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Autologous cell therapy depends on T lymphocyte expansion efficiency and is hindered by suboptimal interactions between T cell receptors (TCR) and peptide-MHC molecules. Various artificial antigen presenting cell systems that enhance these interactions are often labor-intensive, fabrication costly, highly variable, and potentially unscalable toward clinical setting. Here, 3D centrifugation-enabled priming of T cell immune-synapse junctions is performed to generate tight T cell–Dynabead aggregates at a rate 200-fold faster than that of conventional 24-h bulk shaking. Furthermore, by forming T cell–Dynabead aggregates in the starting culture, two- to sixfold greater T cell expansion is achieved over conventional T cell expansion for cancer patient-derived primary T cells while limiting over-activation. Creating 3D T cell–Dynabead aggregates as the “booster” material enables highly efficient polyclonal T cell expansion without the need for complex surface modification of artificial antigen-presenting cells (APCs). This method can be modularly adapted to existing T cell expansion processes for various applications, including adoptive cell therapies (ACTs).

Adoptive T cell therapy (ACT) has achieved remarkable results in the treatment of cancer. Tumor‐antigen specific T cells are the main players in the clearance of cancerous cells, but generating large numbers is a major hurdle in clinical practice. One shortcoming of current expansion procedures is that the artificial presentation of T cell activating signals on rigid surfaces do not recapitulate the physiological presentation on a fluidic membrane. To address this, semi‐flexible poly(isocyanopeptide) (PIC) immunofilaments (IFs) are generated coated on micro‐sized magnetic beads (immunobrushes (IB)). IBs are functionalized with peptide‐loaded major histocompatibility complexes (pMHC, signal 1) and agonistic anti‐CD28 antibodies (αCD28, signal 2) to effectively expand and enrich antigen‐specific T cells. As a direct result of the immunobrush design, strong T cell activation and excellent tumor cell killing capacities are found. More importantly, high selectivity is demonstrated by strong expansion and enrichment of antigen‐specific T cells in a pool of non‐specific cells (93‐fold enrichment of antigen specific T cells in 7 days). The modular character of the immunobrush‐based strategy makes it a great platform for highly effective ACT‐based therapies. [ABSTRACT FROM AUTHOR]

Probabilistic models of adaptive immune repertoire sequence distributions can be used to infer the expansion of immune cells in response to stimulus, differentiate genetic from environmental factors that determine repertoire sharing, and evaluate the suitability of various target immune sequences for stimulation via vaccination. Classically, these models are defined in terms of a probabilistic V(D)J recombination model which is sometimes combined with a selection model. In this paper we take a different approach, fitting variational autoencoder (VAE) models parameterized by deep neural networks to T cell receptor (TCR) repertoires. We show that simple VAE models can perform accurate cohort frequency estimation, learn the rules of VDJ recombination, and generalize well to unseen sequences. Further, we demonstrate that VAE-like models can distinguish between real sequences and sequences generated according to a recombination-selection model, and that many characteristics of VAE-generated sequences are similar to those of real sequences.

Ex vivo expansion conditions used to generate T cells for immunotherapy are thought to adopt metabolic phenotypes that impede therapeutic efficacy in vivo. The comparison of five different culture media used for clinical T cell expansion revealed unique optima based on different output variables, including proliferation, differentiation, function, activation, and mitochondrial phenotypes. The extent of proliferation and function depended on the culture media rather than stimulation conditions. Moreover, the expanded T cell end products adapted their metabolism when switched to a different media formulation, as shown by glucose and glutamine uptake and patterns of glucose isotope labeling. However, adoption of these metabolic phenotypes was uncoupled to T cell function. Expanded T cell products cultured in ascites from ovarian cancer patients displayed suppressed mitochondrial activity and function irrespective of the ex vivo expansion media. Thus, ex vivo T cell expansion media have profound impacts on metabolism and function.

T cells are critical in orchestrating protective immune responses to cancer and an array of pathogens. The interaction between a peptide MHC (pMHC) complex on antigen presenting cells (APCs) and T cell receptors (TCRs) on T cells initiates T cell activation, division, and clonal expansion in secondary lymphoid organs. T cells must also integrate multiple T cell-intrinsic and extrinsic signals to acquire the effector functions essential for the defense against invading microbes. In the case of T helper cell differentiation, while innate cytokines have been demonstrated to shape effector CD4+ T lymphocyte function, the contribution of TCR signaling strength to T helper cell differentiation is less understood. In this review, we summarize the signaling cascades regulated by the strength of TCR stimulation. Various mechanisms in which TCR signal strength controls T helper cell expansion and differentiation are also discussed.

