Memory Recall Responses Research Articles

OCA-B/Pou2af1 is sufficient to promote CD4+ T cell memory and prospectively identifies memory precursors

The molecular mechanisms leading to the establishment of immunological memory are inadequately understood, limiting the development of effective vaccines and durable antitumor immune therapies. Here, we show that ectopic OCA-B expression is sufficient to improve antiviral memory recall responses, while having minimal effects on primary effector responses. At peak viral response, short-lived effector T cell populations are expanded but show increased Gadd45b and Socs2 expression, while memory precursor effector cells show increased expression of Bcl2, Il7r, and Tcf7 on a per-cell basis. Using an OCA-B mCherry reporter mouse line, we observe high OCA-B expression in CD4+ central memory T cells. We show that early in viral infection, endogenously elevated OCA-B expression prospectively identifies memory precursor cells with increased survival capability and memory recall potential. Cumulatively, the results demonstrate that OCA-B is both necessary and sufficient to promote CD4 T cell memory in vivo and can be used to prospectively identify memory precursor cells.

A bispecific antibody AP203 targeting PD-L1 and CD137 exerts potent antitumor activity without toxicity

BackgroundBispecific antibody has garnered considerable attention in the recent years due to its impressive preliminary efficacy in hematological malignancies. For solid tumors, however, the main hindrance is the suppressive tumor microenvironment, which effectively impedes the activation of infiltrating T cells. Herein, we designed a bispecific antibody AP203 with high binding affinity to PD-L1 and CD137 and assessed its safety and anti-tumor efficacy, as well as explored the mechanism of action.MethodsThe optimal antibody binders against PD-L1 and CD137 were screened from the OmniMab phagemid library. The binding affinity of the constructed AP203 were evaluated using enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). T-cell stimulatory capacity was assessed using the allogeneic mixed lymphocyte reaction (MLR), antigen-specific recall response, and coculture with PD-L1-expressing cells. In vivo antitumor efficacy was evaluated using two models of tumor-xenografted humanized mice with profiling of tumor infiltrating lymphocytes (TILs). The possible toxicity of AP203 was examined using in vitro cytokine release assay by human PBMCs.ResultsAP203, which simultaneously targeted PD-L1 and costimulatory CD137, elicit superior agonistic effects over parental antibodies alone or in combination in terms of T cell activation, enhanced memory recall responses, and overcoming Treg-mediated immunosuppression (P < 0.05). The agonistic activity of AP203 was further demonstrated PD-L1-dependent by coculturing T cells with PD-L1-expressing cells. In vivo animal studies using immunodeficient or immunocompetent mice both showed a dose-related antitumor efficacy superior to parental antibodies in combination (P < 0.05). Correspondingly, AP203 significantly increased tumor infiltrating CD8 + T cells, while decreased CD4 + T cells, as well as Treg cells (P < 0.05), resulting in a dose-dependent increase in the CD8 + /CD4 + ratio. Moreover, either soluble or immobilized AP203 did not induce the production of inflammatory cytokines by human PBMCs.ConclusionsAP203 exerts potent antitumor activity not only by blocking PD-1/PD-L1 inhibitory signaling, but also by activating CD137 costimulatory signaling in effector T cells that consequently counteracts Treg-mediated immunosuppression. Based on promising preclinical results, AP203 should be a suitable candidate for clinical treatment of solid tumors.

