Challenges of NK cell-based immunotherapy in the new era / 医学前沿

Natural killer cells (NKs) have a great potential for cancer immunotherapy because they can rapidly and directly kill transformed cells in the absence of antigen presensitization. Various cellular sources, including peripheral blood mononuclear cells (PBMCs), stem cells, and NK cell lines, have been used for producing NK cells. In particular, NK cells that expanded from allogeneic PBMCs exhibit better efficacy than those that did not. However, considering the safety, activities, and reliability of the cell products, researchers must develop an optimal protocol for producing NK cells from PBMCs in the manufacture setting and clinical therapeutic regimen. In this review, the challenges on NK cell-based therapeutic approaches and clinical outcomes are discussed.

Targeting the epitope spreader Pep19 by naïve human CD45RA⁺ regulatory T cells dictates a distinct suppressive T cell fate in a novel form of immunotherapy

PURPOSE: Beyond the limited scope of non-specific polyclonal regulatory T cell (Treg)-based immunotherapy, which depends largely on serendipity, the present study explored a target Treg subset appropriate for the delivery of a novel epitope spreader Pep19 antigen as part of a sophisticated form of immunotherapy with defined antigen specificity that induces immune tolerance. METHODS: Human polyclonal CD4⁺CD25⁺CD127(lo−) Tregs (127-Tregs) and naïve CD4⁺CD25⁺CD45RA⁺ Tregs (45RA-Tregs) were isolated and were stimulated with target peptide 19 (Pep19)-pulsed dendritic cells in a tolerogenic milieu followed by ex vivo expansion. Low-dose interleukin-2 (IL-2) and rapamycin were added to selectively exclude the outgrowth of contaminating effector T cells (Teffs). The following parameters were investigated in the expanded antigen-specific Tregs: the distinct expression of the immunosuppressive Treg marker Foxp3, epigenetic stability (demethylation in the Treg-specific demethylated region), the suppression of Teffs, expression of the homing receptors CD62L/CCR7, and CD95L-mediated apoptosis. The expanded Tregs were adoptively transferred into an NOD/scid/IL-2Rγ(−/−) mouse model of collagen-induced arthritis. RESULTS: Epitope-spreader Pep19 targeting by 45RA-Tregs led to an outstanding in vitro suppressive T cell fate characterized by robust ex vivo expansion, the salient expression of Foxp3, high epigenetic stability, enhanced T cell suppression, modest expression of CD62L/CCR7, and higher resistance to CD95L-mediated apoptosis. After adoptive transfer, the distinct fate of these T cells demonstrated a potent in vivo immunotherapeutic capability, as indicated by the complete elimination of footpad swelling, prolonged survival, minimal histopathological changes, and preferential localization of CD4⁺CD25⁺ Tregs at the articular joints in a mechanistic and orchestrated way. CONCLUSIONS: We propose human naïve CD4⁺CD25⁺CD45RA⁺ Tregs and the epitope spreader Pep19 as cellular and molecular targets for a novel antigen-specific Treg-based vaccination against collagen-induced arthritis.

Alveolar macrophages modulate allergic inflammation in a murine model of asthma

The role of alveolar macrophages (AMs) in the pathogenesis of asthma is still unknown. The aim of the present study was to investigate the effects of AM in the murine model of asthma. AMs were selectively depleted by liposomes containing clodronate just before allergen challenges, and changes in inflammatory cells and cytokine concentrations in bronchoalveolar lavage (BAL) fluid were measured. AMs were then adoptively transferred to AM-depleted sensitized mice and changes were measured. Phenotypic changes in AMs were evaluated after in vitro allergen stimulation. AM-depletion after sensitization significantly increased the number of eosinophils and lymphocytes and the concentrations of IL-4, IL-5 and GM-CSF in BAL fluid. These changes were significantly ameliorated only by adoptive transfer of unsensitized AMs, not by sensitized AMs. In addition, in vitro allergen stimulation of AMs resulted in their gaining the ability to produce inflammatory cytokines, such as IL-1beta, IL-6 and TNF-alpha, and losing the ability to suppress GM-CSF concentrations in BAL fluid. These findings suggested that AMs worked probably through GM-CSF-dependent mechanisms, although further confirmatory experiments are needed. Our results indicate that the role of AMs in the context of airway inflammation should be re-examined.

