bluuebunny/crossref_metadata_2025 huggingface dataset:
Nrf2 expression is upregulated in tumor infiltrating T cells and induces T cell anergy
Authors: JO Yuna , Lee Byunghyuk , Joo Myungsoo , Hong Changwan
Published: January 2023

The transcription factor nuclear factor-erythroid 2-related-2 (Nrf2) controls cellular redox homeostasis and displays immunomodulatory properties. Hypoxic microenvironment in solid tumors generated by their outgrowth activates Nrf2 which induces the expression of anti-oxidant genes and supports continuous growth and proliferation via metabolic reprogramming. It is easy to imagine that hypoxic situation may affect function of immune cells infiltrated into tumor mass. We have thus questioned how hypoxic condition affect T cell immune response against tumor and the role of Nrf2 is involved. First, tumor infiltrating T cells (TILs) were sorted from tumor mass to investigate the alteration of Nrf2 expression. Interestingly, we found that mRNA levels of Nrf2 are significantly increased in TILs compared to control T cells. In order to test whether T cell responses are affected by Nrf2 levels, we stimulated T cells and measured Nrf2 expression. Nrf2 expression and their target genes are significantly downregulated by TCR activation. We further confirmed that IFN-γ production in Nrf2 deficient T cells was dramatically enhanced. Our findings offer novel insights into how immune response and oxidative stress is integrated in tumor cells, and we highlight Nrf2 as candidate of molecular target to control tumor in hypoxic condition.
Low signal strength enhances the antitumor activity of human CAR Th17 cells
Authors: Neal Lilli R , Huff Logan W. , Nelson Michelle , Meek Megan E. , Bailey Stefanie R , Bowers Jacob S. , Kundimi Sreenath , Mehrotra Shikhar , Varela Juan C. , Paulos Chrystal M
Published: January 2023

We reported the antitumor activity of human Th17 cells (engineered to express a chimeric antigen receptor (CAR) that recognizes mesothelin) regresses large human mesothelioma tumors when infused into mice, but it remains unknown what type and what level of co-signal strength impacts their functional fate and antitumor activity. To address this question, human Th17 cells were activated with a high (3 beads: 1 T cell), medium (1:10) or low (1:100) signal via CD3-beads coated with either CD28 or ICOS agonists. We discovered Th17 cells activated with low ICOS signal were highly multifunctional (IL-17A, IFN-γ, IL-2 and IL-22) and possessed a less differentiated phenotype (high CD62L and CD45RA expression) than cells activated with a high ICOS signal. In comparison to low ICOS signal strength, Th17 cells activated with low CD28 signal strength were poorly functional and terminally differentiated. Of clinical significance, in two distinct tumor models (human mesothelioma and murine melanoma), we found that Th17 cells regressed large tumors to a better extent when primed with a low or medium ICOS signal strength compared to if they were activated with a high ICOS signal strength, or with any level of CD28 signal strength. Our data is compelling given that most ACT clinical trials use high CD3/CD28 activation to expand patients’ cells. Specifically, our work suggests the use of a low bead to T cell ratio will result in a more simple and clinically feasible method to expand highly therapeutic T cells for the clinic.
Tonic 4-1BB signaling from chimeric antigen receptors (CARs) impairs expansion of T cells due to Fas-mediated apoptosis
Authors: Mamonkin Maksim , da Silva Diogo Gomes , Mukherjee Malini , Sharma Sandhya , Srinivasan Madhuwanti , Orange Jordan S , Brenner Malcolm K
Published: January 2023

High expression of CARs in T cells or the presence of CAR ligands in cell culture results in tonic CAR signaling that enhances differentiation and exhaustion of T cells and compromises anti-tumor activity. A recent study demonstrated tonic signaling negatively affected T cells expressing a CD28.zeta GD2-specific CAR, indicating 4-1BB costimulation may improve their function. However, whether 4-1BB is a uniformly optimal costimulatory signal for CAR-transduced T cells is unclear.

We found that retroviral expression of several 2nd generation CARs with a 4-1BB domain (4-1BB.zeta) unexpectedly impaired T cell expansion. High expression of 4-1BB.zeta CD19 and kappa CARs led to upregulation of Fas and FasL and enhanced caspase 8-dependent apoptosis. Decreasing CAR expression in retro- or lentiviral systems normalized Fas levels and restored T cell expansion. However, a similar approach did not rescue expansion of 4-1BB.zeta CD5 CAR T cells, likely because the presence of the target antigen CD5 on T cells sustains high tonic signaling. 4-1BB.zeta CD5 CAR T cells recruit Fas and FasL to form stable fratricidal immunologic synapses. Disrupting TRAF2 binding site in the 4-1BB domain prevented Fas upregulation and restored T cell function but compromised costimulation. Therefore, we developed a regulated retroviral expression system that uses a small molecule to reversibly inhibit CAR expression, preventing tonic signaling and the resulting fratricide and differentiation. Withdrawing the inhibitor restores CAR expression and the anti-tumor function of T cells. Hence, we have described a mechanism by which chronic 4-1BB signaling may undermine CAR T cell function and provide a solution that curtails detrimental tonic CAR signaling.
Expression of CLL Upregulated Gene1(CLLU) ,Telomere Reverse Transcriptase (hTERT) ,Zeta -Associated Protein Kinase -70 (ZAP-70) in patients with Chronic Lymphocytic Leukemia
Authors: Abdelrahman Zakia Ahmed , shamaa lobna abou , Sultan Magda , Abdelrahman Mohmed , Almorish Mohmed A
Published: January 2023

Background

Defining prognostic tool may help in B-CLL patient subgroups to benefit from early therapeutic intervention. ZAP-70 expression is one marker for IgVH mutational status. hTERT expression discriminated the Ig-unmutated from Ig-mutated B-CLL and may serve as a molecular prognostic marker. CLLU1 expression holds some prognostic information not offered by other biologic markers in B-CLL. This findings drove us forcibly to investigate their expression in B-CLL.

