bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxidative phosphorylation controls regulatory T cell suppressor activity of in the tumor microenvironment
Authors: Maj Tomasz , Wang Wei , Kryczek Ilona , Zou Weiping
Published: January 2023

Regulatory T cells (Tregs) are important elements of the tumor-associated immunosuppresive network. Here we show that Tregs in the tumor microenvironment are highly apoptotic. Although apoptotic Tregs express classical immunosuppressive proteins including PD-L1, CTLA-4, TGF-β and IL-35, blockade of these mediators does not abolish their suppressive effect. Apoptotic Tregs release a large amount of ATP and suppress T cell responses via adenosine A2A receptor. Furthermore, Tregs have higher mitochondrial load and their survival is associated with OxPhos and fatty acid β-oxidation. Apoptosis of Tregs leads to pannexin-1-dependent ATP release and provide a substrate for immunosuppression. The data support a novel notion that Tregs amplify their suppressor capacity via apoptosis and OxPhos may be a central metabolic pathway controlling Treg functionality in tumor.
Suboptimal influenza vaccination results in higher numbers of pulmonary macrophages upon influenza challenge in mice
Authors: Schotsaert Michael , Garcia-Sastre Adolfo
Published: January 2023

Macrophages are innate immune cells present in the airways and are important for the initial immune answer against invading pathogens. Alveolar macrophages (AM) are transiently lost in the mouse-influenza infection model during the first days of infection. During later stages of infection, AM recover to similar numbers as before challenge. We investigated if AM were protected during experimental sublethal influenza challenge after vaccination with an alum-adjuvanted trivalent inactivated influenza vaccine (TIV) that antigenically matches the challenge virus. TIV vaccination could not prevent morbidity, however, it correlated with lower viral lung titers and faster recovery after challlenge. AM were completely abolished at seven days post infection in negative control mice, but not in TIV-vaccinated mice. Taken together, using the mouse influenza infection model we observed that suboptimal vaccination with TIV results in higher levels of AM in lungs upon sublethal challenge with homologous virus. The possible impact of higher levels of pulmonary macrophages on antigen presentation and disease modulation are further investigated.
A combinatory mucosal vaccine against influenza and botulism
Authors: Yan Yongyong , Li Junwei , Chen Yanping , Wu Lihong , Wang Haiyan , Diaz-Arevalo Diana , Zeng Mingtao
Published: January 2023

Botulinum neurotoxins (BoNTs) are the most poisonous substances known in nature and cause the life-threatening botulism. Currently, there is no FDA licensed botulism vaccine available for public use. Influenza is still an important problem for public health and current influenza vaccines are not optimal for protection against seasonal and potential pandemic influenza. Influenza virus is a negative, segmented RNA virus without DNA intermediate. This makes it safer as a vaccine delivery vector than most DNA viruses that have potential to integrate their genetic elements into host genomes. We hypothesize that a live-attenuated influenza virus expressing botulinum antigens can be developed to a dual use vaccine against both botulism and influenza. Based on influenza virus H1N1 PR8, an engineered influenza viral vector with a 2A cleavage site on NA, was constructed. Using this vector, we generated a recombinant influenza virus, PR8/HC50-rbsd, expressing the receptor binding subdomain of the heavy chain C-terminal receptor binding domain (HC50-rbsd) from BoNT/A. We tested the growth characters of the recombinant PR8/HC50-rbsd virus in chicken eggs and Madin-Darby Canine Kidney epithelial cells (MDCK), and showed that it can be produced to a titer of 5×10E6 plaque forming units/ml in chicken eggs and MDCK cells. Subsequently, mice intranasally vaccinated with the PR8/HC50-rbsd virus conferred protection against challenge with lethal doses of active BoNT/A toxin and wild type H1N1 PR8 influenza virus. Our results demonstrated that it is possible to develop a dual purpose nasal vaccine against both botulism and influenza.
Comparison of influenza vaccine regimens in pediatric patients:immunity and efficacy in 2014–15
Authors: Archibald Crystal A , Elbourne Vicki , Moehling Krissy , Susick Michael , Nowalk Patricia , Zimmerman Richard , Zimmerman Richard , Cole Kelly Stefano
Published: January 2023

Currently, there are two types of influenza vaccines available to children and adolescents, Influenza Inactivated Vaccine (IIV) which is administered intramuscularly, and Live Attenuated Influenza Vaccine (LAIV) which is administered intranasally. The present study was designed to compare the efficacy of the two vaccines in pediatric patients in eliciting immune responses and protecting against infection in the 2014–15 influenza season. To conduct the study, patients ranging from ages 3 to 17 years were recruited for vaccination and evaluation. Patients were segregated by age (3 to 8 vs. 9 to17) and vaccine (IIV vs. LAIV). Peripheral blood samples were collected between the months of August and December, 2014 and processed for collection and storage of serum, plasma and peripheral blood mononuclear cells (PBMC). Each child who agreed to participate in the study was prescreened, consented and provided blood samples at three time points: D0 (blood drawn prior to vaccination), D7 and D21 following influenza vaccination. At the end of the vaccination season and once all enrolled patient samples had been collected, antibody responses were evaluated for reactivity to the vaccine strains as well as against additional circulating or heterogonous strains of influenza. Early in the 2014–15 season, it became apparent that the LAIV strain in the vaccine was not well matched to the circulating strain of influenza, resulting in less-than-effective immunity. Therefore, our analyses focused on IIV only. Our results showed that all participants in the study demonstrated higher titers and increased breadth of reactivity to diverse influenza strains when vaccinated with IIV, regardless of prior vaccination history.
Mucosal Vaccination Primes NK Cell-Dependent Development of CD8+ T Cells for Protection Against Pulmonary Brucella Infection
Authors: wang hongbin , Yang Xinghong , Hoffman Carol , Clapp Beata , Pascual David W.
Published: January 2023

