bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and characterization of lung commensal bacteria that modulate immune response in Tuberculosis
Authors: Bernard Lucie , Colom André , Cescato Margaux , Cougoule Céline , Guilloton Pauline , Ghebrendrias Natsinet , Capilla Florence , Alloy Annie , Remot Aude , Thomas Muriel , Langella Philippe , Neyrolles Olivier , Lugo-Villarino Geanncarlo
Published: January 2023

Tuberculosis (TB) is still one of the deadliest infectious diseases in humans. As TB disease is driven by exacerbation of the immune response, promising approaches include host-targeting therapies aiming at controlling the balance between pathogen eradication and limitation of the inflammation in order to prevent immunopathology. Such immune modulation has been observed through modification of the gut-microbiota compartment with oral administration of specific bacterial strains (i.e. probiotics) in other respiratory diseases. In TB, a dysbiosis of the microbiota is observed in patients, and microbiota perturbation in mice increases susceptibility to Mycobacterium tuberculosis (Mtb). Here, we assessed whether bacterial strains isolated from the recently described lung microbiota could have a local impact on Mtb infection by protecting against immune dysfunction for the host benefit. Through intranasal administration in mice, we identified different microbiota strains isolated from mouse lungs with a strong ability to modulate the lung T cell compartment. The same in vivo approach in Mtb-infected mice revealed one strain with a remarkable capacity to diminish Mtb infection-associated lung immunopathology, accompanied by a specific immune signature characterized by an increase level of regulatory T cells sharing surprisingly T-helper 17 cell markers. Collectively, this work may contribute to the design of host-directed therapies to reduce the destructive inflammatory response in TB, without further fomenting the occurrence of multiple-drug resistant Mtb strains. It will also shed light on poorly described populations in the lungs such as the RORgt+ Foxp3+ biTregs and their interactions with the microbiota.
Repeated respiratory bacterial exposures elicit lung resident memory B cells in the absence of organized tertiary lymphoid tissue
Authors: Barker Kimberly A , Smith Nicole M , Shenoy Anukul T , Martin Ian MC , Jones Matthew R , Quinton Lee J , Mizgerd Joseph P.
Published: January 2023

Recent work has shown that resident memory B cells (BRM) are generated in the lung after flu infection. However, it is unclear whether the existence of lung BRM is unique to influenza, which often elicits iBALT, or alternatively if these cells represent a common feature of the lung immune landscape. We aimed to determine whether bacterial lung exposures experienced by nearly all human children might also elicit lung BRM. To model this scenario, we gave young mice repeated respiratory exposures to low virulence pneumococci. After at least 4 weeks, we then analyzed lung B cells using flow cytometry and an intravital CD45 stain to exclude intravascular leukocytes. Pneumococcus-exposed mice had many more extravascular IgD- lung B cells than controls, and these B cells remained in the lung at least 12 weeks after the last exposure. Although unorganized aggregates of B and T cells were observed in previously exposed lungs, no iBALT structures were generated. Most of the IgD-B cells expressed the memory markers PD-L2, CD80, and CD73; a majority of the B cells expressed two or more of these markers, indicating they are poised to become antibody secreting upon rechallenge. Notably, the hallmark lung TRM phenotype (CD11abrightCD69+CD44brightCD62Llow) was also observed on lung B cells; these markers may represent a common resident memory lymphocyte phenotype. Although all lung B cells were CD20+, they were not affected by αCD20 antibody treatment, which requires circulation of bound cells for depletion to occur. BRM appear to be a common feature of the adaptive immune cell repertoire of experienced lungs. These cells do not require iBALT for their maintenance and represent a previously unrecognized pool of B cells resistant to depletion with αCD20 therapy.
Inducible epithelial resistance protects against acute viral infection and subsequent CD8+Tcell dependent lethal Immunopathology
Authors: Wali Shradha , Flores Jose R , Goldblatt David , Tuvim Michael , Dickey Burton F , Evans Scott E
Published: January 2023

Viral pneumonia leads to significant morbidity and mortality worldwide, demanding better understanding of the host immune response to infections for development of novel strategies to prevent respiratory infections. Our group has shown that mice treated with combination of Toll-like receptor (TLR) 2/6 and 9 agonists (Pam2-ODN) are protected against wide range of respiratory pathogens, including viruses. Mice challenged intra-pharyngeally with Sendai virus showed peak viral burden on day 5 and peak mortality on day 11, overlapping with increased lung CD8+Tcells. We hypothesized that mortality is associated with immunopathology caused by CD8+T cells. Pam2-ODN aerosolization one day prior to Sendai challenge reduced lung viral burden and reduced lung CD8+T cells on day 11 and enhanced mouse survival. Depletion of CD8+T cells before infection increased baseline virus susceptibility and enhanced viral replication in vivo, congruent with the known antiviral function of CD8+T cells. Pam2-ODN pre-treatment protected mice against death following viral challenge, even in the absence of CD8+T cells, reflecting antiviral responses induced directly from epithelial cells. Notably, depletion of CD8+T cells eight days after viral challenge also significantly enhanced survival of sham pre-treated mice, indicating rescue from CD8+T cell-mediated lethal immunopathology. Our findings definitively prove that CD8+T cells, although anti-viral in nature, promote lethal immunopathology that can be prevented by Pam2-ODN pre-treatment. Importantly, Pam2-ODN pre-treatment benefitted survival regardless of when CD8+T cells were depleted and may provide an opportunity to protect vulnerable populations against respiratory infections.
Human IFNe and IFNa modulate immune and anti-HIV functions through the differential involvement of IFNa receptors
Authors: Pizutelli Vanessa , Tasker Carley , Sue Lishan , Lu Wuyuan , Chang Theresa
Published: January 2023

Interferon e (IFNe) is a critical innate immune mediator that protects the host against sexually transmitted infections. Although the current paradigm is that IFNɛ is a type I IFN and signals through IFNa receptors (IFNARs), our and other published results show that IFNɛ has unique gene regulatory and immune functions that are distinct from IFNa. IFNɛ is constitutively expressed in the mucosa, and highly abundant in the female reproductive tract. It exhibits superior mucosal immune activity compared to IFNa/b. We have previously shown that that human IFNe induces an anti-HIV state through a mechanism independent of known type I IFN-induced HIV host restriction factors in primary macrophages. IFNɛ induces phagocytosis and reactive oxygen species that contribute to anti-HIV activity. In this study, we determined the involvement of IFNARs in immune modulation and HIV inhibition by human IFNɛ in comparison to human IFNa.

