Effect of Autoimmune Cell Therapy on Immune Cell Content in Patients with COPD: A Randomized Controlled Trial.

OBJECTIVE: To explore the effect of autoimmune cell therapy on immune cells in patients with chronic obstructive pulmonary disease (COPD) and to provide a reference for clinical treatment of COPD. METHODS: Sixty patients with stable COPD were randomly divided into control group and treatment group (n = 30). The control group was given conventional treatment, and the treatment group was given one autoimmune cell therapy on the basis of conventional treatment. The serum levels of CD3+ T cells, CD4+ T cells, CD8+ cells, B cells, and NK cells in the peripheral blood were detected by flow cytometry. Possible adverse reactions were detected at any time during treatment. RESULTS: There were no significant differences in the contents of CD3+ T cells, CD4+ T cells, CD8+ cells, B cells, and NK cells in the serum of the control group (P > 0.05). Compared with before treatment, the contents of CD3+ T cells, CD4+ T cells, CD8+ cells, B cells, and NK cells in the serum of the treatment group were significantly increased (P < 0.05). The ratio of CD4 + /CD8+ T cells in both control and treatment groups did not change significantly during treatment (P > 0.05). There were no significant differences in serum CD3+ T cells, CD4+ T cells, CD8+ cells, B cells, and NK cells in the treatment group at 30 days and 90 days after treatment (P > 0.05), but they were significantly higher than those in the control group (P < 0.05). CONCLUSION: Autoimmune cell therapy can significantly increase the level of immune cells in the body and can be maintained for a long period of time, which has certain clinical benefits for recurrent respiratory tract infections and acute exacerbation in patients with COPD.

Ex vivo isolation, expansion and bioengineering of CCR7+CD95-/or CD62L+CD45RA+ tumor infiltrating lymphocytes from acute myeloid leukemia patients' bone marrow.

T cell based immunotherapies can be applicable to acute myeloid leukemia (AML). Therefore, the selection of optimal T cells, cell manufacturing, and therapeutic T cell engineering are essential for the development of effective adoptive T cell therapies for AML. Autologous tumor-infiltrating lymphocytes (TILs) have been in clinical trials to treat solid malignancies. Herein, we assessed whether TILs can be isolated from the bone marrow (BM) of AML patients, expanded ex vivo and utilized as a novel therapeutic strategy for AML. To this end, firstly we analyzed the immunophenotypes of a series of primary BM samples from AML patients (N = 10) by flow cytometry. We observed a variable amount of CD3+ TILs (range ∼2.3-∼32.6% of mononuclear cells) among BM samples. We then developed a novel protocol that produced a three-log ex vivo expansion of TILs isolated from AML patient BM (N = 10) and peripheral blood (PB) (N = 10), including from patients with a low number of CD3+ T cells, within 3, 4 weeks. Further, we identified previously described naïve T cells (CCR7+CD95-/or CD62L+CD45RA+) in AML BM and PB samples, which seemed to be required for a successful TILs ex vivo expansion. Finally, we showed that the expanded TILs could: (1) cause cytotoxicity to autologous AML blasts ex vivo (90.6% in control without T cell treatment vs. 1.89% in experimental groups with PB derived T cells and 1.77% in experimental groups with BM derived TILs, p < 0.01), (2) be genetically engineered to express CYP27B1 gene, and (3) infiltrate the BM and reside in close proximity to pre-injected autologous AML blasts of engrafted immunodeficiency mice. Altogether, these results provide a rationale for further studies of the therapeutic use of TILs in AML.

Targeting regulator of G protein signaling 1 in tumor-specific T cells enhances their trafficking to breast cancer.

Reduced infiltration of anti-tumor lymphocytes remains a major cause of tumor immune evasion and is correlated with poor cancer survival. Here, we found that upregulation of regulator of G protein signaling (RGS)1 in helper TH1 cells and cytotoxic T lymphocytes (CTLs) reduced their trafficking to and survival in tumors and was associated with shorter survival of patients with breast and lung cancer. RGS1 was upregulated by type II interferon (IFN)-signal transducer and activator of transcription (STAT)1 signaling and impaired trafficking of circulating T cells to tumors by inhibiting calcium influx and suppressing activation of the kinases ERK and AKT. RGS1 knockdown in adoptively transferred tumor-specific CTLs significantly increased their infiltration and survival in breast and lung tumor grafts and effectively inhibited tumor growth in vivo, which was further improved when combined with programmed death ligand (PD-L)1 checkpoint inhibition. Our findings reveal RGS1 is important for tumor immune evasion and suggest that targeting RGS1 may provide a new strategy for tumor immunotherapy.

