Genetically engineered fixed K562 cells: potent “off-the-shelf” antigen-presenting cells for generating virus-specific T cells

Background aimsThe human leukemia cell line K562 represents an attractive platform for creating artificial antigen-presenting cells (aAPC). It is readily expandable, does not express human leukocyte antigen (HLA) class I and II and can be stably transduced with various genes.MethodsIn order to generate cytomegalovirus (CMV) antigen-specific T cells for adoptive immunotherapy, we transduced K562 with HLA-A10201 in combination with co-stimulatory molecules.ResultsIn preliminary experiments, irradiated K562 expressing HLA-A10201 and 4-1BBL pulsed with CMV pp65 and IE-1 peptide libraries failed to elicit antigen-specific CD8⁺ T cells in HLA-A10201⁺ peripheral blood mononuclear cells (PBMC) or isolated T cells. Both wild-type K562 and aAPC strongly inhibited T cell proliferation to the bacterial superantigen staphylococcal enterotoxin B (SEB) and OKT3 and in mixed lymphocyte reaction (MLR). Transwell experiments suggested that suppression was mediated by a soluble factor; however, MLR inhibition was not reversed using transforming growth factor-β blocking antibody or prostaglandin E₂ inhibitors. Full abrogation of the suppressive activity of K562 on MLR, SEB and OKT3 stimulation was only achieved by brief fixation with 0.1% formaldehyde. Fixed, pp65 and IE-1 peptide-loaded aAPC induced robust expansion of CMV-specific T cells.ConclusionsFixed gene-modified K562 can serve as effective aAPC to expand CMV-specific cytotoxic T lymphocytes for therapeutic use in patients after stem cell transplantation. Our findings have implications for broader understanding of the immune evasion mechanisms used by leukemia and other tumors.     

Gamma irradiation of human dendritic cells influences proliferation and cytokine profile of T cells in autologous mixed lymphocyte reaction

Dendritic cells (DC) are the most potent antigen-presenting cells (APC); their ability to induce proliferation of T cells in a mixed lymphocyte reaction (MLR) assay is commonly used for the evaluation of their function. It is a general thought that gamma irradiation of APC does not influence their ability to activate T-cell proliferation, but the data from several studies are controversial. To further determine the mechanisms involved in DC-induced T-cell activation in MLR assay, human DC induced from peripheral blood mononuclear cells (PBMC) were gamma-irradiated and determine their effects on the proliferation and cytokine profiles of T cells in an autologous MLR. DC were induced from the PBMC of 11 multiple sclerosis (MS) patients with RMPI 640 medium containing recombinant human GM-CSF (rhGM-CSF; 800 U/ml) and recombinant human IL-4 (rhIL-4; 500 U/ml). DC harvested on day 7 were divided into two equal parts. One part was not irradiated (naive DC); the other was gamma-irradiated at a dose of 30 Gy. Cell surface molecules were analyzed by flow cytometry. T-cell proliferation was determined using a beta-scintillation counter. The levels of IL-2, IL-4, IL-6 and IL-10 in co-culture supernatants were measured by ELISA. The results indicated that gamma irradiation reduced expression of CD86, CD80 and HLA-DR molecules on DC, especially CD86 (P=0.0072). DC, irradiated or non-irradiated, effectively stimulated autologous T-cell proliferation. Compared to naive DC, irradiated DC showed a markedly lower capacity to promote T-cell proliferation (P=0.0073), and strikingly up-regulated secretion of IL-4 (P=0.0145) and IL-2 (P=0.0323) by autologous T cells. No significant differences were noted in IL-6 and IL-10 production between T cells co-cultured with naive DC and irradiated DC (P>0.05). It is concluded that gamma irradiation of DC not only influences the phenotype of DC but also alters their capacity to stimulate the proliferation and the cytokine profiles of autologous T cells in a MLR.     

