bluuebunny/crossref_metadata_2025 huggingface dataset:
Beta-glucan-induced trained innate immunity mediates antitumor efficacy in the mouse lung
Authors: Alexander Matthew P , Fiering Steven N , Ostroff Gary R , Cramer Robert A , Mullins David W
Published: January 2023

The innate arm of the vertebrate immune system has classically been regarded as nonspecific and lacking the capacity for adaptive responses, in contrast to the adaptive arm defined by its robust memory responses. Recent high impact work has demonstrated enhanced non-specific responses of innate immune cells to pathogens and malignancy after priming with the fungal cell wall component beta-glucan, a phenomenon termed trained innate immunity (TII). In a pathogen model, the training effect was mediated by epigenetic modifications underlying a metabolic shift to mTOR- and HIF1α-mediated aerobic glycolysis. To investigate whether these same pathways play a role in non-specific enhancement of antitumor responses, we assessed beta-glucan-mediated anti-tumor efficacy in a mouse model of metastatic melanoma. We observed that systemic pretreatment with a particulate beta-glucan significantly diminished the growth of metastatic-like B16 melanoma in the lungs, but not initial tumor cell engraftment. Further, lungs in beta-glucan treated animals had a robust myeloid immune infiltrate, particularly neutrophils and monocytes. Interestingly, initial studies demonstrate that tumor suppressed HIF1α expression, and beta-glucan pretreatment prevented this suppression. These results are consistent with beta-glucan induction of TII, leading to suppression of cancer growth. By extension, TII may modulate the immunogenic nature of the tumor microenvironment, thereby acting as an adjuvant to enhance the efficacy of existing therapies.
Development of a Curative Immunotherapeutic Strategy for the Treatment of Big Established CT26 Tumors
Authors: Nie Yingjie , Oppenheim Joost J , Yang De , Trivett Anna , Xin Haiyun
Published: January 2023

Immunotherapy has demonstrated the ability to cause some complete clinical remissions even in patients with late stage cancers. However, currently used approaches fail in many cases. We hypothesize that a combination of promoting the generation of antitumor immune response and reverting the immunosuppressive environment of cancers would be more effective. In this study, we investigated the therapeutic effect of a combination of cytoxan and HMGN1 on established CT26 tumors. HMGN1 is a Th1-polarizing alarmin capable of promoting antitumor immune protection, whereas cytoxan (CY) reduces regulatory T cells. Treatment with one dose of CY and four doses of HMGN1 completely inhibited tumor growth in Balb/c mice bearing small CT26 tumors, but only partially in mice bearing big (≈1.0 cm diameter) tumors. To improve the efficiency, R848 was added to the treatment combination. The triple combination (‘TheraVac’) eradicated big CT26 tumors. Importantly, the resulting tumor-free mice were resistant to rechallenge with CT26, but not resistant to unrelated 4T1 tumors, indicating the development of protective anti-CT26 immunity. Profiling of tumor infiltrating leukocytes revealed that there was a reduction of dendritic cells and a concomitant increase in T cells in the tumor after TheraVac treatment. Thus, we have developed a curative immunotherapeutic strategy for big established CT26 tumors.
HPV-E1 protein immunization promotes anti-tumoral CTL immune response in C57BL-6 mice.
Authors: Amador Alfredo , Trejo-Moreno Cesar , Sada-Ovalle Isabel , Romero Damaris , Lizano Marcela
Published: January 2023

Cellular immune response is the main immune response to eliminate human papillomavirus (HPV) infected cells. HPV vaccines are effective in preventing HPV infections through humoral response, but fail to protect against many HPV types and established infections. Approximately 15% of women infected with high risk HPV do not have an effective immune response.

In the natural history of HPV infection, the most conserved protein among human papillomavirus, helicase E1, is the first immunological target of HPV infected cells. Therefore, we characterized the cellular immune response against E1 protein in a mouse model.

Immunizations with E1 protein + αGalacsosyl Ceramide (αGalCer) promoted an E1-antigen specific CTL response in C57BL-6 mice. We measured a CD107a/b+Granzime-A kinetic to identify antigen specific populations through flow cytometry.

The anti-tumoral response is only present in those mice immunized along with the αGalCer adjuvant. Within the set of six mice, three of them did not have any tumors and the other three developed small tumors in comparison with the mice in the control group, which were not immunized. In conclusion, the E1-antigen specific CTL plays an important role against tumor growth expressing E1-Carboxyl terminal domain.
Adoptive cell transfer enhances antitumor response generated by Cytomegalovirus-based vaccine
Authors: Grenier Jeremy M , Qiu Zhijuan , Khanna Kamal Mohan
Published: January 2023

Methods for generating long-lived T cell responses to tumor antigen have been highly sought for potential therapy against many cancers. Our group has recently developed a novel vaccine against melanoma using recombinant Mouse Cytomegalovirus (MCMV) expressing a modified tumor antigen which generated life-long CD8+ T cell responses to the melanoma antigen, gp100. While this vaccine delays tumor growth in a mouse model of B16 metastatic melanoma, mice eventually succumb to the disease. Adoptive cell therapy has shown clinical efficacy in some patients with melanoma; however, it requires in vitro activation of large numbers of T cells to be effective. We hypothesized that combining these therapies would result in more effective tumor rejection by 1) increasing precursor frequency of tumor specific T cells, thus inducing a more robust T cell response following vaccination and 2) promoting life-long persistence of transferred cells following transfer. To test this, C57BL/6 mice received 105 CD8+ T cells expressing a transgenic T cell receptor recognizing gp100 (PMEL) followed by vaccination. Five days after transfer, PMEL cells constituted up to 20% of all CD8+ T cells in peripheral blood following MCMV vaccination in contrast to 7.5% following vaccination with another viral vaccine, Vesicular Stomatitis Virus. Our results also show that adoptive transfer and MCMV vaccination delays B16 tumor growth and improves survival in a solid tumor model. In summary, this study tests a novel method for stimulating adoptively transferred antitumor T cells, while avoiding the need for long-term in vitroculture of cells. Future work will also examine if immune checkpoint inhibitors or costimulatory agonists further improve this therapy.
IL-21 dampens NFkB activation to augment efficacy of CD8+ T cell mediated adoptive cell therapy (ACT)
Authors: Shrikant Protul A , Myles Arpita
Published: January 2023

