Immunotherapeutic potential of blinatumomab-secreting γ9δ2 T Cells.

Previous studies have explored the use of engineered blinatumomab-secreting autologous αß T cells for CD19-targeted cancer therapy. To create a more flexible allogeneic delivery system, we utilized γ9δ2 T cells rather than αß T cells in a similar application. First, we showed that γ9δ2 T cells could serve as effector cells for blinatumomab, and these effector memory cells could survive for at least 7 days after infusion. The genetically modified blinatumomab-secreting γ9δ2 T cells induced significant cytotoxicity in CD19+ tumor cell lines and primary cells from chronic lymphocytic leukemia patients. Of note, blinatumomab-secreting γ9δ2 T cells might also exhibit dual-targeting of CD19 and isopentenyl pyrophosphate, a universal tumor-associated antigen. Furthermore, blinatumomab-secreting γ9δ2 T cells killed CD19-transfected adherent cells, suggesting that the γ9δ2 T cells might be effective for treating solid tumors with appropriate cancer antigens. Together, these results demonstrate the promise of blinatumomab-secreting γ9δ2 T cells as a cancer therapy.

The potential of adoptive transfer of γ9δ2 T cells to enhance blinatumomab's antitumor activity against B-cell malignancy.

Blinatumomab, a bispecific T cell engager (BiTE) antibody targeting CD19 and CD3ε, can redirect T cells toward CD19-positive tumor cells and has been approved to treat relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL). However, chemotherapeutic regimens can severely reduce T cells' number and cytotoxic function, leading to an inadequate response to blinatumomab treatment in patients. In addition, it was reported that a substantial portion of R/R B-ALL patients failing blinatumomab treatment had the extramedullary disease, indicating the poor ability of blinatumomab in treating extramedullary disease. In this study, we investigated whether the adoptive transfer of ex vivo expanded γ9δ2 T cells could act as the effector of blinatumomab to enhance blinatumomab's antitumor activity against B-cell malignancies in vivo. Repeated infusion of blinatumomab and human γ9δ2 T cells led to more prolonged survival than that of blinatumomab or human γ9δ2 T cells alone in the mice xenografted with Raji cells. Furthermore, adoptive transfer of γ9δ2 T cells reduced tumor mass outside the bone marrow, indicating the potential of γ9δ2 T cells to eradicate the extramedullary disease. Our results suggest that the addition of γ9δ2 T cells to the blinatumomab treatment regimens could be an effective approach to enhancing blinatumomab's therapeutic efficacy. The concept of this strategy may also be applied to other antigen-specific BiTE therapies for other malignancies.

Human acellular cartilage matrix powders as a biological scaffold for cartilage tissue engineering with synovium-derived mesenchymal stem cells.

In our previous study, we found that cartilage fragments from osteoarthritic knee promoted chondrogenesis of mesenchymal stem cells. In this study, we further transformed the cartilage tissues into acellular cartilage matrix (ACM) and explored the feasibility of using ACM as a biological scaffold. Nonworn parts of cartilage tissues were obtained during total knee arthroplasty (TKA) surgery and were successfully fabricated into ACM powders. The ACM powders and human synovium-derived mesenchymal stem cells (SMSCs) were mixed into collagen gel for in vitro culture. Histological results showed a synergistic effect of ACM powders and chondrogenic growth factors in the formation of engineered cartilage. The findings of real-time polymerase chain reaction (PCR) suggested that ACM powders had the potential of promoting type II collagen gene expression in the growth factors-absent environment. Moreover, with growth factors induction, the ACM powders could reduce the hypertrophy in chondrogenesis of SMSCs. In summary, ACM powders could serve as a functional scaffold that benefited the chondrogenesis of SMSCs for cartilage tissue engineering.

Cartilage fragments from osteoarthritic knee promote chondrogenesis of mesenchymal stem cells without exogenous growth factor induction.