Brucellosis is the most common zoonotic disease worldwide, usually transmitted from Brucella-infected livestock after consumption of contaminated foods or by aerosol exposure. Since no vaccines for humans are available, we developed a live, double-mutant Brucella abortus (BADM) strain, that after mucosal vaccination, confers complete protection, but abated in IFN-γ−/− mice. Pulmonary infection of naive mice with wild-type BA fails to recruit innate and adaptive lymphocytes to the lungs. Thus, we hypothesize that BADM’s protection is tied to innate cell stimulation. To test this hypothesis, 5 days after nasal BADM vaccination, mice showed increased lung IFN-γ-producing ILCs and EOMES+ NK cells by 3- and 6-fold, respectively; CD4+ and CD8+ T cells increased 3- and 5-fold, respectively. By 2 wks, CD8+ T cells were the dominant IFN-γ source, being 15-fold > naive lungs. After pulmonary challenge, BADM-vaccinated lungs showed fewer CD4+ T cells, but a net increase in CD8+ T cells unlike naive mice, showed no differences in their numbers of CD4+ and CD8+ T cells. To assess if NK cells impact CD8+ T cells, vaccinated mice treated with anti-asialo GM1 Ab to deplete their NK cells, resulted in increased splenic weights, but reduced lung neutrophil influx 5 days after vaccination relative to isotype-treated, vaccinated mice. By 14 days, NK cell depletion reduced by 4-fold (P<0.001) the IFN-γ-producing CD8+ T cells. Thus, NK cell recruitment enhances protection to pulmonary BA in BADM-vaccinated, but not naive mice, which fail to activate NK cells. Supported by NIH AI123244.
Recombinant spore delivered M. tuberculosis antigens elicit immune response in mice
Authors: Das Kishore , Thomas Tima , Garnica Omar , Dhandayuthapani Subramanian
Published: January 2023

BCG, the vaccine against tuberculosis (TB), is one of the mandatory vaccines administered to infants in several parts of the world. However, BCG conferred immunity wanes when children reach adulthood. To develop booster vaccines, we engineered recombinant Bacillus subtilis spores to deliver Mycobacterium tuberculosis (Mtb) antigens. MTAG1 recombinant strain was designed to express a fusion protein of CotC-Ag85B-CFP10 on the spore coat, while MTAG2 and MTAG3 strains were designed to express Ag85B-CFP10 fusion protein in vegetative cells. The difference between MTAG2 and MTAG3 strains is that the latter expresses secreted listeriolysin (LLO) in addition to Ag85B-CFP10. Immunoblot analysis revealed that MTAG1 strain showed positive signals for Ag85B and CFP10 in the spore-coat and other strains in the vegetative cell extracts, indicating that antigens are expressed in these strains as expected. Splenocytes of mice immunized with recombinant spores, through intranasal route, displayed significantly higher antigen specific proliferation of IFN-γ producing cells compared to the splenocytes of control mice. Also, the culture fluids of splenocytes from recombinant spores immunized mice exhibited higher antigen specific release of Th1 cytokines than the culture fluids of splenocytes from control mice. These results indicate that antigens delivered via recombinant spores can be processed and presented to the immune cells. Additional in vivo studies may reveal whether these recombinant spores can be used to boost immunity in BCG vaccinated individuals.
The immunotherapeutic GEN-003 as a prophylactic vaccine candidate for genital herpes in guinea pigs
Authors: Kaufmann Johanna K , Cardin Rhonda D , O’Keeffe Michael , Baccari Amy , Le Bonnie , Sanz Vinny , Yu David , Flechtner Jessica B , Flano Emilio , Bernstein David I
Published: January 2023

GEN‑003 is a subunit vaccine candidate containing adjuvanted gD and ICP4 antigens that has shown durable therapeutic efficacy against genital herpes in a phase 2 clinical trial. Here we describe the potential benefit of GEN-003 as a prophylactic vaccine in a guinea pig model of genital herpes.

Animals were immunized three times every two weeks with GEN-003 or control and intravaginally challenged with herpes simplex virus 2 (HSV-2, MS strain) 21 days later. GEN-003 significantly reduced frequency and severity of genital lesions in both acute and early stages of recurrent infection by over 85%, and 60% of vaccinated guinea pigs did not develop any lesions during the 33 day follow-up period. Furthermore, viral genome copy numbers recovered in vaginal swabs from GEN-003-immunized animals were 100-fold lower than from controls. Recurrent shedding frequencies were reduced by 27%.

GEN-003 vaccination induced antigen-specific antibody responses at least as strong as those elicited by viral infection. Driven by gD-specific antibodies, HSV-2 neutralizing antibody titers followed the same pattern. After viral challenge, peripheral antigen-specific T cell responses in spleens of GEN-003-immunized animals were lower than those in controls, as measured by interferon-γ ELISpot. This might have resulted from increased antigen-specific T cell recruitment to the genital mucosa, or decreased exposure to the virus due to fewer recurrences. Additional methods to better understand mucosal mechanisms of protection are in development.