Exploiting mechanisms integrating antigen and cytokine signals to generate tumor-antigen specific CD8+ T cells that are durable and produce low proinflammatory profile can boost efficacy of adoptive cell therapy (ACT) of cancer. Herein, we show that IL-21 via STAT3 induces the atypical IKK family member, Bcl3, to dampen antigen driven NF-kB (p65/p50) activation. The IL-21 induced Bcl3 decreases antigen driven CD8+ proliferation as well as apoptosis to increase clonal expansion. Strikingly, Bcl3 skews expression of master transcriptional factors to facilitate transition of polyfunctional CD8+ effector cells to memory precursor cells (MPCs) with cytotoxicity but reduced IFNg production. The IL-21 conditioned CD8+ T cells are persistent and produce greater tumor efficacy upon adoptive transfer. These studies have revealed a role for NF-kB to serve as a node for integrating antigen and cytokine signals that determine fate of antigen stimulated CD8+ T cells and encourage development of new approaches to target NFkB for T cell mediated immunity.
Targeting T cell senescence in the tumor microenvironment for tumor immunotherapy
Authors: Peng Guangyong , Mo Wei , Li Lingyun , Liu Xia , Zhong Xiaoli , Ye Jian , Hsueh Eddy C.
Published: January 2023

Understanding the molecular mechanisms involved in creating and sustaining the tumor suppressive microenvironment is critical for the development of novel anti-tumor therapeutic strategies. Regulatory T cells (Treg) are a key player for the tumor immunosuppressive microenvironment. However, the molecular processes and functions of suppressed responder T cells mediated by Treg cells remain largely unknown. We discovered a novel suppressive mechanism whereby human Treg cells induce senescence in naïve and effector T cells that then exhibit potent suppressive activity and amplify immune suppression. Treg-induced senescent T cells were distinctly different from the exhausted or anergic T cells based on the transcriptome analyses and phenotypic profiles. We further identified that the nuclear kinase ataxia-telangiectasia mutated protein (ATM)-induced DNA damage was critical and the main cause for the induction of responder T cell senescence and dysfunction mediated by human Treg cells. In addition, MAPK ERK1/2 and p38 were involved in the regulation of senescence process in responder T cells. Importantly, we further revealed that human Treg-induced senescence and suppressor function could be blocked by specific ATM signaling and/or ERK1/2 and p38 signaling inhibition in vitro and in vivo in animal models. Our studies identify a novel molecular mechanism responsible for human Treg cell suppression, and provide new insights relevant for the development of strategies capable of preventing and/or reversing Treg-induced immune suppression for tumor immunotherapy.

Funding Support: Supported by the grants from the Melanoma Research Alliance, American Cancer Society and the National Institutes of Health.
Mice with highly metastatic breast cancer transfer express exosomes in serum which increase metastatic capacity of a poorly metastatic tumor
Authors: Gorczynski R. M , Erin Nuray , Zhu Fang
Published: January 2023

Altered interaction between CD200 and CD200R, represents an example of “checkpoint blockade” disrupting an effective, tumor-directed, host response in murine breast cancer cells. In CD200R1KO mice, long-term cure of EMT6 breast cancer, including metastatic spread to lung and liver, was achieved in BALB/c mice. The reverse was observed with 4THM tumors, an aggressive, inflammatory breast cancer, with increased tumor metastasis in CD200R1KO.

To explore possible explanations for this difference we measured the frequency of circulating tumor cells (CTCs) in peripheral blood of tumor bearers, as well as in lung/liver and draining lymph nodes, and in addition infused exosomes from non-tumor controls, or tumor bearers, with/without additional infusions of anti-cytokine antibodies into tumor bearing mice.

The measured frequency of circulating tumor cells (CTCs) in peripheral blood was equivalent in the two models in both WT and CD200R1KO mice. Increased metastasis in EMT6 tumor bearers was seen following adoptive transfer of serum, or serum-derived exosomes, from 4THM tumor bearers, an effect which was attenuated by anti-IL-6, and anti-IL-17, but not anti-TNFa, antibody. Anti-IL-6 also attenuated enhanced migration of EMT6 cells in vitro induced by 4THM serum or exosomes, or recombinant IL-6. Exosome cytokine proteomic profiles responses in 4THM and EMT6 tumor bearer mice were regulated by CD200:CD200R interactions, with attenuation of both IL-6 and IL-17 in 4THM CD200tg mice, and enhanced levels in 4THM CD200R1KO mice.

