CD8 T-cell Responses Research Articles

Humoral and cellular immune responses in vaccinatedandunvaccinated children following SARS-CoV-2 Omicron infection.

The immune response in children elicited by SARS-CoV-2 Omicron infection alone or in combination with COVID-19 vaccination (hybrid immunity) is poorly understood. We examined the humoral and cellular immune response following SARS-CoV-2 Omicron infection in unvaccinated children and children who were previously vaccinated with COVID-19 mRNA vaccine. Participants were recruited as part of a household cohort study conducted during the Omicron predominant wave (Jan to July 2022) in Victoria, Australia. Blood samples were collected at 1, 3, 6 and 12 months following COVID-19 diagnosis. Humoral immune responses to SARS-CoV-2 Spike proteins from Wuhan, Omicron BA.1, BA.4/5 and JN.1, as well as cellular immune responses to Wuhan and BA.1 were assessed. A total of 43 children and 113 samples were included in the analysis. Following Omicron infection, unvaccinated children generated low antibody responses but elicited Spike-specific CD4 and CD8 T-cell responses. In contrast, vaccinated children infected with the Omicron variant mounted robust humoral and cellular immune responses to both ancestral strain and Omicron subvariants. Hybrid immunity persisted for at least 6 months post infection, with cellular immune memory characterised by the generation of Spike-specific polyfunctional CD8 T-cell responses. SARS-CoV-2 hybrid immunity in children is characterised by persisting SARS-CoV-2 antibodies and robust CD4 and CD8 T-cell activation and polyfunctional responses. Our findings contribute to understanding hybrid immunity in children and may have implications regarding COVID-19 vaccination and SARS-CoV-2 re-infections.

Sirolimus reduces T cell cycling, immune checkpoint marker expression, and HIV-1 DNA in people with HIV

Key HIV cure strategies involve reversing immune dysfunction and limiting the proliferation of infected Tcells. We evaluate the safety of sirolimus, a mammalian target of rapamycin (mTOR) inhibitor, in people with HIV (PWH) and study the impact of sirolimus on HIV-1 reservoir size and HIV-1-specific immunity in a single-arm study of 20weeks of treatment in PWH on antiretroviral therapy (ART). Sirolimus treatment does not impact HIV-1-specific CD8 Tcell responses but leads to a significant decrease in CD4+ Tcell-associated HIV-1 DNA levels at 20weeks of therapy in the primary efficacy population (n= 16; 31% decline, p=0.008). This decline persists for at least 12weeks following cessation of the study drug. Sirolimus treatment also leads to a significant reduction in CD4+ Tcell cycling and PD-1 expression on CD8+ lymphocytes. These data suggest that homeostatic proliferation of infected cells, an important mechanism for HIV persistence, is an intriguing therapeutic target.

Abstract B058: Rewiring CD8+ T cell responses to PD-1 immune checkpoint blockade in PDAC via the inhibitory receptor PSGL-1

