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The influence of recombinant human interleukin-4 (rIL-4) on the proliferation and cytotoxic activity of tumor-infiltrating lymphocytes (TIL) from human colorectal cancers was investigated TIL and peripheral blood lymphocytes (PBL) were cultured for 3-5 weeks. Under simultaneous stimulation with rIL-2 and rIL-4 the proliferation of TIL was less pronounced compared to stimulation with rIL-2 alone. In rIL-2 expanded TIL an outgrowth of CD56+ cells was observed. Concordantly, the expression of CD3+ cells was low. The number of CD56+ cells could be reduced significantly in TIL and PBL by stimulation with rIL-2 and rIL-4 simultaneously while the number of CD3+ cells increased. In TIL and PBL co-stimulation with rIL2 and rIL-4 resulted in higher CD4/CD8 ratios as compared to expansion with rIL-2 alone. In a 72 hour cytotoxicity assay the rIL-2/rIL-4 expanded TIL and PBL showed a lower lytic activity against autologous tumor targets in comparison to the rIL-2 expanded lymphocytes. In contrast, the cytotoxic activity of rIL-2/rIL-4 cultured TIL against allogeneic tumor cells was higher than in rIL-2 stimulated TIL.

Tumor-infiltrating lymphocytes (TILs) from colorectal cancers were separated from tumor cells by enzymatic and mechanical tissue disaggregation and discontinuous density gradients. Peripheral blood lymphocytes (PBLs) were isolated using the same procedure. The freshly separated TILs and PBLs were analyzed phenotypically by flow cytometry. The CD4+/CD8+ ratios of the freshly isolated TILs and PBLs were comparable (> 1 in both lymphocyte populations). CD25+, HLA-DR+ and CD56+ cells were significantly more frequent in the TIL than in the PBL population. However, the number of CD45RA+ cells was lower in the TILs as compared to PBLs, while CD29+ accumulated by about 90% in TILs. TILs and autologous PBLs were expanded in vitro with rIL-2. The mean rate of proliferation after 4 weeks was 642-fold in TIL cultures and 335-fold in PBLs. More than 90% of the rIL-2-expanded lymphocytes expressed CD2 and the great majority was CD29+. Stimulation with rIL-2 in vitro induced an outgrowth of CD56+ cells mainly in the TILs. Accordingly the expression of CD3+ and alpha/beta receptor in the TILs was low. Those cells which phenotypically represented lymphokine-activated killer cells displayed a lytic activity against the autologous tumor as well as against allogeneic K562 and Daudi targets. In accordance with the better proliferative response of TILs in long-term cultures with rIL-2, the lytic activity of TILs against autologous and allogeneic tumor targets was significantly higher as compared to PBLs.

The phenotypical composition of tumor-infiltrating lymphocytes (TIL) from colorectal carcinomas was compared with that of intraepithelial lymphocytes of the autologous normal colon mucosa (IEL) and autologous peripheral blood lymphocytes (PBL). The CD4+/CD8+ ratios of the freshly isolated TIL, IEL and PBL were comparable and always > 1. CD25+, HLA-DR+ and CD56+ cells accumulated significantly in the TIL, while CD45RA+ cells were less frequent compared to the autologous IEL and PBL. CD29+ memory-cells were found with the highest frequency in the TIL. Stimulation with rIL-2 in vitro induced an outgrowth of CD56+ cells in the TIL. Concordantly the expression of CD3+ and the alpha/beta T-cell receptor was low. In a standard 51Cr-release assay these phenotypically LAK-cells representing effectors displayed an unspecific lytic activity against the autologous tumor as well as against allogeneic K562 and Daudi targets. The number of CD56+ cells could be reduced in TIL and PBL by simultaneous stimulation with rIL-2 and rIL-4, while the number of CD3+ cells increased.