In summary, GEN-003 shows promise as a prophylactic vaccine candidate for genital herpes. Ongoing studies will determine if protection can be further enhanced by addition or exchange of antigens and/or adjuvants.
Vaccine-generated lung tissue-resident memory provides heterosubtypic protection to influenza infection
Authors: Zens Kyra D , Chen Jun Kui , Farber Donna L
Published: January 2023

Tissue-resident memory T cells (TRM) comprise a non-circulating subset which provides robust in situ protection upon infection. It is increasingly recognized that vaccination strategies promoting TRM may convey enhanced protection from disease. However, it is unclear whether currently available vaccines elicit TRM and whether this is dependent on vaccine formulation or administration route. In the context of influenza, we assessed lung TRM generation and subsequent protection following vaccination with either injectable inactivated influenza virus (IIV, Fluzone®) or intranasal live-attenuated influenza virus (LAIV, FluMist®) vaccines. We found that IIV induced primarily neutralizing antibodies while LAIV generated robust lung T cell responses. Importantly, LAIV, but not IIV, established persisting lung CD4 and CD8 TRM similar in localization and phenotype to that generated by influenza infection. Furthermore, protection experiments utilizing the immunomodulatory drug FTY720, which sequesters circulating T cells, including TEM and TCM, in secondary lymphoid tissues leaving lung TRM intact, demonstrated that only LAIV provided TRM-mediated protection to viral infection as evidenced by reduced weight loss and lung viral titers in this group following infection. Importantly, TRM generation and protection were dependent on both route and vaccine formulation. The ability of LAIV to generate lung TRM protective against non-vaccine viral strains may have important implications in providing protection in pandemic situations.
How Tolerance Shapes the Development of the HIV-1 Broadly Neutralizing Antibody 2F5
Authors: Finney Joel T , Yang Guang , Kuraoka Masayuki , Nojima Takuya , Verkoczy Laurent , Kitamura Daisuke , Haynes Barton F , Kelsoe Garnett H
Published: January 2023

An effective HIV-1 vaccine is likely to require induction of broadly neutralizing antibodies (bnAbs), which neutralize multiple HIV-1 strains. Generating HIV-1 bnAbs has been challenging due partly to a number of viral immune evasion mechanisms, including viral molecular mimicry of host in order to exploit immunological tolerance. Indeed, generation of neutralizing antibodies to the 2F5 epitope of HIV-1 gp41 is proscribed by immune tolerance. However, the specific mechanisms by which tolerance relieves the autoreactivity of B cells expressing the VH and VL regions (dKI) or the VH region (sKI) of the 2F5 bnAb are not well understood. To compare the reactivity and repertoire of 2F5 dKI and sKI B cells before and after immune tolerance checkpoints, we used a newly developed in vitro single B cell culture system that induces B cell expansion and differentiation into Ab-secreting plasmacytes. The reactivities of expanded B cell clones were determined by ELISA, while repertoire was determined by recovering the V(D)J rearrangements using RT-PCR and Sanger sequencing. In 2F5 dKI mice, the great majority of cells in the small-pre-B compartment express the knock in heavy- and light-chains, and maintain HIV-1 gp41-reactivity. However, 2F5 dKI B cells that survive tolerance checkpoints to become mature B cells have undergone extensive light-chain editing and are purged of gp41- and KYNU-reactivity. Importantly, the peripheral 2F5 dKI IgM−IgD+ (anergic) B cell subset retains a substantial fraction of HIV-1- and KYNU-reactive cells. In conclusion, our study provides mechanistic insights into how immunological tolerance impairs humoral responses to HIV-1, and suggests activation of anergic B cells as a potential target for HIV-1 vaccination.
Early prophylaxis impact for newborns at risk for HIV-1 transmission at birth: viral clearance and attenuation with neutralizing monoclonal antibodies in infant rhesus macaques
Authors: Hessell Ann Jones , Sacha Jonah B. , Kahl Christoph , Graham Barney , Mascola John R , Haigwood Nancy L
Published: January 2023

Our pre-clinical infant macaque model addresses translational implications for early intervention in mother-to-child transmission (MTCT). This model has shown that neutralizing monoclonal antibody (NmAb) therapy given 1 day after SHIVSF162P3 virus exposure intercepts the earliest stages of infection in lymphoid and mucosal tissues. Serial sacrifices revealed that within 2 weeks early viral foci in tissues were eradicated. All animals followed for up to 28 weeks were completely clear of virus infection with no viral rebound after CD8-depletion. Here, twelve 1-month-old rhesus macaques, negative for MHC class I alleles Mamu B*08, B*17 and A*01, were inoculated orally with SHIVSF162P3. Cocktails of NmAbs PGT121 and VRC07-523 were delivered subcutaneously at days 1, 4, 7, and 10 after SHIV exposure. Control groups did not receive NmAbs. Plasma, PBMC, and multiple tissues at necropsy were monitored for viral RNA and DNA. ICS was used to detect Gag and Vif T cell responses. We report that lowering NmAb dosing from 10 to 5 mg/kg given 24 h after virus exposure resulted in only one animal infected, with viremia persisting at about 1 log10 below controls. NmAb treatment at 10 mg/kg delayed for 48 h after oral SHIVSF162P3 exposure resulted in ablated virus infection in 3 of 6 macaques. CD8-depletion resulted in no evidence of virus rebound, indicating the absence of viral latency. In 2 macaques, NmAbs attenuated set-point viremia by 1 – 2 log10, compared to viremia in control animals. These results show that antibodies can have powerful and durable effects on viremia and disease in newborns, but timing and dose are critical. Moreover, post-exposure viral clearance from infected tissue marks a significant advance in pre-clinical platforms to prevent MTCT.
Vaccination route has an impact on level of protection of non-human primates from tuberculosis
Authors: Wong Eileen A , Agger Else Marie , Andersen Peter , Flynn JoAnne L
Published: January 2023

