Hypoimmune anti-CD19 chimeric antigen receptor T cells provide lasting tumor control in fully immunocompetent allogeneic humanized mice.

Manufacturing autologous chimeric antigen receptor (CAR) T cell therapeutics is complex, and many patients experience treatment delays or cannot be treated at all. Although current allogeneic CAR products have the potential to overcome manufacturing bottlenecks, they are subject to immune rejection and failure to persist in the host, and thus do not provide the same level of efficacy as their autologous counterparts. Here, we aimed to develop universal allogeneic CAR T cells that evade the immune system and produce a durable response. We generated human hypoimmune (HIP) T cells with disrupted B2M, CIITA, and TRAC genes using CRISPR-Cas9 editing. In addition, CD47 and anti-CD19 CAR were expressed using lentiviral transduction. These allogeneic HIP CD19 CAR T cells were compared to allogeneic CD19 CAR T cells that only expressed the anti-CD19 CAR (allo CAR T). In vitro assays for cancer killing and exhaustion revealed no differences between allo CAR T and HIP CAR T cells, confirming that the HIP edits did not negatively affect T cell performance. Clearance of CD19+ tumors by HIP CAR T cells in immunodeficient NSG mice was comparable to that of allo CAR T cells. In fully immunocompetent humanized mice, HIP CAR T cells significantly outperformed allo CAR T cells, showed improved persistence and expansion, and provided lasting cancer clearance. Furthermore, CD47-targeting safety strategies reliably and specifically eliminated HIP CAR T cells. These findings suggest that universal allogeneic HIP CAR T cell-based therapeutics might overcome the limitations associated with poor persistence of allogeneic CAR T cells and exert durable anti-tumor responses.

SARS-CoV-2 transmission in classroom settings: Effects of mitigation, age, and Delta variant.

Traditional, in-person classroom settings have been limited during the COVID-19 pandemic due to their potential to transmit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among students, teachers, and other educational workers. Using computational fluid dynamics simulations, mitigation strategies that span approaches using face coverings, various ventilation schemes, air purifiers/cleaners, and desk shields are systematically evaluated in thermally controlled classrooms. Individually, face coverings and source control were the most effective, which was followed by well-designed ventilation systems. The use of desk shields was also studied and appeared to be ineffective. The best mitigation approach is shown to be through multiple measures-using face coverings and ventilation systems combined with air purifiers. The studies were extended to elementary schools and consider Delta variants of SARS-CoV-2. In elementary settings, the reduced pulmonary and viral emission rates of small children are observed to drive reduced transmission rates, to values even lower than those observed with several mitigation methods for classrooms with adults. The Delta variant, with adults, was evaluated by considering an increase in quanta and indicated higher transmission probabilities. These increases are levels that are controllable by increasing the mitigation methods. Results indicate several plans of action for schools to return to in-person schooling in the context of age and new variants.

Stopping the COVID-19 pandemic in dental offices: A review of SARS-CoV-2 transmission and cross-infection prevention.

Due to the essential role of dentists in stopping the COVID-19 pandemic, the purpose of this review is to help dentists to detect any weaknesses in their disinfection and cross-contamination prevention protocols, and to triage dental treatments to meet the needs of patients during the pandemic. We used PRISMA to identify peer-reviewed publications which supplemented guidance from the center for disease control about infection control and guidelines for dentists. Dentists must triage dental treatments to meet the needs of patients during the pandemic. The ongoing pandemic has changed the practice of dentistry forever, the changes make it more cumbersome, time-consuming, and costly due to the possible pathways of transmission and mitigation steps needed to prevent the spread of COVID-19. Dental chairside rapid tests for SARS-CoV-2 are urgently needed. Until then, dentists need to screen patients for COVID-19 even though 75% of people with COVID-19 have no symptoms. Despite the widespread anxiety and fear of the devastating health effects of COVID-19, only 61% of dentists have implemented a change to their treatment protocols. As an urgent matter of public health, all dentists must identify the additional steps they can take to prevent the spread of COVID-19. The most effective steps to stop the pandemic in dental offices are to; vaccinate all dentists, staff, and patients; triage dental treatments for patients, separate vulnerable patients, separate COVID-19 patients, prevent cross-contamination, disinfect areas touched by patients, maintain social distancing, and change personal protective equipment between patients.

