment of cancer
Melanoma induces, and adenosine suppresses, CXCR3-cognate chemokine production and T cell infiltration of lungs bearing metastatic-like disease (TUM7P.1017)
Authors: Mullins David , Perekslis Thomas , Croteau Walburga , Turk Mary Jo , Huang Yina , Clancy-Thompson Eleanor
Published: January 2023

Melanoma-specific vaccines have minimal efficacy in patients with established disease but enhance survival when administered in the adjuvant setting. Therefore, we hypothesized that organs bearing metastatic-like melanoma may differentially produce T cell chemotactic proteins over the course of tumor development. Using an established model of metastatic-like melanoma in lungs, we assessed the production of specific cytokines and chemokines over a time-course of tumor growth, and we correlated chemokine production with chemokine receptor-specific T cell infiltration. CXCR3-cognate chemokines (CXCL9 and CXCL10) were significantly increased in lungs bearing minimal metastatic lesions, but chemokine was at or below basal levels in lungs with substantial disease. Chemokine production correlated with infiltration of the organ compartment by transferred CD8+ tumor antigen-specific T cells in a CXCR3- and host IFN-γ-dependent manner. Adenosine signaling suppressed chemokine production and T cell infiltration in the advanced metastatic lesions, and suppression could be partially reversed by the adenosine receptor antagonist aminophylline. Our data demonstrate that CXCR3-cognate ligand expression is required for efficient T cell access of tumor-infiltrated lungs, and ligands are expressed in a temporally-restricted pattern that is governed, in part, by adenosine. Modulation of adenosine activity may impart therapeutic efficacy to immunogenic but clinically ineffective vaccines.
ImmTACs: bi-specific TCR-based reagents for targeted cancer immunotherapy (VAC7P.1037)
Authors: Jakobsen Bent
Published: January 2023

ImmTACs (immune mobilizing monoclonal T cell receptors against cancer) are a new class of soluble bi-specific reagent that exploit the natural antigen recognition pathway mediated by the T cell receptor (TCR). As TCRs recognise HLA-presented peptides, ImmTACs can access the larger pool of intracellular tumor antigens unavailable to antibody based therapies. ImmTACs target tumors via a soluble monocloncal TCR, engineered to recognise a given tumor associated peptide-HLA complex with exceptionally high sensitivity and specificity. Once bound to their target, ImmTACs redirect host polyclonal T cells via an anti-CD3 antibody fragment. In this way ImmTACs facilitate targeted T cell recognition of tumors leading to potent T cell activation and tumor cell killing, with the potential to break T cell tolerance. ImmTACs are specific for humans, thus efficacy, specificity and off target effects cannot be assessed in animal models. Using antigen positive tumor cells and HLA appropriate primary human cell lines representing a range of tissues, the in vitro pre-clinical package can be predictive of in vivo clinical observations. A first ImmTAC, IMCgp100, has entered phase I/IIa clinical trials for patients with advanced melanoma. The drug is well tolerated and many patients show tumor shrinkage after the first dose of ImmTAC with tumor shrinkage improving with each dose. Emerging data demonstrate durable partial responses in three patients and a complete response in a fourth patient.
Adoptive cellular therapy containing T cells and CD25+ NKT cells modulates myeloid cells and stimulates endogenous anti-tumor immune function (VAC7P.1038)
Authors: Payne Kyle , Graham Laura , Bear Harry , Manjili Masoud
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) are a type of myeloid regulatory cell (Mreg) that suppresses the anti-tumor activity of immune effector cells. We have previously demonstrated that CD25+ NKT cells function as cellular adjuvants for converting MDSC into immune stimulatory myeloid cells, thereby rescuing anti-tumor T cell function from MDSCs in vitro. Here, we sought to characterize the in vivo efficacy of adoptive cellular therapy (ACT) utilizing reprogrammed T cells and CD25+ NKT cells in modulating MDSCs in FVBN202 mice. We demonstrate a significant 2-fold decrease in the frequency and absolute number of splenic MDSCs in recipients of ACT compared with control mice bearing comparable lung metastases. We also observed a significant 2-fold increase in the frequency and absolute number of mature myeloid cells (CD11b-MHC-II+CD40+CD86+). Functional analysis was performed in which CD11b- myeloid cells from ACT-recipient animals bearing lung metastases were cultured with autologous splenocytes. CD11b- MHC II+ cells induced a significant release of splenocyte-derived IFN-γ; this response was enhanced greater than 20-fold upon the addition of tumor cells. Importantly, ACT recipients of mice bearing Ki-67hi circulating tumor cells experienced extended time to progression compared to control animals. These data suggest CD25+ NKT cells may act to promote ‘vaccination in situ’ of endogenous T cells, in addition to boosting anti-tumor efficacy of ACT.
Nasal immunization with M cell-targeting ligand-conjugated ApxIIA toxin fragment induces protective immunity against Actinobacillus pleuropneumoniae infection in a murine model (VAC8P.1056)
Authors: Jang Yong-Suk , Park Jisang , Kim Sae-Hae
Published: January 2023