Tuberculosis (TB) continues to be a major global health problem, causing 1.5 million deaths in 2014. To curb TB, several TB vaccine candidates are currently in various stages of testing. We investigated the importance of vaccination route in immune response and protection against TB in a non-human primate (NHP) model. We vaccinated cynomolgus macaques with a protein subunit vaccine (H56) admixed with a novel adjuvant (CAF09) by three routes—intranasal, subcutaneous, and aerosol. Immunogenicity was measured by intracellular cytokine staining of blood and bronchoalveolar lavage samples during and after vaccination. NHPs were challenged with M. tuberculosis, and their immune recall responses assessed by blood and BAL samples. Lung inflammation was tracked during the course of infection by serial PET-CT scans, and extent of disease was assessed at necropsy. Frequencies of T cells producing pro-inflammatory cytokines increased in the blood and BAL following vaccinations in all vaccinated groups, but NHPs vaccinated by aerosol had an earlier recall of T cells to the airways after the third vaccination. After challenge, aerosol vaccinated NHPs had less inflammation by PET-CT compared to other vaccinated groups and unvaccinated controls. The aerosol cohort had similar gross pathology and total bacterial burden to controls at necropsy, but improved bacterial clearance and higher frequency of IFN-γ-producing T cells in the granulomas compared to controls. Although all vaccinated groups had TB disease, this study suggests that the route of vaccination affects the immune response and extent of disease, with the aerosol group having better outcomes. Further studies into the beneficial effects of aerosolizing TB vaccines are warranted.
Tight regulation of memory CD4 T cells limits immunopathology following chronic viral infection
Authors: Penaloza-MacMaster Pablo A , Barouch Dan H
Published: January 2023

CD4 T cells are critical for the induction of adaptive immune responses. Surprisingly, we published a report showing that memory CD4 T cells could also mediate lethal immunopathology following chronic lymphocytic choriomeningitis virus (LCMV) infection. To interrogate whether naive or memory CD4 T cells can similarly mediate immune dysregulation, we transferred equal numbers of naive or memory virus-specific CD4 T cells into naive mice, and infected these with chronic LCMV. We found that memory CD4 T cells exhausted substantially faster than naive CD4 T cells following chronic viral infection. This downregulation of adoptively transferred memory CD4 T cells was associated with improved adaptive immune responses and survival relative to naive CD4 T cells. Our findings demonstrate the importance of inducing multiple arms of the adaptive immune response and suggest that tight regulation of memory CD4 T cells is necessary for preventing immune disease during chronic viral infection.
The role of MAVS signaling in humoral immune responses to West Nile virus.
Authors: Young Lucy B , Giordano Daniela , Draves Kevin E. , Clark Edward A.
Published: January 2023

MAVS is a key regulator of innate immune responses to RNA viruses, such as West Nile virus (WNV). Previous work identified that MAVS is important for the development of adaptive immunity to WNV. MAVS KO mice are highly susceptible to infection with lethal WNV-Tx. MAVS KO mice produced significantly increased levels of WNV-specific IgM and IgG compared to WT mice, however, a corresponding increase in neutralizing antibodies (NAbs) was not observed. As MAVS KO mice succumb rapidly to WNV-Tx, it was not possible in our initial study to define the role of MAVS in the development of the germinal centers (GCs) as well as long-term plasma cell (PC) and memory B cell responses. Using the less pathogenic WNV-MAD, we examined the development of humoral responses in the absence of MAVS. MAVS KO mice survived infection with WNV-MAD, and as with WNV-Tx infection, produced increased levels of IgM and IgG but comparatively low levels of NAbs. This increase in serum Ab levels was maintained up to 9 weeks post-infection (pi). Moreover, the number of WNV-specific Ab secreting cells in the spleen and bone marrow were increased in MAVS KO mice compared to WT mice. Using flow cytometry we found that MAVS KO mice have a slight delay in expansion of GL7+ GC B cells, but by day 10 pi the number of GC B cells was about 10-fold higher than in WT mice. MAVS KO mice also showed a rapid expansion of IgM−CD138hi PCs that was significantly increased compared to WT mice. We are currently testing the hypothesis that these results are due to an intrinsic dysregulation of MAVS KO B cells rather than simply due to an increase in viral load. We are also examining both extrafollicular and GC responses to identify the basis for the rapid IgG antibody responses in MAVS KO mice. (Supported by NIH grant AI83019).
A tissue-specific phenotype for CD8+ T cells in atherosclerotic lesions: role of the arterial microenvironment
Authors: van Duijn Janine , Jiskoot Wim , Kuiper Johan , Slütter Bram
Published: January 2023

Atherosclerosis is a chronic, inflammatory disorder of the arterial wall that causes millions of deaths worldwide each year. CD8+ T lymphocytes may play an important role in atherogenesis, as they represent 29% of all lymphocytes in early human lesions, increasing up to 50% in advanced plaques, however their contribution to plaque initiation and progression remains unclear. Here we show that CD8+ T cells derived from enzymatically digested atherosclerotic lesions of ApoE−/− mice display a unique phenotype. Most notably, lesion derived CD8+ T cells produce less IFN-γ and TNF-α after PMA/Ionomycin stimulation compared to their counterparts in the spleen, circulation or other non-lymphoid organs. Importantly, cytokine production decreases as the lesion progresses to more advanced stages. This effect is not due to classical exhaustion of the T cells, as we found no difference in the expression of exhaustion markers such as PD-1. Nor is this effect due to an increase in regulatory FoxP3+CD8+ T cells. Together, our results suggest that the specific atherosclerotic environment in the vessel wall decreases CD8+ T cell functionality, which provides a new understanding of CD8+ T cells and their role in the pathogenesis of atherosclerosis.
The contribution of the HSCs niche to bone marrow failure in the IL-2 deficient model
Authors: Al-Kuhlani Mufadhal M , Gravano David M , Manilay Jennifer O , Hoyer Katrina K
Published: January 2023