Estimating COVID-19 exposure in a classroom setting: A comparison between mathematical and numerical models.

The COVID-19 pandemic has driven numerous studies of airborne-driven transmission risk primarily through two methods: Wells-Riley and computational fluid dynamics (CFD) models. This effort provides a detailed comparison of the two methods for a classroom scenario with masked habitants and various ventilation conditions. The results of the studies concluded that (1) the Wells-Riley model agrees with CFD results without forced ventilation (6% error); (2) for the forced ventilation cases, there was a significantly higher error (29% error); (3) ventilation with moderate filtration is shown to significantly reduce infection transmission probability in the context of a classroom scenario; (4) for both cases, there was a significant amount of variation in individual transmission route infection probabilities (up to 220%), local air patterns were the main contributor driving the variation, and the separation distance from infected to susceptible was the secondary contributor; (5) masks are shown to have benefits from interacting with the thermal plume created from natural convection induced from body heat, which pushes aerosols vertically away from adjacent students.

Engineering Tolerance toward Allogeneic CAR-T Cells by Regulation of MHC Surface Expression with Human Herpes Virus-8 Proteins.

Allogeneic, off-the-shelf (OTS) chimeric antigen receptor (CAR) cell therapies have the potential to reduce manufacturing costs and variability while providing broader accessibility to cancer patients and those with other diseases. However, host-versus-graft reactivity can limit the durability and efficacy of OTS cell therapies requiring new strategies to evade adaptive and innate-immune responses. Human herpes virus-8 (HHV8) maintains infection, in part, by evading host T and natural killer (NK) cell attack. The viral K3 gene encodes a membrane-tethered E3 ubiquitin ligase that discretely targets major histocompatibility complex (MHC) class I components, whereas K5 encodes a similar E3 ligase with broader specificity, including MHC-II and the MHC-like MHC class I polypeptide-related sequence A (MIC-A)- and sequence B (MIC-B)-activating ligands of NK cells. We created γ-retroviruses encoding K3 and/or K5 transgenes that efficiently transduce primary human T cells. Expression of K3 or K5 resulted in dramatic downregulation of MHC-IA (human leukocyte antigen [HLA]-A, -B, and -C) and MHC class II (HLA-DR) cell-surface expression. K3 expression was sufficient for T cells to resist exogenously loaded peptide-MHC-specific cytotoxicity, as well as recognition in one-way allogeneic mixed lymphocyte reactions. Further, in immunodeficient mice engrafted with allogeneic T cells, K3-transduced T cells selectively expanded in vivo. Ectopic K5 expression in MHC class I-, MIC-A+/B+ K562 cells also reduced targeting by primary NK cells. Coexpression of K3 in prostate stem cell antigen (PSCA)-directed, inducible MyD88/CD40 (iMC)-enhanced CAR-T cells did not impact cytotoxicity, T cell growth, or cytokine production against HPAC pancreatic tumor target cells, whereas K5-expressing cells showed a modest reduction in interleukin (IL)-2 production without effect on cytotoxicity. Together, these results support application of these E3 ligases to advance development of OTS CAR-T cell products.

Ten-year survival rate of 89% after distal femoral osteotomy surgery for lateral compartment osteoarthritis of the knee.

PURPOSE: The purpose of this study was to assess the accuracy, safety, and survival of distal femoral osteotomy (DFO) surgery for lateral compartment OA of the knee. METHODS: A retrospective cohort study was conducted at a single UK centre, using prospectively collected data over an 8-year period (2009-2017). All patients had pre-operative radiographic analysis and digital planning of their deformity correction in addition to post-operative analysis of the achieved correction and yearly face-to-face follow-up. Complications (defined as an undesirable medical or surgical event as a direct result of the operation), reoperations, and failure (defined as conversion to arthroplasty or revision) were recorded. RESULTS: From a total of 83 patients, 81 patients undergoing 86 primary DFOs were included in this study, with a mean follow-up of 99 months (SD 27 months). The mean pre-operative percentage Mikulicz point was 78.7% (SD 19.1%) and post-operative 35.9% (SD 14.8%). The mean accuracy of correction (intended correction - achieved correction) was an 8.2% overcorrection (SD 13.7%). The complication rate was 4.7%. Using Kaplan-Meier analysis, the mean survival was 113 months (95% CI 106-120) with the probability of surviving 10 years 89%. CONCLUSION: DFO for valgus alignment and lateral compartment arthritis is associated with low complications, long-term joint preservation, and the prevention of arthroplasty surgery. However, the accuracy of correction still requires improvement in intra-operative technique. LEVEL OF EVIDENCE: IV.

