bluuebunny/crossref_metadata_2025 huggingface dataset:
The transcription factor PLZF controls the development of mouse and human innate T cells (64.6)
Authors: Sant'Angelo Derek
Published: January 2023

Natural killer T cells (NKT cells) have an innate immunity-like rapidity of response and the capacity to modulate effector functions of other cells. We have shown that NKT cells express the BTB-ZF transcriptional regulator PLZF. In the absence of PLZF, NKT cells developed, but lacked many innate T cell features. We have also shown that PLZF expression is not restricted to NKT cells, but is also expressed by a subset of NKT cell-like γδ T cells. Interestingly, TCR signal strength and expression of Id3 appear to control the frequency of PLZF-expressing γδ T cells. In human T cells PLZF is expressed in NKT cells, γδ T cells and, unexpectedly, in a population of CD8+ T cells. Analysis of T cells from the only known human with a biallelic loss of functional PLZF demonstrates that the development of invariant NKT cells and other PLZF-expressing T cell subsets is dependent upon expression of PLZF. The PLZF-expressing CD8+ T cells are functionally distinct from both conventional T cells and from invariant NKT cells. Furthermore, this subset of T cells is expanded in the peripheral blood of patients with metastatic melanoma; however it is greatly diminished in that of patients with autoimmune disease. Therefore, the expression of PLZF defines a new human T cell population that is differentially regulated in response to different types of immune stress. Furthermore, these data highlight important differences between the human and mouse immune system.
In vitro production of fully human monoclonal antibodies targeting therapeutic self-antigens (65.10)
Authors: Hoel Brian , Sun Wei , Halstead Lucinda , White David , Fernandes Daniel , Rubinchik Semyon , Sutkowski Natalie
Published: January 2023

Despite their role in autoimmune disease, not all autoreactive antibodies are harmful. Autoantibodies targeting tumor antigens, growth factors and their receptors are currently used therapeutically in cancer and autoimmunity. Ordinarily, healthy subjects do not exhibit strong secondary antibody responses to self-proteins, because most autoreactive B cells do not receive sufficient T cell help required for the germinal center reaction and for differentiation into IgG secreting cells. Our data demonstrate that rare, potentially therapeutic autoreactive B cells can be efficiently rescued from tonsil with EBV immortalization, and manipulated in vitro into secreting IgG, by supplying factors that mimic antigen stimulation and T cell help. Because this process is performed wholly in vitro, it constitutes a rapid and cost effective technology, resulting in the isolation of fully human antibodies. In addition, antibodies can be generated against highly toxic antigens, since immunization is unnecessary. We have developed two panels of human monoclonal antibodies targeting distinct self-antigens for use in cancer immunotherapy, one targeting the tumor antigen nucleolin and the other angiogenic placenta growth factor. We hypothesize that infection of B cells with the ubiquitous herpesvirus EBV might contribute to the persistance of autoreactive memory B cells in vivo, altering the B cell repertoire and resulting in the secretion of destructive and possibly of protective autoantibodies.
Obtain consistent and accurate PBMC cell counting results with Cellometer automatic cell counter (65.14)
Authors: Lin Bo , Chan Leo , Lai Ning , Pirani Alnoor , Smith Tim , Lyettefi Emily , Qiu Jean
Published: January 2023

Traditional methods for fluorescence-based PBMC analysis commonly employ manual hemacytometer and standard flow cytometry for concentration and viability measurement. Manual cell counting requires trained technicians, which is time-consuming and prone to human error. In addition, PBMC samples are usually contaminated with RBCs and platelets, which may increase manual counting difficulty. Standard flow cytometry remains expensive, large in size, and require considerable amount of maintenance, which may not be ideal for research laboratories that need a cost-effective method. In addition, traditional flow cytometers do not have imaging capabilities, which often generate some uncertainties in the fluorescence results obtained. Recently, an imaging cytometry platform has been developed by Nexcelom Bioscience. This system allows automated cell image acquisition and processing with a novel counting algorithm for accurate and consistent measurement of cell population and viability with 20 μl of PBMCs sample. In this work, we demonstrate a rapid and cost-effective method for concentration and viability measurement of PBMCs using the Cellometer imaging cytometry method. This method has the ability to resolve the issues caused by manual hemacytometer and flow cytometer. By using Cellometer imaging cytometry, the assay time for generating concentration and viability result is greatly reduced, which is significant for research development in the biomedical and clinical studies.
A novel ELISA for eosinophil peroxidase provides a sensitive high throughput assay for eosinophil degranulation in either mouse or human biological samples (65.29)
Authors: Ochkur Sergei , Kim John , Protheroe Cheryl , Nair Parameswaran , Furuta Glenn , Lacy Paige , Moqbel Redwan , Lee James , Lee Nancy
Published: January 2023

