Cellular aspects of polyclonal activation of B lymphocytes

immunologytoday, November/980

92 reminiscent of one Fab fragment. In the long run this molecular analysis, in combination with mutational analysis, should be able to identify functionally active parts of the molecule. This is likely to be the golden road to understanding dual recognition by T cells. A problem which the chemists are now getting their teeth into is alien M H C expression. H. Festenstein reported peptide analysis of a D d molecule expressed by an A K R (H-2 k) tumour which strongly supports his contention that tumours synthesize intact alien M H ¢ molecules. The only sour note is news from Heidelberg (heard after the symposium) that one alleged instance of alien M H C expression is now

known to result from contamination. Surely this offers an excellent example of an M H C question which can best be answered by D N A chemistry. N. A. MITCHISON

Turnout Immunology Unit, Department of Zoology, Unive~:ffty College, London WC/E 6BT, U.K. References 1 Seventh International Convocation on Immunology, hnmunobiology of the Major Histocompatibitity Complex (concentrating mainly on sessions II and III) Buffalo,N.Y., 7-10July 1980 2 Cecka, J. M., Kamb, A., Kees, U. and Mitchison, N. A. (1979) Nature (London) 281,336

D3 Cellular aspects of polyclonal activation of B lymphocytes Michael G. Goodman Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, Calitornia 92037, U.S.A. The responses of g lymphocytes to exogenous stimulation can be antigen-spec~c or non-~pec~c. Non-~pedfic stimulation by a single agent activates a multitude of clones of B cells, each secreting antibody of pre-programmed specificity. In this review Michael Goodman examines how polyclonal activation orB cells can be achieved, and the relationship between such stimulation and D N A synthesis, considers the evidence for and against the existence of B-cell subpopulations responsive to different polyclonal activators, and looks at the ways in which polyclonal activation is regulated. Bone-marrow-derived (B) lymphocytes a p p e a r to receive signals from the environment when the signalbearing molecule binds to specific cell-surface receptors. M a n y substances activate B cells to polyclonal Ig synthesis de novo. A partial list of these diverse agents appears in Table I. The capacity of B-lymphocyte-specific mitogens such as bacterial lipopolysaccharide (LPS) to induce the synthesis of 19S i m m u n o g l o b u l i n was first reported by Anderson and co-workers ~. These investigators found that only I g M - a n t i b o d y - p r o d u c i n g cells were induced by exposure to this mitogen, that the n u m b e r of these cells increased in parallel with their stimulation to D N A synthesis, and that there was no demonstrable increase in the synthesis and secretion of 7S (IgG) antibody. Moreover, the specificity of the antibodies produced extended over a broad range, and could be detected by assaying against randomly chosen antigens coupled to indicator red blood cells. © Elsevier/North-Holland Biomedical Press 1980

This activity was not a function of the specific antibody response to LPS, since it occurred in mice . immunologically tolerant to LPS as well as in nontolerant mice. The frequency of murine B cells reactive to LPS has been determined under optimal growth conditions by limiting dilution analysis to be approximately one in three splenic B cells 2. Similar frequencies have been found for B cells responsive to bacterial lipoprotein (LP), for Nocardia mitogen, and for mitogenic compounds in fetal calf serum 3. Interaction of antigen with B cells bearing antigenspecific receptors leads to specific B-cell activation (in the presence of a T-cell signal) but can also evoke polyclonal antibody secretion4,t Polyclonal activation occurred whether antigen was presented in an immunogenic or tolerogenic form. It has been hypothesized that the antibody specificities produced are representative of the host's antigen experience, and that non-specific lymphokines secreted from antigen-

93

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TABLE I. Polyclonal activators of B lymphocytes Lipopolysaccharide (LPS) PPD Staphylococcus aureus protein A Nocardia water-soluble mitogen Lipid-A-associated protein 2-mercaptoethanoI (2-ME) ~-thioglycerol (a-TG) Macrophage- and T-cell-derived lymphokines Fc fragment of |g (in the presence of certain lymphokines) Proteolytic enzymes, eg. trypsin Polyanions, e.g. dextran sulphate, poly IC Antiobiotics, e.g. nystatin, amphotericin B Lanatoside C M ycoplasma pulmonis