Methods

The current study was carried out on newly diagnosed 30 B-CLL patients before treatment. In Binet classification ,23% in stage A, 33.3% in stage B and 43.7% in stage C. lymphoadenopathy was in 76.7% , splenomegaly 66.7% ,hepatomegaly 20% of patients. PBMCs were isolated from B-CLL patients and tested by immunocytochemical staining technique for ZAP-70 expression and QRT-PCR for hTERT and CLLU1 expression using cDNA1 for CLLU1

Results

ZAP-70 in mononuclear cells was positive in 30% of patients . ZAP-70 was significantly increased with Binet stage progression and . It was positively correlated with TLC and lymphocytic count and negatively correlated with the hemoglobin level and platelets count. The expression levels of hTERT mRNA and CLLU1 mRNA were +ive in all BCLL and -ive in normal cells. They were not correlated with Binet stage, hematological parameters ,ZAP-70 . The expression levels of hTERT and CLLU1 were higher in ZAP-70 positive but statistically non significant

Conclusion

ZAP-70 expression by Immunocytochemistry is a standardized, simple and inexpensive cost effective as a prognostic. hTERT and CLLU1 expression were novel prognostic markers in B-CLL that can be measured using a rapid and more sensitive method (QRT-PCR) accessible to routine laboratories.
Leucine metabolism is a promising new approach to study the interplay between T cells and cancer cells
Authors: Ananieva-Stoyanova Elitsa A , Bostic Josh , Torres Ashley , Hutson Susan
Published: January 2023

Cancer and T cells undergo similar metabolic reprogramming during proliferation to support increased biosynthetic and energy demands. They both rely on high rates of glycolysis while retaining mitochondrial respiration, a phenomenon known as the Warburg effect. In the tumor microenvironment, tumor cells can escape immune surveillance by competing with T cells for common metabolites and redirecting nutrients for their own advantage. To gain insight into the relationship between T cells and cancer cells, we studied the effect of leucine metabolism on T cells and their cancerous counterpart, EL-4 lymphoma cells. Our previous results showed that a loss of expression of leucine degrading enzymes (cytosolic or mitochondrial branched chain aminotransferases, BCATc and BCATm, respectively) increased intracellular leucine concentrations and stimulated glycolytic rate in activated T cells. These results correlated with activation of complex 1 of the mammalian target of rapamycin (mTORC1). Continuation of this study with EL-4 lymphoma cells, grown in leucine free media or treated with the leucine antagonist, n-acetyl-leucine amide, revealed a decrease in glycolysis, glycolytic capacity, and glycolytic reserve while mTORC1 was only moderately impacted. The oxygen consumption rate was stimulated in T cells lacking BCATc and/or BCATm but was decreased in the absence of leucine in EL-4 cells. The results suggest that T cells and their cancerous counterpart use leucine in a similar manner and that leucine is essential for the regulation of glucose and oxygen metabolism in either cell type. Future studies targeting leucine metabolism in the tumor microenvironment will aim to improve the effectiveness of T cells to fight cancer cells.
A comprehensive system of congenic mouse melanoma models for evaluation of immune therapies
Authors: Bosenberg Marcus , Meeth Katrina , Damsky William
Published: January 2023

There has been longstanding interest in the relationship between the immune system and malignant melanoma, the deadliest form of skin cancer. Melanoma has been the tumor of choice for the evaluation of a wide variety of therapeutic approaches that rely on stimulation of the immune system to combat cancer. The striking responses to immune checkpoint inhibitors noted in some melanoma patients have now been observed in several other malignancies, including renal cell carcinoma, lung cancer, and prostate cancer. These findings are very encouraging, however the specific mechanisms of response and reasons why only a subset of patients respond to therapy are not known. The extraordinary potential of immune therapies in cancer suggests that human-relevant mouse models of cancer are required to understand mechanisms of response and to optimize therapy so that a larger proportion of patients respond.

We have generated and evaluated a series of genetically engineered mouse models driven by alterations in Braf, Pten, Cdkn2a, beta catenin, p53, and Nras that reflect the genetic diversity of the vast majority of human melanoma. We have backcrossed 7 of these models to C57Bl/6, generated tumors, and isolated a series of over 30 congenic mouse melanoma cell lines. Several independent lines have been isolated for each melanoma genotype generated. We are in the process of characterizing the tumor microenvironment in each of the models using flow cytometry and a novel quantitative imaging approach of histological sections. This series of congenic lines represents an ideal set of models for the study of cancer immunology and responses to immune therapies.
Deciphering the epigenetic programming underlying CD8 T cell dysfunction in solid tumors
Authors: Philip Mary , Fairchild Lauren , Sun Liping , Viale Agnes , Leslie Christina , Schietinger Andrea
Published: January 2023