Bone marrow failure (BMF) is a devastating disease where regeneration of peripheral blood lineages is compromised, resulting in anemia and mortality. In IL-2 KO BALB/c mice, the balance between early myeloid and lymphoid progenitor cell populations is skewed resulting in insufficient myelopoiesis with accumulation of CD8 T cells in the BM. It is widely accepted that crosstalk between cells of the bone microenvironment and hematopoietic cells affects each other’s behavior. However, it remains unknown how the BM niche in this mouse model contribute to the BMF. Multipotent stromal cells (MSCs), osteoblasts (OBs) and endothelial cells (ECs) are the three types of BM stromal cells (BMSCs) that may serve as the distinct functional niche to maintain HSCs. Repots have shown the interplay between CD8 T cells and MSCs cause a change in myelopoiesis and that MSCs promote the formation of Tregs. Our data also indicates that IL-2 KO CD8 T cells are important for the expansion of CD4 T cells in the bone. Since IL-2 KO bones are extremely brittle and CD4 T cells produce RANKL, the ligand needed for osteoclast (OC) differentiation, we evaluated the frequency of BMSCs and OCs. Our preliminary data shows clear differences in BMSCs profiles of IL-2 KO mice, namely elevated stromal cell numbers and a shift in Sca-1 expression in ECs and CD51 expression in OBs. Additionally, OC precursor frequency is elevated in the BM, indicating an increase in bone resorption. The ability of the different subsets of the BMSCs to support the differentiation of HSCs in vitro and the role of CD8 T cells in promoting these changes in vivo is being currently evaluated. Overall, our preliminary data suggests that the change in the bone microenvironment may contribute to the BMF observed in these mice.
Thymic expression of tissue-restricted self-antigens is a highly coordinated and evolutionary conserved process
Authors: Rattay Kristin , Meyer Hannah Verena , Brennecke Philip , Reyes Alejandro , Pinto Sheena , Brors Benedikt , Huber Wolfgang , Steinmetz Lars , Kyewski Bruno
Published: January 2023

Promiscuous gene expression (pGE) of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for tolerance imposition in the thymus. PGE is characterized on the one hand by inclusion of a broad range of TRAs and on the other hand by its mosaic patterns, whereby each antigen is only expressed in 1–3% of mTECs at a given point in time. Yet, this mosaic pattern at the single cell level faithfully adds up to the full repertoire of self-antigens at the population level.

In order to analyze the regulatory mechanisms underlying this transcriptional heterogeneity among mTECs, we applied two complementing approaches, the isolation of minor mTEC subsets as defined by TRA-selected gene co-expression groups in conjunction with single cell mRNA sequencing.

Different TRA-selected mTEC subfractions, each expressing distinct sets of genes in a mutually overlapping fashion, mapped to distinct stages of mTEC development. These co-expression patterns were evolutionary conserved between mouse and human (Rattay et al., J. Autoimmunity 2015).

Applying an unbiased single cell mRNA sequencing approach, we extended these findings to the single cell level and showed that the mouse mTEC population essentially represents a composite of multiple co-expression groups (Brennecke et al., Nat. Immunology 2015).

These co-expression groups may represent only snapshots of a continuum of changing co-expression groups along the lifetime of an individual mTEC, as captured in the model of “sliding co-expression groups” (Pinto et al., PNAS 2013). Continuous genome scanning would potentially enlarge the overall diversity of self-antigens displayed by a single mTEC.
Inhibition of IL-23/IL-23 Receptor signaling ameliorates disease in lupus-prone mice by decreasing the generation of DN T cells
Authors: Dai Hong , Kyttaris Vasileios Christos
Published: January 2023

CD4−CD8−B220+ alpha beta (double negative T cells, DN) T cells are expanded in both SLE patients and lupus-prone mice. Based on our previous data (Zhang et al, J Immunol, 2009), we postulate that interleukin-23 is important for the generation of DN T cells. In the present study, we examined B220+ DN T cells in IL-23p19KO lpr mice and IL-23 receptor KO lpr mice by flow cytometric analysis; We also injected IL-23 into IL23p19KO lpr mice to reconstitute IL-23 expression. We observed that IL23p19KO or IL-23RKO lpr mice had reduced disease severity compared to control mice, accompanied by dramatically decreased CD4−CD8−B220+ alpha beta T cells in the peripheral lymph nodes and spleen. Using BrDU incorporation, we observed that there was enhanced proliferation of CD8+ T cells in the thymus of IL23p19KO or IL-23RKO lpr mice vs. controls. We also observed enhanced apoptosis of thymic DN T cells. IL-23 administration in IL23p19KO B6 lpr mice, but not in IL-23RKO B6 lpr mice, resulted in reconstitution of both CD8+T cells and DN T cell population in the periphery to similar levels as the wild type B6lpr mice. Thus, we conclude that deficiency of IL-23/IL-23R signal ameliorates disease in lupus-prone mice partly by decreasing the generation of DN T cells and normalizing CD8+ T cell levels.
Construction of vector pCAG-Z-eGFP-mIR for T cell specific expression of mouse IR
Authors: Al-Dieri Ali G , Morran Michael , McInerney Marcia F
Published: January 2023

