Transfer Of Lymphocytes Research Articles

Autoimmune-Like Mechanism in Heart Failure Enables Preventive Vaccine Therapy.

Heart failure (HF) is strongly associated with inflammation. In pressure overload (PO)-induced HF, cardiac stress triggers adaptive immunity, ablation, or inhibition, which blocks disease progression. We hypothesized that PO-HF might fulfill the often-used criteria of autoimmunity: if so, the associated adaptive immune response would be not only necessary but also sufficient to induce HF; it should also be possible to identify self-antigens driving the autoimmune response. Finally, we hypothesized that such an antigen-specific response can be manipulated to preventively reduce the severity of PO-HF in a tolerizing vaccine. We used the transfer of lymphocytes or serum from PO-HF mice into healthy recipients to assess whether the adaptive response is sufficient to induce disease. We devised a novel pipeline to identify self-antigens driving the response. We immunized healthy mice with novel antigens to assess whether they induce disease. To determine whether these antigens could be present in human patients, we sought to detect existing responses against these antigens in patients with HF. Finally, we used the antigens in an oral tolerance protocol to preventively protect mice from subsequently induced PO-HF, analyzing the results with next-generation sequencing. We found that PO-HF fulfills the criteria of an autoimmune disease, albeit partially, and identified novel cardiac self-antigens, capable of inducing cardiac dysfunction. The novel antigens in a tolerizing vaccine formulation preemptively reduced the severity of disease triggered by subsequent application of PO, via induction of effector regulatory T cells, enabling a potent reduction of PO-driven loss of systolic function, cardiac inflammation, and proinflammatory CD4+ T-cell clonal expansion. We demonstrate that PO-HF is triggered by hemodynamic stress and then sets off an autoimmune-like response against cardiac self-antigens. The antigens can be used to reduce the severity of future-onset disease, via oral tolerization, effectively acting as a protective vaccine.

IMMU-18. IL-7 MEDIATED UPREGULATION OF VLA-4 INCREASES ACCUMULATION OF ADOPTIVELY TRANSFERRED T LYMPHOCYTES IN MURINE GLIOMA

Abstract We have previously described the development of a novel brain bi-specific T cell engager (BRiTE) targeting EGFRvIII in Glioma. Despite BRiTEs impressive potency, efficacy in solid tumors is dependent on tumor-infiltrating lymphocytes (TILs). We evaluated if peripheral adoptive lymphocyte transfer (ALT) increases TILs in immunocompetent mice, even during glioma-induced sequestration. C57/Bl6 mice were implanted with 3 x 10^4 cells syngeneic glioma (CT-2AvIII) and treated with 1 x 10^7 IV CD45.1+ ALT on Day 15. The brain was divided into tumor and non-tumor bearing hemispheres and analyzed each day for exogenous (CD45.1+) CD3/4/8 counts. We observed that IL-7-expanded ALT accumulated at a higher rate within tumor versus IL-2 ALT (72-hours IL-7 median 26.66x vs IL-2 median 4.291x, p=0.0159*). Exogenous T cell entry into tumor-bearing CNS overwhelmingly consisted of CD8+ T cells, and CD45.1+ IL-7 ALT cells made up ~20% of the CD3+ population in tumor 72-hours following administration (mean 19.4%, SD ±4.53%, n=5). Survival experiments used combination ALT and anti-4-1BB therapy in the same model. Mice were treated with IV ALT on Day 5, along with 200µg of IP anti-4-1BB. IP mAb Injections were given every 3 days for 4 total cycles (D5, D8, D11 & D14). Evaluation at these time points was selected as this correlated with peak sequestration of immune cells as evidenced by splenic involution. For survival studies, only combination ALT and anti-4-1BB produced significantly increased survival benefit in our CT-2AvIII model compared to ALT alone (median survival 45.5 days for combination versus 25 days in ALT only group, p=0.0134, chi-square test). These findings demonstrate the feasibility of combination ALT/antibody-based therapy in pre-clinical glioma, as well as outlining a novel approach for delivering both immune cells and antibody-based immunotherapy. Combination therapy with BRiTE and ALT will be explored in an upcoming clinical trial (NCT04903795).