Intranasal Nanoparticle Vaccination Elicits a Persistent, Polyfunctional CD4 T Cell Response in the Murine Lung Specific for a Highly Conserved Influenza Virus Antigen That Is Sufficient To Mediate Protection from Influenza Virus Challenge.

Lung-localized CD4 T cells play a critical role in the control of influenza virus infection and can provide broadly protective immunity. However, current influenza vaccination strategies primarily target influenza hemagglutinin (HA) and are administered peripherally to induce neutralizing antibodies. We have used an intranasal vaccination strategy targeting the highly conserved influenza nucleoprotein (NP) to elicit broadly protective lung-localized CD4 T cell responses. The vaccine platform consists of a self-assembling nanolipoprotein particle (NLP) linked to NP with an adjuvant. We have evaluated the functionality, in vivo localization, and persistence of the T cells elicited. Our study revealed that intranasal vaccination elicits a polyfunctional subset of lung-localized CD4 T cells that persist long term. A subset of these lung CD4 T cells localize to the airway, where they can act as early responders following encounter with cognate antigen. Polyfunctional CD4 T cells isolated from airway and lung tissue produce significantly more effector cytokines IFN-γ and TNF-α, as well as cytotoxic functionality. When adoptively transferred to naive recipients, CD4 T cells from NLP:NP-immunized lung were sufficient to mediate 100% survival from lethal challenge with H1N1 influenza virus. IMPORTANCE Exploiting new, more efficacious strategies to potentiate influenza virus-specific immune responses is important, particularly for at-risk populations. We have demonstrated the promise of direct intranasal protein vaccination to establish long-lived immunity in the lung with CD4 T cells that possess features and positioning in the lung that are associated with both immediate and long-term immunity, as well as demonstrating direct protective potential.

Treg and IL-1 receptor type 2-expressing CD4+ T cell-deleted CD4+ T cell fraction prevents the progression of age-related hearing loss in a mouse model.

We investigated the association between cellular immunity and age-related hearing loss (ARHL) development using three CD4+ T cell fractions, namely, naturally occurring regulatory T cells (Treg), interleukin 1 receptor type 2-expressing T cells (I1R2), and non-Treg non-I1R2 (nTnI) cells, which comprised Treg and I1R2-deleted CD4+ T cells. Inoculation of the nTnI fraction into a ARHL murine model, not only prevented the development of ARHL and the degeneration of spiral ganglion neurons, but also suppressed serum nitric oxide, a source of oxidative stress. Further investigations on CD4+ T cell fractions could provide novel insights into the prevention of aging, including presbycusis.

Non-transplantable cord blood units as a source for adoptive immunotherapy of leukaemia and a paradigm of circular economy in medicine.

Advances in immunotherapy with T cells armed with chimeric antigen receptors (CAR-Ts), opened up new horizons for the treatment of B-cell lymphoid malignancies. However, the lack of appropriate targetable antigens on the malignant myeloid cell deprives patients with refractory acute myeloid leukaemia of effective CAR-T therapies. Although non-engineered T cells targeting multiple leukaemia-associated antigens [i.e. leukaemia-specific T cells (Leuk-STs)] represent an alternative approach, the prerequisite challenge to obtain high numbers of dendritic cells (DCs) for large-scale Leuk-ST generation, limits their clinical implementation. We explored the feasibility of generating bivalent-Leuk-STs directed against Wilms tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) from umbilical cord blood units (UCBUs) disqualified for allogeneic haematopoietic stem cell transplantation. By repurposing non-transplantable UCBUs and optimising culture conditions, we consistently produced at clinical scale, both cluster of differentiation (CD)34+ cell-derived myeloid DCs and subsequently polyclonal bivalent-Leuk-STs. Those bivalent-Leuk-STs contained CD8+ and CD4+ T cell subsets predominantly of effector memory phenotype and presented high specificity and cytotoxicity against both WT1 and PRAME. In the present study, we provide a paradigm of circular economy by repurposing unusable UCBUs and a platform for future banking of Leuk-STs, as a 'third-party', 'off-the-shelf' T-cell product for the treatment of acute leukaemias.

Identification of Follicular T-Cell Subsets in Murine and Human Tissues.