Eosinophils comprise only 1-3% of circulating leukocytes but are often a diagnostic feature associated with a variety of inflammatory and disease states, including parasitic and fungal infections, allergic diseases (e.g., asthma, rhinitis and sensitivities to specific foods), cancer, and transplant rejection. The accurate evaluations of eosinophilia as well as the release of stored eosinophil granule proteins are important for the monitoring disease progression and/or assessing the effectiveness of a given treatment strategy(ies). Despite the availability of detection methods for each of the eosinophil granule proteins logistical difficulties limiting either the assays specificity and/or sensitivity have prevented their extensive use. We have generated unique eosinophil peroxidase specific monoclonal antibodies (EPX-mAb) and a high throughput sandwich ELISA assay as a means of overcoming these difficulties. Our EPX-mAb based ELISA is ~10 times more sensitive than traditional OPD based activity assays and allows detection of EPX in mouse BAL fluid after an acute OVA protocol as well as EPX release from mouse eosinophils stimulated ex vivo. The assay is highly specific and gives no signal in samples from EPX deficient mice even with massive eosinophilia (e.g., EPX deficient IL-5 transgenic mice). More significantly, this assay detects EPX in human tissue extracts and biological fluids and thus represents novel diagnostic assay previously unavailable in clinical settings.
Optimiser™ microfluidics ELISA plates overcome confounding rheumatoid factor-like and HAMA-like false positive effects in immunoassay detection of IL-17A in serum matrices. (65.35)
Authors: Sehy David , Kai Junhai , Santiago Nelson , Puntambekar Aniruddha , Lee Se Hwan , Durnell Randy , Schultheis Ron , Han Jungyoup , Ahn Chong
Published: January 2023

The measurement of analytes in serum matrices by sandwich ELISA can be confounded by naturally-occurring interfering factors which can cross link capture and detection antibodies, yielding distinct false positives. Such naturally-occurring interfering activities are often attributed to rheumatoid factor or HAMA (Human Anti-Mouse Antibody)-like effects in the serum sample. Rh factor, an autoantibody reactive with the Fc portion of IgG, is often identified in patients suspected of having arthritis, but, notably, this activity is observed in 5-10% of healthy persons. Recently, studies of mouse sera from a panel of outbred mice analyzed by mouse IL-17A sandwich ELISA demonstrated strong false positives when conventional ELISA plates were used. In side-by-side testing, such aberrant false positives were not observed upon running the same samples using the microfluidics-based Optimiser™ ELISA microplate. The absence of false positives in the Optimiser™ ELISA microplate render it an ideal candidate for use in screening serum samples for analytes of interest when naturally-present HAMA-like activity can confound ELISA results and interpretation.
Brilliant Violet 421™ and Brilliant Violet 570™ antibody conjugates deliver consistent and superior performance on violet-laser equipped flow cytometers with varied specifications (65.4)
Authors: Perna Avi , Koh Carolyn , Lundsten Kelly , Nosworthy Lara , Wormsley Susan , Jiang Nan , Lay Gene
Published: January 2023

Brilliant Violet™ derived fluorophores are novel and highly sensitive polymeric molecules that fluoresce upon excitation by a 405 nm laser line. In this study, Brilliant Violet 421™ and Brilliant Violet 570™ antibody conjugates were evaluated for their performance on violet laser equipped flow cytometers with varying instrument configurations. Instrument specific variables included sheath pressure, laser power, PMT gain and position, optical configuration, and filter sets. Cross beam spectral overlap was also evaluated for 488nm and 561nm lasers. All instrument configurations revealed significantly increased signal to noise ratio with Brilliant Violet™ as compared to traditional violet laser dyes. Brilliant Violet 421™ yielded logarithmic improvements in signal to noise ratio, while maintaining manageable spectral compensation properties. Brilliant Violet 421™ antibody conjugates against high and low-density surface markers also increased resolution and accuracy, notably for human CD25, CD56, CD127 and mouse NK1.1 and CD4. The Brilliant Violet™ conjugates were also found to be effective for detection of intracellular cytokines. Overall, Brilliant Violet™ 421 and Brilliant Violet™ 570 were determined to outperform traditional violet-excitable organic dyes on flow cytometers with a variety of specifications and settings.
Tools for enhancement of diagnostic immunoassay applications (65.42)
Authors: Jentz Tim , Nelson Wendy , Opperman Gary
Published: January 2023

Optimization of immunoassay applications is often hampered by issues such as non-specific binding, matrix interferences, destabilization of antibody/antigen interactions, and limited sensitivity. SurModics offers tools to address these problems and provides the framework to develop sensitive, reproducible, and robust immunoassays. Stabilization of antibody-protein interactions is fundamental for achieving a robust and reproducible assay. Novel stabilizer formulations have been developed to provide protein stability in both the dried state as well as in solution. The data presented here show improved stability compared to both common in-house formulations as well as other competitor solutions. Problems with non-specific binding and matrix interferences can cause high background, loss of sensitivity and false positives in many diagnostic applications. These issues were mitigated by use of protein-free blocking solutions, or novel passivating surface chemistry for microarray applications. Data here suggest the TRIDIA™ surface chemistry provides significantly reduced non-specific binding in protein arrays. Shown here are data from a new, protein-free diluent designed to reduce matrix interferences and to be substituted with ease into your existing assay protocol. Whether it is in solution or on a surface, SurModics provides the tools necessary to develop and optimize immunoassays that are sensitive, reproducible, and robust.
Analysis of whole blood immune cell function using imaging flow cytometry. (65.8)
Authors: George Thaddeus , Hall Brian , Friend Sherree , Mordecai Scott , DeMarco Richard , Kong Raymond
Published: January 2023