Enhanced anti-tumor efficacy of electroporation (EP)-mediated DNA vaccine boosted by allogeneic lymphocytes in pre-established tumor models

BackgroundTumor-reactive T cells play a crucial role in anti-tumor responses, but T cells induced by DNA vaccination are time-consuming processes and exhibit limited anti-tumor efficacy. Therefore, we evaluated the anti-tumor effectiveness of reactive T cells elicited by electroporation (EP)-mediated DNA vaccine targeting epidermal growth factor receptor variant III (pEGFRvIII plasmid), in conjunction with adoptive cell therapy (ACT), involving the transfer of lymphocytes from a pEGFRvIII EP-vaccinated healthy donor.MethodsThe validation of the established pEGFRvIII plasmid and EGFRvIII-positive cell model was confirmed through immunofluorescence and western blot analysis. Flow cytometry and cytotoxicity assays were performed to evaluate the functionality of antigen-specific reactive T cells induced by EP-mediated pEGFRvIII vaccines, ACT, or their combination. The anti-tumor effectiveness of EP-mediated pEGFRvIII vaccines alone or combined with ACT was evaluated in the B16F10-EGFRvIII tumor model.ResultsEP-mediated pEGFRvIII vaccines elicited serum antibodies and a robust cellular immune response in both healthy and tumor-bearing mice. However, this response only marginally inhibited early-stage tumor growth in established tumor models. EP-mediated pEGFRvIII vaccination followed by adoptive transfer of lymphocytes from vaccinated healthy donors led to notable anti-tumor efficacy, attributed to the synergistic action of antigen-specific CD4+ Th1 cells supplemented by ACT and antigen-specific CD8+ T cells elicited by the EP-mediated DNA vaccination.ConclusionsOur preclinical studies results demonstrate an enhanced anti-tumor efficacy of EP-mediated DNA vaccination boosted with adoptively transferred, vaccinated healthy donor-derived allogeneic lymphocytes.

Could the Adoptive Transfer of Memory Lymphocytes be an Alternative Treatment for Acinetobacter baumannii Infections?

We evaluated the efficacy of the adoptive transfer of memory B, CD4+, and CD8+ T lymphocytes compared with sulbactam and tigecycline in an experimental murine pneumonia model by two multidrug-resistant Acinetobacter baumannii strains, colistin-susceptible AbCS01 and colistin-resistant AbCR17. Pharmacodynamically optimized antimicrobial dosages were administered for 72 h, and intravenous administration of 2 × 106 of each of the memory cells in a single dose 30 min post-infection. Bacterial lung and blood counts and mortality rates were analyzed. Results showed that a single dose of memory B or CD4+ T cells was as effective as sulbactam in terms of bacterial clearance from the lungs and blood compared with the untreated mice or the tigecycline-treated mice inoculated with the AbCS01 strain. In the pneumonia model by AbCR17, a single dose of memory B or CD4+ T cells also reduced the bacterial load in the lungs compared with both antibiotic groups and was more efficacious than tigecycline in terms of blood clearance. Regarding survival, the adoptive transfer of memory B or CD4+ T cells was as effective as three days of sulbactam treatment for both strains. These data suggest that adoptive memory cell transfer could be a new effective treatment of multidrug-resistant A. baumannii infections.

Interleukin-21 receptor signaling promotes metabolic dysfunction-associated steatohepatitis-driven hepatocellular carcinoma by inducing immunosuppressive IgA+ B cells

BackgroundDysregulation of immune surveillance is tightly linked to the development of metabolic dysfunction-associated steatohepatitis (MASH)-driven hepatocellular carcinoma (HCC); however, its underlying mechanisms remain unclear. Herein, we aimed to determine the role of interleukin-21 receptor (IL-21R) in MASH-driven HCC.MethodsThe clinical significance of IL-21R was assessed in human HCC specimens using immunohistochemistry staining. Furthermore, the expression of IL-21R in mice was assessed in the STAM model. Thereafter, two different MASH-driven HCC mouse models were applied between IL-21R-deficient mice and wild type controls to explore the role of IL-21R in MASH-driven HCC. To further elucidate the potential mechanisms by which IL-21R affected MASH-driven HCC, whole transcriptome sequencing, flow cytometry and adoptive lymphocyte transfer were performed. Finally, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescent staining, chromatin immunoprecipitation assay and western blotting were conducted to explore the mechanism by which IL-21R induced IgA+ B cells.ResultsHCC patients with high IL-21R expression exhibited poor relapse-free survival, advanced TNM stage and severe steatosis. Additionally, IL-21R was demonstrated to be upregulated in mouse liver tumors. Particularly, ablation of IL-21R impeded MASH-driven hepatocarcinogenesis with dramatically reduction of lipid accumulation. Moreover, cytotoxic CD8+ T lymphocyte activation was enhanced in the absence of IL-21R due to the reduction of immunosuppressive IgA+ B cells. Mechanistically, the IL-21R-STAT1-c-Jun/c-Fos regulatory axis was activated in MASH-driven HCC and thus promoted the transcription of Igha, resulting in the induction of IgA+ B cells.ConclusionsIL-21R plays a cancer-promoting role by inducing IgA+ B cells in MASH-driven hepatocarcinogenesis. Targeting IL-21R signaling represents a potential therapeutic strategy for cancer therapy.