We hypothesize that these cytokines act on the microenvironment at sites within the host, and/or directly on tumor cells themselves, to increase metastatic potential.
Mucosal vaccine adjuvant cyclic di-GMP induces STING-dependent pulmonary dendritic cell activation
Authors: Mansouri Samira , Blaauboer Steven , Jin Lei
Published: January 2023

The mucosal surface is the major entry site for many human pathogens, however there are only a dozen of mucosal vaccines approved for human use. The challenge for mucosal vaccines is to develop safe and effective mucosal adjuvants. 3′, 5′-Cyclic di-GMP (CDG) is an attractive mucosal vaccine adjuvant candidate that activates STING (stimulator of interferon genes). However, the in vivo mechanism of action of CDG is not completely understood. Our previous studies found that intranasal immunization with CDG directly activates pulmonary dendritic cells (DCs). We found that CDG adjuvant activity depends on TNFα and not type I interferon. Deletion of STING in pulmonary DCs resulted in the reduction of antigen-specific antibodies and a decrease in Th1/Th2/Th17 responses. Here, we investigated the role of different pulmonary DCs subsets in mediating the mucosal adjuvant activity of CDG. Our results demonstrate that CDG targets unique and specialized functions of pulmonary DC subsets. Using Batf3−/− mice to deplete CD103+ DCs, we found that CD103+ DCs do not mediate the CDG-induced antibody production, but are needed for Th responses as well as the recruitment of the novel CD103+CD11b+ pulmonary DC population. The population of pulmonary CD11b+ DCs was intact in the Batf3−/− mice. CDG-induced humoral immunity completely depends on pulmonary DCs. Our data, thus, suggested that CD11b+ DCs, which were the only pulmonary DCs not depleted in Batf3−/− mice, were sufficient for CDG-induced humoral immunity. How STING mediates CDG responses in these DC subsets will be discussed. Understanding the role of different DC subsets will reveal insight in the mode of action of CDG in vivo and advance the development of CDG as an efficacious mucosal vaccine adjuvant.
An oral prime/boost pediatric vaccine strategy for the prevention of mother-to-child transmission of HIV by breast-feeding
Authors: Curtis Alan Dale , Phillips Bonnie , Choudhary Neelima , Tuck Ryan , Lifson Jeff , Van Rompay Koen , Kozlowski Pamela , De Paris Kristina
Published: January 2023

In 2014, an estimated 220,000 children became newly HIV-infected, with 50% being transmitted via breast-feeding. The present study assessed whether inclusion of an oral route of immunization would increase pediatric vaccine efficacy in the rhesus macaque model of oral SIV infection.

Two groups of 6 neonatal macaques received 2 DNA-SIV immunizations at weeks 0 and 3, followed by 2 boosts with modified vaccinia Ankara virus (MVA)-SIVgag/pol/env at weeks 6 and 9. Group A was immunized by the intramuscular (IM) route. Infants in Group B received the DNA-prime both per-orally (PO) and IM and MVA-SIV was administered both by the sublingual (SL) and IM route. Mock control animals received saline immunizations PO+IM at weeks 0 and 3 and mock MVA vector SL+IM at weeks 6 and 9. At week 12, all infants were challenged once weekly using a low-dose (102 TCID50) oral SIVmac251 challenge regimen until systemic infection was confirmed by PCR. Statistical significance was determined using nonparametric Mann-Whitney or Kruskal-Wallis tests with p<0.05.

Infants vaccinated by the SL/IM route had a significantly lower per-exposure risk of oral SIV infection (p<0.0001; log-ranked Mantel-Cox test) compared to mock- and IM/IM-vaccinated animals and lower peak viremia (p=0.03) compared to mock controls. Control of viremia was associated with higher fecal IgG responses to SIV V1V2 (p=0.0087) and p55 gag (p=0.0087) at the time of oral challenge initiation. In contrast, plasma SIV-specific antibodies did not influence challenge outcome.

These data indicate that the inclusion of an oral route in a pediatric vaccine to prevent oral SIV infection is superior to an IM only regimen and emphasizes the importance of enhancing mucosal immune responses via local immunization.
Deciphering the Role of Suppressor of Cytokine Signaling (SOCS) 1 and 3 in the IL-10 Regulation of Inflammation Induced by Live Chlamydia trachomatis in mouse macrophages
Authors: Duncan Skyla A. , Dixit Saurabh , Sahu Rajnish , Singh Shree R. , Dennis Vida A
Published: January 2023

Chlamydia trachomatis (CT), a bacterial pathogen responsible for sexually transmitted infections worldwide, causes severe inflammation including but not limited to, pelvic inflammatory disease, ectopic pregnancy and infertility in women. Secretion of inflammatory mediators plays a pivotal role in its pathogenesis of disease. Our laboratory published that IL-10 can down-regulate the secretion of inflammatory cytokines triggered by CT in mouse macrophages. Our goal in this study is to decipher the mechanisms of IL-10 down-regulation of CT-induced cytokines by focusing on the intracellular negative regulatory mediators, suppressor of cytokine signaling (SOCS) 1 and 3. We show that live CT and its recombinant major outer membrane protein (rMOMP), in a dose-dependent fashion, markedly induced the transcriptional upregulation of SOCS 1 and 3 in macrophages. In the presence of exogenously added recombinant IL-10, SOCS1 expression was reduced while that of SOCS3 was enhanced, suggesting differential regulation of these regulators by IL-10 to mediate its inhibitory effect. Experiments using various concentrations of exogenously added IL-10 over several time-points further revealed the differential regulation of SOCS 1 and 3 by IL-10 in macrophages. Signal transduction pathway inhibition studies demonstrated that the MAPK pathways including p38, MEK and JNK, particularly the latter are possibly involved in inducing SOCS 1 and 3 by CT and added IL-10, correlating with down-regulation of the cytokines, IL-6 and IL-12p40. Overall, our data helps in understanding the mechanisms of IL-10 regulation of inflammation in CT-infected macrophages by shedding light on the roles of SOCS 1 and 3 in mediating this IL-10 anti-inflammatory effect.
Characterizing the germinal center response to electroporated DNA immunogens
Authors: Gary Ebony Nicole , Bernui Mariana , Kutzler Michele A.
Published: January 2023

