bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of exhausted CD8+ T cells in low tumor mutation burden breast cancer patients
Authors: Egelston Colt , Avalos Christian A , Simons Diana , Huang Yinghui , Lim Min Hui , Lee Peter P.
Published: January 2023

Tumor immunogenicity is generally thought to be correlated with increased tumor mutation burden and the resulting neoantigen driven T cell reactivity to cancer cells. This immunogenicity results in the formation of tumor specific, but exhausted tumor infiltrating CD8+ T cells (TILex). Breast cancer tumors, which are predominantly low mutation burden, are widely viewed as non-immunogenic. Here we demonstrate that subsets of both triple negative and estrogen receptor positive breast cancer patients have primary tumors heavily infiltrated by tumor specific CD8+ TILex. CD8+ TILex in breast cancer patients were demonstrated to coexpress PD-1 and CD39 and showed a significant reduction of IFNγ, TNFα, and IL-2 production capacity. Like melanoma and colorectal CD8+ TILex, CD8+ TILex human breast tumors had increased expression of CD103, CD38, and TIM-3 in addition to loss of KLRG1 and CD127 expression. Similarly, melanoma, colorectal, and breast tumor CD8+ TILex shared transcriptional signatures identified by single cell sequencing. T cell receptor sequencing revealed a distinct repertoire of CD8+ TILex as compared to other CD8+ TILs from the same tumors, suggesting them to be a unique population in response to tumor antigen. Finally, we demonstrated that increased levels of CD8+ TILex in human breast tumors are not associated with higher levels of tumor mutation burden, as assessed by a tumor mutation load oncogene panel. This work suggests that a subset of breast cancer patients have inflamed, tumors associated with the presence of exhausted CD8+ T cells and that these patients may benefit from immunotherapeutic interventions.
Satb1 deficiency licenses TFH-differentiation
Authors: Chaurio Gonzalez Ricardo A , Biswas Subir , Payne Kyle K. , Galindo Carmen Maria Anadon , Costich Tara Lee , Perales-Puchalt Alfredo , Perez-Sanz Jairo , Harro Carly M , Mine Jessica A , Allegrezza Michael J , Svoronos Nikolaos , Kroeger Jodi , Robinson John , Conejo-Garcia Jose R
Published: January 2023

T Follicular Helper cells (TFH) provide both co-stimulation and stimulatory cytokines to B cells to facilitate affinity maturation, class switch recombination, and plasma cell differentiation within the germinal center. However, is not clear how TFH differentiation is regulated. We found that deficiency of the chromatin organizer Satb1 results in increased TFH formation in CD4Cre+Satb1flx/flx mice through up-regulation of the canonical TFH markers ICOS and PD-1 and suppression of Foxp3+PD-1highCXCR5+ T follicular regulatory (TFR) cells as well. Accordingly, CD4Cre+Satb1flx/flx mice, or RAG1−/− mice transferred with Satb1-deficient CD4+ T cells showed a dramatic accumulation of CD4+CXCR5+PD-1high upon ovarian tumor challenge, compared to their Satb1+ counterparts, which was associated with reduced tumor growth. Importantly, intratumoral administration of Satb1-deficient CD4+ T cells re-directed to target ovarian cancer cells through chimeric receptors, but not their Satb1+ counterparts, induce the formation of Tertiary Lymphoid Structures in most tumors.

Conclusion

Satb1 controls three mechanisms relevant for TFH differentiation and, subsequently, antigen-specific humoral responses; namely, PD- 1 expression, ICOS de-repression and TFR formation. Our results suggest a novel role for Satb1 as a major regulator of TFH differentiation and TLS during tumor formation.
Anti-alpha-enolase T cell response in oral squamous cell carcinoma
Authors: Chia Jean-San , Tuan Chih-Hsuan , Lay Fang-Yun , Ye Jung-Hsuan , Chiu Yen-Ling
Published: January 2023

In Taiwan, oral cancer ranks as the 6th highest incidence of cancer in general population. Characterized by high metastatic potency, oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. Alpha-enolase (α-enolase) is a glycolytic enzyme expressed in most tissues. Many researches have pointed out the over-expression of α-enolase in various cancer type. Its role as glycolytic enzyme and plasminogen receptor were reported to promote cancer invasion, proliferation, and metastasis.

Our works focused on the relationship between α-enolase and OSCC. We found the increased α-enolase expression in OSCC and were able to detect anti-α-enolase antibodies in serum of some OSCC patients. These results revealed a non-negligible influence of α-enolase in OSCC. Next, we spliced α-enolase from whole protein into peptide pool and tested T cell response upon its stimulation. Both CD8+ T cells and CD4+ T cells from peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) were able to detect the cytokine production, like interferon gamma (IFN-γ) or tumor necrosis factor (TNF-α), and as well as the expression of activation marker like CD107a. Taken together, our works indicated that α-enolase is a potential antigen that may be involved in anti-tumor response in OSCC.
Metformin potentiates BCG efficacy against Mtb infection
Authors: Haugen Jessica , Obregon-Henao Andres , Ackart David , Todd Alexandra , Basaraba Randall
Published: January 2023