Human IFNɛ elicited a more robust immune response than human IFNa2 in PBMCs and primary macrophages. The immune profiles in response to human IFNɛ were cell-type dependent. Antibodies (Abs) against IFNAR1 or IFNAR2 blocked induction of interferon-stimulated genes (ISGs) by human IFNa2 and murine IFNe. However, anti-IFNAR2 but not anti-IFNAR1 Ab blocked human IFNe-mediated ISG induction. Interestingly, anti-IFNAR1 or anti-IFNAR2 Ab blocked cytokine induction by human IFNa2 but not by human IFNe. Finally, we found that both IFNAR1 and IFNAR2 were involved in anti-HIV activity of human IFNa2 but only IFNAR2 was involved in HIV inhibition by human IFNe. In summary, our results indicate that human IFNe, not a conventional type I IFN, modulates immune and anti-HIV functions through distinct mechanisms from IFNa2.
Novel Vaccine Adjuvants: Exploring the Potential of Rexinoids to Influence Effector T Cell Homing to Mucosal Sites
Authors: Manhas Kavita , Blattman Joseph N , Marshall Pamela A , Wagner Carl E , Jurutka Peter W
Published: January 2023

Vaccines are the most successful defense against infectious diseases. Effective vaccines must target induced immune responses to areas of pathogen entry, a primary site being mucosal surfaces. Certain vitamins can directly impact immune cell migration, indicating potential to function as an adjuvant. We previously showed that all-trans retinoic acid (ATRA), the biologically active form of vitamin A, modifies gut-homing receptor expression on effector T cells upon binding to the retinoic acid receptor (RAR), which joins with the retinoid X receptor (RXR) to function. Giving ATRA during vaccination also resulted in enhanced mucosal protection following viral challenge in murine models. While promising, certain factors limit adding ATRA to a vaccine, including its instability and poor solubility. Rexinoids, synthetic ligands for the RXR, could mimic or better ATRA immune effects by enhancing RAR-mediated transcription. Their improved stability and solubility also allows a greater potential for clinical use. Here we explored the ability of different rexinoids to program effector T cell expression of gut-homing receptors CCR9, α4β7, and CD103. Naïve T cells from transgenic mice were activated with peptide and set concentration of each rexinoid. Flow cytometry showed increased expression of gut-homing receptors in the presence of select rexinoids, indicating their ability to act as an ATRA mimic. A cooperative relationship was also seen between other rexinoids and ATRA as co-culturing with suboptimal doses of each showed enhanced receptor expression, suggesting these rexinoids could potentially act with ATRA normally found in serum. Overall, our findings support the prospective use of rexinoids as adjuvants during vaccination.
In vitro model for assessing the effect of T helper cell cytokines on the barrier function of alveolar type II epithelial cells
Authors: Coello Daniel , Knott Jeffery D. , Porter Edith
Published: January 2023

There are still diseases of global burden for which satisfactory vaccines are unavailable, e.g. tuberculosis. Vaccines against intracellular microbes has relied on the activation of the adaptive T cell response. Our laboratory is interested in investigating whether the innate epithelial host defense could be integrated in a novel approach. In response to airborne pathogens, airway epithelia release antimicrobial peptides like human beta defensin-2 and -3 (HBD2/3), antimicrobial lipids, and chemokines, like IL-8, to attract other immune cells including T helper cells that are integral to the adaptive immune response. We hypothesize that T cell derived cytokines augment the antimicrobial function of airway epithelia and we have developed an in vitro model to generate pilot data. A549 cells, alveolar type II epithelial cells, were grown in air-liquid interface and stimulated with TNF-α & IFN-γ and IL-17 & IL-22, at 5 ng/mL each, for 24 h. IL-8 and HBD2/3 gene expression was quantified by RT-PCR and secretion of antimicrobial peptides was assessed by AU-PAGE and Western blot probing for HBD2/3. Lipid production was assessed with Bodipy and the physical barrier integrity with Phalloidin. IL-8 gene expression was 9-fold elevated when cells were stimulated with TNF-α & IFN-γ. Secretions from stimulated cells appeared to contain more cationic peptides that were not reactive with HBD2/3 polyclonal antibodies. Cellular Bodipy fluorescence was pronounced after IL-17 & IL22 stimulation. Phalloidin staining was consistent with tight junction formation in all samples. This data provides evidence that T helper cells may directly augment epithelial barrier function supporting a novel vaccine design that integrates the epithelial cell response.
EXOSOMES FROM PRIMARY BRONCHIAL EPITHELIAL CELLS INDUCE TOLERANCE IN HUMAN MONOCYTE-DERIVED DENDRITIC CELLS
Authors: Agrawal Anshu , Monterio Clarice , Agrawal Sudhanshu
Published: January 2023