Some viruses and viral components Parasites, e.g. Trypanosoma congolense, T. cruzi activated T lymphocytes are responsible for ensuing polyclonal activation. Andersson's initial observation that murine spleen cells could be activated with LPS to the synthesis of I g M was later extended by Kearney and Lawton 6 who showed that cultured murine lymphocytes from thoracic duct, spleen, lymph node, Peyer's patches, and bone marrow all synthesized intracytoplasmic IgM, IgG1, and IgG2 when cultured at low density in the presence of LPS. Bone-marrow cells could be induced to synthesize IgA in this manner. By the use of cytochalasin B to prevent cytoplasmic division while allowing nuclear division to proceed, Van Der Loo et a[. 7 observed that in the process of switching from IgM to IgG synthesis murine spleen cells cultured with LPS undergo an assymetric division resulting in the production of one IgM- and one IgG-secreting progeny cell. Andersson and his colleagues later reconfirmed the ability of low-density cultures to synthesize and secrete IgG 8. They also verified Kearney and Lawton's observation that IgG secretion can occur independently of the influence of T cells and of external antigen. The ability of LPS to activate murine spleen cells to antibody production against a vast array of antigens has been used to advantage by Goldsby el a/. 9 to fuse LPS-induced lymphoblasts with a non-secreting variant of a B A L B / c myeloma. H y b r i d cells were isolated at limiting dilution, and cells secreting antibody of desired specificities were selectively cloned. Thus individual antigenic specificities were selected from the totality of polyclonally stimulated cells to produce a monoclonat antibody from mice which had never been specifically immunized. The mechanism(s) involved in polyclonal activation of B lymphocytes have not been clearly resolved. LPS has been found to activate small, resting lymphocytes to proliferation and polyclonal activation1°, 11 and Melchers and Andersson have reported that PPD and the mitogen in fetal calf serum (FCS) activate small, resting lymphocytes ~2. However, they more recently have obtained data leading them to contradict these earlier findings and suggest that PPD is a B-cell

mitogen for activated B-cell blasts only and not for resting, small B lymphocytes ~3. In accord with this is Bretscher ~4 who o b s e r v e d an a p p r o x i m a t e proportionality between the magnitude of the background response in untreated mice and the in-vivo polyclonat responses of LPS-treated mice which led him to hypothesize that both the background and the LPS-induced responses are attributable to ongoing antigen-dependent lymphocyte activation in vivo. It has been suggested that the interaction of LPS at the cell surface occurs either through specific LPS receptors or through nonspecific intercalation of LPS into the lipid bilayerlS, 16. The affinity of interaction between LPS and the cell it activates is low in comparison to the affinity of interaction between an antigen and its specific immunoglobulin receptors at the cell surface 17. Despite this difference in affinity, Coutinho and co-workers 18 have recently shown that cell surface immunoglobulin receptors for the antigen dextran B-1355 (which is also a mitogen for B cells) share antigenic determinants with the mitogen receptors for this substance. This was done with an antiserum to the idiotype expressed in over 90% of the anti-dextran B-1355 antibodies produced by high responder mice, an antiserum which stained 10-15% of splenic B cells in all strains examined, and which mimicked the effects of dextran B-1355 as a mitogen and as a polyclonal activator.