The effect of radiation therapy combined with natural killer cells against spontaneous murine fibrosarcoma

The effect of radiation therapy combined with lymphoid cells against spontaneous murine fibrosarcoma (FSa-II) was investigated bothin vivo andin vitro. In thein vivo experiment, syngeneic C3H mice were divided into 3 groups. Animals in the first group were injected with 1 x 10⁵ tumor cells into the right hind leg. Animals in the second and third groups were injected with 1 x 10⁵ tumor cells mixed with 1 x 10⁷ normal lymphoid cells (NLC) or effector lymphoid cells (ELC), respectively. ELC were obtained from spleen and lymph nodes of FSa-II-bearing mice and incubatedin vitro for 40 hr to eliminate suppressor T cell function. NLC were obtained from normal mice and incubated in the same way. Irradiation was given using¹³⁷Cs unit 3 days after cell inoculation. 12 out of 14 mice (85.7%) inoculated with tumor cells mixed with NLC did not show any tumor growth at 60 Gy local irradiation. 12 out of 21 mice (57.1 %) inoculated with tumor cells alone and 6 out of 10 (60%) with tumor cells mixed with ELC rejected tumors at the same radiation dose. This synergistic effect with NLC was not observed when NLC was inoculated after irradiation, indicating that lymphoid cells should be in contact with tumor cells before irradiation. In the⁵¹Cr release assay, lymphoid cells obtained from whole body irradiated (WBI) mice showed 17.8% lysis without irradiation and 28.8% lysis at 5 Gy irradiation. Untreated NLC showed almost no cytotoxic effect at the same radiation dose. This synergistic effect disappeared when WBI lymphoid cells were treated with anti asialo GM1 and complement. These results suggested that NK cells might be important in this synergistic effect with irradiation. To obtain a sufficient level of synergistic effect by in vitro combined treatment of mixed tumor cell - NLC culture and irradiation - incubation for more than 12 hrs and 8 hrs appeared to be necessary before and after irradiation, respectively.     

Stimulatory cells in the generation of T-cell-mediated cytotoxicity: Potent stimulating activity of a small radioresistant spleen cell population (RSCs)

The combined effects of irradiation followed by cultivation on a total spleen cell population in order to study the evolution of the stimulating potential in the in vitro generation of allogeneic cytotoxic T lymphocytes (CTLs) were tested. Results revealed that, after 3 days and up to at least 7 days of cultivating irradiated (1000 rad) spleen cells, the remaining living cells (radioresistant spleen cells or RSC) have the same potential to generate CTLs as irradiated noncultivated spleen cells. RSC can resist a 5000-rad irradiation and induce a primary cytotoxic response pattern similar to that of total spleen cells; they act in primary as well as in secondary cultures with optimal responder to RSC ratios of about 100, but are still stimulatory at MLC ratios up to 1000 or 5000. They are lysed by specific allogeneic CTLs and readily inhibit the specific lysis of H-2-identical labeled targets by CTLs. RSCs do not express unusual levels of H-2 or Ia antigens and do stimulate purified T cells. Alloantisera anti-H-2 are able to completely block the RSC-induced generation of CTL. This RSC population may prove to be a good model to study non-H-2- or H-2-associated, nonserologically detectable determinants interacting in the generation of T-cell-mediated cytotoxicity.     

Dose-Dependency of radiation on enzyme production inTrichoderma reesei

Effect of irradiation dose on the production of cellulase and amylase related enzymes inTrichoderma reesei was studied, in which post-irradiation time responce pattern was measured. The damage of the cells irradiated with certain irradiation doses (1.40±0.20x10⁵, 2.20±0.10x10⁵, 3.00±0.50 x 10 and 3.50±0.20 x 10 rad) was rapidly recovered. The increased enzyme production in the culture of the irradiated cells resulted from the recovery of radiation damage after irradiation. The function of cell growth was not affected by irradiation below dose of 5 x 10⁵ rad, though the function of enzyme synthesis was drastically affected.     

Replication-Dependent Sister Chromatid Recombination in Rad1 Mutants of Saccharomyces Cerevisiae

Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation. In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G(1) or the G(2) phases of the cell cycle than was observed in RAD1/rad1-1 cells. Since sister chromatids are not present during G(1), this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication. The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G(2) phase, but only when they are present during DNA replication. We propose that there are two types of sister chromatid recombination in yeast. In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells. In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G(2). Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination.     

Mixed lymphocyte reactivity in spleen cells from F-9 tumor-bearing ice. I. Suppressor cell induction and characterization

The immune capacity of spleen cells from F-9 tumor-bearing mice was assessed in a one-way mixed leukocyte culture (MLR) and in MLR mixing experiments. Alloreactivity was found to be significantly depressed in mice bearing palpable tumors. Mixing experiments demonstrated that the reduced alloreactivity was due to suppressor cell activity in the spleens of the tumorous mice. Suppressor cells were characterized as adherent, Ly2⁺ T cells. This study provides the basis for an analysis of the cell surface antigens on F-9 embryonal carcinoma cells which lead to the induction of suppressor T cells in these mice.     