Dendritic cells (DCs) act as sentinels by monitoring the tissue environment for microorganisms and other substances. DCs are also critical modulators of tolerance in the periphery as well as in the mucosa including the respiratory mucosa. DCs exist in close proximity to airway epithelial cells (AECs) and both cells influence the functions of each other. Using a 2D model to study the interaction of human primary bronchial epithelial cells (representative of AECs) with DCs we have recently demonstrated that AECs enhance the immune surveillance capacity of myeloid DCs (mDCs) at homeostasis. Culture of mDCs with AECs resulted in the upregulation of pathogen recognition receptors (PRRs) and downstream signaling molecules which was reflected in the enhanced inflammatory cytokine responses to LPS. In contrast to mDCs, culture of monocyte-derived DCs (MoDCs) with AECs led to a significant inhibition of LPS induced pro-inflammatory responses. Thus it seems AECs induce tolerance in MoDCs but enhance the immunogenicity of mDCs suggesting a division of labor between various DC subsets in the respiratory mucosa. Recent studies have indicated a role of exosomes, the endosomal-derived nano-vesicles in influencing the function of a variety of cells. Therefore we investigated whether exosomes play a role in inducing tolerance in MoDCs. MoDCs were cultured with various concentrations of exosomes before activation with LPS. Results indicate that exosomes from AECs conditioned media increased the secretion of IL-10 from MoDCs compared to control media exosome. The expression of CD80/86 was also decreased. In summary these data indicate that exosomes from AECs may be inducing tolerance in MoDCs in the respiratory mucosa.
Genetic and pharmacological modulation of RORα regulates TH17-driven inflammatory disorders
Authors: Solt Laura A , Wang Ran , Amir Mohammed , Chaudhari Sweena , Campbell Sean , Bassette Molly B , Eliason Amber , Sundrud Mark S , Kamenecka Theodore
Published: January 2023

While RORγt has been well characterized as the lineage defining transcription factor for TH17 cell development, TH17 cells are not absent in Rorc-deficient mice, suggesting other factors may be required. RORα, a close family member of RORγt, is also expressed during TH17 cell development but is considered functionally redundant, thus little is known about its function in TH17 cells. Using mouse models of autoimmunity and chronic inflammation, we show that expression of RORα is required for TH17 pathogenicity. T-cell specific deletion of RORα significantly abrogated the development of experimental autoimmune encephalomyelitis (EAE) and colitis, due to decreased development of TH17 cells and expression of homing receptors required for cells to gain access to sites of inflammation. These results were accompanied by increased Foxp3+T regulatory cells. Using a RORα-selective small molecule that we developed, we found that modulation of RORα activity largely phenocopied our genetic data, inhibiting the development of EAE, colitis, and relapse in a relapsing remitting model of multiple sclerosis. Importantly, treatment with a RORα modulator did not affect thymic cellularity. Finally, modulation of RORα activity inhibited the development of human TH17 cells and pro-inflammatory cytokine expression from subsets of CD4+CCR6+memory T cells. Our results establish that RORα has non-redundant functions to RORγt driving TH17 cell development and identifies RORα as a potential therapeutic target for the treatment of TH17-mediated autoimmunity.
Anti-CD4/CD8 antibody therapy induces Foxo1 transcriptional activity promoting T cell egress from inflamed tissue during type 1 diabetes
Authors: Clark Matthew P , Kroger Charles , Martin Aaron , Tisch Roland
Published: January 2023

Type 1 diabetes (T1D) is considered to be a T cell driven autoimmune disease, for which clinical treatment of T1D has been unsuccessful creating a need for immunotherapeutics to match world-wide rising incidence of disease. Pancreatic inflammation in non-obese diabetic (NOD) mice is primarily T cell mediated resulting in immune destruction of the insulin producing β cells in the pancreatic islets of Langerhans, which induces a hyperglycemic state that requires daily insulin injections to maintain homeostasis. The use of nondepleting antibodies provides a strategy to modulate the function of T cells while avoiding their systemic depletion. Our lab has previously reported that treatment of recent onset diabetic NOD mice with a short-course of both monoclonal anti-CD4 and anti-CD8 (anti-CD4/CD8) antibodies rapidly and indefinitely reversed established T1D. Remission of T1D by anti-CD4/CD8 treatment is accompanied by the egress of T cells from the pancreatic islets and pancreatic draining lymph node. Microarray analysis of anti-CD4/CD8 treated T cells revealed increased activity of the Forkhead box protein O1 (Foxo1) transcriptional axis that controls expression of the blood homing chemokine receptor sphingosine-1-phosphate receptor 1 (S1Pr1). In contrast, T cell activation related genes that promote tissue residency of T cells were down regulated. T cell receptor (TCR) signaling negatively regulates Foxo1 transcriptional activity by phosphorylation via the AKT signaling pathway. Our studies have revealed that dampened TCR signaling shortly after anti-CD4/CD8 treatment results in enhanced Foxo1 transcriptional activity promoting S1Pr1-mediated T cell egress from inflamed tissues providing long-term protection from T1D.
ST8Sia6 generated α2,8-disialic acids mitigate hyperglycemia in multiple low dose streptozocin-induced diabetes
Authors: Belmonte Paul , Shapiro Michael , McCue Shaylene , Rajcula Matthew , Shapiro Virginia Smith
Published: January 2023

