bluuebunny/crossref_metadata_2025 huggingface dataset:
E3 ubiquitin ligase NKLAM is important for bacterial killing in a murine model of streptococcal pneumonia
Authors: Lawrence Donald W. , Kornbluth Jacki
Published: January 2023

Natural killer lytic associated molecule (NKLAM) is an important component of the innate immune system. Previous in vitro studies using NKLAM-KO macrophages have demonstrated that NKLAM positively affects the transcriptional activity of STAT1, a transcription factor crucial for proper immune function. Therefore, we hypothesized that NKLAM-KO mice would be unable to mount an effective immune response when exposed to Streptococcus pneumoniae. Using a nasal inhalation infection model we found that the lungs of NKLAM-KO mice had a significantly higher bacterial load than WT mice. This observation was associated with increased lung phosphatase activity and decreased STAT1 phosphorylation. STAT1 regulates a large number of genes, including iNOS and MCP-1, which are important for successful pathogen eradication. In our model, we observed that NKLAM-KO mice expressed less iNOS in their lungs at 24 and 48 hr post-infection in comparison to WT mice. Additionally, infected NKLAM-KO lungs contained less TNF-alpha, IL-12, IFN-gamma and MCP-1 than lungs from WT mice. NKLAM-KO mice had significantly less neutrophil lung infiltration than WT mice, an effect that is likely due to the decreased expression of chemokines including MCP-1. In summary, these novel findings demonstrate the importance of NKLAM in innate immunity and begin to define a role for NKLAM in leukocyte trafficking to the lung upon S. pneumoniae infection.
Autophagy induced by human neutrophil exovesicles controls intracellular growth of Mycobacterium tuberculosis H37Rv in autologous macrophages
Authors: Estrada-Garcia Iris , Alvarez-Jimenez Violeta , Garcia-Martinez Mariano , Vazquez-Flores Luis , Leyva-Paredes Kahiry , Garcia-Perez Blanca , Serafin-Lopez Jeanet , Chacon-Salinas Rommel , Wong-Baeza Isabel , Castañeda-Casimiro Jessica , Ruiz-Sanchez Bibiana Patricia , Estrada-Parra Sergio
Published: January 2023

We have previously shown that human neutrophils (NEU) stimulated with live Mycobacterium tuberculosis H37Rv (Mtb) release exovesicles, named EXO-Mtb. When autologous macrophages were incubated with EXO-Mtb, they increased the expression of CD86 and MHC II and produced inflammatory cytokines. Here we demonstrate that EXO-Mtb can also reduce the intracellular load of Mtb in autologous macrophages. Since autophagy has been shown to control mycobacterial growth, LC3II expression was measured on infected macrophages after incubation with EXO-Mtb (4, 6 and 24h). Control EXO preparations included those released spontaneously from NEU, or after stimulation with PMA or fMLP. Our results showed that of all EXO preparations used, EXO-Mtb induced the largest amounts of LC3II protein, which correlates with a significant reduction of colony forming units (CFU). This phenomenon could be reverted with the addition of Wortmannin, an autophagy inhibitor. It is important to remark that there was not a 100% control of intracellular mycobacterial growth. Further experiments should be done to better understand the role of ectosomes in NEU-macrophage communication through exovesicles.
Adiponectin inhibits eosinophil recruitment and is protective in invasive aspergillosis
Authors: Templeton Steven , Amarsaikhan Nansalmaa
Published: January 2023

Aspergillus fumigatus is a fungal pathogen associated with allergy, asthma, and invasive infection in susceptible individuals. Our previous results suggested that eosinophils may be a novel therapeutic target in invasive aspergillosis (IA) patients that do not respond to conventional pharmacotherapy. Eosinophil recruitment is induced by inhalation of the fungal cell wall polysaccharide chitin. However, the mechanisms of immune regulation that drive this recruitment are not well understood. We observed decreased levels of the metabolic-regulating cytokine adiponectin in the lungs and serum of mice after chitin aspiration, suggesting that adiponectin may be involved in the regulation of type 2 lung immune responses. Co-aspiration of adiponectin with chitin significantly reduced eosinophil accumulation in the lungs, and airway eosinophil recruitment in adiponectin-deficient mice was increased with chitin aspiration. In addition, chitin aspiration resulted in decreased surface expression of the adiponectin receptor AdipoR1 on eosinophils. Most significantly, airway accumulation of eosinophils, disease severity, and fungal burden were increased in adiponectin knockout mice with IA. This may be due to a direct inhibition of eosinophil recruitment, as recombinant adiponectin inhibited chemotaxis of bone marrow-derived eosinophils in vitro. These results suggest that adiponectin inhibits potentially detrimental lung eosinophil recruitment in response to chitin-containing pathogens. Increased understanding of the relationship between metabolic and immune regulation may ultimately result in novel therapies for obesity and allergic and infectious disease.
NK cells require both inhibitory and activation Ly49 receptors to mediate MHC I Dk-dependent MCMV resistance.
Authors: Gamache Awndre Elijah , Nash William , Teoh Jeffrey , Gillespie Alyssa , Brown Michael G
Published: January 2023

