A new approach to CAR T-cell gene engineering and cultivation using piggyBac transposon in the presence of IL-4, IL-7 and IL-21

Background aims

Clinical-grade chimeric antigenic receptor (CAR)19 T cells are routinely manufactured by lentiviral/retroviral (LV/RV) transduction of an anti-CD3/CD28 activated T cells, which are then propagated in a culture medium supplemented with interleukin (IL)-2. The use of LV/RVs for T-cell modification represents a manufacturing challenge due to the complexity of the transduction approach and the necessity of thorough quality control.

Closed-system manufacturing of CD19 and dual-targeted CD20/19 chimeric antigen receptor T cells using the CliniMACS Prodigy device at an academic medical center

Background aims

Multiple steps are required to produce chimeric antigen receptor (CAR)-T cells, involving subset enrichment or depletion, activation, gene transduction and expansion. Open processing steps that increase risk of contamination and production failure are required. This complex process requires skilled personnel and costly clean-room facilities and infrastructure. Simplified, reproducible CAR-T-cell manufacturing with reduced labor intensity within a closed-system is highly desirable for increased availability for patients.

Manufacturing development and clinical production of NKG2D chimeric antigen receptor–expressing T cells for autologous adoptive cell therapy

Background aims

Adoptive cell therapy employing natural killer group 2D (NKG2D) chimeric antigen receptor (CAR)-modified T cells has demonstrated preclinical efficacy in several model systems, including hematological and solid tumors. We present comprehensive data on manufacturing development and clinical production of autologous NKG2D CAR T cells for treatment of acute myeloid leukemia and multiple myeloma (ClinicalTrials.gov Identifier: NCT02203825). An NKG2D CAR was generated by fusing native full-length human NKG2D to the human CD3ζ cytoplasmic signaling domain. NKG2D naturally associates with native costimulatory molecule DAP10, effectively generating a second-generation CAR against multiple ligands upregulated during malignant transformation including MIC-A, MIC-B and the UL-16 binding proteins.

Bispecific chimeric antigen receptors targeting the CD4 binding site and high-mannose Glycans of gp120 optimized for anti–human immunodeficiency virus potency and breadth with minimal immunogenicity

Background aims

Chimeric antigen receptors (CARs) offer great potential toward a functional cure of human immunodeficiency virus (HIV) infection. To achieve the necessary long-term virus suppression, we believe that CARs must be designed for optimal potency and anti-HIV specificity, and also for minimal probability of virus escape and CAR immunogenicity. CARs containing antibody-based motifs are problematic in the latter regard due to epitope mutation and anti-idiotypic immune responses against the variable regions.

Mesenchymal stromal cell exosome–enhanced regulatory T-cell production through an antigen-presenting cell–mediated pathway

Background aims

The immunomodulatory property of mesenchymal stromal cell (MSC) exosomes is well documented. On the basis of our previous report that MSC exosomes increased regulatory T-cell (Treg) production in mice with allogenic skin graft but not in ungrafted mice, we hypothesize that an activated immune system is key to exosome-mediated Treg production.

Generation of alloreactivity-reduced donor lymphocyte products retaining memory function by fully automatic depletion of CD45RA-positive cells

Background aims

For patients needing allogeneic stem cell transplantation but lacking a major histocompatibility complex (MHC)-matched donor, haplo-identical (family) donors may be an alternative. Stringent T-cell depletion required in these cases to avoid lethal graft-versus-host disease (GVHD) can delay immune reconstitution, thus impairing defense against virus reactivation and attenuating graft-versus-leukemia (GVL) activity. Several groups reported that GVHD is caused by cells residing within the naive (CD45RA⁺) T-cell compartment and proposed use of CD45RA-depleted donor lymphocyte infusion (DLI) to accelerate immune reconstitution. We developed and tested the performance of a CD45RA depletion module for the automatic cell-processing device CliniMACS Prodigy and investigated quality attributes of the generated products.

Circulating CD8+CD28− suppressor T cells tied to poorer prognosis among metastatic breast cancer patients receiving adoptive T-cell therapy: A cohort study

Background aims

This study aimed to determine the prognostic value of circulating CD8⁺CD28^? T lymphocytes among breast cancer patients treated with adoptive T-lymphocyte immunotherapy after chemotherapy.

Altered expression of T cell Immunoglobulin-Mucin (TIM) molecules in bronchoalveolar lavage CD4+ T cells in sarcoidosis

Background

Activated T helper (Th)-1 pulmonary CD4⁺ cells and their mediators are essential for the inflammation and granulomatous process in sarcoidosis. Recently, T-cell immunoglobulin and mucin domain (TIM) molecules were suggested to be important regulators of immune function. In this study, we wanted to investigate whether TIM molecules could play a role in sarcoidosis.

