Puncturing the Perforin Problem
Cytotoxic T lymphocyte (CTL) release of perforin from secretory granules into a target cell is a primary immune defense mechanism. Perforin deficiency or dysfunction in humans results in a predisposition to developing immune-mediated diseases and malignancies. Despite the known importance of this molecule in immunity, a reliable method to detect perforin in experimental assays has not been developed. In this issue, Brennan et al. (p. 5744) describe a technique that enables the detection of perforin by confocal microscopy and flow cytometry. They fixed activated OT-I CTLs expressing perforin (Prf1+/+) with Bouin’s solution, a reagent used to preserve cellular morphology and intracellular organelles in immunohistochemistry, and permeabilized these cells in a 0.1% Triton X solution. Short term incubation with an anti-perforin Ab did not result in detectable perforin in fixed/permeabilized cells; however, some, but not all, cells incubated for 16 h with anti-perforin Ab at 4°C displayed a robust intracellular perforin signal when analyzed via confocal microscopy or flow cytometry. Perforin was not detected in cells fixed with paraformaldehyde. In confocal microscopy experiments, perforin was found to colocalize with granzyme B and lysosomal-associated membrane protein 1, and granzyme B and perforin also colocalized at the immune synapse between OT-I Prf1+/+ CTLs and target cells. In flow cytometry experiments, the authors demonstrated that the sensitivity of the perforin staining protocol was sufficient to detect decreased perforin abundance in OT-I Prf1+/− CTLs compared with OT-I Prf1+/+ CTLs. These data suggest that this staining protocol represents a reliable method to detect perforin in CTLs and has the potential to help resolve many unanswered questions in immunology research.
Immunotherapy Enhancement
Epithelial ovarian cancer (EOC) is considered one of the most lethal gynecological cancers and effective treatments are actively being sought. Parente-Pereira et al. (p. 5557) examined the use of immunotherapeutic Vγ9Vδ2 γδ T cells in conjunction with liposome-encapsulated aminobisphosphonates (NBPs) for the treatment of ovarian cancer in hopes of improving the pharmacokinetics of NBPs in this therapeutic context. NBPs cause an accumulation of nonpeptide phosphoantigens (PAgs) in tumor cells, which sensitizes them to destruction by γδ T cells. In this study, liposome-encapsulated zoledronic acid (L-ZA) or liposome-encapsulated alendronic acid (L-AA) sensitized autologous or immortalized ovarian tumor cells to destruction in vitro by Vγ9Vδ2 T cells from healthy donors or ovarian cancer patients. Addition of folate to either of these formulations targeted these molecules to folate receptor-α+ ovarian tumor cells, and further improved their efficacy in vitro. In a mouse model of ovarian cancer, L-ZA was highly toxic, but free AA and L-AA were well tolerated and sensitized ovarian tumors to destruction by infused γδ T cells. I.v. delivery of L-AA was more effective than free AA in sensitizing advanced tumors to γδ T cell destruction, but addition of folic acid to L-AA did not significantly improve its efficacy in vivo. Together, these results suggest that liposome-encapsulated NBPs combined with γδ T cells may represent a promising avenue of immunotherapy for ovarian cancer.
Blanket Attack on HCV
Seventy-five percent of individuals infected with hepatitis C virus (HCV) develop chronic infection, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. In previous studies, it was found that CD8+ T cells directed against a relatively conserved HCV epitope exhibited broad TCR diversity, whereas CD8+ T cells reactive with the hypervariable immunodominant HLA-B*0801-restricted epitope, 1395HSKKKCDEL1403 (HSK), tended toward decreased TCR variability that was associated with chronic infection. A limited TCR repertoire could potentially aid the ability of hypervariable viruses like HCV to escape immune clearance (resulting in chronic infection) by mutating T cell–targeted residues. To explore whether limited TCR diversity impacts HCV viral escape, Nivarthi et al. (p. 5402) conducted functional and structural studies on TCRs reactive with HSK and its intergenotypic variants and other mutants. They used polyclonal HSK-specific T cell lines derived from an HCV-infected individual (D#1) to assess reactivity of these cells to alanine substitutions at different positions in the HSK peptide and found that peptide residues P4, P6, and P7 were integral to T cell recognition. They also found that most HSK mutant peptides and intergenotypic variants elicited IFN-γ production and cytolytic activity by D#1 HSK-specific T cell lines in functional assays, suggesting that these T cells were cross-reactive with several HSK variants. Structural studies revealed that HSK-specific TCRs in complexes with HSK and HLA-B*0801 contacted all available surface-exposed residues on the peptide, providing maximum coverage of the hypervariable epitope and suggesting a rationale for the ability of this limited TCR repertoire to recognize intergenotypic and quasispecies variants. Together, these results suggest that limited TCR diversity in HSK-reactive T cells is compensated by “blanketing” the hypervariable epitope during TCR–HLA-B*0801–HSK interactions and does not preclude the ability of these cells to mount an effective immune response.
Biography of a Thymic B Cell
Many studies have detailed the thymic selection process that T cells undergo, but less is known about the development and selection of thymic B cells. Fujihara et al. (p. 5534) examined the role of CD40 in bidirectional T cell–B cell cross-talk in the thymus and how CD40-initiated signals influence the maintenance and function of thymic B cells. Previous studies have shown that thymic B cells can present Ag to shape the T cell repertoire during thymic selection. In this study, Ag recognition by the TCRs of single positive thymocytes was shown to positively regulate the maintenance of thymic B cells, and CD40–CD40L signaling was crucial for supporting thymic B cell proliferation. Further investigation revealed that cell-autonomous expression of CD40 on thymic B cells was crucial to the maintenance of this cell population. T cell–dependent activation of peripheral B cells is known to require the expression of both CD40 and MHCII on B cells, but this study showed that thymic B cell maintenance did not require cell-autonomous expression of MHCII. In addition, thymic B cell expression of CD40 was required for negative selection of superantigen-reactive T cells. These results provide greater insight into the selection and maintenance processes of thymic B cells, particularly how thymic B cell–autonomous expression of CD40 is involved in cross-talk with and negative selection of T cells.
- Copyright © 2014 by The American Association of Immunologists, Inc.
