bluuebunny/crossref_metadata_2025 huggingface dataset:
cDC1 prime and receive help from CD4 T cells to promote anti-tumor responses
Authors: Ferris Stephen T , Wu Renee , Durai Vivek , Murphy Theresa L , Murphy Kenneth M
Published: January 2023

CD4 T cells are thought to help promote anti-tumour responses by ‘licensing’ antigen presenting cells (APCs) that activate CD8 T cells. Conventional type 1 dendritic cells (cDC1s) are responsible for cross-presentation of tumour-derived antigens to CD8 T cells. Prevailing models presume that the cDC1 is licensed by CD4 T cells that are themselves activated by a distinct cDC subset, the cDC2. The recent finding that neoantigens presented by major histocompatibility complex (MHC) class II molecules can promote rejection of tumours that lack MHC class II (MHC-II) surface expression is consistent with an indirect action of CD4 T cells, such as cDC1 licensing. However, no study has directly identified the APC that primes the CD4 T cells responsible for licensing or clearly identified the target of CD4 licensing in vivo. Here, we generated cDC1-specific Cre expressing mouse strain to inactivate or induce expression of MHC-I, MHC-II, or CD40 specifically within the cDC1 lineage. Using a tumour model that relies on CD8 T cells and CD4 T cells for rejection, we discovered that priming of CD4 T cells against tumour-derived antigens, in contrast to soluble antigens, relied overwhelmingly on the cDC1 and not the cDC2. cDC1 do not simply transport antigen to lymph nodes for processing by cDC2, since lack of MHC-II expression on cDC1 prevented CD4 T cell priming. We also found that CD40 signaling not only affects licensing of cDC1 for CD8 T cell priming, but is also critical for the activation of CD4 T cells. Thus, in the setting of tumour-derived antigens, cDC1 function as an autonomous platform capable of priming both CD4 and CD8 T cells and orchestrating their cross-talk required for optimal anti-tumour immunity.
Granuloma in tumor-draining lymph nodes impairs tumor-reactive CD8+ T cell trafficking in immune-checkpoint therapy
Authors: Cho Eunjung , Han Chungyong , Kim Seon-Hee , Choi Beom K.
Published: January 2023

Based on the unique properties of 4-1BB (CD137) signaling – strong enhancer of CD8+ T cell proliferation, agonistic anti-4-1BB mAbs are under evaluation as cancer therapeutics in the clinic. However, there are concerns about adverse effects in using 4-1BB agonist. Here we found that hyper-activation of 4-1BB signals induced the accumulation of IFN-γ-producing PD-1+CD8+ T cells in secondary lymphoid organs of tumor-bearing mice by increasing the numbers of each dividing CD8+ T cells, and which were beneficial in suppressing tumor growth in the early phase of anti-4-1BB triggering. However, when the tumor-bearing mice were repeatedly exposed to anti-4-1BB mAb, the mice developed granuloma in tumor draining lymph nodes (TDLNs) by recruiting and accumulating macrophages via CD8+ T cell-IFN-γ axis, which accompanied the excessive LN swelling and eventually the impaired proliferation and trafficking of CD8+ T cells in TDLNs. Our data not only reveals the granuloma in TDLN as unexpected adverse effects of anti-4-1BB mAb, but also provides an insight that the granuloma in TDLN is the other side of the enhanced CD8+ T cell responses.
NF-kB1 induces T cell dysfunction to promote colon tumorigenesis
Authors: Lu Chunwan , Klement John D. , Xiao Wei , Ibrahim Mohammed L. , Smith Alyssa D. , Yang Dafeng , Browning Daren , Munn David H. , Liu Kebin
Published: January 2023

Background and aims

Chronic colonic inflammation drives colon tumorigenesis. Chronic infection-associated inflammation causes T cell exhaustion/dysfunction. NF-kB is a molecular link between colonic inflammation and colorectal cancer. We aimed at testing the hypothesis that the p50 NF-kB (NF-kB1) regulates T cell dysfunction to promote colon tumor immune evasion and development.

Methods

Nfkb1 knock out (p50 KO), Nfkb1 KO chimera mice, Nfkb1 KO colon tumor cells were used to determine p50 NF-kB function in colon tumor development. RNA-Seq, chromatin immunoprecipitation, and electrophoresis mobility shift assay were used to elucidate molecular mechanism of NF-kB1 action. Flow cytometry was used to analyze cytotoxic T lymphocytes.

Results

p50 KO mice showed a significantly decrease in tumorigenesis. However, mice with p50 deficiency only in immune cells also exhibited a significant decrease in tumorigenesis, and p50-sufficient colon tumor cells also grew significantly slower in p50 KO mice than in WT mice. RNA-Seq identified Gzmb as a p50 NF-kB target gene. p50 KO mice have significantly higher tumor-infiltrating granzyme B+ CTLs than WT mice, and CTL granzyme B protein level is also significantly higher in p50 KO mice than in WT mice. A kB sequence element was identified at the Gzmb promoter and p50 NF-kB directly binds to this kB element in T cells to repress Gzmb expression.

Conclusion

NF-kB1 has opposing functions in tumor cells and T cells in vivo. The p50 NF-kB represses Gzmb expression to confer colon tumor-infiltrating CTL a dysfunctional phenotype to promote colon tumor immune evasion, resulting in enhanced colon tumorigenesis and growth.
PD1 and LAG3 converge to limit polyfunctionality and systemic immunity
Authors: Andrews Lawrence Peter , Manne Sasikanth , Wherry E. John , Workman Creg , Vignali Dario
Published: January 2023

Targeting PD1 has yielded clinical success across many tumor types, yet a significant proportion of patients remain unresponsive to treatment. Co-expression of PD1 and LAG3 inhibitory receptors (IRs) on CD8+ tumor-infiltrating T cells (TIL) is associated with an exhausted phenotype and dual blockade synergistically limits tumor growth, greater than targeting PD1 alone.