Tumor-specific CD8 T cells in progressive solid tumors express inhibitory receptors and fail to proliferate and produce effector cytokines. Using a mouse model (ASTxCre-ERT2) in which tamoxifen (TAM) treatment induces an oncogenic driver neoantigen, SV40 large T antigen (Tag) in hepatocytes, we previously showed that tumor-specific CD8 T cells (TCRSV40-I) become unresponsive quite early during the pre-/early malignant phase. While the dysfunctional state is initially plastic, TCRSV40-I cells soon enter a fixed dysfunctional state with the phenotypic, functional, and molecular hallmarks of T cells from late-stage human tumors. To define the epigenetic programs associated with plasticity and imprinting of tumor-specific T cell dysfunction, we used the “Assay for Transposase-Accessible Chromatin using Sequencing” (ATAC-Seq) to map chromatin accessibility in TCRSV40-I cells isolated from pre/early malignant lesions as well as in TCRSV40-I differentiating to the normal effector and memory states. Chromatin accessibility changes underlying TCRSV40-I activation and differentiation in pre-malignant lesions diverged from that in normal differentiation, and we observed 2 distinct chromatin accessibility patterns in dysfunctional TCRSV40-I correlating with the plastic and fixed dysfunctional states. Surprisingly, memory TCRSV40-I transferred into mice with late-stage established Tag-expressing hepatocellular carcinomas displayed these same 2 chromatin accessibility profiles, thus regardless of the initial CD8 T cell epigenetic state, epigenetic remodeling drives tumor-specific T cell differentiation to the dysfunctional state in both pre-malignant lesions and late established solid tumors.
Identification of flu-specific ICOS+CD38+ cTfh after influenza vaccination
Authors: Muselman Alexander D , Herati Ramin Sedaghat , Vella Laura , Su Laura , Tebas Pablo , Wherry E. John
Published: January 2023

The foundation of successful vaccines lies in the ability to prime the immune system to target a specific pathogen. The interaction between follicular helper T cells (Tfh) and B cells within germinal centers is necessary for the production of class-switched, affinity-matured antibodies. However, the size, diversity, and specificity of the T cell receptor (TCR) repertoire of the Tfh during an immune response remains unknown. We identified a subset of circulating Tfh (cTfh) coexpressing ICOS and CD38 that expresses markers of recent activation, such as the proliferation marker Ki67 and the transcription factor Helios. This activated cTfh subset increases following influenza vaccination. To begin to interrogate how the TCR repertoire changes in human cTfh cells responding to a vaccine, we performed T-cell receptor beta chain sequencing on cTfh from individuals before and after influenza vaccination. Although the ICOS−CD38− cTfh maintained a stable TCR repertoire pre- and 7 days post vaccination, the ICOS+CD38+ cTfh exhibited increased clonality indicative of antigen-driven activation and expansion. Using HLA class II HA306- and HA40-specific tetramer staining on peripheral blood from HLA-DR0401 individuals, we then further assessed the specificity of the responding cTfh at day 7 after vaccination. We found many tetramer-positive cells that expressed ICOS and CD38 in the cTfh subset, confirming that the ICOS+CD38+ response after vaccination contains influenza-specific cells. Ultimately, rational vaccine design will depend on better understanding of the clonotypic response elicited in response to various pathogens and their relevance to the overall immune response.
A computationally designed hemagglutinin stem-binding protein provides in vivo protection from influenza in the ferret model.
Authors: Brichacek Allison , Treants Merika , Hartman Amy , Reed Douglas , Baker David , Fuller Deborah Heydenburg , Cole Kelly Stefano
Published: January 2023

Influenza viruses cause thousands of deaths worldwide and remain a prominent public health issue. Currently, the influenza vaccine is the best tool available at protecting against infection, but the correct strains are hard to predict and the vaccines do not always work. More effective antiviral drugs are needed. Protection against influenza by broadly neutralizing antibodies targeted to a highly conserved region in the hemagglutinin (HA) stem shows great potential for immunotherapy. We have investigated the protective efficacy of an engineered protein, HB36.6, that was computationally designed to bind with high affinity to the same region in the HA stem as targeted by broadly neutralizing antibodies. Studies in the mouse model demonstrated that intranasal delivery of HB36.6 affords protection in mice lethally challenged with diverse strains of influenza, both when administered as a single dose of 6.0 mg/kg up to 48 hrs prior to challenge and significantly reduces disease when administered as a daily therapeutic after challenge. The current study was designed to evaluate HB36.6 in the ferret model, comparing prophylactic and therapeutic efficacy of this new antiviral against high dose influenza aerosol challenge. Similar to data obtained in the mouse model, results from these studies demonstrated significant reduction in viral load and clinical signs of disease in ferrets treated with HB36.6 compared to untreated controls or ferrets treated with Tamiflu. Together, these results show that binding of HB36.6 to the influenza HA stem region alone was sufficient to reduce the viral infection and disease process in vivo. These studies demonstrate a potential new class of antivirals for influenza.
Lactococcus lactis expressing HIV antigens on a Group A Streptococcus (GAS) pilus serves as potential dual mucosal vaccine against HIV and GAS
Authors: Jones Andrew , Chamcha Venkateswarlu , Quigley Bernard , Scott June , Amara Rama Rao
Published: January 2023