Tumor-specific tumor-infiltrating lymphocytes (TILs) can be in vitro expanded and have the ability to induce complete and durable tumor regression in some patients with melanoma following adoptive cell therapy (ACT). In this preclinical study, we investigated the feasibility of expanding TIL from sarcomas, as well as performing functional in vitro analyses on these. TILs were expanded in vitro by the use of IL2 stimulation with or without the addition of 4-1BB and CD3 antibodies. Phenotypical and functional analyses were mainly performed by flow cytometry. TILs were expanded from 25 of 28 (89%) tumor samples from patients with 9 different sarcoma subtypes. TILs were predominantly αβ T-cells of effector memory subtype with CD4+ dominance. In particular, CD8+ TIL highly expressed LAG3 and to a lesser degree PD-1 and BTLA. In total, 10 of 20 TIL cultures demonstrated in vitro recognition of autologous tumor. In some cases, the fraction of tumor-reactive T cells was more than 20%. 4-1BB stimulation augmented expansion kinetics and favored CD8+ occurrence. In conclusion, TIL expansion from sarcoma is feasible and expanded TILs highly express LAG3 and comprise multifunctional tumor-reactive T-cells. [ABSTRACT FROM AUTHOR]

T cells are critical in orchestrating protective immune responses to cancer and an array of pathogens. The interaction between a peptide MHC (pMHC) complex on antigen presenting cells (APCs) and T cell receptors (TCRs) on T cells initiates T cell activation, division, and clonal expansion in secondary lymphoid organs. T cells must also integrate multiple T cell-intrinsic and extrinsic signals to acquire the effector functions essential for the defense against invading microbes. In the case of T helper cell differentiation, while innate cytokines have been demonstrated to shape effector CD4+ T lymphocyte function, the contribution of TCR signaling strength to T helper cell differentiation is less understood. In this review, we summarize the signaling cascades regulated by the strength of TCR stimulation. Various mechanisms in which TCR signal strength controls T helper cell expansion and differentiation are also discussed. [ABSTRACT FROM AUTHOR]

Probabilistic models of adaptive immune repertoire sequence distributions can be used to infer the expansion of immune cells in response to stimulus, differentiate genetic from environmental factors that determine repertoire sharing, and evaluate the suitability of various target immune sequences for stimulation via vaccination. Classically, these models are defined in terms of a probabilistic V(D)J recombination model which is sometimes combined with a selection model. In this paper we take a different approach, fitting variational autoencoder (VAE) models parameterized by deep neural networks to T cell receptor (TCR) repertoires. We show that simple VAE models can perform accurate cohort frequency estimation, learn the rules of VDJ recombination, and generalize well to unseen sequences. Further, we demonstrate that VAE-like models can distinguish between real sequences and sequences generated according to a recombination-selection model, and that many characteristics of VAE-generated sequences are similar to those of real sequences.

Ipilimumab, in combination with nivolumab, is one of the promising drugs that enhance the anti-tumor immune response of patients with advanced melanoma. Since the co-administration of nivolumab with ipilimumab in the neoadjuvant setting expands melanoma-reactive T cells at the primary site of melanoma and has a high rate of histological complete response, the pre-surgical administration of this combination could be the optimal therapy for unresectable advanced melanoma. In this report, a case of unresectable advanced melanoma treated successfully with administration of nivolumab with ipilimumab before primary tumor resection is presented. In addition, CD8+ T cells increased among the tumor-infiltrating lymphocytes that were surrounding melanoma cells and caspase 3+ cells. The present case suggests that pre-surgical administration of nivolumab with ipilimumab could be the optimal therapy for the treatment of unresectable advanced melanoma.

T cell-based adoptive cell therapy (ACT) has emerged as a promising treatment for various diseases, particularly cancers. Unlike other immunotherapy modalities, ACT involves directly transferring engineered T cells into patients to eradicate diseased cells; hence, it necessitates methods for effectively activating and expanding T cells in vitro . Artificial antigen-presenting cells (aAPCs) have been widely developed based on biomaterials, particularly micro- and nanoparticles, and functionalized with T cell stimulatory antibodies to closely mimic the natural T cell-APC interactions. Due to their vast clinical utility, aAPCs have been employed as an off-the-shelf technology for T cell activation in FDA-approved ACTs, and the development of aAPCs is constantly advancing with the emergence of aAPCs with more sophisticated designs and additional functionalities. Here, we review the recent advancements in particle-based aAPCs for T cell activation in ACTs. Following a brief introduction, we first describe the manufacturing processes of ACT products. Next, the design and synthetic strategies for micro- and nanoparticle-based aAPCs are discussed separately to emphasize their features, advantages, and limitations. Then, the impact of design parameters of aAPCs, such as size, shape, ligand density/mobility, and stiffness, on their functionality and biomedical performance is explored to provide deeper insights into the design concepts and principles for more efficient and safer aAPCs. The review concludes by discussing current challenges and proposing future perspectives for the development of more advanced aAPCs.

