Biology subjects

Ferreira, L. M. R.

Publications and source records attributed to Ferreira, L. M. R..

13 recordsLinked to original sources

bioRxiv2026

Acute and Lifelong Exercise Modulate the Tumorigenic Potential of Human Lung Cancer Cells and Their Susceptibility to Cisplatin

The association between higher levels of physical activity and lower cancer risk and mortality is well established. However, a causal link is yet to be proven. Recent studies showed a decrease in the proliferation rates of cultured human cancer cells when the human serum employed to stimulate them was conditioned by acute exercise. Here, we tested the hypothesis that serum mediates some of the putative benefits of exercise on cancer through alterations to the growth pattern and susceptibility to chemotherapy agents of cancer cells. To this end, human non-small cell lung cancer (NSCLC) cells were exposed to serum from two cohorts that differed significantly on their levels of physical activity and, accordingly, cardiorespiratory fitness, but were otherwise identical (master athletes and non-exercisers), collected before and after an acute exercise intervention. Serum levels of glucose, lipids, albumin, C-reactive protein and cytokines were determined and the impact of the serum responses to acute and lifelong exercise on the above-mentioned parameters were analyzed. We found that acute exercise decreased the cells proliferation rate, yet shortened the cells lag phase after detachment, whereas lifelong exercise had the opposite effects. Significantly, we showed, for the first time, that lifelong exercise increased susceptibility to a chemotherapy agent (cisplatin), which may contribute to the decreased cancer mortality rates found among those who exercise regularly. Similar to the cellular effects, changes to serum cytokine levels - several of them linked to the senescence-associated secretory phenotype - depended on whether serum was conditioned by acute or by chronic exercise. Key pointsChronic exercise increased the in vitro susceptibility of lung cancer cells to cisplatin. Acute and chronic exercise modulated the in vitro tumorigenic potential of lung cancer cells. Effects were mediated by serological changes produced by exercise. Acute and chronic exercise had distinct impacts on serological cytokine levels.

cancer biology↗

bioRxiv2026

MUC4-targeted CAR-T Cells Restrain Chemotherapy-resistant Colorectal Cancer

Colorectal cancer (CRC) is the leading cause of cancer mortality in young adults, with chemoresistance and metastatic progression severely limiting treatment options. MUC4, a membrane-bound mucin normally confined to the apical surface of epithelial cells, becomes aberrantly overexpressed across the tumor cell membrane in CRC and other cancer types, promoting cancer survival, immune evasion, and metastasis. Therefore, MUC4 is a promising candidate for the development of targeted therapies. We detected MUC4 cell surface expression in a panel of human CRC cell lines. We engineered MUC4-specific chimeric antigen receptor (CAR) T cells and evaluated their activity against methotrexate-resistant MUC4-expressing human CRC cell lines HT29-MTX and T84. MUC4 CAR-T cells efficiently eliminated HT29-MTX and T84 cells in vitro and delayed HT29-MTX subcutaneous tumor growth in vivo. Importantly, MUC4 CAR-T cell treatment significantly reduced tumor burden and improved survival in a highly aggressive and lethal CRC model, intraperitoneal HT29-MTX CRC metastatic dissemination in NSG mice. Mouse necropsies revealed no off-target in vivo toxicities associated with MUC4 CAR-T cell therapy. Altogether, our findings establish MUC4 as an important and clinically relevant CAR-T cell target and demonstrate therapeutic efficacy of MUC4 CAR-T cell therapy for invasive CRC, a setting where chemotherapy typically fails. MUC4-targeted CAR-T cell therapy has the potential to fill a critical treatment gap for patients with chemoresistant and invasive CRC and may extend to other MUC4-expressing solid tumors. Statement of PriorityColorectal cancer is increasingly diagnosed in younger individuals, a trend accompanied by a higher prevalence of aggressive and chemotherapy-resistant disease. Despite advances in systemic therapy, outcomes for patients with refractory or metastatic colorectal cancer remain poor, underscoring a critical unmet clinical need. CAR-T cell therapy offers a mechanism to selectively target tumor cells that evade conventional treatments and may thus be particularly valuable in mucinous, chemotherapy-resistant subsets. Developing effective CAR-T strategies for new targets in colorectal carcinoma, such as MUC4, is an urgent priority in this era of rising early-onset disease.

immunology↗

bioRxiv2025

Hypoimmunogenic hPSC-derived cardiac organoids for immune evasion and heart repair