Relation to D N A synthesis The dual effects of B-lymphocyte mitogens, as activators of D N A synthesis and of polyclonal antibody synthesis and secretion, has provided an opportunity to investigate the relationship between the requirement for D N A synthesis and subsequent induction of polyclonal immun0globulin secretion. Early studies of thymus-independent antigens led to the conclusion that their inductive properties were directly related to their ability to act as B-lymphocyte mitogens. Quintans and Lefkovits ~9first examined this question by an analysis of precursor frequency of cells responsive to LPS as opposed to those responsive to specific antigen. These authors found that LPS was capable of activating responsive B cells to polyclonal antibody production without significant cellular proliferation. Re-examining the question in a more traditional assay by adding hydroxyurea and cytosine arabinoside, known inhibitors of D N A synthesis, to lymphocytes cultured in the presence of LPS 2°, Andersson and Melchers distinguished an early stage d u r i n g which i m m a t u r e l y m p h o b l a s t s m a t u r e d independently of D N A synthesis and a later stage during which the development of mature plasma cells was dependent upon D N A synthesis. Poe e t a [ . 21 con.firmed that both hydroxyurea and cytosine arabino.side failed to inhibit the LPS-induced polyclonal response, further substantiating a dissociation between proliferative a n d p o l y c l o n a l responses to LPS.

94 Moreover, a subpopulation of lymphocytes larger than resting cells can be stimulated to polyclonal antibody production by LPS even after exposure to high doses of irradiation z2. The radio-resistance of this activity was felt to be related more closely to the position of the cell in its cycle than to its state of differentiation. Further dissociation of polyclonal activation from the initiation of D N A synthesis has been accomplished by Poe and Michael 23 who used serum inhibitory factors to suppress the mitogenic response to bacterial antigens while leaving the polyclonal antibody response intact. N a r i u c h i and A d l e r 24 d i s s o c i a t e d these responses by age, showing that whereas no significant change in the proliferative response occurred with age, the polyclonal response was significantly diminished in old mice.

B-cell subsets in the polyclonal response Several different approaches have been used to investigate the existence of discrete B-lymphocyte subpopulations responsive to certain polyclonal B-cell activators. These include suicide techniques, in which cells responsive to a given mitogen are killed and the residual non-responsive cells are then evaluated; study of the acquisition of mitogen responsiveness during ontogeny; examination of the ability of different activators to evoke additive or non-additive responses, and others. Melchers and Andersson ~° reported that LPS, PPD, and the mitogen present in FCS all stimulate largely identical B-cell subpopulations. In support of this thesis, they found that the continued growth and maturation of a single B cell requires the continued presence of the polyclonal activator 3. However, growth and maturation continue for a large percentage of B cells if a second mitogen is substituted for the original one. These authors thus propose that most B-cell blasts are responsive to more than one mitogen, probably expressing receptors at their cell surfaces for several different mitogens. It must be noted, however, that these investigators routinely include in their culture medium more mitogens than the one they are proposing to study: either 2-ME or TG, frequently FCS, and helper signals from thymus 'filler' cells (see below and ref. 25). In contrast with these results, Gronowicz and Coutinho 26 found that dextran sulfate, LPS and PPD each appeared to activate a distinct B-lymphocyte subpopulation into proliferation and polyclonal antibody secretion. They have s'hown furthermore that responsiveness to these mitogens emerges sequentially in ontogeny - first to dextran sulfate, later to LPS, and lastly to PPD. Moreover, LPS-unresponsive bonemarrow cells acquired responsiveness after induction with dextran sulfate, whereas pretreatment with LPS could inhibit the dextran sulfate response2L Sequential appearance of responsiveness in different B-cell subpopulations was not attributable to suppressive influences or to limiting accessory-cell development

immunology loday, November 1980

during the early stages of maturation. This viewpoint has been corroborated by G o o d m a n et al. 2s who found that LPS and poly IC activate essentially the same Bcell subset, that B cells activated by PPD are distinct and mature later than those of the first subset, and that those responsive to 2-ME are distinct from either of the first two groups and mature latest of all. Bona and co-workers >, using an in vivo suicide technique, produced further evidence that the cells responsive to Nocardia water-soluble mitogen, LPS, and dextran sulfate belong to distinct subsets.