The cellular and mechanistic basis for PD1/LAG3 synergy is unknown. We have generated conditional knockin mice that facilitates the CD8+ T cell-restricted deletion of Pdcd1 and/or Lag3 when crossed to E8ICre.GFP. These mice have also been crossed with a pMEL TCR transgenic mouse, congenically marked to allow intrinsic analysis of PD1 and/or LAG3-deficient CD8+ T cells, and controls, in a ‘quad’ co-adoptive transfer system.

Pdcd1L/L and/or Lag3L/L-yfp E8ICre.GFP mice show reduced B16-F10 tumor growth with improved survival, compared to Lag3L/L-yfp E8ICre.GFP mice and controls. CD8+ TIL frequency is increased with loss of both IRs, as a result of enhanced proliferation. Although expression of TIM3, TIGIT and 2B4 IRs is maintained, CD8+ TIL isolated from Pdcd1L/LE8ICre.GFP and Pdcd1L/ Lag3L/L-yfp E8ICre.GFP mice show increased polyfunctionality, by IFNg, TNFa and GzmB release. This was confirmed to be largely driven by effector and chemoattractive secretions by analysis with a 28-plex single-cell cytokine response panel (Isoplexis).

Overall, these data suggest that PD1 and LAG3 synergize to have a dominant effect on CD8+ TIL and limit antitumor immune effects, as intrinsic removal of both IRs results in reduced tumor growth that substantially impacts anti-tumor immunity. These results are encouraging for the development of co-targeting LAG3 and PD1 in the clinic.
Evaluation of immune checkpoints in small cell lung cancer: implications for immunotherapy
Authors: Thomas Portia , Maddox Sarah , Iams Wade , Quaranta Vito , Tyson Darren , Lovly Christine
Published: January 2023

Introduction

The FDA recently approved Atezolizumab, a programmed death-ligand 1 (PD-L1) inhibitor, for first-line combinatory treatment in patients with extensive-stage small cell lung cancer. Across several cancer types, immunotherapy has been shown to provide durable benefit in patients with metastatic disease. However, Atezolizumab only improves overall survival by 2 months. This could be due to the fact that <20% of patients express >1% tumor PD-L1 positivity. We assert that a better understanding of this immune contexture – defined as abundance and type of checkpoint molecules in SCLC – may provide important criteria for patient stratification.

Objectives

In these studies, we will test the hypothesis that immune checkpoints in addition to the PD-1/PD-L1 axis play a role in immune evasion in SCLC.

Methods

To evaluate expression of immune checkpoint molecules, we acquired bulk RNA-Seq data of SCLC cell lines and primary tumor specimens. Further, we generated tissue-microarrays containing >20 unique cell lines and >200 unique patient specimens with fully annotated clinical data to confirm expression of these molecules by immunohistochemistry.

Results

Preliminary RNA-Seq analyses show low PD-L1 expression in SCLC cell lines and primary tumors. Further, ubiquitously high expression of certain checkpoint molecules (B7-H3) is seen. Future analysis will confirm abundance and type of checkpoint molecules as well as capture interpatient heterogeneity.

Conclusions

Understanding the checkpoint molecules present in SCLC could further understanding of SCLC immune suppression, ultimately leading to optimal single-agent immunotherapy deployment and rational, effective combination therapy for patients with SCLC.
Pembrolizumab treatment increases K+ channel function and calcium fluxes in cytotoxic T cells of head and neck cancer patients
Authors: Newton Hannah , Gawali Vaibhavkumar S , Chimote Ameet A , Lehn Maria , Palackdharry Sarah , Hildeman David , Janssen Edith , Wise-Draper Trisha , Conforti Laura
Published: January 2023

Head and neck cancer (HNC) is the sixth most common cancer worldwide with a five-year survival of 50%. Immunotherapy is emerging as a promising treatment modality in HNC. Pembrolizumab, an anti-PD-1 antibody, is an immunotherapy currently approved in metastatic HNC and in clinical trials for curative intent. Although there are promising responses to Pembrolizumab, its effect on cytotoxic CD8+ T cell function is not understood. T cell cytotoxicity is critically important in the elimination of tumors and in the tumor microenvironment, cytotoxicity depends on ion channel activity which is defective in HNC patients. Therefore, to elucidate mechanisms of Pembrolizumab, we studied potassium channel (KCa3.1 and Kv1.3) functionality and Ca2+ fluxes in CD8+ peripheral blood T cells (PBTs) of naïve HNC patients before/after Pembrolizumab treatment (n=22) as well as tumor infiltrating lymphocytes (TILs) functionality. Untreated HNC patients were controls (n= 13). Pembrolizumab-treated patients were categorized as responders (R) or non-responders (NR) based on pathological response. We then performed electrophysiological and Ca2+ flux experiments. We observed that Pembrolizumab increased KCa3.1 currents in PBTs (29.8%, p=0.026). Moreover, after treatment, R PBTs also had increases in Kv1.3 currents and Ca2+ fluxes (22.1%, p=0.047; 16.5%, p<0.001; respectively). In TILs, Kv1.3 currents and Ca2+ fluxes were increased in Pembrolizumab-treated patients (64%, p=0.002; 34.8%, p=0.004; respectively). These data show that Pembrolizumab increases the fitness of CD8+PBTs and TILs in relation to ion channel functionality, especially in R, and thus could lead to better understanding of the Pembrolizumab mechanism of action/resistance.
Novel non-segmented negative-sense RNA virus-based vaccine platforms for Zika virus
Authors: Li Anzhong , Lu Mijia , Yu Jingyou , Harder Olivia , Attia Zayed , Shan Chao , Liang Xueya , Xue Miaoge , Shi Pei-Yong , Peeples Mark E. , Liu Shan-Lu , Boyaka Prosper N , Niewiesk Stefan , Li Jianrong
Published: January 2023