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, poorly immunogenic cancer and non-responsive to all currently available treatments, including immune checkpoint blockade (ICB) therapies. As survival rates remain low (13%), there is an urgent need for new therapeutic options. A hallmark of PDAC is the limited infiltration of anti-tumor cytotoxic CD8+ T cells. Analysis of single-cell RNA-sequencing data of PDAC tumors from treatment naïve patients reveals high expression of P-selectin glycoprotein ligand 1 (PSGL-1) on tumor-infiltrating CD8+ T cells. PSGL-1 is an intrinsic negative regulator of CD8+ T cells that promotes the development of functional exhaustion. We therefore hypothesized that PSGL-1-mediated immune suppression is a critical barrier to effective anti-tumor immunity in PDAC. To test this, we used preclinical models of orthotopic injection PDAC tumor cells into pancreata, recapitulating patient responses to PDAC with limited T cell infiltrates and uncontrolled tumor growth in C57BL/6 (wildtype) control mice and compared anti-tumor immune responses in PSGL-1KO (C57BL/6 background) mice. At 28 days post-injection of KPC.4662 PDAC tumor cells, tumor burden in PSGL-1KO mice was reduced by >50% compared to controls, with 45% of PSGL-1KO animals exhibiting >70% reduction. Total immune infiltration (CD45+) was 2-fold greater in tumors from PSGL-1KO mice, with significant increases in infiltrating CD4+ and CD8+ T cells. Detailed analyses revealed that infiltrating CD8+ T cells from PSGL-1KO mice were less terminally differentiated towards exhaustion. PSGL-1KO PD-1+ CD8+ T cells expressed less TOX expression and fewer cells co-expressed CD38 and CD101 while more expressed CX3CR1 compared to controls. Intratumoral PSGL-1KO CD8+ T cells also retained greater functionality as well as indicated by inflammatory cytokine production (IFNg, TNFa) and Granzyme B expression. Moreover, PSGL-1KO mice showed protection against metastatic disease to the lungs. We find that protection by PSGL-1KO mice is T cell dependent as transient depletion of CD4+ and CD8+ T cells prior to tumor cell implantation results in the loss of PDAC tumor growth inhibition by PSGL-1KO mice. Less-differentiated, proliferation-competent precursors of exhausted CD8+ T cells (TPEX) are essential to PD-1 ICB responsiveness in patients and preclinical models. Given that PSGL-1-deficiency limits terminal differentiation of CD8+ T cells in PDAC, we hypothesized that PSGL-1-deficiency would promote responsiveness to PD-1 ICB, which is ineffective as a monotherapy in PDAC patients and mouse models. While therapeutic treatment with anti-PD-1 had no effect on the tumor growth in the control animals, PD-1 ICB resulted in tumor ablation in 85% of PSGL-1KO animals, demonstrating a profound synergistic effect of PD-1 inhibition in the absence of PSGL-1. From these studies, we conclude that PSGL- 1 is a critical inhibitor anti-tumor T cell immunity in PDAC and represents a novel ICB target with high translational potential for promoting immune responses to PDAC tumors. Citation Format: Jennifer L Hope, Yijuan Zhang, Hannah A. F. Hetrick, Evelyn S. Sanchez Hernandez, Gabriele Romano, Sreeja Roy, Michelle Lin, Ashley B Palete, Swetha Maganti, Dennis C Otero, Katelyn T Byrne, Cosimo Commisso, Linda M Bradley. Rewiring CD8T cell responses to PD-1 immune checkpoint blockade in PDAC via the inhibitory receptor PSGL-1 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B058.

Antiviral capacity of the early CD8 T-cell response is predictive of natural control of SIV infection: Learning in vivo dynamics using ex vivo data.

While most individuals suffer progressive disease following HIV infection, a small fraction spontaneously controls the infection. Although CD8 T-cells have been implicated in this natural control, their mechanistic roles are yet to be established. Here, we combined mathematical modeling and analysis of previously published data from 16 SIV-infected macaques, of which 12 were natural controllers, to elucidate the role of CD8 T-cells in natural control. For each macaque, we considered, in addition to the canonical in vivo plasma viral load and SIV DNA data, longitudinal ex vivo measurements of the virus suppressive capacity of CD8 T-cells. Available mathematical models do not allow analysis of such combined in vivo-ex vivo datasets. We explicitly modeled the ex vivo assay, derived analytical approximations that link the ex vivo measurements with the in vivo effector function of CD8-T cells, and integrated them with an in vivo model of virus dynamics, thus developing a new learning framework that enabled the analysis. Our model fit the data well and estimated the recruitment rate and/or maximal killing rate of CD8 T-cells to be up to 2-fold higher in controllers than non-controllers (p = 0.013). Importantly, the cumulative suppressive capacity of CD8 T-cells over the first 4-6 weeks of infection was associated with virus control (Spearman's ρ = -0.51; p = 0.05). Thus, our analysis identified the early cumulative suppressive capacity of CD8 T-cells as a predictor of natural control. Furthermore, simulating a large virtual population, our model quantified the minimum capacity of this early CD8 T-cell response necessary for long-term control. Our study presents new, quantitative insights into the role of CD8 T-cells in the natural control of HIV infection and has implications for remission strategies.

Cytomegalovirus inhibitors of programmed cell death restrict antigen cross-presentation in the priming of antiviral CD8 T cells.