Regulation of polyclonal B cell activation The ability of T lymphocytes to regulate the humoral immune response to specific antigen has been appreciated since Claman e t a [ . 3° and Miller and MitchelP ~ first described the absolute requirement for T helper cells in the generation of an antigen-specific response. Modulation of the humoral response by T suppressor cells32, 33 and by T amplifier cells34, 35 has been studied extensively. The regulatory effect of T cells on the non-specific responses of B cells evoked by polyclonal B cell activators are much less thoroughly understood. Shinohara and Kern 36 reported that whereas depletion of T cells from rabbit splenocyte populations failed to alter their responsiveness to LPS in a proliferative assay, the production of immunoglobulin in response to LPS was significantly diminished. This B-cell response could be enhanced by adding back thymocytes. R e g u l a t i o n of the p o l y c l o n a l response of h u m a n peripheral blood lymphocytes to pokeweed mitogen has been studied more extensively than the regulation of specific immune responses due to the difficulty of obtaining specific responses irz vitro until recently3L However, a review of the mechanisms involved in the h u m a n system are beyond the scope of the present review. Because of the ubiquitous nature of polyclonal stimulants and the unusually large proportion of B lymphocytes which respond to such mitogens 2, it a p p e a r e d probable that this arm of the humoral immune system be subject to some type of regulatory influence in the mouse. Recent studies in this laboratory have demonstrated that polyclonal activation of murine splenic B lymphocytes to secrete immunoglobulin is in fact subject to regulation by splenic T cells 3s. Admixture of separated B and T lymphocyte populations led to significantly augmented polyclonal B-cell responsiveness to LPS. Optimal collaboration between these two cell types followed when they were co-cultured at nearly equivalent numbers. T-ceil-mediated enhancement of polyclonal B cell responses was manitested upon the interaction of these two cell types soon after culture initiation, since exposure of B cells to helper T cells after the first 24 h of culture failed to result in any regulatory effects; removal of these cells before but not after the first 24 h of culture consistently abrogated their helper activity. The observation that T cells from

immunology today, November 1980

an LPS-non-responsive strain of mice were deficient in their capacity to enhance polyclonal responsiveness of B cells derived from a responder strain implied either direct action of LPS on the involved T cells or a conjoint actio~l of LPS and a B cell-derived factor. Theofilopoulos eta[. 39 have made parallel observations with respect to the effect of T cells on polyclonal responses of normal and a u t o i m m u n e murine strains. In further studies from this laboratory, polyclonal activation of murine splenic B lymphocytes by LPS was found to be subject to regulation by both helper and suppressor influences from T lymphocytes 4°. In the normal adult spleen, only helper influences were exercised over polyclonal B-cell activation; this influence is a property of Lyt 1 + 2 3 - , slowly sedimenting T cells. Suppressive influence evidently is latent, for it exists at such a low level (or the cells are so few in number) that its effects are difficult to detect. Suppressor T cell function may be evoked by culturing spleen cells at high ratios of T to B cells, by activating splenic T cells with concanavalin A, or by sonicating unstimulated splenic T cells in order to liberate a suppressive potential which is not expressed by these cells when intact. The soluble fraction of resident splenic T-cell sonicates exerts both helper and suppressor regulatory influences. The soluble helper activity is derived from Lyt 1+23-, slowly sedimenting T cells, whereas suppressor activity (which has no adverse effect on B-cell viability) is generated from a distinct subpopulation of L y t l - 2 3 +, rapidly sedimenting T cells. The thymus evidently contains cells capable only of helping but not of suppressing polyclonal activation of splenic B cells. This is in accord with the observations of Rocklin et al. 41 who have described a histamine-induced suppressor factor which is present in spleen and lymph-node T cells, but not in thymus. The helper and suppressor activities contained in splenic T-cell sonicates appear to be distinct molecular moieties which can be separated by gel chromatography; the suppressive activity eluted with a mol.wt between 68,000 and 84,000 daltons, and the helper activity eluted with a mol.wt between 15,000 and 23,000 daltons. It is reasonable to suspect that these factors, isolated from unstimulated splenic T lymphocytes, may well prove to be either precursors or structural components of other T cell regulatory factors. As factor precursors, they may be subject to enzymatic cleavage before generating non-specific regulatory activity. However, if they are components of other factors, they may be conjugated to additional components (such as antigen a n d / o r I-region-specific components) and thus participate in antigen-specific a n d / o r non-specific T-cell regulation. Primi el al. 42 have recently reported that polyclonal activation by a n u m b e r of different polyclonal B-cell stimulants are subject to suppression by con-A-activated T cells. Suppression acted upon a proliferation-dependent stage of induction.