Zika virus (ZIKV) is a mosquito-borne flavivirus that can cause Congenital Zika Syndrome (including microcephaly), Guillain-Barré syndrome, and other severe neurological disorders. Despite major efforts, there is no FDA-approved vaccine for ZIKV. Current efforts to develop ZIKV subunit vaccines have been exclusively focused on pre-membrane (prM) and envelope (E) proteins. However, the role of other viral proteins such as nonstructural protein 1 (NS1) in protection is poorly understood. Here, we developed two novel ZIKV vaccine candidates using non-segmented negative-sense RNA viruses (vesicular stomatitis virus, VSV; measles virus, MeV) as a vector. ZIKV prM-E-NS1 or prM-E gene was inserted into the attenuated VSV or MeV vector, and recombinant viruses expressing prM-E-NS1 or prM-E were recovered. The VSV- and MeV-based vaccine candidates were highly attenuated, induced strong ZIKV-specific antibody and T cell immune responses in animal models, and provided complete protection against ZIKV challenge in both immunocompetent and immunodeficient mice. Compared with vaccine candidates expressing only prM-E, ones with NS1 induced significantly higher levels of antibody and/or T cell responses and provided better protection against ZIKV infection, suggesting that NS1 plays a regulatory role in immune response and protection. In addition, we found that NS1, alone, is capable of conferring protection against ZIKV infection even though it did not induce neutralizing antibody. Our results demonstrated that prM-E-NS1 was superior to prM-E as a vaccine antigen. In summary, we have developed safe and highly efficacious ZIKV vaccine candidates, which are ready for pre-clinical trials in nonhuman primates and humans in the future.
Mucosal Vaccination with a Novel Live Attenuated Brucella melitensis Mutant Protects via IFN-γ-producing Memory CD8+ T Cells in Concert with B Cells
Authors: Goodwin Zakia I , Yang Xinghong , Hoffman Carol , Pascual David W
Published: January 2023

A B. melitensis mutant vaccine expressing mCherry (znBM-mC), when applied mucosally, conferred nearly complete protection against pulmonary challenge with wild-type (wt) BM 16M than did BM Rev-1 vaccine. To identify cellular subsets and mechanisms involved in inducing and maintaining protection post-BMDM vaccination, we hypothesize that both arms of the adaptive immune system are required for optimal protection. Mucosal vaccination with znBM-mC induced both effector memory and resident memory CD8+ T cells in the lungs, which were the primary source of IFN-γ. In the absence of IFN-γ, IL-17 significantly increased, but was insufficient for protection. In the absence of B cells, the total number of lung CD8+ T cells and proinflammatory cytokine production were significantly reduced resulting in diminished systemic protection. Reconstitution with naïve or immune B cells restored some protection in znBM-mC-vaccinated B cell-deficient mice. B cell involvement begins as early as 5 days post-nasal vaccination. Total CD19+ B cells significantly increased in BALB/c head and neck lymph nodes (LNs), lower respiratory LNs, and bronchio-alveolar lavage fluid. mCherry+ BALB/c B cells were elevated in the lungs and spleen correlating to a significant increase in CD8+ T cell expansion in the spleen compared to B cell-deficient mice. In-vitro uptake of znBM-mC by B cells resulted in their activation evidenced by increased CD80, CD86, CD11b, and CD24 expression. Moreover, purified immune CD8+ T cells co-cultured with znBM-mC-infected B cells showed enhanced T cell proliferation. Although B cells become infected with Brucella, these are important for optimal T cell immunity. Work is supported by NIH AI123244 & AI125516.
Vaccine adjuvant choice regulates antigen-specific B cell activation and phenotype
Authors: Prior John Timothy , Taylor Justin J. , McLachlan James B
Published: January 2023

There has been a global rise of antibiotic resistant bacteria that are causing a significant increase in morbidity and mortality. Vaccines offer one of the best ways to combat and prevent these infections. Antibody responses are often the most effective outcomes of vaccine-induced immunity; however, the role of different vaccine adjuvants has not been fully explored in terms of antigen-specific B cell responses. Using a B cell antigen tetramer, we sought to determine how a novel adjuvant, double mutant heat labile toxin from enterotoxigenic E. Coli (dmLT), compared with those currently used in vaccines. We show that either intradermal or intramuscular immunization of mice with a model vaccine formulated using dmLT induces greater antigen specific B cell numbers in the injection site draining lymph nodes and spleen compared to antigen alone. These B cells were also antibody isotype switched and were phenotypically germinal center B cells. We further demonstrate that vaccine-specific B cells migrate from the injection site draining lymph nodes to more distal lymphoid organs. When we compared dmLT to the most commonly used human vaccine adjuvant, alum, we found dmLT was better at inducing a B cell response. Lastly, we show that dmLT is a more potent B cell adjuvant compared to the TLR4 agonist monophosphoryl lipid A (MPL-A) and that the combination of these two adjuvants is superior to either adjuvant alone. Interestingly, MPL-A induced greater antigen specific IgG compared to dmLT despite dmLT inducing a more robust B cell expansion. These results demonstrate that dmLT is a potent B cell activating adjuvant and that combining dmLT with existing adjuvants has the potential to greatly improve vaccine responses.
Vaccine and abscopal effects result from in situ vaccination with nanosecond electric pulse treatment for breast cancer in mice
Authors: Guo Siqi , Nanajian Anthony , Scott Megan , Beebe Stephen J. , Ruedlinger Brittaney
Published: January 2023