Tuberculosis (TB) is a chronic inflammatory disease that is caused by the bacterium Mycobacterium tuberculosis (Mtb) and is currently the leading cause of death by an infectious disease worldwide. Presently, TB therapeutic strategies include prolonged treatments with antimicrobial drugs, which often produce toxic side-effects, and vaccination with Bacille Calmette-Guérin (BCG), the only available and only partially protective TB vaccine. Considering the critical need for better prevention strategies, there have been multiple attempts to enhance the efficacy of BCG, yet none have significantly outperformed BCG alone. Previously, we have demonstrated that the anti-diabetic drug, metformin, has immunomodulatory effects and is associated with increased host resistance against TB. Based on our findings we hypothesized that following BCG vaccination, treatment with metformin would also enhance the vaccine response to Mtb. To test our hypothesis, we vaccinated guinea pigs with BCG Pasteur, dosed them with metformin or mock treatment, and performed low dose aerosol infection with Mtb H37Rv. At 90 days following infection, histological evaluation revealed that guinea pigs that received metformin- BCG had significantly reduced lung lesion burden compared to BCG alone. In addition, we evaluated the effects of metformin on T cell differentiation using an in vitro model of anti-CD3/anti-CD28 activation and found that metformin enhances memory T cell marker expression. Moreover, we show that mitochondrial hyperpolarization, a critical component of T cell activation, is dampened by metformin, which may ultimately alter T cell differentiation towards a memory-like phenotype and help explain metformin’s protective efficacy.
CD40-targeted S1 subunit vaccine protects against MERS-CoV and S1-associated pulmonary immunopathology in transgenic human DPP4 mouse model
Authors: Hashem Anwar M , Algaissi Abdullah , Agrawal Anurodh , Al-amri Sawsan , Almasoud Abdulrahman , Alharbi Naif , Peng Bi-Hung , Li Xuguang , Tseng Chien-Te
Published: January 2023

Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic respiratory virus that emerged in 2012. While infection control measures have played a major role in limiting human/camel-to-human transmissions, development of safe and effective human or camel vaccines is warranted. Here, we extended and optimized our previous rAd5-based vaccine platform characterized by in vivo amplified and CD40-mediated specific responses to generate MERS-CoV S1 subunit-based vaccine. We generated rAd5 constructs expressing CD40-targeted S1 fusion protein (rAd5-S1/F/CD40L), untargeted S1 (rAd5-S1), and GFP (rAd5-GFP), and evaluated their efficacy and safety in human DPP4 transgenic (hDPP4 Tg+) mice. Immunization of hDPP4 Tg+ mice showed that a single dose of rAd5-S1/F/CD40L elicited robust and significant specific IgG and neutralizing antibody responses as those induced in mice immunized with two-doses of rAd5-S1. After MERS-CoV challenge, both vaccines conferred complete protection against morbidity and mortality, as evidenced by significantly undetectable/reduced pulmonary viral loads as compared to control group. However, rAd5-S1 but not rAd5- S1/F/CD40L immunized mice exhibited marked pulmonary perivascular hemorrhage post MERS-CoV challenge despite the observed protection. Collectively, these data indicate that incorporating CD40L into this Ad5-based MERS-CoV S1 subunit vaccine as targeting molecule and molecular adjuvant can not only enhance immunogenicity and efficacy but also prevents induction of inadvertent pulmonary pathology in immunized and challenged mice, thereby offering a promising strategy to enhance the safety and potency of antigen-specific immune responses.
Recombinant live attenuated influenza virus expressing G protein fragment in a chimeric hemagglutinin molecule induces G-specific antibodies and confers protection against respiratory syncytial virus
Authors: Jung Yu-Jin , Lee Yu-Na , Kim Ki-Hye , Lee Youri , Lee Jongsang , Kim Cheol , Kang Sang-Moo
Published: January 2023

Respiratory syncytial virus (RSV) is one of the most important pathogens associated with significant morbidity and mortality in infants and the elderly. Live attenuated influenza vaccine (LAIV) is a licensed platform for vaccination in humans and known to induce broader immune responses locally and systemically. RSV G attachment proteins mediate virus binding to the target cells and contain a conserved central domain with neutralizing epitopes. Here, we generated recombinant LAIV based on the attenuated A/PR8/34 virus backbone, expressing a RSV G conserved domain in a chimeric hemagglutinin (HA) fusion molecule (HA-G). The attenuated phenotypes of chimeric HA-G LAIV were evident by restricted replication in the upper respiratory (nose) but not in the lower respiratory track lung tissues of the mice. Chimeric HA-G LAIV grew better at low temperature 33□. Prime and boost intranasal vaccination of mice with HA-G LAIV induced G-protein specific IgG and IgG2a (T helper type 1) isotype antibodies. Immunization of mice with chimeric HA-G LAIV showed significant increases in G-protein specific IgG and antibody-secreting cell responses in lung, bronchioalveolar fluid, bone marrow, and spleens after challenge. Vaccine-enhanced disease typically caused by inactivated-RSV vaccination was not observed in chimeric HA-G LAIV as analyzed by lung histopathology and infiltrating innate and adaptive immune cells after RSV challenge. Immunity of chimeric HA-G LAIV against influenza virus was not compromised. These results in this study suggest a novel approach of developing RSV vaccine candidates using LAIV, potentially dual vaccines conferring protection against both pathogens.
A Computationally Optimized Broadly Reactive Antigen (COBRA) Elicits Broadly Neutralizing Antibodies Against a Conserved Influenza Virus Hemagglutinin B-Cell Epitope
Authors: Sautto Giuseppe Andrea , Kirchenbaum Greg A. , Ecker Jeffrey William , Abreu Rodrigo Bessa , Pierce Spencer Reynolds , Ross Ted M.
Published: January 2023

The development of broadly protective influenza vaccines will represent the main countermeasure to overcome influenza virus spread and improve the coverage offered by the current standard of care.

A computationally optimized broadly reactive influenza virus hemagglutinin (HA) antigen (COBRA), named P1, elicits a broadly reactive antibody (Ab) response against multiple H1N1 strains.

In order to understand the mechanism of P1-conferred breadth of response, we first characterized the breadth of the Ab response at the B-cell level. Specifically, the reactivity of secreted Abs from mice immunized with P1 or seasonal/pandemic vaccine strains was assessed against a panel of H1N1 recombinant HA. Interestingly, while Abs from seasonal/pandemic immunized mice showed a predominant homologous response, those from P1- immunized animals exhibited a broader recognition.

