Secondary response of in vitro primed human lymphocytes to allogeneic cells

The secondary response of human lymphocytes primed in vitro with allogeneic lymphocytes is reported. Accelerated proliferation is observed ' both against the specific priming cell and against unrelated third party cells, but the intensity of proliferation against the specific cell is usually, but not always, higher than that against third party cells. To clarify the respective roles ofHL-A andMLR-S in the development of this secondary proliferative response, three kinds of cells were used from which MLR-S activity was supposed to have been abolished while serologically-defined HL-A antigens were present: (a) heattreated cells, (b) UV-treated cells, and (c) a recombinant betweenHL-A andMLR-S. Heat treated cells were unsatisfactory for this study, but UV-treated and recombinant cells showed thatMLR-S was sufficient and necessary both for priming and for eliciting a secondary proliferative response. No role could be found forHL-A or for a secondMLR-S locus positioned between the first and secondHL-A loci.     

Secondary delayed-type hypersensitivity to sheep red blood cells in mice: a long-lived memory phenomenon

Immunization of mice with sheep red blood cells (SRBC) can induce the capacity to react with a secondary delayed-type hypersensitivity (DTH) immune response upon a booster injection of the antigen. In this paper the kinetics of secondary DTH after intravenous (iv) immunization with various doses of SRBC was studied by means of the foot swelling test. Dose-response studies showed that maximal secondary DTH responsiveness was obtained by iv administration of a priming dose of 3 × 10⁴ SRBC and a booster dose of 3 × 10⁵ SRBC 2 months later. Secondary DTH in such treated mice was characterized by an earlier appearance of the state of DTH, an earlier peak reactivity, and an increased intensity of the DTH response as compared to the primary DTH response. Up to 1 year after priming, a secondary DTH could be elicited, indicating the long-lived character of this memory phenomenon. With increasing intervals between the priming and booster injection, a gradual shift to a later time, of the peak secondary DTH reactivity was found. The capacity of primed mice to react with an increased intensity upon a booster injection could be adoptively transferred into lethally irradiated recipients by means of spleen cells obtained from primed mice. This phenomenon appeared to be highly dependent on Thy 1.2⁺ cells and on the booster dose of SRBC. The DTH reaction, evoked in such recipients, showed a prolonged time course.     

Cooperation between Carrier-reactive and Hapten-sensitive Cells <Emphasis Type="Italic">in vitro</Emphasis>

THE mechanism, known as the carrier effect, whereby immunity to one or more determinant groups enhances the response to other determinants on the same multivalent antigen, was first recognized in delayed hypersensitivity to haptens, in which, for an appreciable response, the hapten must be coupled to the same protein carrier for priming and challenge^{1, 2}. Carrier specificity has also been demonstrated in the secondary antibody responses to hapten protein conjugates³. Two alternative hypotheses have been advanced to explain this specificity. The “local environment” hypothesis supposes that the hapten-sensitive cell recognizes both the hapten and the carrier determinants. However, the antihapten antibodies produced do not distinguish details of the carrier molecule and so do not reflect the specificity of the cellular receptor. Furthermore, inert spacer molecules inserted between hapten and carrier do not interfere with carrier specificity in the antibody response³. Reflecting current views on the cooperation between thymus-derived (T) and bone marrow derived (B) lymphocytes in the antibody response to various antigens⁴, the second hypothesis invokes two or more cells, one with receptors directed towards the hapten (hapten-sensitive cell), the others specific for the carrier molecule proper (carrier-reactive cells). Supporting this is the observation that pre-immunization to a particular protein carrier alone could potentiate the primary or secondary antihapten response to a hapten conjugated to that protein⁵. In an adoptive transfer system, moreover, the efficiency of antihapten antibody production by cells primed to a particular hapten-protein conjugate and stimulated with the hapten conjugated on a heterologous protein, is significantly enhanced by the introduction of cells primed to the heterologous carrier alone. Anti-carrier serum antibody does not cause such enhancement⁶. The carrier-reactive cells must therefore cooperate in increasing the efficiency of the hapten-sensitive cells in some way other than by providing humoral anti-carrier antibody. Recent work strongly suggests that carrier reactive cells are thymus-derived^{6, 7}.     