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and severe economic loss in the swine industry has been caused by the infection. Therefore, the development of an effective vaccine against the bacteria is necessary. ApxII toxin, among virulence factors expressed by the bacteria, is considered to be a promising vaccine candidate because ApxII toxin not only accompanies cytotoxic and hemolytic activities, but is also expressed in all 15 serotypes of bacteria except serotypes 10 and 14. In this study, we identified the peptide ligand capable of targeting the ligand-conjugated ApxIIA#5 fragment antigen to nasopharynx-associated lymphoid tissue. It was found that nasal immunization with ligand-conjugated ApxIIA#5 induced efficient mucosal and systemic immune responses measured at the levels of antigen-specific antibodies, cytokine-secreting cells after antigen exposure, and antigen-specific lymphocyte proliferation. More importantly, the nasal immunization induced protective immunity against nasal challenge infection of the bacteria, which was confirmed by histopathological studies and bacterial clearance after challenge infection. Collectively, we confirmed that the ligand capable of targeting the ligand-conjugated antigen to nasopharynx-associated lymphoid tissue can be used as an effective nasal vaccine adjuvant to induce protective immunity against A. pleuropneumoniae infection.
Enhancing immunogenicity of recombinant protein vaccines in mice with vaccine self-assembling immune matrix, a non-viral delivery platform (VAC8P.1059)
Authors: Shollenberger Lisa , Grenfell Rafaella , Samli E. , Harn Donald
Published: January 2023

Vaccination remains the most effective public health tool to prevent infectious disease. Many vaccines are marginally effective in general, or are less efficacious when used in immune-compromised populations. To enhance vaccine immunogenicity, we exploited the biphasic property of the (RADA)4 synthetic oligopeptide to create a new vaccine delivery method, VacSIM (vaccine self-assembling immune matrix). Vaccine components are mixed with the VacSIM solution for injection, after which the (RADA)4 peptides self-assemble into hydrated nanofiber gel-matrices, forming a vaccine depot with the vaccine antigens in the aqueous phase, allowing for slow-release of vaccine components over time. Thus, we have a non-viral, inert, biodegradable delivery system, not requiring formulation that can deliver a multitude of vaccines. We hypothesize that slow-release of vaccine components provides antigen persistence, driving enhanced vaccine-specific responses that are improved in both quantity and quality of the response. We have tested VacSIM with live bacterial vectors, inactivated virus and multiple recombinant protein vaccines. Shown here, VacSIM improves vaccine responses to protein immunogens in 2 strains of mice and remains stable after long-term storage. In addition, VacSIM by itself does not activate antigen presenting cells. Enhancement of vaccine responses by VacSIM administration could represent a fundamental paradigm shift in how vaccines are delivered.
Therapeutic role of interleukin 22 in abdominal bacterial infection in mice (VAC8P.1049)
Authors: Zheng Mingquan , Kumar Pawan , Stefanovski Matthew , Pociask Derek , Good Misty
Published: January 2023