The MHC class I molecule, Dk, is a key genetic factor of murine cytomegalovirus (MCMV) resistance. NK cells expressing the C57L-derived inhibitory receptor Ly49G2 (G2) mediate Dk-dependent MCMV control, however, a precise in vivo role for activation receptors has not been delineated. We studied a role for activation receptors in Dk-disparate B6.NKCc57l congenic mice by injecting neutralizing mAbs prior to infection. We found that only Ly49R (R) blockade specifically, and completely, abrogated host resistance. R+ NK cells had a differentiated phenotype (CD27−, CD11b+, KLRG1+, DNAM1−) and displayed higher granzyme B during MCMV infection, in comparison to R− NK cells. R+ G2+ NK cells responding to MCMV also showed higher proliferative potential and cytokine responsiveness compared to R+ G2− NK cells. To further interrogate the role of these NK cell subsets in vivo, we exploited differences in expression of Nkrp1 (NK1.1) and Ly49 receptor alleles in our NKC congenic mouse strains. Endogenous NK cells in H-2 matched host B6.Dk mice were either selectively depleted with mAb PK136 (NK1.1) or neutralized with mAb 3D10 (a-Ly49H), rendering them susceptible to MCMV infection, prior to adoptive transfer of donor B6.NKCc57l NK cells. We found that responding donor NK cells were mostly R+ G2+ and actively expanding in contrast to R+ G2− NK cells. Ongoing adoptive transfer experiments with sorted NK cell populations suggest MCMV control coincides with donor R+ G2+ NK cells. We are continuing to investigate how inhibitory Ly49 receptors modify NK cell responsiveness via activation Ly49 receptors. We infer from these data that inhibitory and activation Ly49 receptors work in tandem to deliver specific NK cell-dependent virus control.
Targeting stem cell-derived viral antigen-specific T lymphocytes for HBV immunotherapy
Authors: Haque Mohammad Shamsul
Published: January 2023

Adoptive cell transfer (ACT) of hepatitis B virus (HBV)-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for chronic HBV infection. Naive or central memory T cell-derived effector CTLs known as “highly reactive” cells are optimal populations for cell-based therapies. However, such ACT is often not feasible due to difficulties in obtaining sufficient numbers of CTLs from patients. Pluripotent stem cells (PSCs) have the capacity to be reprogrammed into functional viral antigen (Ag)-specific CTLs, which have a potential to be used for ACT-based immunotherapy. However, the optimal approach to differentiate viral Ag -specific CTLs from PSCs remains unclear. In this study, we generated HBV-specific CTLs from induced PSCs (iPSCs), i.e., iPSC-CTLs, and utilized a mouse model of chronic HBV infection to test ACT of iPSC-CTLs for HBV immunotherapy. Murine iPSCs that genetically modified with HBV-specific T cell receptor (TCR) were co-cultured with OP9 cell line expressing Notch ligands Delta-like 1 and 4 (OP9-DL1/DL4 cell) for a week and adoptively transferred into recipient C67BL/6 mice for in vivo development. The combination of Notch signaling and incorporation of HBV-specific TCR genes in iPSCs drove the development of HBV-specific iPSC-CTLs. In addition, In a mouse model of chronic HBV infection in which the HBV viral genome is delivered into the mouse liver through hydrodynamic injection, HBV-specific iPSC-CTLs infiltrated into liver tissue, and inhibited HBV replication after ACT. These results confirm that ACT of viral Ag-specific iPSC-CTLs can substantially suppress HBV replication, and indicate that stem cell-derived highly reactive viral Ag-specific CTLs may be used for HBV immunotherapy.
Loss of CCR5 signaling results in early NK cell and neutrophil recruitment into the liver and increased hepatic inflammation in a mouse model of acute hepatitis B
Authors: Stevens Kathleen , Thio Chloe , Osburn William
Published: January 2023

Human genetic studies demonstrate that a non-functional CCR5 receptor sequence variant is associated with enhanced control of acute hepatitis B virus (HBV) infection. To better understand the role of CCR5 in immune responses to HBV, we employed a mouse model of acute HBV in which Ccr5 +/+(WT) or Ccr5 −/−(KO) C57BL/6 mice were intravenously infected with adenovirus containing an overlapping HBV1.3 construct (AdHBV) or vector control (AdShuttle). Mice (N=4–6/group) were euthanized at days 2, 3, 7, 10, and 14 post injection. Increased plasma ALT levels were observed in both Ccr5 WT and KO AdHBV mice at day 7, but levels were significantly higher in the Ccr5 KO compared to WT mice by day 14 (554 vs 274 U/L, p < 0.05). Plasma ALT levels were not significantly different between Ccr5 WT and KO AdShuttle mice. Analysis of intrahepatic leukocytes by flow cytometry revealed significantly increased intrahepatic CD11b+ NK cells (26.3 vs 6.9, % of live leukocytes, p< 0.01) and neutrophils (7.3 vs 1.3, % of live leukocytes, p< 0.05) at day 3 in Ccr5 KO compared to WT AdHBV mice. By day 7, these differences in cell populations were no longer present. At Day 14, significantly higher levels of intrahepatic Ly6c+ macrophages were detected in Ccr5 KO compared to WT AdHBV mice (17.0 vs 6.4, % of live leukocytes, p< 0.01). These differences in intrahepatic leukocytes were not observed in AdShuttle mice. Loss of Ccr5 function did not significantly alter intrahepatic CD4+ and CD8+ populations at the time points investigated. Taken together, these findings suggest that loss of Ccr5 signaling increases intrahepatic innate cells and inflammation during acute HBV infection, providing evidence for a role of CCR5 in regulation of innate immune responses to acute HBV infection.
Role of CD28 superagonist in preventing HSK lesions in mice
Authors: Jaggi Ujjaldeep , Rouse Barry T
Published: January 2023

