Normal Mouse Thymocytes Research Articles

The Mechanism of Epipodophyllotoxin-Induced Thymocyte Apoptosis: Possible Role of a Novel Ca 2+-Independent Protein Kinase

The epipodophyllotoxins, etoposide (VP-16) and teniposide (VM-26), inhibit topoisomerase II activity by stabilization of the cleavable complex between the enzyme and DNA and formation of protein-bound double-stranded DNA breaks. While it is thought that these agents are cytotoxic by preventing cells from completing the S phase or undergoing mitosis, recent evidence suggests that these agents are also potent inducers of programmed cell death or apoptosis in both normal and malignant cells. We have examined the intracellular pathway leading to epipodophyllotoxin-induced apoptosis in normal mouse thymocytes. Epipodophyllotoxin-induced apoptosis may proceed via a mechanism that is independent of inhibition of topoisomerase activity per se because novobiocin and coumermycin, which inhibit the ATPase subunit of topoisomerase II, were relatively inefficient inducers of apoptosis in these cells, under conditions where strong apoptosis by the epipodophyllotoxins and dexamethasone could be observed. In addition, camptothecin, which inhibits topoisomerase 1 by stabilization of the cleavable complex between that enzyme and DNA, was also a poor inducer of apoptosis in these cells. Our data suggest that epipodophyllotoxin-induced mouse thymocyte apoptosis, like that induced by dexamethasone, proceeds via a mechanism that involves protein kinase C (PKC) or a similar enzyme. Apoptosis induced by VM-26 or by dexamethasone was inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H7), an inhibitor of both PKC and cAMP-dependent protein kinases, but was relatively unaffected by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a more specific inhibitor of cAMP-dependent protein kinases. A more specific inhibitor of PKC, sangivamycin, also inhibited both VM-26-induced and dexamethasone-induced apoptosis. Both VM-26- and dexamethasone-induced apoptosis were unaffected by EGTA, a calcium (Ca 2+) chelator, under conditions that inhibited apoptosis induced by the Ca 2+ ionophore A23187. Moreover, while strong increases in intracellular Ca 2+ were observed in thymocytes treated with A23187, we failed to detect increases in intracellular Ca 2+ in cells induced to apoptose with either VM-26 or dexamethasone within the first 2 hr of culture. These results suggest that in mouse thymocytes there are at least two intracellular pathways leading to apoptosis: one, utilized by glucocorticoid and the epipodophyllotoxins, that proceeds in the absence of detectable increases in intracellular Ca 2+ and possibly requires a novel Ca 2+-independent PKC-like enzyme and another, utilized by Ca 2+ ionophores, that is at least partially dependent on increased intracellular Ca 2+.

Prolactin receptor gene expression in lymphoid cells

To understand the role of pituitary prolactin (PRL) and its receptor (PRL-R) in the growth and differentiation of lymphoid cells, PRL-R gene expression was analyzed in various lymphoid tissues and in a rat T lymphoma cell line, Nb2, which requires PRL for growth. The technique of reverse transcription coupled to polymerase chain reaction (RT-PCR) was used to detect the low abundance PRL-R transcripts. Within 30 min to 1 h, PRL stimulates a rapid but transient increase in PRL-R mRNA levels in Nb2 T cells. By 4 h, PRL-R mRNA returned to near basal levels and then gradually declined to a new steady-state level by 12 h. Significant increases in receptor RNA levels were observed in the presence of protein synthesis inhibitors, which suggests that PRL-R mRNA levels are under negative regulation. PRL-R gene expression was also demonstrated in normal mouse thymocytes, splenocytes, and in several lymphoid cell lines. The expression of the PRL-R gene in stimulated lymphoid cells provides additional evidence for the role of PRL as an immunomodulatory molecule.

Lymphocyte-specific Ca2+-binding protein LSP1 is associated with the cytoplasmic face of the plasma membrane.