Human pluripotent stem cell (hPSC)-derived cardiac therapies hold great promise for heart regeneration but face major translational barriers due to allogeneic immune rejection. Here, we engineered hypoimmunogenic hPSCs using a two-step CRISPR-Cas9 strategy: (1) B2M knockout, eliminating HLA class I surface expression, and (2) knock-in of HLA-E or HLA-G trimer constructs in the AAVS1 safe harbor locus to confer robust immune evasion. Hypoimmunogenic hPSCs maintained pluripotency, efficiently differentiated into cardiac cell types that resisted both T and NK cell-mediated cytotoxicity in vitro, and self-assembled into engineered cardiac organoids. Comprehensive analyses of the hypoimmunogenic cells and organoids revealed preservation of transcriptomic, structural, and functional properties with minimal off-target effects from gene editing. In vivo, hypoimmunogenic cardiac organoids restored contractile function in infarcted rat hearts and demonstrated superior graft retention and immune evasion in humanized mice compared to wild-type counterparts. These findings establish the therapeutic potential of hypoimmunogenic hPSC-CMs in the cardiac organoid platform, laying the foundation for off-the-shelf cardiac cell therapies to treat cardiovascular disease, the leading cause of death worldwide.

bioengineering↗

bioRxiv2025

Chimeric anti-HLA antibody receptor engineered human regulatory T cells suppress alloantigen-specific B cells from pre-sensitized transplant recipients

Organ transplantation is a lifesaving procedure, with 50,000 transplants happening every year in the United States. However, many patients harbor antibodies and B cells directed against allogeneic human leukocyte antigen (HLA) molecules, notably HLA-A2, greatly decreasing their likelihood of receiving a compatible organ. Moreover, antibody-mediated rejection is a significant contributor to chronic transplant rejection. Current strategies to desensitize patients non- specifically target circulating antibodies and B cells, resulting in poor efficacy and complications. Regulatory T cells (Tregs) are immune cells dedicated to suppressing specific immune responses by interacting with both innate and adaptive immune cells. Here, we genetically modified human Tregs with a chimeric anti-HLA antibody receptor (CHAR) consisting of an extracellular HLA-A2 protein fused to a CD28-CD3zeta intracellular signaling domain, driving Treg activation upon recognition of anti-HLA-A2 antibodies on the surface of alloreactive B cells. We find that HLA-A2 CHAR Tregs get activated specifically by anti-HLA-A2 antibody-producing cells. Of note, HLA-A2 CHAR activation does not negatively affect Treg stability, as measured by expression of the Treg lineage transcription factors FOXP3 and HELIOS. Interestingly, HLA-A2 CHAR Tregs are not cytotoxic towards anti-HLA-A2 antibody-producing cells, unlike HLA-A2 CHAR modified conventional CD4+ T cells. Importantly, HLA-A2 CHAR Tregs recognize and significantly suppress high affinity IgG antibody production by B cells from HLA-A2 sensitized patients. Altogether, our results provide proof-of-concept of a new strategy to specifically inhibit alloreactive B cells to desensitize transplant recipients.

immunology↗

bioRxiv2024

Scalable intracellular delivery via microfluidic vortex shedding enhances the function of chimeric antigen receptor T-cells

Adoptive chimeric antigen receptor T-cell (CAR-T) therapy is transformative and approved for hematologic malignancies. It is also being developed for the treatment of solid tumors, autoimmune disorders, heart disease, and aging. Despite unprecedented clinical outcomes, CAR-T and other engineered cell therapies face a variety of manufacturing and safety challenges. Traditional methods, such as lentivirus transduction and electroporation, result in random integration or cause significant cellular damage, which can limit the safety and efficacy of engineered cell therapies. We present hydroporation as a gentle and effective alternative for intracellular delivery. Hydroporation resulted in 1.7- to 2-fold higher CAR-T yields compared to electroporation with superior cell viability and recovery. Hydroporated cells exhibited rapid proliferation, robust target cell lysis, and increased pro-inflammatory and regulatory cytokine secretion in addition to improved CAR-T yield by day 5 post-transfection. We demonstrate that scaled-up hydroporation can process 5 x 108 cells in less than 10 s, showcasing the platform as a viable solution for high-yield CAR-T manufacturing with the potential for improved therapeutic outcomes.

bioengineering↗

bioRxiv2024

High affinity chimeric antigen receptor signaling induces an inflammatory program in human regulatory T cells