CD8 T cells are the predominant effector cells of adaptive immunity in preventing cytomegalovirus (CMV) multiple-organ disease caused by cytopathogenic tissue infection. The mechanism by which CMV-specific, naïve CD8 T cells become primed and clonally expand is of fundamental importance for our understanding of CMV immune control. For CD8 T-cell priming, two pathways have been identified: direct antigen presentation by infected professional antigen-presenting cells (pAPCs) and antigen cross-presentation by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Studies in mouse models using murine CMV (mCMV) and precluding either pathway genetically or experimentally have shown that, in principle, both pathways can congruently generate the mouse MHC/H-2 class-I-determined epitope-specificity repertoire of the CD8 T-cell response. Recent studies, however, have shown that direct antigen presentation is the canonical pathway when both are accessible. This raised the question of why antigen cross-presentation is ineffective even under conditions of high virus replication thought to provide high amounts of antigenic material for feeding cross-presenting pAPCs. As delivery of antigenic material for cross-presentation is associated with programmed cell death, and as CMVs encode inhibitors of different cell death pathways, we pursued the idea that these inhibitors restrict antigen delivery and thus CD8 T-cell priming by cross-presentation. To test this hypothesis, we compared the CD8 T-cell responses to recombinant mCMVs lacking expression of the apoptosis-inhibiting protein M36 or the necroptosis-inhibiting protein M45 with responses to wild-type mCMV and revertant viruses expressing the respective cell death inhibitors. The data reveal that increased programmed cell death improves CD8 T-cell priming in mice capable of antigen cross-presentation but not in a mutant mouse strain unable to cross-present. These findings strongly support the conclusion that CMV cell death inhibitors restrict the priming of CD8 T cells by antigen cross-presentation.

CSF1R inhibition by PLX5622 reduces pulmonary fungal infection by depleting MHCIIhi interstitial lung macrophages

PLX5622 is a small molecular inhibitor of the CSF1 receptor (CSF1R) and is widely used to deplete macrophages within the central nervous system (CNS). We investigated the impact of PLX5622 treatment in wild-type C57BL/6 mice and discovered that one-week treatment with PLX5622 was sufficient to deplete interstitial macrophages in the lung and brain-infiltrating Ly6Clow patrolling monocytes, in addition to CNS-resident macrophages. These cell types were previously indicated to act as infection reservoirs for the pathogenic fungus Cryptococcus neoformans. We found that PLX5622-treated mice had significantly reduced fungal lung infection and reduced extrapulmonary dissemination to the CNS but not to the spleen or liver. Fungal lung infection mapped to MHCIIhi interstitial lung macrophages, which underwent significant expansion during infection following monocyte replenishment and not local division. Although PLX5622 depleted CNS infiltrating patrolling monocytes, these cells did not accumulate in the fungal-infected CNS following pulmonary infection. In addition, Nr4a1-deficient mice, which lack patrolling monocytes, had similar control and dissemination of C. neoformans infection to wild-type controls. PLX5622 did not directly affect CD4 T-cell responses, or significantly affect production of antibody in the lung during infection. However, we found that mice lacking lymphocytes had reduced numbers of MHCIIhi interstitial macrophages in the lung, which correlated with reduced infection load. Accordingly, PLX5622 treatment did not alter fungal burdens in the lungs of lymphocyte-deficient mice. Our data demonstrate that PLX5622 may help reduce lung burden of pathogenic fungi that utilise CSF1R-dependent myeloid cells as infection reservoirs, an effect which is dependent on the presence of lymphocytes.

PD-1 regulation of pathogenic IL-17-secreting γδ T cells in experimental autoimmune encephalomyelitis.

The PD-1-PD-L1 immune checkpoint helps to maintain self-tolerance and prevent the development of autoimmune diseases. Immune checkpoint inhibitors are successful immunotherapeutics for several cancers, but responding patients can develop immune-mediated adverse events. It is well established that PD-1 regulates CD4 and CD8 T-cell responses, but its role in controlling the activation of pathogenic γδ T cells is less clear. Here we examined the role of PD-1 in regulating γδ T cells in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. We found that PD-1 was highly expressed on CD27- Vγ4 γδ T cells in the lymph node (LN) and CNS of mice with EAE. Treatment of mice with anti-PD-1 significantly augmented IL-17A-producing CD27- Vγ4 γδ T cells in the LN and CNS and enhanced the severity of EAE. The exacerbating effect of anti-PD-1 on EAE was lost in Tcrd-/- mice. Conversely, ligation of PD-1 suppressed Il17a and Rorc gene expression and IL-17A production by purified Vγ4 γδ T cells stimulated via the TCR, but not with IL-1β and IL-23. Our study demonstrates that PD-1 regulates TCR-activated CD27- Vγ4 γδ T cells, but that cytokine-activated IL-17A producing γδ T cells escape the regulatory effects of the PD-1-PD-L1 pathway.