Analysis of immune function directly in human whole blood samples has several advantages over analysis in processed samples. First, leucocytes are activated in conditions closely matching their natural environment in the bloodstream, complete with all cofactors and cell types necessary for normal function. Second, purification processes are time consuming, technically challenging, and can themselves be a source of stress (temperature, physical, pH, etc) that modulates immune cell function. Furthermore, whole blood is the most obtainable source of clinically-relevant material from patients and healthy controls. Whole blood imaging assays are challenging due to the need for simultaneous multispectral immunophenotyping and in many cases due to the rarity of the cell type of interest. However, recent advancements in high speed flow imaging uniquely enable the study of leukocyte function directly in whole blood samples in three image-based applications that we present here. These include measurement of: 1) chemokine-induced receptor internalization in whole blood T cells; 2) chemokine-induced shape change in whole blood monocytes; and 3) dose and time kinetic activation of NFkB translocation in monocytes and plasmacytoid dendritic cells, the latter of which represent less than 0.1% of whole blood leukocytes. These results uniquely demonstrate methods for statistically robust image-based measurement of rare immune cell function directly in whole blood samples.
Enhancement of DC vaccine efficacy via regulation of interleukin-6 receptor signaling (66.2)
Authors: Choi Inhak , Hwang Wonchan , Jung Keunok , Jean You-Kyoung
Published: January 2023

Tumor microenvironment has emerged as one of the major obstacles against the clinical efficacy of dendritic cell (DC) vaccines. Tumor-derived IL-6 inhibits DC maturation, leading to suppression of T cell-mediated immune response. Based on the findings showing that infusion of blocking anti-IL-6 mAb regressed tumor growth, we reasoned that knockdown of IL-6 receptor signaling could improve the capacity of DC vaccine in tumor-specific T cell activation. We injected mice with a cervical cancer line TC-1 that are expressing human papilloma virus-16 E7 oncogene and IL-6 in vivo, and immunized them with E7 peptide-pulsed bone-marrow-derived DC (BMDC) transfected with IL-6Rα siRNA (siIL-6Rα DC) or treated with IL-6 receptor signal competitor (IL-6RC DC). TC-1-bearing mice immunized with siIL-6Rα DC or IL-6RC showed an obvious decrease in tumor growth and prolonged survival compared to the control mice. siIL-6Rα DC and IL-6RC DC vaccines significantly enhanced the frequency of tumor-specific CD8+ CTLs- producing effector molecules such as IFN-γ, TNF-α, FasL, perforin, and granzyme B, and generated more CD8+ memory T cells. Our results suggest that DC vaccine with regulation of IL-6R signaling may potentiate the therapeutic efficacy of conventional DC vaccines.
Establishment of a positive feedback between PGE2 and COX2 diverts DC differentiation into MDSCs and sustains their immunosuppressive functions in ovarian cancer (66.28)
Authors: Obermajer Natasa , Muthuswamy Ravikumar , Lesnock Jamie , Edwards Robert , Kalinski Pawel
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) are essential players in cancer-associated immune dysfunction. We report that the differentiation and functional stability of human monocytic MDSCs is driven by a single cancer-associated inflammatory mediator, prostaglandin E2 (PGE2), and depends on the establishment of a positive feedback between PGE2 and COX-2. PGE2 abrogates the differentiation of CD1a+ DC from monocytes, redirecting it towards CD14+CD33+CD34+ cells expressing all MDSC-associated suppressive factors, and inducing their CTL-suppressive functions. Moreover, PGE2 promotes the expression of endogenous COX2 in MDSCs, establishing a positive feedback loop that stabilizes the MDSC phenotype. The frequencies of CD11b+ CD33+ MDSCs in the ovarian cancer (OvCa) ascites correlate with the local production of PGE2, while the OvCa ascites environment can promote the induction of MDSCs from differentiating monocytes in a COX2-dependent manner. Importantly. disruption of COX2-PGE2 feedback in fully-developed MDSCs isolated from cancer patients, using COX2 inhibitors or inhibitors of EP2/EP4-dependent PGE2 signaling, eliminates the production of all other suppressive factors and terminates the CTL-suppressive function of MDSCs. The key role of the positive COX2-PGE2 feedback loop in the differentiation and functional stability of MDSCs facilitates the targeting of MDSCs in cancer immunotherapy.
Impaired antigen presentation and potent phagocytic activity identifying tumor-tolerant human monocytes: demonstration in isolated cells from Mature B-cell neoplasms. (66.47)
Authors: Fernandez-Ruiz Irene , Jurado Teresa , Siliceo Maria , Soares-Schanoski Alessandra , Gomez-Piña Vanesa , Lopez-Collazo Eduardo
Published: January 2023

Monocyte exposure to tumor cells induces a transient state in which these cells are refractory to further exposition to cancer. This phenomenon, termed “tumor tolerance”, is characterized by a decreased production of proinflammatory cytokines in response to tumors. We have established a human model of tumor tolerance and have observed a marked downregulation of MHCII molecules as well as the MHCII master regulator, CIITA. These cells combine an impaired capability for antigen presentation, with potent phagocytic activity and exhibit an M2-like phenotype. We also show that circulating monocytes, isolated from patients who suffer from mature B-cell neoplasm, share all the determinants that characterize cells locked in a tumor tolerance state. In addition, lymphocytes from these patients and supernatants from their in vitro culture, induced a tumor tolerance state in control monocytes. Finally, our results indicated that both, serum from mature B-cell neoplasm patients and supernatants from tumor cells exhibit high levels of Hyaluronic Acid in contrast to control serum from healthy voluteers. Hyaluronic Acid was able to induce a tumor tolerance phenotype in human naïve monocytes.
Role of acute B cell response in dengue disease severity and long-term immunity (67.19)
Authors: Montoya Magelda , Simona Zompi , Pohl Marie , Balmaseda Angel , Harris Eva
Published: January 2023