Due to the majority of HIV/SIV transmissions occurring via the mucosal route, a mucosal vaccine that generates immune responses along the genitorectal tract may provide the tools needed to combat HIV/SIV in its earliest stages of infection. We recently demonstrated that an orally delivered mucosal vaccine based around the probiotic bacterium Lactococcus lactis expressing HIV/SIV antigens fused to a cell wall anchored Group A Streptococcus (GAS) pilus induces a strong systemic and mucosal immunity in mice. To test the mechanisms behind immunogenicity, we gavaged mice and examined the activation level of various dendritic cells in the intestinal Peyer’s patches and found increased activation of CD11b+CD8− DC compared to wild-type L. lactis, indicating some inherent adjuvant activity of the GAS pilus. As there is evidence that GAS pilus specific antibodies may aid in the protection from GAS infection, we evaluated the ability of our L. lactis vectors to generate these pilus-targeting antibodies. We observed a strong induction of GAS-pilus specific antibodies in both mice and rhesus macaques orally immunized with our L. lactis vectors. We were unable to detect antibody responses generated against the backbone L. lactis bacterium, underlining the immunodominance of the GAS pilus in these vectors. These data suggest that these constructs may serve a dual role in generating protective immune responses against HIV and GAS infections.
Non-specific activation of autoreactive B cells after anthrax vaccination delays protection
Authors: Sawatzki Kaitlin , Ataca Sila , Watanabe Akiko , Kuraoka Masayuki , Walter Emmanuel , Kelsoe Garnett H. , Feng Feng , Kepler Thomas B
Published: January 2023

Anthrax Vaccine Adsorbed (AVA) vaccination protects against anthrax infection by eliciting a neutralizing antibody response. However, protective antibody titers are not observed in human sera until at least three vaccinations have been administered over six months. Even then, humoral responses to AVA do not provide long-term immunity without an annual booster, presenting a serious challenge in public health.

We followed six healthy volunteers over the standard five-dose, 18-month AVA schedule to characterize immunoglobulin genetics during vaccination. Plasmablasts were isolated one week after each vaccination and natively-paired heavy- and light-chain immunoglobulin mRNA were sequenced. Immunoglobulin sequences were analyzed for mutations and clonal evolution.

After primary vaccination, the average mutation frequency of observed plasmablast lineages indicated they were derived from previously affinity matured B cells (6.1–7.7%). Many sequences had very high mutation frequencies (>10%), a characteristic that has been linked to multiple antigen exposures. We synthesized antibodies from these lineages and tested them against a panel of human antigens. While they had little to no affinity to anthrax antigens, they displayed significant affinity to a variety of autoantigens. These memory lineages were non-persistent, and we observed naïve B cell activation after secondary vaccination. We tested antibodies from naïve-derived lineages first observed after secondary vaccination and found these to be anthrax-specific and non-autoreactive. Thus, while primary AVA vaccination elicits a robust memory response, it is non-protective and delays anthrax-specific naïve B cell recruitment until secondary vaccination.
Vaccinated female mice are better protected against Tularemia infection compared to their male counterparts
Authors: Sunagar Raju , Kumar Sudeep , Franz Brian J , Gosselin Edmung J
Published: January 2023

Francisella tularensis (Ft) is a Category A biothreat agent for which there currently is no FDA-approved vaccine. Thus, there is a substantial effort underway to develop an effective tularemia vaccine. While it is well established that gender can significantly impact susceptibility to primary infection, the impact of gender on vaccine efficacy is not well established. Thus, development of a successful vaccine against tularemia will require an understanding of this impact. In this study, a role for gender in vaccine-induced protection against tularemia is identified for the first time. Mucosal vaccination with inactivated Ft (iFt) LVS elicited gender-based protection in C57BL/6 mice against respiratory challenge with Ft LVS. Specifically, vaccinated male mice were more susceptible to subsequent Ft LVS challenge. This increased susceptibility in male mice correlated with increased bacterial burden, tissue inflammation, and elevated proinflammatory cytokine production late in infection. In contrast, improved survival of iFt-vaccinated female mice correlated with reduced bacterial burden and enhanced levels of Ft-specific Abs in serum and broncho-alveolar lavage (BAL) fluid. Furthermore, vaccination with a live attenuated vaccine consisting of an Ft LVS super oxide dismutase (SodB) mutant demonstrated a similar gender bias in protection following Ft SchuS4 challenge. Of particular significance is the fact that these are the first studies to demonstrate that gender differences impact disease outcome in the case of lethal tularemia following mucosal vaccination. In addition, these studies further emphasize the fact that gender differences must be a serious consideration in any future tularemia vaccine development studies
The common human HAQ STING variant affects Pneumovax® 23 efficacy
Authors: Patel Seema , Tucker Heidi , Jin Lei
Published: January 2023

Multiple polysaccharide vaccines have been licensed in U.S including the pneumococcal vaccine Pneumovax® 23, meningococcal vaccine Menomune, and Salmonella typhi vaccine Typhim Vi. Polysaccharides antigens activate the Stimulator of Interferon Gene (STING/MPYS) pathway in B cells. We previously found that ~3% of Caucasian and ~16% of Asian are homozygous for a STING variant containing three non-synonymous SNPs, R71H-G230A-R293Q (HAQ). Here, using human B cells derived from HAQ individuals and a knock-in mouse expressing mouse-equivalent of human HAQ variant, we investigated the impact of the HAQ variant on the effectiveness of Pneumovax® 23. Our results suggest that Pneumovax® 23 is likely less effective in HAQ individuals than the wt (R232) STING carriers. Our novel finding revealed, for the first time, the impact of the common HAQ human STING variant on Pneumovax® 23 efficacy and illustrated the need to develop personalized STING-targeting immunotherapy.
Obesity outweighs antibody responses generated by an aduvanted H7N9 influenza vaccine and fails to protect mice from influenza infection
Authors: Hertz Tomer , Karlsson Erik , Krammer Florian , Schultz-Cherry Stacey
Published: January 2023