Boosting location of COVID-19 mRNA vaccine regulates the germinal center reaction

Abstract Germinal centers (GCs) are the specialized sites for B cells to differentiate into long-lived plasma cells or memory B cells during vaccinations. COVID-19 mRNA vaccines require boosting to mount high levels of neutralizing antibody responses. mRNA vaccines induce persistent GCs in the draining lymph nodes (dLNs), which might change the microenvironment for subsequent immunizations. It is unclear whether the site of booster immunization (ipsilateral versus contralateral) will affect the GC responses in the dLNs. To test this, we compared GC responses induced by ipsilateral and contralateral booster vaccination three weeks post the primary immunization in mice. Although these two boosting strategies induce similar numbers of GC B cells in the dLNs, we discovered that ipsilateral boosting elicits more SARS-CoV-2 RBD-specific GC B cells than contralateral boosting. However, very few RBD-specific GC B cells were differentiating into plasma cells as probed by BCL6 lowIRF4 highPC precursors within the GC B cell population. Serum antibody levels were comparable between these boosting strategies, suggesting the memory recall response was not different. These studies suggest boosting location of COVID19 mRNA vaccines might affect RBD-specific GC B cell responses. Future studies are needed to investigate whether boosting location may impact the persistence of neutralizing antibody response induced by COVID19 mRNA vaccines.

Investigating the Distinct Tissue Repair and Immunosuppressive Roles of Regulatory T cells in HSV-2 Infection

Abstract Herpes Simplex Virus 2 (HSV-2) is the primary cause of genital herpes, affecting 500 million people worldwide of which approximately 64% are women. Despite this disproportionately high burden, the female reproductive tract and its unique mucosal immune environment is surprisingly understudied. Tregs are necessary for CD4 +T cell priming in the draining lymph node (dLN), though their role in shaping tissue-resident memory T cell (T rm) responses during HSV-2 reactivation is unclear. Our preliminary data indicate that activated Tregs accumulate and persist in mouse vaginal tissue (VT) after above baseline out to at least 90 days post-infection (p.i.), thus implicating Tregs in memory recall responses to reinfection. We hypothesize that Tregs restrain robust T rmrecall responses to HSV-2 challenge. Additionally, we hypothesize that vaginal Tregs promote tissue healing in the VT in response to inflammatory alarmins through the expression of the epidermal growth factor ligand, amphiregulin (Areg). Female FoxP3 DTRmice were infected intravaginally with attenuated HSV-2, then systemically depleted of Tregs via intraperitoneal injection of diphtheria toxin and challenged with WT HSV-2 on day 30 p.i. Systemically Treg-depleted mice show increased activation and proliferation of T cells, and increased frequency of cytotoxic HSV-2-specific CD8 +T cells in the VT by day 3 post-challenge. Treg-depleted mice also have significantly higher pathology scores in H&amp;E-stained VT. We also report that VT Tregs from infected mice express more Areg than dLN Tregs via flow cytometry and single cell RNAseq. Ongoing work includes characterization of Areg expressing VT Tregs and further dissection of the T rmrecall responses in Treg-depleted mice. NIH (T32 AI07140, R01 AI141435)

The impact of post-encoding alcohol consumption on episodic memory recall and remember-know responses in heavy drinkers.

People often consume alcohol following trauma, particularly in response to distressing memories. To date, little is known about how post-encoding alcohol consumption influences episodic memory recall for negative events. Understanding these effects may help to improve support for trauma victims - for example, witnesses and victims of crimes. We tested 60 participants who self-described as heavy drinkers. After watching an analog trauma film, half were allocated to consuming a moderate dose of alcohol (Alcohol-Exposed group), while half received a placebo drink (Placebo-Control group). Immediately and after a one-week delay, participants recalled the event via free and cued recall tasks. Participants also gave remember-know responses and confidence ratings, elucidating alcohol's effect on experiential memory. Free recall performance was similar for the Alcohol-Exposed group and the Placebo-Control group during Sessions 1 and 2. The Alcohol-Exposed group benefitted more from the delayed repeated retrieval attempt. For the cued recall task, the Alcohol-Exposed group provided more "Do not Know" responses compared to the Placebo-Control group in both sessions. For the Alcohol-Exposed group only "Correct Know" responses increased from Session 1 to 2. Although memory performance improved across sessions, confidence levels decreased from Session 1 to 2 in the Alcohol-Exposed group. Post-encoding alcohol consumption appears to impact immediate episodic memory retrieval; however, this effect is only temporary in nature. No evidence was found that alcohol primarily reduces remembering responses. Much like previous findings focusing on pre-encoding alcohol consumption (Hagsand et al., 2017), current findings suggest that providing individuals who drank alcohol after witnessing an incident with a delayed repeated retrieval attempt can lead to more complete and accurate testimonies.