The four serotypes of dengue virus (DENV) cause dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), the most prevalent mosquito-borne viral disease in humans. Little is known about the acute B cell response during DENV infection and its impact on the breadth and quality of the long-term humoral response. We have measured by flow cytometry the percent of naïve, memory B cells and plasmablasts (PB)/plasma cells (PC) in fresh whole blood from acutely infected patients from a hospital-based study in Managua, Nicaragua. DENV-positive cases demonstrate a significant increase in PB/PCs as compared to DENV-negative cases. The percent of PB/PC in DENV-negative samples ranges from 0.004-5.44% (mean 0.6 +/- 0.19; n=40) compared to 0.04-7.68% (mean 1.5 +/- 0.21; n=83) in DENV-positive samples (p=0.003). No significant difference was found among naïve and memory B cell compartments. Serum avidity is greater in secondary as compared to primary DENV infections. The percent of PB/PC at day 5 post-onset of symptoms appears to positively correlate with serum avidity as measured by ELISA at convalescence (r2=0.83, p=0.015). These data will be correlated with the number of DENV-specific Ab-secreting cells (ASC) measured by ELISpot, as well as with disease severity and the DENV-specific neutralization capacities of the serum. This study will set the basis for future studies and could ultimately impact the design of safe and effective dengue vaccines.
Protein energy malnutrition decreases immunity and increases susceptibility to influenza infection. (67.5)
Authors: Taylor Andrew , Vora Keyur , Cao Weiping , Shieh Wun-Ju , Zaki Sherif , Katz Jacqueline , Sambhara Suryaprakash , Gangappa Shivaprakash
Published: January 2023

Protein energy malnutrition (PEM), a common cause of secondary immune deficiency in children, is associated with an increased risk of infection. We investigated the influence of PEM on susceptibility and immune responses to influenza virus infection using a mouse model. Groups of weanling mice maintained on isocaloric diets providing varying levels of protein energy [18%/adequate protein (AP), 5%/low protein (LP), and 2%/very low protein (VLP)] were infected with either 2009 A(H1N1) or laboratory-adapted H1N1 virus and assessed for disease severity and immune responsiveness. We found that compared to mice fed with AP and LP diets, mice maintained on the VLP diet exhibited an increase in virus-induced mortality and morbidity. Mice maintained on the VLP diet demonstrated diminished IFNγ levels and increased virus titer and inflammatory cell types in lung tissue, compared with mice on higher protein content diets. Moreover, groups of mice maintained on the VLP diet showed a lower hemagglutination-inhibition antibody response and reduced total numbers of splenic NP-specific CD8+ T cells compared with mice on higher protein content diets. Following re-feeding of the VLP group with the AP diet, post-infection morbidity, mortality and virus titer were all improved. Our results highlight the impact of protein energy on immunity to influenza infection and suggest balanced protein energy replenishment may be one strategy to boost immunity against influenza viral infections.
CCL21 dependent CD4+ T cell migration plays a role in protective immune responses against Toxoplasma infection in the brain. (99.22)
Authors: Noor Shahani , Nance J. , Lo David , Carson Monica , Wilson Emma
Published: January 2023

The chemokine receptor, CCR7 and interactions with its ligands CCL19 and CCL21 are well known to facilitate priming of T cells in lymphoid tissue. Our data demonstrate upregulation of CCR7, CCL19 and CCL21 in the brain during Toxoplasma gondii infection at a time point coincident with the peak of T cell infiltration in the CNS. CNS resident astrocytes significantly upregulate CCL21 following direct infection with Toxoplasma tachyzoites and in the absence of CCL21, plt/plt mice were less efficient in controlling parasite replication during chronic infection. Although T cells enter the infected brain in a CCR7 independent manner, migration of extravasated CD4+T cells from the perivascular area into the CNS parenchyma is CCL21-dependent. Our data also demonstrate accumulation of CD4+CCR7 expressing T cells in the infected brain. A significant proportion of these CCR7+T cells express memory T cell markers including IL-7R (Interleukin 7 Receptor) and CD103 (tissue resident memory phenotype). These data indicate CCR7-CCL21 interactions play a role in guiding CD4+CCR7+ memory T cell migration in the brain to prevent parasite reactivation and disease. Analysis of the behavior and phenotype of this memory T cell population in the absence of CCR7 or CCL21 will be presented. These data address the potential contribution of CCR7/CCL21 mediated interactions to maximize memory responses in the brain during Toxoplasma infection.
Cellular and soluble requirements for survival of pulmonary infection with virulent Francisella tularensis (99.29)
Authors: Bosio Catharine , Crane Deborah
Published: January 2023

Francisella tularensis is a facultative intracellular bacterium and the causative agent of tularemia. Inhalation of as few as 15 bacteria can cause lethal disease in humans. Development of novel vaccines and therapeutics for tularemia has been hampered by the lack of understanding of what immune components are required to survive infection. Defining requirements of immunity against virulent F. tularensis, such as strain SchuS4, has been difficult since animals typically die within 5 days after exposure to as few as 10 bacteria. To enable reliable identification of the components of the immune system that are important for defense against infection with F. tularensis we developed a convalescent model of tularemia in C57Bl/6 mice. We show that administration of a low dose of antibiotic over the course of 14 days resulted in clearance of SchuS4 from all organs within 21 days, regardless of the route of infection. However, only 60% of mice infected intranasally survived more than 30 days of infection, whereas 100% of mice infected intradermally survived past this time point. Using mice deficient of specific immune components in this model revealed that both T cells and B cells, are necessary to control SchuS4. Similarly, IL-12p40 and IFN-gamma are also critical for host defense against SchuS4 infection. Together this data provides a novel mechanism by which to identify host factors involved in both resolution and exacerbation of tularemia.
Th17 mediated recurrence of virus-induced immunopathology following discontinued FTY720 treatment (105.10)
Authors: Sehrawat Sharvan , Reddy Pradeep , Suryawanshi Amol , Khatri Madhu , Rouse Barry
Published: January 2023