An electrical engineering technology, nanosecond electric pulse (nsEP), has been studied to ablate local tumor in a non-thermal and minimally invasive manner. Recently, several groups including our lab discovered that animals with complete tumor ablation following nsEP treatment were protected from a second tumor challenge. Therefore, nsEP tumor ablation is also a novel in situ vaccination approach. Breast cancer is considered a low immunogenic tumor with only a small subset of patients responsive to immune checkpoint inhibitors. We found nsEP treatment resulted in not only a potent in situ vaccine effect in a poorly immunogenic 4T1 mouse breast cancer model but also an abscopal effect, a rejection of distant untreated tumor lesion. We further examined the impact of nsEP on cancer cell death, tumor microenvironment (TME) modification, the dynamic changes of local and systemic immune response. 4T1 cells treated with nsEP did not show apoptotic death markers but released damage-associated molecular patterns, including calreticulin, high mobility group protein B1, and ATP. In contrast to abundant immune suppressor cells in untreated breast TME, nsEP treatment led to a significant reduction of both myeloid-derived suppressor cells and regulatory T cells. Conversely, the ratio of tumor associated macrophages M1-like verse M2-like was reversed. An increase of tissue-resident memory T cells was observed in draining lymph nodes. In summary, our results suggest that nsEP is a novel non-drug immunogenic cell death inducer and in situ vaccination approach. Further elucidation of its mechanisms and the development of an in vivo nsEP delivery system suitable for patients is critical to warrant this novel technology toward a clinical trial.
Secreted heat shock protein gp96-Ig vaccine induces malaria specific intrahepatic CD8 T cell responses
Authors: Strbo Natasa , Padula Laura , Fisher Eva , Wijayalath Wathsala , Patterson Noelle B. , Huang Jun , Ganeshan Harini , O’Neill Katelyn , Garcia Denisse , Etobayeva Irina V. , Edgel Kimberly A. , Podack Eckhard R , Sedegah Martha , Villasante Eileen F.
Published: January 2023

A highly-efficacious and durable malaria vaccine is an essential tool for global malaria eradication. One promising strategy to develop such a vaccine is to induce robust CD8+ T cell-mediated protective immunity against malaria liver stage parasites.

We developed and tested a novel malaria vaccine platform based on the secreted form of the heat shock protein gp96 immunoglobulin, (gp96-Ig) to induce malaria antigen-specific liver resident memory CD8+ T cells. Gp96-Ig has ideal properties as an antigen carrier and vaccine adjuvant to activate antigen presenting cells (APCs) and effectively chaperone antigens for cross presentation via MHC I to CD8+ T lymphocytes. Our study shows that vaccination of mice and non-human primate (NHP) animal models with HEK-293 cells transfected with gp96-Ig and two well-known Plasmodium falciparum (Pf) vaccine candidate antigens, circumsporozoite protein (CSP) and apical membrane antigen 1 (AMA1), induce liver infiltrating, antigen-specific, cytotoxic memory CD8+ T cell responses. The majority of the CSP and AMA specific intrahepatic CD8+ T cells expressed CD69 and CXCR3, hallmarks of tissue resident memory T cells (TRM). Also, we found an increased frequency of intrahepatic CD8+ T cells secreting IL-2, which is relevant for maintenance of effective memory responses.

Our findings are strongly supportive of a novel gp96-Ig-based malaria vaccine as a unique systemic and liver-homing, liver-stage antigen-specific CD8+ cytotoxic T lymphocyte (CTL) vaccine strategy.
Superior CD8 T cell-mediated protection against malaria parasite liver stage infection after vaccination with Replication Competent Genetically Attenuated Parasites (RC-GAP) and impact of blood stage infection on immunity
Authors: Patel Hardik , Minkah Nana K. , Sheikh Amina A. , Reynolds Laura , Vaughan Ashley M. , Kappe Stefan H.I.
Published: January 2023

Plasmodium sporozoites (SPZ) are transmitted by mosquito bite, infect the liver, grow and differentiate as intrahepatocytic liver stages (LS) and ultimately form the first generation of red blood cell-infectious merozoites, which initiate the symptomatic blood stage infection. A vaccine against the clinically silent pre-erythrocytic SPZ and LS stages is attractive as it confers protection against infection before onset of symptomatic blood stage infection. Immunization with replication-deficient attenuated SPZ constitutes a promising vaccination strategy to engender protective humoral and CD8 T cell responses, with tissue resident memory CD8 T cell (TRMs) mediating sterilizing immunity in animal models. Attenuated parasite vaccines have undergone clinical testing and it was observed that sterilizing immunity is high in malaria naïve subjects but lower in malaria pre-exposed subjects residing in malaria-endemic regions. Previous studies in rodent models indicate that the ongoing blood stage infection suppresses protection after immunization with replication-deficient irradiated sporozoites. We have shown that replication competent late liver stage arresting genetically attenuated parasites (RC-GAP) confer superior protection when compared to irradiated sporozoites. We show that this protection is dependent on CD8 TRMs in the liver. However, whether the blood stage infection also suppresses the RC-GAP parasite vaccine mediated protective immunity remains unknown. Here, we examined whether blood stage infection either before or during RC-GAP immunization affects vaccine induced protection in the Plasmodium yoelii/Balb/c mouse model and how this correlated with perturbation of memory T and B cell responses.
A small molecule based immunotherapy targeting IL-10/STAT3 praxis to augment the potential of rBCG30 vaccine against experimental tuberculosis
Authors: Ahmad Faraz , Umar Mohd Saad , Khan Nazoora , Gupta Pushpa , Gupta Umesh Datta , Owais Mohammad
Published: January 2023