Discovery of epitope-specific T-cell receptors (TCRs) for cancer therapies is a time consuming and expensive procedure that usually requires a large amount of patient cells. To maximize information from and minimize the need of precious samples in cancer research, prediction models have been developed to identify in silico epitope-specific TCRs. In this chapter, we provide a step-by-step protocol to train a prediction model using the user-friendly TCRex webtool for the nearly universal tumor-associated antigen Wilms' tumor 1 (WT1)-specific TCR repertoire. WT1 is a self-antigen overexpressed in numerous solid and hematological malignancies with a high clinical relevance. Training of computational models starts from a list of known epitope-specific TCRs which is often not available for new cancer epitopes. Therefore, we describe a workflow to assemble a training data set consisting of TCR sequences obtained from WT1 37-45 -reactive CD8 T cell clones expanded and sorted from healthy donor peripheral blood mononuclear cells., Competing Interests: Conflicts of interest K.L., P.M. and B.O. hold shares in ImmuneWatch BV, an immunoinformatics company., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Introduction: Therapy with chimeric antigen receptor T (CART) cells for hematological malignancies has shown promising results. Effectiveness of CART cells may depend on the ratio of Tn (naive) vs. effector (Te) T cells, Tn cells being responsible for an enduring antitumor activity through maturation. Therefore, we investigated factors influencing the TrVTE ratio of CART cells. Materials and methods: CART cells were generated upon transduction of peripheral blood mononuclear cells with a CD19.CAR-CD28-CD137zeta third generation retroviral vector under two different stimulating culture conditions: anti-CD3/anti-CD28 antibodies adding either interleukin (IL)-7/IL-15 or IL-2. CART cells were maintained in culture for 20 days. We evaluated 24 healthy donors (HDs) and 11 patients with chronic lymphocytic leukemia (CLL) for the composition of cell subsets and produced CART cells. Phenotype and functionality were tested using flow cytometry and chromium release assays. Results: IL-7/IL-15 preferentially induced differentiation into TN, stem cell memory (Tscm: naive CD27+ CD95+), CD4+ and CXCR3+ CART cells, while IL-2 increased effector memory (Tem), CD56+ and CD4+ T regulatory (TReg) CART cells. The net amplification of different CART subpopulations derived from HDs and untreated CLL patients was compared. Particularly the expansion of CD4+ CARTn cells differed significantly between the two groups. For HDs, this subtype expanded >60-fold, whereas CD4+ CARTN cells of untreated CLL patients expanded less than 10-fold. Expression of exhaustion marker programmed cell death 1 on CARTN cells on day 10 of culture was significantly higher in patient samples compared to HD samples. As the percentage of malignant B cells was expectedly higher within patient samples, an excessive amount of B cells during culture could account for the reduced expansion potential of CARTn cells in untreated CLL patients. Final TN/TE ratio stayed <0.3 despite stimulation condition for patients, whereas this ratio was >2 in samples from HDs stimulated with IL-7/IL-15, thus demonstrating efficient CARTN expansion. conclusion: Untreated CLL patients might constitute a challenge for long-lasting CART effects in vivo since only a low number of TN among the CART product could be generated. Depletion of malignant B cells before starting CART production might be considered to increase the TN/TE ratio within the CART product. [ABSTRACT FROM AUTHOR]

LPS‐induced chemokine, CXCL9 promotes Ag‐specific CD4 T cell accumulation in both lymphoid (lymph node) and non‐lymphoid tissues (liver). LPS is a powerful adjuvant, and although LPS‐mediated TLR4 signaling has been exquisitely delineated, the in vivo mechanism of how TLR4 responses impact T cell priming is far less clear. Besides costimulation, TNF and type 1 IFN are dominant cytokines released after TLR4 activation and can shape T cell responses, but other downstream factors have not been examined extensively. Depending on context, we show that IFNαR1 blockade resulted in minor to major effects on specific CD4 T cell clonal expansion. To help explain these differences, it was hypothesized that IFNαR1 blockade would inhibit specific T cell migration by reducing chemokine receptor signaling, but specific CD4 T cells from IFNαR1‐blocked mice were readily able to migrate in response to specific chemokines. Next, we examined downstream factors and found that type 1 IFN signaling was necessary for chemokine production, even when mice were immunized with specific Ag with LPS and CD134 costimulation. IFNαR1 signaling promoted CXCL9 and CXCL10 synthesis, suggesting that these chemokines might be involved in the LPS and CD134 costimulation response. After immunization, we show that CXCL9 blockade inhibited CD4 T cell accumulation in the liver but also in LNs, even in the presence of elevated serum IFN‐β levels. Thus, whereas type 1 IFN might have direct effects on primed CD4 T cells, the downstream chemokines that play a role during migration also impact accumulation. In sum, CXCL9 production is a key benchmark for productive CD4 T cell vaccination strategies. [ABSTRACT FROM AUTHOR]