Type 1 diabetes is an autoimmune disease that destroys insulin-producing beta cells in the pancreatic islets. Although exogenous insulin replacement therapy is lifesaving, precise blood glucose management remains a significant challenge. Experimental islet transplantation restores normoglycemia, but the lack of available islets and graft rejection present major barriers to widespread adoption as standard therapy. The islet resident macrophage (IRM) is in close contact with beta cells, efficiently presents islet antigen to autoreactive T cells, and is critical to disease occurrence. We have demonstrated that murine IRMs express high levels of Siglec-E, an immune-regulatory receptor that preferentially binds α2,8-disialic acids in cis or in trans. When islets were exposed to streptozocin (STZ)-induced stress in culture, beta cells upregulated ligands for Siglec-E. To determine the biological significance of this, we generated a mouse model where ST8Sia6, the sialyltransferase that produces α2,8-disialic acids and thus Siglec-E ligands, is constitutively overexpressed in beta cells. This increased trans Siglec-E binding affinity to the beta cell surface, and mitigated hyperglycemia in the multiple low-dose STZ model of diabetes. Significant reduction in blood sugar was evident at 14 days post-initial injection of STZ, coinciding with T cell infiltration of islets. T cells from ST8Sia6+ islets were less activated when compared to littermates. This work reveals a novel and important role for Siglec-E in autoimmune diabetes and provides insight into how rational targeting of this immune receptor may instruct islet-residing antigen presenting cells to provide local tolerance signals in the setting of islet transplantation.
Kidney Allograft Recipient Absence of Myeloperoxidase Decreases Donor-Specific Antibody Titers and Attenuates Antibody-Mediated Allograft Rejection
Authors: Fairchild Robert L , Miyairi Satoshi , Dvorina Nina , Valujskikh Anna , Baldwin William M
Published: January 2023

The incidence of antibody-mediated rejection (AMR) in clinical kidney transplants is increasingly observed. We have reported that strong donor-specific antibody (DSA) responses are induced in B6.CCR5−/−mice transplanted with complete MHC mismatched A/J kidney allografts and that NK cells play a critical role in acute injury and graft failure. Since this acute AMR is accompanied by intense macrophage infiltration into the allograft, we tested the role of recipient-derived myeloperoxidase (MPO) production on kidney allograft survival. B6.CCR5−/−and B6.CCR5−/−MPO−/−mice were transplanted with complete MHC mismatched A/J kidney grafts. Allografts were rejected between days 18 and 25 post-transplant in B6.CCR5−/−recipients but not until days 46–54 in B6.CCR5−/−MPO−/− recipients. DSA titers in B6.CCR5−/−MPO−/−recipients were 4–5 fold lower than those induced in the B6.CCR5−/− recipients. There was also a 60% decrease in the number of donor-reactive T cells producing IFN-g in the spleens of the B6.CCR5−/−MPO−/− vs. B6.CCR5−/−recipients on day 7 post-transplant. Despite the extended survival, qPCR analyses indicated slight increases in mRNA encoding TNFa, IL-6 and FasL in kidney allografts on day 14 post-transplant in B6.CCR5−/−MPO−/− vs. B6.CCR5−/−recipients as well as the pro-fibrogenic factors P-selectin and connective tissue growth factor on day 50 post-transplant. These results suggest MPO regulates the magnitude of the DSA and T cell response in kidney transplant recipients and that the decreased DSA titers attenuate acute AMR but induce the indolent development of interstitial fibrosis and glomerular injury that will eventually lead to graft dysfunction and failure at later times.
Human Immune Cells Directly Recognize Xenogeneic MHCs
Authors: Nouri-Shirazi Mahyar A , Katz Haley , Victor Lisa
Published: January 2023

The advancement in tissue typing and the use of immunosuppressive drugs has overcome major complications in organ transplantation, therefore, making this therapy a viable option for irreversible organ diseases. However, organ shortage continues to be the forefront of problems facing clinical transplantation. To compensate for the disparity between organ supply and demand, xenografts have become a promising alternative. Nevertheless, the success of xenotransplants needs further investigation into the contribution of cell-mediated rejection. Here, we explored the direct and indirect contribution of human immune cells in xenorecognition using human and murine in vitro coculture systems. Our data shows that human T cells directly recognized the xenogeneic MHC molecules since blocking of MHCs suppressed their proliferative response and cytokines production, IL-2 and IFN-γ. Furthermore, while B and NK cells alone did not generate significant response, the combination of B and T cells promoted indirect xenorecognition by T cells as evidenced by an increase in their proliferative response. In contrast, the murine immune cells showed limited response to human MHCs. Our study suggests that human T cells have a plasticity to recognize xenogeneic MHCs and contribute to xenograft rejection.
High Stearic Acid diet aggravates aGVHD by promoting Th17 cells through Akkermansia muciniphila
Authors: Ma Shoubao , Yang Bingyu , Gong Huanle , Wu Xiaojin , Wu Depei
Published: January 2023

T-cell metabolism is a promising target for controlling aGVHD. By using GC-MS technique, we previously identified that serum stearic acid/palmitic acid ratio on day 7 was an excellent biomarker to predict aGVHD after allo-HSCT, suggesting an important role of saturated fatty acid intake in the pathogenesis of aGVHD. However, the mechanism remains unknown. By using mouse aGVHD model, here we found that high stearic acid diet (HSAD), but not palmitic acid diet, significantly aggravated aGVHD, and its promoting role was mediated by gut microbiota. 16S rRNA sequencing identified Akkermansia muciniphila (Akk) was obviously increased in HSAD recipient. Add back Akk in WT mice ameliorated aGVHD-related mortality. Furthermore, depleting CD4 T cells significantly rescued HSAD induced aGVHD mortality, whereas depleting CD8 T cells could not. In addition, HSAD recipients of RORgt−/− allografts had improved survival compared with recipients of WT allografts, indicating that Th17 cells were involved in HSAD induced aGVHD. Meanwhile, we found that Th17 cells were significantly increased in aGVHD target tissues in HSAD recipients. Functional studies showed that stearic acid could promote Akk growth in vitro, and extracts from Akk significantly promoted Th17 cells differentiation in vitro, suggesting that Akk increased the proportion of Th17 cells, thereby exacerbating aGVHD.
Inhibition of DNA-PK(cs) Prevents Graft Rejection in a Murine Allograft Skin Graft Study
Authors: Burdine Marie , Harrison David , Waldrip Zachary , Burdine Lyle
Published: January 2023