Obesity is a risk factor for developing severe influenza infection making vaccination of utmost importance for this high-risk population. Yet, vaccinated obese animals and adults have decreased neutralizing antibody responses. We tested the hypothesis that addition of a squalene-based adjuvant to an influenza H7N9 vaccine would improve neutralizing antibody responses and protect obese mice from challenge. Adjuvanted vaccine increased both neutralizing and non-neutralizing antibody levels as compared to vaccine alone; however, obese mice were not protected from lethal H7N9 challenge. Increasing the antigen dose affords no added protection and decreasing the viral dose did not fully mitigate the increased mortality seen in obese mice. Although obese mice did mount virus-specific antibody responses; both the breadth and the magnitude of the response was decreased as compared to lean mice. Furthermore, antigen microarrays spotted with overlapping 20mer peptides (with 15 overlap) of the hemgaluttinin and neuraminidase H7N9 vaccine strain revealed that lean mice generated stronger and broader antibody responses to the H7 HA and N9. Interestingly, several specific antigens that were targeted in lean mice were not targeted by the obese mice, including some located around the sialic acid recptor binding domain. Overall, these studies highlight that while adjuvant does improve seroconversion, vaccination does not fully protect obese mice from influenza challenge, possibly due to the increased sensitivity of obese animals to infection. Given the continued increase in the global obesity epidemic, our findings have important implications for public health.
Herpes simplex virus-2 ΔgD vaccination primes robust cellular immunity with memory Th17 cell recall following challenge
Authors: Weiss Kayla A , Petro Christopher , Herold Betsy C. , Jacobs William R.
Published: January 2023

Approximately 25 percent of women are infected with herpes simplex virus (HSV)-2 in the United States. Even with anti-viral treatments, the rate of HSV transmission has increased 30 percent in the last 20 years. Furthermore the risk of acquiring an HIV infection increases 2–4 fold for HSV-2-infected individuals. Therefore, the need to have an FDA-approved HSV vaccine is immediate. Recently we have developed a single-cycle HSV-2 vaccine strain genetically deficient in glycoprotein D (HSV-2 ΔgD). Complete protection from disease and the establishment of latency is observed following either HSV-1 or HSV-2 challenge of HSV-ΔgD-vaccinated mice. We have also previously demonstrated that HSV-2 ΔgD elicits a protective, broadly functional antibody response. We hypothesized that HSV-2 ΔgD vaccination also primed T cell immunity, contributing to vaccine-induced protection. We observe both CD4 and CD8 T cells are activated by vaccination, and contract to establish a stable memory population. Vaccine-induced memory CD8 T cells are polyfunctional cytokine-producers with 55 percent specific for the immunodominant HSV gB498-505 epitope. Memory CD4 T cells primed by vaccination are quickly recalled to the infection site following challenge and exhibit a Th17-dominant phenotype with 60 percent of recalled cells producing IL-17A. We also determined that CD4 T cell help is required to stabilize the germinal center B cell population and to elicit protective antibodies following vaccination. Together, our data demonstrate HSV-2 ΔgD vaccination induces robust cellular, as well as humoral, immunity and likely an ideal vaccine vector.
Redistribution of T follicular helper cells to the lungs of neonatal mice infected with influenza
Authors: Fuller Michael John , Bradley John E. , Randall Troy D
Published: January 2023

The infant immune system responds poorly to both infection and vaccination, however the mechanisms responsible for impaired immune responses in neonates are incompletely understood. Here we analyzed T and B cell responses to influenza infection in neonatal mice. We found that neonatal mice were much more susceptible to primary influenza infection and cleared virus with delayed kinetics. Although CD8 T cell responses were slightly delayed in neonates compared to adults, neonates ultimately generated robust CD8 T cell responses. Following resolution of infection, however, the accumulation of tissue-resident memory (Trm) CD8 T cell responses was impaired in mice infected as neonates. In contrast, the appearance of influenza-specific CD4 T cells, including T follicular helper (Tfh) cells, was not delayed. Surprisingly, Tfh cells appeared in the lungs of neonatal mice, but not the lungs of adult mice. Tfh cells in the lungs of neonates correlated with the appearance of germinal center (GC) B cells and the formation of inducible bronchus-associated lymphoid tissue (iBALT) in the lungs. However, despite the appearance of local Tfh and GC B cells, the production of influenza-specific antibody was severely impaired in neonatal mice. Thus, despite the differentiation of influenza-specific Tfh in both lymphoid organs and lungs of neonatal mice, B cell-mediated immunity was still profoundly impaired. Nevertheless, while mice given a primary influenza infection as neonates had diminished Trm CD8 T cell responses and impaired B cell-mediated immunity, they were able to survive lethal rechallenge as adults, indicating that protective immunity could be generated in neonates.
Virtual memory cells make a major contribution to the response of aged influenza-naive mice to influenza virus infection
Authors: Blackman Marcia A , Lanzer Kathleen G , Cookenham Tres , Reiley William W
Published: January 2023