Ocular infection with herpes simplex virus (HSV) can result in a chronic immune-inflammatory lesion that is a significant cause of human blindness. A key to controlling stromal keratitis (SK) lesion severity is to identify cellular and molecular events responsible for tissue damage. Regulatory T cells (Tregs) are known for preventing autoimmune diseases like systemic lupus erythematosus (SLE), multiple sclerosis (MS), Type 1 diabetes (T1D), rheumatoid arthritis (RA) and Inflammatory bowel disease (IBD). Furthermore, Tregs play an important role in reducing the severity of SK lesions by inhibiting the immune responses which cause immunopathology. In T cell activation and clonal expansion, the costimulatory receptor CD28 and its closest structural relative, the de-activating receptor CTLA-4 (CD152) are of central importance. Both bind to CD80 and CD86 expressed by APC. Interestingly, there is a class of “superagonistic” CD28 specific mAb which are able to preferentially expand and activate Tregs. Previous studies have shown that treating mice with a CD28 superagonistic antibody (CD28SA) ameliorated the acute signs of arthritis and completely prevented the development of chronic destructive arthritis in a mouse model. In the present study, we report that a single injection with CD28SA mAb given at day 3 post infection, reduced the severity of SK lesions. We found increased numbers of Tregs in mice treated with CD28SA. Additional mechanisms involved will be further evaluated during the course of the study. Thus, our studies demonstrate the usefulness of CD28SA as a therapeutic agent to control virus induced immunopathology.
Late-rising CD4 T cells resolve viral persistence.
Authors: Benedict Chris A , McDonald Bryan , Seumois Gregory , El Morabiti Rachid , Ghosh Raima , Fu Zheng , Adler Barbara , Vijayanand Pandurangan , Gupta Ankan
Published: January 2023

Robust CD4 T cell responses help to control chronic infection and restrict disease, but their specific requirements for resolving viral persistence remain poorly defined. During mouse cytomegalovirus (MCMV) infection, CD4 T cells recognizing an epitope derived from the viral M09 protein expand at late times of infection and display a unique phenotype compared to their conventional counterparts, including high IFNγ-production and low expression of the activated CD43 isoform. Ablating these late-rising CD4 T cells by mutating the MCMV M09 genomic epitope, or inducing them through vaccination, revealed their critical role in resolving persistent replication by overcoming IL-10 mediated immune suppression. Together, these data show that unique subsets of late-rising CD4 T cells are qualitatively superior in resolving chronic infection when compared to those that expand earlier, results that should be considered for vaccine development.
IL-33 promotes antiviral CD8+ T cells through the mTORC1 pathway and limits liver injury via recruiting immunosuppressive neutrophils
Authors: Liang Yuejin , Yi Panpan , Jie Zuliang , Yuan Denley Denley Ming Kee , Soong Lynn , Cong Yingzi , Sun Jiaren
Published: January 2023

Viral hepatitis is a major public health problem worldwide. The multifunctional IL-33 is critical for the activity of antiviral cytotoxic T lymphocytes (CTLs) in persistent viral infection; however, the signaling mechanism defining how IL-33 promotes immune cell activation is unclear. In addition, IL-33 can also recruit neutrophils, suggesting that it may regulate liver injury through modulating myeloid cell functions. In this study, we revealed an essential role for endogenous IL-33 regarding antiviral T cell responses in lymphocytic choriomeningitis virus (LCMV) infection. Deficiency of CTL effector activity in IL-33 knockout (KO) mice was restored by recombinant IL-33 treatment. Mechanistically, IL-33 induced CD8+ T cell activation and proliferation through the PI3K/mTORC1 singling pathway. Interestingly, IL-33 treatment also induced significant neutrophil accumulation, but decreased CD8+ T cell numbers, in infected liver. By depleting neutrophils in vivo, we demonstrated that neutrophils played an immunomodulatory role and attenuated liver injury by inhibiting infiltration and the activity of CTLs at the contraction stage of immune response. IL-33-induced immunosuppressive neutrophils expressed higher levels of arginase-1 through type 2 innate lymphoid cells (ILC2)/IL-13/STAT6 axis. This study, therefore, demonstrates a dual role of IL-33 in promoting antiviral CD8+ T cell response at the initial stage, but inducing immunomodulatory neutrophils through induction of ILC2 to limit liver injury during the contraction stage of immune response.
The cytotoxic formulation, INT230-6, induces protective T cell immunity
Authors: Bloom Anja , Bender Lewis H , Walters Ian B , Clark Katharine T , Terabe Masaki , Berzofsky Jay A
Published: January 2023

The benefits of anticancer agents extend beyond direct tumor cell lysis. One major aspect of cell death is the potential to release antigen and induce a long lasting immune response. In this study, a novel tissue and cell diffusive cytotoxic formulation, INT230-6, was administered intratumorally over 5 sequential days into subcutaneous 300mm3 murine Colon26 tumors. Treatment resulted in regression from baseline of 100% of the tumors and up to 80% complete response (CR) with protection from re-challenge. We then analyzed the role of T cells in the protection induced by INT230-6. After CD8 or CD8/CD4 double-depletion at treatment onset no animals achieved a CR, indicating a critical role of T-cells in complete tumor regression. Mice with CRs challenged by subcutaneous re-inoculation of the Colon26 cells also lost protection following CD4/CD8 double-depletion prior to the re-challenge. This result indicates that immunological T cell memory was induced by INT230-6. Colon26 tumors express the endogenous retroviral protein gp70 containing the AH-1 CD8 T cell epitope. AH-1-specific CD8 T cells were detected in tumors of mice without treatment. However, these cells highly expressed PD-1, and did not lead to tumor regression. Hence, INT230-6 given locally to treat tumors appears to induce tumor-specific protective T cell immunity that may overcome suppressive signals or exhaustion. INT230-6 is currently in clinical testing as monotherapy and will be tested in combination with anti-PD1 compounds.
Efficacy of combining radiotherapy and immunotherapy for metastatic colorectal cancer and the modulation of tumor microenvironment
Authors: Wu Chia-Jen , Tsai Yi-Ting , Chang Ching-Cheng , Lee I-Jung , Wu Ping-Yi , Tao Mi-Hua
Published: January 2023