Methods

We used real-time polymerase chain reaction to investigate the differential gene expression of TIM-1 and TIM-3 as well as a few Th1 and Th2 cytokines (IL-2, IFN-γ, IL-4, IL-5 and IL-13) in CD4⁺ T cells isolated from bronchoalveolar lavage fluid (BALF) of patients (n = 28) and healthy controls (n = 8). Using flow cytometry, we were also able to analyse TIM-3 protein expression in 10 patients and 6 healthy controls.

Results

A decreased TIM-3 mRNA (p < 0.05) and protein (p < 0.05) expression was observed in patients, and the level of TIM-3 mRNA correlated negatively with the CD4/CD8 T cell ratio in BALF cells of patients. Compared to a distinct subgroup of patients i.e. those with Löfgren''s syndrome, BALF CD4⁺ T cells from non- Löfgren''s patients expressed decreased mRNA levels of TIM-1 (p < 0.05). mRNA expression of IL-2 was increased in patients (p < 0.01) and non-Löfgren''s patients expressed significantly higher levels of IFN-γ mRNA (p < 0.05) versus patients with Löfgren''s syndrome.

Conclusion

These findings are the first data on the expression of TIM-1 and TIM-3 molecules in sarcoidosis. The reduced TIM-3 expression in the lungs of patients may result in a defective T cell ability to control the Th1 immune response and could thus contribute to the pathogenesis of sarcoidosis. The down-regulated TIM-1 expression in non-Löfgren''spatients is in agreement with an exaggerated Th1 response in these patients.     

A role for B cells in organic dust induced lung inflammation

Background

Agriculture organic dust exposures induce lung disease with lymphoid aggregates comprised of both T and B cells. The precise role of B cells in mediating lung inflammation is unknown, yet might be relevant given the emerging role of B cells in obstructive pulmonary disease and associated autoimmunity.

Methods

Using an established animal model, C57BL/6 wild-type (WT) and B-cell receptor (BCR) knock-out (KO) mice were repetitively treated with intranasal inhalation of swine confinement organic dust extract (ODE) daily for 3 weeks and lavage fluid, lung tissues, and serum were collected.

Results

ODE-induced neutrophil influx in lavage fluid was not reduced in BCR KO animals, but there was reduction in TNF-α, IL-6, CXCL1, and CXCL2 release. ODE-induced lymphoid aggregates failed to develop in BCR KO mice. There was a decrease in ODE-induced lung tissue CD11c⁺CD11b⁺ exudative macrophages and compensatory increase in CD8⁺ T cells in lavage fluid of BCR KO animals. Compared to saline, there was an expansion of conventional B2-, innate B1 (CD19⁺CD11b⁺CD5^+/?)-, and memory (CD19⁺CD273^+/-CD73^+/?) B cells following ODE exposure in WT mice. Autoreactive responses including serum IgG anti-citrullinated protein antibody (ACPA) and anti-malondialdehyde-acetaldehyde (MAA) autoantibodies were increased in ODE treated WT mice as compared to saline control. B cells and serum immunoglobulins were not detected in BCR KO animals.

Conclusions

Lung tissue staining for citrullinated and MAA modified proteins were increased in ODE-treated WT animals, but not BCR KO mice. These studies show that agriculture organic dust induced lung inflammation is dependent upon B cells, and dust exposure induces an autoreactive response.

     

Oncoretroviral gene transfer to NOD/SCID repopulating cells using three different viral envelopes

Background

The aim of this study was to investigate gene transfer to human umbilical cord blood (CB) CD34⁺/CD38^low and NOD/SCID repopulating cells using oncoretroviral vectors and to compare the transduction efficiency using three different viral envelopes.

Methods

CB cells were transduced on Retronectin using an MSCV‐based vector with the gene for GFP (MGIN), which was packaged into three different cell lines giving different envelopes: PG13‐MGIN (GALV), 293GPG‐MGIN (VSV‐G) or AM12‐MGIN (amphotropic).

