Ronald B. Faanes

Ultrastructural changes during T-lymphocyte-mediated cytolysis

The cytological changes that occur during the process of T-cell-mediated cytolysis (CMC) were studied in correlation with the 51Cr release assay. Our data show that both target P-815 and the effector cytolytic T-lymphocytes (CTL), when adequately purified, have a well-defined and distinguishable surface morphology which remained unaltered after lymphocyte-target cell binding. At subsequent stages of the CMC process when specific 51Cr release became significant, some target cells lost their surface microvilli and developed large surface protrusions or blebs. CTL remained bound to target cells undergoing the blebbing process with only minor changes in their surface morphology. Quantitation of the cellular interactions revealed that the frequency of target cells blebbing and binding of specific CTL rose concomitantly with the specific 51Cr released. Transmission electron microscopy indicated that the earliest cytological damage occurred apparently in the mitochondria of the target cells at a stage of the CMC process preceding significant 51Cr release.

Immunoenhancing activity of NPT 15392: A potential immune response modifier☆

NPT 15392, a new immunomodulating compound related to inosine in structure and isoprinosine in action, enhances T-cell dependent immune responses. Antibody responses to sheep red blood cells are augmented two to threefold in mice receiving NPT 15392 while T-cell independent antibody responses to TNP-LPS are unaffected. NPT 15392 does not enhance or alter the number of clonable B cells. This drug also increases cytotoxic T-lymphocyte responses to allogeneic tumor cells but does not alter the number of cytotoxic precursor cells. Immature hematopoietic cell classes (clonable progenitor cells) were also monitored and found not to be influenced by NPT 15392.

T-lymphocyte mediated lysis of tumor cells in the presence of alloantiserum

Abstract The blocking effects of tumor alloantiserum (AS) on the process of T-lymphocyte mediated cytolysis of target cells was investigated with the scanning electron microscope by analyzing the frequency of lymphocyte-target cell interactions and the respective changes in target cell morphology at various time intervals of the cytolytic process. Our results demonstrate that in the presence of AS the frequencies of lymphocyte-target cell conjugates in which the target cells were undergoing active blebbing correlated with the delayed kinetics of 51Cr release. Our data also show that the AS did not interfere significantly with the binding of lymphocytes to target cells, but delayed the appearance of surface blebbing of the target cells. Thus, effector lymphocytes required a prolonged time of continuous interaction with the target cells in order to exert their cytolytic effects in the presence of AS. This conclusion was further confirmed by experiments in which lymphocyte-target cell interactions were interrupted by the addition of EDTA to the culture medium.

Activation of specific suppressor cells with heat-treated allogeneic tumor cells

Abstract The ability of heat-treated allogeneic cells to induce suppressor cells was examined. The tumor cell lines EL-4 (H-2 b ) and P815-X2 (H-2 d ), were heated to 56 °C for 10 min and injected intravenously into mice of the DBA/2J (H-2 d ) and C57BL/6J (H-2 b ) strains, respectively. After 4 days, the splenocytes of the treated mice were mixed with normal spleen cells and cultured for 5 days with allogeneic tumor cells. The cytotoxic T-cell response was reduced in cultures of these cell mixtures. An allogeneic difference was required to induce suppression because the syngeneic combination did not induce suppressor cell activity. Furthermore, the induction of cytotoxic T cells to the C118 cell line (H-2 k ) was not suppressed by this procedure, which suggests that the suppression was haplotype specific. These suppressor cells were sensitive to anti-Thy 1.2 and complement, cortisone, and cyclophosphamide, but insensitive to irradiation. These are characteristics similar to suppressor cells activated by intact cells. Heat treatment abrogated the tumor cells ability to induce a proliferative and a primary, but not a secondary, cytotoxic T-cell response. The heat-treated cells also lost their ability to function as cold target inhibitor cells, but retained the same quantity of serologically detected antigens as the intact cells. These results suggest that the serologically detected antigens are responsible for the activation of the suppressor cells of the cytotoxic T-cell response.

Recovery of the capacity for cytotoxic T cell generation in cyclophosphamide-treated mice by the addition of Lyt-1+2− helper cells

Spleen cells from mice treated with cyclophosphamide (150 mg/kg) and cultured at suboptimal concentrations do not generate cytotoxic T lymphocytes to allogeneic tumor cells. The reduced response of spleen cells from cyclophosphamide-treated mice is not due to the elimination of cytotoxic T cell precursors because normal responses are obtained by the addition of Lyt-1+2- T helper cells to the culture system. These results indicate that helper cells, required for the development of cytotoxic T cell responses to tumor alloantigens, are eliminated by cyclophosphamide in the absence of evident toxicity to cytotoxic T cell precursors.

Anomalous killer cells: Thymus cell dependency, precursor frequency, and response to immunosuppressive therapy☆

Abstract Human peripheral blood lymphocytes (HPBL) cultured with the Daudi tumor cell line develop into nonspecific cytotoxic cells. These cytotoxic cells, anomalous killer (AK) cells, are dependent upon thymus-derived cells and monocytes in the generation phase and bear serologically determined T-cell antigens in the effector cell stage. Precursor frequency determinations of these killer cells range from 1 166 to 1 689 in untreated patients and drop considerably after treatment with fractionated X irradiation or cytoreductive therapy. Lymphokine (LK)-containing supernatants partially restore cytotoxic activity of AK cells after X irradiation, indicating a second signal requirement for precursor differentiation into lytic effector cells.

The existence of antibody-like activities against the weakly immunogenic minor histocompatibility antigens.

Abstract BALB/c mice develop cytotoxic lymphocytes as well as produce specific antibodies against the minor histocompatibility antigens when injected with DBA/2 P815 cells. P815 cells grown in BALB/c mice have IgG antibodies on their surface as demonstrated by the binding of 125 I-labeled goat anti-mouse IgG and by complement-dependent cytotoxicity. Serum from BALB/c mice hyperimmunized with P815 cells blocked lymphocyte-mediated cytotoxicity by BALB/c immune peritoneal exudate cells. This blocking activity was removed by absorbing hyperimmune serum with DBA/2 spleen cells or P815 cells. This result suggests that specific antibodies were generated against the minor histocompatibility differences between BALB/c and DBA/2 mice. The experimental procedures described may be very useful in demonstrating minute quantities of antibody against minor histocompatibility antigens on tumor cells.