Selective addition of CXCR3+CCR4-CD4+ Th1 cells enhances generation of cytotoxic T cells by dendritic cells in vitro

Increasing importance is being given to the stimulation of Th1 response in cancer immunotherapy because its presence can shift the direction of adaptive immune responses toward protective immunity. Based on chemokine receptor expression, CXCR3+CCR4-CD4+ T cells as Th1-type cells were investigated its capacity in monocyte-derived dendritic cell (DC) maturation and polarization, and induction of antigen specific cytotoxic T lymphocytes (CTL) in vitro. The levels of IL-4, IL-5 and IL-10 were decreased to the basal level compared with high production of IFN-gamma, TNF-alpha, and IL-2 in CXCR3+CCR4-CD4+ T cells stimulated with anti-CD3 and anti-CD28 antibodies. Co-incubation of activated CD4+ or CXCR3+CCR4-CD4+ T cells with DC (CD4+/DC or CXCR3+CD4+/DC, respectively) particularly up-regulated IL-12 and CD80 expression compared with DC matured with TNF-alpha and LPS (mDC). Although there was no significant difference between the effects of the CXCR3+CCR4-CD4+ and CD4+ T cells on DC phenotype expression, CXCR3+CD4+/DC in CTL culture were able to expand number of CD8+ T cells and increased frequencies of IFN-gamma secreting cells and overall cytolytic activity against tumor antigen WT-1. These results demonstrated that the selective addition of CXCR3+CCR4-CD4+ T cells to CTL cultures could enhance the induction of CTLs by DC in vitro, and implicated on a novel strategy for adoptive T cell therapy.

Adoptive Transfer of Colon Cancer Derived Peptide-specific CD8+ T Cells in HHD Mice

BACKGROUND: 1-8D gene is a member of human 1-8 interferon inducible gene family and is shown to be overexpressed in fresh colon cancer tissues. Three peptides 1-6, 3-5 and 3-7 derived from 1-8D gene were shown to have immunogenicity against colon cancer. METHODS: To study tumor immunotherapy of these peptides we established an adoptive transfer model. D(b-/-)Xbeta2 microglobulin (beta2m) null mice transgenic for a chimeric HLA-A2.1/D(b)-beta2m single chain (HHD mice) were immunized with irradiated peptide-loaded RMA-S/HHD/B7.1 transfectants. Spleens were removed after last immunization, and splenocytes were re-stimulated in vitro. Lymphocytes from vaccinated HHD mice were transferred together with IL-2 to the tumor bearing nude mice that were challenged S.C. with the HCT/HHD/B7 colon carcinoma cell line that was found to grow in these mice. RESULTS: Peptide 3-5 was found to be highly effective in CTL activity. Adoptively transferred anti-peptide 3-5 cytolytic T lymphocytes caused significant retardation in tumor growth. CONCLUSION: This study shows that peptide 3-5 can be the most effective candidate for the vaccine of adoptive immunotherapy against colon cancer.

Study on a type Ⅱ collagen-specific T cell line and the pathogenesis of arthritis / 中华风湿病学杂志

Objective To establish the type Ⅱ collagen specific T cell line of Wistar rat and observe its effect on transferring arthritis.Methods The Wistar rats were immunized with emulsified chicken type Ⅱ collagen (CCⅡ) in complete Freund′s adjuvant by intradermal injection to induce the rat model of collagen induced arthritis (CIA).The lymphocytes were obtained from mesenteric lymph nodes of CIA rats,and the type Ⅱ collagen reactive T cell line was selected and propagated by CCⅡ stimulating in vitro .The proliferation response and phenotype were analyzed by 3 H TdR incorporation and fluorescence activated cell sorter (FACS).The onset of arthritis and pathological characteristic in ankle joints of recipient rats were observed with naked eye and histochemical examination.Anti CCⅡ antibody in serum was assayed by enzyme linked immunosorbent assay (ELISA).Results A T cell line was successfully established.The results of FACS labeled with fluorescent antibodies showed that 98 2% of the line cells were T cells,of which 89 7% were CD4 + T cells.The results of adoptive transfer showed that the incidence of arthritis was 50% when the injected cell number was 5?10 7,meanwhile the level of anti CCⅡ antibody in serum was elevated more than that of the control.Conclusion A cell line has been successfully established.The result of arthritis transferring by T cell line shows that the T cell plays a great role in the pathogenesis of CIA and provides a research datum for rheumatoid arthritis therapy with T cell vaccine.