Cytomegalovirus Seropositivity in Older Adults Changes the T Cell Repertoire but Does Not Prevent Antibody or Cellular Responses to SARS-CoV-2 Vaccination.

Chronic infection with human CMV may contribute to poor vaccine efficacy in older adults. We assessed the effects of CMV serostatus on Ab quantity and quality, as well as cellular memory recall responses, after two and three SARS-CoV-2 mRNA vaccine doses, in older adults in assisted living facilities. CMV serostatus did not affect anti-Spike and anti-receptor-binding domain IgG Ab levels, nor neutralization capacity against wild-type or β variants of SARS-CoV-2 several months after vaccination. CMV seropositivity altered T cell expression of senescence-associated markers and increased effector memory re-expressing CD45RA T cell numbers, as has been previously reported; however, this did not impact Spike-specific CD4+ T cell memory recall responses. CMV-seropositive individuals did not have a higher incidence of COVID-19, although prior infection influenced humoral immunity. Therefore, CMV seropositivity may alter T cell composition but does not impede the durability of humoral protection or cellular memory responses after SARS-CoV-2 mRNA vaccination in older adults.

Investigation of the Optimal Prime Boost Spacing Regimen for a Cancer Therapeutic Vaccine Targeting Human Papillomavirus.

Simple SummaryThe selection of a therapeutic vaccine schedule can influence the magnitude, efficacy, and durability of immune responses. This study aims to test different prime-boost intervals using a model vaccine in a well-established tumor model system to investigate how the timing of repetitive antigen exposure impacts the induction of effector and memory T cells. Identifying the vaccine schedule most likely to induce durable protective anti-tumor immunity will facilitate decisions made to balance the induction of highly cytotoxic effector T cells and the generation of long-term immunologic memory.Therapeutic vaccine studies should be designed to elicit durable, high magnitude, and efficacious T cell responses, all of which can be impacted by the choice of the vaccination schedule. Here, we compare different prime-boost intervals (PBI) in a human papillomavirus (HPV) model using a HPV16E7E6 Venezuelan equine encephalitis virus replicon particle (VRP) vaccination to address the optimal boosting schedule, quality of immune response, and overall in vivo efficacy. Six different vaccine regimens were tested with each group receiving booster vaccinations at different time intervals. Analysis of T-cell responses demonstrated a significant HPV16 E7 specific CD8+ T cell response with at minimum a one-week PBI between antigen re-exposure. Significant E7-specific in vivo cytotoxicity was also observed with longer PBIs. Additionally, longer PBIs led to an enhanced memory recall response to tumor challenge, which correlated with differential expansion of T cell memory subsets. Our findings imply that when using alphavirus vector platforms as a vaccination strategy, a one-week PBI is sufficient to induce high magnitude effector T cells with potent anti-tumor activity. However, longer PBIs lead to enhanced long-term protective anti-tumor immunity. These findings have implications for therapeutic vaccine clinical trials in which shorter intervals of prime-boost regimens may lead to suboptimal durable immune responses.

Human T-bet+ B cell development is associated with BTK activity and suppressed by evobrutinib.