Most licensed vaccines rely on the production of antibodies that mediate protection from disease. The germinal center (GC) is a dynamic cellular substructure found in lymphoid organs where follicular helper T cells (TFH) provide the necessary proliferation stimuli and cytokine signaling that promote antibody class-switching, somatic hypermutation, and affinity maturation of B cell receptors. Here, mature B cells differentiate into long-lived antibody-secreting cells and memory B cells, instituting the characteristic humoral memory response to vaccination. Although HIV-1 DNA vaccines have been shown to be highly immunogenic, leading to enhanced HIV-1 env epitope breadth and titers in pre-clinical and clinical studies, many questions remain regarding the quality of the humoral immune response and the ability of the DNA antigen/molecular adjuvant to induce long-lived anti-viral antibody that seeds mucosal secondary immune tissues. However, the magnitude and kinetics of the GC response to EP DNA has not been elucidated. Our results indicate that DNA antigens induce a GC response. Antigen-specific GC B cells (B220+CD138−IgM−IgD−PNA+CXCR5+gp120+) are successfully detected by flow cytometry using fluorophore-conjugated HIV-1 gp120 protein after two immunizations in the spleens and draining lymph nodes of vaccinated animals and by antigen-specific ELISpot assay. The presence of antigen-specific antibodies of multiple isotypes is successfully detected in the serum of vaccinated animals via ELISA. These results represent, to our knowledge, the first reports on the magnitude and kinetics of the germinal center response to EP DNA. This work is funded by a W.W. Smith Charitable Foundation grant to MK.
Mature B cell deficient-BAFFR−/− mice can be protected from a lethal WNV-Tx (WNV) infection by vaccination with an anti-CD180 (RP105) antibody - WNVEnv protein conjugate (αCD180-WNVE)
Authors: Giordano Daniela , Draves Kevin E , Bryan Marianne A , Dresch Christiane , Young Lucy B , Roe Kelsey , Richner Justin M , Diamond Michael S , Gale Michael J , Clark Edward A
Published: January 2023

BAFFR−/− mice are a model of humoral immunodeficiency since they lack mature B cells. We found that in contrast to WT mice BAFFR−/− mice were highly susceptible to WNV. BAFFR−/− mice infected with 100pfu WNV succumbed to infection between 8 to 12 days post-infection and higher virus titers were found in their brains compared to WT mice. Infected BAFFR−/− mice had substantially reduced WNVE-specific IgG responses and delayed but still detectable WNV neutralizing Ab compared to WT mice. Passive transfer of immune sera from previously infected WT mice rescued BAFFR−/− mice as well as μMT mice lacking all B cells, but in contrast to μMT mice that all died around 30 days post-infection, BAFFR−/− mice survived and were able to overcome a second WNV challenge. Furthermore, rescued BAFFR−/− mice receiving immune sera later produced their own WNVE-specific IgG, although at much lower levels than WT mice. These data suggested that protective immunity could be induced in these mature B cell-deficient mice. Indeed, one injection of αCD180-WNVE 30 days prior to WNV infection induced strong neutralizing Ab responses in BAFFR−/− mice and significant protective immunity against a normally lethal infection. In contrast, μMT mice could not be protected by αCD180-WNVE immunization confirming that the vaccination requires B cells to induce protective immunity to WNV infection. BAFFR−/− mice have a profound reduction in mature B cells but, unlike μMT mice, have normal numbers of T1 B cells, which our laboratory has shown previously can make IgG Abs. Interestingly, αCD180 immunization rapidly expands T1/T2 B cells. Thus a CD180-based vaccine may be useful to induce protective immunity in immune compromised individuals. (Supported by NIH grants AI83109 and AI52203).
CD4 T cell-mediated immunopathology of a Staphylococcus aureus whole cell vaccine during systemic infection
Authors: Karauzum Hatice , Haudenschild Christian , Mahmoudieh Mahta , Moore Ian N. , Datta Sandip K.
Published: January 2023

Staphylococcus aureus causes a wide array of diseases, ranging from superficial skin infections to life threatening diseases like sepsis. Treatment is difficult due to emerging multidrug-resistant strains and importantly, several approaches to develop an efficacious vaccine have either failed or augmented disease in clinical trials. In the present study, gamma-irradiated S. aureus USA300 (IRS) was used as a whole-cell vaccine to evaluate the immune response towards vaccination and subsequent infection. C57BL/6 mice were vaccinated thrice intramuscularly, adjuvanted with or without Al(OH)3, at two-week intervals and challenged with USA300 intravenously 2 weeks after the last vaccination. Vaccination enhanced mortality and failed to control bacterial growth in blood and organs when compared to PBS controls and was worse without Al(OH)3. USA300-specific antibodies were induced by vaccination but did not cause enhanced mortality when tested in serum transfer studies. Furthermore, B cell deficient and WT mice were similarly susceptible to infection after vaccination. Infection increased frequency of CD44hiTbet+ and CD44hiIFNg+ (Th1 effector) cells in spleens of both vaccinated groups but was higher in mice vaccinated without Al(OH)3. Frequency of CD44hiRORgt+ and CD44hiIL-17+ (Th17 effector) cells increased only in groups vaccinated with Al(OH)3. Depletion of CD4 T cells during infection reversed the lethal phenotype. Similarly, neutralization of IFNg but not IL-17 during infection improved survival and reduced organ pathology. Taken together, our findings show a detrimental role for IFNg+CD4 Teff cells generated by vaccination with IRS, and might shed some light on recent vaccine failures.
ICOS+CD38+ cTfh in peripheral blood and thoracic duct lymph.
Authors: Vella Laura A , Buggert Marcus , Herati Ramin S , Itkin Maxim , Dori Yoav , Betts Michael R , Wherry E. John
Published: January 2023