Abstract LB073: Adoptive transfer of one CD4TCR and one CD8TCR targeting autochthonous neoantigens are essential and sufficient for eradication of naturally heterogeneous solid tumors

Abstract Despite advances in checkpoint blockade therapy or adoptive transfer of tumor-infiltrating lymphocytes (TILs), achieving cure remains challenging, but success of immunotherapy seems to depend on recognition of tumor-specific antigens (neoantigens). We aimed to determine how many neoantigens need to be recognized by how many different TCRs for eradication of solid tumors. Unmanipulated, naturally expressed (autochthonous) neoantigens were targeted with adoptively transferred TCR-engineered autologous T cells (TCR-therapy). Investigated were effects of TCR-engineered CD8+ T cells, TCR-engineered CD4+ T cells or a combination of both. The targeted tumors were established for at least three weeks and derived from primary autochthonous cancer cell cultures, resembling natural solid tumors and their heterogeneity as found in humans. CD8TCR-therapy was only effective against homogenous tumors and relapse was common in heterogeneous tumors, even when targeting multiple different autochthonous neoantigens. By contrast, a combination of CD8TCR-therapy with CD4TCR-therapy, each targeting an independent neoantigen, eradicated large and established solid tumors of natural heterogeneity. CD4TCR-therapy targeted a mutant neoantigen on tumor stroma while direct cancer cell recognition by CD8TCR-therapy was essential for cure. Thus, two cancer-specific TCRs can be essential and sufficient to eradicate heterogeneous solid tumors expressing unmanipulated, autochthonous neoantigens. Citation Format: Steven Wolf, Vasiliki Anastasopoulou, Kimberley Drousch, Markus Diehl, Boris Engels, Poh Yin Yew, Kazuma Kiyotani, Yusuke Nakamura, Karin Schreiber, Hans Schreiber, Matthias Leisegang. Adoptive transfer of one CD4TCR and one CD8TCR targeting autochthonous neoantigens are essential and sufficient for eradication of naturally heterogeneous solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB073.

Abstract 15: Massively parallel base-editing screens to map variant effects on anti-tumor hallmarks of primary human T cells and improve cell-based cancer immunotherapies

Abstract Base editing enables precise generation of single nucleotide variants, but large-scale screening in primary human T cells has been limited by low editing efficiency. Here, we developed a high-throughput approach for highly efficient, massively parallel adenine and cytosine base-editor screening in primary human T cells. We performed multiple large-scale pooled screens editing 108 genes with central functions in T cells and whole-gene tiling mutagenesis of selected genes. All screens were functionally read out to capture the effect of variants on hallmarks of T cell anti-tumor immunity, including activation, proliferation, long-term persistence, and cytokine production. We discovered a broad landscape of gain-of-function and loss-of-function mutations and characterized their impact across multiple axes of T cell functions. Among others, we identified gain-of-function mutations in PIK3CD and its regulatory subunit PIK3R1, LCK, AKT1, and loss-of-function mutants of CTLA-4, driving increased T cell polyfunctionality. We validated several of these mutations by probing downstream signaling and functional outputs. To demonstrate the feasibility of using such screen results for improving cell-based cancer immunotherapies, we used human T cells with engineered T cell receptors recognizing specific epitopes in melanoma models. We base-edited these T cells with mutations identified in our screens and demonstrate that these mutations indeed significantly enhance cytotoxic cytokine production and tumor-lytic capacity with minimal off-target activity. Lastly, we also provide proof-of-feasibility for high-efficiency combinatorial base-editing in T cells. This study presents a framework for large-scale base editing screens with multi-dimensional functional readouts in cells that are typically difficult to molecularly engineer in a precise manner. This work also has important therapeutic implications: T cells are the substrate for clinically active cell-based therapies, such as adoptive transfer of tumor-infiltrating lymphocytes (TILs) or chimeric-receptor antigen T (CAR-T) cells. Our screen-informed base editing to achieve an improved T cell product against melanoma exemplifies the therapeutic power of generating synthetic protein variants that improve a wide range of existing and future T cell-based cancer immunotherapies. Citation Format: Zachary Hudson Walsh, Parin Shah, Neeha Kothapalli, Gergo Nikolenyi, Shivem Shah, Giuseppe Leuzzi, Neil Vasan, Mohammed AlQuarishi, Alberto Ciccia, Johannes Melms, Benjamin Izar. Massively parallel base-editing screens to map variant effects on anti-tumor hallmarks of primary human T cells and improve cell-based cancer immunotherapies [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 15.