95 The Fc h-agment of h u m a n IgM, IgG, IgA, and IgD antibodies are potent mitogens for murine spleen cells 43. Fc fragments obtained from h u m a n IgG have been shown to activate murine spleen cell cultures to polyclonal antibody synthesis and secretion 44. Activation is dependent upon the presence of T cells 45. Morgan et al. have obtained evidence that macrophages are required to process the Fc fragment into a 14,000 dalton subfragment 46. The T-cell signal required for polyclonal activation can be provided by interleukin II, a T-cell helper factor of approximately 30,000 daltons isolated trom supernates of concanavalin A-activated spleen cells 47. Here again, regulation of polyclonal B cell activation appears to be T-cell mediated. The author wishes to express his appreciation to Mrs Barbara Marchand for excellent secretarial work in the preparation of the manuscript. This is publication no. 2153 from the Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, California. Supported in part by United States Public Health Service grant AI-07007, National Institute of Health grant AI15284, and a fellowshipfrom The Arthritis Foundation.

References i Andersson, J., Sjoberg, O. and Moiler, G. (1972) Eur. J. Immunol. 2, 349 2 Andersson,J., Coutinho, A., Lernhardt, W. and Melehers, F. (1977) Cell 10, 27 3 Andersson,J., Coutinho, A. and Melchers, F. (1979) J. Exp. Med. 149, 553 4"Eichmann, K., Braun, D.G., Feizi, T. and Krause, R.M. (1970) J. Exp. Med. 131, 1169 5 Moticka, E.J. (1975) Cell. Immunol. 19, 32 6 Kearney,J.S. and Lawton, A.R. (1975)J. Immunol. 115,671 7 Van Der Loo, W., Gronowicz, E.S., Strober, S. and Herzenberg, L.A. (1979)J. Immunol. 122, 1203 8 Andersson,J., Coutinho, A. and Melchers, F. (1978) Eur. J. lmmunol. 8, 336 9 Goldsby, R.A., Osborne, B.A., Suri, D., Mandel, A., Williams, J., Gronowicz, E. and tlerzenberg, L.A. (1978) Curr. Top. Mierobiol. lmmunol. 81, 149 10 Melchers, F., Von Boehmer, H. and Phillips, R.A. (1975) Transplant Bey. 25, 26 11 Hecht, T.T., Ruddle, N.H. and Ruddle, F.H. (1976) Cell. lmmunol. 22, 193 12 Melchers, F. and Andersson,J. (1974) Eur. J. Immunol. 4, 687 13 Andersson,J., Lernhardt, W. and Melchers, F. (1979)J. Exp. Med. 150, 1339 14 Bretscher, P.A. (1978) Eur. J. lmmunol. 8,534 15 ForM, L. and Coutinho, A. (1978) Eur.J. Immunot. 8, 56 16 Kabir, S. and Rosenstreich, D.L. (1977) Infect. Immun. 15,156 17 Morgan, E.L., Speigleberg, H.L. and Weigle, W.O. (1979) Scand. J. hnmunol. 10, 395 18 Coutinho, A., ForM, L. and Blomberg, B. (1978)57. Exp. Med. 148,862 19 Quintans, J. and Lefkovits, I. (1974)J. [mmunol. 113, 1373 20 Andersson,J. and Melehers, F. (1974) Eur. J. Immunol. 4, 533 21 Poe, W.J., Riedel, S.H. and Michael,J.G. (1978)J. Reticuloend. Soc. 23, 411 22 Lafleur, L. (1978) Cell. Immunol. 38, 181 23 Poe, W.J. and Michael,J.G. (1976)J, lmmunol. 116,1129 24 Nariuchi, H. and Adler, W.H. (1979) Cell. lmmunoL'45, 295 25 Goodman, M.G. and Weigle, W.O. (1980) Clin. lmmunoL Immunopath. 15, 375