While the number of patients needing solid organ transplants has steadily increased over the years, the development of safer, more effective transplant drugs has stagnated. Current therapy has been largely successful at thwarting acute cellular rejection; however, antibody-mediated rejection (AMR) remains difficult to treat in the clinic making it the primary cause of graft loss. In this study, we provide proof of principle experiments that DNA-dependent protein kinase catalytic subunit DNA-PK(cs) is a worthwhile target for transplant immunosuppression. In our previous studies, we confirmed that DNA-PK(cs) controls NFAT-induced IL-2 expression, in part, through a mechanism that promotes calcineurin activity by preventing expression of the endogenous calcineurin inhibitor Cabin1. Here, we show that treatment with DNA-PK(cs) inhibitors prevents rejection in a murine skin allograft model by decreasing the production of cytokines including IL2 and IL6 and the infiltration of lymphocytes into the graft. Most importantly, our studies showed that DNA-PK(cs) inhibitors prevented the development of donor-specific antibodies, a driving force behind AMR. These results highlight novel functions for DNA-PK(cs) in the immune system and the potential use of DNA-PK(cs) inhibitors to prevent organ rejection there-bye warranting further investigation.
IL-1β And IL-6 Facilitate T Cell Reconstitution In Heart Allograft Recipients Treated With Anti-Thymocyte Globulin
Authors: Hasgur Suheyla , Fan Ran , Zwick Danny , Valujskikh Anna
Published: January 2023

Antibody-mediated lymphocyte depletion is used in solid organ transplantation. T cell reconstitution after lymphoablation may lead to acute rejection and poor graft outcome. We investigated the mechanisms of T cell reconstitution in heart allograft recipients treated with murine anti-thymocyte globulin (mATG).

B cell depletion with anti-mouse CD20 mAb significantly diminished T cell reconstitution in B6 (H-2b) recipients of BALB/c (H-2d) heart allografts treated with mATG. However, cognate T/B cell interactions were dispensable for T cell recovery suggesting the potential role of B cell derived cytokines. The NanoString gene expression analysis of isolated B cells showed that mATG upregulated IL-1β and IL-6 expression in CD4 T cell-dependent manner. Intracellular flow cytometry confirmed that B cells are the major source of IL-1β and IL-6 following mATG treatment. The proportion of IL-1β secreting B cells was increased in mATG treated compared to untreated recipients, whereas IL-6 producing B cells were not significantly affected by lymphoablation. Neutralization of either IL-1β or IL-6 with mAb significantly decreased T cell recovery and extended heart allograft survival in mATG treated recipients. To further test the role of B cell derived IL-1β, we injected either WT or Caspase-1−/− B cells into B cell deficient μMT mice followed by BALB/C heart transplantation and mATG treatment. Caspase-1 deficiency in injected B cells resulted in significantly lower CD8 T cell recovery.

Our results show the novel functions of IL-1β and IL-6 in regulating homeostatic T cell recovery following mATG treatment. IL-1β and IL-6 neutralization may be a promising strategy for enhancing the efficacy of mATG lymphoablation in transplant recipients.
Albumin-Flt3L-induced cross-presenting dendritic cells promote neoantigen-specific antitumor immunity and subsequent tumor control in murine models of cancer
Authors: Lam Brandon Krishna , Esquivel Daniel , Lee Brandon , Tan Marietta , Wu T.C. , Hung Chien-Fu
Published: January 2023

Central to the long-term success of essentially all cancer treatment strategies is the generation of durable tumor specific cytotoxic CD8+ T cell immunity, which requires uptake and potent antigen cross-presentation by dendritic cells (DCs) since tumor cells do not efficiently present relevant CD8+ T cell epitopes. FMS-like tyrosine kinase 3 ligand (Flt3L) is a cytokine that expands and differentiates DC precursors to murine cross-presenting CD103+ and CD8a+ DCs, but the therapeutic potential of Flt3L is limited because of its short half-life and global distribution in vivo. We have overcome the described issues by generating a novel genetic fusion of Albumin (Alb) to Flt3L, named Albumin-Flt3L (Alb-Flt3L), since Alb has a long half-life and exhibits trafficking to the lymph node (LN) and tumor (TM) as a serum protein. The novel immunotherapeutic Alb-Flt3L fusion protein exhibits increased half-life measured by ELISA, selective accumulation in LN and TM by IVIS imaging compared to native Flt3L, and potently expands cross-presenting DCs with no detectable toxicities. Challenging mice with HPV-associated TC-1 and MC38 adenocarcinoma tumor model, Alb-Flt3L + targeted radiation therapy to release tumor antigen and enhance tumor immunogenicity is able to control tumor progression and extend survival. When analyzing the immune response in mice challenged with MC38 tumor, significant intratumoral neoantigen specific CD8+ T cell responses are seen by tetramer staining, suggesting that Alb-Flt3L expanded DCs are able to engender neoantigen specific T cells without additional peptide vaccination. The therapeutic potential, mechanism of action, and subsequent implications of Alb-Flt3L use are under further investigation.
Mass spectroscopy-defined neoepitopes are a rich source of tumor rejection-mediating neoepitopes in a mouse sarcoma
Authors: Ebrahimi-Nik Hakimeh , Shcheglova Tatiana , Michaux Justine , Pak HuiSong , Sherafat Elham , Al Seesi Sahar , Mandoiu Ion I , Bassani-Sternberg Michal , Srivastava Pramod K
Published: January 2023

Using tandem mass spectroscopy (MS), we identified 3646 unique sequences among peptides eluted from purified Kd and Dd MHC I molecules of the BALB/c fibrosarcoma Meth A. These peptides were cross-referenced with the output of neoepitopes predicted for this tumor by our prediction pipeline CCCP (Cross Consensus Calling Platform). Eleven (11) of the eluted peptides were identified as neoepitopes and eight neoepitopes (of 11) were confirmed by targeted MS.