Recent clinical trials have shown promising results for the next-generation Bruton’s tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of multiple sclerosis (MS). BTK has a central role in signaling pathways that govern the development of B cells. Whether and how BTK activity shapes B cells as key drivers of MS is currently unclear. Compared with levels of BTK protein, we found higher levels of phospho-BTK in ex vivo blood memory B cells from patients with relapsing-remitting MS and secondary progressive MS compared with controls. In these MS groups, BTK activity was induced to a lesser extent after anti-IgM stimulation. BTK positively correlated with CXCR3 expression, both of which were increased in blood B cells from clinical responders to natalizumab (anti–VLA-4 antibody) treatment. Under in vitro T follicular helper–like conditions, BTK phosphorylation was enhanced by T-bet–inducing stimuli, IFN-γ and CpG-ODN, while the expression of T-bet and T-bet–associated molecules CXCR3, CD21, and CD11c was affected by evobrutinib. Furthermore, evobrutinib interfered with in vitro class switching, as well as memory recall responses, and disturbed CXCL10-mediated migration of CXCR3+ switched B cells through human brain endothelial monolayers. These findings demonstrate a functional link between BTK activity and disease-relevant B cells and offer valuable insights into how next-generation BTK inhibitors could modulate the clinical course of patients with MS.

B cell intrinsic and extrinsic factors impacting memory recall responses to SRBC challenge.

MBCs (MBCs) generated in T-dependent immune responses can persist for a lifetime and rapidly react upon secondary antigen exposure to differentiate into plasma cells (PCs) and/or to improve the affinity of their BCR through new rounds of hypermutation in germinal centers (GCs). The fate of a MBC in secondary immune reactions appears to depend upon multiple parameters, whose understanding is mandatory for the design of efficient vaccine strategies. We followed the behavior of MBCs in recall responses to SRBCs using an inducible AID fate mapping mouse model in which B cells engaged in a germinal center (GC) response are irreversibly labeled upon simultaneous tamoxifen ingestion and immunization. We used different schemes of mouse immunization and tamoxifen feeding in adoptive-transfer experiments of total splenic B cells into congenic mice that have been pre-immunized or not, to assess the contribution of the different effector subsets in a physiological competitive context. We were able to show that naive B cells can differentiate into GC B cells with kinetics similar to MBCs in the presence of previously activated T follicular helper (TFH) cells and a primed microenvironment. We also showed that MBCs are recruited into secondary GCs, together with naive B cells. In contrast, PC differentiation, which dominated secondary MBC responses, was not dependent upon a previous TFH activation. We observed that the presence of persisting germinal centers and circulating antibody levels are key factors determining the germinal center versus plasma cell fate in a recall response. Notably, disruption of persistent germinal center structures by a lymphotoxin beta-receptor fusion protein or a longer timing between the prime and the boost, which correlated with reduced antigen-specific immunoglobulin levels in serum, were two conditions with an opposite impact, respectively inhibiting or promoting a GC fate for MBCs. Altogether, these studies highlight the complexity of recall responses, whose outcome varies according to immunization contexts.

ABCB10 Loss Reduces CD4+ T Cell Activation and Memory Formation.

T cells must shift their metabolism to respond to infections and tumors and to undergo memory formation. The ATP-binding cassette transporter ABCB10 localizes to the mitochondrial inner membrane, where it is thought to export a substrate important in heme biosynthesis and metabolism, but its role in T cell development and activation is unknown. In this article, we use a combination of methods to study the effect of ABCB10 loss in primary and malignantly transformed T cells. Although Abcb10 is dispensable for development of both CD4+ and CD8+ T cells, it is required for expression of specific cytokines in CD4+, but not CD8+, T cells activated in vitro. These defects in cytokine expression are magnified on repeated stimulation. In vivo, CD8+ cells lacking ABCB10 expand more in response to viral infection than their control counterparts, while CD4+ cells show reductions in both number and percentage. CD4+ cells lacking ABCB10 show impairment in Ag-specific memory formation and recall responses that become more severe with time. In malignant human CD4+ Jurkat T cells, we find that CRISPR-mediated ABCB10 disruption recapitulates the same cytokine expression defects upon activation as observed in primary mouse T cells. Mechanistically, ABCB10 deletion in Jurkat T cells disrupts the ability to switch to aerobic glycolysis upon activation. Cumulatively, these results show that ABCB10 is selectively required for specific cytokine responses and memory formation in CD4+ T cells, suggesting that targeting this molecule could be used to mitigate aberrant T cell activation.