Suppression of mixed lymphocyte reactions by alloantigen-activated spleen-localizing thymocytes.

Heavily irradiated BALB/c and C57BL/6 mice were injected intravenously with BALB/c thymocytes. At varying times thereafter spleen and lymph node cell suspensions from these animals were treated with mitomycin C and added as regulator populations to mixed lymphocyte reactions (MLR) with syngeneic responder cells. Alloantigen-activated spleen-localizing thymocytes suppressed MLR responses 40 to 95%. Suppressor activitity, manifested as a quantitative reduction in peak proliferative responses, was maximal 4 days after cell transfer. Antigenic specificity for the stimulator cell strain in MLR was not demonstrated. The effect of lymph nodelocalizing thymocytes as regulators in MLR was variable, but in most experiments these cells slightly enhanced responses. We conclude that splenic localization is an intrinsic property of the thymocyte subpopulation capable of suppressing MLR responses, and that the suppressor activity of this subpopulation is substantially enhanced by activation in allogeneic hosts.     

Radiation-induced molecular changes inEscherichia coli B and its S-30 and DNA-plus-membrane fractions

Oxygenated aqueous suspensions ofEscherichia coli B cells in the resting state were irradiated with 0.8-MeV electrons. Dried films of whole cells, the S-30 fraction, and the DNA-plus-membrane fraction were studied by using infrared spectroscopy in conjunction with the technique of attenuated total reflectance (ATR) in the range from 4000 cm^?1 to 800 cm^?1. Cells irradiated in the oxygenated or the anoxic state yield the same kind of molecular damage, the main difference being the lower doses (by a factor 4 or 5) required in well oxygenated systems. Results show that some bonds are more sensitive to radiation than others. Decreases in the PO₂ bands (1225 and 1084 cm^?1) indicate radiation-induced degradation of the DNA-RNA backbone. The increase in absorption between 1700 cm^?1 and 1750 cm^?1 indicates formation of C=O bonds upon exposure to ionizing radiation. Most of the radiation damage occurs in cells that have undergone lysis during irradiation, but the process of cell lysis, by itself, does not cause appreciable molecular bond damage as measured by ATR. Doses ranged from 0.1 Mrad to 1.1. Mrad.     

Suppression of cell-mediated immune responses after total lymphoid irradiation (TLI). I. Characterization of suppressor cells of the mixed lymphocyte reaction

Total lymphoid irradiation (TLI) was administered to (BALB/c X C57BL/6)F1 mice in eight daily doses of 200 rad (total 1600 rad). Spleen cells isolated from mice after treatment with TLI do not respond to alloantigens in vitro in a one-way mixed lymphocyte reaction (MLR), but normal reactivity recovers after approximately 2 mo. Radioresistant, antigen-nonspecific suppressor cells are documented in the spleens of TLI-treated mice immediately after radiotherapy, but suppressive capacity gradually disappears within 30 days. After TLI, the spleen is repopulated with large cells, the proportion of which is greatest at a time when theta-bearing cells are still depleted. Radioresistant suppression is mediated predominantly by the large cell subset and is thymus independent. Suppressor function can be abolished by lethal physicochemical procedures including formaldehyde fixation, multiple freeze-thawing, and heating to 56 degrees C, and it cannot be conferred by supernatants of TLI-suppressed MLR suspensions. Suppression cannot be overcome by adding various cell factors including T cell growth factor (TCGF) and lymphocyte-activating factor (LAF), nor is it affected by a prostaglandin inhibitor. Equally potent radioresistant suppressive activity is documented by co-culturing cells derived from other sources enriched in large, immature hematopoietic cells, including fetal liver cells and bone marrow cells obtained from normal and congenitally athymic mice. The presence of a large cell population and MLR suppressor function is also documented in the spleens of mice treated with single dose or fractionated doses of lethal whole body irradiation, followed by reconstitution with bone marrow cells obtained from normal mice. The data suggest that MLR suppressor cells, which are large, immature and predominantly radioresistant, can be induced after a short and well-tolerated TLI regimen.     