T follicular helper cells provide essential B cell help within the germinal center (GC), and these cells have been shown to populate the follicular mantel once the GC reaction is complete. The significance and ontology of circulating Tfh (cTfh) is less clear. Studies in humans have suggested subsets of cTfh that correlate with humoral responses in chronic infection (HIV, malaria) and in response to acute challenge (influenza vaccination). We have previously described a subset of CXCR5+PD1+ cTfh that expresses both ICOS and CD38. This population was significantly increased 7 days after influenza vaccination as well as in patients infected with HIV. Single cell transcriptional analysis of the ICOS+CD38+ cTfh after influenza vaccination demonstrated more frequent expression of the GC Tfh transcripts CMAF, BCL6, and CD200 than was found in the ICOS−CD38− cTfh, and bulk transcriptional analysis indicated higher expression of IL-21. These data suggested that ICOS and CD38 mark the rare population of cTfh that are more recent émigrés from the lymph nodes. To evaluate this possibility, we analyzed Tfh in an immune compartment that is more proximal to the lymph nodes than peripheral blood: thoracic duct lymph (TDL). As expected, TDL Tfh had a higher frequency of ICOS+CD38+ cells than cTfh in matched peripheral blood. In addition, TDL had a population of CXCR5+PD1bright Tfh, previously only identified in lymphoid tissues. CXCR5+PD1bright Tfh in the TDL had a significantly increased frequency of ICOS+CD38+ cells. These data provide a critical missing link in the understanding of cTfh and suggest that the ICOS+CD38+ cTfh subset warrants further exploration as peripheral surrogates of the active immune responses within secondary lymphoid tissues.
BscF from Bordetella pertussis provides advantageous adjuvant activity when paired with the acellular pertussis vaccine: enhanced Th1/Th17 pertussis-specific immune response
Authors: Alvine Travis D , Knopick Peter L , Kumar Meera Ajeet , Nilles Matthew L , Bradley David S
Published: January 2023

Incidence of pertussis, a severe respiratory disease caused by Bordetella pertussis, have been on the rise. This resurgence has been linked to antigenic divergence in circulating pertussis strains as well as waning or ineffective immunity induced by the current acellular pertussis (aP) vaccine. The current aP vaccine replaced a whole cell pertussis (wP) vaccine in the 1990s due to adverse events associated with the wP vaccine. Alum-absorbed aP vaccine has been shown to elicit a strong antibody response and considerable Th2 type CD4+ T cells. In contrast, wP vaccine promotes Th1/Th17 type cellular immunity and associated opsonizing antibodies presumably via its PAMPs. We have previously demonstrated that BscF, a B. pertussis needle protein, acts as a strong TLR agonist. Here we characterized the contribution of BscF to a laboratory prepared aP vaccine-induced response. aP + BscF resulted in statistically greater pertussis-specific antibody titers, compared to sham aP + PBS. BscF enhanced long-term aP-specific immunity, compared to either the alum or sham controls, as measured by increased numbers of central memory T cells. Moreover, the addition of BscF resulted in skewing the pertussis-specific immunity toward Th1 and Th17 responses, compared to the addition of alum or PBS which skewed pertussis-specific immunity away from Th1/Th17 responses. Mice immunized with aP + BscF demonstrated significantly reduced bacterial burden in their lungs 5 dpi. These findings suggest that BscF induced a strong Th1 and Th17 anti-pertussis response, potentially may provide protective BscF-specific immunity, and could be a novel additional component in the next generation aP vaccine.
Pathogenic chikungunya virus evades B cell responses to establish persistence
Authors: Morrison Thomas , Fox Julie M. , Ashbrook Alison W. , May Nick A. , Schroeder Kristin M. , Torres Raul M , Dermody Terence S. , Diamond Michael S , Hawman David W.
Published: January 2023

Chikungunya virus (CHIKV) causes large outbreaks of acute and chronic musculoskeletal disease. To investigate mechanisms of CHIKV clearance and persistence, we performed studies in mice with an attenuated CHIKV strain (181/25) and its pathogenic parent strain (AF15561). Whereas AF15561 infection of WT mice resulted in viral persistence in joint-associated tissue, 181/25 was cleared within 4 weeks. In contrast to WT mice, 181/25 infection of Rag1−/−mice resulted in viral persistence in joint tissue at levels similar to that detected in AF15561-infected Rag1−/− mice, suggesting an important role for adaptive immunity in the clearance of 181/25 infection. Indeed, 181/25 infection of B cell-deficient μMT mice or B cell receptor transgenic mice that cannot produce virus-specific antibody resulted in viral persistence that was equivalent to AF15561-infected WT and immunodeficient mice. In WT mice, 181/25 infection resulted in a higher quality neutralizing antibody response at early times post-infection. In addition, detailed mapping studies demonstrated that the amino acid at position 82 in the A domain of the E2 glycoprotein influenced clearance and serum neutralization of CHIKV. Neutralization assays revealed that inhibition of AF15561 by monoclonal antibodies specifically targeting E2 domain B was impaired relative to that observed with 181/25. Collectively, our data suggest that virus-specific antibody responses contribute to the clearance of 181/25 infection and that AF15561 evades this response by minimizing the impact of B domain neutralizing antibodies to establish persistent infection.
Regulatory CD4+ T cells differentially modulate HIV-1 infection of polarized M1 and M2 macrophages
Authors: Robinson Tanya O , Zhang Mingce , Cron Randy
Published: January 2023