Immune checkpoint blockade antibodies specific to programed death-1/ligand-1 (PD-1/PD-L1) shows durable clinical responses in various cancers but benefit only to a small portion of patients. The failure of immunotherapy is likely due to the complex network of immunosuppressive mechanisms present in advanced tumors. Radiation (RT) modifies tumor microenvironment and causes tumor antigen release, and thus augment efficacy of immunotherapy. Interleukin 12 (IL-12) and granulocyte macrophage colony-stimulator factor (GM-CSF) have shown potent antitumor activities through activating innate and adaptive immunities, as well as reprogramming myeloid cells to a phenotype that favors immune activation. The aim of this project is to compare the therapeutic efficacy of two types of combination therapy: RT combined with IL-12/GM-CSF (RT/12GM) versus RT combined with anti-PD-L1 antibody (RT/PD-L1), and to dissect the molecular mechanisms of the antitumor responses. Our data showed that RT/12GM led to tumor regression in a majority of mice with large established tumors and suppressed liver metastasis, while RT/PD-L1 could only suppress primary tumors but not metastatic tumor. Mechanistic analysis showed that RT/12GM was superior to RT/PD-L1 in inducing accumulation of CD8+ T cells in tumor and in augmenting both NK and CD8+ T cells cytotoxicity in tumor and liver. Interestingly, RT/12GM also induced infiltration of tumor-associated neutrophils (TAN) and impaired their immunosuppressive functions. Depletion experiments showed that both CD8+ T cell and TAN were required for tumor regression in RT/12GM. Together, our results demonstrate that RT/12GM represent a powerful alternative therapy against both primary and metastatic tumors.
Development of a curative therapeutic vaccine (TheraVac) for the treatment of large established tumors
Authors: Yang De , Nie Yingjie , Trivett Anna L , Han Zhen , Chen Xin , Oppenheim Joost J
Published: January 2023

Harnessing immune system to treat cancer requires simultaneous generation of tumor-specific CTLs and curtailment of tumor immunosuppressive environment. Here, we developed an immunotherapeutic regimen capable of eliminating large established mouse tumors using HMGN1, a DC-activating TLR4 agonist capable of inducing anti-tumor immunity. Intratumoral delivery of HMGN1 with low dose of Cytoxan that acts as a functional checkpoint inhibitor cured mice bearing small (diameter ~ 0.5 cm), but not large (diameter ~ 1.0 cm) CT26 tumors. Screening for activators capable of synergizing with HMGN1 in activating DC identified R848. Intratumoral delivery of HMGN1 and R848 plus Cytoxan eradicated large established CT26 tumors. The resultant tumor-free mice were resistant to subsequent challenge with CT26, indicating the generation of CT26-specific protective immunity. This immunotherapeutic regimen caused homing of tumor-infiltrating DC to draining lymph nodes and increased infiltration of T cells into tumor tissues. Cytoxan in this regimen could be replaced by anti-CTLA4) or anti-PD-L1. Importantly, this immunotherapeutic regimen was also curative for large established mouse Renca and EG7 tumors. Thus, we have developed a curative therapeutic vaccination regimen dubbed ‘TheraVac’ consisting of HMGN1 and R848 plus a checkpoint inhibitor, that can, without using exogenous tumor-associated antigen(s), eliminate various large tumors and induce tumor-specific immunity.
Dual antigen transfer pathways mediate highly efficacious anti-tumor immunity induced by intravenously injected antigen-loaded monocytes
Authors: Huang Min-Nung , Nicholson Lowell , Batich Kristen , Kopin David , Prabhakar Vijay , Lipes Barbara , Sampson John , Gunn Michael
Published: January 2023

Dendritic cells (DC) have been key elements in developing effective cancer vaccines to induce potent anti-tumor immunity. However, the common approach adopted in clinical trials using ex vivo generated DC loaded with tumor antigens (Ag) has been challenged by its limited clinical response, complexity, and quality of the manufacturing process. Alternative efforts focused on in vivo Ag loading on endogenous DCs have not yet been well validated in their efficacy for cancer treatment, suggesting the efficiency of in vivo Ag transfer from Ag-carrying vehicles to DCs needs to be further improved. As an attempt to bypass the aforementioned problems, we are testing the hypothesis that Ag-loaded monocytes can induce efficacious anti-tumor T cell responses by directly presenting Ag to T cells through in vivo monocyte-to-DC differentiation. We find that intravenously (IV) injected monocytes loaded with either model or known tumor Ag induce robust Ag-specific CD4 and CD8 T cell responses in mice without triggering antibody responses, display anti-tumor efficacy superior to that seen with traditional adjuvants or RNA-pulsed DC vaccines, and can be combined with checkpoint blockade to increase their efficacy. Unexpectedly, monocyte-induced T cell responses do not require Ag presentation by monocytes themselves, but rather the transfer of Ag to endogenous splenic DC. This transfer occurs via gap junctions for CD8 T cell responses and via macrophages for CD4 T cell responses. These findings demonstrate the existence of efficient dual Ag transfer pathways between monocytes and splenic DCs and suggest that the administration of Ag-loaded monocytes may serve as a simple and efficacious immunotherapeutic platform for the treatment of human cancers.
PCI-based vaccination: a novel method for targeting of MHC class I antigen presentation and stimulation of antigen-specific cytotoxic T cells
Authors: Varypataki Eleni Maria , Kündig Thomas M. , Johansen Pål
Published: January 2023

Cytotoxic CD8+ T cells are key players in fighting cancer and infectious diseases, hence, the design of therapeutic vaccines focuses on ways to prime strong cytotoxic immune responses. Yet, current vaccines efficacy is limited mainly due to the default processing of exogenous antigens via the MHC class II pathway and the activation of CD4+ and not CD8+ T cells. Photochemical internalization (PCI) is a novel intracellular delivery approach, able to circumvent the MHC class II processing. Briefly, after endocytosis of antigen and photosensitizer by antigen presenting cells, light activation of the photosensitizer causes disruption of endosomal membrane and antigen release into the cytosol, resulting in stimulation of MHC class I-restricted CD8+ T cells.