Hypocortisolemic ASIA: a vaccine- and chronic infection-induced syndrome behind the origin of long COVID and myalgic encephalomyelitis.

Myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS), long COVID (LC) and post-COVID-19 vaccine syndrome show similarities in their pathophysiology and clinical manifestations. These disorders are related to viral or adjuvant persistence, immunological alterations, autoimmune diseases and hormonal imbalances. A developmental model is postulated that involves the interaction between immune hyperactivation, autoimmune hypophysitis or pituitary hypophysitis, and immune depletion. This process might begin with a deficient CD4 T-cell response to viral infections in genetically predisposed individuals (HLA-DRB1), followed by an uncontrolled immune response with CD8 T-cell hyperactivation and elevated antibody production, some of which may be directed against autoantigens, which can trigger autoimmune hypophysitis or direct damage to the pituitary, resulting in decreased production of pituitary hormones, such as ACTH. As the disease progresses, prolonged exposure to viral antigens can lead to exhaustion of the immune system, exacerbating symptoms and pathology. It is suggested that these disorders could be included in the autoimmune/adjuvant-induced inflammatory syndrome (ASIA) because of their similar clinical manifestations and possible relationship to genetic factors, such as polymorphisms in the HLA-DRB1 gene. In addition, it is proposed that treatment with antivirals, corticosteroids/ginseng, antioxidants, and metabolic precursors could improve symptoms by modulating the immune response, pituitary function, inflammation and oxidative stress. Therefore, the purpose of this review is to suggest a possible autoimmune origin against the adenohypophysis and a possible improvement of symptoms after treatment with corticosteroid replacement therapy.

Arenavirus-Based Vectors Generate Robust SIV Immunity in Non-Human Primates.

Arenavirus-based vectors are being investigated as therapeutic vaccine candidates with the potential to elicit robust CD8 T-cell responses. We compared the immunogenicity of replicating (artPICV and artLCMV) and non-replicating (rPICV and rLCMV) arenavirus-based vectors expressing simian immunodeficiency virus (SIV) Gag and Envelope (Env) immunogens in treatment-naïve non-human primates. Heterologous regimens with non-replicating and replicating vectors elicited more robust SIV IFN-γ responses than a homologous regimen, and replicating vectors elicited significantly higher cellular immunogenicity than non-replicating vectors. The heterologous regimen elicited high anti-Env antibody titers when administered intravenously, with replicating vectors inducing significantly higher titers than non-replicating vectors. Intramuscular immunization resulted in more durable antibody responses than intravenous immunization for both vector platforms, with no difference between the replicating and non-replicating vectors. Overall, both replicating and non-replicating arenavirus vectors generated robust T- and B-cell-mediated immunity to SIV antigens in treatment-naïve non-human primates, supporting further evaluation of these vectors in a clinical setting for HIV therapy.

HB-200 arenavirus-based immunotherapy plus pembrolizumab as first-line treatment of patients with recurrent/metastatic HPV16-positive head and neck cancer: Updated results.