Follicular helper T (Tfh) cells play a key role in B cell activation and differentiation. Within recent years, distinct subsets of follicular T cells, including regulatory and cytotoxic T cells, have been identified. Apart from classical Tfh cells in secondary lymphoid organs, Tfh-like cells are found in chronically inflamed nonlymphoid tissues. Here, we provide protocols to identify different follicular T cell subsets in murine and human tissues by flow cytometry. This chapter also contains an immunization protocol for the induction of large numbers of Tfh cells in mice.

Analysis of T-Cells in Inflamed Nonlymphoid Tissues.

Under chronic inflammatory conditions, T and B cells are frequently found in nonlymphoid tissues. We recently identified a follicular helper-like T cell population in inflamed lung tissue, which drives the local differentiation of antigen-specific B cells into germinal center-like cells and plasma blasts. Here, we describe a lung inflammation mouse model, which is ideally suited to analyze antigen-specific T and B cells in secondary lymphoid organs and inflamed nonlymphoid tissue in parallel.

Separating the wheat from the chaff: Making sense of Treg heterogeneity for better adoptive cellular therapy.

Regulatory T (Treg) cells are essential for immunological tolerance and can be used to suppress unwanted or excessive immune responses through adoptive cellular therapy. It is increasingly clear that many subsets of Treg cells exist, which have different functions and reside in different locations. Treg cell therapies may benefit from tailoring the selected subset to the tissue that must be protected as well as to characteristics of the immune response that must be suppressed, but little attention is given to this topic in current therapies. Here, we will discuss how three major axes of heterogeneity can be discerned among the Treg cell population, which determine function and lineage fidelity. A first axis relates to the developmental route, as Treg cells can be generated from immature T cells in the thymus or from already mature Tconv cells in the immunological periphery. Heterogeneity furthermore stems from activation history (naïve or effector) and location (lymphoid or peripheral tissues). Each of these axes bestows specific properties on Treg cells, which are further refined by additional processes leading to yet further variation. A critical aspect impacting on Treg cell heterogeneity is TCR specificity, which determines when and where Treg cells are generated as well as where they exhibit their effector functions. We will discuss the implications of this heterogeneity and the role of the TCR for the design of next generation adoptive cellular therapy with Treg cells.

A Functionally Distinct CXCR3+/IFN-γ+/IL-10+ Subset Defines Disease-Suppressive Myelin-Specific CD8 T Cells.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS. We have previously demonstrated that CNS-specific CD8 T cells possess a disease-suppressive function in MS and variations of its animal model, experimental autoimmune encephalomyelitis (EAE), including the highly clinically relevant relapsing-remitting EAE disease course. Regulatory CD8 T cell subsets have been identified in EAE and other autoimmune diseases, but studies vary in defining phenotypic properties of these cells. In relapsing-remitting EAE, PLP178-191 CD8 T cells suppress disease, whereas PLP139-151 CD8 T cells lack this function. In this study, we used this model to delineate the unique phenotypic properties of CNS-specific regulatory PLP178-191 CD8 T cells versus nonregulatory PLP139-151 or OVA323-339 CD8 T cells. Using multiparametric flow cytometric analyses of phenotypic marker expression, we identified a CXCR3+ subpopulation among activated regulatory CD8 T cells, relative to nonregulatory counterparts. This subset exhibited increased degranulation and IFN-γ and IL-10 coproduction. A similar subset was also identified in C57BL/6 mice within autoregulatory PLP178-191 CD8 T cells but not within nonregulatory OVA323-339 CD8 T cells. This disease-suppressing CD8 T cell subpopulation provides better insights into functional regulatory mechanisms, and targeted enhancement of this subset could represent a novel immunotherapeutic approach for MS.

Distinct metabolic programs established in the thymus control effector functions of γδ T cell subsets in tumor microenvironments.

Metabolic programming controls immune cell lineages and functions, but little is known about γδ T cell metabolism. Here, we found that γδ T cell subsets making either interferon-γ (IFN-γ) or interleukin (IL)-17 have intrinsically distinct metabolic requirements. Whereas IFN-γ+ γδ T cells were almost exclusively dependent on glycolysis, IL-17+ γδ T cells strongly engaged oxidative metabolism, with increased mitochondrial mass and activity. These distinct metabolic signatures were surprisingly imprinted early during thymic development and were stably maintained in the periphery and within tumors. Moreover, pro-tumoral IL-17+ γδ T cells selectively showed high lipid uptake and intracellular lipid storage and were expanded in obesity and in tumors of obese mice. Conversely, glucose supplementation enhanced the antitumor functions of IFN-γ+ γδ T cells and reduced tumor growth upon adoptive transfer. These findings have important implications for the differentiation of effector γδ T cells and their manipulation in cancer immunotherapy.