Effect of hypoxia-induced mIL15 expression on expansion and memory progenitor stem-like TILs in vitro.

The adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) has proven clinically beneficial in patients with non-small cell lung cancer refractory to checkpoint blockade immunotherapy, which has prompted interest in TIL-adoptive cell transfer. The transgenic expression of IL15 can promote the expansion, survival, and function of T cells ex vivo and in vivo and enhance their anti-tumor activity. The effect of expressing mIL15 regulated by hypoxia in the tumor microenvironment on the expansion, survival, and stem-like properties of TILs has not been explored. Using TILs expanded from the tumor tissues of lung cancer patients, TILs with or without mIL15 expression (TIL-mIL15 or UN-TIL) were generated by lentiviral transduction. To reflect the advantages of mTIL15, the cells were divided into groups with IL2 (TIL-mIL15+IL2) or without IL2 (TIL-mIL15-IL2). Compared to UN-TIL cells, mIL15 expression had a similar capacity for promoting TIL proliferation and maintaining cell viability. Our experimental findings indicate that, compared to UN-TIL and TIL-mIL15+IL2 cells, the expression of mIL15 in TIL-mIL15-IL2 cells promoted the formation of stem-like TILs (CD8+CD39-CD69-) and led to significant decreases in the proportion and absolute number of terminally differentiated TILs (CD8+CD39+CD69+). RNA-Seq data revealed that in TIL-mIL15-IL2 cells, the expression of genes related to T cell differentiation and effector function, including PRDM1, ID2, EOMES, IFNG, GZMB, and TNF, were significantly decreased, whereas the expression of the memory stem-like T cell marker TCF7 was significantly increased. Furthermore, compared to UN-TIL and TIL-mIL15+IL2 cells, TIL-mIL15-IL2 cells showed significantly lower expression levels of inhibitory receptors LAG3, TIGIT, and TIM3, which was consistent with the RNA-Seq results. This study demonstrates the superior persistence of TIL-mIL15-IL2 cells, which may serve as a novel treatment strategy for lung cancer patients.

T Cell Antigen Recognition and Discrimination by Electrochemiluminescence Imaging.

Adoptive T lymphocyte (T cell) transfer and tumour-specific peptide vaccines are innovative cancer therapies. An accurate assessment of the specific reactivity of T cell receptors (TCRs) to tumour antigens is required because of the high heterogeneity of tumour cells and the immunosuppressive tumour microenvironment. In this study, we report a label-free electrochemiluminescence (ECL) imaging approach for recognising and discriminating between TCRs and tumour-specific antigens by imaging the immune synapses of T cells. Various T cell stimuli, including agonistic antibodies, auxiliary molecules, and tumour-specific antigens, were modified on the electrode's surface to allow for their interaction with T cells bearing different TCRs. The formation of immune synapses activated by specific stimuli produced a negative (shadow) ECL image, from which T cell antigen recognition and discrimination were evaluated by analysing the spreading area and the recognition intensity of T cells. This approach provides an easy way to assess TCR-antigen specificity and screen both of them for immunotherapies.

T Cell Antigen Recognition and Discrimination by Electrochemiluminescence Imaging

AbstractAdoptive T lymphocyte (T cell) transfer and tumour‐specific peptide vaccines are innovative cancer therapies. An accurate assessment of the specific reactivity of T cell receptors (TCRs) to tumour antigens is required because of the high heterogeneity of tumour cells and the immunosuppressive tumour microenvironment. In this study, we report a label‐free electrochemiluminescence (ECL) imaging approach for recognising and discriminating between TCRs and tumour‐specific antigens by imaging the immune synapses of T cells. Various T cell stimuli, including agonistic antibodies, auxiliary molecules, and tumour‐specific antigens, were modified on the electrode's surface to allow for their interaction with T cells bearing different TCRs. The formation of immune synapses activated by specific stimuli produced a negative (shadow) ECL image, from which T cell antigen recognition and discrimination were evaluated by analysing the spreading area and the recognition intensity of T cells. This approach provides an easy way to assess TCR‐antigen specificity and screen both of them for immunotherapies.