In this report we show that the anti-inflammatory effect of FTY720 is lost on the discontinuation of treatment in Herpes Simplex Virus (HSV-I) induced ocular inflammation. The lesions developed after FTY720 treatment withdrawals were characterized by the predominance of Th17 cells over Th1 cells. Th17 but not Th1 cells expressed higher levels of surface CD103, an integrin that permits migration to inflammatory sites. Furthermore, we demonstrate that CD4+ T cells isolated from FTY720 treated but uninfected mice could be efficiently polarized towards Th17 and Treg but to a lesser extent towards Th1 phenotype. The hyper acute emergence of inflammation in FTY720 treated animals could be slowed down in IL-17RKO mice and upon administration of neutralizing antibody for IL-6, a proinflammatory cytokine involved in the generation of Th17 cells. These results suggested that approaches such as neutralization of proinflammatory cytokines might be considered along with FTY720 if interruption of the therapy is required to achieve the anti-inflammatory effects of the drug.
Pre-existing viral antigens change the dynamics of CTL response to secondary influenza virus infection (105.24)
Authors: Wu Tao , Lee Young-Tae , Bouchard Keith , Cauley Linda
Published: January 2023

Cytotoxic T lymphocytes (CTL) can provide immunity between different strains of influenza virus, but the immunity declines over the course of a few months and does not correlate with the number of surviving virus-specific memory CD8 T cells. We previously found that processed peptide antigens were presented to naïve CD8 T cells in the draining lymph nodes of the respiratory tract until ~2 months after primary influenza virus infection, which helped some activated virus-specific CTL colonize the lung airways between repeated infections. Similar results were obtained with multiple strains of influenza virus, which indicates that antigen-persistence is a common characteristic of this pathogen. Confocal imaging has been used to examine the distribution of virus-specific CTL in vivo as they response to reinfection in the presence and absence of the pre-existing viral peptides. Our data show that residual peptides alter the local inflammatory environment and augment the response to reinfection. The gradual disappearance of the viral peptides may account for the early loss of heterosubtypic immunity.
Generation of protective memory CD8 T cells specific for a retroviral cryptic epitope (105.25)
Authors: Stevens Cynthia , Rutkowski Melanie , Green William
Published: January 2023

Our lab has shown that cytotoxic CD8 T cells specific for immunodominant cryptic retroviral epitopes are capable of protecting against retrovirus-induced disease. LP-BM5 murine retrovirus causes a profound and progressive immunodeficiency in strains of mice that are unable to produce a protective CD8-T cell response. In resistant BALB/c mice, our lab has identified a Kd-restricted LP-BM5 immunodominant epitope, SYNTGRFPPL, within a previously unrecognized alternative open reading frame of the retroviral gag gene. Upon adoptive transfer, CD8 T cells which recognize SYNTGRFPPL mediate an acute, effector-phase response to LP-BM5, but are inefficient at generating long-term immunological memory to LP-BM5-induced disease. Rapamycin, IL-15 and high-dose IL-2 have been shown to increase the generation and survival of memory CD8 T cells. Using the CD8 T-cell adoptive transfer system employed in our lab to demonstrate acute protection to LP-BM5-induced disease, the aim was to engineer a protective, SYNTGRFPPL-specific memory response by including rapamycin, IL-15, or high-dose IL-2 at the in vitro CD8 T-cell expansion phase of adoptive cellular immunotherapy. The optimization of both acute and long-term memory responses via adoptive transfer of protective CD8 T cells specific for cryptic retroviral epitopes has implications for immunotherapeutic treatment of retrovirus-induced disease.
Virus-specific CD62L- memory CD8 T cells are preferentially retained in the draining lymph node following systemic viral infection (105.28)
Authors: McDermott Daniel , Olson Matthew , Varga Steven
Published: January 2023

Lymphocytic choriomeningitis virus (LCMV) induces a systemic infection in mice with virus replication occurring in both peripheral tissues and secondary lymphoid organs. Because of the rapid dissemination of the virus, the secondary lymphoid organs responsible for the induction of the LCMV-specific CD8 T cell response are poorly defined. We show that the mediastinal lymph nodes (MedLN) serve as the primary draining lymph node following intraperitoneal LCMV infection and that the majority of the LCMV-specific CD8 T cells are primed within the MedLN. Following the resolution of the acute infection (i.e. day 34), we observe that the majority of the memory (KLRG-1-CD127+) LCMV-specific CD8 T cells within the draining-MedLN are CD62L- in contrast to the non-draining LNs where the memory LCMV-specific CD8 T cells are predominantly CD62L+. Importantly, this reduced frequency of LCMV-specific CD62L+ memory CD8 T cells in the MedLN is independent of residual antigen. Furthermore, we demonstrate that the prolonged presence of CD62L- CD8 T cells is not a unique feature of the MedLN as a localized LCMV-footpad infection leads to a similar increase of virus-specific CD62L- memory CD8 T cells in the draining ipsilateral popliteal lymph node vs. the non-draining contralateral popliteal lymph node. These data indicate that CD62L- memory CD8 T cells are preferentially retained in the draining lymph nodes for an extended time following the resolution of a systemic viral infection.
Treating retroviral immunodeficiency-associated tumor by CD8 adoptive T-cell therapy (105.29)
Authors: Li Wen , Green William
Published: January 2023