Conversion of type III pneumococcal polysaccharide low responders to high responders by immunization with a thymus-dependent form of antigen

C57BL/Ks mice immunized with 0.6 μg Type III pneumococcal polysaccharide (S3) or with 10⁹ S3 conjugated sheep erythrocytes (S3-SRBC) produced 5–7 times fewer S3-specific plaque-forming cells than similarly immunized BALB/c mice. However, when mice were primed with the SRBC carrier prior to challenge with S3-SRBC the low responder C57BL/Ks mice responded as well as the high-responder BALBc strain. The cell activated by the carrier priming was shown to be a thymus-derived (T) cell and the antibody produced by primed mice was mercaptoethanol sensitive (presumably IgM). Nonspecific T cell activation by unrelated antigens did not enhance C57BL/Ks responses to the same degree as specific carrier priming. These findings are discussed in relation to the possible cellular basis for genetic control of the S3 immune response.     

In vitro secondary MLR. I. Kinetics of proliferation and specificity of in vitro primed responder cells.

We have examined the kinetics and specificity of secondary in vitro mixed lymphocyte reactions (MLR). With limited numbers of primed responder cells (PRC) in the presence of "excess antigen" it was possible to obtain proliferative responses that were proportional to the number of PRC initially placed in culture. The responding cells, after an initial lag period, seem to grow exponentially until day 3 of culture. The responses of PRC (with the strain combinations and culture conditions described in this report) seemed to be directed toward stimulator cell determinants whose expression was determined by genes in the I region of the MHC. In one case, the relevant incompatibilities could be further restricted to the I-A region. Although PRC responded best to stimulator cells sharing the I region with the priming stimulator cell, apparent cross-reactivity could be observed by restimulating PRC with stimulator cells that did not carry the MHC haplotype of the priming stimulator cell. The rate of proliferation (measured as 3H-thymidine incorporation) in these apparent cross-reactions was reproducible and comparable to the rate observed in response to the priming stimulator cell. It was possible, therefore, to estimate the proportion of PRC that reacted in the presence of third party stimulator cells compared to the response of these PRC to the priming stimulator cells. We have estimated that the response of A (B6) PRC against H-2d and H-2s haplotype stimulator cells is about half of the response of these PRC to H-2b, the priming stimulator cell.     

Human allospecific TLCs generated against HLA antigens associated with DR1 through DRw8

Serologic, cellular, and molecular evidence supports the concept of extreme complexity within the HLA-D region. To study the complexity and fine specificity of the HLA-D region at the level of T -cell recognition, a panel of T-cell clones was generated against alloantigens associated with HLA-DRI through -DRw8. After initial screening of more than 800 clones, 89 representative lines were selected for extensive testing against 204 unrelated stimulator cells. Clone-by-clone correlation analyses were performed to test whether any clones recognized similar or identical epitopes. In addition, clonal reactivity patterns were correlated with known HLA specificities. Twelve clusters of clones were identified with similar reactivity patterns using clone-by-clone correlation analysis. Some groups were significantly correlated with specificities associated with various D-region haplotypes; others had no significant correlation with any defined D-region specificity. Five general types of clones obtained in our study can be categorized as follows: (1) Those recognizing epitopes clearly demonstrating a primary association with the classically defined D-region molecules against which the clones were primed. (2) Clones recognizing epitopes associated with one of the priming antigens and also with another unrelated D-region specificity. (3) Clones detecting epitopes which showed significant correlation with D-region molecules totally different from those against which they were originally primed. (4) Clones with limited reactivity in population studies and no correlation with defined D-region molecules. (5) Clones recognizing class I-associated epitopes.Abbreviations used in this paper cpm counts per minute - DNV double normalized value - EBV Epstein-Barr virus - FCS fetal calf serum - HLA human MHC - HTC homozygous typing cell - LCL lymphoblastoid cell line - MHC major histocompatibility complex - MLC mixed lymphocyte culture - MoAB monoclonal antibody - PLT primed lymphocyte typing - T-max maximized T test - TCGF T-cell growth factor - TLC T-lymphocyte clone     

In vivo immune response to TNP hapten coupled to thymus-independent carrier lipopolysaccharide