Abstract 13821: Induction of Immune Tolerance to Intramyocardial Transplantation of Cardiac Progenitor Cells by Regulatory T Lymphocytes

Introduction: The clinical use of human embryonic stem cell (hESCs)-derived cardiac cells is still hampered by rejection which is managed by immunosuppressive drugs and exposes to their side-effects. Hypothesis: We evaluated an alternate strategy based on the induction of immune tolerance by adoptive regulatory T lymphocytes (Treg) transfer. Methods: Human hTreg were isolated from peripheral blood mononuclear cells, expanded for 9 days and phenotyped. Immune-competent mice were then subjected to a myocardial infarction. Three weeks later (baseline), mice with an echocardiographically-measured ejection fraction (EF) ≤50% underwent trans-cutaneous echo-guided peri-infarct injections of 1.4x10 6 hESC-cardiovascular progenitor cells (CPC; n=11) or an equivalent volume of PBS (controls, n=4). Among the CPC-transplanted mice, 6 were intravenously injected with 2x10 6 hTreg at the time of transplantation and 3 days later and 5 only received the vehicle (PBS). Functional and histological end points were assessed 3 weeks after treatments (final) and spleen tissue was processed by flow cytometry for the evaluation of immune cell populations. Results: Three weeks after treatments, all CPC-transplanted hearts had a significantly better preservation of EF [m±SEM], compared with their respective baseline values, than PBS-injected hearts (35.4% ± 5.4% in CPC and 37.5% ± 6.8% in CPC+hTreg vs. -1.3 ± 7.3% in PBS, p=0.004) which was mainly driven by a reduction in LV end-systolic volumes (p=0.004) but only in the hTreg group global longitudinal strain improved by 15.7% ± 13.3% from baseline while it worsened by 36.5% ± 18.8% in their non-hTreg counterparts (p=0.037). Infarct size did not differ among the 3 groups but Treg reduced interstitial fibrosis in remote non infarcted myocardium (0.6% ± 0.1%, p=0.007 vs. CPC-non hTreg and PBS, respectively) and, in spleen tissue, induced significant decreases in CD80+ M1 macrophages (p=0.010) and NKG2D+ NK cells (p=0.013) and an increase in CD206+ M2 macrophages (p=0.004) compared with the other 2 groups. Conclusions: Systemic hTreg administration mitigates adverse heart remodeling by modulating the immune response, potentially facilitating transplantation of hESC-CPC with reduced immunosuppression requirements.

Exploring lncRNAs associated with human pancreatic islet cell death induced by transfer of adoptive lymphocytes in a humanized mouse model.

Long noncoding RNA (lncRNA)-mediated posttranscriptional and epigenetic landscapes of gene regulation are associated with numerous human diseases. However, the regulatory mechanisms governing human β-cell function and survival remain unknown. Owing to technical and ethical constraints, studying the direct role of lncRNAs in β-cell function and survival in humans in vivo is difficult. Therefore, we utilized humanized mice with human islets to investigate lncRNA expression using whole transcriptome shotgun sequencing. Our study aimed to characterize lncRNAs that may be crucial for human islet cell function and survival. Human β-cell death was induced in humanized mice engrafted with functional human islets. Using these humanized mice harboring human islets with induced β-cell death, we investigated lncRNA expression through whole transcriptome shotgun sequencing. Additionally, we systematically identified, characterized, and explored the regulatory functions of lncRNAs that are potentially important for human pancreatic islet cell function and survival. Human islet cell death was induced in humanized mice engrafted with functional human islets. RNA sequencing analysis of isolated human islets, islet grafts from humanized mice with and without induced cell death, revealed aberrant expression of a distinct set of lncRNAs that are associated with the deregulated mRNAs important for cellular processes and molecular pathways related to β-cell function and survival. A total of 10 lncRNA isoforms (SCYL1-1:22, POLG2-1:1, CTRB1-1:1, SRPK1-1:1, GTF3C5-1:1, PPY-1:1, CTRB1-1:5, CPA5-1:1, BCAR1-2:1, and CTRB1-1:4) were identified as highly enriched and specific to human islets. These lncRNAs were deregulated in human islets from donors with different BMIs and with type 2 diabetes (T2D), as well as in cultured human islets with glucose stimulation and induced cell death induced by cytokines. Aberrant expression of these lncRNAs was detected in the exosomes from the medium used to culture islets with cytokines. Islet-enriched and specific human lncRNAs are deregulated in human islet grafts and cultured human islets with induced cell death. These lncRNAs may be crucial for human β-cell function and survival and could have an impact on identifying biomarkers for β-cell loss and discovering novel therapeutic targets to enhance β-cell function and survival.