In order to dissect the antibody response, a panel of P1-specific B-cell hybridomas was generated. Subsequently, the corresponding purified monoclonal Abs (mAbs) were assessed for the breadth of HA binding and hemagglutination inhibition (HAI) activity against different H1N1 viruses. Collectively, P1-specific mAbs exhibited a broad spectrum of binding and functional activities, spanning from strain-specific to broadly reactive and neutralizing mAbs, while seasonal/pandemic-specific mAbs displayed a narrower spectrum of activity. Epitope mapping studies revealed that P1-elicited broadly reactive and neutralizing Abs recognize unique conserved epitopes within the HA receptor binding site. Collectively, these studies shed light on the mechanism of breadth conferred by P1 and leverage its optimization for the development of a more effective influenza vaccine.
Computer-Optimized Immunization Strategy to Boost Highly Cross-Reactive Antibodies to RSV
Authors: Anderson Christopher S , Mariani Thomas J.
Published: January 2023

Respiratory Syncytial Virus (RSV) is a tremendous burden on public health. RSV infects humans within two years of life and causes continual infections throughout their lifetime. RSV is estimated to cause approximately 30 million episodes of acute lower respiratory infection resulting in over 100,000 deaths annually (in children ages 0 – 5 alone). Despite this burden, no vaccine is currently available for RSV. RSV has been classified into two subtypes (A and B) based on genomic and antigenic properties. Evolutionary rates are similar to other RNA viruses with estimates of 1.83–1.95 x 10−3 nucleotide substitutions/site/year. The high mutation rates of RSV produce an evolutionary landscape that has led to genetic and antigenic variation across and within subtypes. The attachment glycoprotein (G-protein) is must abundant on the surface of the virus and contains an almost completely conserved region, the central conserved domain (CCD). We developed a computational model of the humoral immune system that simulates B cell responses to G-protein antigen. We use a sequence-based approach to approximate antigenic differences across (>1000) RSV strains. We then use computer simulations to identify an immunization strategy that can boost antibody levels to the CCD region. Last, we demonstrate that the immunization strategy results in a highly cross-reactive antibody response.
A knock-in mouse to test vaccines for the broadly neutralizing influenza response
Authors: Li Wenjuan , Ota Takayuki , Nemazee David
Published: January 2023

Influenza virus is a major public health burden controlled by vaccination. However, influenza viruses develop mutants at a high frequency. The yearly vaccine elicits a specific response that fails to protect against most influenza strains, and its efficacy and production is risky every flu season. It would be ideal to develop a universal vaccine conferring life-long, broad protection. So far, the academic field has isolated several broadly neutralizing antibodies that target conserved regions of viral proteins from donors. However, how to elicit these antibodies in human through designed vaccines remains challenging and inefficient. Herein we generated humanized BCR mice to facilitate testing of strategies to induce broadly neutralizing antibodies in humans. CR9114 is one of the most broadly neutralizing antibodies known with activity against group 1 and group 2 influenza A and also influenza B virus. Most contacts are with the H-chain only, which uses VH1-69. We knocked in a promoter-germline VDJ element to the physiological Igh locus, allowing analysis of the response of B cells capable in principle of broad neutralization and the testing of vaccine candidates.
Intravenous Bacille Calmette-Guerin provides protection across a dose spectrum in a Rhesus macaque model of tuberculosis
Authors: Zeppa Joseph John , Darrah Patricia A , Pokkali Supriya , Hackney Joshua A , Maiello Pauline A , Scanga Charles A. , Laddy Dominick J , Lin Philiana Ling , Seder Robert A , Roederer Mario , Flynn JoAnne L
Published: January 2023

Our laboratory has demonstrated that intravenous (IV) vaccination with Bacille Calmette-Guerin (BCG; 5x107CFU) provides remarkably robust protection against low-dose Mycobacterium tuberculosis (Mtb) infection in Rhesus macaques (9/10 animals protected [<50 thoracic Mtb CFU]; 100,000-fold reduction compared to intradermal [ID] BCG). IV BCG also resulted in a 100-fold increase in mycobacterium-specific T cells in the bronchoalveolar lavage (BAL) of animals compared to other routes of administration (ID or aerosol). These results led us to hypothesize that significant protection could be observed at lower IV BCG doses. We therefore vaccinated macaques with decreasing doses of BCG (5x107, 1.6x107, 5x106, 1.6x106, 5x105and 1.6 x105CFU) followed by six months of immune monitoring and a three-month low-dose Mtb challenge. As expected, a mycobacterium-specific T cell dose-dependent response was observed in both the BAL (FACS) and PBMCs (IFNgELIspot) prior to infection, as well as in the lung at time of necropsy (FACS). Remarkably, 17/25 animals had no thoracic Mtb CFU across all groups, and at least one animal from each group was protected (30–100% protection). Mycobacterium-specific CD4 T cell numbers in the BAL appear to be a correlate of protection. These results demonstrate that even at low doses IV BCG can be efficacious in preventing tuberculosis with a potential role for T cell-dependent immunity.
SOCS1 antagonist-expressing recombinant BCG enhances anti-tuberculosis protection in a mouse model
Authors: Yasutomi Yasuhiro
Published: January 2023