In vivo clearance of Japanese encephalitis virus by adoptively transferred virus specific cytotoxic T lymphocytes.

Japanese encephalitis virus (JEV) is a positive stranded RNA virus that belongs to the flavivirus group. JEV infection damages the central nervous system (CNS) and is one of the main causative agents of acute encephalitis. H-2 restricted virus-specific cytotoxic T lymphocytes (CTL) have been generated specifically against JEV in our laboratory and these CTL have been shown to protect mice against lethal challenge with JEV. Virus replication was found to be inhibited in the brains of animals that were adoptively transferred with JEV specific CTL as revealed by immunohistological staining as well as viral plaque assays. We further show that virus specific CTL could be recovered from such protected mice as long as 45 days after adoptive transfer.

in vivo and in vitro Anti-Tumor Efficiency by Tumor-Specific CD8+ CTL Induced with Tumor Associated Peptide / 中国肿瘤生物治疗杂志

The aim of the current study was to determine whether tumor-specific T cells can be primed in and obtained from sponge implants loaded with tumor associated peptides. Naive C57BL/6 mice as well as C57BL/6 mice previously primed with FBL-3 tumor cells were implanted with small polyurethane sponges containing FBL-3 gag peptides CCLCLTVFL(gPr80 gag 85-93)or RSPTNLAKV(Pr65 gag p30 131-139). Both FBL-3 gag peptides were shown could bind to H-2 Db molecules. Ten days later, cells that had accumulated in the sponges were harvested, stimulated in vitro with the immunizing peptide, and tested for cytolytic activity against FBL-3 tumor and FBL-3 gag peptides. The results demonstrated that peptide-specific CD8+ CTL could be elicited and obtained from the sponge implants of both naive and immune mice. The FBL-3 gag p85-93 peptide induced CTL could specifically lyse syngeneic targets pulsed with the FBL-3 gag p85-93 peptide as well as FBL-3 tumor. However, the FBL-3 gag p131-139 peptide induced CTL lysed only the FBL-3 gag p131- 139 peptide pulsed syngeneic targets but not the FBL- 3 tumor. Tumor-specific T cells obtained from peptide-loaded sponge implants could be induced to grow to large numbers in vitro by periodic restimulation with the immunizing peptide plus syngeneic APC and low concentrations of IL- 2. Adoptive transfer of the resultant expanded FBL-3 gag p85-93 peptide-induced CTL into mice with disseminated FBL-3 could mediate effective anti-tumor therapy. Thus,in vivo immunization with peptide-loaded sponges provides a potentially useful technique for procuring primed peptide- specific T cells for tumor therapy.

Autoimmune reaction mediates myocardial injury after acute myocardial infarction in rats / 中国病理生理杂志

AIM: The study was designed to explore the autoimmune mechanism of myocardial injury and ventricular remodeling after acute myocardial infarction (AMI). METHODS: An experimental animal model of AMI was adopted in Wistar rats. After 6 weeks, splenocytes were freshly transferred to syngeneic inbred rats. Four weeks later, these recipient rats were anesthetized for hemodynamics analysis by catheter technique. Serum antibody against cardiac myosin heavy chanin (MHC) was screened by ELISA. Histopathological studies were performed on all hearts. The phenotypes of T lymphocytes in myocardium were analyzed by histocytochemistry stain. RESULTS: Histopathological studies showed the lymphocytes infiltration in non-infarction myocardium in AMI rats and the organ specific inflammation of myocardium in all succedent recipient (AMI-T) rats. Histocytochemistry stain revealed the predominant CD4+T cells infiltration in myocardium. The antibody against MHC was examined in 8/22 cases of AMI rats and AMI-T rats, but none in sham-T rats. The left ventricular dysfunction was found in AMI-T rats, which was characterized by slight decline of ~+dp/dt_~max. CONCLUSIONS: The study showed inflammatory response of non-infarction myocardium in AMI rats and demonstrated the lymphocytes-mediated myocardial injury and cardiac dysfunction by adoptive transfer of splenocytes of AMI rats. The autoimmune-mediated myocardial injury might be a novel mechanism of ventricular remodeling after AMI. [