Macrophages are HIV-1 targets but relatively resistant to HIV-1 cytotoxicity. Effector macrophages can be functionally polarized into M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes. M1 monocyte-derived macrophages (MDM) suppress CCR5 (R5) HIV-1 replication, while M2 MDM promote it. While CD4 regulatory T cells (Tregs) inhibit HIV-1 infection of primary CD4 T cells in co-culture, their effect on MDM infection is unknown. We generated M1 (GM-CSF+IFNγ+LPS) and M2 (M-CSF+IL-4) MDM with predicted phenotypes and infected them with R5 HIV-1BAL reporter viruses. M2 MDM had notably higher infection than M1 MDM. To explore acute stages of infection, we infected M1 and M2 MDM with 3 different R5 HIV-1 transmitted founder (T/F) viruses. Similarly, M2 MDM had higher levels of T/F HIV-1 infection than M1 MDM but lower compared to HIV-1BAL. To investigate the role of Tregs on HIV-1 infection of MDM, we co-cultured Tregs with HIV-1 infected MDM. Treg co-cultures yielded decreased HIV-1 infection of M1 but increased infection of M2 MDM. Surprisingly, Tregs mediate divergent effects on HIV-1 infection of M1 and M2 MDM. A transwell system was used to explore if Treg effects on HIV-1 infection of MDM were cell contact and/or cytokine dependent. Similar to co-culture, Tregs (compared to effector T cells) in the transwell system with HIV-1 infected polarized MDM led to decreased HIV-1 infection of M1 but increased infection of M2 MDM. However, the level of infection was significantly lower in the transwell system compared to co-culture, suggesting optimal Treg mediated effects require cell-contact. We are currently studying the roles of CTLA-4, IL-10, and TGFβ in Treg suppressive activity on HIV-1 infection of MDM to explore the mechanism.
BCG-vaccination in SIV-infected infant macaques is associated with enhanced B cell, myeloid dendritic cell and CD4+ T cell activation.
Authors: Wood Matthew P , Wood Lianna , Sodora Donald L.
Published: January 2023

Active tuberculosis infection is the leading cause of death for people living with HIV. HIV+ infants in resource poor settings are exposed to tuberculosis and the vaccine M. bovis BCG, which can become disseminated in immune-compromised hosts. How BCG impacts the HIV infected immune system is poorly understood. Here, we used the SIV-macaque model to evaluate BCG vaccination in infant Rhesus macaques infected with SIV (N=7) or uninfected controls (N=5). Blood was obtained from macaques (0–10 months of age) that were either SIV-infected or uninfected, and assessed at multiple timepoints following intradermal vaccination with BCG (Danish strain). The frequency of CCR5+ CD4+ T cells significantly increased following BCG, with a greater difference observed in SIV+ (33.9 fold change compared to pre-BCG vaccination) compared to uninfected (2.7 fold) infants. Markers of activation were assessed within the B cells (CD20), monocytes (CD14), mDCs (CD11c), and pDCs (CD123). We observed an increase in the costimulatory molecule CD86 in both SIV-infected and uninfected macaques following BCG vaccination, with higher overall levels observed SIV+ infants. These elevated levels of CD86+ cells were observed for both B cells (1.4% uninfected and 4% in SIV+) and mDCs (4.1% uninfected and 16.1% SIV+) after BCG vaccination, which is in part a reflection of higher baseline immune activation in the SIV+ infants. These studies identify changes associated with BCG vaccination in SIV+ infant macaques, which include an increase in potential SIV target cells (CCR5+/CD4+) and elevated activation of B cells and mDCs in the blood. Studies are now underway to evaluate these findings in tissues from these infants.
Antisense transcript derived HIV-1 cryptic epitopes are recognized by T cells during chronic infection.
Authors: Peng Binghao J , Carlson Johnathan M , Hunter Eric , Bansal Anju , Goepfert Paul A
Published: January 2023

Alternative reading frames (ARF) of HIV, both sense and antisense directions, have shown to produce polypeptides of unknown functions. Although the roles of these polypeptides are unclear, they can still potentially act as HIV derived antigens, eliciting host T cell responses. These ARF derived epitopes, known as cryptic epitopes (CE), occur commonly in HIV, SIV, and other retroviruses through ribosomal frame-shifting, internal ribosomal entry sites, and alternative start codon initiation.

We hypothesize that T cell responses to CE are capable of driving viral escape mutations at a population level. In order to identify CE of HIV capable of driving viral escape, sequence changes in gag, pol, and nef of HIV were identified during acute infection in a cohort of Zambian serodiscordant couples. HLA-I associated polymorphisms in ARF can thus be identified, predicting potentially immunogenic CE. Several CE were identified as a result (N=66), in which the majority appears to have been derived from antisense ARF. These CE were used to stimulate cryopreserved PBMC samples from HIV-1 patients in the chronic stage of infection.

We found 8/32 patients responded to at least one predicted CE as shown by IFNγ ELISpot. Interestingly, all 10 immunogenic CEs found in this study were derived from a single antisense reading frame in the pol coding region. Furthermore, these CE were not only capable of eliciting CD8, but also CD4 responses. When examining alternative start codons in each reading frame, we found an enrichment of CUG in the antisense frame containing the immunogenic CE. Together, our results show HIV antisense CE can be targeted by T cells and may therefore have implications for T-cell vaccines.
Omega-3 rich diet alters T cell affinity and decreases anti-viral immunity
Authors: Kolawole Elizabeth Motunrayo , Evavold Brian D
Published: January 2023