Suppressor of cytokine signaling 1 (SOCS1) plays a key role in the negative regulation of JAK/STAT signaling, which is involved in innate immunity and subsequent adaptive immunity. Bacillus Calmette-Guerin (BCG) induces up-regulation of SOCS1 expression in host cells, which may lead to the suppression of immune responses by BCG via inhibition of the JAK/STAT signaling pathway. This might cause reduction in the protective effect of a BCG vaccine. In the present study, we assessed the immune responses and protective efficacy of a recombinant BCG vaccine expressing an SOCS1 antagonist molecule (rBCG-SOCS1DN). C57BL/6 mice were immunized with rBCG-SOCS1DN or parental BCG Tokyo vaccine strain harboring an empty plasmid vector (rBCG-pSO). rBCG-SOCS1DN enhanced induction bone marrow-derived dendritic cell activation and T cells activation compared to rBCG-pSO. The amount of IFN-g, TNF-a and IL-6 produced by splenocytes of rBCG-SOCS1DN-immunized mice were larger than those produced by splenocytes of rBCG-pSO-immunized mice. Moreover, the rBCG-SOCS1DN-immunized mice showed a substantial reduction in the number of colony-forming units (CFU) of Mycobacterium tuberculosis (Mtb) in the lungs and spleens in comparison with that in control BCG-immunized mice when the immunized mice were infected with a highly pathogenic Mtb strain by inhalation. These findings provide evidence for the possibility of rBCG-SOCS1DN being an effective Mtb vaccine with a novel concept of rBCG as a tool for immunomodulation in host cells.
Immunity of a Klebsiella Pneumoniae Vaccine Independent of Antigen Specific Antibody
Authors: Iwanaga Naoki , Sandquist Ivy , Chen Kong , Norton Elizabeth B , Moreno Javier Rangel , Kolls Jay K
Published: January 2023

Objective

We demonstrated the serotype independent immunity of Klebsiella pneumoniae (K. pneumoniae) vaccine. We would like to declare next if the noble effect is dependent on the antibody specific manner.

Methods

Mice were vaccinated via intranasal with Kp serotype 2 OMPx adjuvanted with heat-labile enterotoxin A1 domain (LTA1) and boosted at 3 weeks after the initial immunization. Protective immunity was compared among the C57Bl/6 mice, STAT3 fl/fl×CD4 Cre mice, and Il21r −/− mice by infecting with K1 strain of K. pneumoniae. Lung T-cell responses were evaluated by FACS and ELISPOT as well as K1 strain specific antibodies were assessed by ELISA.

Results

CD4+ T-cells of the vaccinated lungs in the CD4+ STAT3 deficient mice were significantly decreased compared to the WT mice and the Il21r −/− mice, and IL17A response by ELISPOT were significantly reduced as well. Meanwhile, K1 strain specific IgG in the serum and IgA in the lung homogenates of vaccinated WT mice were significantly increased in vaccinated mice and these responses were reduced in vaccinated CD4+ STAT3 deficient mice and Il21r −/− mice. Furthermore, vaccine induced protection was compromised in vaccinated CD4+ STAT3 deficient mice whereas the protection as well as antigen elicited lung IL-17 responses were maintained in Il21r −/− mice.

Conclusions

STAT3 activation in CD4+ T-cells regulated by IL-21R signaling are required for the K. pneumoniae specific antibodies, but these antibody responses were dispensable for the serotype independent immunity LTA1 adjuvanted K. pneumoniae vaccine. However, CD4 STAT3 was essential for Th17 immunity and therapeutic efficacy of the vaccine.
Phospholipase A2 group IID is indispensable for coronavirus-specific humoral immune memory in middle aged mice
Authors: Zheng Jian , Perlman Stanley
Published: January 2023

Phospholipases hydrolyze phospholipids to release long-chain fatty acids that are further metabolized sequentially by cyclooxygenases and synthases to yield various lipid mediators. Phospholipase A2 group IID (PLA2G2D) is a member of phospholipases family and shows age-dependent increases in the lungs. In our previous study, we found that infection of mice lacking PLA2G2D expression (Pla2g2d(−/−) mice) converted a lethal severe acute respiratory syndrome-coronavirus (SARS-CoV) infection to a nonlethal one (>80% survival), subsequent to enhanced respiratory dendritic cells (DCs) migration to the draining lymph nodes, augmented antivirus T cell responses, and diminished lung damage. However, the long-term effects of PLA2G2D on host immune system against coronavirus remained unknow. In this study, we compared coronavirus-specific cellular and humoral immune memory induced by immunization or infection in middle-aged Pla2g2d−/− mice and wild type mice. Surprisingly, virus-derived replicon (VRP)-immunized or sublethal dose-infected Pla2g2d−/− mice failed to generate virus-specific neutralizing antibodies, which was accompanied by a lack of antibody-producing plasma cells and reduced follicular T cells (Tfh) compared to wild type mice. We hypothesize that PLA2G2D deficiency in myeloid cells, likely DCs, play a critical role in the formation of CD4 Tfh cells and humoral immune memory against respiratory coronavirus infection.
Characterization of HLA-DP and DQ restricted dengue virus-specific CD4+T cell responses reveals lower magnitude responses associated with a differential pattern of immunodominance as compared to DR restricted responses
Authors: Weiskopf Daniela , Grifoni Alba , Angelo Michael , Moore Eugene , Sidney John , Phillips Elizabeth J , Mallal Simon Alexander , de Silva Aruna D , Peters Bjoern , Sette Alessandro
Published: January 2023

Background

Assessment of adaptive immunity is an important element to understand immunopathology and to define the correlates of protection against dengue virus (DENV). While previous studies characterized responses restricted by the HLA DRB1 locus, the responses associated with the DP and DQ loci have not been characterized to date. Accordingly, here we mapped HLA DP and DQ restricted DENV-specific CD4 T cell epitopes in PBMCs from individuals previously exposed to DENV from the general population of the Sri Lanka.

Methods

We studied the DP (*0101, *0201, *0401, *0402) and DQ (*0501, *0602, *0301, *0302, *0201) allelic variants which provide a greater than 80% population coverage worldwide. CD4+ T cells purified by magnetic bead negative selection were cultured for two weeks with autologous APC and pools of HLA –specific predicted binders and tested for reactivity in a standard IFNγ ELISPOT assay.