Stimulation of Autochthonous Lymphocytes by Cells from Normal and Leukaemic Lines

THE mixed lymphocyte reaction (MLR) can possibly be regarded as an in vitro form of an in vivo phenomenon reflecting the recognition of “non-self” tumour specific or neo-antigens on the surface of lymphoid cells. A reaction similar to the normal MLR but of greater magnitude occurs when irradiated lymphoid cells from lymphoblastoid cell lines (LCL) are added to freshly isolated peripheral lymphocytes from allogeneic individuals^1,2. The intense stimulation which occurred in every case when irradiated cells from various LCL were added to lymphocytes from a large number of individuals³ suggested the presence of extra surface determinants on the cells, which are not present on normal freshly isolated cells. We have investigated whether freshly isolated lymphoid cells could detect and respond to extra antigenic determinants on the surface of cell lines derived more than 3 months earlier from their own lymphoid cells.     

Rabbit antiserum to human B-cell alloantigens. II. Mechanism of inhibition of stimulation in the mixed lymphocyte response.

The process by which a rabbit antiserum to human B-cell alloantigens blocks stimulation in the mixed lymphocyte response (MLR) was investigated. A functional mammalian Fc region was necessary for the antiserum to be inhibitory, since F(ab′)₂ fragments failed to inhibit and a chicken antiserum with similar specificity to the rabbit anti-B-cell serum did not effectively block the response. Immune elimination of the stimulating cell population possibly via antibody dependent cell-mediated cytotoxicity (ADCC) or phagocytosis by macrophages was suggested by the observation that the addition of aggregated IgG to the MLR reduced the level of inhibition. It was also found that the number of immunoglobulin positive cells decreased in cultures treated with intact rabbit anti-B-cell serum, but not the corresponding F(ab′)₂ fragments, whether the cells were from a single individual or an allogeneic cell mixture. ADCC appears to be involved in the blocking process, as demonstrated by the marked reduction in MLR suppression when the MLR was initiated in the absence of ADCC effector cells. Removal or inhibition of monocytes in the MLR partially restored the response in experiments where the stimulator cells were pretreated with the antiserum, but not when the antiserum was present throughout the MLR.     

Genetic Effects of Uv Irradiation on Excision-Proficient and -Deficient Yeast during Meiosis

The lethal and recombinational responses to ultraviolet light irradiation (UV) by excision-proficient (RAD⁺) and deficient strains (rad1) of Saccharomyces cerevisiae has been examined in cells undergoing meiosis. Cells that exhibit high levels of meiotic synchrony were irradiated either at the beginning or at various times during meiosis and allowed to proceed through meiosis. Based on survival responses, the only excision repair mechanism for UV damage available during meiosis is that controlled by the RAD1 pathway. The presence of pyrimidine dimers at the beginning of meiosis does not prevent cells from undergoing meiosis; however, the spore products exhibit much lower survival than cells from earlier stages of meiosis. The reduced survival is probably due to effects of UV on recombination. Meiotic levels of gene conversion are reduced only two to three times in these experiments; however, intergenic recombination is nearly abolished after a dose of 4 J/m ² to the rad1 strain. Exposure to 25 J/m² had little effect on the wild-type strain. Since normal meiotic reciprocal recombination is generally considered to involve gene conversion-type intermediates, it appears that unrepaired UV damage dissociates the two processes. These results complement those obtained with the mei-9 mutants of Drosophila which also demonstrate a dissociation between gene conversion and reciprocal recombination. These results are consistent with molecular observations on the UV-irradiated rad1 strain in that there is no excision of pyrimidine dimers or exchange of dimers during meiosis.     

The effects of phorbol ester on alloantigen presentation

B cells and Ia+ thyroid cells fail to stimulate alloreactive T cells in a primary mixed leukocyte reaction (MLR) and fail to activate some allo-class II (I-A) reactive T cell hybridomas. We now demonstrate that B cells can specifically stimulate a primary MLR in combination with the phorbol ester, PMA, but not with interleukin 1 (IL 1) or calcium ionophore. The primary MLR induced with B cells plus PMA can be blocked by either monoclonal anti-I-A or anti-L3T4 antibodies. In contrast, thyroid cells that can be induced to express Ia antigens after incubation with interferon-gamma fail to stimulate a primary MLR even in the presence of PMA or IL 1. We confirmed these observations by using the alloreactive T cell hybridoma, HTB-9.3, which does not react to stimulator B cells. In the presence of PMA, however, this I-Ab-specific hybridoma line was able to respond to relevant but not to control stimulator B cells. Furthermore, the response of HTB-9.3 to B cells plus PMA was also blocked by anti-I-A or anti-L3T4 antibody. In contrast to B cells, Ia+ thyroid cells could not activate HTB-9.3 even in the presence of PMA or IL 1. The data indicate that for primary class II restricted allo-responses, B cells provide signals that can be complemented with the phorbol ester PMA, whereas Ia+ thyroid cells do not, suggesting the existence of additional requirements for T cell activation.     