Type 1 diabetes is a T cell mediated autoimmune disease. The insulin receptor (IR) is a chemotactic molecule capable of cell movement toward an insulin gradient. In NOD mice, T cells expressing IR can move into the islet causing insulitis and diabetes, whereas IR negative cells do neither. Our aim was to determine if a high density of IR expression is a viable mechanism to deliver T cells to the beta cells regardless of antigen specificity, in strains of mice that do not normally become diabetic. Therefore, we will use a Cre-Lox system with a strong CAG viral promoter and eGFP reporter to drive high IR expression on all T cells. To this end, a viable plasmid was constructed by sub cloning a mIR fragment into a lox vector with CRE recombinase restriction to ensure specific expression. Preliminary data shows successful transfection of this novel plasmid into a HEK293T CRE and NIH/3T3 cell line.
Listeria monocytogenes-encoded expression of a neuroantigen induces CD8 T-cell-mediated inhibition of autoimmune demyelinating disease
Authors: Itani Farah R. , Brate Ashley , Sinha Sushmita , Epping Lecia , Harty John T , Karandikar Nitin J
Published: January 2023

CD8 T-cells predominate in the central nervous system (CNS) lesions of multiple sclerosis (MS), an autoimmune demyelinating disease, yet their function remains controversial. We have previously shown that myelin-specific CD8 T-cells suppress experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Here, we describe a novel system to probe the role of endogenously generated myelin specific-CD8 T-cell responses in CNS autoimmunity. A sequence encoding proteolipid protein peptide PLP178-191 was inserted into attenuated Listeria monocytogenes (LM), deleted for the actA and inlB genes. PLP178-191-specific CD8 T-cell responses were confirmed by infecting B6 mice with the LM-PLP strain and assessing peptide-induced IFN-γ production. We tested the effect of LMP-PLP infection on chronic (B6 mice) and relapsing-remitting (SJL mice) forms of EAE. In both systems, infection with LM-PLP did not result in disease induction. In contrast, LM-PLP infection resulted in significant amelioration of clinical disease in PLP178-191-immunized B6 and SJL mice, compared to LM-OVA-infected controls. LM-PLP infection also effectively suppressed relapses in SJL/J mice. Interestingly, LM-PLP-induced protection was not observed in CD8(−/−) B6 mice, indicating the importance of CD8 T-cells in mediating this effect. Our studies indicate that myelin-specific CD8 T-cell responses generated following LM-PLP175-194 infection are not encephalitogenic and attenuate CNS autoimmunity in multiple mouse models of EAE, further strengthening their role as regulators of disease and their potential as a novel immunotherapeutic strategy.
The role of c-Ski and TGFβ signaling in autoimmunity
Authors: Ong SuFey , Hauri-Hohl Mathias , Ziegler Steven F
Published: January 2023

TGFβ is involved in both Treg and Th17 differentiation via its induction of Foxp3 and RORγt. The oncogene c-ski regulates TGFβ function by inhibiting SMAD2/3 from both binding to SMAD4 and to DNA, repressing the transcription of TGFβ-inducible genes. Previously, we have shown that the absence of ski in medullary thymic epithelial cells (mTECs) limits cellular growth due to unchecked TGFβ signaling. Given that central role of Treg and Th17 cells in the development of autoimmune disease, we explored the role of Ski and TGFβ signaling in T cell function and differentiation. We demonstrate that the absence of Ski in CD4+ T cells leads to amplified TGFβ sensitivity as shown by increased Th17 differentiation at low levels of TGFβ in vitro. Additionally, Foxp3CreSkifl/flT cells polarized in the presence of TGFβ and IL-2 in vitro lead to increased Treg conversion in culture, suggesting that Ski may also play a role in Treg stability. To determine the role of Ski in an autoimmune disease model in vivo, we transferred Ski-deficient or WT naïve CD4+ T cells into Rag1−/− host mice. Ski-deficient T cells induced significantly greater colitis disease severity that was mirrored by increases in double-producing IL-17A and IFNγ pathogenic CD4 T cells. In conclusion, we have shown that Ski functions as a modifier of TGFβ signaling in CD4+ T cells and can directly influence the outcome of autoimmune disease pathogenesis.
Modelling endothelial cells to investigate lymphocyte proliferation under inflammatory conditions
Authors: Kulkarni Aneesha , Westfall Jessica , Segal-Abramson Tzvia
Published: January 2023

Bordetella pertussis is a gram-negative bacteria and the causative agent for whooping cough, a toxin mediated respiratory disease, which causes extensive damage to epithelium in the trachea and bronchial mucosa.

In the absence of a human model to study vascular leukocyte recruitment to the respiratory system, and tissue injury repair, we propose an in vitro model of endothelial cells (ECs) derived from embryonic stem cells (ESCs). We hypothesize that this model may be used to study the proliferation of Th17 and Treg cells during respiratory infection induced with B. pertussis antigens. Previous studies have shown that ECs constitutively express MHC II molecules allowing antigen presentation to recipient CD4+ T cells leading to a pro-inflammatory response. In addition, it has been demonstrated that human allogeneic EC induce Treg and Th17 proliferation.

In this study, we differentiated mouse ESCs in vitro to endothelial cells in the presence of VEGF and bFGF. The cells were enriched for endothelial cell surface adhesion markers CD31 and CD144. Using flow cytometry, qPCR and immunofluorescence, we confirmed the expression of endothelial markers vWF and CD144 with 8% of our population double-positive for CD31 and CD144.

The current model will be used to study pro-inflammatory response and T cell polarization will be assessed by activating endothelial cells with B. pertussis antigens or IFN-γ and then co-culturing with CFSE stained CD4+ T cells in polarizing conditions for 4 days.