Extracellular matrix (ECM) is thought to participate significantly in guiding the differentiation process of mesenchymal stem cells (MSCs). In this study, we hypothesized that cartilage fragments from osteoarthritic knee could promote chondrogenesis of MSCs. Nonworn parts of cartilage tissues were obtained during total knee arthroplasty (TKA) surgery. Cartilage fragments and MSCs were wrapped into fibrin glue; and the constructs were implanted subcutaneously into nude mice. Histological analysis showed neocartilage-like structure with positive Alcian blue staining in the cartilage fragment-fibrin-MSC constructs. However, constructs with only MSCs in fibrin showed condensed appearance like MSCs in the pellet culture. Gene expression of type II collagen in the constructs with 60 mg cartilage fragments were significantly elevated after 4 weeks of implantation. Conversely, the constructs without cartilage fragments failed to express type II collagen, which indicated MSCs did not differentiate into a chondrogenic lineage. In conclusion, we demonstrated the effect of cartilage fragments from osteoarthritic knee in promoting chondrogenic differentiation of MSCs. This may be a favorable strategy for MSC chondrogenesis without exogenous growth factor induction.

Ex vivo expansion of dendritic-cell-activated antigen-specific CD4+ T cells with anti-CD3/CD28, interleukin-7, and interleukin-15: potential for adoptive T cell immunotherapy.

There is an increasing realization that the failure of adoptive therapy with cytotoxic T lymphocytes in the autologous setting, at least in part, results from the lack of help from antigen-specific CD4+ T cells. To incorporate these cells into this treatment strategy, it is not known whether currently used ex vivo culture conditions are adequate for expanding and charting these T cells with the desired qualities for optimal in vivo activity. In this study, we show that stimulation with agonistic antibodies to CD3 plus CD28 (anti-CD3/CD28), a commonly used method for CD4+ T cell expansion, is unable to expand dendritic-cell-activated hepatitis B virus (HBV)-specific CD4+ T cells to clinical relevant numbers. Whereas, in combination with interleukin(IL)-7 and IL-15, it leads to a 4000-fold expansion of HBV-specific CD4+ T cells in 2 weeks. This outcome is correlated with the anti-apoptosis effect of IL-7 and IL-15. Importantly, antigen specificity is preserved during expansion. Although a late addition of IL-2 to the anti-CD3/CD28-expanding cultures also results in robust expansion, this expansion condition renders HBV-specific CD4+ T cells more sensitive to cytokine withdrawal-, activation-, and transforming growth factor-beta-induced cell death compared to those expanded in IL-7 and IL-15. Moreover, NKG2D rather than 4-1BB, whose ligands are constitutively expressed on tumor cells, is significantly up-regulated on IL-7/IL-15-expanded HBV-specific CD4+ T cells, and its engagement promotes expansion and interferon-gamma production by these cells and thus may serve to provide co-stimulation to T cells once they reach tumor tissues. Collectively, these results may have important therapeutic implications for adoptive T cell therapy.

Transforming growth factor-beta induces apoptosis in antigen-specific CD4+ T cells prepared for adoptive immunotherapy.

Transforming growth factor-beta (TGF-beta), found at the site of most tumors, has been recognized as one of the mechanisms involved in tumor immunological escape. To evaluate its impact on adoptive immunotherapy against cancer, we examined the susceptibility of tumor-specific T cells to TGF-beta in the setting of these T cells being prepared for adoptive transfer. Hepatitis B virus (HBV)-specific CD4(+) T cells were ex vivo generated by activating with HBV-transfected dendritic cells and selecting with antibodies to CD25 activation molecules, and then expanded with antibodies to CD3/CD28. These T cells expressed higher levels of the type II TGF-beta receptor than nai;ve T cells and exhibited enhanced apoptosis when exposed to TGF-beta. The underlying apoptotic pathway was linked to the dissipation of the mitochondrial inner membrane potential and activation of caspase-9. The absence of caspase-8 activity in TGF-beta-treated T cells suggests that the death receptor system may not be involved in this type of apoptosis. Interleukin-2 (IL-2), which is concomitantly administered with tumor-specific T cells in adoptive immunotherapy, was unable to protect HBV-specific CD4(+) T cells from the pro-apoptotic effect of TGF-beta when added simultaneously with TGF-beta. Interesting, IL-2-pretreated T cells displayed the type II TGF-beta receptor at lower levels and were more resistant to TGF-beta. Together, our findings indicate that the effectiveness of adoptive cancer immunotherapy may be impaired by tumor-derived TGF-beta and appropriate manipulation of exogenous IL-2 might overcome this hurdle.