6005 Background: Treatment options are limited for patients with recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) with no approved treatment specifically targeting human papillomavirus (HPV) 16-positive tumors. In the first-line (1L) R/M setting, only a minority of patients (~20%) respond to pembrolizumab monotherapy, including those with high programmed death ligand 1 (PD-L1) expression in the tumor. HB-200 comprises an alternating sequence of two replicating attenuated arenavirus vectors derived from LCMV (HB-201) and Pichinde virus (HB-202), respectively. HB-200 vectors express a non-oncogenic HPV16 E7E6 fusion protein and induce E6 and E7-specific CD8 T-cell responses. Previously, we reported promising preliminary clinical activity of HB-200 in combination with pembrolizumab as 1L treatment for patients with HPV16+ PD-L1+ R/M HNSCC. Here, we report updated results with a focus on PD-L1 status. Methods: In this non-randomized Phase 2 part of the study, participants with HPV16+ PD-L1 combined positive score (CPS) ≥1 R/M HNSCC were treated with HB-200 intravenously (every 3 weeks [Q3W] then Q6W) in combination with pembrolizumab (200 mg Q3W or 400 mg Q6W). Safety, T cell response, and preliminary antitumor activity were assessed. Results: As of 12 January 2024, 42 patients with HPV16+ PD-L1+ R/M HNSCC (41 of oropharynx as primary cancer site) were treated with HB-200 plus pembrolizumab in the 1L setting. Median follow-up time was 5.6 months. Characteristics of this cohort included: median age 66 years (range 50-77), 41 (98%) male, 37 (88%) White, 14 (33%) with ≥ 10 pack/year smoking history, 40 (95%) check point inhibitor (CPI) naïve (2 received CPI in the adjuvant setting), and 21 (50%) with PD-L1 CPS ≥ 20. HB-200 + pembrolizumab were generally well tolerated. Grade ≥3 treatment-related adverse events (TRAEs) were reported in 6 (14%) patients, serious TRAEs in 3 (7%) patients, and TRAEs leading to treatment discontinuation in 2 (5%) patients. No treatment-related death were reported. Among 35 evaluable patients (those with ≥ 1 tumor response assessments), the overall response rate (ORR) was 43% (3 complete response [CR], 9 partial response [PR], 3 unconfirmed PR) and the disease control rate (DCR) was 71%. Notably, among patients with PD-L1 CPS ≥20 (N = 17), ORR was 59% (3 CR, 6 PR, 1 unconfirmed PR) and DCR was 88%. Conclusions: Updated data of HB-200 arenavirus-based immunotherapy plus pembrolizumab continued to demonstrate a favorable safety profile and promising clinical activity as 1L treatment in patients with HPV16+ PD-L1+ R/M HNSCC and confirms previously reported data. The results suggest that the subgroup of patients with PD-L1 CPS ≥20 may benefit more from this treatment, which warrants further development in a randomized pivotal study. Clinical trial# NCT04180215. Clinical trial information: NCT04180215 .

SARS-CoV-2 breakthrough infections enhance T cell response magnitude, breadth, and epitope repertoire

Little is known about the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or SARS2) vaccine breakthrough infections (BTIs) on the magnitude and breadth of the Tcell repertoire after exposure to different variants. We studied samples from individuals who experienced symptomatic BTIs during Delta or Omicron waves. In the pre-BTI samples, 30% of the donors exhibited substantial immune memory against non-S (spike) SARS2 antigens, consistent with previous undiagnosed asymptomatic SARS2 infections. Following symptomatic BTI, we observed (1) enhanced S-specific CD4 and CD8 Tcell responses in donors without previous asymptomatic infection, (2) expansion of CD4 and CD8 Tcell responses to non-S targets (M,N, and nsps) independent of SARS2 variant, and (3) generation of novel epitopes recognizing variant-specificmutations. These variant-specific Tcell responses accounted for 9%-15% of the total epitope repertoire. Overall, BTIs boost vaccine-induced immune responses by increasing the magnitude and by broadening the repertoire of Tcell antigens and epitopes recognized.

Immunogenicity and immunodominant linear B-cell epitopes of a new DNA-based tetravalent vaccine against four major enteroviruses causing hand, foot, and mouth disease

Hand, foot, and mouth disease (HFMD) poses a significant public health threat primarily caused by four major enteroviruses: enterovirus 71 (EV71), coxsackieviruses A16, A10, and A6. Broadly protective immune responses are essential for complete protection against these major enteroviruses. In this study, we designed a new tetravalent immunogen for HFMD, validated it in silico, in vivo evaluated the immunogenicity of the DNA-based tetravalent vaccine in mice, and identified immunogenic B-cell and T-cell epitopes. A new tetravalent immunogen, VP1me, was designed based on the chimeric protein and epitope-based vaccine principles. It contains a complete EV71 VP1 protein and six reported neutralizing B-cell epitopes derived from the four major enteroviruses causing HFMD. In silico validation using multiple immunoinformatic tools indicated good attributes of the VP1me immunogen suitable for vaccine development. The VP1me-based DNA vaccine efficiently induced both humoral and cellular immune responses in BALB/cAJcl mice. A combination of in silico prediction and immunoassays enabled the identification of immunogenic linear B-cell and CD8 T-cell epitopes within the VP1me immunogen. Immunodominant linear B-cell epitopes were identified in six regions of VP1me, with one epitope located at the N-terminus of the VP1 protein (aa 9–23) regarded as a novel epitope. Interestingly, some B-cell epitopes could also induce the CD8 T-cell response, suggesting their dual functions in immune stimulation. These results lay the groundwork for further development of VP1me as a new vaccine candidate.