We anticipate that this study will contribute important information about endothelial regulatory response to injury and inflammation during whooping cough.
Activation and infiltration of neutrophils in the cerebellum and brainstem is associated with the atypical experimental autoimmune encephalomyelitis phenotype
Authors: Yan Zhaoqi , Liu Yudong , Gibson Sara A. , Lee Kevin , Benveniste Etty N. , Qin Hongwei
Published: January 2023

The JAK/STAT signaling is critical for the initiation, regulation and termination of immune responses, and dysregulation of JAK/STAT signaling is associated with many pathological conditions such as multiple sclerosis. We previously demonstrated that myeloid lineage–specific deletion of SOCS3, a negative regulator of STAT3, resulted in a severe, non-resolving atypical form of experimental autoimmune encephalomyelitis (EAE). Mice with atypical EAE exhibit ataxia and tremors and are characterized by lesions, inflammatory infiltrates, elevated STAT3 activation, and increased cytokine and chemokine expression in the cerebellum. In the current study, our data indicate that the atypical EAE observed in LysMCre-SOCS3fl/fl mice is associated with elevated pro-inflammatory cytokines and chemokines in the cerebellum and brainstem. Greater numbers of infiltrating neutrophils were found in the cerebellum and brainstem of LysMCre-SOCS3fl/fl mice compared to SOCS3fl/fl mice, however, neutrophil numbers in the periphery were comparable. Infiltrating neutrophils in LysMCre-SOCS3fl/fl mice have hyper-activated STAT3 and express higher levels of IL-6 and CCL2. Recruitment of Ly6Chi monocytes in LysMCre-SOCS3fl/flmice is comparable to SOCS3fl/fl mice. During the peak stage of disease, LysMCre-SOCS3fl/fl mice had increased numbers of both Th1 and Th17 cells, and significantly increased numbers of IFN-γ+IL-17+ T cells in the cerebellum and brainstem, compared to SOCS3fl/fl mice. Depletion of neutrophils in LysMCre-SOCS3fl/fl mice prevented the atypical EAE phenotype, rather, classical EAE was observed. This study illustrates the critical function of neutrophils in the pathogenesis of atypical EAE.
Persistence of inflammatory adipose tissue immune cells despite weight loss in obese mice
Authors: Zamarron Brian F , Mergian Taleen , Martinez-Santibanez Gabriel , Luan Danny , Lumeng Carey N
Published: January 2023

Obesity causes dramatic changes to the adipose tissue immune environment including the recruitment and activation of macrophages. The inflammatory polarization of these cells is thought to play a critical role in the severity of insulin resistance associated with obesity. Weight loss can improve insulin resistance, but little is known concerning the effects of weight loss on immune activity in adipose. Diet switch from a 60% high-fat diet to 13% normal-fat diet was used to induce weight loss in diet-induced obese C57Bl/6 mice. Adipose tissue structural changes were evaluated by histology. Leukocyte populations were analyzed by flow cytometry, including sorting and comparing cytokine expression of adipose tissue macrophages before and after weight loss. Weight loss improved glucose tolerance and reduced serum insulin. However, extensive adipose fibrosis developed during weight loss and was associated with macrophage accumulation. Analysis of adipose macrophages after weight loss showed they retained an inflammatory gene expression profile similar to those found during obesity. These macrophages were derived primarily from older cells present during obesity. These macrophages were maintained though high proliferation and low apoptosis. The data indicate that inflammatory activation of adipose macrophages is not sufficient for whole body metabolic dysfunction, but the retained macrophages may promote adipose tissue fibrosis while impacting adipocyte health and function.
Serum amyloid A induces T lymphocyte migration through chemokine receptor CXCR3
Authors: Thompson Aaron J. , Omer Linda , He Rong Lucy
Published: January 2023

Serum amyloid A (SAA) is a major acute-phase protein with cytokine/chemokine-like properties. It can be expressed at sites of inflammation and induce the migration of immune cells including T lymphocytes to the inflammatory sites. SAA plasma concentration can be 1000 times higher in patients with autoimmune diseases such rheumatoid arthritis, than in healthy people. However, its precise role in inflammation and infection remains unclear. SAA is known to bind to several receptors such as FPRL1, TLR2, and TLR4, etc. It is possible that SAA also use other chemokine receptors to recruit immune cells during the inflammation. In our previous study, we used a mathematical approach called Natural Vector Method (NVM) to analyze the genetic information of all major human chemokines and SAA and found that chemokine receptor CXCR3 may be a new receptor for SAA. We reported here that SAA indeed can induce MAP kinase ERK activation in the CXCR3 over-expressed cell line, and trigger the receptor-mediated intracellular calcium mobilization. Our chemotaxis experiments also showed that SAA promotes CXCR3-overexpressed cell migration. These findings indicate that SAA may use lymphocyte chemokine receptor CXCR3 to recruit activated lymphocytes to the inflammation sites. We will further investigate the signaling pathways involved in the CXCR3 mediated SAA-induced cell migration. We hope that our research work not only helps to understand the function of SAA, but also introduces a new way of finding receptors for the endogenous molecules. [This study is supported by NSF grant DMS-1119612 and NIH grant 1SC3GM098180 awarded to Rong L He]
IL-7 receptor pathway in Xenopus laevis
Authors: Coyne William E , Foulkrod Ashley M , Lambert Lisa A , Appasamy Pierette M
Published: January 2023