Each neoepitope was used to immunize BALB/c mice (twice, one week apart, using precise neoepitopes along with bone marrow-derived dendritic cells); mice were challenged with Meth A cells one week after the last immunization, and tumor growth was monitored in individual mice. In parallel, immunized mice were tested for CD8+ T cells to the neoepitopes using tetramer staining and interferon g secretion by CD8 cells.

Four of the eight neoepitopes elicited rejection of Meth A fibrosarcoma; two of the four neoepitopes elicited highly potent tumor rejection, while the other two elicited statistically significant but weaker tumor rejection. Of the two strong neoepitopes, only one elicited a measurable CD8 response. Both weak neoepitopes elicited measurable CD8 responses. Of the four neoepitopes that did not elicit tumor rejection, only one elicited a measurable CD8 response; this CD8 response was the strongest of all CD8 responses detected.

These observations indicate that MS-defined neoepitopes can be a rich source of neoepitopes that can mediate tumor rejection. Further, they highlight the fact that CD8 responses are not a good predictive surrogates for tumor rejection.
Active cancer vaccine targeting carbohydrates for immunotherapy
Authors: Padler-Karavani Vered , Reuven Eliran Moshe , Ben-Arye Shani Leviatan , Yu Hai , Duchi Roberto , Perota Andrea , Conchon Sophie , Soulillou Jean-Paul , Galli Cesare , Chen Xi
Published: January 2023

Transformed cells present cell surface neoantigens, including aberrantly expressed carbohydrates. Targeting tumor-associated carbohydrate antigens could be useful for cancer immunotherapy. N-glycolylneuraminic acid (Neu5Gc) is a non-human carbohydrate consumed in diet and accumulates on human cancer cells, leading to expression of neoantigens. Passive immunotherapy with anti-Neu5Gc antibodies in mice inhibits tumor growth. Here we describe an active cancer vaccination immunotherapy strategy to target Neu5Gc-positive tumors. We generated glyconanoparticles from engineered red blood cells to form nano-ghosts (NGs) that either express (NGpos) or lack expression (NGneg) of Neu5Gc-glycoconjugates. We then show that optimized immunization of Neu5Gc-deficient Cmah-KO mice with NGpos glyconanoparticles induce a robust, diverse and long-lasting anti- Neu5Gc IgG immune response. Anti-Neu5Gc IgG antibodies also migrated into Neu5Gc-positive tumors and inhibited tumor growth in-vivo. Using glycan microarrays we also show that the kinetics and quality of the immune responses influence the efficacy of the vaccine. Hence, immunotherapy targeting Neu5Gc-carbohydrate neoantigens is promising.
Induction of immune responses and clinical efficacy in a pilot personalized trial of 26 multi-epitope immunotherapy using long peptides Th1 and CD8 simultaneously in progressive tumors
Authors: Marquez-Manriquez Juan P , MATUTE-BRISENO JOSE ANTONIO , ICEDO-ZAMORA SALVADOR , DURAZO-ACUNA ALBERTO , Alejandro Lucero-Diaz PEDRO ALEJANDRO , ROSAS-DELGADO MARTIN ORLANDO , CAMACHO-HERNANDEZ ALEJANDRO
Published: January 2023

Background

Most of the immunotherapy trials in progressive cancer are limited targeting few antigens and most of them are not clinically relevant as they are not important for tumor survival. For such reason our approach is to develop multi peptide immunotherapy in order to potentially avoid chemoresistance and target simultaneously several biologically relevant pathways. We treated patients with progressive cancer after failure of the standard of care treatment with this methodology.

Methods

N=12 patients with progressive tumors such as pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), epithelial ovarian cancer (EOC) and sarcomas (ES) were treated with 26 long-peptides for 3 months until progressive disease. The patients were analyzed initially for several immune parameters such as DTH in both tumor area and non-tumor area, Granzyme B ELISPOT, Antigen-specific ELISA, T-cell expansion using individual peptides and immunohistochemistry.

Results

We were able to improve significant the mOS (38 months) with absence of toxicity. The proteins that we targeted were previously showed that were biologically relevant according with the siRNA studies we were able to silence and kill permanently in vitro different chemoresistant cell lines from the types of tumors treated as following PDAC (n=0.001), CRC (n=p0.005), EOC (P=0.001) and sarcomas (n=0.0001).

Discussion

After we validate this data with a large cohort of progressive tumors we will be able to prepare a clinical trial phase/II I order to accelerate the potential addition of these type of antigen active specific immunotherapy with other treatment modalities such as immunogenic chemotherapy, cytotoxic chemotherapy, radiotherapy, etc.
IL17 depletion enhances the DNA vaccine efficacy in pancreatic ductal adenocarcinoma treatment
Authors: Cappello Paola , Mucciolo Gianluca , Curcio Claudia , Roux Cecilia , Curto Roberta , Barutello Giuseppina , Novelli Francesco
Published: January 2023