Characterization of the Protective Cellular Immune Response in Pigs Immunized Intradermally with the Live Attenuated African Swine Fever Virus (ASFV) Lv17/WB/Rie1.

Candidate vaccines against African swine fever virus (ASFV) based on naturally attenuated or genetically modified viruses have the potential to generate protective immune responses, although there is no consensus on what defines a protective immune response against ASFV. Studies, especially in sensitive host species and focused on unravelling protective mechanisms, will contribute to the development of safer and more effective vaccines. The present study provides a detailed analysis of phenotypic and functional data on cellular responses induced by intradermal immunization and subsequent boosting of domestic pigs with the naturally attenuated field strain Lv17/WB/Rie1, as well as the mechanisms underlying protection against intramuscular challenge with the virulent genotype II Armenia/07 strain. The transient increase in IL-8 and IL-10 in serum observed after immunization might be correlated with survival. Protection was also associated with a robust ASFV-specific polyfunctional memory T-cell response, where CD4CD8 and CD8 T cells were identified as the main cellular sources of virus-specific IFNγ and TNFα. In parallel with the cytokine response, these T-cell subsets also showed specific cytotoxic activity as evidenced by the increased expression of the CD107a degranulation marker. Along with virus-specific multifunctional CD4CD8 and CD8 T-cell responses, the increased levels of antigen experienced in cytotoxic CD4 T cells observed after the challenge in immunized pigs might also contribute to controlling virulent infection by killing mechanisms targeting infected antigen-presenting cells. Future studies should elucidate whether the memory T-cell responses evidenced in the present study persist and provide long-term protection against further ASFV infections.

Delay induced stability switch in a mathematical model of CD8 T-cell response to SARS-CoV-2 mediated by receptor ACE2.

The pathogen SARS-CoV-2 binds to the receptor angiotensin-converting enzyme 2 (ACE2) of the target cells and then replicates itself through the host, eventually releasing free virus particles. After infection, the CD8 T-cell response is triggered and appears to play a critical role in the defense against virus infections. Infected cells and their activated CD8 T-cells can cause tissue damage. Here, we established a mathematical model of within-host SARS-CoV-2 infection that incorporates the receptor ACE2, the CD8 T-cell response, and the damaged tissues. According to this model, we can get the basic reproduction number R0 and the immune reproduction number R1. We provide the theoretical proof for the stability of the disease-free equilibrium, immune-inactivated equilibrium, and immune-activated equilibrium. Finally, our numerical simulations show that the time delay in CD8 T-cell production can induce complex dynamics such as stability switching. These results provide insights into the mechanisms of SARS-CoV-2 infection and may help in the development of effective drugs against COVID-19.

Characterisation of clinical response and transcriptional profiling of proliferating CD8 T cells in the blood of cancer patients after PD-1 monotherapy or combination therapy

ObjectiveImmune checkpoint inhibitors (ICI) that block the programmed cell death 1 (PD-1) pathway have shown promise with limited benefit. We and others have shown in small patient cohorts that an...

Abstract 6704: Development and characterization of a tri-specific selective CD8 T cell engager (CD8xCD3xCD19) for treatment of B cell lymphoma