Wild type and MHC class II-deficient mice were immunized intradermally with protein antigens and photosensitizer, followed by controlled light exposure for activation of the photosensitizer. The effectiveness of the PCI to stimulate CD8+ T cells was analyzed by measuring frequency, phenotype, and function of antigen-specific T cells in blood and spleens. PCI-treated mice showed a strong activation and proliferation of specific CD8+ T cells, with improved functions in terms of IFN-g production and cytotoxic capacity. The CD8+ T cell effector functions were not impaired in MHC class II-deficient or in CD4+ T cell-depleted mice, suggesting that the PCI-mediated antigen processing is independent of MHC class II and CD4+ T helper cells.

Our data demonstrate that PCI–based vaccination can stimulation strong and functional cytotoxic CD8+ T cells, making PCI a potential method for use in therapeutic vaccines against cancer and infectious diseases.
Humanized HLA-DQ8αβ transgenic mice treated with SEG/SEI superantigens exhibit long-term CD4+-mediated anti-tumor responses.
Authors: Knopick Peter Leo , Alvine Travis D , Biswas Nivedita , Nilles Matthew L , Terman David S , Bradley David S
Published: January 2023

SEG and SEI are primordial S. aureus superantigens (SAgs) notably unaccompanied by human neutralizing antibodies that have hampered the use of classic SAgs as cancer therapeutics. Here we show SEG/SEI presented from a HLA-DQ8αβ (HLA-DQA*0301 and HLA-DQB*0302) tg platform outperform C57BL/6 mice in achieving long term survival in vaccinated and established Lewis lung carcinoma (LLC) and B16-F10 melanoma. Vaccination of DQ8 tg mice with LLC or B16-F10 melanoma followed by SEG/SEI immunization and subsequent tumor challenge resulted in 100% and 83% survival for >150 days respectively, compared to a median survival of 14 days in unvaccinated DQ8 tg controls (p<0.001). Likewise, SEG/SEI treatment of established melanoma in DQ8 tg mice resulted in 100% survival for 70 days compared to a median survival of 20 days in untreated DQ8 tg controls. Similar vaccinations/challenges in C57BL/6 mice produced a median survival of 23–28 days. SEG/SEI-activated splenocytes from DQ8 tg mice displayed a TH-1/TH-17 cytokine phenotype with a threefold greater T cell proliferative and CD4+-mediated cytotoxicity responses, compared to C57BL/6 mice. These surprising data delineate a clear pathway to translation of an SEG/SEI-HLA-DQ8αβ tg platform for melanoma/lung cancer prophylaxis and treatment.
Probing changes to local and systemic immunity following intranodal injection
Authors: Andorko James I , Gammon Joshua M , Jewell Christopher M
Published: January 2023

Polymeric vaccine carriers protect and control delivery of encapsulated immune cues to target organs such as lymph nodes (LNs), tissues that coordinate adaptive immunity. Our lab recently established a non-surgical intranodal injection (i.LN.) technique allowing for control of the dose and combination of signals that reach inguinal LNs of mice. We discovered that after treating both inguinal LNs with a model antigen (Ovalbumin) and polymeric particles encapsulating a TLR3 agonist PolyIC (PolyIC MP), strikingly large increases to the percentage and number of activated dendritic cells and macrophages led to increases in B and T cells after 7 days. This treatment generated antigen-specific CD8+ T cells within the treated LNs (~3%) and blood (~13%) leading to memory phenotypes 28 days after vaccination. We used this system as a tool to study how local vaccine exposure and persistence impact systemic immunity by priming of one or more immune sites. Matching vaccine doses were deposited via i.LN. injection into one LN, two LNs, or with the antigen and adjuvant split into separate LNs. LNs receiving PolyIC MPs increased dendritic cell activation. Within the treated LNs, the highest percentage of antigen-specific CD8+ T cells occurred after treating one LN while treating two LNs resulted in half as many antigen-specific cells per LN. Interesting, while all treatments increased blood antigen-specific responses, the response to treating two LNs was double that of one LN treatment and six-fold higher than splitting the signals. These results indicate that the localization and kinetics of immune signals influences the magnitude of systemic immunity and could inform the design of future vaccine strategies to mount robust, antigen-specific responses.
Pro-inflammatory properties of orally delivered calcium-encapsulated CpG Oligodeoxynucleotides
Authors: Kayraklioglu Neslihan , Parker Katherine H , Scheiermann Julia , Klinman Dennis M
Published: January 2023