Results

Sorted CD34⁺/CD38^low cells were efficiently transduced after 3 days of cytokine stimulation and the percentage of GFP‐positive cells was 61.8±6.6% (PG13‐MGIN), 26.9±3.5% (293GPG‐MGIN), and 39.3±4.8% (AM12‐MGIN). For transplantation experiments, CD34⁺ cells were pre‐stimulated for 2 days before transduction on Retronectin preloaded with vector and with the addition of 1/10th volume of viral supernatant on day 3. On day 4, the expanded equivalent of 2.5×10⁵ cells was injected into irradiated NOD/SCID mice. All three pseudotypes transduced NOD/SCID repopulating cells (SRCs) equally well in the presence of serum, but engraftment was reduced when compared with freshly thawed cells. Simultaneous transduction with all three vector pseudotypes increased the gene transfer efficiency to SRCs but engraftment was significantly impaired. There were difficulties in producing amphotropic vectors at high titers in serum‐free medium and transduction of CD34⁺ cells using VSV‐G‐pseudotyped vectors under serum‐free conditions was very inefficient. In contrast, transduction with PG13‐MGIN under serum‐free conditions resulted in the maintenance of SRCs during transduction, high levels of engraftment (29.3±6.6%), and efficient gene transfer to SRCs (46.2±4.8%).

Conclusions

The best conditions for transduction and engraftment of CB SRCs were obtained with GALV‐pseudotyped vectors using serum‐free conditions. Copyright © 2002 John Wiley & Sons, Ltd.

     

Cord blood–derived cytokine-induced killer cells combined with blinatumomab as a therapeutic strategy for CD19+ tumors

Background

Cytokine-induced killer cells (CIKs) are an advanced therapeutic medicinal product (ATMP) that has shown therapeutic activity in clinical trials but needs optimization. We developed a novel strategy using CIKs from banked cryopreserved cord blood units (CBUs) combined with bispecific antibody (BsAb) blinatumomab to treat CD19⁺ malignancies.

TIM-3 expression characterizes regulatory T cells in tumor tissues and is associated with lung cancer progression

Background

T cell immunoglobulin-3 (TIM-3) has been established as a negative regulatory molecule and plays a critical role in immune tolerance. TIM-3 is upregulated in exhausted CD8⁺ T cells in both chronic infection and tumor. However, the nature of TIM-3⁺CD4⁺ T cells in the tumor microenvironment is unclear. This study is to characterize TIM-3 expressing lymphocytes within human lung cancer tissues and establish clinical significance of TIM-3 expression in lung cancer progression.

Methodology

A total of 51 human lung cancer tissue specimens were obtained from pathologically confirmed and newly diagnosed non-small cell lung cancer (NSCLC) patients. Leukocytes from tumor tissues, distal normal lung tissues, and peripheral blood mononuclear cells (PBMC) were analyzed for TIM-3 surface expression by flow cytometry. TIM-3 expression on tumor-infiltrating lymphocytes (TILs) was correlated with clinicopathological parameters.

Conclusions

TIM-3 is highly upregulated on both CD4⁺ and CD8⁺ TILs from human lung cancer tissues but negligibly expressed on T cells from patients'' peripheral blood. Frequencies of IFN-γ⁺ cells were reduced in TIM-3⁺CD8⁺ TILs compared to TIM-3⁻CD8⁺ TILs. However, the level of TIM-3 expression on CD8⁺ TILs failed to associate with any clinical pathological parameter. Interestingly, we found that approximately 70% of TIM-3⁺CD4⁺ TILs expressed FOXP3 and about 60% of FOXP3⁺ TILs were TIM-3⁺. Importantly, TIM-3 expression on CD4⁺ T cells correlated with poor clinicopathological parameters of NSCLC such as nodal metastasis and advanced cancer stages. Our study reveals a new role of TIM-3 as an important immune regulator in the tumor microenvironment via its predominant expression in regulatory T cells.     

Expansion of CD4+CD25+ helper T cells without regulatory function in smoking and COPD

Background

Regulatory T cells have been implicated in the pathogenesis of COPD by the increased expression of CD25 on helper T cells along with enhanced intracellular expression of FoxP3 and low/absent CD127 expression on the cell surface.

Method

Regulatory T cells were investigated in BALF from nine COPD subjects and compared to fourteen smokers with normal lung function and nine never-smokers.

Results

In smokers with normal lung function, the expression of CD25⁺CD4⁺ was increased, whereas the proportions of FoxP3⁺ and CD127⁺ were unchanged compared to never-smokers. Among CD4⁺ cells expressing high levels of CD25, the proportion of FoxP3⁺ cells was decreased and the percentage of CD127⁺ was increased in smokers with normal lung function. CD4⁺CD25⁺ cells with low/absent CD127 expression were increased in smokers with normal lung function, but not in COPD, when compared to never smokers.

Conclusion

The reduction of FoxP3 expression in BALF from smokers with normal lung function indicates that the increase in CD25 expression is not associated with the expansion of regulatory T cells. Instead, the high CD127 and low FoxP3 expressions implicate a predominantly non-regulatory CD25⁺ helper T-cell population in smokers and stable COPD. Therefore, we suggest a smoking-induced expansion of predominantly activated airway helper T cells that seem to persist after COPD development.     