The ability to mount adaptive immune responses is impaired with age. One important contributing factor is the dramatic decline in repertoire and number of naïve T cells, raising the possibility that the response to new infections relies largely on cross-reactive memory T cells. We have analyzed the CD8 T cell response to influenza virus in influenza-naïve aged mice and confirmed that memory phenotype cells do make a major contribution to the response to new infections. There are two possible origins of the responding memory T cells. The response may be mediated by fortuitously cross reactive memory cells that were generated by earlier heterologous infections, which can be defined as “true memory” (TM). This possibility is supported by the observations that T cell recognition is highly degenerate and many unexpected cross-reactivities have been defined. Alternatively, the response may be mediated by antigen-specific memory cells that have been generated in the absence of antigen exposure, termed “virtual memory” (VM) cells. This possibility is supported by the observation that the ratio of VM:TM increases dramatically with age. To determine whether TM or VM respond to influenza in aged mice we used dual adoptive transfer with VM and TM cells. The data show that the response to influenza virus is dominated by memory CD8 T cells with a VM phenotype. The ability of these cells to afford protective immunity and develop into long-lived memory cells is under investigation and has implications for our understanding of immune function in the aged.
Tissue resident memory CD8 T cells augment innate immunity for tissue-wide antiviral protection
Authors: Zhu Jia , Peng Tao , Chang Yon , Layton Erik , Sun Sijie , Phasouk Khamsone , Johnston Christine , Halford William P , wald Anna , Corey Lawrence
Published: January 2023

Tissue-resident memory (TRM) CD8+ T cells provide effective, localized protection against pathogen re-encountering at barrier surfaces. During human herpes simplex virus 2 (HSV-2) infection, TRM CD8+ T cells persist at dermal-epidermal junction, interacting with basal keratinocytes and performing immune containment. The precise mechanism of how a few TRM CD8+ T cells convey broad tissue-wide protection is unclear. This study investigated the effects of TRM CD8+ T cells on tissue microenvironment by laser microdissecting and transcription profiling keratinocytes during human genital HSV-2 infection. In asymptomatic reactivation and lesion forming recurrences, keratinocytes exhibited a cell-intrinsic antiviral signature. Genes involved in RNA trafficking and protein translation were down-regulated, while a subset of interferon stimulated genes (ISGs) was elevated. The ISG expression was widely spread throughout the epidermis in the affected area. IFN-γ was expressed in TRM CD8+ T cells, but IFN-α and IFN-β were undetectable in keratinocytes and whole tissue. In primary cultured keratinocyte and fibroblast cells, pretreatment of IFN-γ delayed and restricted HSV gene expression at immediate-early stage of viral transcription. The inhibition on viral gene expression was dose-dependent and relied on IFN-γ receptor signaling. Thus, by harnessing cell-intrinsic innate immunity, TRM CD8+ T cells convey a tissue-wide antiviral protection in a non-cytolytic manner.
Accumulation of Th1 biased Tfh in lymphoid tissues during chronic SIV infection defines functionally distinct germinal center Tfh cells
Authors: Velu Vijayakumar , Mylvaganam Geetha H , Gangadhara Sailaja , Iyer Smita S , Hong Jung Joo , Ibegbu Chris , Villinger Francois , Amara Rama Rao
Published: January 2023

Follicular helper CD4T cells (Tfh) play a critical role in determining the magnitude and quality of antibody (Ab) responses. Chronic HIV infection is associated with hypergammaglobulinemia and germinal center Tfh (GC-Tfh) in lymphoid tissue may contribute to this process. However, the heterogeneous nature of GC-Tfh cells and the relative contribution of specific GC-Tfh subsets to viral persistence and Ab production is not understood in HIV/SIV infections. Here, we characterized the phenotype and function of GC-Tfh cells based on the expression of chemokine receptors associated with Th1 (CXCR3), Th2 (CCR4) and Th17 (CCR6) in lymph nodes following SIV251 infection in rhesus macaques (RMs). In SIV naïve RM, only a small fraction of GC-Tfh expressed CXCR3, CCR4 and CCR6. During chronic SIV infection, despite a global depletion of CD4 T cells, there was an increase in total GC-Tfh cells. Interestingly the majority of expanded GC-Tfh cells expressed CXCR3, while the proportion of CCR4+ cells did not change but CCR6+ cells decreased. CXCR3+ but not CXCR3−GC-Tfh produced IFN-g and IL-21 while both expressed CD40L following stimulation. IHC analysis demonstrated an accumulation of IFN-g+ T cells within the follicles during chronic infection. CXCR3+GC-Tfh also expressed higher levels of ICOS, CCR5 and a4b7, contained more copies of SIV DNA compared to CXCR3−GC-Tfh cells. Both CXCR3+ and CXCR3−GC-Tfh delivered help to B cells in vitro, but CXCR3+ GC-Tfh favored the production of IgG and IgG1. These data demonstrate that chronic SIV infection promotes expansion of Th1-biased GC-Tfh cells, which are phenotypically and functionally distinct from conventional GC-Tfh cells and contribute to IgG1 biased hypergammaglobulinemia and viral reservoirs
Combinatorial ex vivo analysis of beta cell specific CD4+ T-cell responses reveals a predominance of proinsulin specific cells
Authors: Blahnik-Fagan Gabriele , Uchtenhagen Hannes , Chow I-Ting , Kwok William W , James Eddie A
Published: January 2023