The gene for LSP1 is a lymphocyte-specific gene previously isolated by us using a subtractive hybridization technique. LSP1 mRNA is found in normal and transformed B lymphocytes and in normal T lymphocytes but not in transformed T lymphocytes. To study the expression of the mouse LSP1 protein, we prepared a polyclonal antiserum specific for the LSP1 protein. Here we report that the gene for LSP1 was expressed in transformed B-lymphoma cell lines and in normal mouse thymocytes as a protein doublet with apparent molecular masses of 52 and 50.5 kilodaltons when analyzed on a sodium dodecyl sulfate-10% polyacrylamide gel. BW5147 cells transfected with an LSP1 cDNA clone expressed only the 52-kilodalton protein. No LSP1 protein was expressed in nine T-lymphoma cell lines tested. Immunofluorescence studies of intact and permeabilized cells and subcellular fractionation experiments showed that the LSP1 protein was associated with the cytoplasmic side of the plasma membrane in transformed B-lymphoma cell lines and in normal thymocytes. Using a simple filter-binding assay, we showed that recombinant LSP1 protein was Ca2+ binding, as predicted on the basis of its deduced amino acid sequence. On the basis of the particular expression pattern, the subcellular localization, and the Ca2+-binding property of the LSP1 protein, we hypothesize that the LSP1 protein is a lymphocyte-specific component of a signal transduction pathway involved in the regulation of lymphocyte growth.

Lymphocyte-specific Ca2+-binding protein LSP1 is associated with the cytoplasmic face of the plasma membrane.

The gene for LSP1 is a lymphocyte-specific gene previously isolated by us using a subtractive hybridization technique. LSP1 mRNA is found in normal and transformed B lymphocytes and in normal T lymphocytes but not in transformed T lymphocytes. To study the expression of the mouse LSP1 protein, we prepared a polyclonal antiserum specific for the LSP1 protein. Here we report that the gene for LSP1 was expressed in transformed B-lymphoma cell lines and in normal mouse thymocytes as a protein doublet with apparent molecular masses of 52 and 50.5 kilodaltons when analyzed on a sodium dodecyl sulfate-10% polyacrylamide gel. BW5147 cells transfected with an LSP1 cDNA clone expressed only the 52-kilodalton protein. No LSP1 protein was expressed in nine T-lymphoma cell lines tested. Immunofluorescence studies of intact and permeabilized cells and subcellular fractionation experiments showed that the LSP1 protein was associated with the cytoplasmic side of the plasma membrane in transformed B-lymphoma cell lines and in normal thymocytes. Using a simple filter-binding assay, we showed that recombinant LSP1 protein was Ca2+ binding, as predicted on the basis of its deduced amino acid sequence. On the basis of the particular expression pattern, the subcellular localization, and the Ca2+-binding property of the LSP1 protein, we hypothesize that the LSP1 protein is a lymphocyte-specific component of a signal transduction pathway involved in the regulation of lymphocyte growth.

Preferential usage of JH2 in D-J joinings with DQ52 in murine lymphocytes

Southern blot analysis of genomic DNA of normal mouse thymocytes with a JH4 probe, a probe for JH4 of Igh genes, consistently showed a second band in addition to a germline band. The same band was also observed in analysis with a 5' DQ probe, a 5'-flanking sequence of DQ52, thus suggesting the use of DQ52. By analyzing EcoRI- and PvuII-digested genomic DNA, the above-observed band was demonstrated to be the result of DQ52-JH2 joining. This was further confirmed utilizing a T cell leukemia line with known DQ52-JH joinings. We then quantitatively estimated the frequencies of JH segment usages in joining with DQ52, through plaque hybridization assays. Three hundred and fifty JH4-positive clones were obtained from 7 x 10(5) plaques by plaque hybridization. Forty-eight randomly selected non-germline clones were purified and the use of JH segments was determined through Southern blot analysis. Nine clones used JH1, 29 used JH2, 9 used JH3, and 1 used JH4, thus indicating an apparent preferential usage of JH2 segment. Southern blot analysis of genomic DNA of progenitor B cells in culture showed a dominant band of DQ52-JH2 joining 1 week after initiation of culture. This may indicate that the dominant DQ52-JH2 joining in thymocytes is representative of early D-J joinings in progenitor B cells.