A human CD137\xd7PD-L1 bispecific antibody promotes anti-tumor immunity via context-dependent T cell costimulation and checkpoint blockade

Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages.

Mechanisms for the regulation of memory B-cell recall responses in mice.

Upon infection by pathogens or vaccination, the adaptive immune system rapidly but transiently produces antibodies. Some weeks later, however, long-lasting immunity is established that protects the host against the same pathogens almost for life through continuous production of antibodies on one hand and the maintenance of cytotoxic T cells on the other, collectively called immunological memory. The antibody-mediated arm, also called serological memory, is mainly exerted by long-lived plasma cells and memory B cells (MBCs). MBCs express receptors for the specific pathogens and circulate to survey the body for almost a life-long period. Upon recognizing the pathogen, MBCs clonally expand and produce a large amount of the specific antibodies to stop the infection, the process called a (memory) recall response. Although such a function of MBCs has long been known, the mechanism of how their performance is regulated has been obscure. This is due to their paucity in the body, lack of definitive surface markers and obscure ontogeny. However, recent studies have revealed the multifold mechanisms by which the recall response of MBCs is regulated: rapid and enhanced antibody production is due to a mechanism intrinsic to MBCs, namely, up-regulated expression levels of surface molecules interacting with T cells and the property of IgG-class antigen receptors; to a property of the responsible subset of MBCs; and to co-stimulation through innate receptors and cytokines. It has also been unveiled that the recall response is negatively regulated by an inhibitory receptor on MBCs and by antigens with repetitive epitopes.

Abstract 292: A dual CTLA4 and PD1 microbially derived inhibitor shows efficacy in anti-PD1 mAb unresponsive tumor models through a unique allosteric mechanism of action

Abstract Background Maintenance of a symbiotic relationship with microbes has led to the evolution of a complex host immune recognition system. How microbes achieve homeostasis with their hosts is an unexploited source of novel chemistry and targets for cancer immunotherapy. Peptides are a proven and powerful medium through which microbes communicate amongst themselves and with the host immune system [Hilchie et al. Nat Chem Biol. 2013 and Hancock Nat Rev Microbiol 2012]. Accordingly, we have developed a platform to mine bioactive peptides from microbial databases/libraries. As proof of concept of our screening technology we targeted PD1 to (A) determine if the PD1 “rheostat” is a target of the microbial immune manipulation, and (B) characterize the activity of the peptide(s) towards developing a novel checkpoint inhibitor therapeutic modality. Methods A microbiome database was queried against previously identified PD1 binding peptides [Kotraiah V Front. Immunol. 2020] and identified peptides were computationally modeled to determine binding site. Reporter assays and ex vivo models were used to measure binding and quantify receptor antagonism. Syngeneic tumor models responsive and unresponsive to anti-PD1 monoclonal antibodies were used to test the efficacy of the peptides. Further, an infectious disease therapeutic model was used to measure and characterize the T-cell response of the peptides in vivo. Results A 22-amino acid peptide, BtLD01, from Bacillus thuringiensis was computationally shown to target a novel allosteric binding site on PD1 which is shared by the related CD28 family receptor, CTLA4. Indeed, BtLD01 antagonizes PD1 and CTLA4 simultaneously as measured in a cell-based reporter assay. Using ex vivo assays we show that BtLD reverses exhaustion-like phenotype in human PBMC recall and that they can stimulate T-Cell proliferation in the presence of PD-L1 blockade. Intriguingly, BtLD checkpoint inhibition is not dependent on PD-L1 blockade but by potentially recruiting or stabilizing the PD1:PD-L1 complex. In vivo studies with sygeneic, mouse tumor models displayed activity in both anti-PD1 mAb responsive and unresponsive tumors. Further, in a lethal malaria model of T-cell activation, we saw that BtLD01 not only protects mice and forms a memory recall response, but also suppresses Tregs. Conclusions Novel Microbially-Derived BtLD peptides target PD1/CTLA4 receptors. The unique allosteric binding site allows peptides to function non-competitively with PD-L1. This novel combined checkpoint inhibition mechanism of activating Tcells and suppressing Tregs along with the rapid and transient modulating nature of peptide may provide a safe and potent means to treat tumors refractory to anti-PD1 mAb therapy alone in clinic. Citation Format: Gabriel M. Gutierrez, Timothy W. Phares, Vinayaka Kotraiah, Peter Buontempo, James Pannucci, Llew Keltner. A dual CTLA4 and PD1 microbially derived inhibitor shows efficacy in anti-PD1 mAb unresponsive tumor models through a unique allosteric mechanism of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 292.