Quasi-stiffness of the knee joint is influenced by walking on a destabilising terrain.

BACKGROUND: Predictive models have been devised to estimate the necessary quasi-stiffness that a transfemoral prosthesis should be set to aligning the body and gait parameters of the user. Current recommendations exist only for walking over level ground. This study aimed to ascertain whether walking across destabilising terrain influences the quasi-stiffness of the knee joint thus influencing prosthetic engineering. METHODS: Ten healthy males (age: 25.1 ± 2.5 years; mean ± sd, height: 1.78 ± 0.05 m, weight: 84.40 ± 11.02 kg) performed 14 gait trials. Seven trials were conducted over even ground and seven over 20 mm ballast. Three-dimensional motion capture and ground reaction force were collected. Paired samples t-tests and Wilcoxon signed ranked test compared variables including; quasi-stiffness, gait speed, stride length and stride width. RESULTS: Quasi-stiffness (d = 0.562, P = 0.001) and stride width (d = 0.909, P < 0.001) were significantly greater in the destabilising terrain condition. Gait speed (r = -0.731, P = 0.001) was significantly greater in the control condition. No significant difference was seen in stride length (d = 0.583, P = 0.016). CONCLUSIONS: An increase in quasi-stiffness when walking across destabilising terrain was attributed to a magnified shock absorption mechanism, facilitating an increased flexion angle during the stance phase. This causes a lower centre of mass resulting in the musculoskeletal system having to produce a greater knee extensor moment to prevent the knee collapsing. Therefore, transfemoral prostheses should be tuned to apply increased extension moments if ambulation is to occur on a destabilising terrain.

Inducible MyD88/CD40 synergizes with IL-15 to enhance antitumor efficacy of CAR-NK cells.

Natural killer (NK) cells expressing chimeric antigen receptors (CARs) are a promising anticancer immunotherapy, leveraging both innate NK cell antitumor activity and target-specific cytotoxicity. Inducible MyD88/CD40 (iMC) is a potent, rimiducid-regulated protein switch that has been deployed previously as a T-cell activator to enhance proliferation and persistence of CAR-modified T cells. In this study, iMC was extended to CAR-NK cells to enhance their growth and augment cytotoxicity against tumor cells. iMC-activated NK cells substantially increased cytokine and chemokine secretion and displayed higher levels of perforin and granzyme B degranulation. In addition, iMC activation could be coupled with ectopic interleukin-15 (IL-15) to further enhance NK cell proliferation. When coexpressed with a target-specific CAR (CD123 or BCMA), this IL-15/iMC system showed further augmented antitumor activity through enhanced CAR-NK cell expansion and cytolytic activity. To protect against potential toxicity from engineered NK cells, an orthogonal rapamycin-regulated Caspase-9 (iRC9) was included in a 4-gene, dual-switch platform. After infusion of dual-switch NK cells, pharmacologic iRC9 dimerization led to rapid elimination of a majority of expanded transduced NK cells. Thus, CAR-NK cells utilizing dual molecular switches provide an innovative and effective approach to cancer immunotherapy with controlled specificity, efficacy, and safety.

Correction to: Paediatric proximal ACL tears managed with direct ACL repair is safe, effective and has excellent short-term outcomes.

Authors would like to update the full author names.

Paediatric proximal ACL tears managed with direct ACL repair is safe, effective and has excellent short-term outcomes.