There has been much investigation into the influence of dietary fatty acids with regard to modulation of immune function. Omega-3 polyunsaturated fatty acids have been shown to exert beneficial anti-inflammatory effects for both chronic and acute inflammatory disease. Acute viral infection can be controlled and eliminated with an effective CD8 response. Despite studies suggesting the incorporation of omega-3 fatty acids into lipid membranes leading to the production of less inflammatory metabolites, little is known about T cell engagement with peptide-MHC (pMHC) antigen and whether omega-3 fatty acids modulate T cell responses. Here using the novel two dimensional (2D) micropipette adhesion frequency assay we determine T cell frequency and affinity for antigen on a single cell basis. Our data indicates that dietary fish oil reduces antigen affinity in T cell receptor transgenic cells. Moreover, we demonstrate decreased polyclonal numbers of antigen specific (gp-33) T cells at peak anti viral immunity to lymphocytic choriomeningitis (LCMV) infection. These data indicate that omega-3 fatty acids decrease antigen recognition to viral antigens.
Increasing strength of antigenic stimulation negatively impacts memory CD8 T cell formation and function during persistent viral infection
Authors: Maru Saumya , Haley Jeremy S , Schell Todd D , Lukacher Aron
Published: January 2023

Memory CD8 T cells are critical for keeping persistent viral infections in check. Here, we used a novel approach to directly study the impact of T cell stimulation strength on memory T cell differentiation in the setting of a persistent viral infection. Using site-directed mutagenesis, we generated a series of MPyV variants with substitutions in a subdominant Db-restricted epitope that modulated level of activation of TCR transgenic CD8 T cells specific for a corresponding epitope in SV40 Large T Antigen. By altering a subdominant epitope constituting approximately 1% of the total anti-MPyV CD8 T cell response, we avoided perturbing MPyV-associated inflammation, virus infection levels, antiviral CD4 T cell responses, and the dominant anti-MPyV CD8 T cell response. Moreover, by using physiologic numbers of naïve donor monoclonal CD8 T cells for adoptive transfer into immunocompetent C57BL/6 recipients, we also controlled for variability in naïve T cell precursor frequency and TCR affinity inherent in T cell recruitment to infection. Our data demonstrate an inverse relationship between antigenic stimulation strength and expression of memory precursor effector phenotype (CD127highKLRG1low) by virus-specific CD8 T cells during acute infection. Although stronger antigenic stimulation augmented expansion of functional CD8 T cells in acute infection, lower stimulation favored functional competence of memory antiviral CD8 T cells during persistent infection. Together, these data provide clear in vivo evidence that the strength of antigenic stimulation during recruitment of MPyV-specific CD8 T cells dictates the functional integrity of memory T cells during persistent infection.
Acute virus-specific responses by NK cells shape CD8+ memory T cells and protective immunity
Authors: Teoh Jeffrey , Gamache Awndre , Bullock Timothy N , Brown Michael G
Published: January 2023

NK cells are cytotoxic to CD8+ T cell effectors following certain viral infections, such that transient NK depletion can increase T cell effector activities. Previous studies have shown that licensed Ly49G2+ (G2) NK cells confer MCMV resistance in mice expressing MHC I Dk, a specific ligand of the inhibitory G2 receptor, which resulted in enhanced adaptive immunity due to accelerated accumulation of virus-specific CD8+ T cells. However, the longer-term impact of licensed NK-mediated virus resistance on adaptive T cell immunity was not previously investigated. Hence, we examined CD8+ T cells for KLRG1, CD127, CD62L and cytokine production at discrete times post-infection. Early on (6-10d), licensed G2+ NK-mediated resistance in Dk mice led to a profound skewing of CD8+ T cells from short-lived effectors (SLEC) into memory precursor effector cells (MPEC). By comparison, mice without Dk-licensed G2+ NK cells were biased towards SLECs throughout 10d, and upon contraction. This T cell differentiation effect endured through 21-56d postinfection such that the frequencies of memory and effector cells reflected differences observed for the strains at d10. We then transferred CD8+ T cells from latently MCMV-infected mice into naïve, G2+ NK cell-depleted hosts and challenged them with high dose MCMV. Remarkably, recipients of CD8+ T cells primed in the presence of licensed G2+ NK cells exhibited significantly lower viral titers than those that received CD8+ T cells primed in non-Dk mice. We infer that licensed NK-mediated viral resistance significantly boosted the formation of memory CD8+ T cells, and that the means of early NK cell activation and virus resistance shapes adaptive immunity.
Overcoming CD4 T helper cell fate restrictions to enhance CD8 T cell and B cell immunity to control persistent LCMV infection
Authors: Snell Laura M , Osokine Ivan , Yamada Douglas H , Elsaesser Heidi , Brooks David G
Published: January 2023

Persistent viral infection inhibits CD4 Th1 responses, resulting in loss of the CD8 T cell helping Th1 subset and accumulation of B cell helping Tfh cells. Yet, the mechanisms that inhibit CD4 Th1 differentiation and the repercussions of loss of Th1 cells on viral control are unclear. Herein, we demonstrate that the dual upregulation of PD-L1 and IL-10 by chronic type I interferon (IFN-I) signaling during persistent infection suppresses CD4 Th1 priming. Therapeutic blockade of IL-10R and PD-L1 could restore the differentiation of new CD4 Th1 cells, highlighting a new function for PD-L1 to co-inhibit CD4 Th1 priming and identifying a new combinatorial suppressive mechanism during persistent infection. However, CD4 Th1 cells generated by blocking IFN-I signaling in vivo could not be sustained, giving way to a Tfh based response and predominant help to B cells. In vitro polarized virus-specific CD4 Th1 cells also converted towards Tfh cells, however maintained a population of Th1 cells that could enhance exhausted CD8 T cell responses and promote augmented viral clearance. Interestingly, virus-specific CD4 Th17 and Treg cells were also redirected towards Tfh as persistent infection progressed. Thus, our studies have important implications for stability of de novo activated and immunotherapeutic T cells and demonstrate that the redirection away from Th1 responses is a mechanism of immunosuppression to limit the control of persistent viral infection.
IL-1-induced Bhlhe40 identifies pathogenic TH cells in a model of autoimmune neuroinflammation
Authors: Lin Chih-Chung , Bradstreet Tara R , Schwarzkopf Elizabeth A , Jarjour Nicholas N , Chou Chun Jim , Archambault Angela S , Sim Julia , Zinselmeyer Bernd H , Carrero Javier , Wu Gregory F , Taneja Reshma , Artyomov Maxim , Russell John H , Edelson Brian T
Published: January 2023