IL-21 is necessary to both induce and sustain humoral immunity to Plasmodium

Abstract Resistance to malaria depends on the generation of antibodies derived from germinal center (GC) responses. T follicular helper (Tfh) cells play a critical role in the selection of high-affinity GC B cells that can differentiate into either memory B cells or antibody secreting plasma cells. Mice deficient in IL-21 or its receptor fail to mount GC responses, secrete parasite-specific IgG and control primary Plasmodium infections. However, whether IL-21 exerts temporally distinct contributions to either the maintenance of GC reactions or memory, during primary anti-Plasmodium humoral immunity or memory recall responses, is not known. We hypothesize that CD4+ T cell derived IL-21 acts to support the maintenance of GC B cells and is critical for secondary humoral immune reactions orchestrated by memory Tfh. To test this hypothesis, we utilized IL-21-VFP reporters and developed cell type-specific and conditional IL-21 knockout systems. Our data show that Tfh-derived IL-21 expression peaks during the first week of infection as GC are initially forming. Following tamoxifen mediated deletion of CD4 derived IL-21 during an established GC reaction (days 16–18 p.i.), we observed significant reductions in GC B cells and loss of memory B cell-derived, parasite-specific IgG production following challenge. Thus, in addition to inducing GC responses, our experiments show that IL-21 is critical for sustaining GC responses and the formation of functional memory B cell responses.

Local immune responses to tuberculin skin challenge in Mycobacterium bovis BCG-vaccinated baboons: a pilot study of younger and older animals

Individuals over the age of 65 are highly susceptible to infectious diseases, which account for one-third of deaths in this age group. Vaccines are a primary tool to combat infection, yet they are less effective in the elderly population. While many groups have aimed to address this problem by studying vaccine-induced peripheral blood responses in the elderly, work from our lab and others demonstrate that immune responses to vaccination and infectious challenge may differ between tissue sites and the periphery. In this pilot study, we established an in vivo delayed-type hypersensitivity model of Mycobacterium bovis BCG vaccination and tuberculin skin test in two adult and two aged baboons. Vaccination generates BCG-specific immune cells that are recruited to the skin upon tuberculin challenge. We tested short term recall responses (8 weeks post-vaccination) and long term recall responses (25 weeks post-vaccination) by performing skin punch biopsies around the site of tuberculin injection. In short term recall responses, we found increased oxidation and decreased production of immune proteins in aged baboon skin at the site of TST challenge, in comparison to adult skin. Differences between adult and aged animals normalized in the long term response to tuberculin. In vitro, aged peripheral blood mononuclear cells had increased migration and functional responses to antigen-specific stimulation, suggesting that age-related changes in the tissue in vivo impairs aged immune recall responses to antigenic challenge. These findings highlight the impact of age-associated changes in the local tissue environment in memory recall responses, which may be more broadly applied to the study of other tissues. Moreover, these findings should be considered in future studies aimed at understanding and improving aging immune responses to vaccination and tissue challenge.