PURPOSE: Anterior cruciate ligament (ACL) surgery in the paediatric population has long been a challenge. Non-operative treatment will result in persistent instability which can lead to chondral and meniscal injuries. The results of primary open ACL repair are poor. Concerns of growth plate disturbance with transphyseal techniques and issues with relatively small-diameter grafts in Tanner 1 and 2 patients, which are inadequate, have contributed to these challenges. With advancing instrumentation, there is renewed interest in ACL repair. The minimally invasive approach of arthroscopic primary ACL repair retains the native ligament. The objective and subjective outcomes at 2 years are presented. METHODS: Paediatric patients, less than 16 years of age, presenting acutely with complete proximal ACL ruptures underwent direct arthroscopic ACL repair, reinforced by a temporary internal brace, which was subsequently removed after 3 months. Patient-reported outcome measures including the Lysholm, Tegner and KOOS scores were collected at 6 months, 1 year and 2 years post-operatively. RESULTS: Twenty patients (age 6-16) completed data at 2 years post-operatively. There were no failures, no complications and no growth disturbance out to 2 years. The 2-year postoperative outcomes; Lysholm 95 (90-100), Tegner 7 (6-10), KOOS-Child 96.5 (88.9-100) demonstrated statistically significant improvements following surgery (p < 0.001). Objective measurements with an accelerometer did not demonstrate any significant side-to-side difference. CONCLUSION: ACL repair for proximal ACL tears in the paediatric population demonstrates the potential for excellent outcomes at short-term follow-up. This presents an attractive alternative to ACL reconstruction when an adequate ACL remnant permits direct repair. Our results demonstrate that paediatric ACL repair is safe and effective.

Streamlined production of genetically modified T cells with activation, transduction and expansion in closed-system G-Rex bioreactors.

BACKGROUND: Gas Permeable Rapid Expansion (G-Rex) bioreactors have been shown to efficiently expand immune cells intended for therapeutic use, but do not address the complexity of the viral transduction step required for many engineered T-cell products. Here we demonstrate a novel method for transduction of activated T cells with Vectofusin-1 reagent. Transduction is accomplished in suspension, in G-Rex bioreactors. The simplified transduction step is integrated into a streamlined process that uses a single bioreactor with limited operator intervention. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy donors were thawed, washed and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags or in G-Rex bioreactors. Cells were cultured in TexMACS GMP medium with interleukin (IL)-7 and IL-15 and transduced with RetroNectin in bags or Vectorfusin-1 in the G-Rex. Total viable cell number, fold expansion, viability, transduction efficiency, phenotype and function were compared between the two processes. RESULTS: The simplified process uses a single vessel from activation through harvest and achieves 56% transduction with 29-fold expansion in 11 days. The cells generated in the simplified process do not differ from cells produced in the conventional bag-based process functionally or phenotypically. DISCUSSION: This study demonstrates that T cells can be transduced in suspension. Further, the conventional method of generating engineered T cells in bags for clinical use can be streamlined to a much simpler, less-expensive process without compromising the quality or function of the cell product.

Constitutively active MyD88/CD40 costimulation enhances expansion and efficacy of chimeric antigen receptor T cells targeting hematological malignancies.

Successful adoptive chimeric antigen receptor (CAR) T-cell therapies against hematological malignancies require CAR-T expansion and durable persistence following infusion. Balancing increased CAR-T potency with safety, including severe cytokine-release syndrome (sCRS) and neurotoxicity, warrants inclusion of safety mechanisms to control in vivo CAR-T activity. Here, we describe a novel CAR-T cell platform that utilizes expression of the toll-like receptor (TLR) adaptor molecule, MyD88, and tumor-necrosis factor family member, CD40 (MC), tethered to the CAR molecule through an intentionally inefficient 2A linker system, providing a constitutive signal that drives CAR-T survival, proliferation, and antitumor activity against CD19+ and CD123+ hematological cancers. Robust activity of MC-enhanced CAR-T cells was associated with cachexia in animal models that corresponded with high levels of human cytokine production. However, toxicity could be successfully resolved by using the inducible caspase-9 (iC9) safety switch to reduce serum cytokines, by administration of a neutralizing antibody against TNF-α, or by selecting "low" cytokine-producing CD8+ T cells, without loss of antitumor activity. Interestingly, high basal activity was essential for in vivo CAR-T expansion. This study shows that co-opting novel signaling elements (i.e., MyD88 and CD40) and development of a unique CAR-T architecture can drive T-cell proliferation in vivo to enhance CAR-T therapies.

Two-Dimensional Regulation of CAR-T Cell Therapy with Orthogonal Switches.