Several recent studies have contributed to our understanding of the need for host directed immunotherapeutic strategies against Mycobacterium tuberculosis (Mtb), either concomitantly with classical chemotherapy or as standalone intervention. Nevertheless, most of these studies were focused on enhancing chemotherapeutic potential of standard anti-tuberculosis chemotherapy and only few were aimed towards improving prophylactic immunity offered either by BCG or its potential replacements. In the present study we tried to address the potential gap with the aim to improve anti-TB immunity offered by 2nd generation rBCG30 vaccine. We attempted to bolster rBCG30 invoked anti-TB immunity in mice through modulation of the IL-10/STAT3 interaction mediated anti inflammatory program. An immunomodulator of IL-10/STAT3 signaling axis was administered in immunized mice to reinforce the anti-pathogen capabilities of mononuclear phagocyte system with expectation to improve T cell mediated secondary immune responses. We envisage that modulation of this crucial praxis may result in reduced expansion of pathogen permissive AAMs/AADCs (Alternatively Activated Monocytes/Macrophages/DCs) and augmented abundance of pro-inflammatory CAMs/CADCs (Classically Activated Monocytes/Macrophages/DCs), the cells that resist establishment of successful Mtb infection. The study was also aimed towards generating optimum activated APCs early either after vaccination or infection, which can process and present mycobacterial antigens more efficiently to T cells.
Th17 responses to Pseudomonas aeruginosa vaccines are defective in cystic fibrosis mice
Authors: Merakou Christina , Bonney Sarah , Liao Tiffany , Priebe Gregory P.
Published: January 2023

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) associated with lung function decline. Since levels of IL-17 are high in the CF airway, it is unclear whether vaccines that induce Th17 responses in CF hosts would be effective, or potentially harmful. Our prior work showed that vaccine-induced Th17 cells confer antibody-independent protection against acute P. aeruginosa pneumonia in mice and that the P. aeruginosa protein PopB stimulates protective Th17 responses. In this study, we tested whether vaccine-induced Th17 responses are abnormal in CF mice.

CF mice (delF508 in FVB/N background) and littermate controls were immunized intranasally either with live-attenuated P. aeruginosa vaccines or with PopB mixed with curdlan as adjuvant. We tested Th17 responses by measuring IL-17 secretion from splenocytes stimulated with antigens in vitro. Protective efficacy was assessed using a murine acute pneumonia model.

Our results demonstrate that CF mice show 2-fold lower IL-17 recall responses in the spleen after IN immunization with a live-attenuated vaccine in comparison to wild-type mice (P<0.05 by ANOVA). CF mice were not protected when challenged with a LPS-heterologous strain, suggesting the absence of Th17-mediated immunity when LPS-directed opsonic antibodies are not available. CF mice immunized with PopB also showed defective Th17 responses in the spleen compared to wild-type or heterozygous mice (2-fold lower, P<0.05 by ANOVA).

CF mice have low Th17 responses to P. aeruginosa vaccines. Correction of the underlying CF defect may be needed to achieve vaccine-induced protection against P. aeruginosa in the setting of CF.
The regulation of transcription factor T cell factor 1 (TCF1) in vaccine-elicited CD8+ T cells
Authors: Ivanova Daria Leonidovna , Kilgore Augustus , Thompson Scott , Kedl Ross
Published: January 2023

Our lab has developed a subunit vaccination regimen that induces a highly robust T cell response. These vaccine-elicited CD8+ T cells (Tvac) are phenotypically, functionally and metabolically distinct from infection-elicited CD8+ T cells (Tinf). In contrast to Tinf, almost all Tvac rapidly acquire a memory phenotype (CD127hi, KLRG1lo). This suggests that Tvac could be differentially transcriptionally programmed to commit to a memory, rather than effector, cell fate. The transcription factor TCF1 (encoded by tcf7) is essential for CD8+ T cell memory in response to infection. Since high TCF1 expression is also important for the generation of stem-like T cells with long-term survival capacity and anti-tumor properties, it is now recognized as a promising target for immunotherapies. However, little is known about how TCF1 is regulated in response to subunit vaccination. We find that, in striking contrast to Tinf, TCF1 expression is actually increased in Tvac after vaccination. As others have shown, we also find that increasing inflammation during vaccination by the addition of TLR9 agonist CpG reduces TCF1 expression in Tvac, skewing their memory phenotype towards the effector phenotype (CD127lo, KLRG1hi) and compromising their long-term memory. While the loss of TCF1 in this context has conventionally been ascribed to inflammatory receptors, utilizing reporter Nur77GFP mice, we also observe an altered duration and frequency of TCR triggering in the context of inflammation. Our data indicates a complex, previously unappreciated interplay between inflammatory and TCR-mediated signals in the regulation of TCF1.
Immunomodulatory effects of topotecan augment the effectiveness of chemotherapy-immunotherapy combination
Authors: Lee Jeongmi , Shin Kwang-Soo , Koh Choong-Hyun , Jeon Insu , Kang Tae-Seung , Song Boyeong , Kang Chang-Yuil
Published: January 2023

Combination therapy is a cornerstone of anticancer therapy, suggesting potential therapeutic benefit to nonresponders to single-agent treatment. BVAC, a B-cell-and-monocyte-based vaccine loaded with alpha-galactosylceramide (α-GC) and tumor antigen as reported in our previous studies, can be successfully combined with other treatments.

Here we found out a novel synergistic regimen in TC-1 tumor model which combines BVAC with topotecan, a chemotherapeutic agent as a topoisomerase I inhibitor. We demonstrated some significant alteration in the immunoprofiles of topotecan-treated mice. We observed increased tumor recruitment of monocytes and CD8+ T cells, and decreased frequencies in regulatory T cells in tumor. Accordingly, we are currently investigating how topotecan-induced changes in immune cell composition could lead to improvement of antitumor effect. These data indicate that topotecan not only has cytotoxicity to tumor but can contribute to modulating tumor immune microenvironment.