Regulatory T cells (Tregs) are promising cellular therapies to induce immune tolerance in organ transplantation and autoimmune disease. The success of chimeric antigen receptor (CAR) T-cell therapy for cancer has sparked interest in using CARs to generate antigen-specific Tregs. Here, we compared CAR with endogenous T cell receptor (TCR)/CD28 activation in human Tregs. Strikingly, CAR Tregs displayed increased cytotoxicity and diminished suppression of antigen-presenting cells and effector T (Teff) cells compared with TCR/CD28 activated Tregs. RNA sequencing revealed that CAR Tregs activate Teff cell gene programs. Indeed, CAR Tregs secreted high levels of inflammatory cytokines, with a subset of FOXP3+ CAR Tregs uniquely acquiring CD40L surface expression and producing IFN{gamma}. Interestingly, decreasing CAR antigen affinity reduced Teff cell gene expression and inflammatory cytokine production by CAR Tregs. Our findings showcase the impact of engineered receptor activation on Treg biology and support tailoring CAR constructs to Tregs for maximal therapeutic efficacy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=54 SRC="FIGDIR/small/587467v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@11e5bd8org.highwire.dtl.DTLVardef@1fe86forg.highwire.dtl.DTLVardef@d4eb2borg.highwire.dtl.DTLVardef@9b89d1_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Precision engineering of an anti-HLA-A2 chimeric antigen receptor in regulatory T cells for transplant immune tolerance

Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-{zeta} signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR+TCRdeficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR+TCRdeficient Tregs did not impair the function of these HLA-A2+ islets, whereas similarly engineered A2-CAR+TCRdeficientCD4+ conventional T cells rejected the islets in less than 2 weeks. A2-CAR+TCRdeficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.

immunology↗

bioRxiv2021

TNFa and IL-6 promote ex-vivo proliferation of lineage-committed human regulatory T cells

Treg therapy is being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs is unclear. In this study, we challenged human Tregs ex-vivo with pro-inflammatory cytokines, TNF and IL-6. These cytokines enhanced Treg proliferation induced by anti-CD3 and anti-CD28 or CD28 superagonist (CD28SA) while maintaining high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low expression of cytokines IFNg, IL-4 and IL-17. Blocking TNF receptor signaling using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression, revealing the importance of TNFR2 signaling in Treg proliferation and lineage stability. The robust proliferation induced by CD28SA with IL-6 and TNF may be adopted for the expansion of therapeutic Tregs. Metabolomics analysis showed that Tregs expanded with CD28SA plus cytokines had more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential. Finally, CD28SA plus cytokine-expanded Tregs had comparable suppressive activity in vitro and in vivo in a humanized mouse model of graft-versus-host-disease when compared to Tregs expanded using the conventional protocol. These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function.

immunology↗

bioRxiv2020

The CD28-transmembrane domain mediates chimeric antigen receptor heterodimerization with CD28

Anti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge (HD) and transmembrane (TMD) domains between the extracellular antigen-targeting and the intracellular signaling modalities of CARs has not been systemically studied. Here, a series of CD19-CARs differing only by their HD (CD8/CD28/IgG4) and TMD (CD8/CD28) was generated. CARs containing a CD28-TMD, but not a CD8-TMD, formed heterodimers with the endogenous CD28 in human T cells, as shown by co-immunoprecipitation and CAR-dependent proliferation to anti-CD28 stimulation. This dimerization depended on polar amino-acids in the CD28-TMD. CD28-CAR heterodimerization was more efficient in CARs containing a CD8-HD or CD28-HD as compared to an IgG4-HD. CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but led to a significant reduction of CD28 cell-surface expression. These data unveil a new property of the CD28-TMD and suggest that TMDs can modulate CAR T-cell activities by engaging endogenous partners. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/296913v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@bcd17aorg.highwire.dtl.DTLVardef@5cbca9org.highwire.dtl.DTLVardef@660eecorg.highwire.dtl.DTLVardef@5db163_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Peptide Antidotes to SARS-CoV-2 (COVID-19)