Eomes Impedes Durable Response to Tumor Immunotherapy by Inhibiting Stemness, Tissue Residency, and Promoting the Dysfunctional State of Intratumoral CD8+ T Cells.

Sustaining efficacious T cell-mediated antitumor immune responses in the tumor tissues is the key to the success of cancer immunotherapy. Current strategies leverage altering the signals T cells sense in the tumor microenvironment (TME). Checkpoint inhibitor-based approaches block inhibitory signals such as PD-1 whereas cytokine-based therapies increase the level of immune-stimulatory cytokines such as IL-2. Besides extrinsic signals, the genetic circuit within T cells also participates in determining the nature and trajectory of antitumor immune responses. Here, we showed that efficacy of the IL33-based tumor immunotherapy was greatly enhanced in mice with T cell-specific Eomes deficiency. Mechanistically, we demonstrated that Eomes deficient mice had diminished proportions of exhausted/dysfunctional CD8+ T cells but increased percentages of tissue resident and stem-like CD8+ T cells in the TME. In addition, the IFNγ+TCF1+ CD8+ T cell subset was markedly increased in the Eomes deficient mice. We further demonstrated that Eomes bound directly to the transcription regulatory regions of exhaustion and tissue residency genes. In contrast to its role in inhibiting T cell immune responses at the tumor site, Eomes promoted generation of central memory T cells in the peripheral lymphoid system and memory recall responses against tumor growth at a distal tissue site. Finally, we showed that Eomes deficiency in T cells also resulted in increased efficacy of PD-1-blockade tumor immunotherapy. In all, our study indicates that Eomes plays a critical role in restricting prolonged T cell-mediated antitumor immune responses in the TME whereas promoting adaptive immunity in peripheral lymphoid organs.

Macrophages in Organ Transplantation

Current immunosuppressive therapy has led to excellent short-term survival rates in organ transplantation. However, long-term graft survival rates are suboptimal, and a vast number of allografts are gradually lost in the clinic. An increasing number of animal and clinical studies have demonstrated that monocytes and macrophages play a pivotal role in graft rejection, as these mononuclear phagocytic cells recognize alloantigens and trigger an inflammatory cascade that activate the adaptive immune response. Moreover, recent studies suggest that monocytes acquire a feature of memory recall response that is associated with a potent immune response. This form of memory is called “trained immunity,” and it is retained by mechanisms of epigenetic and metabolic changes in innate immune cells after exposure to particular ligands, which have a direct impact in allograft rejection. In this review article, we highlight the role of monocytes and macrophages in organ transplantation and summarize therapeutic approaches to promote tolerance through manipulation of monocytes and macrophages. These strategies may open new therapeutic opportunities to increase long-term transplant survival rates in the clinic.