Clonal analysis of the T lymphocytes involved in parent versus Fn1 graft-versus-host reaction

A systemic graft-versus-host reaction (GVHR) leading to 50% mortality by day 20 was elicited by the injection of CBA (10⁵) or B10 (10⁶) parental T lymphocytes into irradiated (750 rad) and bone marrow protected (CBA x B10)F₁ recipients. Between days 12 and 28 the spleens of the sick mice were analyzed by limiting dilution, performed with irradiated F₁ cells and a source of interleukin-2 (IL-2), to determine the frequency of cells with an antihost proliferative or cytolytic activity and to derive T lymphocyte clones. The frequency of cells with antihost proliferative or cytolytic activity was approximately 10^–3 in either combination. In the CBA vs F₁ GVHR, all eight clones isolated with anti-F₁ activity were Lyt-2^–, noncytolytic, mixed lymphocyte reaction (MLR) responders and IL-2 producers, three of which mapped to the A ^b locus, while in the B10 anti-F1 combination, eight of the nine anti-F₁ clones isolated were Lyt-2⁺, poor MLR responders and non-IL-2 producers, but cytolytic and mapping to K _k . These findings suggest a much higher frequency of T cells recognizing the A-locus antigens in the CBA than in the B10 strain.     

Effects of226Ra and X-irradiation on the proliferative and differentiative ability of mouse hemopoietic stem cells

Summary The ability of hemopoietic stem cells to repopulate spleens of heavily irradiated syngeneic hosts in form of macroscopically visible clonal colonies of differentiating cells was studied in mice exposed for 32 and 4 weeks to internally deposited²²⁶Ra (0.56 and 0.46 µCi per mouse respectively) or to 100 rad X-irradiation. Exocolonizing test and cytological techniques were used for quantitative evaluation. The size of stem cell compartment was reduced and the function of the surviving stem cells was altered by radium and X-ray irradiation. The proliferation and maintenance of hemopoietic cell populations were found to depend not only on the numbers of stem cells but also on their multiplicative and differentiative capability.     

Induction of immunoglobulin-secreting cells in the allogeneic mixed leukocyte reaction: regulation by helper and suppressor lymphocyte subsets in man

The existence of functionally distinct T lymphocyte subsets in man, initially demonstrated with heteroantisera, has been confirmed with monoclonal reagents. Two major subsets have been defined: Leu-2 cells, which effect cell mediated lymphocytotoxicity (CML), and Leu-3 cells, which amplify CML and other T cell functions. This study is an effort to determine the effects of these subsets on the immunoglobulin secretion induced in the human mixed leukocyte reaction (MLR). T cells were separated from non-T cells by rosetting with sheep erythrocytes and were fractionated into Leu-2 and Leu-3 subsets by solid phase immunoabsorption with monoclonal antibodies. T cell subsets were cultured with autologous non-T cells and irradiated allogeneic stimulator non-T cells. Secretion of IgM and IgG was measured by a reverse hemolytic plaque assay. MLR-induced antibody secretion was specifically dependent on the Leu-3 T lymphocyte subset. The Leu-2 subset was incapable of generating large numbers of IgM- or IgG-secreting cells, and, in fact, suppressed the Leu-3-induced response. Exposure of Leu-3 cells to a dose of irradiation sufficient to prevent their proliferation in MLR did not reduce induced immunoglobulin secretion. Leu-2-mediated suppression, however, was sensitive to low dose irradiation. Thus Ig secretion in human MLR is regulated by a balance of helper activity from the Leu-3 subset and suppressor activity from the Leu-2 subset.     