Use of chimeric antigen receptors (CARs) as the basis of targeted adoptive T cell therapies has enabled dramatic efficacy against multiple hematopoietic malignancies, but potency against bulky and solid tumors has lagged, potentially due to insufficient CAR-T cell expansion and persistence. To improve CAR-T cell efficacy, we utilized a potent activation switch based on rimiducid-inducible MyD88 and CD40 (iMC)-signaling elements. To offset potential toxicity risks by this enhanced CAR, an orthogonally regulated, rapamycin-induced, caspase-9-based safety switch (iRC9) was developed to allow in vivo elimination of CAR-T cells. iMC costimulation induced by systemic rimiducid administration enhanced CAR-T cell proliferation, cytokine secretion, and antitumor efficacy in both in vitro assays and xenograft tumor models. Conversely, rapamycin-mediated iRC9 dimerization rapidly induced apoptosis in a dose-dependent fashion as an approach to mitigate therapy-related toxicity. This novel, regulatable dual-switch system may promote greater CAR-T cell expansion and prolonged persistence in a drug-dependent manner while providing a safety switch to mitigate toxicity concerns.

Non-Identical Bilateral Rupture of the Extensor Mechanism of the Knee in a Patient with Hyperlipidemia: A Case Study.

INTRODUCTION: Rupture of the extensor mechanism is a relatively common injury, most frequently occurring as a result of patella fracture, while ruptures of the quadriceps tendon and patellar ligament are less common. Extensor mechanisms of healthy knees are able to tolerate large forces before rupturing; therefore, complete ruptures without significant trauma are due to minor injury to an already degenerate or attenuated tendon. Hyperlipidemia has been linked as a cause of tendon degeneration due to the systemic biological effect that it has on tenocytes. Non-identical bilateral ruptures are rare. To the best of our knowledge, this is the only case report of bilateral ruptures involving the quadriceps tendon of one knee and patellar ligament of the contralateral knee simultaneously. CASE REPORT: A 42-year-old man presented to our department with bilateral traumatic rupture of the extensor mechanism of the knee. He had no medical history, was not taking any regular medications, and had no significant family history but a 15-year history of anterior knee pain. Both of his legs gave way on landing from a jump. Radiographs demonstrated a knee effusion with normal patella height on the left and a knee effusion with an elevated patella on the right. A diagnosis of quadriceps tendon rupture on the left and patellar ligament rupture on the right was made. CONCLUSION: Hyperlipidemia has been associated with ruptures of the Achilles tendon but has not been reported in association with failure of the extensor mechanism of the knee. We suggest that all patients presenting with bilateral tendon ruptures, especially in the absence of systemic disease or corticosteroid therapy, are investigated for hyperlipidemia and treated accordingly.

Regulated Expansion and Survival of Chimeric Antigen Receptor-Modified T Cells Using Small Molecule-Dependent Inducible MyD88/CD40.

Anti-tumor efficacy of T cells engineered to express chimeric antigen receptors (CARs) is dependent on their specificity, survival, and in vivo expansion following adoptive transfer. Toll-like receptor (TLR) and CD40 signaling in T cells can improve persistence and drive proliferation of antigen-specific CD4+ and CD8+ T cells following pathogen challenge or in graft-versus-host disease (GvHD) settings, suggesting that these costimulatory pathways may be co-opted to improve CAR-T cell persistence and function. Here, we present a novel strategy to activate TLR and CD40 signaling in human T cells using inducible MyD88/CD40 (iMC), which can be triggered in vivo via the synthetic dimerizing ligand, rimiducid, to provide potent costimulation to CAR-modified T cells. Importantly, the concurrent activation of iMC (with rimiducid) and CAR (by antigen recognition) is required for interleukin (IL)-2 production and robust CAR-T cell expansion and may provide a user-controlled mechanism to amplify CAR-T cell levels in vivo and augment anti-tumor efficacy.

Systematically probing the bottom-up synthesis of AuPAMAM conjugates for enhanced transfection efficiency.

BACKGROUND: Gold nanoparticles (AuNPs) have shown great promise as scaffolds for gene therapy vectors due to their attractive physiochemical properties which include biocompatibility, ease of functionalization via the nearly covalent gold-sulfur dative bond, and surface plasmon optical properties. Previously, we synthesized stable AuNP-polyamidoamine (AuPAMAM) conjugates and showed their success in vitro as non-viral gene delivery vectors. RESULTS: In this study, we systematically perturbed each component of the AuPAMAM conjugates and analyzed the resulting effect on transfection efficiency. Due to the modular, bottom-up nature of the AuPAMAM synthesis, we were able to probe each step of the fabrication process. The relationship between each conjugation parameter and the function of the final vector were investigated. More than fourfold enhanced transfection efficiency was achieved by modifying the PAMAM concentration, PAMAM core chemistry, PAMAM terminus chemistry, and self-assembled monolayer composition of the AuPAMAM conjugates. CONCLUSIONS: This work suggest that AuPAMAM synthesis platform is a promising non-viral gene therapy approach and highlights the importance of inspecting the role of each individual constituent in all nanotechnology hybrid materials.