CD160 Plays a Protective Role During Chronic Infection by Enhancing Both Functionalities and Proliferative Capacity of CD8+ T Cells.

The understanding of protective immunity during HIV infection remains elusive. Here we showed that CD160 defines a polyfunctional and proliferative CD8+ T cell subset with a protective role during chronic HIV-1 infection. CD160+ CD8+ T cells derived from HIV+ patients correlated with slow progressions both in a cross-sectional study and in a 60-month longitudinal cohort, displaying enhanced cytotoxicity and proliferative capacity in response to HIV Gag stimulation; triggering CD160 promoted their functionalities through MEK-ERK and PI3K-AKT pathways. These observations were corroborated by studying chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. The genetic ablation of CD160 severely impaired LCMV-specific CD8+ T cell functionalities and thereby resulted in loss of virus control. Interestingly, transcriptional profiling showed multiple costimulatory and survival pathways likely to be involved in CD160+ T cell development. Our data demonstrated that CD160 acts as a costimulatory molecule positively regulating CD8+ T cells during chronic viral infections, thus representing a potential target for immune intervention.

Sublingual Omp16-driven redirection of the allergic intestinal response in a pre-clinical model of food allergy.

BACKGROUND: IgE-mediated food allergy remains a significant and growing worldwide problem. Sublingual immunotherapy (SLIT) shows an excellent safety profile for food allergy, but the clinical efficacy needs to be improved. This study assessed the effects of the Toll-like receptor 4 agonist outer membrane protein (Omp) 16 from Brucella abortus combined with cow´s milk proteins (CMP) through the sublingual route to modulate cow's milk allergy in an experimental model. METHODS: Mice sensitized with cholera toxin and CMP were orally challenged with the allergen to elicit hypersensitivity reactions. Then, mice were treated with a very low amount of CMP along with Omp16 as a mucosal adjuvant, and finally, animals were re-exposed to CMP. Systemic and mucosal immune parameters were assessed in vivo and in vitro. RESULTS: We found that the sublingual administration of Omp16 + CMP induced a buccal Th1 immune response that modulated the intestinal allergic response with the suppression of symptoms, reduction of IgE and IL-5, and up-regulation of IgG2a and IFN-γ. The adoptive transfer of submandibular IFN-γ-producing α4ß7+ CD4+ and CD8+ cells conferred protection against allergic sensitization. The use of Omp16 + CMP promoted enhanced protection compared to CMP alone. CONCLUSION: In conclusion, Omp16 represents a promising mucosal adjuvant that can be used to improve the clinical and immune efficacy of SLIT for food allergy.

TCR+CD4-CD8- (double negative) T cells protect from cisplatin-induced renal epithelial cell apoptosis and acute kidney injury.

Acute kidney injury (AKI) due to cisplatin is a significant problem that limits its use as an effective chemotherapeutic agent. T cell receptor+CD4-CD8- double negative (DN) T cells constitute the major T cell population in the human and mouse kidney, express programmed cell death protein (PD)-1, and protect from ischemic AKI. However, the pathophysiological roles of DN T cells in cisplatin-induced AKI is unknown. In this study, wild-type mice were treated with cisplatin (30 mg/kg) or vehicle, and the effects on kidney DN T cell numbers and function were measured. In vitro experiments evaluated effects of kidney DN T cells on cisplatin-induced apoptosis and PD ligand 1 (PD-L1) in renal epithelial cells. Adoptive transfer experiments assessed the therapeutic potential of DN T cells during cisplatin-induced AKI. Our results show that kidney DN T cell population increased at 24 h and declined by 72 h after cisplatin treatment. Cisplatin treatment increased kidney DN T cell proliferation, apoptosis, CD69, and IL-10 expression, whereas CD62L, CD44, IL-17A, interferon-γ, and TNF-α were downregulated. Cisplatin treatment decreased both PD-1 and natural killer 1.1 subsets of kidney DN T cells with a pronounced effect on the PD-1 subset. In vitro kidney DN T cell coculture decreased cisplatin-induced apoptosis in kidney proximal tubular epithelial cells, increased Bcl-2, and decreased cleaved caspase 3 expression. Cisplatin-induced expression of PD ligand 1 was reduced in proximal tubular epithelial cells cocultured with DN T cells. Adoptive transfer of DN T cells attenuated kidney dysfunction and structural damage from cisplatin-induced AKI. These results demonstrate that kidney DN T cells respond rapidly and play a protective role during cisplatin-induced AKI.