Abstract The recent development of CD3 bi-specific antibodies (BsAbs) targeting CD20, BCMA, GPRC5D and CD19 to treat B cell malignancies have resulted in significant clinical benefit to patients. However, long-term remission rates and survival remain to be demonstrated. Current CD3 BsAbs preferentially bind to and activate CD4 T cells. However, CD8 cytotoxic T cells also play the key role in anti-tumor response and T cell exhaustion in cancer immunity. To potentially enhance anti-tumor efficacy by augmenting CD8 T-cell response, we developed a novel tri-specific T cell engager (TriTE) IM-8319, which simultaneously binds human CD3, CD8 and CD19 epitopes with KD at 14.2 nM, 3.3 nM and 0.3 nM, respectively. The binding affinity to human CD8 T cells and CD4 T cells was measured with EC50 at 0.02 nM and 1.2 nM, respectively. In human PBMC-killing assays, IM-8319 was found to preferentially activate CD8 T cells with EC50 of 6.0 pM, compared to an EC50 of 94 pM for activating CD4 T cells. Unique cytokine release responses were seen with IM-8319 treatment. Compared to bi-specific TCE controls without CD8 binding domain, IM-8319 in human PBMC assays increased INFγ and TNFα in CD8 T cells and blunted IL-2 release in CD4 T cells. We observed robust cytotoxic killing of CD19+ B cells with IM-8319. Compared the bi-specific TCE controls lacking CD8 binding domain, in vitro CD19+ B-cell depletion was increased 6-fold with IM-8319. In addition, we observed notable structure activity relationships, with >100-fold increases in TriTE cytotoxicity with a weak CD3 binding domain. T-cell fratricide has hindered the development of antibodies containing 2+ T-cell epitopes. We observed correctable, dose-dependent CD8 T cell depletion at high concentrations of CD8xCD3xCD19 TriTEs in vitro and xenograft mouse models. This unwanted biological effect was not a surprise when the TriTE binds to CD8 T cells in trans. However, by structure engineering IM-8319 to allow for cis binding of CD3 and CD8, we effectively eliminated CD8 T cell fratricide at therapeutic doses. In a Raji-inoculated murine xenograft that was reconstituted with human PBMC, IM-8319 significantly inhibited tumor growth without obvious CD8 T cell depletion. In summary, we report a novel TriTE (IM-8319) that selectively binds to and activates CD8 T cells. IM-8319 results in robust on-target cytotoxicty with specific activation of CD8 T cells, suggesting potential to reduce systemic toxicities such as cytokine release syndrome (CRS) while augmenting direct antitumor efficacy. Further preclinical evaluation of IM-8319 is warranted in patients with B-cell leukemias and lymphomas. Citation Format: Zheng Yang, Wuzhong Shen, Guiyun Tu, Shenjie Xue, Ying Tan, Bingbin Xie, Jason Xu, Hanyang Chen, Shui Liu, Xiaoqiang Yan. Development and characterization of a tri-specific selective CD8 T cell engager (CD8xCD3xCD19) for treatment of B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6704.

DNAJB1-PRKACA fusion neoantigens elicit rare endogenous T cell responses that potentiate cell therapy for fibrolamellar carcinoma

Fibrolamellar carcinoma (FLC) is a liver tumor with a high mortality burden and few treatment options. A promising therapeutic vulnerability in FLC is its driver mutation, a conserved DNAJB1-PRKACA gene fusion that could be an ideal target neoantigen for immunotherapy. In this study, we aim to define endogenous CD8 Tcell responses to this fusion in FLC patients and evaluate fusion-specific Tcell receptors (TCRs) for use in cellular immunotherapies. We observe that fusion-specific CD8 Tcells are rare and that FLC patient TCR repertoires lack large clusters of related TCR sequences characteristic of potent antigen-specific responses, potentially explaining why endogenous immune responses are insufficient to clear FLC tumors. Nevertheless, we define two functional fusion-specific TCRs, one of which has strong anti-tumor activity invivo. Together, our results provide insights into the fragmented nature of neoantigen-specific repertoires in humans and indicate routes for clinical development of successful immunotherapies for FLC.

Immunotherapy targeting tumor-associated antigen in a mouse model of head and neck cancer.

The identification of epitope peptides from tumor-associated antigens (TAAs) is informative for developing tumor-specific immunotherapy. However, only a few epitopes have been detected in mouse TAAs of head and neck cancer (HNSCC). Novel mouse c-Met-derived T-cell epitopes were predicted by computer-based algorithms. Mouse HNSCC cell line-bearing mice were treated with a c-Met peptide vaccine. The effects of CD8 and/or CD4 T-cell depletion, and vaccine combination with immune checkpoint inhibitors (ICIs) were evaluated. Tumor re-inoculation was performed to assess T-cell memory. We identified c-Met-derived short and long epitopes that elicited c-Met-reactive antitumor CD8 and/or CD4 T-cell responses. Vaccination using these peptides showed remarkable antitumor responses via T cells in which ICIs were not required. The c-Met peptide-vaccinated mice rejected the re-inoculated tumors. We demonstrated that novel c-Met peptide vaccines can induce antitumor T-cell response, and could be a potent immunotherapy in a syngeneic mouse HNSCC model.