Lipopolysaccharide has been utilized as a carrier for the TNP hapten, producing an antigen which induces an in vivo thymus-independent antibody response to TNP as determined using athymic nude mice and their normal littermates. The immune response to TNP-LPS was investigated at both the antibody-forming cell and the serum antibody levels.The primary response to an optimal dose of TNP-LPS (1.0 μg) exhibited unusual kinetics reaching a sharp peak on day 3 of 58,000 anti-TNP PFC/spleen. Serum antibody to TNP was first detected on day 3 and reached a maximum log₂ titer of 17.5 on day 5, an uncommonly high level for hapten-carrier conjugates and most carriers. Both the anti-TNP serum antibody and PFCs were exclusively IgM. No IgG antibody was detected in the primary response through 28 days postimmunization, nor was any detected in any experiment described in this paper. The primary PFC response to 1.0 μg of TNP-LPS was specific for TNP, producing no evidence of polyclonal antibody synthesis. The relative affinities of PFC-secreted antibody were investigated using hapten inhibition. The hapten inhibition curves for TNP-LPS and TNP-SRBC were very similar, indicating that relatively high affinity antibody was elicited by TNP-LPS. The secondary response to this dose following priming with TNP-SRBC or TNP-LPS was similar to the primary response, though the peak was less sharp in both cases. The response to the homologous secondary challenge shifted somewhat, reaching a peak on days 3–4. The effect of various doses in priming or challenging for the secondary response to TNP-LPS was investigated. Using an increased PFC response as a criterion, no dose was optimal for priming or immunological memory to TNP-LPS. While the adoptive primary response to TNP-LPS reached a low level peak on day 7, the adoptive secondary attained a maximum on day 6. This shift in kinetics in intact mice and in adoptive hosts in comparing primary to secondary responses indicated that a state of B cell priming may be induced. However, its full expression may be suppressed by endogenous factors at the time of priming, such as the high level of circulating anti-TNP antibody or residual antigen. Adoptive transfer would remove the cells from these influences, allowing such B cell priming to manifest itself fully.     

Cellular requirements for the expression of IgM. Immunological memory in vitro

In vitro culture techniques have been used to compare the direct (IgM) plaqueforming cell (PFC) response to heterologous erythrocytes (RBC) by normal mouse spleen cells and spleen cells from mice injected intravenously with 5 × 10⁴ RBC ten days previously [low dose primed (LDP)]. Although LDP mice fail to undergo a significant primary PFC response, their spleen cells are capable of a secondary or enhanced PFC response in vitro. The secondary PFC response is shown to be a function of: (A) an increase in the frequency of immunocompetent cells or units (IU) due to in vivo priming, and (B) an increased number of PFC generated per IU subsequent to in vitro stimulation. The latter increase is shown to be mediated through a shorter PFC doubling-time during logarithmic expansion of the PFC population. Analysis of nonadherent spleen cell dose response experiments indicate that two nonadherent cell types interact in the secondary response. Subsequent cocultivation experiments suggested that both of these cell types must be “primed” to allow induction of a secondary response. Although adherent cells are required for the secondary response, normal splenic adherent cells serve as equivalent substitutes for LDP adherent cells.     

Primed lymphocyte test in dogs: Restimulation by non-DLA-D determinants and failure to detect lymphoma associated antigens

The primed lymphocyte test (PLT) has been adapted to the dog and utilized in histocompatibility typing and in an attempt to detect lymphoma associated antigens (LAA) in dogs with spontaneously occurring lymphoma. After primary culture lasting 12–14 days, primed lymphocytes could be stimulated by the priming cells to undergo blastogenesis within 2–4 days of secondary culture. Dogs sharing defined DLA-D determinants always showed cross-reactivity in PLT, but dogs showing cross-reactivity in PLT did not necessarily share DLA-D determinants defined by homozygous typing cells. After using two variations of the test, no evidence for reactivity of presumed LAA in the PLT could be found.     

