Freshly isolated tumor-infiltrating lymphocytes (TIL) are often functionally deficient. Since one of the key functional parameters of an immune response is the local production of cytokines, we studied the expression of cytokine genes in freshly isolated renal cancer tissue. Using a PCR-assisted mRNA amplification assay, the constitutive expression of mRNA for 10 different cytokines was assessed in renal cancer tissue. We compared the cytokine mRNA expression in freshly isolated samples of renal carcinomas, renal cancer cell lines established from the tumor samples, peripheral blood mononuclear cells (PBMC) and non-tumor kidney tissue isolated from the same patients. IL-10 mRNA expression was detected only in tumor samples, while renal cancer lines, PBMC and non-tumorous kidney tissues were devoid of this cytokine. One-third of the tumor samples but none of the normal kidney samples also expressed G-CSF mRNA. IL-6, TNF-alpha and IFN-gamma mRNA were expressed non-selectively in tumors, PBMC and normal rental tissue. Expression of IL-2, IL-3 and IL-4 mRNA was not detected in any of the tissues analyzed. Established renal cancer lines exhibited expression of IL-1 alpha, IL-6, TNF-alpha and GM-CSF. Culture of tumor-derived T cells with anti-CD3 monoclonal antibody (MAb) resulted in expression of IL-2, IL-3 and IL-4 mRNA. In contrast, none of these cytokines was detected in culture with recombinant human IL-2 alone. Since IL-10 is known to suppress antigen presentation, these findings have important implications for the possible in vivo role of IL-10 as a suppressor of local anti-tumor response.
Tumour-infiltrating lymphocytes (TIL) are often observed in human tumours and their presence has been correlated with a better prognosis. It has been suggested that TIL are enriched for tumour-specific cytotoxic cells, and TIL activated and expanded in vitro by interleukin-2 (IL-2) are currently used in the therapy of human cancer. We have studied the T cell repertoire in IL-2-expanded TIL cells from patients with ovarian and renal carcinoma using T-cell-receptor-V-beta-specific monoclonal antibodies and a polymerase-chain-reaction-based Southern blot technique for analysis of J-beta usage. In TIL lines derived from three of nine patients with ovarian carcinomas and from two of eight patients with renal carcinomas, selective usage of the V-beta 6 or V-beta 5 T-cell receptor gene products was found. The majority of the cells were CD4+, with up to 40% of the T cells utilizing the same V-beta gene. T-cell lines derived from peripheral blood lymphocytes from patients or healthy donors contained normal levels of V-beta subsets. Only moderate levels of V-beta 6+ T cells were detected from freshly isolated TIL and the increase of this subpopulation appeared as a result of in vitro culture. The level of clonal restriction, as measured by the usage of J-beta gene segments within the V-beta 5 or V-beta 6 families, was analysed using a recently developed technique based on the polymerase chain reaction. Evidence for restricted J-beta usage was detected only in TIL expanded in vitro, while this was not the case in freshly isolated tumour-derived lymphocytes or T cell lines obtained from peripheral blood lymphocytes. The presence of a population with biased T cell receptor expression in cells derived from tumour tissue could be explained by their activation in vivo as a result of contact with tumour antigens and should be taken into consideration when discussing the therapeutic efficiency of IL-2-expanded TIL.
The cytotoxic activity and T cell receptor (TCR) V beta repertoire in tumor-infiltrating lymphocytes (TIL) of three primary adrenal cell carcinomas were analyzed. Fresh, non-cultured TIL from two of the three tumors showed low but significant lysis of the autologous tumor, and for one of the patients this activity was strongly enhanced upon culture in interleukin-2. An allogeneic adrenal cell carcinoma line and the K562 or Daudi targets included as controls were not killed. Phenotypic analysis of freshly isolated TIL demonstrated that the cells from the two patients that demonstrated cytolytic capacity mainly consisted of CD45RO+ T cells. In vitro cultured TIL lines from these patients demonstrated a high percentage of CD8+ cells expressing either the V beta 6 gene or the V beta 8 gene product, as measured with a panel of mAb specific for TCR V alpha and V beta gene products. Analysis of the TCR V beta gene mRNA expression in freshly isolated non-cultured TIL, using a polymerase-chain-reaction-assisted cDNA-amplification assay, confirmed the strong expression of the genes coding for the TCR V beta 6 or the V beta 8. This assay also demonstrated a more restricted TCR V beta gene usage in the TIL as compared to peripheral blood lymphocytes from the same patient.
To study the resolution required for simulating gravitational fragmentation with newly developed Lagrangian hydrodynamic schemes, meshless finite-volume method (MFV) and meshless finite-mass method, we have performed a number of simulations of the Jeans test and compared the results with both the expected analytical solution and results from the more standard Lagrangian approach: smoothed particle hydrodynamics (SPH). We find that the different schemes converge to the analytical solution when the diameter of a fluid element is smaller than a quarter of the Jeans wavelength, λJ. Among the three schemes, SPH/MFV shows the fastest/slowest convergence to the analytical solution. Unlike the well-known behaviour of Eulerian schemes, none of the Lagrangian schemes investigated displays artificial fragmentation when the perturbation wavelength, λ, is shorter than λJ, even at low numerical resolution. For larger wavelengths (λ > λJ), the growth of the perturbation is delayed when it is not well resolved. Furthermore, with poor resolution, the fragmentation seen with the MFV scheme proceeds very differently compared to the converged solution. All these results suggest that, when unresolved, the ratio of the magnitude of hydrodynamic force to that of self-gravity at the sub-resolution scale is the largest/smallest in MFV/SPH, the reasons for which we have discussed in detail. These tests are repeated to investigate the effect of kernels of higher order than the fiducial cubic spline. Our results indicate that the standard deviation of the kernel is a more appropriate definition of the ‘size’ of a fluid element than its compact support radius.
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