Early expression of a T-cell receptor beta-chain transgene suppresses rearrangement of the V gamma 4 gene segment.

beta transgenic mice have a T-cell receptor beta-chain gene that is prematurely expressed on the surface of CD4- CD8- thymocytes and paired with an uncharacterized non-T-cell receptor alpha-chain polypeptide. The rearrangement of the T-cell receptor variable region gamma chain gene segment V gamma 4, a component of the gamma-chain gene that is rearranged and expressed preferentially on thymocytes of normal adult mice, is severely repressed in beta transgenic mice. Consequently no gamma delta T-cell receptor heterodimers are detectable on the surface of adult thymocytes or splenic T cells. These results indicate that cells expressing alpha beta or gamma (V gamma 4)-delta TCRs originate from a common precursor in which the first productive rearrangement of either the beta or gamma locus determines the further differentiation pathway into either alpha beta or gamma delta T cells. The repression of V gamma 4 rearrangement by a preexisting beta-chain gene may be indicative of one of several mechanisms which ensure that gamma delta and alpha beta receptors do not as a rule appear on the surface of the same cell.

Formation of IgM-binding factors by murine thymocytes

Normal mouse thymocytes activated with concanavalin A (Con A) released soluble factors which selectively inhibited rosette formation between human peripheral blood lymphocytes (PBL) and ox erythrocytes (E O) sensitized with rabbit IgM antibodies. The factors were removed by absorption with mouse IgM-coupled Sepharose, and were recovered from the beads by elution at acid pH. They neither bound to mouse IgG-Sepharose nor inhibited rosette formation of PBL with E o sensitized with rabbit IgG antibodies. Mouse IgM enhanced the formation of IgM-binding factors by Con A-activated thymocytes. Unstimulated normal mouse thymocytes also released IgM-binding factors upon incubation with mouse IgM. The molecular weights of IgM-binding factors were approximately 90–110 and 35–50 kDa as estimated by gel filtration. Each species of IgM-binding factors markedly suppressed the IgM-plaque-forming cell (PFC) response of sheep red blood cell-primed spleen cells and slightly suppressed the IgG PFC response.

Natural killer cells in murine muscular dystrophy: IV. Characterization of percoll fractionated splenic and thymic natural killer cells and natural killer-sensitive thymocyte targets

The Natural Killer (NK) activity in the thymus and NK-sensitive thymocyte targets of dystrophic mice was investigated. Dystrophic and normal mouse thymocytes or spleen cells were layered on discontinuous Percoll gradients (5 or 10% increments, respectively) between 40 and 70% and centrifuged at 1700g for 30 min. All fractions were tested for either NK activity or used as 51Cr-labeled NK-sensitive targets in a 6-hr 51Cr release assay. The density interface between the 50% (1.060 g/ml) and 60% (1.075 g/ml) Percoll fractions of either dystrophic or normal mouse spleen cells and the 40% (1.050 g/ml) and 50% (1.060 g/ml) Percoll fractions of either dystrophic or normal mouse thymocytes were found to contain the largest proportion of NK activity using YAC-1 lymphoma tumor cells as targets. In addition, the NK activity in dystrophic mouse spleen cells and thymocytes was significantly greater when compared with normal mouse controls. Target binding cell studies revealed that these Percoll fractions of dystrophic mouse spleen cells and thymocytes had greater numbers of conjugate-forming cells when compared with normal control groups. Cell depletion experiments using either anti-Thy 1.2, anti-asialo-GM 1 or anti-NK-1 plus complement treatment revealed that the cell responsible for NK activity in the 50% Percoll fraction interface of dystrophic mouse spleen cells was asialo-GM 1 positive, NK-1 positive, and partially Thy 1.2 positive. However, the cells displaying NK-activity in the thymus of normal or dystrophic mice were found to be highly Thy-1.2 positive and peanut agglutinin (PNA) negative. The density interface between the 60% (1.075 g/ml) and 65% (1.081 g/ml) Percoll fractions of either normal or dystrophic mouse thymocytes contained the largest proportion of NK-sensitive target cells. Interestingly, the 60% Percoll fraction of dystrophic mouse thymocyte targets was significantly more susceptible to NK-mediated lysis than that of the normal mouse thymocyte population. Cell depletion experiments revealed that the NK-sensitive thymocyte population was similar in both mice, that is. Thy-1.2 positive, cortisone sensitive, PNA positive. Dolichos biflorus (DBA) negative and asialo GM-1 negative. The results indicate that there are density differences between splenic and thymic NK cells. In addition, there are density and phenotypic differences between thymic NK cells and thymic NK-sensitive target cells. The findings support the hypothesis that there are different populations of NK cells.