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG ODN) stimulate immune cells via TLR9. CpG ODN are being evaluated clinically for use as vaccine adjuvants and in cancer immunotherapy. CpG ODNs are generally delivered parenterally as they are susceptible to degradation by acids and nucleases in the GI tract if delivered orally. Wang et al. developed a method to protect ODNs from digestion by encapsulating them in calcium (ODNcaps). They reported that orally delivered ODNcaps could alter susceptibility to atopic dermatitis in mice. To understand the effect of orally delivered CpG ODNcap on local and systemic immune responses, their in vivo and in vitro activity was evaluated in comparison to free (unencapsulated) ODN. TLR9-expressing spleen cells stimulated with CpG ODNcaps in vitro responded by producing cytokines. Oral delivery of CpG ODNcaps increased cytokine production in the Peyer’s patches and mesenteric lymph nodes. Oral administration of CpG ODNcaps exacerbated disease severity in a murine model of dextran sulfate induced inflammatory colitis similar to oral CpG ODN. Oral CpG ODNcap had no detectable systemic effects: they induced no change in serum cytokine levels or spleen cell activation. However, oral CpG ODNcap did enhance host responsiveness to subsequent LPS challenge while reducing susceptibility to L. monocytogenes challenge. Of note, the protection conferred by CpG ODNcap did not exceed that provided when free CpG ODN were delivered orally. These findings indicate that ODNcaps retain biological activity but that calcium encapsulation does not improve their bioavailability or activity in vivo.
Modulation of the energy metabolism inhibits plasmacytoid dendritic cell activation and delays onset of autoantibody production in murine models of systemic lupus erythematosus
Authors: Qiu Connie Claire , Lee Michael Hweemoon , Gallucci Stefania
Published: January 2023

Despite the heterogeneity of systemic lupus erythematosus (SLE) clinical manifestations, an increased expression of type I interferon (IFN) regulated genes, termed the interferon signature, has been reported in most SLE patients. Long recognized to facilitate the progression of SLE, Type I IFNs can be produced in small amounts by most cell types, but the plasmacytoid dendritic cells (pDCs) are the primary producers. Since murine models of SLE have shown that depletion of pDCs stop the onset and progression of autoimmunity, we speculated that it is important to suppress pDC activities that go beyond the production of type I IFNs. We have confirmed that changes in fatty acid metabolism are essential for pDC activation. Using inhibitors of fatty acid oxidation, oxidative phosphorylation, and glycolysis, we have found that metabolic modulation prevents pDC activation upon stimulation with TLR ligands CpG and R848. This metabolic inhibition did not affect pDC viability. Moreover, in vivo treatment of mice with metabolic inhibitors delayed the onset of autoantibody production in murine models of SLE. Indeed, the inhibition of energy metabolism strongly reduced the production of anti-dsDNA and antichromatin autoanti-bodies in i) a model of lupus induced by Graft versus Host Disease (GvHD) and ii) in lupus-prone mice accelerated by TLR stimulation. Therefore, our findings suggest that the in vivo suppression of the major producers of type I IFN through inhibition of energy metabolism may therapeutically downregulate the production of autoantibodies in murine lupus. Our data raises the possibility of manipulating cellular metabolic pathways to regulate the detrimental type I IFN effects in SLE.
Rapid Immunoassay Development using the U-PLEX® Assay Platform.
Authors: Yatsula Victoria , Shelburne Christopher , Ning Qian , Krai Priscilla , Cheo David , Ranganathan Sripriya , Davydov Ilia V. , Liu Pu , Stewart David , Oberoi Pankaj , Wilbur James , Wohlstadter Jacob N.
Published: January 2023

In the expanding field of biomarker research, methods to identify immunoassay reagents and optimize their use can be rate limiting for product development programs. Flexible tools that accelerate assay development, from antibody screening and selection to assay feasibility, can accelerate these programs. Here we demonstrate the use of the MSD® U-PLEX platform to conduct early assay development steps in parallel in a multiplex format. This resulted in rapid identification of a multiplexed panel of compatible assays.

Unbiased pairwise screening of 59 antibodies was performed on MULTI-SPOT® U-PLEX plates using biotinylated capture antibodies and detection antibodies conjugated with SULFO-TAG™ label. Feasible antibody pairs were identified based on high signal and low background, then ranked using parameters such as dynamic range, sensitivity, specificity, sample recognition, and matrix tolerance. Within four weeks, two prototype immunoassays were developed and characterized. This screening time frame can range from days to weeks depending on the number of starting antibodies (typically 10–50) and the level of characterization required. Due to the flexible nature of the platform and the ability to mix and match reagents, the same conjugated reagents identified in screening were used to create and characterize multiplex assay panels. Parameters such as calibration curve, assay protocol, detection antibody concentration, and non-specific binding were characterized with up to ten assays simultaneously in a multiplex.

In conclusion, we demonstrate the utility of a flexible assay development system that allows rapid identification and optimization of assays for stand-alone use or in combination with existing MSD assays.
Suction blistering of vitiligo lesional skin provides insights into the pathophysiology of organ specific autoimmunity
Authors: Strassner James P , Rashighi Mehdi , Refat Maggi Ahmed , Richmond Jillian M , Harris John E
Published: January 2023

Vitiligo is an autoimmune disease of the skin in which CD8+ T cells destroy the pigment producing cells of the skin: melanocytes. To study the immune infiltrate that propagates vitiligo directly in human skin, we employed a novel suction blistering technique to study skin interstitial fluid by ELISA and flow cytometry. Consistent with previous publications demonstrating IFNγ signatures in vitiligo, CD8+ T cell number and CXCL9 protein concentration were elevated in vitiligo lesional skin compared to normal control skin. Receiver operator characteristic analysis revealed that these measurements may be used to distinguish active from stable disease. Phenotypic analysis of T cells in the skin interstitial fluid showed that the majority of CD4+ T cells in vitiligo skin express CXCR3, the receptor for CXCL9, whereas only 47% of CD4+ T cells expressing CXCR3 in the skin of healthy subjects. The frequency of CD8+ T cells expressing CXCR3 was high in both vitiligo patients and healthy donors. Higher CXCR3+ T cell recruitment via elevated CXCL9 expression may contribute to an observed increase in CD8+ T cell to Treg ratio observed active vitiligo skin (4.1 to 2.5 non-lesional). Additionally, all T cells in the skin microenvironment highly express PD-1. These results support the hypothesis that vitiligo is driven by IFNγ dependent signaling, and targeting components downstream of IFNγ may be useful in treating vitiligo. Moreover, suction blistering human skin is a powerful tool that can be used to aid the study of other inflammatory skin diseases.
Topology of multiple antigen expression in tonsillar lymphoid follicles and stroma analyzed by imaging mass cytometry.
Authors: Ornatsky Olga , Allo Bedilu , Talaei Nafiseh , Bouzekri Alexandre , Baranov Vladimir
Published: January 2023