Restimulation properties of human alloreactive cloned t-cell lines. Dissection ofHLA-D-Region alleles in population studies and in family segregation analysis

Alloactivated human lymphocytes were cloned by limiting dilution. After 1 month in culture with T-cell growth factor several clones incorporated tritiated thymidine when stimulated with the appropriate allogeneic cells. Specificity of restimulation of two primed lymphocyte clones, designated 12-2 and 12-8, was studied in detail after varying periods of culture (up to 50 days). Clone 12-2 cells were stimulated only by cells expressing the HLA-Dw antigens of the original priming cells (Dw3); furthermore, this primed lymphocyte reagent specifically recognized antigens associated with only one of the three distinct Dw3-bearing haplotypes from an informative family (KOH). Clone 12-8 cells, on the other hand, failed to recognize Dw3 antigens in the random panel or on homozygous typing cells (including the original priming cell), but were strongly restimulated by certain cells expressing Dw4 antigens. In addition, within family KOH, these restimulating products segregated with another one of the three Dw3-bearing haplotypes but with none of the three Dw4-bearing haplotypes. These two clones exemplify a hitherto unknown precision in cellular typing of theHLA-D region. Clone 12-2 allows the discrimination of a probably rare and as yet undetected HLA-Dw3 subtypic specificity. Clone 12-8, on the other hand, apparently identifies an allelic system segregating withHLA but distinct from the HLA-D determinants definable by HTC-typing.Abbreviations used in this paper MHC major histocompatibility complex - HLA human leukocyte antigens - PBL peripheral blood leukocytes - HTC homozygous typing cells - MLC mixed leukocyte culture - PLT primed lymphocyte testing - TCGF T-cell growth factor - CTC cultured T cells - Tdr tritiated thymidine     

The secondary antibody response induced in tissue cultures

The conditions for induction of memory cells (B-MC) and evocation of the secondary antibody (Ab) response in tissue cultures (TC) were estimated.(1) In vivo primed B-MC cells were isolated 6–150 d after priming and stimulated in TC with different doses of sheep red blood cell (SRBC) antigen. The Ab response has a strict time and dose dependence: only small doses (10⁵) evoke a secondary response, high doses (10⁸, 10⁹) a state of immediate tolerance. (2) Antigen added to TC directly with B-MC rescued their Ab production for a long period. Addition of the antigen 1 or 2 d after setting the TC, follows the Ab-response decay, comparable with virgin cells (B-ICC). (3) Primed B-MC stimulated in TC responded preferentially with an IgM secondary response; the same cell suspension adoptively transferred into isologous recipients switched into IgG cells. (4) Virgin, immunocompetent, B-ICC were primarily stimulated in TC with a small dose of antigen (10⁵ SRBC); after 7 d of cultivation the cells were transferred into isologous recipients, SCID mice and into TC. In all cases, the secondary response of IgM was determined, 10 times higher than in the primary controls.     

Priming with Secreted Glycoprotein G of Respiratory Syncytial Virus (RSV) Augments Interleukin-5 Production and Tissue Eosinophilia after RSV Challenge

The respiratory syncytial virus (RSV) G glycoprotein promotes differentiation of type 2 CD4⁺ T lymphocytes and induces an eosinophilic response in lungs of RSV-infected mice. A unique feature of G is that a second initiation codon in the transmembrane region of the glycoprotein results in secretion of soluble protein from infected cells. Recombinant vaccinia viruses that express wild-type G (vvWT G), only secreted G (vvM48), or only membrane-anchored G (vvM48I) were used to define the influence of G priming on immunopathogenesis. Mice immunized with vvM48 had more severe illness following RSV challenge than did mice primed with vvWT G or vvM48I. Coadministration of purified G during priming with the construct expressing membrane-anchored G shifted immune responses following RSV challenge to a more Th2-like response. This was characterized by increased interleukin-5 in lung supernatants and an increase in G-specific immunoglobulin G1 antibodies. Eosinophils were present in the infiltrate of all mice primed with G-containing vectors but were greatest in mice primed with regimens including secreted G. These data suggest the form of G protein available for initial antigen processing and presentation is an important factor in promoting Th2-like immune responses, including the induction of lung eosinophilia. The ability of RSV to secrete G protein may therefore represent a viral strategy for immunomodulation and be a key determinant of disease pathogenesis.     

Secondary IgG responses to type III pneumococcal polysaccharide. I. Kinetics and antigen requirements.

Mice primed with a thymus- (T)3 dependent form of Type III pneumococcal polysaccharide (S3), i.e., S3 coupled to sheep or horse erythrocytes (S3-RBC), produce S3-specific IgG antibody after secondary challenge with either the T-dependent (S3-RBC) or T-independent (S3) form of the antigen. The potential to produce IgG antibody after challenge with S3-RBC appears earlier after priming than the potential to produce IgG after challenge with S3, suggesting that different "memory" cells may be involved in the two responses. The "memory" cells were shown to be S3-specific since S3 had to be present on the carrier in order for priming to occur and carrier specificity was not required for elicitation of the secondary response by S3-RBC.     