Natural killer (NK) cell activity in murine muscular dystrophy: II. Age-related tissue distribution and enhanced NK activity in the thymus of dystrophic mice

The age-related tissue distribution of natural killer (NK) cell activity in murine muscular dystrophy was investigated. Lymphoid tissues including the spleen, thymus, mediastinal (or bronchial) lymph nodes (BLN), mesenteric lymph nodes (MLN), inguinal/popliteal lymph nodes (PLN1), and axillary/brachial lymph nodes (PLN2) were obtained from various aged normal (+/+) and dystrophic (dy 2J/dy 2J) C57BL/6J mice. Cell suspensions were incubated with 51Cr-labeled YAC-1 lymphoma target cells in a 4-hr 51Cr-release assay. The data indicated that dystrophic mice, at all ages studied, had elevated levels of NK activity in the spleen, BLN, MLN, PLN1, and PLN2 as compared with the normal age- and sex-matched control group. The NK activity in the thymocyte population from dystrophic mice at 2 weeks of age was found to be negligible, while at 8 weeks of age it was two-fold higher than that for the normal control group. In addition, dystrophic mice had an age-related decline in NK activity in all tissues after 10 weeks of age but the activity was still elevated at 40 weeks of age as compared with the normal control group. Target cell binding studies revealed that the number of conjugate-forming cells in thymocytes from 8-week-old dystrophic mice were found to be significantly higher than that found in normal mouse thymocytes using NK-sensitive YAC-1 tumor target cells. The number of cells bound per YAC-1 target cell ranged from 1 to 3 for dystrophic mouse thymocytes as compared with 1 to 2 for the normal control group. Thus, the data indicate an elevated NK activity in all lymphoid tissues studied from dystrophic mice of different ages. In addition, the thymus from dystrophic mice at 8 weeks of age contains an enhanced number of conjugate-forming NK cells and NK activity.

Enhanced natural killer (NK) cell activity and NK-sensitive thymic cells in murine muscular dystrophy