We anticipate that this study could provide a rationale for combination of cancer vaccine and chemotherapy and define future optimal treatment regimens in cancer patients.
Novel minigene vaccine stimulates CD8+ memory inflation to control growth of murine colorectal tumor
Authors: Kwok Jonathan , Kroon Anna Maria Theresa , Chinnakannan Senthil , de Lara Cathy , Gileadi Uzi , Cerundolo Vincenzo , Klenerman Paul , Lee Lian Ni
Published: January 2023

Fundamental science

Memory “inflation” describes the sustained accumulation of expanded pools of antigen-specific CD8+ T-cells, which occurs in certain persistent viral infections e.g. cytomegalovirus, and after vaccination with replication deficient adenoviral vectors. Inflating T cells are sustained at high frequencies, possess an effector memory phenotype and home to non-lymphoid tissues – characteristics which are desirable in anti-tumor responses. Use of minigene (epitope only) vaccines allows targeting of responses and reliable generation of strong memory inflation. To explore this, adenoviral vectors were designed to stimulate an anti-tumor response against the dominant antigen in CT26 tumors, AH1, either as a full length or peptide immunogen. Groups of mice were immunized with the minigene AH1 vector. Tumor growth and immune responses were monitored using prophylactic or therapeutic regimens.

Results

Our lab has previously demonstrated that minigene vaccines generate strong inflationary responses compared to a full-length vector. Compared to the controls, tumors from minigene-immunized mice grew at a significantly slower rate, and the intra-tumoral environment was enriched for functional effector cells. The presence of AH1-specific CD8+ T cell populations in peripheral blood correlated overall with the anti-tumor effect.

Conclusions

Inflationary CD8+ T cells can be targeted against tumor antigens using our adenovirus minigene approach. Such responses show anti-tumor activity in prophylactic and therapeutic settings. Further studies to assess the impact of increasing the breadth of targeting and the impact of checkpoint inhibition are ongoing.
Application of individual HLA-binding prediction thresholds increases the detection of target cell surface-presented HPV16 E6- and E7-derived epitopes as a basis for therapeutic HPV vaccine design
Authors: Bonsack Maria , Mohan Nitya , Öhlenschläger Kathrin , Lotsch Catharina , Heinze Julia M. , Schmitt Janika , Hoppe Stephanie , Förster Jonas , Blatnik Renata , Riemer Angelika B.
Published: January 2023

For the development of a therapeutic vaccine against human papillomavirus (HPV), viral epitopes bona fide presented on the surface of HPV-transformed cells need to be identified. The constitutively expressed HPV oncoproteins E6 and E7 represent ideal targets for immunotherapeutic vaccination approaches. However, viral epitopes are low abundant and human leukocyte antigen (HLA)-restricted, posing a challenge for developing peptide-based vaccines.

For the identification of novel epitopes, in silico HLA-binding prediction was used to preselect HPV16 E6- and E7-derived peptides for 7 major HLA-types representing 5 HLA supertypes. Based on performance evaluation of predictors, new individual binding affinity thresholds were applied. HLA-binding of synthesized predicted binders was validated in vitro in competitive cellular binding assays. Interferon-γ ELISpot assays performed with blood samples from HLA-characterized healthy donors identified memory T cell responses against HLA-binding HPV peptides. T cells of responding donors were further characterized for epitope-specific mediation of specific target cell lysis by CD8+ CTLs using a flow cytometry based cytotoxicity assay. Complementary, an immunopeptidomics approach was used to determine the surface presentation of epitopes on HPV16-transformed cancer cells. Targeted liquid chromatography-mass spectrometry was capable of detecting epitopes predicted within the new individual affinity thresholds but missed by common thresholds.

Completing the identification of bona fide target cell surface-presented E6- and E7-derived T cell epitopes will provide a map of targets for the development of a therapeutic vaccine to treat HPV16-induced malignancies.
Preclinical studies of the off-target reactivity of AFP158-specific TCR engineered T cells
Authors: He Yukai , Cai Lun , Caraballo-Galva Leidy D , Peng Yibing
Published: January 2023

Autologous T cells engineered with T receptor genes (TCR-T) are being studied to treat cancers. We have recently identified a panel of mouse TCRs specific for the HLA-A0201/alpha fetoprotein epitope (AFP158) complex and have shown that the TCR-engineered human T cells can eradicate hepatocellular carcinoma xenografts in NSG mice. In this current study, we evaluate their off-target toxicity. We found that 3 AFP158-specific TCR-Ts could be cross-activated by ENPP1436 peptide and that the TCR3-Ts could also be activated by another off-target peptide, RCL1215. However, compared to AFP158, it requires 250 times more ENPP1436 and 10,000 times more RCL1215 peptides to achieve the same level of activation. The EC50 of ENPP1436 peptide for activating TCR-Ts is approximately 17–33 times higher than AFP158. Importantly, the ENPP1+ tumor cells did not activate TCR1-Ts and TCR2-Ts, and only weakly activated TCR3-Ts. The IFNγ produced by TCR3-Ts after ENPP1+ cell stimulation was 22 times lower than that after HepG2 cells. And, all TCR-Ts did not kill ENPP1+ tumor cells. Furthermore, ectopic over-expression of ENPP1 protein in HLA-A0201+ tumor cells did not activate TCR-Ts. In silico analysis showed that the ENPP1436 peptide affinity for HLA-A0201 was ranked 40 times lower than AFP158 and the chance of ENPP1436 peptide being processed and presented by HLA-A0201 was 100 times less likely than AFP158. In contrast, the two off-targets (Titin and MAGE-A3) that did cause severe toxicity in previous trials have the same or higher MHC-binding affinity and the same or higher chance of being processed and presented. In conclusion, our data showed that these TCR-Ts, especially TCR1-Ts and TCR2-Ts, will unlikely cause significant off-target toxicity.
Improved trafficking and tumor control with regional, pressure-enabled drug delivery of anti-CEA CAR-T cells for colorectal liver metastases in a murine model
Authors: Chai Louis F. , Hardaway John C. , Heatherton Kara R. , O’Connell Kyle P. , LaPorte Jason P. , Guha Prajna , Saied Abdul , Knight Robert , Jaroch David , Cox Bryan F. , Katz Steven C.
Published: January 2023