Secondary response on in vitro primed human lymphocytes to allogeneic cells

In vitro primed human cells have been shown to proliferate and to generate cytotoxic effector cells only when triggered by MLR-S determinants; they do not respond to HL-A antigens alone (i.e., they behave in this respect as unprimed cells). In contrast, in vivo-primed mouse spleen cells acquire the ability to proliferate and to generate cytotoxic effector cells even when triggered by cells artifically depleted by physical means of MLR-S activity or by cells, such as fibroblasts, normally devoid of MLR-S activity. For this reason, peripheral blood lymphocytes (PBL) from immunized volunteers were studied and the immunogenetic requirements of such in vivo-primed cells were compared to those of the in vitro-primed cells.Both in vivo- and in vitro-primed PBL were found to obey the same Laws: (a) proliferation is induced only by MLR-S disparities and is not induced by HL-A disparities alone; (b) proliferation appears to be specific for a given MLR-S; (c) specific cytotoxic effectors are generated by either a specific MLR-S stimulus or a third party-cell stimulus; (d) nonspecific mitogens can also, generate memory cytotoxic effector cells from a preimmunized cell population. However, the expression of such an immune status by PBL is short-lived, suggesting homing in privileged sites of the immune memory cells.     

The genetics of PLT response. II. HLA-DRw is a major PLT-stimulating determinant.

PLT response is restricted by the HLA-D region. The present study was undertaken to help define the role of HLA-DRw in PLT restimulation. Haplotype-primed intrafamily PLT cells were made against specificities HLA-DRw1, HLA-DRw3, and HLA-DRw7; each PLT was then restimulated with cells from a 35-member unrelated panel. Restimulation values for each PLT were subjected to bimodal clustering analysis. In addition, blocking experiments were performed with other intrafamily and homozygous typing cell PLT after preincubation with B cell alloantisera. The results show a high correlation (0.881 less than or equal to r less than or equal to 1.00) between the HLA-DRw specificity of the priming haplotype and the HLA-DRw specificity of unrelated panel cells that restimulate in PLT. When stimulating cells were absorbed with the corresponding DRw alloantisera or p29,34 heteroantiserum (against B cell specific antigens), PLT restimulation was significantly blocked. However, the PLT cells treated with antisera showed no effect. The results strongly suggest that HLA-DRw is the principal PLT-stimulating determinant.     

Characterization of human T-lymphocyte clones (TLCs) specific for HLA-region gene products

Clones of lymphocytes, primed in vitro to HLA-DR1; Dw1, were tested for allospecific proliferation on a panel of thirty-one HLA-phenotyped stimulating cells. No clone was restimulated exclusively by cells sharing the DR1; Dw1 priming antigens and most clones were restimulated by subsets of cells bearing DR1; Dw1. Generally, positive responses were at least 20-fold higher than autologous negative controls. Peak proliferative responses occurred around 72 h and varied, depending on the stimulating cell as well as the responding clone. Selected clones were induced to proliferate only by cells incapable of forming rosettes with sheep erythrocytes. Specific proliferation by TLCs was blocked by monoclonal DR-specific antibodies, but not by monoclonal anti-Thy 1.2. Genetic studies demonstrated that TLCs detected some cell-surface determinants that are encoded by genes in linkage disequilibrium with HLA and others that may not be linked to the human major histocompatibility complex.Abbreviations used in this paper ³HTdR tritiated methylthymidine - HTC homozygous typing cell - MHC major histocompatibility complex - HLA human MHC - MLC mixed leukocyte culture - PBL peripheral blood lymphocytes - PLT primed lymphocyte typing - TCGF T-cell growth factor - IL-2 interleukin 2 - TLC T-lymphocyte close     

Effect of bacterial lipopolysaccharide on the in vitro secondary antibody response in mice. I. Description of the suppressive capacity of lipopolysaccharide.