Anti-Tumor Effects after Adoptive Transfer of IL-12 Transposon-Modified Murine Splenocytes in the OT-I-Melanoma Mouse Model.

Adoptive transfer of gene modified T cells provides possible immunotherapy for patients with cancers refractory to other treatments. We have previously used the non-viral piggyBac transposon system to gene modify human T cells for potential immunotherapy. However, these previous studies utilized adoptive transfer of modified human T cells to target cancer xenografts in highly immunodeficient (NOD-SCID) mice that do not recapitulate an intact immune system. Currently, only viral vectors have shown efficacy in permanently gene-modifying mouse T cells for immunotherapy applications. Therefore, we sought to determine if piggyBac could effectively gene modify mouse T cells to target cancer cells in a mouse cancer model. We first demonstrated that we could gene modify cells to express murine interleukin-12 (p35/p40 mIL-12), a transgene with proven efficacy in melanoma immunotherapy. The OT-I melanoma mouse model provides a well-established T cell mediated immune response to ovalbumin (OVA) positive B16 melanoma cells. B16/OVA melanoma cells were implanted in wild type C57Bl6 mice. Mouse splenocytes were isolated from C57Bl6 OT-I mice and were gene modified using piggyBac to express luciferase. Adoptive transfer of luciferase-modified OT-I splenocytes demonstrated homing to B16/OVA melanoma tumors in vivo. We next gene-modified OT-I cells to express mIL-12. Adoptive transfer of mIL-12-modified mouse OT-I splenocytes delayed B16/OVA melanoma tumor growth in vivo compared to control OT-I splenocytes and improved mouse survival. Our results demonstrate that the piggyBac transposon system can be used to gene modify splenocytes and mouse T cells for evaluating adoptive immunotherapy strategies in immunocompetent mouse tumor models that may more directly mimic immunotherapy applications in humans.

In vivo gold nanoparticle delivery of peptide vaccine induces anti-tumor immune response in prophylactic and therapeutic tumor models.

Gold nanoparticles (AuNPs) are promising vehicles for cancer immunotherapy, with demonstrated efficacy in immune delivery and innate cell stimulation. Nevertheless, their potential has yet to be assessed in the in vivo application of peptide cancer vaccines. In this study, it is hypothesized that the immune distribution and adjuvant qualities of AuNPs could be leveraged to facilitate delivery of the ovalbumin (OVA) peptide antigen and the CpG adjuvant and enhance their therapeutic effect in a B16-OVA tumor model. AuNP delivery of OVA (AuNP-OVA) and of CpG (AuNP-CpG) enhanced the efficacy of both agents and induced strong antigen-specific responses. In addition, it is found that AuNP-OVA delivery alone, without CpG, is sufficient to promote significant antigen-specific responses, leading to subsequent anti-tumor activity and prolonged survival in both prophylactic and therapeutic in vivo tumor models. This enhanced therapeutic efficacy is likely due to the adjuvant effect of peptide coated AuNPs, as they induce inflammatory cytokine release when cultured with bone marrow dendritic cells. Overall, AuNP-mediated OVA peptide delivery can produce significant therapeutic benefits without the need of adjuvant, indicating that AuNPs are effective peptide vaccine carriers with the potential to permit the use of lower and safer adjuvant doses during vaccination.

Modification of T lymphocytes to express tumor antigens.

Human T cells can be genetically modified to express tumor-associated antigens (TAA) for the induction of tumor-specific immunity, suggesting that T cells may be alternative candidates of effective antigen-presenting cells (TAPC) and may be useful in vivo as cellular cancer vaccines. The effective induction of TAA-specific T cell immune responses requires activation of T cells by CD3/CD28 antibodies and the presence of proinflammatory cytokines such as interleukin-7 (IL-7) and interleukin-12 (IL-12). Here, we describe the technique of preparing activated human TAPC pulsed with TAA peptides for the induction of tumor antigen-specific T cell immunity in vitro.