Adoptive T cell therapy has successfully been implemented for the treatment of cancer. Nevertheless, ex vivo expansion of T cells by artificial antigen-presenting cells (aAPCs) remains cumbersome and can compromise T cell functionality, thereby limiting their therapeutic potential. We propose a radically different approach aimed at direct expansion of T cells in vivo, thereby omitting the need for large-scale ex vivo T cell production. We engineered nanosized immunofilaments (IFs), with a soluble semiflexible polyisocyanopeptide backbone that presents peptide-loaded major histocompatibility complexes and costimulatory molecules multivalently. IFs readily activated and expanded antigen-specific T cells like natural APCs, as evidenced by transcriptomic analyses of T cells. Upon intravenous injection, IFs reach the spleen and lymph nodes and induce antigen-specific T cell responses in vivo. Moreover, IFs display strong antitumor efficacy resulting in inhibition of the formation of melanoma metastases and reduction of primary tumor growth in synergy with immune checkpoint blockade. In conclusion, nanosized IFs represent a powerful modular platform for direct activation and expansion of antigen-specific T cells in vivo, which can greatly contribute to cancer immunotherapy.

Autologous T cells were genetically modified to express a chimeric antigen receptor (CAR) directed toward carboxy-anhydrase-IX (CAIX) and used to treat patients with CAIX-positive metastatic renal cell carcinoma. In this study, we questioned whether the T cell maturation stage in the pre-infusion product affected CAIX CAR expression and function in vitro as well as in vivo CAR T cell numbers and expansion. During the 14 days expansion of CAR T cells prior to administration, we observed shifts from a predominant CD4 to a CD8 T cell phenotype and from a significant fraction of naïve to central effector T cells. Surface expression of the CAR was equally distributed among different T cell subsets and T cell maturation stages. During T cell culture days 14-18 (which covered patient treatment days 1-5), T cells demonstrated a decline in CAR expression level per cell irrespective of T cell maturation stage, although the proportion of CAR-positive T cells and CAR-mediated T cell effector functions remained similar for both CD4 and CD8 T cell populations. Notably, patients with a higher fraction of naïve CD8 T cells at baseline (prior to genetic modification) or central effector CD8 T cells at 2 weeks of CAR T cell culture demonstrated a higher fold expansion and absolute numbers of circulating CAR T cells at 1 month after start of therapy. We conclude that the T cell maturation stage prior to and during CAR T cell expansion culture is related to in vivo CAR T cell expansion. [ABSTRACT FROM AUTHOR]

Adoptive T cell therapy has demonstrated great promise for treating cancer and other diseases. While extensive effort has been made to improve ex vivo expansion of T cells, strategies for maintaining the proliferation and function of T cells post adoptive transfer are still lacking. Here we report an injectable T cell-responsive macroporous hydrogel that enables in situ activation and expansion of T cells. The macroporous gel is composed of a polymeric network with dispersed macropores (∼150 μm) that are large enough to home T cells. In the presence of T cells that can gradually disrupt the gel network surrounding the macropores, activation cues can be gradually released for in situ activation and expansion of T cells. This T cell-responsive macroporous gel enables expansion of effector T cells in vivo, is stable over weeks upon subcutaneous injection, and results in enhanced CD8+ T cell response and antitumor efficacy. We further show that the T cell-responsive macroporous gel could achieve comparable antitumor efficacy to conventional T cell therapy with a much lower cell dose. This injectable, T cell-responsive macroporous gel provides a platform for in vivo expansion of engineered T cells in a controlled manner, for timely and effective treatment of diseases. [ABSTRACT FROM AUTHOR]

Abstract: Background and aims: The aim of the present study was to compare several in vitro anti-CD3/CD28-coated magnetic bead expansion methods as a means of improving T cell recovery and to evaluate the impact of the respective methods on T cell viability, differentiation and function. The effect of the respective expansion protocols on cytokine production and cytotoxicity was also characterized. Methods: Peripheral blood mononuclear cells were isolated from healthy donors, expanded for 7days by different methods in vitro and then counted. T cell viability, phenotype and differentiation status were determined by flow cytometry. The cytotoxicity of collected cells was evaluated by a non-radioactive assay. Results: An equal bead-to-cell ratio in the presence of IL-2, IL-7 and IL-15 generated the highest T cell yields (median 4.75-fold increase [range 4.10–6.25], P=0.043) with a median of 79.20% viable cells (range 70.00%–80.30%). By contrast, a high bead-to-cell ratio (3:1) favored the selection of central memory T cells (CD4: 0.44 [0.40–1.69]; CD8: 0.77 [0.42–1.19], P=0.043) with increased interferon-gamma (IFN-γ) production following re-stimulation (48.5% [42.1%–68.2%], P=0.043) and also a trend towards enhanced cytotoxicity against target cells (10:1 ratio: 61.80% [40.80%–80.00%], P=0.068). Conclusions: An equal bead-to-cell ratio is optimal in keeping the balance between promoting proliferations and preserving cellular vitality and the combination of homeostatic cytokines further improved cell output, whereas a high bead-to-cell ratio favored the production of bioactive cells. [Copyright &y& Elsevier]