Results

Magnitude of responses for both the DP and DQ loci were significantly lower than previously observed for the DRB1 locus. This was correlated with lower membrane expression of HLA- DP and DQ molecules on the PBMCs as compared to HLA DR. While responses against Capsid were dominant for the various DR and DP alleles this protein was hardly recognized by DQ alleles. Responses against NS3 were dominant in the case of DR and DQ but absent in the case of DP, and response to NS1 were prominent in the case of the DP alleles, but negligible in the case of the DR and DQ alleles tested.

Conclusion

The HLA DP and DQ loci mediate DENV-CD4 specific immune responses of lower magnitude as compared to HLA DRB1. The responses mediated by the DP and DQ loci are also associated with distinct and characteristic patterns of immunodominance.
EVALUATION TH17 AND TREG CELLS DURING CHRONIC CHIKUNGUNYA INFECTION
Authors: Guerra-Gomes Isabel Cristina , Leite Jefferson , Santana Alynne , Macêdo Bruna , Fonseca Rephany , Keesen Tatjana , Carregaro Vanessa , Silva João
Published: January 2023

Our aim was to evaluate the role of Th17 and its balance with Treg cells in defining CHIK chronification pathogenesis, as well as its association with clinical scores. We hypothesized that given that Chikungunya (CHIK) is caused by an arthritogenic alphavirus and possess several similarities with rheumatoid arthritis (RA), both characterized by Th17 cells playing a pathogenic role, in association with limited studies on the role of T cells regarding this CHIK chronification process. To reach this aim, without suitable animal models of CHIKV to properly assess the development of disease clinical signs, we developed a model of CHIK in DBA1/J mice associated with collagen-induced arthritis (CIA). Virus titration was obtained through One Step qPCR. Flow cytometry analyses were performed through cells isolation of draining lymph nodes from CHIKV previously infected (n=6), CIA (n=6) and CHIKV+CIA (n=7). As for humans, peripheral blood samples were obtained, controls (n=6), acute patients (n=6) and chronic patients (n=6). It was observed that our model was able to address clinical scores, such as develop joint swelling. Results showed that association with CHIKV infection increased clinical scores, number of paws involved, and the incidence of arthritis. Analyses of transcripts demonstrated an increased IL-17a and FoxP3 mRNA, however anti-inflammatory cytokines, such as IL-10, was diminished in animals infected with CHIKV. In humans, Treg frequencies presented diminished Treg number and percentage in patients in the chronic and acute stage compared with control individuals. Therefore, our results suggest an important role of T cell response in CHIK infection chronification.
Cytotoxic CD4 T cells control host immunity during viral persistence
Authors: Thiault Nicolas , Husain Mushtaq , Chen Angeline , Verstichel Greet , Larange Alexandre , Patil Veena Shivagouda , Vijayanand Pandurangan , Kronenberg Mitchell , Cheroutre Hilde
Published: January 2023

The CD4 T helper cell lineage is initially established and controlled in the thymus by the transcription factor, ThPOK. Sustained expression of ThPOK by mature T cells in the periphery maintains the CD4 T helper commitment and allows for plasticity and differentiation to different helper fates (Th1, Th2, Th17...) in response to stimulation with cognate antigens. Our group demonstrated before that in the intestine, repeated stimulation of mature CD4 T cells results in the loss of ThPOK expression followed by the reprogramming of the T helper cells to CD4 cytotoxic T lymphocytes (CTL), expressing notably CD8 T cells attributes (such as Runx3, granzyme B).

Continuous activation is also characteristic of chronic viral infections. We therefore investigated if viral infections lead to the generation of reprogrammed CD4 CTL and moreover if these reprogrammed CD4 CTL play essential roles for the organism’s protection. Using the well-defined lymphocytic choriomeningitis virus (LCMV) acute (strain LCMV-Amstrong) versus chronic (LCMV-Clone 13) infection model, we observed that solely chronic infection enables activated CD4 T helper cells to lose ThPOK and convert to CTL with potent killer activity. Moreover, viral-induced CD4 CTL precursors produce various cytokines, notably Il-10, which is required for the induction of the reprogramming process. Finally, we engineered two unique mouse model systems, in which either all the CD4 T cells are forced to reprogram to CTL (gain of function) or else, prevented from reprogramming to CTL (loss of function). Using these models, we show that the reprogrammed CD4 CTL generated under chronic infection conditions, play critical roles in protective immunity.
Investigating contributions of the accessory molecule HLA-DO to development of autoimmune diseases
Authors: Welsh Robin A , Song Nianbin , Foss Catherine , Boronina Tatiana , Cole Robert , Sadegh-Nasseri Scheherazade
Published: January 2023

Used biochemical and biophysical studies, our laboratory has previously shown that HLA-DO (DO) enhances binding of DM-insensitive peptides, and reduces binding of DM-sensitive peptides to HLA-DR1 molecules. We hypothesized that these properties of DO might be important for ensuring proper presentation of immunodominant epitopes in the thymic medulla, a main DO expressing area; thereby ensuring proper negative selection. To test this hypothesis, we investigated the development of experimental autoimmune encephalomyelitis (EAE) in mice that were H2-O wild type (DO-WT), or knock-out (DO-KO). With this model, we found that DO-KO mice displayed faster disease onset in comparison to DO-WT mice. Accelerated disease onset correlated with increased recovery of MOG specific CD4 cells at all stages of disease in DO-KO mice. More recently, these observations were confirmed with a second autoimmune disease model, collagen induced arthritis (CIA), in transgenic HLA-DR1 expressing mouse. Using in vivo Near Infrared Fluorescence (NIRF) imaging, we found that DR1+DO-KO mice had increased levels of active collagen degradation relative to DR1+DO-WT mice. Diseased DR1+DO-KO mice were also found to have a greater accumulation of CD4 T cells in the affected areas. These observations suggest that DR1+DO-KO mice have larger pools of collagen specific CD4 T cells. Indeed, DR1+DO-KO mice immunized with the immunodominant collagen epitope showed an increased percentage of collagen specific CD4 T cells as compared to DR1+DO-WT mice. As a whole, data from both autoimmune models suggest that loss of DO function leads to an increased peripheral pool of autoreactive CD4 T cells, likely due to faulty negative selection.
CpG-A and CpG-B oligonucleotides differentially regulate MR1-mediated microbial Ag presentation
Authors: Liu Jianyun , Brutkiewicz Randy , Liu Sheng , Wan Jun
Published: January 2023