Bacterial lipopolysaccharide (LPS) suppressed the in vitro secondary antibody response in mice to the protein antigens human gamma globulin (HGG) and turkey gamma globulin (TGG). This is the first report of LPS inhibiting a secondary antibody response. Consistent suppression was dependent on the time of LPS addition; LPS added at culture initiation was less effective than LPS added 12 to 48 hr later. The mitogenic moiety of LPS was the inhibitory principle, as shown by the lack of suppression of spleen cells from C3H/HeJ mice, and the inability of the polysaccharide component, but not the lipid component of LPS, to suppress A/J spleen cells. The mechanism of suppression by LPS was not due to large numbers of B cells proliferating in response to LPS, since removal of B cells not bearing specificity for the priming antigen did not reduce suppression by LPS. However, the possibility exists that LPS may act through B cells specific for the priming antigen.     

DNA-synthesizing cells in oral epithelium have a range of cell cycle durations: evidence from double-labelling studies using tritiated thymidine and bromodeoxyuridine

Abstract Mouse tongue epithelium is characterized by a circadian variation in the number of DNA-synthesizing cells (labelling index, LI). Cells undergoing DNA synthesis were labelled with tritiated thymidine ([³H]TdR) at 0300 (peak LI) or 1200 h (low LI). The fate of these cells was assessed by injecting animals with bromodeoxyuridine (BrdU) at intervals from 12–48 h after [³H]TdR, to follow them from one cell cycle to the next. Labelling was revealed by combining [³H]TdR autoradiography with immunoperoxidase detection of BrdU in the same sections.
A single peak in the appearance of double-labelled cells was seen at 44 h, if [³H]TdR was given at 1200 h; following [3H]TdR at 0300 h, a peak of double labelling was seen at 48 h with the possibility of smaller peaks at 24 h and 36 h.
These results show that the 24 h periodicity in LI in this tissue is associated with a predominant cell cycle duration of 44–48 h, but that a few cells cycle more quickly. Double labelling with [³H]TdR and BrdU provides a useful method for establishing cell cycle duration by labelling S-phase cells in successive cell cycles.     

Cellular origin of cytotoxic effectors and secondary educated lymphocytes in human mixed leukocyte reaction

Human lymphocytes sensitized in vitro during a mixed leucocyte reaction (MLR) against an allogeneic-stimulating cell respond by blast transformation and generation of specific cytotoxic effector cells. Both proliferation and cytotoxicity are maximum on Days 6 and 7 of culture. On Day 14, no more dividing cells or cytotoxic cells are detected in such primary cultures. Restimulation by the specific priming cell triggers a secondary proliferative response and rapid reappearance of specific cytotoxic effector cells. The velocity sedimentation cell separation method which separates cells according to their size was applied to human lymphocytes sensitized in vitro during an MLR on Day 7 of culture. Blast cells were separated from nondividing small lymphocytes. It was shown that: (1) cytotoxic effectors generated at the peak of a primary response are exclusively present in the isolated blast population; (2) highly cytotoxic secondary effector cells are induced to reappear mainly from the blast-derived population upon restimulation; and (3) secondary educated proliferative cells mainly derive from the blast population. Conversely, the blast-depleted small lymphocyte population is operationally depleted of cells able to respond by proliferation to the priming cell while responding normally against third party control cells. HLA-D region specificity of the secondary proliferative response is suggested.     

DNA-synthesizing cells in oral epithelium have a range of cell cycle durations: evidence from double-labelling studies using tritiated thymidine and bromodeoxyuridine

Mouse tongue epithelium is characterized by a circadian variation in the number of DNA-synthesizing cells (labelling index, LI). Cells undergoing DNA synthesis were labelled with tritiated thymidine [( 3H]TdR) at 0300 (peak LI) or 1200 h (low LI). The fate of these cells was assessed by injecting animals with bromodeoxyuridine (BrdU) at intervals from 12-48 h after [3H]TdR, to follow them from one cell cycle to the next. Labelling was revealed by combining [3H]TdR autoradiography with immunoperoxidase detection of BrdU in the same sections. A single peak in the appearance of double-labelled cells was seen at 44 h, if [3H]TdR was given at 1200 h; following [3H]TdR at 0300 h, a peak of double labelling was seen at 48 h with the possibility of smaller peaks at 24 h and 36 h. These results show that the 24 h periodicity in LI in this tissue is associated with a predominant cell cycle duration of 44-48 h, but that a few cells cycle more quickly. Double labelling with [3H]TdR and BrdU provides a useful method for establishing cell cycle duration by labelling S-phase cells in successive cell cycles.