Abstract: Pertussis toxin (PTX) has pronounced adjuvant activity and strongly enhances innate and adaptive immune responses, including increased antibody production and Th1/Th2 cytokine production. Adjuvant effects of PTX on Th1 and Th2 cells are primarily mediated via CD80/86 costimulation via enhanced expression of these molecules by APCs. However, it has remained unresolved whether PTX modulates the expression of costimulatory and inhibitory molecules on CD4+ and CD8+ T cells. To address this question, we determined the expression kinetics of CD28, CTLA-4, and CD40L on spleen CD4+ and CD8+ T cells after incubation with PTX. The results show that PTX upregulated the expression of CD28 by CD8+ T cells, but not by CD4+ T cells. In contrast, the expression of CTLA-4 and CD40L was not substantially altered on CD4+ or CD8+ T cells. CD28 upregulation by CD8+ T cells was paralleled by upregulation of CD69 and the induction of IFN-γ, Granzyme B (GrB), and IL-17. CD8+ T cell activation and cytokine production could be substantially blocked with anti-CD80 and CD86 antibodies, consistent with CD28 mediated signaling. Treatment of highly purified CD8+ T cells with PTX resulted in upregulation of CD28 and CD69, and production of IFN-γ. Incubation with CD28 mAb further enhanced this effect, suggesting that PTX has direct effects on CD8+ T cells which are enhanced by CD80/86-mediated costimulation provided by APCs. [ABSTRACT FROM AUTHOR]

Abstract: We have been studying the re-activation of tumor-associated antigen (TAA)-specific CD8+ T cells in sentinel lymph nodes (SLN) of melanoma patients upon intradermal administration of the CpG-B oligodeoxynucleotide PF-3512676. To facilitate functional testing of T cells from small SLN samples, high-efficiency polyclonal T cell expansion is required. In this study, SLN cells were expanded via classic methodologies with plate- or bead-bound anti-CD3/CD28 antibodies and with the K562/CD32/4-1BBL artificial APC system (K32/4-1BBL aAPC) and analyzed for responsiveness to common recall or TAA-derived peptides. K32/4-1BBL-expanded T cell populations contained significantly more effector/memory CD8+ T cells. Moreover, recall and melanoma antigen-specific CD8+ T cells were more frequently detected in K32/4-1BBL-expanded samples as compared with anti-CD3/CD28-expanded samples. We conclude that K32/4-1BBL aAPC are superior to anti-CD3/CD28 antibodies for the expansion of in vivo-primed specific CD8+ T cells and that their use facilitates the sensitive monitoring of functional anti-tumor T cell immunity in SLN. [ABSTRACT FROM AUTHOR]

Abstract: Adoptive T cell therapy is a promising treatment strategy for patients with different types of cancer. The methods used for generation of high numbers of tumor specific T cells usually require long-term ex vivo culture, which frequently lead to generation of terminally differentiated effector cells, demonstrating low persistence in vivo. Therefore, optimization of protocols for generation of T cells for adoptive cell therapy is warranted. The aim of this work was to develop a protocol for expansion of antigen-specific T cells using Dynabeads CD3/CD28 to obtain T cells expressing markers important for in vivo persistence and survival. To achieve high numbers of antigen-specific T cells following expansion, we have tested the effect of depleting regulatory T cells using Dynabeads CD25 and including a pre-stimulation step with peptide prior to the non-specific expansion with Dynabeads. Our data demonstrate that virus- and tumor specific T cells can be expanded to high numbers using Dynabeads CD3/CD28 following optimization of the culture conditions. The expansion protocol presented here results in enrichment of antigen-specific CD8+ T cells with an early/intermediate memory phenotype. This is observed even when the antigen-specific CD8+ T cells demonstrated a terminal effector phenotype prior to expansion. This protocol thus results in expanded T cells with a phenotypic profile which may increase the chance of retaining long-term persistence following adoptive transfer. Based on these data we have developed a cGMP protocol for expansion of tumor specific T cells for adoptive T cell therapy. [Copyright &y& Elsevier]

Abstract: Development of methods for efficient in vitro stimulation and expansion of peptide specific CD8+ T cells is compelling not only with respect to adoptive T cell therapy but also regarding analysis of T cell responses and search for new immunogenic peptides. In the present study, a new approach to in vitro T cell stimulation was investigated. By use of an antigenic peptide derived from the cytomegalovirus (CMVp) we tested the stimulatory efficacy of recombinant plate bound MHC molecules (PB-MHC), being immobilized in culture plates. A single stimulation of non-adherent peripheral blood mononuclear cells (NA-PBMCs) with PB-MHC/CMVp resulted in significant expansion of CMVp specific CD8+ T cells, which was comparable to that achieved by CMVp pulsed mature dendritic cells (DCs). By repeated exposure of NA-PBMCs to PB-MHC/CMVp more than 60% CMVp specific CD8+ T cells, representing a 240-fold expansion, were reached after only two stimulations. Although stimulation with PB-MHC/CMVp clearly demonstrated efficient peptide specific expansion of CD8+ T cells, there was a tendency to proliferative exhaustion of the cells after 3–4 stimulations. Thus, it will be of interest to examine the effect of new stimulatory cocktails, e.g. cytokines and co-stimulatory molecules, by use of the present rapid and easy-to-use method of expanding peptide specific T cells. [Copyright &y& Elsevier]