Mucosal-associated invariant T (MAIT) cells are conserved innate T cells that express a semi-invariant T cell receptor (TCR; Vα7.2 in human and Vα19 in mice). MAIT cells are activated by microbial Ags presented by the major histocompatibility complex (MHC)-related molecule, MR1. Little is known how MR1 molecules are processed and loaded with microbial Ag in APCs. We have previously found that Toll-like Receptor 9 (TLR9) is important in regulating MR1-mediated bacterial Ag presentation to MAIT cells in B cells. We further demonstrated that, similar to human B cells, CpG-A (but not CpG-B) upregulated MR1 expression on mouse B cells as well. CpG-A treatment increased MR1 expression on WT mouse B cells but not MR1 KO B cells, further confirming that activation of B cells by CpG-A specifically enhances MR1 surface expression. Initial RNAseq analysis has indicated that B cells activated by CpG-A are distinctively different from those activated by CpG-B or the vehicle control. In conclusion, CpG-A and CpG-B differentially impact MR1 expression and microbial Ag presentation. We have successfully deleted the expression of TLR9 and its downstream mediators in a human B lymphoblastoid cell line (B-LCL) using CRISPR technology. Future work will focus on how genes altered by the activation of CpG-A (but not CpG-B) regulate MR1-mediated microbial Ag presentation.
Depletion of embryonic macrophages impairs vascular development in the murine neonatal small intestine and increases mortality in an experimental model of Necrotizing Enterocolitis
Authors: Managlia Elizabeth , Yan Xiaocai , De Plaen Isabelle G
Published: January 2023

Necrotizing enterocolitis (NEC) is a devastating disease common in premature infants. We previously found that the Cx3cr1+ population and the microvasculature were disrupted in the small intestine (SI) of pups subjected to an experimental NEC model. In neonates, embryonic macrophages (Mφ) dominate the SI Cx3cr1+ population while the adult SI Mφ population expands at weaning to include bone marrow derived-Mφ. Here, we investigated the Cx3cr1+ population in the neonatal (24hr) SI and evaluated its role in vascular development and NEC. We confirmed the Cx3cr1+ population was predominately embryonic by performing flow cytometry on the Lamina Propria (LP) of pups compared to adults using the recently described CD11b+CD64+Tim4+ embryonic phenotype (67.67±8.6 vs 28.35±1.44; p≤0.05). Subsequently, we isolated Cx3cr1+ cells and used real time RT-PCR to establish greater expression of the embryonic-associated genes Tim4 (0.74±0.19 vs 0.22±0.09; p≤0.05), IGF-1 (0.69±0.13 vs 0.26±0.14; p≤0.05) and Lyve (0.49±0.1 vs 0.04±0.02; p≤0.01) in the Cx3cr1+ LP population of neonates compared to adults, while monocyte-expressed L-selectin was heightened in the adult Cx3cr1+ LP population (0.91±0.24 vs 3.2±1.1; p≤0.05). We next found that isolated neonatal SI CD31+ endothelial cells formed tubules on matrigel when co-cultured with Cx3cr1+ cells from the neonatal SI but not from the adult. Finally, in utero depletion of embryonic Mφ via anti-CD115 antibody injection at E6.5, resulted in disorganized vasculature and increased susceptibility to NEC mortality. These data show that the embryonic Cx3cr1+ population plays a role in the development of the SI vasculature and that disrupting this compartment enhances NEC.
Mapping the generation and fate of Tregs associated with recovery of Autoimmune Disease
Authors: Lee Darren J , Muhammad Fauziyya
Published: January 2023

Experimental autoimmune uveitis (EAU) is used to gain a better understanding of human autoimmune uveitis. Resolution of EAU is in part due to emergence of ocular antigen specific regulatory T cells (Post-EAU Tregs) that are found in the spleen of post-EAU mice. However, the stability of these cells and in what tissues these cells migrate to suppress disease is not known. Therefore, we assessed the stability of post-EAU Tregs, asked where these cells migrate to suppress disease, and determined when and where they emerge during EAU. FoxP3GFP-Cre; Rosa26stopfl/fl-Tom mice were used to identify FoxP3 and exFoxP3 T cells. Post-EAU FoxP3 or exFoxP3 cells were adoptively transferred into recipient EAU mice. The post-EAU exFoxP3 cells did not suppress EAU in the recipient mice, but Tregs capable of suppressing uveitis had stable FoxP3 expression and were found in both the eye and lymphoid tissues at the onset of EAU in recipient mice. In donor mice, both FoxP3 and exFoxP3 cells emerged in the eye and were found in the lymphoid tissues throughout EAU. In contrast, neither FoxP3 nor exFoxP3 T cells were found in the eyes of post-EAU mice, rather were found in lymphoid tissues alone. This study elucidates a dynamic relationship of Treg migration from the eye to secondary lymphoid tissues that coincides with the resolution of EAU. Also, this study highlights that FoxP3 is a requirement for the suppressive function of post-EAU Tregs, and these Tregs traffic to the eye to prevent a uveitic relapse but migrate out after resolution. This work suggests that unstable FoxP3 expression or aberrant tissue trafficking may provide an explanation for chronic and relapsing autoimmune uveitis.
Osteopontin promotes survival of intestinal intraepithelial lymphocytes and protects against colitis
Authors: Nazmi Ali , Greer Michael J. , Hoek Kristen L. , Piazuelo M. Blanca , Weitkamp Joern-Hendrik , Olivares-Villagomez Danyvid
Published: January 2023