Investigation of co-distribution of 35 biomarkers simultaneously (major cell type and immune-oncology, stromal and tissue architecture markers, as well as cell proliferation and nuclear markers) in normal lymphoid sections using Imaging Mass Cytometry (Qing et al, Cytometry: Part A, 2017) is presented. Tonsil tissue revealed primary lymph follicles as dense clusters of lymphocytes expressing B-cell markers, surrounded by CD3, CD4, and CD8 T-cells. Secondary lymph follicles could be distinguished by proliferating B-cells with high Ki-67 expression. Strong expression of Bcl-6 was detected in germinal-center B-cells. Tonsillar FoxP3 CD8 T-cells exhibit a Treg phenotype with high CTLA-4 and CD45RO. CD68+ mac/mono were found in germinal centers and rarely in stroma. Squamous epithelium of crypts was beta-catenin positive and infiltrated by PD-L1+ T-cells, while PD-1+ cells were also found within the follicle centers. Images acquired on the Hyperion™ Imaging System (Fluidigm Inc.) were compared to immunofluorescence (IF) of sequential sections stained with the same antibodies (Ab) conjugated to fluorophores (FL). For direct comparison of IF and IMC, dual tagged Abs were created by attaching a metal tag first (Maxpar®, Fluidigm Inc.), then conjugating to FL using click chemistry. CD3 and CD19 were labeled with metals and FL, and tested in combination with the full panel on 8 μm frozen tonsil sections. CD3+ and CD19+ lymphs were identified by IF first, followed by IMC analysis. Results show that IMC is comparable to IF images and allows identification, characterization and localization of cell populations and tissue architecture in the absence of autofluorescence, photobleaching, with low background for acquisition of up to 50 targets.
Platelet Detection in Unaltered Whole Blood
Authors: Bradford Jolene A , Ward Michael
Published: January 2023

Platelets are non-nucleated cellular fragments circulating in the peripheral blood, derived from megakaryocytes. They are critical in the maintenance of hemostasis. Alterations in their function are associated with a range of clinical conditions. Using flow cytometry, light scatter signals from platelets typically overlap signals from red blood cells (RBC). Since RBC outnumber platelets by more than an order of magnitude in whole blood, platelets are typically studied by depleting samples of RBCs by centrifugation methods or selective RBC cell lysis. These sample preparation procedures require manipulations that may affect platelet health and function, and result in loss of cells. Additionally, lysed samples require labeling of the platelets with a fluorescent marker to adequately resolve the platelets from debris/noise. We present a novel technique for resolving leukocytes and platelets from RBC in whole blood using orthogonal light scatter from 2 lasers. This method exploits the differences in light-scattering properties between RBCs and platelets and white blood cells (WBC). Hemoglobin readily absorbs 405 nm laser light, reducing the scatter signal from RBC relative to WBCs and platelets, resulting in a reproducible scatter pattern when analyzing human whole blood with both blue (488 nm) and violet (405 nm) side scatter. Using this approach, a distinct platelet population can be discerned in unaltered whole blood, requiring only dilution of whole blood in buffer. Combining this approach with acoustic cytometry allows great flexibility in determining optimal dilution and sample collection rates for minimal coincidence and rapid analysis. For research use only. Not for use in diagnostic procedures.
IL-17 plays a critical role in airway epithelial injury after transplantation
Authors: Xu Jiangnan , Zhang Yamei , Ochando Jordi , Li Liwu , Ding Yaozhong
Published: January 2023

Bronchiolitis obliterans syndrome (BOS) is the major obstacle limiting long-term allograft survival of lung transplantation. Airway epithelium is the primary target in obliterative airway diseases, and the degree of epithelial injury is closely correlated with the development of BOS. IL-17 plays a critical role in mediating inflammatory responses and is involved in transplant graft rejection. In this study, we performed murine orthotopic allogeneic trachea transplantations and examined the role of IL-17 in airway epithelial injury after transplantation. Expression of Th1 and Th17 cytokines, IFNγ, IL-6 and IL-17, was significantly increased in allografts of wild type recipients, and the airway pseudostratified epithelia of allografts changed into flat epithelia with inflammation and fibroblast proliferation in submucosa tissue. Deficiency in Th17 essential transcription factor, RORγt, completely restored normal airway epithelial morphology in trachea grafts, while absence of Th1 transcription factor, T-bet, resulted in exacerbated airway inflammation and epithelial injury with elevated IL-17 expression. Administration of anti-IL-17, anti-IL-6 or Stat3 activation inhibitor (C188-9), but not anti-IFNγ, improved airway epithelial integrity. Our results demonstrate that IL-17 is crucial to post-transplant airway inflammation and epithelial damage, and devoid of IL-17 production diminishes epithelial injury and prolongs graft survival. Our findings provide important implications to understand the role of IL-17 in lung transplant immunity and further indicate the necessity of suppressing IL-17 for airway epithelial protection and allograft survival after transplantation.
Cross-dressing of recipient Ag-presenting cells with donor exosomes trigger direct T-cell allosensitization in transplantation
Authors: Morelli Adrian E , Zeng Furong , Rojas-Canales Darling M , Divito Sherrie , Shufesky William J , Larregina Adriana T , Liu Quan
Published: January 2023

The idea that donor dendritic cells (DCs) from the graft present donor MHC Ag to naïve T cells has been challenged. Evidence suggests that donor DCs are undetectable or found are low numbers in graft-draining lymphoid organs. Thus, the aim of our study is to elucidate how donor MHC molecules are recognized so efficiently by recipient naïve T cells.