Abstract: Pertussis toxin (PTX) has potent immunologic adjuvant activity in vivo and concomitantly enhances both T helper type (Th1) and Th2 cytokine responses. The PTX-induced enhancement of Th1 and Th2 immunity is mediated via the activation of antigen presenting cells (APCs), but the underlying mechanism is not known. Here we asked whether the adjuvant activity of PTX on T cell immunity was mediated by cytokines and/or costimulatory signals. The results show that in vivo blockade of CD28–CD80/86 costimulation essentially abrogated PTX-mediated enhancement of antigen-specific Th1 and Th2 responses. Blockade of CD40L–CD40 interactions was less efficient in inhibiting PTX-mediated enhancement of Th1 and Th2 responses. In contrast, the adjuvant activity of PTX was not mediated via cytokines, because neither Th1 nor Th2 responses were substantially impaired in mice deficient for IL-12, IFN-γ, IL-4, IL-5, or IL-6. Collectively, the data suggest that PTX mediates its adjuvant effects on T cell cytokine differentiation and clonal expansion via the modulation of costimulatory molecules on APCs. Understanding the costimulatory pathways targeted by PTX could lead to the design of novel adjuvants that selectively induce Th1 or Th2 immunity. [Copyright &y& Elsevier]

Aim: To identify T cell expansions, i.e. increased frequencies of T cells using a particular T cell receptor (TCR) Vα or Vβ gene segment, in patients with immune thrombocytopenic purpura (ITP). Methods: The TCR repertoires of CD4+ and CD8+ peripheral blood lymphocytes of 16 patients with chronic ITP were analysed by staining with a panel of anti-TCR Vα and Vβ antibodies followed by flow cytometry. Results: Four of the 16 patients exhibited a total of 6 expansions of CD8+ T cells using a particular Vβ segment, but no expansions were detected in the CD4+ subset. For three of the expansions where a follow-up blood sample after treatment with intravenous immunoglobulin was available, only one expansion remained. IConclusion: Overall T cell expansion frequency was the same as in healthy individuals. However, the presence of expansions that normalized with treatment suggests the presence of specific T cells implicated in the pathogenesis of ITP. [ABSTRACT FROM AUTHOR]

Background Lymphocyte-derived malignancies can be treated with combinations of drugs that efficiently eradicate normal and malignant lymphocytes. Lack of T lymphocytes after treatment of B lymphocyte CLL (B-CLL) makes the patients susceptible to serious infections and may limit the benefit of the therapy. The aim of the study was to purify and culture-expand normal T lymphocytes from B-CLL patients prior to therapy. These cells could be frozen and given to the patients in the lymphopenic period post-chemotherapy. Methods: T lymphocytes were isolated from the mononuclear cell apheresis products from five patients with previously untreated B-CLL. The apheresis products were red-cell depleted by density gradient centrifugation. B-lymphocyte purging was performed by incubating with MAbs to four different B-cell epitopes, followed by magnetic-bead depletion. One round of negative selection removed >90% of the B lymphocytes. The T-lymphocyte enriched cell suspension was cultured for 10/11 days in the presence of IL-2 and the anti-T cell receptor Ab OKT3. In addition, in some cultures anti-CD22 ricin immunotoxin was added. Results: T cells from CLL patients expanded 4.7-21-fold over a 10/11 days culture interval. After culture, CLL cells could no longer be identified by flow cytometric evaluation. The cultured T lymphocytes were predominately CD8[sup +], and were capable of lysing autologous CLL cells through a fas-dependent mechanism. Discussion: Selection and expansion of T lymphocytes by this method may represent a strategy for enhancing immunity in the lymphopenic period following CLL treatment. [ABSTRACT FROM AUTHOR]