Studies have shown that there is an abnormality in the thymus of dystrophic mice with respect to age-dependent thymus weight changes and altered morphology (T. DeKretser and B. Livett, Nature (London), 263, 682, 1976). Recently, others have shown that natural killer (NK) cells can lyse cells of a large, immature, rapidly dividing cell subpopulation within the thymus of normal young (3 weeks of age) mice (M. Hansson, K. Karre, R. Kiessling, J. Roder, B. Anderson, and P. Hayry, J. Immunol., 123, 765, 1979). The NK susceptibility of dystrophic mouse thymocytes as targets was therefore studied. Spleen cells from normal (+/+) and dystrophic ( dy 2J dy 2J ) male C57BL/6J mice 8–10 weeks old were passed over nylon wool and the nonadherent cells were incubated with 51Cr-labeled YAC-1 lymphoma target cells or thymocytes in a 51Cr-release assay. Spleen cells from dystrophic mice killed twofold more YAC-1 target cells than did spleen cells from normal mice. Thymocytes from 3- to 4-week-old dystrophic mice were three to four times more susceptible to NK lysis by dystrophic mouse spleen cells as compared with normal mouse spleen cells. Spleen cells from dystrophic mice had the same NK activity against dystrophic and normal mouse thymocytes as targets. Normal mouse spleen cells killed three- to fourfold more dystrophic mouse thymocytes than that of normal mouse thymocytes as targets. Target cellbinding studies revealed that conjugate-forming cells from nylon nonadherent dystrophic mouse spleen cells were found to be two- to fourfold greater than for normal mouse spleen cells using YAC-1 tumor cells as targets. The number of lymphocytes bound per YAC-1 target cell ranged from 2 to 5 for dystrophic mouse spleen cells as compared with 1 to 2 for the normal control group. Using both normal and dystrophic mouse thymocytes as targets, the conjugate-forming cells from dystrophic mouse spleen cells were also found to be twofold greater than in the normal control group. Cold target inhibition studies revealed that the natural killing of dystrophic mouse thymocytes was due to a YAC-1-reactive NK cell. Effector cell depletion studies using monoclonal anti-Thy-1.2 plus complement treatment and plastic petri dish adherence also revealed that the natural killing of dystrophic mouse thymocytes was not due to either T lymphocytes or macrophages. Taken together, these results show an increase in NK-sensitive thymocyte targets in dystrophic mice, in combination with an increase in splenic NK activity.

Effect of Toxoplasma infection on the sensitivity of mouse thymocytes to natural killer cells.

Thymocytes from mice 2 weeks after infection with Toxoplasma gondii resisted natural killer (NK) cell-mediated cytolysis in contrast to the high sensitivity of normal mouse thymocytes. The infected mouse thymocytes also failed to form conjugates with effector cells and to compete for cytolysis of NK sensitive targets. These effects were mediated, at least in part, by interferon-gamma because normal thymocytes became NK insensitive after incubation in the infected mouse serum which contained significant amount of interferon-gamma, and pH 2 treatment of the serum abolished the effect. An alternate possibility for the reduced NK sensitivity of the infected mouse thymocytes was the elimination of NK-sensitive cells from the thymus, since histopathological studies showed marked atrophy and clearance of NK-sensitive thymocytes in the cortex of thymuses of infected mice. Although T. gondii induced augmentation followed by suppression of the host splenic NK activity, it seems unlikely that this altered NK activity was responsible for the lowered NK sensitivity of the thymocytes.

Delineation of membrane-bound phosphatase activities in normal and leukemic thymocytes

The activities of the various phosphatases on the plasma membrane of normal and leukemic mouse thymocytes were delineated using the specific inhibitors, α, β-methylene ADP, which inhibits 5′-nucleotidase while not affecting nonspecific phosphatases, (−)- p-bromotetramisole, which inhibits nonspecific alkaline phosphatase, and the competitive substrate p-nitrophenylphosphate. The K m for AMP and the K i for the inhibition of AMPase by α, β-methylene ADP at pH 7.5 were 13 and 38 μM, respectively. The activity also shows substrate inhibition at this pH. 30% of the AMPase activity could not be inhibited, and an Eadie-Hofstee plot shows mixed inhibition. Inability of the combined use of α, β-methylene ADP and (−)- p-bromotetramisole to totally suppress activity indicates the presence of a third phosphatase enzyme. Subsequent analysis at various pH values indicates that this third enzyme has an activity peak in the acid range. Using β-glycerophosphate as a substrate, 81% of the activity at pH 9.5 is due to alkaline phosphatase and none is due to 5′-nucleotidase. Likewise virtually none of the β-glycerophosphatase activity at pH 7.5 is due to alkaline phosphatase. With both Swiss albino and AKR mice, activity of 5′-nucleotidase increased markedly in adult male mice when compared to younger immature animals whereas alkaline phosphatase showed little change. In leukemic AKR mice, 5′-nucleotidase activity was sharply lower in the cells of thymomas when compared to nonleukemic AKR mice of comparable age. Alkaline phosphatase activity in the thymus of leukemic AKR mice was either increased markedly or remained unchanged. Thus the specificity of three distinct phosphatase activities in thymocytes are clearly defined and monitored during developmental and pathological alterations of the cell: (1) 5′-nucleotidase, (2) alkaline phosphatase, and (3) a third phosphatase with a lower pH optimum.