Introduction

Efficacy of cellular immunotherapy for liver metastases (LM) has been limited by poor trafficking and elevated intra-tumoral pressures. In a phase 1b study, we found that regional, pressure-enabled drug delivery (PEDD, TriSalus Life Sciences, Inc.) improved LM CAR-T delivery. We hypothesized that PEDD of CAR-T in a murine model can improve LM control.

Methods

LM bearing CD45.2 mice were treated with CD45.1 anti-CEA CAR-T via systemic delivery (SD, tail vein) or regional delivery (RD, portal vein) at low pressure (LPRD) vs. high pressure (HPRD) to model PEDD. Flow rates were calibrated to achieve LPRD or HPRD. Tumor bioluminescence (TB), flow cytometry (FC), immunofluorescence (IF), & quantitative polymerase chain reaction (qPCR) were assessed on various post-treatment days (PTD).

Results

FC of liver non-parenchymal cells on PTD7 showed increased CAR-T density in HPRD & LPRD (9.4% & 11.0%, p = 0.03 & 0.002) vs. SD (3.7%). qPCR of the CAR transgene supported this observation, but was not significant (HPRD 135.8, p = 0.76, LPRD 108.6, p = 0.40 vs. SD 69.8). TB was lower in RD vs. SD up to PTD7 (log2 fold-change HPRD −1.3 & LPRD −0.9 vs. SD 4.3, p = 0.005 & 0.006). Tumor proliferation by Ki67 IF was significantly reduced with RD vs. control (HPRD & LPRD, p < 0.001 & p = 0.05) with HPRD superior to LPRD (p = 0.03). Serum liver enzymes were not significantly elevated using HPRD vs. LPRD at PTD7 (AST/ALT 154.7/36.3 vs. 139.1/29.1, p = 0.84/0.67), but did rise at PTD17, correlating with tumor progression (AST/ALT 800.7/229.3 vs. 387.5/139.5, p = 0.10/0.28).

Conclusions

RD increased LM CAR-T delivery and HPRD showed evidence of improved CAR-T trafficking and tumor control relative to LPRD. Further clinical testing of PEDD is planned for LM cellular therapy.
Engineering CAR T cells with a GM-CSF:IL-18 cytokine switch receptor results in potent antitumor activity in solid tumors
Authors: Lange Shannon Marie , Sand Laurens , Gottschalk Stephen
Published: January 2023

Immunotherapy in the form of chimeric antigen receptor (CAR) T cells has emerged as a promising treatment option for various cancers following its success for the treatment of B cell hematological malignancies. However, the solid tumor microenvironment (TME) has presented many challenges to CAR T cell efficacy and persistence, in part due to lack of T cell-supportive cytokines at the tumor site. We hypothesized that we could improve the efficacy of CAR T cell infusion by engineering a self-sustaining cytokine receptor that would fuel CAR T cells in the TME. One cytokine that is abundantly produced by CAR T cells upon stimulation and is abundant in the TME is the myeloid cytokine GM-CSF. We exploited this biology to design a chimeric cytokine switch receptor that would bind GM-CSF, but signal intracellularly through the IL-18 receptor (GM18). This switch receptor encoded by a retroviral vector was transduced into second generation CAR T cells targeting different solid tumor antigens in order to test the effect of GM18 in multiple model systems. In culture, GM18-expressing CAR T cells initially killed tumor cells to the same degree as unmodified CAR T cells, but displayed greater expansion when repeatedly challenged with tumor cells over time. In vivo, GM18+ CAR T cells exhibited enhanced antitumor activity compared to unmodified CAR T cells in NSG experimental models of solid tumors. These findings paralleled greater expansion and persistence by GM18+ CAR T cells, leading to improved survival. Overall, this work demonstrates the GM18 cytokine switch receptor as an advantageous modification to CAR T cells in solid tumors, suggesting this receptor could be a novel mechanism to modify existing cellular therapies to enhance efficacy in solid tumors.
Type I interferons mediate pathogenic reactive oxygen species during neonatal influenza virus infection
Authors: Kumova Ogan K , Carey Alison J
Published: January 2023

Infants less than six months of age are particularly susceptible to respiratory viral infection-associated morbidity and mortality. Type I Interferons, IFN I, a potent anti-viral cytokine which controls viral replication, can also result in the production of pathogenic reactive oxygen species, ROS. Infants are particularly susceptible to ROS because of immature antioxidant production, such as superoxide dismutase 3, and demonstrate increased pathology under oxidative stress conditions. Using our previously established neonatal mouse influenza virus model, we sought to determine if IFN I was protective or deleterious in neonatal influenza virus infection. Three-day old neonatal IFN I receptor, IFNAR-deficient and C57Bl/6 mice were infected with PR8, H1N1 influenza virus and tracked for survival. IFNAR deficient neonatal mice had improved survival, 80% survival versus 20% survival in C57Bl/6 pups. To determine the mechanism of IFN I toxicity, pulmonary type II epithelial cells were isolated 48 hours post-infection and IFNb was applied to the epithelial cells ex vivo. A flow cytometric CellROX assay was performed to determine production of ROS. When exposed to IFNb, neonatal lung epithelial cells produce more ROS than adult lung cells. To determine if an antioxidant could mitigate oxidative stress during in vivo neonatal influenza virus infection, 3 day old murine neonates were treated with an antioxidant, N-acetyl cysteine, NAC, after influenza virus infection. Survival improved from 10% in sham-treated pups to 60% in NAC-treated pups. We speculate that IFN I-induced ROS, coupled with the neonatal inability to neutralize ROS, increases mortality in this vulnerable population.
Early IFN-I limits virus-specific immunity
Authors: Palacio Nicole M , Dangi Tanushree , Chung Young Rock , Wang Yidan , Loredo Juan , Zhang Zhongyao , Penaloza-MacMaster Pablo
Published: January 2023