Results

After transplantation of CD45.2 BALB/c hearts in CD45.1 B6 mice, very few donor DCs were detected in the spleen, and no donor DCs were found in lymph nodes draining BALB/c skin grafted in B6 mice. In both cases, donor intact MHC molecules were detected by electron microscopy on exosomes attached to recipient conventional DCs in graft-draining lymphoid organs. The transferred MHC Ag induced proliferation and differentiation of CD8 T cells against the BALB/c H2Ld Ag. Cultures of BALB/c DCs with B6 DCs with inhibitors of exosome release (Rab27a siRNA) confirmed that passage of BALB/c MHC occurred via exosomes. Importantly, transfer of exosomes released by donor DCs (unlike other types of vesicles) promoted maturation of recipient DCs. By high resolution confocal microscopy, BALB/c DCs, engineered to release RFP+ exosomes and injected in CD11c-YFP B6 mice, transferred RFP+ exosomes to recipient YFP+ DCs in lymph nodes and spleen. Accordingly, depletion of recipient DCs in CD11c-DTR B6 recipients prevented presentation of donor intact MHC Ag to T cells and delayed heart allograft rejection.

Conclusion

Our results elucidate the ultrastructural basis of the still elusive semi-direct pathway of allorecognition, by demonstrating that donor exosomes (released by the graft or donor migrating DCs) cross-dress recipient APCs and promote the potent T-cell allosensitization seen in transplantation.
Sirolimus, Post–Transplant Cyclophosphamide, and CTLA4-Ig Maintain Stable Mixed Chimerism In a Mismatched Murine Model
Authors: Fisher Cameron , Xu Xin , Phang Oswald , Fitzhugh Courtney , Olkhanud Purevdorj B
Published: January 2023

Hematopoietic stem cell transplantation (HSCT) is the only curative option for patients with sickle cell disease (SCD). We sought to extend our haploidentical HSCT regimen with nonmyeloablative conditioning. The objective of our study was to assess the ability of the immunosuppressive agent CTLA4-Ig to induce mixed chimerism in the haploidentical HSCT setting when combined with sirolimus, post-transplant cyclophosphamide (PT-Cy), or both.

We used a mismatched mouse model with BALB/c donors and C57BL/6 recipients. Recipient mice received 200 cGy TBI on day of the transplant and were conditioned with or without sirolimus, PT-Cy, and/or CTLA4-Ig.

Our data show that when CTLA4-Ig alone or CTLA4-Ig/PT-Cy are given, donor cells are not detected. However, mixed chimerism is maintained in mice who received either sirolimus/PT-Cy or sirolimus/PT-Cy given together with CTLA4-Ig over 60 days. Additionally our data indicate a significant increase in recovery of donor cells within CD8 T-cell, CD19 B-cell, and CD11b myeloid cell populations in groups conditioned with sirolimus/PT-Cy, or sirolimus/PT-Cy/CTLA4-Ig as compared to groups conditioned with CTLA4-Ig (p=0.003; p=0.002; p=0.049) alone or CTLA4-Ig/PT-Cy (p=0.001; p<0.0001; p=0.001) at 30 days, respectively.

These data demonstrate that sirolimus in combination with PT-Cy and CTLA4-Ig is synergistic in an MHC-mismatched mouse model. The novel tri-drug group establishes mixed chimerism while conditioning with CTLA4-Ig alone or CTLA4-Ig/PT-Cy does not. The data also identify induction of stable mixed chimerism in specific cellular subpopulations within the sirolimus/PT-Cy and sirolimus/PT-Cy/CTLA4-Ig groups, indicating that these cells may play an important role in HCST.
Characterization of human preformed antibodies to non-SLA xenoantigens on RBC from genetically-engineering pigs
Authors: Wang Zheng-Yu , Martens Gregory , Tector Matthew , Tector A. Joseph
Published: January 2023

BACKGROUND

Genetic engineering pig models of pig-to-human xenotransplantation may overcome a growing shortage of available solid organs. Development of pigs deficient in galactose α1,3 galactose and N-glycolylneuraminic acid have significantly improved cell survival when challenged by human antibody and complement in vitro. The b4GalNT2 glycosyltransferase may also synthesize xenoantigens in pigs. In this study, we used RBC, as a cell model, to assess human antibody-mediated response to non-SLA xenoantigens of pigs deficient in these carbohydrate-modifying genes.

MATHODS AND MATERIALS

RBC were isolated from wild type pigs and pigs deficient in GGTA1/CMAH knockout (DKO) and GGTA1/CMAH/β4GalNT2 knockout (TKO). Human ABO sera were purchased from Valley Biomedical. Human ABO RBC were isolated from healthy people. Total antibody binding was assessed by hemagglutination assay; human IgG or IgM binding to RBC was characterized by flow cytometry; opsonized RBC phagocytosis by THP-1 macrophages was analyzed using confocal microscope.

RESULTS

Phenotype of TKO RBC was characterized by flow cytometry. Silencing the three genes reduced hemagglutination levels of TKO RBC compared with that of DKO RBC by all three human blood-type A, O, and AB sera but except blood-type B sera, and decreased both of IgG and IgM binding to TKO RBC compared with DKO RBC. Confocal image data showed that phagocytosis of opsonized TKO RBC was lower than that of opsonized DKO RBC by THP-1 cells.

DISCUSSION

Non-SLA xenoantigenicity of pig RBC can be disrupted using gene engineering tools, which greatly reduced the humoral barrier to xenotransplantation