CD4+ T cell responses are thought to play a key role in the progression of type 1 diabetes (T1D). However, effective monitoring and characterization of T cells that respond to beta cell epitopes in subjects with T1D has been limited by technical obstacles, including the inherently low frequencies of autoreactive CD4+ T cells and the variable responsiveness of individual subjects to single epitopes. We have recently implemented a combinatorial staining approach that allows direct ex vivo characterization of multiple CD4+ T-cell specificities in a single sample. We applied this combinatorial HLA class II multimer assay to directly measure the frequency and phenotype of beta cell specific CD4+ T cells. For this work we utilized five peptides that were previously identified as naturally processed DRB1*04:01 restricted epitopes from proinsulin, GAD65, IA-2, and IGRP. Although responses to each of these peptides can be readily detected after in vitro expansion culture, our results indicated that only proinsulin specific T cells were consistently present at high frequencies in subjects with T1D. Phenotypic analysis of beta cell specific CD4+ T cells revealed that subjects with T1D had a higher frequency of proinsulin specific T cells with a memory phenotype than HLA matched controls. The majority of these memory cells were CXCR3 positive and an increased percentage were CCR7 negative, suggesting that Th1-like effector memory responses are present in subjects with T1D. Finally, this approach is compatible with combinatorial class I multimer analysis, facilitating the characterization of self-reactive CD4+ and CD8+ T cells using a single sample.
T Cells derived from Peripheral lymph nodes have different activation requirements than T cells isolated from spleen.
Authors: Elhofy Aelhofy
Published: January 2023

Primary murine T cells have been used as a model for human immune function. Animal models like EAE have shown disparate activities between splenic T cells and T cells derived from peripheral lymph nodes (PLN T cells). In this study we investigated if PLN T cells had the same activation requirements as splenic T cells in a serum free environment. PLN T cells activated in did not proliferate or secrete cytokines serum free conditions either by the non-specific stimuli of anti-CD3 and anti-CD28, or by PLP139-151 specific antigen re-stimulation. Conversely, splenic T cells cultured in serum free or FBS conditions proliferated and secreted cytokines equally in both activation models. The activation of serum free PLN T cells can be rescued with the addition of serum albumin as proliferation and cytokine production in both activation models was equivalent to those cultured in FBS. The activation requirement was specific to serum albumin, as the addition of other proteins including ovalbumin and serum proteins; gamma globulin, and collagen did not result in PLN T cell activation. Furthermore full antigen specific activity was returned to the PLN T cells with the addition of serum albumin demonstrated by the ability to induce adoptive transfer of EAE. The use of FBS in culture has masked the differential activation requirement of T cells depending on where they were derived and this studied showed serum albumin is requisite for the activation of T cells derived from PLN but not from spleen.
Dual T cell receptor-expressing CD8 T cells potentiate autoreactivity
Authors: Tsai Kevin , Huang Yu-Hsuan , Wang Xiaoxia , Priatel John J
Published: January 2023

Central tolerance serves to eliminate newly developing T cells that express strongly autoreactive T cell receptors. Although central tolerance is efficient in deleting high avidity autoreactive T cells, some lower avidity autoreactive T cells escape negative selection to cause autoimmune diseases. Although tight allelic exclusion limits thymocytes to expressing a single TCRbeta chain, rearrangement of the TCRalpha chain continues unabated until halted by positive selection, enabling thymocytes to express up to two TCRalpha chains and thus two TCRs. Moreover, it has been postulated that pathogenic low avidity autoreactive CD8 T cells may escape central tolerance through expression of a secondary benign TCR that mediates positive selection. To determine the role of dual TCR expressing CD8 T cells in autoreactivity and autoimmunity, we have compared CD8 T cell autoreactivity against the model autoantigen ovalbumin between T cells capable of expressing two TCRs (TCRalpha+/+) versus T cells capable of expressing a single TCR (TCRalpha+/−). TCRalpha+/− CD8 T cells exhibited reduced proliferative capacity upon OVA stimulation relative to TCRalpha+/+ CD8 T cells. In addition, a lower frequency of TCRalpha+/− CD8 T cell effectors produced IFN-gamma upon activation with OVA compared to TCRalpha+/+ CD8 T cell effectors. Taken together, we shown that dual TCR expression by CD8 T cells reduces the efficiency of T cell tolerance and may potentiate T cell autoreactivity. We are investigating whether dual TCR-expressing T cells are key to the pathogenesis of autoimmune diabetes in our mouse model system. Our results will provide valuable insight into the escape mechanisms exploited by pathogenic autoreactive CD8 T cells.
Type I interferon signaling is essential for cytotoxic T lymphocytes to infiltrate into the pancreatic islet and cause type I diabetes
Authors: Marro Brett S. , Ware Brian C. , Oldstone Michael B.A.
Published: January 2023

Type I diabetes (T1D) is characterized by elevated blood glucose, low pancreatic insulin and lymphocyte migration into Islets of Langerhans with destruction of insulin-producing β-cells. A seminal question in the pathogenesis of T1D is the signal(s) that control lymphocyte entry and attack on insulin-producing β-cells. Numerous epidemiological studies in humans suggest that infectious agents may initiate T1D. To model T1D in the mouse and probe the underlying mechanisms governing disease, several years ago we engineered Tg mice to solely express the glycoprotein gene of Lymphocytic Choriomeningitis Virus within β-cells using the rat insulin promoter (RIP-LCMV-Gp Tg mice). In this Tg model, infection with LCMV generates Gp-specific CD8+ CTL that target β-cells, leading to T1D by day 14–20 post-infection. We now report that blockade of Type I interferon signaling via antibody-mediated neutralization of the Type I interferon receptor (IFNAR) or its cognant ligands interferon-α (IFNα) or interferon-β (IFNβ) suppress the selective infiltration of virus (autoimmune) specific CD8+ T cells into the islet, thereby sparing islets from destruction and preventing T1D. During this early phase of viral challenge (up to day 20), neutralization of either IFNα or IFNβ did not affect the expansion of virus (autoimmune) specific CD8+ T cells or impact viral clearance, but nevertheless abrogated disease by restricting CD8+ T cells to the rim of the islets, thus preventing access of T cells into the islet. These results demonstrate that IFNα and IFNβ are central orchestrators in developing diabetes by allowing the entry of lymphoid cells into the islet that then target and kill insulin-producing β-cells.
The pathogenic effect of TNF-α on development of Sjögren’s syndrome in non-obese diabetic mice
Authors: Zhou Jing , Yu Qing
Published: January 2023