Modulation of cytomegalovirus immune evasion identifies direct antigen presentation as the predominant mode of CD8 T-cell priming during immune reconstitution after hematopoietic cell transplantation.

Cytomegalovirus (CMV) infection is the most critical infectious complication in recipients of hematopoietic cell transplantation (HCT) in the period between a therapeutic hematoablative treatment and the hematopoietic reconstitution of the immune system. Clinical investigation as well as the mouse model of experimental HCT have consistently shown that timely reconstitution of antiviral CD8 T cells is critical for preventing CMV disease in HCT recipients. Reconstitution of cells of the T-cell lineage generates naïve CD8 T cells with random specificities among which CMV-specific cells need to be primed by presentation of viral antigen for antigen-specific clonal expansion and generation of protective antiviral effector CD8 T cells. For CD8 T-cell priming two pathways are discussed: "direct antigen presentation" by infected professional antigen-presenting cells (pAPCs) and "antigen cross-presentation" by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Current view in CMV immunology favors the cross-priming hypothesis with the argument that viral immune evasion proteins, known to interfere with the MHC class-I pathway of direct antigen presentation by infected cells, would inhibit the CD8 T-cell response. While the mode of antigen presentation in the mouse model of CMV infection has been studied in the immunocompetent host under genetic or experimental conditions excluding either pathway of antigen presentation, we are not aware of any study addressing the medically relevant question of how newly generated naïve CD8 T cells become primed in the phase of lympho-hematopoietic reconstitution after HCT. Here we used the well-established mouse model of experimental HCT and infection with murine CMV (mCMV) and pursued the recently described approach of up- or down-modulating direct antigen presentation by using recombinant viruses lacking or overexpressing the central immune evasion protein m152 of mCMV, respectively. Our data reveal that the magnitude of the CD8 T-cell response directly reflects the level of direct antigen presentation.

Batf3-dependent orchestration of the robust Th1 responses and fungal control during cryptococcal infection, the role of cDC1.

While type I conventional dendritic cells (cDC1s) are vital for generating adaptive immunity against intracellular pathogens and tumors, their role in defense against fungal pathogen Cryptococcus neoformans remains unclear. We investigated the role of the cDC1 subset in a fungus-restricting mouse model of cryptococcal infection. The cDC1 subset displayed a unique transcriptional signature with highly upregulated T-cell recruitment, polarization, and activation pathways compared to other DC subsets. Using Batf3-/- mice, which lack the cDC1 population, our results support that Batf3-dependent cDC1s are pivotal for the development of the effective immune response against cryptococcal infection, particularly within the lung and brain. Deficiency in Batf3 cDC1 led to diminished CD4 accumulation and decreased IFNγ production across multiple organs, supporting that cDC1s are a major driver of potent Th1 responses during cryptococcal infection. Consistently, mice lacking Batf3-cDC1 demonstrated markedly diminished fungicidal activity and weaker containment of the fungal pathogen. In conclusion, Batf3-dependent cDC1 can function as a linchpin in mounting Th1 response, ensuring effective fungal control during cryptococcal infection. Harnessing cDC1 pathways may present a promising strategy for interventions against this pathogen.IMPORTANCECryptococcus neoformans causes severe meningoencephalitis, accounting for an estimated 200,000 deaths each year. Central to mounting an effective defense against these infections is T-cell-mediated immunity, which is orchestrated by dendritic cells (DCs). The knowledge about the role of specific DC subsets in shaping anti-cryptococcal immunity is limited. Here, we demonstrate that Batf3 cDC1s are important drivers of protective Th1 CD4 T-cell responses required for clearance of cryptococcal infection. Deficiency of Batf3 cDC1 in the infected mice leads to significantly reduced Th1 response and exacerbated fungal growth to the point where depleting the remaining CD4 T cells no longer affects fungal burden. Unveiling this pivotal role of cDC1 in antifungal defense is likely to be important for the development of vaccines and therapies against life-threatening fungal pathogens.