LP-BM5 virus infection in susceptible B6 mice induces a HIV-like immunodeficiency syndrome, termed “murine acquired immunodeficiency syndrome (MAIDS)”. We have previously shown that highly active, LP-BM5-specific CD8 cytolytic T lymphocytes (CTLs) can be raised against LP-BM5-associated B-cell lymphomas through in vivo priming and in vitro restimulation with syngeneic LP-BM5-associated B-cell lymphoma line. Adoptive transfer of such polyclonal anti-LP-BM5 tumor CTLs inhibited the development of MAIDS. To identify new potential immunotherapeutic approaches to promote protective CTL-mediated responses, thus to treat LP-BM5-associated B-cell lymphomas, we examined the effects of specific blockadge of the PD-1 mediated negative signaling pathway in CD8 T cells following tomor inoculation. Adoptive transfer experiments using CD8 T cells from B6.PD-1-/- mice were performed, and then tumor growth, and retrovirus-induced tumor specific effector CD8 T-cell responses were measured. Our results showed that LP-BM5-specific CTL responses were significantly enhanced in mice adoptively transferred with PD-1-/- CD8 T cells than that in miced transferred with wild type CD8 T cells. Tumor growth was also significantly inhibited in mice transferred with PD-1-/- CD8 T cells. Altogether, manipulation of PD-1-associated down-regulation of CD8 T cell responses may provide novel immunotherapies in treating retroviral infection-associated lyphomas.
Increased immune activation during chronic SIV infection drives the TFH dynamics. (105.33)
Authors: Petrovas Constantinos , Yamamoto Takuya , Gerner Michael , Boswell Kristin , Roederer Mario , Seder Robert , Germain Ronald , Haddas Elias , Koup Richard
Published: January 2023

We have investigated the phenotype, location, molecular signature and function of follicular CD4 T helper cells (TFH) in rhesus macaques. Similar to human TFH, they are characterized by a CCR7lowPD-1highICOShighCTLA-4highCXCR4high phenotype and represent a heterogeneous population with respect to cytokine function. PD-1high CD4 cells were localized closer to the follicle center than PD-1low CD4 cells. This relative cellular distribution was not affected by the SIV infection. Production of Th1 cytokines was compromised while IL-4 was secreted predominantly by a highly differentiated (CD150low) subpopulation of TFH cells. Compared to non-TFH cells, TFH cells exhibited a distinct gene profile that was significantly altered by SIV infection. A significant reduction in the expression of the IL-4 gene in TFH was found implying a defective help for the development of B cell responses during chronic SIV. CD150low TFH cells were characterized by defective survival while in vivo BrdU integration studies revealed a defective cycling capacity of these cells. Preferential infection of TFH cells was found only during acute SIV yet progression to chronic SIV resulted in their accumulation, which was associated with high immune activation and altered IL-6-induced signaling. Thus, the preservation of TFH in the face of high rates of infection can be explained by their normally short half-life and the diverse populations of CD4 T cells that can provide to their continual replenishment.
Effects of Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) on cytokine production in primary and transformed B cells (105.40)
Authors: Swanson-Mungerson Michelle , Incrocci Ryan , McCormack Molly , Leof Caroline
Published: January 2023

Epstein-Barr Virus (EBV) latently-infected B cells are precursors of EBV-associated malignancies. However, the transition from a latently-infected cell to a malignant tumor cell is poorly understood. Studies show that EBV modulates pro-survival and anti-inflammatory cytokine levels in EBV-transformed cell lines. However, gene expression in these cell lines may not reflect gene expression found in latent B cells and lymphomas. One EBV protein, LMP2A, is consistently detected in both latently-infected resting B cells, as well as in EBV-associated malignancies. Therefore, we hypothesized that LMP2A may modulate cytokines that aid in the transition between latency and malignancy. Cytokine array data analyzing B cells from LMP2A-transgenic mice and B cell lymphomas expressing LMP2A indicate that LMP2A does not globally alter B cell-produced cytokine levels, but rather targets a limited number of cytokines. Additional studies using ELISA and Real-time RT-PCR confirm that LMP2A increases IL-10 levels and flow cytometry reveals that LMP2A increases the amount of IL-10 produced per B cell. Inhibitors of the LMP2A signaling pathway are being tested to determine the signaling requirements for LMP2A-mediated increases in IL-10. These data identify a novel function of LMP2A in the alteration of a cytokine that is important for both tumor survival and anti-tumor responses.
Premature senescence of circulating T cells in HCV-chronically infected patients (105.41)
Authors: Yu Qigui , Amet Tohti , Byrd Daniel
Published: January 2023

Persistent HCV infection is characterized by chronic immune system activation and inflammatory cytokine production. Thus, HCV infection may result in features characteristic of premature aging of immune cells (immune senescence), driven by chronic antigen exposure and immune system activation. To determine if untreated HCV infection is associated with premature aging of naïve and memory CD4+ and CD8+ T cells, peripheral blood mononuclear cells (PBMCs) from 24 individuals chronically infected with HCV and 13 healthy blood donors were stained with fluorochrome-conjugated antibodies, and then subjected to multiple-color flow cytometric analysis. We found that chronic HCV infection was associated with a decreased CD28 median florescence intensity on both CD4+ (CD3+/CD4+/CD45RA+CD27+CD28+) and CD8+ (CD3+/CD8+/CD45RA+CD27+CD28+) T cells. An increased proportion of intermediate- and late-differentiated CD4+ and CD8+ T cells was also observed in PBMCs from HCV chronically-infected individuals. In addition, higher levels of activated monocytes (CD14+/CD16+ or CD14+/CD69+) were detected in HCV chronically-infected individuals than that in healthy blood donors. Conclusions: The overall change during chronic HCV infection is associated with a shift in the T-cell population toward an aged confirmation. Our data suggest that HCV-infected individuals experience similar immunologic changes as the individuals infected with HIV-1.
The polyfunctionality of memory CD8+ T cells in response to chronic and acute viral infections is not influenced by age (105.49)
Authors: Lelic Alina , Ventresca Mario , Verschoor Chris , Parsons Robin , Evelegh Carol , Bowdish Dawn , Betts Michael , Loeb Mark , Bramson Jonathan
Published: January 2023