Bacterial ghost cell based bivalent candidate vaccine against Salmonella Typhi and Salmonella Paratyphi A: A prophylactic study in BALB/c mice

Typhoid and emerging paratyphoid fever are a severe enteric disease worldwide with high morbidity and mortality. Licensed typhoid vaccines are in the market, but no paratyphoid vaccine is currently available. In the present study we developed a bivalent vaccine against Salmonella Typhi and Paratyphi A using a bacterial ghost platform. Bacterial ghost cells (BGs) are bacteria-derived cell membranes without cytoplasmic contents that retain their cellular morphology, including all cell surface features. Furthermore, BGs have inherent adjuvant properties that promote an enhanced humoral and cellular immune reaction to the target antigen. Sodium hydroxide was used to prepare ghost cells of Salmonella Typhi and Paratyphi A. The bacterial ghost cells were characterised using electron microscopy. Then BALB/c mice were immunized three times (0th, 14th and 28th day) with the bivalent typhoidal bacterial ghost cells. Haematological study of adult mice throughout immunization period reflected that the immunogen was safe to administer and does not affect the animals’ health. After complete immunization, we found significant serum antibody titter against whole cell lysate, outer membrane protein and lipopolysaccharide of both bacteria, and cell-mediated immunity was observed in an ex-vivo experiment. CD4+, CD8a+ and CD19+ splenic cell populations were increased in immunized animals. Bivalent Typhoidal ghost cell immunized mice showed better survival, less bacterial colonization in systemic organs, and less inflammation and/or destruction of tissue in histopathological analysis than non-immunized control mice.Serum antibodies of immunized animals can significantly inhibit bacterial motility and mucin penetration ability with better killing properties against Salmonella Typhi and Paratyphi A. Furthermore, significant passive protection was observed through the adoptive transfer of serum antibody and lymphocytes of immunized animals to naïve animals after bacterial infection. In summary, Bivalent Typhoidal Bacterial Ghost cells (BTBGs) enhances immunogenic properties and serves as a safe and effective prevention strategy against Salmonella Typhi and Paratyphi A.

Using patient-derived tumor organoids from common epithelial cancers to analyze personalized T-cell responses to neoantigens

Adoptive cell transfer of tumor-infiltrating lymphocytes (TIL) can mediate durable complete responses in some patients with common epithelial cancers but does so infrequently. A better understanding of T-cell responses to neoantigens and tumor-related immune evasion mechanisms requires having the autologous tumor as a reagent. We investigated the ability of patient-derived tumor organoids (PDTO) to fulfill this need and evaluated their utility as a tool for selecting T-cells for adoptive cell therapy. PDTO established from metastases from patients with colorectal, breast, pancreatic, bile duct, esophageal, lung, and kidney cancers underwent whole exomic sequencing (WES), to define mutations. Organoids were then evaluated for recognition by autologous TIL or T-cells transduced with cloned T-cell receptors recognizing defined neoantigens. PDTO were also used to identify and clone TCRs from TIL targeting private neoantigens and define those tumor-specific targets. PDTO were successfully established in 38/47 attempts. 75% were available within 2 months, a timeframe compatible with screening TIL for clinical administration. These lines exhibited good genetic fidelity with their parental tumors, especially for mutations with higher clonality. Immunologic recognition assays demonstrated instances of HLA allelic loss not found by pan-HLA immunohistochemistry and in some cases WES of fresh tumor. PDTO could also be used to show differences between TCRs recognizing the same antigen and to find and clone TCRs recognizing private neoantigens. PDTO can detect tumor-specific defects blocking T-cell recognition and may have a role as a selection tool for TCRs and TIL used in adoptive cell therapy.

Neutrophils infiltrate sensory ganglia and mediate chronic widespread pain in fibromyalgia

Fibromyalgia is a debilitating widespread chronic pain syndrome that occurs in 2 to 4% of the population. The prevailing view that fibromyalgia results from central nervous system dysfunction has recently been challenged with data showing changes in peripheral nervous system activity. Using a mouse model of chronic widespread pain through hyperalgesic priming of muscle, we show that neutrophils invade sensory ganglia and confer mechanical hypersensitivity on recipient mice, while adoptive transfer of immunoglobulin, serum, lymphocytes, or monocytes has no effect on pain behavior. Neutrophil depletion abolishes the establishment of chronic widespread pain in mice. Neutrophils from patients with fibromyalgia also confer pain on mice. A link between neutrophil-derived mediators and peripheral nerve sensitization is already established. Our observations suggest approaches for targeting fibromyalgia pain via mechanisms that cause altered neutrophil activity and interactions with sensory neurons.