Intestinal intraepithelial lymphocytes (IEL) comprise a diverse population of cells that reside in the epithelium at the interface between the contents of the intestinal lumen and the sterile environment of the lamina propria. Because of this anatomical location, IEL are considered critical components of intestinal immune responses. Indeed, IEL are involved in many different immunological processes ranging from pathogen control to tissue stability. However, maintenance of IEL homeostasis is incompletely understood. In this report, we present evidence that osteopontin, a glycophosphoprotein with diverse roles in biomineralization, cell-mediated immunity, and inflammation, is important for maintaining normal levels of IEL. Mice in which the osteopontin gene (Spp-1) is disrupted present decreased levels of IEL subtypes, such as TCRαβ and TCRγδ IEL in the intestine, an effect not observed for lymphocytes in other immune compartments such as spleen or lamina propria, indicating an epithelium-specific effect. In vitro experiments show that mouse and human IEL survival is improved by culture with recombinant osteopontin. In vivo and in vitro IEL survival studies show that CD44, a ligand for osteopontin, is critical for the capacity of osteopontin to promote IEL survival. Adoptive transfer of total T cells (including Tregs) from wild type mice into Spp-1−/−Rag-2−/− mice results in the development of exacerbated intestinal inflammation, as compared with Rag-2−/− recipient mice. Further analyses show that osteopontin-deficiency results in decreased survival of Foxp3-expressing T cells. These findings support osteopontin’s critical role in IEL homeostasis and make it a potential target for curbing intestinal inflammation.
Short-term antibiotic treatment immediately after weaning prevents spontaneous ileocolitis
Authors: De Ciantis Christopher L , Jeschke Jonathan C , Hadiono Matthew A , Ziegelbauer Jennifer , Singh Selina , Suchi Mariko , Salzman Nita H , Williams Calvin B
Published: January 2023

A spontaneous model of ileocolitis, a condition found in a majority of cases of Crohn’s Disease, allows for the study of processes that promote homeostasis within the ileum. Our system uses a “Bigenic” approach that combines TCR transgene mice with a transgenic line expressing the cognate antigen in ileal crypts. Bigenic mice exhibit decreased nTreg production that predisposes them to developing a chronic ileocolitic inflammatory disease in the weeks after weaning. Half of Bigenic mice accumulate ileal-reactive Treg cells and fail to develop colitis. In contrast, Ifng−/− Bigenic mice are unable to establish this homeostatic state, resulting in a fully penetrant and acute colitis. We aimed to identify time sensitive interventions capable of preventing colitis and restoring mucosal homeostasis in Ifng−/− Bigenic mice. Mice were started at weaning on a prophylactic treatment with ad libidum non-absorbable antibiotics in their drinking water and continued for three weeks. Alternately, mice were allowed to naturally progress into a disease state at which time the same antibiotic treatments were initiated and continued for nine weeks. We found that antibiotics prevented ileocolitic disease when initiated at weaning but were insufficient to reverse established disease measured by weight loss. Interestingly, this resistance to ileocolitis mediated by short-term early use of antibiotics was maintained, as these mice did not subsequently manifest either ileocolitis or the degree of breakthrough weight loss observed in antibiotic-rescued mice. Our findings suggest that in our model of ilieocolitis, there is a critical window of time after weaning where manipulation of the microbiome alone can facilitate resistance to spontaneous disease.
Targeting gut inflammation inhibits alcohol-induced liver disease
Authors: Deng Zhongbin , Chu Shenghui , Gu Xuemei , Liu Min
Published: January 2023

Alcoholic liver disease (ALD) is one of the leading causes of liver diseases and liver-related death worldwide, but little is known about its influence on the intestinal immune system. Here we show that the gut immune system is altered during alcohol intake and is a functional regulator of alcohol-related liver injury that can be exploited therapeutically. Alcohol intake induces a chronic phenotypic pro-inflammatory shift in immune cell populations in gut lamina propria and results in the disruption of intestinal barrier. Treatment of wild-type C57BL/6 mice with the local gut anti-inflammatory, mesalamine, decreases alcohol-induced liver injury, reverses gut inflammation and improves metabolic parameters. Mesalamine reduced gut permeability and endotoxemia, increased IgA secretion and is associated with reduced Akt activation and P-b-catenin levels in the ileum and large intestine. These beneficial effects are dependent on adaptive and gut immunity. Thus, targeting gut inflammation with mesalamine represents a novel treatment approach for alcoholic liver disease.
Lingo3 interacts with TFF2 to control mucosal integrity, Type 1 inflammation, and colitic tissue repair
Authors: Zullo Kelly , Ji Yingbiao , Wei Yun , Herbine Karl , Maloney Nicole , Cohen Rachel , Pastore Christopher , Somsouk Ma , Srivatsa Sriram , Hung Li-Yin , Kohanski Michael , Cohen Noam , Herbert De’Broski
Published: January 2023