H-2 restriction as a consequence of intentional priming. Frequency analysis of alloantigen-restricted, trinitrophenyl-specific cytotoxic T lymphocyte precursors within thymocytes of normal mice.

An in vitro acute-depletion protocol was used to detect trinitrophenyl (TNP)-specific, allo-major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) within thymocytes of inbred mice. After removal of alloreactivity, the negatively selected cells could be sensitized to become TNP-specific, allo-MHC-restricted cytotoxic T cells. A precursors frequency analysis revealed a three- to ninefold lower frequency of allo-MHC-restricted CTL precursors (CTL-P) as compared to self-MHC-restricted CTL-P. The specificity analysis of clonally distributed allo-MHC-restricted CTL-P excluded cross-reactivity as an explanation of allo-MHC restriction. These results provide direct evidence that thymic T cells are composed of a mixture of self-MHC- and allo-MHC-restricted immunocompetent T cells and that antigen-driven selection of precommitted T cells dictates the H-2-restriction phenotype, i.e., H-2 restriction is a consequence of priming.

Effect of interferon and interferon inducers on the NK sensitivity of normal mouse thymocytes.

Effect of interferon and interferon inducers on the NK sensitivity of normal mouse thymocytes.

Killing of normal cells by activated mouse natural killer cells: evidence for two patterns of genetic regulation of lysis.

Normal primary mouse thymocytes and peritoneal cells are sensitive for lysis by mouse spleen cells activated by in vivo infection with lymphocytic choriomeningitis virus (LCMV). With both thymocytes and peritoneal cells as targets, the active killer cell seemed to be an NK cell, as judged by high activity in the mouse mutant nude and low activity in the mouse mutant beige. Thymocytes from 1- to 3-week-old mice were found to be most NK-sensitive, while peritoneal cells showed highest sensitivity for lysis when harvested from older (6- to 8-week-old) animals. In a direct comparison the genetic regulation of NK activity against thymocytes and peritoneal cells was analyzed. The same strain origin of both target cells and effector cells in either system was used. With thymocytes as target cells, spleen cells from LCMV-infected C3H/St mice invariably showed the highest activity closely followed by cells from the BALB/c strain, while the SWR/J and the A/J strains both were considerably less reactive. The same pattern of high- or low-reactive strains was seen regardless of the genotype of the thymocyte target donor, and the same pattern of cytotoxicity was also seen against the YAC-I lymphoma target. An entirely different genetic regulation was active in the lysis of peritoneal cells, where a unique pattern of reactivity was seen for each effector cell genotype, dependent on the strain origin of the peritoneal target cell. In line with previous findings, syngeneic combinations of effector-target cells yielded little or no reactivity, while various allogeneic combinations showed considerable levels of activity. Also, primary cultures of embryonic fibroblasts were used as target cells. The genetics of the fibroblast target system was more similar to that seen in the thymocyte-YAC-I assay since no evidence for a preferential killing of allogeneic target cells was seen.

Mitogen-induced interferon production by normal and steroid-resistant mouse thymocytes.