IL7/IL7 receptor signaling is essential for development of mammalian B and T cells, in particular γδ T cells. The IL7 receptor is a heterodimer comprised of the IL7R α chain and the γ common chain that is shared by other IL2 family cytokines that signal through the JAK-STAT pathway. Since the SCID-like phenotype observed in IL7-knockout mice is less severe than that seen in IL7R knockout mice, there may be other cytokines that signal through the IL7R. In fact, thymic stromal lymphopoietin (TSLP) can substitute for IL7 and signal through the IL7R, at least in mice and humans. We have begun to address the role of IL7/IL7R signaling in Xenopus laevis, in which γδ T cells are abundant, particularly at the larval stages. Although an IL7R α ortholog is present in frogs, BLAST searches of the Xenopus tropicalis genome and gene synteny analyses strongly suggest the absence of IL7. To better understand the function of the IL7 heterodimer receptor in frogs, we have created protein models of both IL7R and the γ common chain (γc). We identified regions of the predicted X. tropicalis IL-7R cDNA that are well-conserved among lower organisms and created primers for use in PCR analyses of cDNA products from oligo-dT primed RNA isolated from the Xenopus laevis thymus. Analysis of the PCR products is currently underway.
IL-6 induces IL-21 and B cell helper function in CD8 cells
Authors: Yang Rui , Rincon Mercedes
Published: January 2023

Interleukin 6 (IL-6) is a pro-inflammatory produced by various cell types in response to external stimuli including trauma, stress and infection. IL-6 signaling plays an important role in immune reaction and progression of several autoimmune diseases by programing CD4 cell differentiation and effector functions. CD8 cells was first believed to primarily function as cytotoxic effector cells, their helper cell effector function has been better understood till recently. During activation, CD8 cells can be primed in different cytokine environments to differentiate into different CD8 effector subtypes, including Tc1, Tc2 and recently identified Tc17. Although the role of IL-6 in CD4 cell differentiation and effector function is well known, how IL-6 may regulate CD8 cell differentiation is less studied. In this study, we show that IL-6 induces IL-21 production in CD8 cells during activation through the transcription factor Stat3. Importantly, IL-6 also differentiates CD8 cells to an effector subset producing high IL-21 but low IFN-γ and IL-2. IL-6R expression level in CD8 cells determines the subset of cells that capable of making IL-21. Similar to IL-21 producing CD4 cells, surprisingly, IL-21 producing CD8 cells provides B cell help by inducing isotype switching and promoting antibody production in vitro and in vivo during influenza A flu infection. Thus, our study suggests that IL-6 could differentiate CD8 cells into IL-21 producing B cell helper subset that may play a unique role in restricting infection and the progression of autoimmune diseases.
Notch signaling directs splenic resident hematopoietic stem cell fate decisions
Authors: Vanderbeck Ashley Nicole , Land Ruben H. , Mumau Melanie D. , Lynch Elizabeth D. , Golec Sophia B. , Punt Jennifer A. , Emerson Stephen G.
Published: January 2023

Adult hematopoiesis occurs primarily in the bone marrow (BM), generating mature blood cells as well as maintaining a heterogeneous pool of self-renewing hematopoietic stem cells (HSCs). However, studies suggest that the BM may not be the only site of adult hematopoiesis: during times of BM hematopoietic distress stemming from radiation or disease, the spleen can independently reconstitute the blood. Given that the spleen does indeed contain a small, resident population of lineage-sca1+ckit+CD48-CD150+ long-term (LT)-HSCs with potent reconstitution ability, we investigated the peripheral blood cell contribution of these extra-medullary HSCs by transplanting UBC-GFP Tg reporter splenic fragments into a splenectomized syngenic wildtype mouse and tracking the blood cell production over time. While the spleen gives rise to both mature myeloid and lymphoid cells, we find that the proportion is skewed in favor of T cells. We see spleen-derived donor HSCs generating CD4+CD8+ double positive thymocytes for over five months post-transplant, suggesting that this peripheral T cell population derives from a long-lived stem or progenitor cell resident in the spleen. RNA sequencing demonstrates that splenic LT-HSCs are more responsive to Notch signaling than BM LT-HSCS, as indicated by increased levels of Hes1 transcription and decreased levels of the granulocyte/monocyte (GM) lineage priming genes Mpo, Gfi1, and Fcgr2b. Taken together with our finding that splenic HSCs have higher levels of cytoplasmic Notch2, we suggest that the Notch2-Hes1 axis is simultaneously repressing GM priming and enforcing T cell programming even among the most immature HSCs resident in the spleen.
Irritant contact dermatitis is regulated by antioxidative enzyme NAD(P)H:quinone oxidoreductase (Nqo1).
Authors: Kitajima Masayuki , Kimura Akihiro , Suzuki Harumi
Published: January 2023

Irritant contact dermatitis (ICD) is the most common occupational skin inflammatory disorder caused by the exposure of chemical or physical irritants. Local release of inflammatory mediators such as cytokines, lipid mediators, and reactive oxygen species (ROS) are known to be important in the establishment of ICD. Antioxidative enzymes such as Nqo1 are known to be responsible for the degradation of ROS. Therefore, in the current study, we focused on the involvement of Nqo1 in ICD.

In the mouse ICD model using croton oil, we found that Nqo1-deficient mice exhibited increased ear swelling and augmented expression of proinflammatory cytokines such as IL-6 and IL-17A in the skin. As we expected, it is indicated that Nqo1 has protective role against ICD. However, it is not applicable for general inflammatory responses, because the knockout mice showed unaltered response in the allergic contact dermatitis mouse model. Interestingly, we found that severe reduction of Vγ5Vδ1+ dendritic epidermal T cells (DETCs) in the skin of Nqo1 deficient mice, which are reported to inhibit ICD. Transfer of DETCs into RAG−/− mice revealed impaired survival of Nqo1-deficient DETCs. In addition, Nqo1-deficient DETCs were sensitive to oxidative stress-induced cell death, and anti-oxidant NAC treatment rescued decreased DETCs in Nqo1-deficient mice. These results suggest that Nqo1 regulates ICD through DETC development and/or homeostasis, possibly by antioxidative function.