IL-22 is an IL-10-related cytokine and produced by Th17 and other immune cells that signals via IL-22Ra1 and IL-10R2. IL-22RA is widely expressed on non-immune tissues. IL-22 binding to IL-22 receptor complex leads to STAT3 activation. IL-22 has shown to have hepatoprotective effects through STAT3 in liver injury model. However it is unclear if IL-22 can directly regulate antimicrobial programs in the liver. Using intra-abdominal infection with Klebsiella pneumoniae (KP) where recombinant IL-22 (rIL-22) was administered 2 hours after infection, we found that rIL-22 reduced bacterial burdens in liver. This was associated with reduced liver necrosis and reduced serum ALT levels. rIL-22 required hepatic IL-22Ra1 as rIL-22 therapy was ineffective in liver specific IL-22Ra1 KO mice in terms of controlling bacterial burden or liver injury. rIL-22 also required hepatic STAT3 as rIL-22 therapy was also ineffective in liver specific STAT3 KO mice. Administration of anti-gp-130 conferred a mark increase in KP growth in the liver but this could be rescued by rIL-22, demonstrating that the effect of rIL-22 is independent of gp130. Serum from rIL-22 treated mice showed potent bacteriostatic activity against KP and this activity was dependent on lipocalin-2 (Lcn2). However, in vivo, the antimicrobial activity was independent of Lcn2 as rIL-22 was still therapeutic in Lcn2-/- mice. We are currently investigating the mechanisms underlying the lcn-2 independent activity of rIL-22.
Induction of anti-microbial immune responses in severely immunodeficient hosts by IFNγ-expressing Salmonella enterica serovar Typhimurium correlates with efficient activation of macrophage effectors (VAC8P.1050)
Authors: Al-Ramadi Basel , Al-Sbiei Ashraf , Mohamed Yassir , Bashir Ghada , Al-Ojali Samia , Fernandez-Cabezudo Maria
Published: January 2023

Susceptibility to infection by the intracellular bacterial pathogen, Salmonella enterica serovar Typhimurium (S. typhimurium), is controlled by many genes of innate and adaptive immunity. One of the most critical genes is IFN-γ and mice deficient in IFN-γ synthesis are highly susceptible to Salmonella. Previously, we also demonstrated that mice deficient in MyD88, an adaptor that regulates TLR signaling, are susceptible to Salmonella infection. In the current study, we compared immune responses in mice deficient in IFN-γ or MyD88 with wild-type controls following infection with an attenuated strain of S. typhimurium (designated BRD509) or a recombinant derivative engineered to express murine IFN-γ (GIDIFN). Infection studies with BRD509 or GIDIFN revealed that the latter strain was significantly less virulent in immunodeficient mice than BRD509 and correlated with decreased bacterial loads in systemic organs. Enhanced responsiveness was due to GIDIFN strain’s ability to activate effector macrophages, as shown by increased synthesis of inflammatory cytokines and anti-microbial effector molecules, including NO. Gene expression profiling by qPCR demonstrated stronger induction of key inflammatory modulators by GIDIFN in macrophages of immunodeficient animals. These findings suggest that immunotherapeutic approaches using attenuated bacterial strains expressing immunomodulatory genes is more efficacious and offers a superior safety profile even in severely immunodeficient hosts.
A novel recombinant adjuvant replacing the CD4+ T helper cell functions in inducing IgG isotype antibodies and protection by T cell dependent influenza split vaccines (VAC8P.1052)
Authors: Ko Eun-Ju , Lee Young-tae , Jung Yu-Jin , Lee Youri , Kim Min-Chul , Kang Sang
Published: January 2023

The action mechanisms of adjuvants have not been well understood yet. We hypothesized that an effective adjuvant would overcome a defect in CD4+ T cell help for the induction of immune responses and protection by T cell dependent vaccines. We found that current influenza split vaccine was not able to induce antibodies and protection in a CD4+ T cell deficient condition. In an attempt to develop an effective adjuvant, we developed a recombinant virus-like particle expressing flagellin (FliC-VLP) and investigated its adjuvant effects and mechanisms by comparing alum adjuvant. CD4 knockout (CD4KO) mice that were immunized with influenza split vaccine plus FliC-VLP (or MF59, AS04 human adjuvant) induced IgG isotype-switched antibodies, germinal center formation, and virus-specific memory cells, all of which were not induced in CD4KO mice with split vaccine alone. Levels of antibodies and protective efficacy in CD4KO mice with split vaccine plus FliC-VLP were significantly higher than those in C57BL/6 wild type mice without adjuvant and CD4KO mice adjuvanted by alum. The adjuvanticity of FliC-VLP was partially dependent on TLR5 when tested in TLR5KO mice. Taken together, the results provide evidence that FliC-VLP adjuvant can stimulate the induction of antigen-specific immune responses and improve protection against lethal infection in CD4KO mice immunized with T cell dependent split vaccines, indicating CD4+ T cell independent action mechanisms of adjuvant.
Effects of Ag/adjuvant ratio on the systemic and mucosal antibody responses of BALB/c mice immunized intradermally with CfaEB, a minor-major subunit fusion of the enterotoxigenic E. coli CFA/I fimbriae, and LT(R192G/L211A) (VAC9P.1104)
Authors: Maciel Milton , Vaidyanathan Deepika , Hulsey William , Savarino Stephen
Published: January 2023