Pancreatic ductal adenocarcinoma (PDA) remains one of the most aggressive cancer with a 5-year survival rate of ~8%. PDA is considered an “immunologically-cold” tumor because of the abundant desmoplastic immune-suppressive microenvironment and low presence of neo-antigens. Moreover, it is characterised by a complex inflammatory response in which the role of the IL17 and T helper-17 remain controversial. We have demonstrated that a DNA-based vaccination strategy targeting a PDA-associated antigen, namely alpha-enolase (ENO1), significantly prolongs survival in genetically engineered mice (GEM). We have also observed that the absence of IL17 strongly modified PDA microenvironment, improving CD8 T cell recruitment. Therefore, the aim of this study is to assess the role of IL17-targeting in combination with the DNA-vaccination. GEM crossed with IL17 knockout mice (GEM/IL17−/−) were vaccinated and monitored weekly to obtain a Kaplan-Meier survival curve. GEM vaccinated in absence of IL17 showed higher level of anti-ENO1 antibodies and IFNγ-secreting ENO1-specific T cells. The analysis of the tumor infiltrating cells revealed an increase of antigen-presenting cells and effector/memory T cells. To translate the experimental model into a clinical setting, mice orthotopically injected with PDA cells, were treated with anti-IL17 mAb in combination or not with ENO1-DNA vaccine. Only the combination elicited a significantly reduction in tumor growth that paralleled the induction of cytotoxic T cells not observed after ENO1-vaccine or anti-IL17 Ab alone. Overall, the depletion of IL17 opens new possibilities for combination to design more effective immunotherapeutic strategies to be associated with conventional one.
A powerful ICOS agonist that enhances anti-tumor immune responses restored by immune checkpoint inhibitors
Authors: Gariepy Jean , Prodeus Aaron , Sparkes Amanda , Fischer Nicholas , Saha Shravasee
Published: January 2023

The Inducible T cell Costimulator (ICOS, CD278) is a receptor in the CD28 family of B7-binding proteins expressed mostly by activated T cells. Upon binding to its ligand ICOS-L expressed on APCs,T cells are further activated by ICOS, resulting in their expansion and production of effector cytokines. Clinical data in patients treated with anti-CTLA-4 or anti-PD-1 mAbs have shown that ICOShiT cells correlated with an increased treatment response in these patients. Here, we report the design of a high avidity pentavalent form of the human ICOS-L extracellular domain fused to a short α-helical peptide from the human cartilage oligomeric matrix protein (COMP). This ICOS agonist, termed ICOS-L.COMP, spontaneously assembles into stable pentamers, binds tightly to both human and murine ICOS (SPR; Kd<10 nM) and co-stimulates (with anti CD3 mAb) the proliferation and cytokine release of both murine and human CD4+ and CD8+ T cells when added as a soluble factor (unlike ICOS-L.Fc). Soluble ICOS-L.COMP also phosphorylates Akt in activated T cells and enhances anti-tumor immune responses in tumor-bearing mice [murine MC38 or CT26 tumors] treated with either anti-murine PD-1 or anti-murine CTLA-4 mAb. Impressively, most mice treated with ICOS-L.COMP combined with an immune checkpoint inhibitor were either cured or displayed a stable tumor burden in contrast to checkpoint monotherapies (delayed tumor progression) or when ICOS-L.COMP was given as a monotherapy (comparable to no treatment). Importantly, ICOS-L.COMP lacks a Fc domain thus eliminating Fc-associated off-target adverse effects. In summary, our results suggest that ICOS-L.COMP represents a new and powerful biologic to be used in combination with existing immune checkpoint inhibitors.
Rejection-resistant off-the-shelf T cells for adoptive cell therapy
Authors: Mo Feiyan , Srinivasan Madhuwanti , Ma Royce , Smith Tyler S , McKenna Mary K , Atilla Erden , Atilla Pinar Ataca , Heslop Helen E , Brenner Malcolm K , Mamonkin Maksim
Published: January 2023

‘Off-the-shelf’ (OTS) chimeric antigen receptor (CAR) T cells pre-manufactured from healthy donors are a readily available and less expensive alternative to autologous products. However, immune rejection of OTS cells by host T- and NK-cells may limit their persistence and reduce therapeutic effect. Here, we engineered rejection-resistant OTS T cells that recognize and eliminate alloreactive lymphocytes while retaining desired anti-tumor activity. As T- and NK-cells transiently upregulate 4-1BB after activation, T cells expressing a 4-1BB-specific alloimmune defense receptor (ADR) selectively eliminated activated T- and NK-cells while sparing resting lymphocytes. Using this mechanism, ADR-expressing T cells suppressed alloimmune activation and resisted rejection in a mixed lymphocyte reaction (MLR) model in vitro. Further, T cells co-expressing the ADR and a CD19 CAR retained undiminished activity through both receptors in vitro and in vivo. We established a mouse model of allogeneic cell therapy in which NSG mice were engrafted with systemic CD19+ leukemia and normal human T cells. In this model, adoptively transferred unmodified CD19 CAR T cells from an HLA mismatched donor produced only transient anti-tumor activity and were rapidly rejected by pre-engrafted alloreactive T cells within 7 days, leading to fatal leukemia relapse. In contrast, T cells co-expressing both CAR and ADR were protected from immune rejection, resulting in long-term persistence (>9 weeks) and durable leukemia eradication in most animals. These data support the feasibility of using ADR to generate highly potent OTS CAR T cell products that suppress immune rejection to produce long-term therapeutic benefit even in immunocompetent patients.
Modulating Interlukin-7 (IL-7) cytokine receptor signaling to enhance the persistence and anti-tumor efficacy of Epstein-Barr Virus (EBV) specific T-cells in EBV positive malignancies
Authors: Sharma Sandhya , Shum Thomas , Parikh Kathan , Omer Bilal , Rooney Cliona
Published: January 2023

EBV-specific T-cells (EBVSTs) targeting viral proteins in EBV+ lymphoma patients have produced 50% clinical response. Limited T-cell expansion and persistence attributed to lack of cytokines in the tumor microenvironment (TME) remains a major challenge. IL-7 signaling has survival and anti-apoptotic effects on T-cells, however, IL-7 availability in vivo is limited and the IL7 receptor is downregulated on activated T-cells. We aim to overcome this limitation of suppressive TME by augmenting IL-7 signaling.