Abstract 4061: Systemic autologous lymphocyte transfer can enhance tumor-infiltrating lymphocyte infiltration in glioblastoma and license co-stimulatory immunotherapy

Abstract Glioblastoma is an immunologically ‘cold’ malignancy where few tumor infiltrating lymphocytes (TILs) are present. We sought to overcome this by ‘seeding’ the tumor with TILs via administration of autologous lymphocytes (ALT). We hypothesized that prior administration of ALT will enhance subsequent immunomodulatory therapies (e.g. immune checkpoint blockade (ICB) or co-stimulation). To evaluate this, we initially generated a pro-infiltrative TIL phenotype. CD45.1 splenocytes (C57/Bl6 background) were co-cultured with either IL2 and Concanavalin A (Con A) or IL7 and Con A for 5 days. Flow cytometric analysis found that both conditions resulted in similar CD4:CD8 ratios (20:80%) but IL7 co-culture upregulated expression of VLA-4, a pro migratory integrin. We then compared the entry of TILs into tumor bearing CNS when co-cultured with either IL2 or IL7. 8–10-week-old C57/Bl6 mice (n=5-6 per group) were implanted orthotopically with 30,000 CT2AvIII cells (syngeneic glioma) which established for 14 days. Mice received (1) CD45.1 lymphocytes activated with IL-7 and Con A (single intravenous (IV) injection, 1 × 107 cells) or (2) CD45.1 lymphocytes activated with IL2 and serial Con-A stimulation. Mice were sacrificed 3- and 48-hours following ALT and brains analysed for CD45.1+CD3+ populations. Groups were compared using a Mann-Whitney U test. Mice in Group 1 demonstrated significantly enhanced entry of CD8+ effector and memory T cells into tumor bearing hemispheres at both time points following administration compared to Group 2 (p = 0.005, p = 0.0159 respectively). By 48 hours post ALT, IL7 co-culture resulted in an average 810.1% expansion of CD8 T cell numbers in tumor bearing CNS compared to counts at Day 0. We then evaluated if mice with a TIL enriched CNS might be more responsive to ICB or co-stimulatory therapy. 8–10-week-old C57/Bl6 mice (n=5-7 per group) were implanted with 30,000 CT2AvIII cells that established over 10 days. On day 10, mice received IV ALT of either IL7 T cells only, or IL-7 and treatment courses of IP ICB (anti-PD1, anti-CTLA 4 or anti-41BB). Mice given ICB following IL7 ALT demonstrated a median survival (aPD1: 27 days, aCTLA-4: 25 days) similar to mice given IL7 ALT alone (25 days). However, 4/7 mice who received IL7 ALT and anti-41BB were still alive 45 days post tumor implantation (p=0.0004). Taken together, we demonstrated that certain ex vivo autologous lymphocyte co-culture conditions can generate a T cell population which infiltrates tumor bearing CNS in significant numbers. Further, we found that seeding a tumor with TILs prior to stimulatory therapy yields long term survival in vivo. Future work will focus on optimizing ex vivo culturing approaches for ALT and determine whether therapeutic efficacy is due to expansion of resident TILs at the tumor site, or ongoing recruitment of endogenous T cells from the periphery. Citation Format: Kirit Singh, Kelly M. Hotchkiss, Eliese Moelker, Gary E. Archer, John H. Sampson, Mustafa Khasraw. Systemic autologous lymphocyte transfer can enhance tumor-infiltrating lymphocyte infiltration in glioblastoma and license co-stimulatory immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4061.

Engineered Tcell therapy for viral and non-viral epithelial cancers.

Engineered Tcell therapy has shown remarkable efficacy in hematologic malignancies and has the potential for application to common epithelial cancers. Diverse Tcell therapy strategies including adoptive transfer of tumor-infiltrating lymphocytes, chimeric antigen receptor (CAR)-T cells, and Tcell receptor (TCR)-T cells have been studied in clinical trials. Recent research has established treatment of human papillomavirus (HPV)-associated cancers with TCR-T cells as a model for proof-of-principle studies in epithelial cancers. These studies and others have provided critical insight into mechanisms of tumor regression, therapeutic targets, treatment safety, treatment design, and barriers to curative cell therapies for common types of cancer. This perspective will review and consolidate understanding gained from clinical trials to treat viral and non-viral epithelial cancers with cell and gene therapy and will examine how past experience may guide future strategy in treatment and biomarker discovery.

Adoptive cell transfer therapy for melanoma.

Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) for melanoma is an example of the most successful cancer immune therapy. It achieves a durable complete response about ~20% of patients, and they might be cured. However, the ratio of patients with durable benefits is not high, and its complicated procedure prevents its diffusion. Therefore, many efforts to enhance the effect and simplify the protocol of TIL therapy have been made so far, resulting in the establishment of a simple and effective current TIL therapy that has been propagated to other institutes and countries. Moreover, TIL therapy and translational research using clinical samples derived from durable responders elucidate the important element for developing more effective cancer immune therapies in the future. This review introduced the brief history, attempts for the improvement and important findings elucidated by translational research of ACT for melanoma.

CTIM-31. IMMUNOLOGIC MONITORING AFTER ADOPTIVE CELL THERAPY IN PEDIATRIC PATIENTS WITH RECURRENT MEDULLOBLASTOMA USING IMMUNOGENOMICS APPROACH

Abstract BACKGROUND Adoptive cellular therapy (ACT) using transfer of tumor-specific lymphocytes has emerged as a potent strategy for treatment of advanced and refractory malignancies. We have employed the use of total tumor RNA (TTRNA)-pulsed DCs in the ex vivo expansion of tumor-specific lymphocytes for use in adoptive cellular therapy targeting pediatric malignant brain tumors. METHODS Total RNA was isolated from patient PBMC samples collected prior to adoptive cellular therapy and after 2-weeks and 4-weeks following immunotherapy treatment from 18 patients (Re-MATCH protocol, FDA IND BB-14058). We applied bulk RNA-Seq and high-throughput T cell receptor sequencing to monitor changes in blood draws after ACT. RESULTS We observed an increase of expression level of genes responsible for T cell activation in PBMC after adoptive cell therapy. We found that these genes were involved in T cell signaling pathways, proliferative signals, and cytokine production. The data revealed that certain T-cells were clonally expanded after ACT, with an increasing number of “hyper-expanded” TCR clones (comprising > 1% of all TCR beta or alpha sequences) after adoptive cellular therapy. These TCR clones established a new balance in the peripheral blood that was stable over the month following the completion of cellular immunotherapy. Hyper-expanded TCR clones and increased TCR diversity following ACT was associated with radiographic response to ACT and prolonged progression-free and overall survival. Limited TCR expansion and diversity following ACT was observed in patients with short overall and progression-free survival. CONCLUSIONS These results emphasize the importance of gene expression profiling to achieve higher resolution in monitoring immune responses in patients receiving immunotherapeutic treatment. Also, these findings support further study of the use of TCR sequencing to monitor responses to adoptive cellular therapy and suggest that TCR clonal expansion and increasing TCR diversity following treatment may be associated with positive clinical responses.

EXTH-98. ENHANCING T CELL TRAFFICKING OF CD3-ENGAGING IMMUNOTHERAPY TO TUMORS OF THE CENTRAL NERVOUS SYSTEM

Abstract BACKGROUND Effective immunotherapy against tumors of the central nervous system (CNS) requires that the drug the blood brain barrier (BBB) and encounter immune cells. We have previously described a mechanism which may facilitate transport of CD3-engaging therapeutics into the CNS, via carriage on activated T cells. Building on this, we sought to produce specific T cell phenotypes that rapidly enter and accumulate in the CNS. METHODS 8–10-week-old C57/Bl6 mice (n=5-6 per group) were implanted with 30,000 CT2AvIII cells which established over 14 days. Mice received (1) CD45.1 lymphocytes activated with IL-7 and Concanavalin-A (single intravenous (IV) injection, 1 x 107 adoptive lymphocyte transfer (ALT)) or (2) CD45.1 lymphocytes activated with IL-2 and serial Con-A stimulation. Mice were sacrificed 3 hours following ALT and their brains analysed via flow cytometry. A follow-up timepoint of 48 hours was also analysed. Groups were compared using a Mann-Whitney U test. RESULTS Ex vivo culture in group 1 yielded a mixed population of T effector and T central memory cells whereas group 2 resulted in terminally differentiated T effector memory cells only. VLA-4, a migratory integrin involved in T cell entry into the CNS was upregulated in the IL-7 group. Mice in Group 1 demonstrated significantly enhanced entry of CD8+ effector and memory T cells into tumor bearing hemispheres 3- and 48-hours following administration compared to Group 2 (p = 0.005, p = 0.0159 respectively). CONCLUSIONS Varying the cytokine cocktail used for ex vivo activation and expansion of T cells results in markedly different trafficking properties and phenotype composition. Ongoing work will determine how enhanced T cell localization to tumor affects accumulation of immunotherapy via the hitchhiking mechanism. Further, we will evaluate the safety of combinatorial CD3-engaging immunotherapy and ALT in Phase I trials (NCT04903795) using a cGMP Brain Bi-specific T cell engager (BRiTE).