Emerging Approaches for Regulation and Control of CAR T Cells: A Mini Review.

Chimeric antigen receptor (CAR) T cells have emerged as a promising treatment for patients with advanced B-cell cancers. However, widespread application of the therapy is currently limited by potentially life-threatening toxicities due to a lack of control of the highly potent transfused cells. Researchers have therefore developed several regulatory mechanisms in order to control CAR T cells in vivo. Clinical adoption of these control systems will depend on several factors, including the need for temporal and spatial control, the immunogenicity of the requisite components as well as whether the system allows reversible control or induces permanent elimination. Here we describe currently available and emerging control methods and review their function, advantages, and limitations.

Adoptive T-Cell Transfer to Treat Lymphangioleiomyomatosis.

Patients with lymphangioleiomyomatosis (LAM) develop pulmonary cysts associated with neoplastic, smooth muscle-like cells that feature neuroendocrine cell markers. The disease preferentially affects premenopausal women. Existing therapeutics do not cure LAM. As gp100 is a diagnostic marker expressed by LAM lesions, we proposed to target this immunogenic glycoprotein using TCR transgenic T cells. To reproduce the genetic mutations underlying LAM, we cultured Tsc2-/- kidney tumor cells from aged Tsc2 heterozygous mice and generated a stable gp100-expressing cell line by lentiviral transduction. T cells were isolated from major histocompatibility complex-matched TCR transgenic pmel-1 mice to measure cytotoxicity in vitro, and 80% cytotoxicity was observed within 48 hours. Antigen-specific cytotoxicity was likewise observed using pmel-1 TCR-transduced mouse T cells, suggesting that transgenic T cells may likewise be useful to treat LAM in vivo. On intravenous injection, slow-growing gp100+ LAM-like cells formed lung nodules that were readily detectable in severe combined immunodeficient/beige mice. Adoptive transfer of gp100-reactive but not wild-type T cells into mice significantly shrunk established lung tumors, even in the absence of anti-PD-1 therapy. These results demonstrate the treatment potential of adoptively transferred T cells to eliminate pulmonary lesions in LAM.

Metcalf Lecture Award: Applying niche biology to engineer T-cell regenerative therapies.

The processes generating cells of adaptive immunity render them less amenable to the single cytokine signals used so effectively to regenerate myeloid cells. T-cell neogenesis begins in the bone marrow, where specific sets of late osteolineage cells govern the specification of hematopoietic cells capable of migrating to the thymus where differentiation is completed. Osteocalcin-expressing bone marrow stromal cells producing Dll4 serve as a progenitor niche enabling this T-competent cell production. Biocompatible alginate-based cryogels containing bone morphogenetic proteins (BMP-2) and the Notch ligand Dll4 were engineered to recapitulate the endogenous niche. These cryogels are highly pliable and can be injected under the skin of animals undergoing bone marrow transplantation. The result in mice is an ectopic niche fostering T-competent progenitor generation that results in improved T-cell numbers and receptor diversity. The recipients can generate neoantigen vaccine responses while having improved tolerance manifest by reduced graft-versus-host disease upon allogeneic transplant. Through emerging details of niches in the bone marrow, therapeutics more complex than those necessary for myeloid reconstitution are possible. Niche biology-guided bioengineered design offers the possibility of regenerative therapies for T lymphoid cells.

Humanized mice in cutaneous leishmaniasis-Suitability analysis of human PBMC transfer into immunodeficient mice.

Humanized mice represent a suitable preclinical test system for example therapeutic interventions in various disease settings, including infections. Here, we intended to establish such system for cutaneous leishmaniasis by infecting T, B and NK cell-deficient mice adoptively transferred with human peripheral blood mononuclear cells (PBMC). L major infection led to the establishment of parasite lesions harbouring viable parasites and human T cells, but parasite elimination was not seen due to a species-specific activity of T cell-derived human IFNγ. In addition, up to 50% of infected mice succumbed to severe graft-versus-host disease. In summary, even though long-term disease outcome assessments are impossible, this model of humanized mice can be used for studying lesion development and generation of oligoclonal anti-parasite human T cell responses in vivo.