The features that define autoreactive TH cell pathogenicity remain obscure. We have previously shown that TH cells require the transcription factor Bhlhe40 to mediate experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Here, using Bhlhe40-EGFP reporter mice and analyzing both polyclonal and TCR transgenic CD4+ T cells, we found that Bhlhe40 expression was heterogeneous after EAE induction. Bhlhe40-expressing CD4+ T cells displayed marked production of IFN-γ, IL-17A, and GM-CSF, while exhibiting reduced expression of the anti-inflammatory cytokine IL-10 and the regulatory T cell transcription factor Foxp3. In adoptive transfer EAE models Bhlhe40-deficient TH1 and TH17 cells were both nonencephalitogenic. Pertussis toxin (PTX), a classical coadjuvant for actively induced EAE, promoted IL-1β production by myeloid cells in the draining lymph node and served as a strong stimulus for Bhlhe40 expression in TH cells. Furthermore, PTX coadjuvanticity was Bhlhe40 dependent. IL-1β induced Bhlhe40 expression in polarized TH17 cells, and Bhlhe40-expressing cells exhibited an encephalitogenic transcriptional signature. In vivo, IL-1R signaling was required for full Bhlhe40 expression by TH cells after immunization. Overall, we demonstrate that Bhlhe40 expression identifies encephalitogenic TH cells and define a PTX-IL-1-Bhlhe40 pathway active in EAE.
Absence of dual TCR T cells protects NOD mice from diabetes due to an increased insulin-specific Treg:Teff ratio
Authors: Schuldt Nathan J , Spanier Justin A , Auger Jennifer L , Hogquist Kristin A , Fife Brian T , Binstadt Bryce Anthony
Published: January 2023

Due to allelic exclusion, most αβ T cells express only a single αβ TCR specificity. However, around 10% of human and mouse T cells express two different α chains with the potential for dual specificities. Dual β-expressing T cells also exist and account for ~1% of all T cells. Expression of two different TCR specificities by a single T cell is one hypothesized mechanism by which self-reactive TCRs escape central tolerance to initiate autoimmunity. While peripheral tolerance mechanisms typically control any self-reactive T cells in healthy animals, these mechanisms are hypo-functional in NOD mice. We hypothesized that eliminating dual TCR T cells would protect NOD mice from diabetes. To test this hypothesis we generated NOD mice hemizygous at both the TCRα and β loci (TCRα+/−β+/−). Indeed, we found that NOD mice incapable of generating dual TCR T cells were protected from diabetes with both lower diabetes incidence than wildtype (WT) NOD mice (0% versus 70% at 30 weeks of age) and also lower insulitis scores at 10 weeks of age. Examination of the insulin-specific T cell population in the pancreas-draining lymph node revealed a lower Treg:Teff ratio in WT NOD mice compared to single TCR T cell NOD mice. Anti-PD-1 treatment resulted in the rapid development of diabetes in 100% of WT NOD mice but in only 30% of single TCR T cell mice, suggesting that the diabetes resistance phenotype of single TCR T cell mice is not primarily due to increased anergy. These data support the hypothesis that the presence of dual TCR T cells increases the risk of diabetes in NOD mice by altering the insulin-specific Treg:Teff ratio.
Dependence of spontaneous autoimmunity to neuroretina on Th1 and Th17 effector cytokines
Authors: Horai Reiko , Bing So Jin , Silver Phyllis B , Jittayasothorn Yingyos , Caspi Rachel R
Published: January 2023

Autoimmune uveitis is a group of sight-threatening diseases caused by retina-specific autoreactive T cells. Upon activation, these retina-specific T cells differentiate into effector cells that can cross the blood-retinal barrier to cause uveitis. IFN-γ-secreting Th1 cells and IL-17A-secreting Th17 cells are thought to be pathogenic populations in tissue-specific autoimmune diseases, including uveitis. However, the contribution of either subset to disease pathogenesis is still controversial. In this study, we analyzed the role of Th1 and Th17 subsets in the R161H mice, which express a transgenic T cell receptor specific for the retinal protein IRBP and develop spontaneous uveitis with high penetrance. To understand how these effectors and their respective signature cytokines contribute to pathogenesis, we crossed R161H mice with IL-17A−/− and/or IFN-γ−/− (GKO) mice. R161H-GKO mice developed significantly attenuated disease with delayed onset, whereas disease in R161H-IL-17A−/− mice was essentially undiminished, suggesting IFN-γ, but not IL-17A, is critical for pathogenesis. Although the number of ocular infiltrating cells in R161H-GKO mice was decreased, the proportions of Th17 cells and granulocytes were increased, in line with an enhanced Th17 response in the absence of IFN-γ. Surprisingly, R161H mice made deficient in both IL-17A and IFN-γ developed more severe disease than R161H-GKO mice, although its onset was also delayed. These results suggest that ‘classic’ Th1 and Th17 cells are not the only pathogenic effectors in uveitis, and that inflammatory cytokine(s) other than IFN-γor IL-17A can also drive disease. Further mechanistic studies may uncover novel effector pathways in pathogenesis of ocular autoimmunity.