Tumor necrosis factor-alpha (TNF-α), is a proinflammatory cytokine involved in the pathogenesis of various autoimmune diseases including rheumatoid arthristis, inflammatory bowel disease and psoriasis. Previous studies showed that patients with Sjögren’s syndrome(SS), a prevalent systemic autoimmune disease primarily affecting tear and saliva secretion, exhibit enhanced expression of TNF-α in saliva and salivary gland. However, the exact role of TNF-α in SS disease development is not yet fully determined. Our current study employed non-obese diabetic (NOD) mice, a model of SS-like diseases, to investigate the function of endogenous TNF-α in pathogenesis of SS. We observed that administration of a neutralizing anti-TNF-α antibody significantly improved salivary secretion in female NOD mice, indicating a remission of clinical symptom of SS. TNF-α neutralization also decreased the number of leukocytic foci and reduced the number of total and activated CD4 T cells and B cells in the submandibular gland of NOD mice. Moreover, neutralization of TNF-α reduced the number of CXCR3-expressing CD4 T cells and T-bet protein level in the submandibular gland, suggesting a decrease in the infiltrating T helper 1 and T cytotoxic 1 cells. In addition, the decrease in CXCR3+ CD4 T cells was associated with reduced expression of CXCR3 ligand CXCL9. Overall, the results presented here indicate that endogenous TNF-α plays a pathogenic role in the initiation and development of SS disease and neutralization of TNF-α may be greatly beneficial for the prevention and treatment of this prevalent autoimmune disease.
S1P1 deletion differentially affects TH17 and Regulatory T cells
Authors: Eken Ahmet , Duhen Rebekka , Singh Akhilesh K , Fry Mallory , Buckner Jane H , Kita Mariko , Bettelli Estelle , Oukka Mohamed
Published: January 2023

Sphingosine-1 phosphate receptor 1 (S1P1) is a G-protein coupled receptor critical for the egress of T and B cells out of lymphoid organs. Although S1P1 agonists (such as fingolimod) are currently used for the treatment of multiple sclerosis (MS) little is known how S1P1 signaling regulates Th17 and Treg cell homeostasis. To study the impact of S1P1 signaling on Th17 and Treg biology, we specifically deleted S1P1 in Th17 and Treg cells using IL-17ACre and Foxp3Cremice, respectively. Deletion of S1P1 in Th17 cells conferred resistance to experimental autoimmune encephalomyelitis (EAE) characterized by reduced Th17 cell distribution across peripheral organs and diminished Th17 cell generation. On the other hand, permanent deletion of S1P1 in Treg cells resulted in autoimmunity and acute deletion rendered mice more susceptible to EAE. Importantly, our study revealed that S1P1 not only regulated the egress of Treg cells out of lymphoid organs and subsequent non-lymphoid tissue distribution but also their phenotypic diversity. Most of the Treg cells found in S1P1-deficient mice had an activated phenotype and were more prone to apoptosis, thus converted to effector Treg. The comparison of Treg cells obtained from MS patients treated with fingolimod to those treated with other oral drugs confirmed the switch of Treg cells into effector memory phenotype. Our results provide novel insight into the functions of S1P1 and potential impact of long term fingolimod use on Th17 and Treg cell biology and general health in MS patients.
CNS tissue response to Multiple Sclerosis
Authors: Berl Sabina , Waisman Ari
Published: January 2023

A well known inflammatory, autoimmune disease of the central nervous system (CNS) is multiple sclerosis (MS), where cells of the immune system invade the CNS, causing demyelination and neurodegeneration and leading to paralysis. The most common animal model of MS is experimental autoimmune encephalomyelitis (EAE). Not much is known about the response of neurons to the inflammatory triggers, although damage to them is the main initiator for paralysis in MS patients and EAE, due to the difficulty to separate and analyze them for gene expression on single cell level.

The aim of this project is to isolate and analyze neuronal mRNA expression level from adult mice. Isolated neuronal mRNA of naïve mice and mice with EAE will be sequenced by Next Generation Sequencing (NGS) to better understand how neurons respond to damage induced by inflammation.

RiboTag mice have a modified ribosomal protein L22 including a HA-tag. After crossing the RiboTag mice to the neuron-specific NFH-Cre mice, HA-tagged ribosomes will be specifically expressed in neurons giving the possibility to immunoprecipitate only neuronal RNA.

Immunoprecipitated neuronal mRNA of the RiboTag mice will be further analyzed by deep sequencing using NGS technology.

The final goal of this study is to analyze gene expression pattern of neurons that are attacked by pro-inflammatory cells and mediators during EAE. Understanding how neurons respond during autoimmune disease should allow to design better therapies that will protect these cells under such pathogenic conditions. This work has implications not only for patients of MS, but also for other neurological diseases, including Alzheimer’s disease and stroke.