Deficits in T helper cells after total lymphoid irradiation (TLI): reduced IL-2 secretion and normal IL-2 receptor expression in the mixed leukocyte reaction (MLR)

Spleen cells from BALB/c mice treated with total lymphoid irradiation (TLI) and from normal, unirradiated mice were compared in the mixed leukocyte reaction (MLR). Although the percentage of CD4+ cells in the spleen was close to normal, 4 to 6 weeks after TLI, the MLR of unfractionated spleen cells from irradiated mice was more than 10-fold lower than controls. A similar reduction was observed when purified CD4+ cells were used as responders in the MLR. Secretion of IL-2 by cells from irradiated mice was also about 10-fold lower than controls. However, the percentage of CD4+ and CD8+ cells which expressed IL-2 surface receptors during the MLR was similar using spleen cells from irradiated and control mice. Addition of an exogenous source of IL-2 restored the proliferative capacity of the irradiated cells and suggests that the lack of IL-2 secretion is the likely explanation of the marked deficit in the MLR of CD4+ spleen cells after TLI.     

Recognition and response to alloantigens in vivo. I. Negative and positive selection of MLR reactivity in murine peripheral blood lymphocytes to major histocompatibility complex and Mls antigens

Specific mixed lymphocyte reaction (MLR) responsiveness to allogeneic major histocompatibility complex (MHC), or minor lymphocyte-stimulating (Mls) determinants, was depleted in the peripheral blood lymphocytes (PBL) obtained from mice 24 to 48 hr after i.v. injection of 5 to 7.5 X 10(7) MHC or Mlsa-incompatible spleen cells, respectively. Results of cell mixture experiments suggest that the generation of suppressor cells was not the explanation for this specific reduction in MLR proliferation occurring with these PBL responder cells. To gain additional insight into parameters involved in the recognition of allodeterminants in vivo, experimental manipulations of the host environment and donor cell inoculum utilized in the negative selection procedure were employed. For example, removal of the spleen in the recipient animal, an anatomic site in which injected allogeneic cells and corresponding host antigen-reactive cells (ARC) are trapped, still permitted the specific depletion in murine PBL of host ARC for donor foreign MHC antigens. This finding may implicate other sites such as the liver where unprimed host alloreactive clones are trapped. In addition, irradiation of allogeneic donor cells significantly reduced their capacity to trap alloreactive T cell clones in vivo, whereas heat treatment of the donor cells completely eliminated this ability, even though the Ia determinants were still expressed, measured by flow cytometry. After the negative selection period, kinetic analysis of proliferation showed that 3, 4, or 5 days after injection of MHC-incompatible allogeneic spleen cells, the PBL of the recipient showed specific hyperresponsiveness to the MHC-haplotype of the donor cells. Interestingly, these primed PBL responder cells had the volume distribution of small resting cells; thoracic duct lymphocytes (TDL), positively selected by adoptive transfer of T cells to irradiated semiallogeneic recipients, are reported to be mainly blast cells. In contrast to the MLR hyperresponsiveness that results from priming with MHC-incompatible splenocytes, PBL, obtained at these later time points from mice primed with Mlsa-incompatible, H-2-compatible splenocytes, showed complete unresponsiveness in MLR to these Mlsa-bearing stimulator cells, as well as some nonspecific reduction in proliferation to MHC-incompatible stimulator cells regardless of their Mls genotype.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Effect of X-Irradiation On Cell Loss In Five Solid Murine Tumours, As Determined By the 125Iudr Method

The rate of cell loss in irradiated RIF-1, EMT6, KHJJ, B16 and KHT tumours was studied using the ¹²⁵IUdR loss technique. Administration of ¹²⁵IUdR preceded localized tumour irradiation by 2 days. Loss of tumour radioactivity was measured for 6–8 days after irradiation. the blood flow to some tumours was occluded during, and for 30 min following, injection of the label to measure the amount of radioactivity entering the tumour as a result of reutilization of label from the gut epithelia and influx of labelled host cells. Irradiation did not significantly alter the amount of radioactivity entering these clamped tumours during the 8–10 days after injection of ¹²⁵IUdR. This permitted comparison of irradiated and control groups based on the loss of radioactivity from the non-occluded tumours. Irradiation of RIF-1, EMT6, KHJJ or B16 tumours with doses of 600, 1400, 2400 or 4400 rads produced no significant increase in the rate of loss of tumour radioactivity. This suggested that, in the population of labelled cells, cell lysis following irradiation proceeded slowly. In contrast, KHT tumours showed a significant increase in loss rate following each radiation dose, although the increase was dose-independent. In all tumour systems, the constant rate of cell loss after radiation appeared to coincide with published reports of tumour growth responses after irradiation. the present data suggest that the manner of expression of radiation-induced cell killing results from the cellular proliferative status, i.e. whether a cell is cycling or non-cycling.