As humans age, they experience a progressive loss of thymic function and a corresponding shift in the nature of the circulating CD8+ T cell population from naïve to memory phenotype. These alterations are believed to result in impaired development of CD8+ T cell immunity in older individuals; however evidence that these global changes impair antigen-specific CD8+ T cell responses in the elderly is lacking. To address this possibility, we studied CD8+ T cell memory development following primary exposure to West Nile virus (WNV) in a cohort of young (<40 yrs), middle-aged (41-60 yrs) and aged (>61 yrs) individuals using polychromatic flow cytometry. Although the aged cohort displayed the previously described alterations in the CD8+ T cell compartment, including a marked loss of naïve CD8+ T cells, accumulation of CD28- and CD57+ T cells, we did not observe any influence of age on either the breadth, magnitude or functionality of antigen-specific CD8+ T cells following WNV, cytomegalovirus (CMV) or Epstein-Barr virus (EBV) peptide stimulation. We did however observe virus-specific CD8+ T cell functional differences between WNV, CMV and EBV. Collectively these data fail to support the hypothesis that immunosenescence leads to defective CD8+ T cell immunity and suggest that it should be possible to develop CD8+ T cell vaccines to protect aged individuals from infections with novel and emerging viruses.
Inverse relationship between T-cell receptor αβ repertoire diversity and pathogen-specific antibody levels in human cytomegalovirus infection (105.51)
Authors: Wang George , Dash Pradyot , McCullers Jonathan , Doherty Peter , Thomas Paul
Published: January 2023

A diverse T-cell receptor (TCR) repertoire capable of recognizing a broad range of antigenic peptides is thought to be central to effective pathogen-specific immunity, but evidence that TCR diversity is important for the successful control of human infections is limited. A novel, single-cell strategy for the clonotypic analysis of human CD8+ TCRαβ repertoires was used to probe the diversity and magnitude of individual human cytomegalovirus (CMV)-specific CD8+ T-cells recovered directly ex vivo. We found that CD8+ TCRαβ repertoire diversity, but not the size of the CD8+ T-cell response, was inversely related to circulating CMV-specific antibody levels, a measure that has been correlated epidemiologically with differential mortality risks and found here to be higher in persons with detectable (versus undetectable) CMV viral loads. Overall, these findings indicate that CD8+ T-cell diversity may be more important than T-cell abundance in limiting the negative consequences of CMV persistence, and provides insights into the selection of TCRαβ public motifs in a life-long virus infection.
IFN-γ secretion by memory CD4 T cells mediates immunopathology during secondary influenza infection by enhancing local lymphoid responses (105.6)
Authors: Bickham Kara , Turner Damian , Farber Donna
Published: January 2023

During secondary flu infection, flu-specific memory CD4 T cells induce protection and exacerbate cellular infiltration and inflammation. We investigated the role of IFN-γ production by memory CD4 T cells in the development of lung immunopathology during secondary flu infection by generating and analyzing in vivo responses of memory IFN-γ deficient CD4 T cells. We found similar precursor frequencies of flu-specific CD4 T cells after primary influenza infection of IFN-γ-/- and WT mice, with IFN-γ-/- memory CD4 T cells producing rapid IL-2 and TNF-α compared to the predominant IFN-γ and IL-2 production by WT memory CD4 T cells. Adoptive transfer of equal numbers of flu-specific WT and IFN-γ-/- memory CD4 T cells into naïve mouse hosts resulted in increased pulmonary infiltration and hypoxia 7 days after flu challenge only in recipients of WT memory CD4 T cells. Notably, flu-specific WT cells in the MLN exhibited increased expansion and activation compared to flu-specific IFN-γ-/- CD4 T cells. Furthermore, lung homogenates from recipients of WT memory CD4 T cells had significantly higher levels of the chemokines, CCL4 and CCL5, compared to recipients of IFN-γ-/- CD4 T cells. These findings suggest that IFN-γ secretion by memory CD4 T cells acts as a potential switch for the development of immunopathology, possibly by recruiting DCs to the lung, which in turn migrate to the MLN for activation and priming of large numbers of CD4 and CD8 cells
Neutralizing anti-HIV antibodies develop in a humanized mouse model of HIV-1 infection (105.8)
Authors: Seung Edward , Dudek Timothy , Dugast Anne-Sophie , Mattoo Hamid , Vrbanac Vladimir , Tivey Trevor , Cariappa Annaiah , Pillai Shiv , Luster Andrew , Tager Andrew
Published: January 2023