The design of an immunogenic scaffold that serves a role in treating a pathogen, and can be rapidly and predictively modeled, has remained an elusive feat. Here, we demonstrate that SARS-BLOCK synthetic peptide scaffolds act as antidotes to SARS-CoV-2 spike protein-mediated infection of human ACE2-expressing cells. Critically, SARS-BLOCK peptides are able to potently and competitively inhibit SARS-CoV-2 S1 spike protein receptor binding domain (RBD) binding to ACE2, the main cellular entry pathway for SARS-CoV-2, while also binding to neutralizing antibodies against SARS-CoV-2. In order to create this potential therapeutic antidote-vaccine, we designed, simulated, synthesized, modeled epitopes, predicted peptide folding, and characterized behavior of a novel set of synthetic peptides. The biomimetic technology is modeled off the receptor binding motif of the SARS-CoV-2 coronavirus, and modified to provide enhanced stability and folding versus the truncated wildtype sequence. These novel peptides attain single-micromolar binding affinities for ACE2 and a neutralizing antibody against the SARS-CoV-2 receptor binding domain (RBD), and demonstrate significant reduction of infection in nanomolar doses. We also demonstrate that soluble ACE2 abrogates binding of RBD to neutralizing antibodies, which we posit is an essential immune-evasive mechanism of the virus. SARS-BLOCK is designed to "uncloak" the viral ACE2 coating mechanism, while also binding to neutralizing antibodies with the intention of stimulating a specific neutralizing antibody response. Our peptide scaffolds demonstrate promise for future studies evaluating specificity and sensitivity of immune responses to our antidote-vaccine. In summary, SARS-BLOCK peptides are a promising COVID-19 antidote designed to combine the benefits of a therapeutic and vaccine, effectively creating a new generation of prophylactic and reactive antiviral therapeutics whereby immune responses can be enhanced rather than blunted.

bioengineering↗

bioRxiv2020

Immunodietica: interrogating the role of diet in autoimmune disease

Diet is an environmental factor in autoimmune disorders, where the immune system erroneously destroys ones own tissues. Yet, interactions between diet and autoimmunity remain largely unexplored, particularly the impact of immunogenetics, ones human leukocyte antigen (HLA) allele make-up, in this interplay. Here, we interrogated animals and plants for the presence of epitopes implicated in human autoimmune diseases. We mapped autoimmune epitope distribution across organisms and determined their tissue expression pattern. Interestingly, diet-derived epitopes implicated in a disease were more likely to bind to HLA alleles associated with that disease than to protective alleles, with visible differences between organisms with similar autoimmune epitope content. We then analyzed an individuals HLA haplotype, generating a personalized heatmap of potential dietary autoimmune triggers. Our work uncovered differences in autoimmunogenic potential across food sources and revealed differential binding of diet-derived epitopes to autoimmune disease-associated HLA alleles, shedding light on the impact of diet on autoimmunity.

immunology↗

bioRxiv2019

Immunodietica: A data-driven approach to investigate interactions between diet and autoimmune disorders

Autoimmunity is on the rise around the globe. Diet has been proposed as a risk factor for autoimmunity and shown to modulate the severity of several autoimmune disorders. Yet, the interaction between diet and autoimmunity in humans remains largely unstudied. Here, we systematically interrogated commonly consumed animals and plants for peptide epitopes previously implicated in human autoimmune disease. A total of fourteen species investigated could be divided into three broad categories regarding their content in human autoimmune epitopes, which we represented using a new metric, the Gershteyn-Ferreira index (GF index). Strikingly, pig contains a disproportionately high number of unique autoimmune epitopes compared to all other species analyzed. This work uncovers a potential new link between pork consumption and autoimmunity in humans and lays the foundation for future studies on the impact of diet on the pathogenesis and progression of autoimmune disorders.

immunology↗

bioRxiv2019

Perceptions and Prospects in Life Sciences in a Heterogenous Latin American Population

Particular challenges exist for science education in the developing world, where limited resources beget curricula designed to balance state-of-the-art knowledge with practical and political considerations in region-specific contexts. Project-based biology teaching is particularly difficult to execute due to high infrastructural costs and limited teacher training. Here, we report our results implementing short, challenging, and low-cost biology courses to high school and college students in Bolivia, designed and taught in collaboration between scientists from developed nations and local science instructors. We find our approach to be effective at transmitting advanced topics in disease modeling, microscopy, genome engineering, neuroscience, microbiology, and regenerative biology. Importantly, this approach was unaffected by the students backgrounds, education level, socioeconomic status, or initial interest in the course, and increased participants interest in pursuing scientific careers. These results demonstrate efficacy of participatory learning in a developing nation, and suggest that such techniques could drive scientific engagement in other developing economies.

scientific communication and education↗