COVD-02. ADAPTING RNA-NANOPARTICLE VACCINES FROM GLIOBLASTOMA TO SARS-COV-2

BACKGROUNDGlioblastoma (GBM) can be an effective teacher in the war on COVID-19, as an operative vaccine for either must elicit near-immediate protective responses that overcomes disease heterogeneity and immune suppression. Current prophylactic strategies against COVID-19 utilize mRNA vaccines targeting small fragments of the SARS-CoV-2 genome, but these may not induce robust T cell responses or elicit immunity quickly enough.OBJECTIVEWe sought to adapt an FDA-IND approved mRNA vaccine in GBM against COVID-19 for: 1) activation of near immediate immune responses, 2) targeting of full-length SARS-CoV-2 structural proteins, and 3) induction of bidirectional (B and T cell) adaptive immunity.METHODSWe utilized a novel engineering design that layers mRNA into a lipid-nanoparticle (NP) shell (much like an onion); this allows greater packaging of mRNA per particle to quickly boost innate/adaptive immune responses against full-length glioblastoma antigens or SARS-CoV-2 structural proteins.RESULTSIn small and large animal models, RNA-NPs safely mimic viremia activating the quiescent immune system in only a few hours for induction of protective immunity against its mRNA payload. RNA-NPs activate dendritic cells (DCs), upregulate critical innate gene signatures, and induce antigen-specific cellular and humoral immunity. We found that mice receiving SARS-CoV-2 spike RNA-NPs had more effector T cells after vaccination with significant memory recall expansion after in vitro re-stimulation with overlapping SARS-CoV-2 spike peptide mix. We also found increased release of MIP-1-alpha (i.e. CCL3) previously shown by our group (Mitchell et al. Nature 2015) to be responsible for Th1 mediated memory recall to infectious vaccine antigens in GBM patients.CONCLUSIONSARS-CoV-2 RNA-NPs elicit memory recall response after vaccination. We have obtained FDA-IND approval (BB-19304, Sayour) in GBM with SARS-CoV-2 specific amendment (BB-20871) underway to support first-in-human trials of RNA-NPs targeting both GBM and COVID-19.

Identification of a Protective Leishmania Antigen Dihydrolipoyl Dehydrogenase and Its Responding CD4+ T Cells at Clonal Level.

There is currently no clinically effective vaccine against cutaneous leishmaniasis because of poor understanding of the Ags that elicit protective CD4+ T cell immunity. In this study, we identified a naturally processed peptide (DLD63-79) that is derived from Leishmania dihydrolipoyl dehydrogenase (DLD) protein. DLD is conserved in all pathogenic Leishmania species, is expressed by both the promastigote and amastigote stages of the parasite, and elicits strong CD4+ T cell responses in mice infected with L. major We generated I-Ab-DLD63-79 tetramer and identified DLD-specific CD4+ T cells at clonal level. Following L. major infection, DLD63-79-specific CD4+ T cells massively expanded and produced effector cytokines (IFN-γ and TNF). This was followed by a gradual contraction, stable maintenance following lesion resolution, and display of memory (recall) response following secondary challenge. Vaccination with rDLD protein induced strong protection in mice against virulent L. major challenge. Identification of Ags that elicit protective immunity and their responding Ag-specific T cells are critical steps necessary for developing effective vaccines and vaccination strategies against infectious agents, including protozoan parasites.

Reduced T cell priming to vaccination in microbially-experienced (‘dirty’) mice

Abstract Successful vaccination strategies can provide lifelong immunity against infectious diseases and cancer. Vaccines are widely considered one of the greatest advancements in public health; however, recent failures in vaccine development indicate our inadequate understanding of the immune response to vaccination. There has been increased attention recently regarding the limited translation of preclinical findings generated using specific pathogen free (SPF) laboratory mice to humans. One potential reason for the difference between laboratory mice and humans lies in maturation status of the immune system at the time of challenge. We have been using a ‘dirty’ mouse model, where SPF laboratory mice are cohoused with pet store mice to permit pathogen transfer and immune system maturation, to investigate the naive T cell response after peptide vaccination with Trivax (polyI:C and anti-CD40 mAb) or Complete Freund’s Adjuvant (CFA). Despite having no differences in the number of antigen-specific naïve CD4 and CD8 T cell precursors, the expansion of these CD4 and CD8 T cell populations in cohoused (CoH) B6 mice was significantly reduced (compared to SPF B6 mice) following vaccination with either Trivax or CFA. Furthermore, memory recall responses to cognate antigen – as measured by proliferative capacity or cytokine production – were reduced in the CoH mice. Evaluation of the dendritic cell compartment revealed reduced expression of costimulatory molecules (e.g., CD80/CD86) on DC from CoH mice shortly after vaccination. One consequence of the muted T cell priming in CoH mice was reduced antitumor immunity after vaccination. Collectively, our data show the potential limitation of exclusive use of SPF mice when testing vaccine efficacy.