We recently reported the transgenic expression of a constitutively active IL-7 receptor (C7R) to provide the proliferative and survival advantage in T-cells. Hence, we hypothesize that EBVSTs endowed with cytokine-independent C7R signaling will have resistance to the immunosuppressive TME and increased persistence and anti-tumor potency.

C7R expressing EBVSTs demonstrated enhanced antigen-specific IFN-γ release and superior target specific cytotoxicity in-vitro. In contrast to unmodified EBVSTs, C7R-EBVSTs demonstrated EBV antigen dependent, cytokine-independent proliferation and survival, associated with increased phosphorylated STAT5. Furthermore, C7R signaling provided resistance against TGFβ, Arginase-I, and M2 macrophages, as demonstrated by increased proliferation attributed to decreased apoptosis in C7R-EBVSTs. Following adoptive transfer in an EBV+ murine xenograft model, C7R-EBVSTs persisted significantly longer and demonstrated tumor clearance, while unmodified EBVSTs failed.

C7R signaling augments EBVSTs potency and persistence against suppressive components of TME in our preclinical models. Once effective, the findings will be translated to clinical trials in EBV+ malignancies.
Assessing the in-vitro effects of anti-PD-L1 (Avelumab) and recombinant human rhIL-15 in CD8 T cell function from HIV infected patients
Authors: Goshu Bruktawit , Lane Clifford , Catalfamo Marta
Published: January 2023

Persistent immune activation in chronic HIV infection leads to exhaustion/activation of virus-specific CD8 T cells accompanied by expression of checkpoint receptors such as PD1. Here, we hypothesized that blockade of the PD1/PD-L1 pathway in combination with IL-15 (known to induce proliferation of CD8 T cells) may have a higher impact in improving HIV-specific responses. We assessed the in vitro effects of a checkpoint inhibitor, anti-PD-L1 (Avelumab), in combination with rhIL15 in HIV-specific CD8 T cell proliferation and cytokine secretion. Cell Trace Violet (CTV) labeled PBMCs from HIV infected patients (HIV+, n= 15) and healthy controls (HC, n= 7) were stimulated with HIVGag and CEF-peptides in the following conditions: rhIL-15, anti-PD-L1, and combination rhIL- 15/anti-PD-L1. After 5 days of culture, cells were re-stimulated and analyzed by flow cytometry. We found that rhIL-15 promoted higher antigen-independent proliferation (CTVlow) and cytokine secretion IFNg+ (p=0.009) and TNFa+ (p=0.01) in CD8 T cells from HIV+ patients than HC. In addition, rhIL-15 induced an increased trend in HIVGag-specific CD8 T cells (CTVlowIFNg+) but did not reach significance (p=0.1250). In HC, rhIL15 increased CEF-specific CD8 T cell CTVlowIFNg+ (p=0.03) and TNFa+ (p=0.03). Anti-PD-L1 alone did not show any effect in CD8 T cells from HIV+ patients and HC. In contrast, the combined treatment increased the CTVlowIFNg+ HIVGag-specific CD8 T cells in HIV+ patients (p=0.039) and CTVlowIFNg+ CEF-specific CD8 T cells in HC (p= 0.031). Overall, there was a synergistic effect in HIV-specific CD8 T cell function with the combined treatment. Further studies should assess the potential use of this combined treatment in the context of HIV infection.
Passive infusion of Fc-modified neutralizing antibodies does not affect the dynamics of plasma virus decay in SHIV-infected macaques
Authors: Asokan Mangaiarkarasi , Maximova Anna , Dias Joana , Crowley Andrew R , Pegu Amarendra , Ambrozak David , McKee Krisha , Shi Wei , Todd John-Paul , Ackerman Margaret E , Gama Lucio , Keele Brandon F , Lifson Jeffrey D , Perelson Alan S , Mascola John R , Koup Richard
Published: January 2023

Passive neutralizing antibody (NAb) infusion leads to a reduction of HIV plasma viremia in infected people as well as in SHIV-infected rhesus macaques. Potential mechanisms of viral reduction include neutralization of free virus as well as Fc-dependent effector functions that can clear infected cells. We generated several Fc variants of the human IgG1 NAb VRC07-523LS and characterized them for neutralization, complement binding, ADCC, phagocytosis, and binding to rhesus FcgR. Based on these assays, we down selected two variants, LALA and DEL, that showed knock-out or increase in ADCC and phagocytosis respectively, with complement binding knocked out in both. The parental, LALA and DEL variants of VRC07-523LS were administered at a single dose of 20 mg/kg i.v. to rhesus macaques chronically infected with SHIV-SF162P3 for 6–14 weeks (n=6 to 10 per group). Animals were followed for rate of plasma virus decay and antibody PK. All groups showed similar characteristics: 1) plasma virus decay was delayed for 24h after NAb infusion 2) the rate of plasma virus decay was the same between day 1 and day 5, and 3) plasma virus decay was independent of FcgRIII genotype. Pharmacokinetic analysis showed that serum NAb concentrations in the VRC07-523LS, LALA and DEL groups were maintained at greater than ten-fold excess of the in vitro IC80 against SHIV SF162P3 throughout the period of investigation. Further, unlike VRC07-523LS-LALA, VRC07-523LS-DEL was able to engage natural killer cells and monocytes and mediate both HIV envelope-dependent ADCC and phagocytosis. These results show that the initial impact on plasma viremia by passive NAb therapy is predominantly mediated by virus neutralization rather than ADCC or phagocytosis.