HBsAg-redirected T cells exhibit antiviral activity in HBV-infected human liver chimeric mice

Background

Chronic hepatitis B virus (HBV) infection remains incurable. Although HBsAg-specific chimeric antigen receptor (HBsAg-CAR) T cells have been generated, they have not been tested in animal models with authentic HBV infection.

Involvement of Galectin-9/TIM-3 Pathway in the Systemic Inflammatory Response in Early-Onset Preeclampsia

Background

Preeclampsia is a common obstetrical disease affecting 3-5% of pregnancies and representing one of the leading causes of both maternal and fetal mortality. Maternal symptoms occur as an excessive systemic inflammatory reaction in response to the placental factors released by the oxidatively stressed and functional impaired placenta. The T-cell immunoglobulin domain and mucin domain (TIM) family is a relatively newly described group of molecules with a conserved structure and important immunological functions. Identification of Galectin-9 as a ligand for TIM-3 has established the Galectin-9/TIM-3 pathway as an important regulator of Th1 immunity and tolerance induction.

Methods

The aim of our study was to investigate the expression and function of Galectin-9 and TIM-3 molecules by peripheral blood mononuclear cells and the possible role of Galectin-9/TIM-3 pathway in the immunoregulation of healthy pregnancy and early-onset preeclampsia. We determined TIM-3 and Gal-9 expression and cytotoxicicty of peripheral lymphocytes of early-onset preeclamptic women and healthy pregnant woman using flow cytometry.

Results

Investigating peripheral lymphocytes of women with early-onset preeclampsia, our results showed a decreased TIM-3 expression by T cells, cytotoxic T cells, NK cells and CD56^dim NK cells compared to healthy pregnant women. Interestingly, we found a notably increased frequency of Galectin-9 positive cells in each investigated lymphocyte population in the case of early-onset preeclamptic patients. We further demonstrated increased cytotoxic activity by cytotoxic T and CD56^dim NK cells in women with early-onset preeclampsia. Our findings showed that the strongest cellular cytotoxic response of lymphocytes occurred in the TIM-3 positive subpopulations of different lymphocytes subsets in early-onset preeclampsia.

Conclusion

These data suggest that Gal-9/TIM-3 pathway could play an important role in the immune regulation during pregnancy and the altered Galectin-9 and TIM-3 expression could result an enhanced systemic inflammatory response including the activation of Th1 lymphocytes in preeclampsia.     

A Lower Proportion of Regulatory B Cells in Patients with Henoch–Schoenlein Purpura Nephritis

Background

Henoch—Schoenlein purpura is the one of most common types of systemic vasculitis that involves impaired renal function and Henoch-Schoenlein purpura nephritis (HSPN). The diagnosis of this condition is largely based on immunohistologic detection of immunoglobulin A1-containing immune complex in the glomerular deposits of mesangium. Despite clinical advances, the etiopathogenesis of HSPN is still largely unknown.

Methods

In this study, we enrolled 25 newly diagnosed HSPN patients and 14 healthy controls. Then, fractions of B cell subtypes were determined in venous blood using flow cytometry. The serum interleukin (IL)-10 concentration was determined by enzyme-linked immunosorbent assay.

Results

Compared to those in healthy controls, the numbers of CD38⁺CD19⁺, CD86⁺CD19⁺, CD38⁺CD86⁺CD19⁺, and CD95⁺CD19⁺ B cells per microliter of blood were significantly higher in HSPN patients. In contrast, the numbers of CD5⁺CD19⁺, IL-10⁺CD19⁺, CD5⁺CD1d⁺CD19⁺, and IL-10⁺CD5⁺CD1d⁺CD19⁺ B cells per microliter of blood and the serum IL-10 concentration were significantly lower in HSPN patients. Following treatment, the numbers of CD38⁺CD19⁺ and CD86⁺CD19⁺ B cells per microliter of blood were significantly reduced in HSPN patients. However, the numbers of CD5⁺CD1d⁺CD19⁺, CD5⁺CD1d⁺IL-10⁺CD19⁺, and IL-10⁺CD19⁺ B cells per microliter of blood and the serum IL-10 concentration were significantly increased in HSPN patients following treatment. The estimated glomerular filtration rate (eGFR) was negatively correlated with the number of CD38⁺CD19⁺ B cells but positively correlated with the numbers of IL-10⁺CD19⁺, CD1d⁺CD5⁺CD19⁺, and IL-10⁺CD1d⁺CD5⁺CD19⁺B cells per microliter of blood and the serum IL-10 concentration. The 24-h urinary protein concentration was positively correlated with the number of CD38⁺CD19⁺B cells but negatively correlated with the numbers of IL-10⁺CD19⁺, CD1d⁺CD5⁺CD19⁺, and IL-10⁺CD1d⁺CD5⁺CD19⁺B cells per microliter of blood and the serum IL-10 concentration.