Robust expansion of adoptively transferred T cells is a prerequisite for effective cancer immunotherapy, but how many genes in the genome modulate T cell expansion remains unknown. Here, we perform in vivo and in vitro CRISPR screens to systematically identify genes influencing CD8 T cell expansion. In the mouse genome, ∼2,600 and ∼1,500 genes are required for optimal CD8 T cell expansion in vivo and in vitro , respectively. In vivo -specific CD8 T cell essential genes are enriched in metabolic pathways, including mitochondrial metabolism. The strongest repressor of CD8 T cell expansion is Roquin, the ablation of which drastically boosts T cell proliferation by enhancing cell-cycle progression and upregulation of IRF4. Roquin deficiency or IRF4 overexpression potently enhances anti-tumor immunity. These data provide a functional catalog of CD8 T cell fitness genes and suggest that targeting the Roquin-IRF4 axis is an effective strategy to enhance efficacy of adoptive transfer therapy for cancer. [Display omitted] • Genome-wide identification of CD8 T cell fitness genes in vitro and in vivo • Expansion of CD8 T cells in vivo requires many more genes than in vitro expansion • Roquin is a potent repressor of CD8 T cell expansion and anti-tumor immunity • Roquin target IRF4 overexpression boosts CD8 T cell expansion and anti-tumor immunity A canonical feature of adaptive immunity is clonal expansion. Through genome-wide CRISPR screens in vitro and in vivo , Zhao et al. systematically identify genes that modulate CD8 T cell expansion and reveal a key role of the Roquin-IRF4 axis in T cell expansion and anti-tumor immunity. [ABSTRACT FROM AUTHOR]

Simple Summary: Adoptively transferred T cells expressing recombinant chimeric antigen receptors (CAR T cells) have been approved for the therapy of hematological malignancies of the B cell lineage. However, CAR T cell therapy for patients with solid tumors so far has shown limited benefits. Correlative clinical studies of patients with hematological malignancies have suggested that less differentiated CAR T cells have improved anti-leukemic activity. We have therefore investigated the role of differentiation on the anti-tumor activity of CAR T cells targeting the solid tumor antigen HER2 in preclinical models. We utilized different activation/expansion protocols, and explored whether different co-stimulatory domains in the CAR construct influence the short- and long-term efficacy of HER2-CAR T cells. We demonstrate that the CAR T cell product with the highest proportion of effector cells and maintaining a good balance of CD4+/CD8+ cells is the most effective against solid tumors both in vitro and in vivo. Correlative clinical studies of hematological malignancies have implicated that less differentiated, CD8+-dominant CAR T cell products have greater antitumor activity. Here, we have investigated whether the differentiation status of CAR T cell products affects their antitumor activity in preclinical models of solid tumors. We explored if different activation/expansion protocols, as well as different co-stimulatory domains in the CAR construct, influence the short- and long-term efficacy of CAR T cells against HER2-positive tumors. We generated T cell products that range from the most differentiated (CD28.z; OKT3-antiCD28/RPMI expansion) to the least differentiated (41BB.z; OKT3-RetroNectin/LymphoONE expansion), as judged by cell surface expression of the differentiation markers CCR7 and CD45RA. While the effect of differentiation status was variable with regard to antigen-specific cytokine production, the most differentiated CD28.z CAR T cell products, which were enriched in effector memory T cells, had the greatest target-specific cytolytic activity in vitro. These products also had a greater proliferative capacity and maintained CD4+ T cells upon repeated stimulation in vitro. In vivo, differentiated CD28.z CAR T cells also had the greatest antitumor activity, resulting in complete response. Our results highlight that it is critical to optimize CAR T cell production and that optimal product characteristics might depend on the targeted antigen and/or cancer. [ABSTRACT FROM AUTHOR]

Our objective was to study whether CD4+ or CD8+ T cells expressing particular T cell receptors (TCR) would accumulate in the lungs of patients with allergic asthma following allergen exposure. We thus analysed the TCR Vα and Vβ gene usage of CD4+ and CD8+ lung and peripheral blood lymphocytes (PBL) of eight patients with allergic asthma before and 4 days after inhalation challenge with the relevant allergen. Lung cells obtained by bronchoalveolar lavage (BAL) and paired PBL samples were analysed by flow cytometry using a panel of anti-TCR V-specific monoclonal antibodies that encompass ≈ 50% of the T cell repertoire. Lung-limited T cell expansions were recorded in both the CD4+ and the CD8+ subsets. In BAL CD8+, out of a total of 126 analyses, the number of T cell expansions increased from two to 11 after challenge, some of them dramatic. In BAL CD4+ the frequency of expansions was moderately increased already before challenge, but remained unchanged. A few expansions that tended to persist were noted in PBL CD8+. When analysing the overall change in TCR V gene usage the largest changes were also recorded in the BAL CD8+ subset. Specific interactions between T cells and antigens may lead to an increased frequency of T cells using selected TCR V gene segments. In this study we demonstrate that following allergen bronchoprovocation in allergic asthmatic subjects, T cell expansions preferentially emerge in the lung CD8+ T cell subset. [ABSTRACT FROM AUTHOR]

