Routine evaluation of HBV-specific T cell reactivity in chronic hepatitis B using a broad-spectrum T-cell epitope peptide library and ELISpot assay.

BACKGROUND: The clinical routine test of HBV-specific T cell reactivity is still limited due to the high polymorphisms of human leukocyte antigens (HLA) in patient cohort and the lack of universal detection kit, thus the clinical implication remains disputed. METHODS: A broad-spectrum peptide library, which consists of 103 functionally validated CD8+ T-cell epitopes spanning overall HBsAg, HBeAg, HBx and HBpol proteins and fits to the HLA polymorphisms of Chinese and Northeast Asian populations, was grouped into eight peptide pools and was used to establish an ELISpot assay for enumerating the reactive HBV-specific T cells in PBMCs. Totally 294 HBV-infected patients including 203 ones with chronic hepatitis B (CHB), 13 ones in acute resolved stage (R), 52 ones with liver cirrhosis (LC) and 26 ones with hepatocellular carcinoma (HCC) were detected, and 33 CHB patients were longitudinally monitored for 3 times with an interval of 3-5 months. RESULTS: The numbers of reactive HBV-specific T cells were significantly correlated with ALT level, HBsAg level, and disease stage (R, CHB, LC and HCC), and R patients displayed the strongest HBV-specific T cell reactivity while CHB patients showed the weakest one. For 203 CHB patients, the numbers of reactive HBV-specific T cells presented a significantly declined trend when the serum viral DNA load, HBsAg, HBeAg or ALT level gradually increased, but only a very low negative correlation coefficient was defined (r = - 0.21, - 0.21, - 0.27, - 0.079, respectively). Different Nucleotide Analogs (NUCs) did not bring difference on HBV-specific T cell reactivity in the same duration of treatment. NUCs/pegIFN-α combination led to much more reactive HBV-specific T cells than NUCs monotherapy. The dynamic numbers of reactive HBV-specific T cells were obviously increasing in most CHB patients undergoing routine treatment, and the longitudinal trend possess a high predictive power for the hepatitis progression 6 or 12 months later. CONCLUSION: The presented method could be developed into an efficient reference method for the clinical evaluation of cellular immunity. The CHB patients presenting low reactivity of HBV-specific T cells have a worse prognosis for hepatitis progression and should be treated using pegIFN-α to improve host T-cell immunity.

Frequency and reactivity of antigen-specific T cells were concurrently measured through the combination of artificial antigen-presenting cell, MACS and ELISPOT.

Conventional peptide-major histocompatibility complex (pMHC) multimer staining, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay cannot concurrently determine the frequency and reactivity of antigen-specific T cells (AST) in a single assay. In this report, pMHC multimer, magnetic-activated cell sorting (MACS), and ELISPOT techniques have been integrated into a micro well by coupling pMHC multimers onto cell-sized magnetic beads to characterize AST cell populations in a 96-well microplate which pre-coated with cytokine-capture antibodies. This method, termed AAPC-microplate, allows the enumeration and local cytokine production of AST cells in a single assay without using flow cytometry or fluorescence intensity scanning, thus will be widely applicable. Here, ovalbumin257-264-specific CD8+ T cells from OT-1 T cell receptor (TCR) transgenic mice were measured. The methodological accuracy, specificity, reproducibility, and sensitivity in enumerating AST cells compared well with conventional pMHC multimer staining. Furthermore, the AAPC-microplate was applied to detect the frequency and reactivity of Hepatitis B virus (HBV) core antigen18-27- and surface antigen183-191-specific CD8+ T cells for the patients, and was compared with conventional method. This method without the need of high-end instruments may facilitate the routine analysis of patient-specific cellular immune response pattern to a given antigen in translational studies.

Induction of tumor antigen-specific cytotoxic T cell responses in naïve mice by latex microspheres-based artificial antigen-presenting cell constructs.

Latex microspheres-based artificial antigen-presenting cell constructs (aAPCs) are proved to be valuable tools to expand T cells ex vivo for adoptive cell therapy, but little is known about their potential for active immunization. In this report, HLA-A2/peptide tetramers were generated and co-coated with anti-mouse CD28 monoclonal antibody onto surface of cell-sized latex microspheres followed by immunization of naïve HLA-A2/K(b) transgenic mice. Five- to six-fold expansion of tumor antigen-specific CTLs was observed in the spleen after three rounds of immunization. The consequent splenocytes can efficiently recognize endogenously expressed tumor antigen on the surface of human target cells and cytolyze the tumor cells in an antigen-specific manner. This report provides initially the experimental evidence that latex microspheres-based aAPCs can effectively prime antigen-specific CTL proliferation and cytolysis in naïve mice. This may contribute to a better insight into the potential of microspheres-based aAPCs for active immunization.

Structural and functional characterization of peptide-beta2m fused HLA-A2/MART1(27-35) complexes.

The uses of soluble HLA class I/peptide complexes to monitor antigen reactive T cells are often hampered by their low-yield and high-cost production. As an alternative strategy, the peptide-beta(2)m fused, 2-component (2C) HLA class I/peptide complex has been developed, but its application is limited due to the lack of the comparison of its structural and functional characteristics with those of its conventional 3-component (3C) counterpart. In this study, we have demonstrated that the 2C and 3C HLA-A2/MART1(27-35) complexes have a similar chromatographical profile and comparable stability, but the former has 2.5 times higher yield and significantly higher binding ability with HLA-A2/MART1(27-35) complex-specific receptors than the latter. Furthermore, the 2C complex has a comparable ability to stimulate specific CTL proliferation, but appears to be more effective in eliciting the cytotoxicity of antigen-specific CTL, as compared to its 3C counterpart.

[Immune reaction in the mixed culture of host lymphocytes with allogenic and host epithelial cells].

OBJECTIVE: To observe the immune reaction in the mixed culture of host lymphocytes with allogenic and host endothelial cells. METHODS: The host epithelial cells and lymphocytes from burn patients and allogenic epithelial cells were mix-cultured in different ratios, so as to simulate the local immune micro-environment of host skin island in intermingled skin grafting. In addition, the cells from normal human subjects were also mix-cultured as control. The lymphocyte cpm values were detected by (3)H-TdR and HLA molecules and T cell subgroup were determined by immunohistological technique. RESULTS: (1) The lymphocyte proliferation reaction could be effectively inhibited by the epithelial cells from burn patients but not from normal control. (2) The inhibition of host lymphocyte proliferation could not be mediated by the HLA-DQ molecules of epithelium from burn patients. (3) The positive expression rate of HLA-DR of epithelia from burn patients was evidently higher that that from normal control (P < 0.05), (4) The CD8 expression of lymphocyte in burn patients was significantly higher than that in normal control (P < 0.01), while the CD4 expression in burn patients was lower than that in normal control (P < 0.01). But there was no obvious difference of the CD3 expression between patients and normal subjects (P > 0.05). CONCLUSION: The lymphocyte proliferation reaction could be obviously inhibited by the host epithelium, which might be related to the specific immune state of the host lymphocytes and epithelium of burn patients.