The ratio of Ag/adjuvant in a vaccine can modulate the immune response as well as side effects. We studied the effects of different doses of CfaEB, a minor-major subunit fusion of the enterotoxigenic E. coli (ETEC) CFA/I fimbriae, given ID with the adjuvant LT(R192G/L211A), or dmLT, an attenuated form of the ETEC heat-labile enterotoxin. BALB/c mice were immunized with 0.1, 1, 10 or 100 mg of CfaEB alone or with 10 or 100 ng of dmLT on days 0 and 21. Sera from days 21 and 28 were tested by ELISA and hemagglutination inhibition assay. Mucosal responses were assessed in day 28 fecal pellets. Serum anti-CfaE IgG Ab titers directly correlated with the dose in groups receiving no adjuvant but the highest titers were seen in the two groups receiving 10 mg of CfaEB plus 10 or 100 ng dmLT. A similar pattern was observed with serum anti-CfaE IgA responses, although 100 mg of CfaEB plus 10 or 100 ng of adjuvant elicited the highest levels. Groups receiving 10 or 100 mg of CfaEB plus 100 ng dmLT manifested the highest functional Ab titers. The adjuvant at either dose was required to elicit a strong fecal anti-CfaE IgA response, and it directly correlated with CfaEB dose in the adjuvanted groups. Our results demonstrate the importance of dmLT in promoting mucosal Ab responses to our vaccine when given by the ID route. Since optimal mucosal vs. systemic functional Ab responses are elicited by different Ag/adjuvant ratios, the ratio should be selected to elicit the desired immune response.
Humoral and cellular immune responses to Mycobacterium tuberculosis antigens delivered through recombinant DNA plasmids and live mycobacteria (VAC9P.1105)
Authors: Mustafa Abu
Published: January 2023

The failures to provide consistent protection by the currently available vaccine against tuberculosis, i.e. Mycobacterium bovis BCG, have led to efforts to develop alternative vaccines. The three low molecular weight major antigenic proteins, i.e. PE35, CFP10 and ESAT6, encoded by Mycobacterium tuberculosis-specific region are among the candidates to develop new vaccines. In this study, two DNA vaccine vectors and three live mycobacterial vectors were evaluated for delivery of these antigens. DNA corresponding to the genes of above proteins were amplified from the genomic DNA of M. tuberculosis and cloned into two DNA vaccine vectors and a shuttle plasmid capable of transforming mycobacteria. The recombinant shuttle plasmids were electroporated into M. bovis BCG, M. vaccae and M. smegmatis. The recombinant DNA any mycobacterial constructs were tested for protein expression in vitro and for the induction of antigen-specific antibody and cellular immune responses in mice, guinea-pigs or rabbits. The results showed that the in vitro expression of PE35 and CFP10 was observed by one or more recombinant constructs. Furthermore, the induction of both humoral (antibody) and cellular immune responses (cell proliferation, protective cytokine secretion and induction of delayed-type hypersensitivity responses) were consistently observed only with the vaccine constructs expressing PE35. These results suggest the superiority of PE35 antigen as a new vaccine candidate against tuberculosis.
Vaccination does not protect aged mice from influenza-induced lung inflammation (VAC9P.1062)
Authors: Lorenzo Erica , Hopkins Jacob , Lefebvre Julie , Haynes Laura
Published: January 2023

Aging predisposes individuals to increased susceptibility to influenza infection and delays in viral clearance. Importantly, we show that age-related delays in viral clearance are correlated with lingering inflammation in the lungs of aged mice. This is critical since inflammation in the lungs is associated with an enhanced susceptibility to secondary bacterial infection, which is a significant sequela to flu infection and often causes death. Inflammation in young lungs following flu infection can be significantly reduced by prior flu immunity, such as that generated following vaccination with recombinant influenza nucleoprotein (rNP). This protection is characterized by reduced lung inflammation, reduced lung damage, reduced susceptibility to flu challenge and also correlates with increased levels of IL-10 and anti-NP antibodies. In contrast, vaccination of aged mice with rNP does not reduce lung inflammation and shows no protection from weight loss and no reduction in viral titers in the lungs following subsequent flu infection. Additionally, aged mice generate lower levels of NP-specific antibodies, which are absolutely critical for reducing lung inflammation. Consequently, unlike young mice, these NP vaccinated aged mice are not protected from death due to secondary bacterial infection. Thus, when considering a universal flu vaccine such as rNP, it is important to understand how efficacious it can be in highly susceptible populations such as the elderly.