Conclusion

Our results suggest that CD38⁺CD19⁺ and CD1d⁺CD5⁺CD19⁺ B cells (Bregs) contribute to the pathogenesis of HSPN.     

The doubling potential of T lymphocytes allows clinical-grade production of a bank of genetically modified monoclonal T-cell populations

Background aims

To produce an anti-leukemic effect after hematopoietic stem cell transplantation we have long considered the theoretical possibility of using banks of HLA-DP specific T-cell clones transduced with a suicide gene. For that application as for any others, a clonal strategy is constrained by the population doubling (PD) potential of T cells, which has been rarely explored or exploited.

Inhibitory Phenotype of HBV-Specific CD4+ T-Cells Is Characterized by High PD-1 Expression but Absent Coregulation of Multiple Inhibitory Molecules

Background

T-cell exhaustion seems to play a critical role in CD8⁺ T-cell dysfunction during chronic viral infections. However, up to now little is known about the mechanisms underlying CD4⁺ T-cell dysfunction during chronic hepatitis B virus (CHB) infection and the role of inhibitory molecules such as programmed death 1 (PD-1) for CD4⁺ T-cell failure.

Methods

The expression of multiple inhibitory molecules such as PD-1, CTLA-4, TIM-3, CD244, KLRG1 and markers defining the grade of T-cell differentiation as CCR7, CD45RA, CD57 and CD127 were analyzed on virus-specific CD4⁺ T-cells from peripheral blood using a newly established DRB1*01-restricted MHC class II Tetramer. Effects of in vitro PD-L1/2 blockade were defined by investigating changes in CD4⁺ T-cell proliferation and cytokine production.

Results

CD4⁺ T-cell responses during chronic HBV infection was characterized by reduced Tetramer⁺CD4⁺ T-cell frequencies, effector memory phenotype, sustained PD-1 but low levels of CTLA-4, TIM-3, KLRG1 and CD244 expression. PD-1 blockade revealed individualized patterns of in vitro responsiveness with partly increased IFN-γ, IL-2 and TNF-α secretion as well as enhanced CD4⁺ T-cell expansion almost in treated patients with viral control.

Conclusion

HBV-specific CD4⁺ T-cells are reliably detectable during different courses of HBV infection by MHC class II Tetramer technology. CD4⁺ T-cell dysfunction during chronic HBV is basically linked to strong PD-1 upregulation but absent coregulation of multiple inhibitory receptors. PD-L1/2 neutralization partly leads to enhanced CD4⁺ T-cell functionality with heterogeneous patterns of CD4⁺ T-cell rejunivation.     

The Immunology of a Healing Response in Cutaneous Leishmaniasis Treated with Localized Heat or Systemic Antimonial Therapy

Background

The effectiveness of systemic antimonial (sodium stibogluconate, Pentostam, SSG) treatment versus local heat therapy (Thermomed) for cutaneous leishmaniasis was studied previously and showed similar healing rates. We hypothesized that different curative immune responses might develop with systemic and local treatment modalities.

Methods

We studied the peripheral blood immune cells in a cohort of 54 cutaneous Leishmania major subjects treated with SSG or TM. Multiparameter flow cytometry, lymphoproliferative assays and cytokine production were analyzed in order to investigate the differences in the immune responses of subjects before, on and after treatment.

Results

Healing cutaneous leishmaniasis lead to a significant decline in circulating T cells and NKT-like cells, accompanied by an expansion in NK cells, regardless of treatment modality. Functional changes involved decreased antigen specific CD4⁺ T cell proliferation (hyporesponsiveness) seen with CD8⁺ T cell depletion. Moreover, the healing (or healed) state was characterized by fewer circulating regulatory T cells, reduced IFN-γ production and an overall contraction in polyfunctional CD4⁺ T cells.

Conclusion

Healing from cutaneous Leishmaniasis is a dynamic process that alters circulating lymphocyte populations and subsets of T, NK and NKT-like cells. Immunology of healing, through local or systemic treatments, culminated in similar changes in frequency, quality, and antigen specific responsiveness with immunomodulation possibly via a CD8⁺ T cell dependent mechanism. Understanding the evolving immunologic changes during healing of human leishmaniasis informs protective immune mechanisms.