A dramatic and persistent T cell expansion in a healthy adult male was initially identified, using anti-T cell receptor for antigen (TCR)-specific MoAbs. The expanded T cells were found to be expressing TCR containing Vα 12.1 and Vβ 5.2, and they composed approximately one third of all the CD8+ T cells. The cells were shown to be not only non-activated (HLA-DR-, IL-2R-) but also of 'virgin' cell type (CD45RA+/CD45RO-) and they persisted over the observation period of more than one and a half years. Various T and B cell markers, and all other laboratory and physical parameters analysed, were normal. The expanded CD8+ T cells were further characterized by polymerase chain reaction (PCR) amplification, using Vβ- and Cβ-specific primers, followed by hybridization with Jβ-specific probes. Close to 90% of the Vα 12.1+ Vβ+ 5.2 + T cells were found to utilize the Jβ 2.5 gene segment, thus strongly suggesting the expanded T cells to be monoclonal. The condition may constitute a T cell counterpart to 'monoclonal gammopathy of undetermined significance' (MGUS), and by analogy we suggest it should be designated 'monoclonal T cell expansion of undetermined significance' (MTUS). [ABSTRACT FROM AUTHOR]

In recent years, the molecular mechanism by which natural killer cells lyze, or fail to lyze, different target cells has been elucidated. Natural killer cells express receptors which recognize MHC class I molecules on target cells. This interaction leads to inhibition of cytolytic activity, thus preventing lysis of target cells. The receptors belong to two distinct molecular types: (1) the Ig superfamily which includes receptors (p58.1, p58.2, p70, and p140) which recognize specific HLA allotypes; (2) CD94 molecules which display a broad specificity for HLA class I molecules. Recently, a subset of cytolytic T lymphocytes has been shown to express the various natural killer cell receptors. Such T cells are detectable in peripheral blood, spleen, tonsils, and lymph nodes, but not in the thymus and cord blood. In some instances, two or more natural killer receptors can be coexpressed at the single cell level. Surface marker analysis has revealed that natural killer cell receptor-positive T cells always express a memory phenotype. Moreover, they are characterized by a skewed T cell receptor VΒ repertoire. Further analysis of the T cell receptor VDJ sequences revealed that natural killer cell receptor-positive, CD3-positive cells isolated from a given individual are oligoclonal or monoclonal in nature. Crosslinking of natural killer receptors leads to inhibition of different T cell functions, including non-specific lysis of appropriate HLA class I-negative target cells, T cell receptor mediated cytotoxicity, and cytokine production. The inhibitory effect on T cell receptor-mediated function has important implications. Thus, the expression of natural killer cell receptors as a consequence of chronic antigen stimulation may result in functional impairment of specific cytolytic T lymphocytes. Preliminary data indicate that this phenomenon may occur in tumor or virally infected patients. Remarkably, various patients with large granular lymphocyte expansions characterized by a CD3-/ natural killer receptor-positive phenotype had chronic viral infections. The fact that antigen-specific cytolytic T lymphocytes may simultaneously express T cell and natural killer cell receptors, both recognizing HLA class I molecules but mediating opposite signals, offers new perspectives in our appreciation of the regulation of T cell responses and offers new clues for understanding the immunopathological events involved in certain diseases. [ABSTRACT FROM AUTHOR]

• Monocyte seeding density affects phenotype of generated immature DCs. • Immature DC phenotype affects extent of T cell proliferation. • MicroDEN successfully generates functionally competent immature DCs. • MicroDEN immature DCs induce greater T cell proliferation than well plate DCs. • MicroDEN generates similar DC counts to well plates at highest MO seeding density. Dendritic cells (DCs) are increasingly important for research and clinical use but obtaining sufficient numbers of dendritic cells is a growing challenge. We systemically investigated the effect of monocyte (MO) seeding density on the generation of monocyte-derived immature DCs (iDCs) in MicroDEN, a perfusion-based culture system, as well as 6-well plates. Cell surface markers and the ability of the iDCs to induce proliferation of allogeneic T cells were examined. The data shows a strong relationship between iDC phenotype, specifically CD80/83/86 expression, and T cell proliferation. MicroDEN generated iDCs proved better than well plate generated iDCs at inducing T cell proliferation within the 200k-600k MO/cm2 seeding density range studied. We attribute this to perfusion in MicroDEN which supplies fresh differentiation medium continuously to the differentiating MOs while concurrently removing depleted medium and toxic byproducts of cellular respiration. MicroDEN generated fewer iDCs on a normalized basis than the well plates at lower MO seeding densities but generated equivalent numbers of iDCs at 600k MO seeding density. These results demonstrate that MicroDEN is capable of generating greater numbers of iDCs with less manual work than standard well plate culture and the MicroDEN generated iDCs have greater ability to induce T cell proliferation. [ABSTRACT FROM AUTHOR]

Adoptive transfer of genetically engineered T cells can lead to profound and durable responses in patients with hematologic malignancies, generating enormous enthusiasm among scientists, clinicians, patients, and biotechnology companies. The success of adoptive cellular immunotherapy depends upon the ability to manufacture good quality T cells. We discuss here the methodologies and reagents that are used to generate T cells for the preclinical study of chimeric antigen receptor T cell therapy for acute myeloid leukemia (AML).