Type I interferons (IFN-I) play a major role in the innate immune response. These cytokines are induced within hours of infection, conferring rapid protection before the onset of adaptive immunity. IFN-I are also critical for the priming of adaptive immune responses, but it is not clear if early antiviral restriction by IFN-I can also limit the amount of antigen available to prime the adaptive immune system. Here we demonstrate that reinforcement of IFN-I signaling early after viral immunization limits the priming of adaptive immunity, whereas blocking IFN-I signaling improves adaptive immunity and enhances subsequent host protection following viral re-infection. Paradoxically, short-term blockade of IFN-I improved the efficacy of clinically approved vaccines, such as the yellow fever virus vaccine (YFV-17D) and the smallpox virus vaccine, as well as experimental HIV vaccines in mice. Short-term blockade of IFN-I also improved immune responses following acute viral infections, such as those with Zika virus, lymphocytic choriomeningitis virus (LCMV), and vesicular stomatitis virus (VSV). This unexpected adjuvant effect of blocking early IFN-I responses was associated with a hyperacute increase in antigen availability, which could not be recapitulated by increasing the dose of the virus inoculum. The improvement of virus-specific immunity following acute IFN-I blockade was mechanistically due to an increase in antigen presentation and costimulation. These findings warrant a re-evaluation of the immunoregulatory roles of IFN-I during immune memory differentiation, which may provide a framework for rational vaccine design.
GILT in thymic epithelial cells facilitates central CD4 T cell tolerance to a tissue-restricted, melanoma-associated self antigen
Authors: Hastings Karen , Rausch Matthew P. , Meador Lydia R. , Metzger Todd C. , Li Handong , Qiu Shenfeng , Anderson Mark S
Published: January 2023

Central tolerance prevents autoimmunity, but also limits T cell responses to potentially immunodominant tumor epitopes with limited expression in healthy tissues. In peripheral antigen presenting cells (APCs), gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of disulfide bond-containing proteins, including the self and melanoma antigen tyrosinase-related protein 1 (TRP1). The role of GILT in thymic antigen processing and generation of central tolerance has not been investigated. We found that GILT enhanced the negative selection of TRP1-specific thymocytes. GILT expression was enriched in thymic APCs capable of mediating deletion, namely medullary thymic epithelial cells (mTECs) and dendritic cells, while TRP1 expression was restricted solely to mTECs. GILT facilitated MHC class II-restricted presentation of endogenous TRP1 by pooled thymic APCs. Using bone marrow chimeras, GILT expression in thymic epithelial cells (TECs), but not hematopoietic cells, was sufficient for complete deletion of TRP1-specific thymocytes. An increased frequency of TRP1-specific regulatory T (Treg) cells was present in chimeras with increased deletion of TRP1-specific thymocytes. Only chimeras that lacked GILT in both TECs and hematopoietic cells had a high conventional T:Treg cell ratio and were protected from melanoma challenge. Thus, GILT expression in thymic APCs, and mTECs in particular, preferentially facilitates MHC class II-restricted presentation, negative selection and increased Treg cells, resulting in a diminished anti-tumor response to a tissue-restricted, melanoma-associated self antigen.
TLR9 dependent regulation of AXL expression impacts migration of autoreactive B cells
Authors: Nundel Kerstin (Kiefer) , Moses Stephanie , Richmond Jillian M , Rothlin Carla V. , Marshak-Rothstein Ann
Published: January 2023

Migration of autoreactive B cells to target tissues and the formation of tertiary lymphoid structures (TLS) is an important step in end-organ damage in autoimmune disease. TLSs are thought to be the main site of autoantibody production by autoreactive B cells, leading to immune complex formation and deposition in the affected organ. The formation of TLS was reported to be dependent on B cells as well as chemokines like CXCL13. Hence it is of importance to understand the migratory behavior of autoreactive B cells to these sites and how it is regulated. The receptor tyrosine kinase AXL was shown to play a role in the migration of cancer cells and expression of AXL in cancer cells is usually associated with worse prognoses. We found that AXL expression on B cells is highly induced by crosslinking of the B cell receptor (BCR) and not by TLR ligation as in dendritic cells and macrophages. Intriguingly, co-engagement of TLR9 together with the BCR inhibits AXL upregulation. This is unique to TLR9 and does not occur following co-engagement with other TLRs. Furthermore, we have found that AXL deficient B cells have a diminished potential to migrate in response to the B cell chemokine CXCL13. Also, in the pristane induced model of SLE, mice with AXL deficient B cells have lower numbers of TLS in the peritoneum compared to mice with AXL sufficient B cells, further supporting a role of AXL in the migration of B cells. Our data suggests that TLR9 plays an important role in suppressing AXL expression and the concurrent inhibition of B cell migration to TLS. Our studies have the potential to identify a novel and pharmacologically tractable mechanism for restricting the migration of autoreactive B cells and ensuing tissue damage.