Administration of human recombinant IL-7 to normal and irradiated mice increases the numbers of lymphocytes and some immature cells of the myeloid lineage.

In vitro experiments performed by several investigators have demonstrated that IL-7 is a growth factor for immature B lymphocytes, thymocytes, and mature T lymphocytes. To evaluate the potential therapeutic use for human rIL-7 (rhuIL-7) as a hematopoietin, we have studied the in vivo hematopoietic effects of rhuIL-7 in mice. In these experiments, sublethally irradiated and normal mice were treated with or without rhuIL-7 for up to 26 days. Administration of rhuIL-7 significantly increased the white blood cell count in the peripheral blood and spleen in both normal and irradiated mice. Treatment with rhuIL-7 also accelerated lymphocytic recovery in irradiated mice. Precursor and mature B lymphocytes showed the greatest expansion in response to rhuIL-7 administration, with smaller increases in T lymphocytes being observed. In mice recovering from high dose irradiation, rhuIL-7 treatment resulted in preferential expansion of CD8+ T lymphocytes and more rapid normalization of the CD4/CD8 ratios. Differential analysis of peripheral blood smears demonstrated that rhuIL-7 also increased the numbers of immature granulocytes in both normal and irradiated mice. Moreover, administration of rhuIL-7 to normal, irradiated, cyclophosphamide-pretreated, or 5-fluorouracil-pretreated mice increased the number of acetylcholinesterase-positive megakaryocytes in the spleen, but not the bone marrow. Therefore, although the major in vivo effects of rhuIL-7 were on cells of the lymphocytic lineage, rhuIL-7 also increased the numbers of some immature cells of the myeloid lineage.

Modulation of human leukocyte antigen class I expression by gamma interferon in head and neck cancer cell lines.

One possible mechanism explaining the action of interferon (IFN) on squamous cell carcinoma (SqCC) of the head and neck is the modulation of major histocompatibility antigen expression on tumor cells. We tested the ability of gamma interferon (gamma-IFN) to modulate major histocompatibility class I antigens and beta 2-microglobulin (beta 2-M) on two carcinoma cell lines derived from SqCC of the head and neck. Major histocompatibility class I antigens and beta 2-M were detected using a two-step immunochemical stain; antigen expression was quantified using flow cytometry. gamma-IFN increased constitutive antigen expression by as much as five times on both cell lines. Maximum modulation was seen within 72 hours of exposure to gamma-IFN at clinically attainable doses (10 U/mL to 100 U/mL). The presence of gamma-IFN in cell cultures was necessary for continued modulation of surface antigens. These findings suggest a possible mechanism of action and encourage further clinical trials with gamma-IFN.

Proliferative response of the Dunning R3327H experimental model of prostatic adenocarcinoma to conditions of androgen depletion and repletion.

Administration of androgen to the castrate rat elicits a pronounced wave of proliferative activity in the ventral prostate gland. We wished to determine if this phenomenon could also be demonstrated in malignant prostate tissue. An experimental protocol consisting of 12 days of androgen depletion induced by castration followed by 7 days of androgen repletion was utilized in animals bearing the androgen-dependent Dunning R3327H prostatic adenocarcinoma. Repletion of androgen levels was achieved by daily s.c. injection of testosterone or dihydrotestosterone at 8, 16, or 32 mg/kg of animal body weight. The proliferative response of the tumor to conditions of depletion as well as repletion on Days 3, 5, and 7 was determined using [3H] thymidine autoradiography, quantitative morphometry, and flow cytometric analysis. The autoradiographic and flow cytometric data were complementary and indicated that androgen depletion caused a slight reduction in the percentage of S-phase nuclei. Repletion initiated a highly reproducible, significant increase in both the S-phase compartment as well as tritiated thymidine incorporation into DNA, and of the days quantitated, the greatest values were obtained on Day 3. The response patterns were nearly identical for both testosterone and dihydrotestosterone, and no significant differences were detected among the doses used. Quantitation of the autoradiographs revealed a striking disparity in the response of the cell types. The labeling indices of nonepithelial cells increased only minimally during repletion, whereas the epithelial cells responded consistently and reached levels 2- to 4-fold over intact values. These data indicate that protocols of androgen depletion/repletion have the capacity to elicit a significant wave of cell proliferation. These manipulations support the feasibility of transiently increasing the number of cancer cells in S phase as a means of potentiating cytotoxic chemotherapy for treatment of adenocarcinoma of the prostate.

Effect of herpes simplex virus types 1 and 2 on surface expression of class I major histocompatibility complex antigens on infected cells.

Cytotoxic T lymphocytes (CTL) generated in C57BL/6 (H-2b) mice in response to infection with the serologically distinct herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) were cross-reactive against target cells infected with either serotype. However, HSV-2-infected cells were shown to be much less susceptible to CTL-mediated lysis, and analysis through the use of HSV-1 X HSV-2 intertypic recombinants mapped the reduced susceptibility to a region contained within 0.82 to 1.00 map units of the HSV-2 genome. The study reported here was undertaken to determine the possible reasons for the reduced susceptibility of HSV-2-infected cells to lysis by CTL. Competition for the specific lysis of labeled HSV-1-infected cells by either HSV-1- or HSV-2-infected, unlabeled inhibitor cells and frequency analysis of the CTL precursor able to recognize HSV-1- and HSV-2-infected cells suggested that the reduced susceptibility of HSV-2-infected cells to lysis could be explained, at least in part, by reduced levels of target cell recognition. A determination of the surface expression of the critical elements involved in target cell recognition by CTL following infection with HSV-1 or HSV-2 revealed that all the major HSV-specific glycoprotein species were expressed. Infection with both HSV-1 and HSV-2 caused a reduction in the expression of the class I H-2 antigens. However, this reduction was much greater following infection with HSV-2. This suggested that one important factor contributing to reduced lysis of HSV-2-infected cells may be the altered or reduced expression of the class I H-2 self-antigens.

Identification of live cells for flow cytometric analysis of lymphoid subset proliferation in low viability populations.

Combined analysis of single cell DNA content and immunofluorescence by flow cytometry is complementary to tritiated thymidine analysis of cellular proliferation, allowing detailed dissection of particular cell types in a mixed population which respond proliferatively to selective stimuli. However, in vitro culture of primary immune cells (e.g., mouse spleen or lymph node) for periods of 24-72 h frequently results in a considerable fraction of non-viable cells which bind antibodies non-specifically, resulting in altered immunofluorescence distributions, inaccurate distinctions between positive and negative cells, and sometimes in misleading DNA distributions. Forward angle light scatter cannot readily be used to distinguish live from dead cells in this case because of the heterogeneous size distributions characteristic of cultured populations. We describe a method which uses treatment with DNAase prior to immunofluorescence staining to allow more accurate distinction between live and dead cells. This treatment markedly reduces the intensity of DNA staining for non-viable cells, providing complete live/dead discrimination and improved ability to analyze the proliferative status of specific cell subtypes in low viability cultures.