Constant exposure of intestinal epithelial cells (IEC) to potentially damaging stimuli makes mucosal repair programs essential for gut homeostasis, immunological quiescence and resistance to colitis. Trefoil factor 2 (TFF2) is a mucus associated protein that promotes epithelial barrier integrity in the lung and intestine, but whether a bona-fide TFF2 receptor exists remains controversial. Herein, we provide evidence that leucine rich repeat nogo interacting protein 3 (LINGO3) is a transmembrane component of TFF2 signaling and proliferation within IEC. TFF2 requires LINGO3 for barrier recovery in scratch-wound assays and mice lacking LINGO3 (LINGO3KO) have impaired intestinal barrier function under steady state conditions. Interestingly, LINGO3KO mice phenocopy TFF2KO mice, with both having a significant accumulation of mucosal CD4+TH1 cells expressing IFNg+ TNFa+ under steady-state conditions and impaired recovery from DSS-induced colitis. Impaired recovery in LINGO3KO animals was marked by reduced intestinal crypt regeneration and reduced expression of the stem cell marker LGR5 compared to WT counterparts. Additionally, TFF2 agonist administration (TFF2-Fc) promoted enhanced recovery and limited inflammatory cell recruitment in WT mice during DSS-induced colitis whereas TFF2-Fc treatment was ineffective in LINGO3KO animals. Combined, these data support our contention that a TFF2-LINGO3 ligand/receptor axis regulates tissue repair and inflammation within the gastrointestinal tract through regulation of IEC function.
Human neural stem cells induce central nervous system specific regulatory T cells from the ex Treg pool and promote repair in models of multiple sclerosis
Authors: Greilach Scott A , McIntyre Laura L. , Hasselmann Jonathan , Othy Shivashankar , Nguyen Quy , Kessenbrock Kai , Cahalan Michael D , Blurton-Jones Mathew , Lane Thomas , Walsh Craig M
Published: January 2023

Multiple Sclerosis (MS) is a chronic inflammatory autoimmune disease affecting the central nervous system (CNS) and for which there is no cure. Current treatments focus on suppression of the immune system but fail to repair the resulting damage to the CNS. Neural stem cell (NSC) transplantation is a promising therapeutic strategy for treating neurodegenerative diseases through cell replacement and repair however it is unclear how these cells would mediate repair in MS. We report that human NSCs promote CNS specific T regulatory cells (Tregs) which activate endogenous repair pathways and promote remyelination in a murine model of MS. We observed remyelination, decreased inflammation and an increase in (CNS)-infiltrating CD4+CD25+FoxP3+ Tregs in EAE mice receiving an intra-spinal transplant of NSCs. Recovery was not a result of cell replacement, as NSCs underwent xenograft rejection, and was Treg dependent, as ablation of Tregs abrogated histopathological improvement. RAG2−/−2D2 (R2D2) mice, which bear a TCR repertoire restricted to myelin oligodendrocyte glycoprotein (MOG) and neurofilament, lack CD25+FoxP3+ Tregs under homeostatic conditions; however, upon exposure to MOG, R2D2 mice developed CD25+FoxP3+ Tregs in cervical lymph nodes and the spinal cord. hNSCs also promoted Tregs in vitro in co-cultures with wild type B6 and R2D2 splenocytes, but not with RAG2−/− OT-II+ splenocytes. Additionally, hNSC-Tregs also appear to derive from the exTreg pool suggesting both antigen specific expansion and antigen dependent maintenance of FOXP3 in CNS-specific Tregs. hNSC Tregs also have a unique expression profile and express transglutimase-2 which is implicated in oligodendrocyte dependent repair in the CNS.
Non-redundant requirement for CXCR3 signaling for effective treatment of CNS autoimmunity with type I interferon
Authors: Chen Jun , Chong Wai Po , Wang WeiWei , Li Chunmei , Gery Igal , Caspi Rachel R
Published: January 2023

Type I interferons (IFNs) have shown therapeutic potential in treating CNS autoimmune diseases (IFN-β for multiple sclerosis and IFN-α for uveitis), but treatment is not always effective. Here, we describe a non-redundant requirement for GαI-coupled CXCR3 in the immunomodulatory actions of type I IFNs that culminates in the suppression of human uveitis and experimental autoimmune uveitis (EAU). Effective treatment with IFN-α/β inhibited autopathogenic CD4+ T cell migration to effector sites in mice by upregulating expression of the cognate ligands CXCL9, CXCL10 and CXCL11, causing ligand-mediated downregulation of CXCR3 expression and effector T cell retention in the spleen. These effects of IFN-α/β also required IFN-γ. In the absence of CXCR3, type I IFNs were ineffective in treating active EAU. In patients with uveitis, disease exacerbations associated with reduced serum IFN-α concentrations. Importantly, IFN-α/β reduced CXCR3 expression and human effector T cell migration, and these parameters markedly associated with IFN-α therapeutic efficacy in uveitis patients. Our findings provide new insights into the molecular basis of type I IFN therapy for CNS autoimmune diseases and identify CXCR3 as a critical biomarker for effective immunotherapy with type I IFNs.
Antitumor effect of metformin: what about Burkett’s lymphoma?
Authors: Wan Xiaochun , Zhang Guizhong , Huang Shiran , Liu Zhao , Liu Lvyan , Lu Zhen , Tang Chao
Published: January 2023

Metformin, the first-line drug in the treatment of type 2 diabetes, has been reported to possess antitumor activity. However, the roles and detailed mechanisms of metformin in Burkett’s lymphoma are unclear, though it has been suggested to act, at least partially, by mitochondrial complex I inhibition in several tumors. Here, we show that metformin promotes cell death of Burkett’s lymphoma cells but has no effect on the survival of healthy human PBMCs. The pro-apoptotic activity of metformin does not depend on AMPK activation and ROS generation because neither inhibition of AMPK by Dorsomorphin nor scavenging ROS by NAC can block metformin-induced cell death. Moreover, metformin impairs mitochondrial function and induces mitophagy. However, inhibition of the autophagic process by 3-MA promotes the death of these cells, indicating that mitophagy may be a protective factor in cell survival after metformin treatment. Overall, metformin induces cell death via an AMPK- and ROS-independent, mitochondria-mediated manner, which provides new insights into the mechanism of metformin’s antitumor function and suggests that metformin represents a promising and attractive agent for Burkett’s lymphoma therapy.