We have investigated the proliferative response and interferon production in cultures of mouse thymocytes stimulated with two different mitogens, PHA (phytohemagglutinin), or Con A (concanavalin A). Normal thymocytes proliferated weakly and did not produce detectable interferon levels in response to both mitogens. Supplementing this cell population with macrophages or adding 2-mercaptoethanol to the culture medium strongly enhanced the proliferative response to both mitogens, but only in response to PHA marginal levels of interferon could be detected. When the steroid-resistant population was tested, both PHA and Con A induced strong proliferative responses; in this case significant interferon levels could be obtained after stimulation with PHA, but only borderline levels with Con A. Peripheral lymphocytes from the spleen responded identically to both mitogens with respect to interferon production as well as proliferation. The data suggest that distinct differentiation pathways may exist for T lymphocytes producing interferon in response to different mitogens.

Expression of viral antigens on the membrane of normal and leukemic thymocytes of AKR mice

Expression of antigenous determinants of structural proteins G-MuLV (p10, p12, p30, gp14, gp17) and R-MuLV (gp69/71, p15) on thymocytes of normal and leukemic AKR mice was studied by membrane immunofluorescence. From this sign sharp difference between normal and malignant thymocytes was shown. A possible role in the antitumour immunity of antigens to structural viral proteins MuLV expressed on the leukemic cell membrane is discussed.

Induction of B lymphocyte colony growth in vitro by thymus‐derived stimulating factor

Murine lymphoid cells which were stimulated in liquid culture containing thymus culture fluid (Thy-CF) and seeded in a soft agar culture system, proliferated and developed into B cell colonies. Two types of colonies were formed: large colonies within the upper layer and small flat colonies on the surface of the upper layer. Thy-CF prepared from cells of normal hydrocortisone-treated mice had a higher cloning potential than Thy-CF prepared from untreated mice. At concentrations of Thy-CF in culture medium greater than 35%, Thy-CF prepared from normal mice had an inhibitory effect on colony formation. Cells of nude mice were also able to form B cell colonies if thymocytes of normal mice were mixed with lymphoid cells in the culture medium. Thymocytes elaborate a B lymphocyte colony-stimulating factor which, with the help of T cells, triggers a B cell population into colony formation and immunoglobulin production.

Activation of complement by spontaneous leukemic cells of AKR mice

AbstractComplement activation via the alternative pathway by tumor cells was tested by rosette formation of human erythrocytes (HuE). Test cells were treated with C4‐deficient guinea‐pig serum (C4DS) in the presence of Mg++ and absence of Ca++, following which 10–30% of lymphoma cells and 10–17% of thymoma cells of spontaneous leukemia in AKR mice formed rosettes. The incidence of rosette formation did not change significantly after transplantation of these cells to other non‐leukemic AKR mice or after in vitro cultivation. Following treatment with fresh C4DS, however, normal AKR mouse thymocytes as well as lymph‐node cells contained only 3–4% HuE‐positive cells. These findings suggest that the ability of non‐specific activation of complement (NAC) may be increased when normal AKR mouse cells become transformed into leukemic cells and/or that the leukemic cells arise from thymus cells which possess NAC ability. This interpretation was further confirmed by the finding that a higher proportion of leukemic cells than of normal thymocytes were lysed by mouse complement in Mg++‐EGTA. The fact that NAC‐positive AKR thymoma cells were lysed effectively by homologous C3H/He plasma agrees with earlier findings that in leukemic AKR mice supplemented with C5 a temporary regression of leukemia occurs.

Effect of a human serum thymic factor on hydrocortisone‐treated thymocytes

A human serum thymic factors (SF) stimulated cyclic adenosine-3' ,5'-monophosphate (cAMP) synthesis in normal mouse thymocytes. Such stimulation was no longer observed when thymocytes were depleted of hydrocortisone (HC)-sensitive cells. It was concluded that SF selectively stimulate cAMP in HC-sensitive cells. Furthermore, incubation of thymocytes with SF enlarged the population of HC-resistant thymocytes. These results suggest that SF might act on HC-sensitive thymocytes increasing their cellular cAMP level and inducing their transformation in HC-resistant cells.