In BLT (bone marrow-liver-thymus) humanized mice, human thymocytes are educated by autologous human thymic tissue, resulting in functional human T cells. In contrast, limitations to B cell maturation have been noted. But despite this, we show for the first time that HIV infected BLT mice can produce class-switched anti-HIV antibodies with neutralizing activities. Human transitional B cells were present in greater frequencies in BLT mice than adult humans. Most of these cells had a T1 phenotype in the blood and spleen. But despite this B cell maturation defect, class-switched IgG Abs against various HIV proteins were detected by Western Blot in HIV-infected BLT mice. Using ELISA to determine anti-p24 IgG Ab titers, Abs were present as early as 8 weeks post infection (p.i.), with peak Ab titers seen after 15 weeks. One infected mouse demonstrated a peak titer similar to that seen in a chronically infected human. Finally, plasma samples from infected BLT mice after 22 weeks p.i. demonstrated neutralizing activities against the challenge virus. IC50 neutralizing titers in these mice were greater than those from humans infected for up to 5 months, but less than one infected for several years. The ability of humanized BLT mice to generate functional humoral immune responses may be further improved by strategies to improve their B cell maturation, which will further improve the potential of these mice to become a model system to study candidate HIV vaccines and therapies.
In vivo analysis of antigen presentation during acute CNS viral infection (106.11)
Authors: Fan Ying , Nayak Debasis , McGavern Dorian
Published: January 2023

Antigen presenting cells (APCs) upon activation break down proteins into peptides that are presented on major histocompatibility complexes (MHC). APCs help shape immune responses at sites of viral infection and can foster the development of immunopathology. Intracerebral infection of mice with lymphocytic choriomeningitis virus (LCMV) induces a CD8 T cell-dependent fatal meningitis. Because little is known about the dynamics of antigen-presentation in the brain, we set out to identify the APCs that influence CD8 T cell motility and function during the development of meningitis. This was accomplished by generating a LCMV recombinant expressing ovalbumin (OVA), which enabled us to quantify in vivo presentation of OVA257-264 (SIINFEKL) peptides by H-2Kb. We also used two-photon (2P) microscopy to study interactions between virus-specific CD8 T cells and different APC populations in the virally infected brain. Surprisingly, our results revealed that all brain resident APCs (dendritic cells, macrophages, and microglia) contained LCMV antigen during the development of meningitis, but only microglia had detectable H-2Kb-OVA257-264 complexes on their cell surface. The presentation capacity of microglia was confirmed by 2P data showing extensive CD8 T cell interactions and clustering around microglia. During LCMV meningitis, our findings suggest that CD8 T cell interactions with brain resident microglia are favored over those with peripherally-derived professional APCs.
Cutaneous delivery of Poly(dG) conjugated protein antigens targets and activates dendritic cells in vivo. (106.13)
Authors: Toussaint Hélène , Erdos Geza , Larregina Adriana , Donahue Cara , Falo Louis
Published: January 2023

Efficient and specific targeting of antigen (Ag) to Ag presenting cells (APCs) in vivo is a critical challenge for vaccine design. Ag delivery and formulation strategies have been directly linked to type and the quality of induced immune responses. APCs acquire Ags in a variety of forms from a variety of sources and endocytic mechanisms can predetermine intracellular Ag routing, processing and presentation to CD4 or CD8 T cells. We have designed an APCs targeting strategy based on simple and direct coupling of Poly(dG) oligo deoxynucleotides to protein Ags. In comparison to soluble OVA, we find that conjugates of Poly(dG) and OVA (Poly(dG)-OVA) are rapidly internalized by DCs and follow distinct intracellular processing pathways. Importantly, immunization of naïve animals by direct injection of Poly(dG)-OVA induces protective multifunctional T cell immunity. This includes efficient priming of potent antigen specific CTL responses, differentiation of naïve CD4 T cells into Th1 CD4 T cells, induction of specific memory T cell responses, and generation of Ag-specific antibody responses. Extending our findings to human skin, we show that intradermal injection of Poly(dG)-Ag results in efficient uptake by human skin migratory DCs, preferentially dermal CD14+ DCs, that also acquire potent T cells stimulatory functions. Our data suggest that Poly(dG) can both target Ags to human skin immune APCs and function as an adjuvant to induce potent CD4 and CD8 mediated T cell immunity.
CD13 regulates dendritic cell cross-presentation and t cell responses by inhibiting receptor-mediated antigen uptake (106.16)
Authors: Ghosh Mallika , McAuliffe Beata , Subramani Jaganathan , Basu Sreyashi , Shapiro Linda H
Published: January 2023

Dendritic cell (DC) antigen cross-presentation is generally associated with immune responses to tumors and viral antigens and enhancing this process is a focus of tumor vaccine design. In this study, we found that the myeloid cell surface peptidase CD13 is highly and specifically expressed on the subset of DCs responsible for cross-presentation, the CD8+ murine splenic DCs. In vivo studies indicated that lack of CD13 significantly enhanced T cell responses to soluble OVA antigen, although the development, maturation, antigen processing and presentation of DCs is normal in CD13KO mice. In vitro studies showed that CD13 regulates receptor-mediated, dynamin-dependent endocytosis of antigens such as OVA and transferrin but not fluid-phase or phagocytic antigen uptake. CD13 and antigen are co-internalized in DCs but CD13 did not co- immunoprecipitate with antigen receptors, suggesting that CD13 does not control internalization of specific receptors but regulates endocytosis at a more universal level. Mechanistically, we found that phosphorylation of the endocytic regulators p38MAPK and Akt was dysregulated in CD13KO DCs and blocking these kinases perturbed CD13-dependent endocytic uptake. Therefore, CD13 is a novel endocytic regulator that may be exploited to enhance antigen uptake and T cell activation to improve the efficacy of tumor-targeted vaccines.