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96 26 Gronowicz,E. and Coutinho, A. (1974) Eur. J. lmmunol. 4, 771 27 Gronowicz, E. and Coutinho, A. (1975) Scand..7" lmmunol. 4, 429 28 Goodman, M.G., Fidler, J.M. and Weigle, W.O. (1978) .7. Immunol. 121, 1905 29 Bona, C., Yano, A., Dimitriu, A. and Miller, R.G. (1978) J. Exp. Med. 148, 136 30 Claman, H.N., Chaperon, E.A. and Triplet, R.F. (i966) Proc. Soc. Exp. Biol. (NI') 122, 1167 31 Miller, J.F.A.P. and Mitchell, G.F. (1967) Nature (London) 216, 659 32 Gershon. R.K. and Kondo, K. (197l) Immunology21,903 33 Gershon, R.K. (1974) (.Snlemp. Top. Immunobiol. 3, 1 34 Tada, T., Takemori, T., Okumura, K., Nonaka, M. and Tokuhisa, T. (1978)J. Exp. Med. 147, 446 35 Muirhead, D.Y. and Cudkowicz, G. (1978) J. Immunol. 121, 130 36 Shinohara, N. and Kern, M. (1976)J. Immunol. 116, 1607

37 Ballieux, R.E., Heijnen, C.J., Uytdehaag, F. and Zegers, B.J.M. (1979) lmmunol. Rev. 45, 3 38 Goodman, M.G. and Weigle, W.O. (1979) J. lmm~mol. 122, .2548 39 Theofilopoulos, A.N., Shawler, D.L., Eisenberg, R.A. and Dixon, FJ. (1980)j . Exp. Med. 151,446 40 Goodman, M.G. and Weigle, W.O. (1980) Fed. Proe.39, 459 41 Rocklin, R.E., Greineder, D.K. and Melmon, K.L. (1979) Cell. Immunol. 44, 404 42 Primi, D., Hammarstrom, L. and Smith, C.I.E. (1979) (,'ell. Irnmunol. 42, 90 43 Berman, M.A., Speigleberg, H.L. and Weigle, W.O. (1979)J. Immunol. 122, 89 44 Berman, M.A. and Weigle, W.O. (1977)J. Exp. Med. 146, 241 45 Morgan, E.L. and Weigle, W.O. (1980)J. Immunol. 124, 1330 46 Morgan, E.L. and Weigle, W.O. (1980)J. Exp. Med. 151, 1 47 Thoman, M.L., Morgan, E.L. and Weigle, W.O. (1980) Fed. Proc. 39, 665

D o N K cells play a role in antitumor surveillance ? John C. Roder and Tina Haliotis Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada KTL 3N6

A class of lyrnphocytes found in several mammalian species including man will kill cells of many lumor lines in vitro 1-4. There is growing evidence, derived mainly from the work of Kiessling and co-workers, that these natural killer (NK) cells play a role in surveillance against tumor development in vivo. In this article John Roder and Tina Haliotis discuss a hypothesis for anti-tumor surveillance which integrates all of the potentially important immunological systems in the host and gives to N K cells the role of foremost barrier against developing tumors.

The NK hypothesis of tumor s u r v e i l l a n c e N K cells exist in the unstimulated host at high frequency (0.6-2.497o of all lymphocytes s) and do not require a lengthy period of preactivation. O n the basis of these characteristics, one can hypothesize that N K cells provide a first line of defence against a newly arising malignancy or its metastases (Table I). Other potential effector cells such as T cells, activated macrophages or K cells participating in antibodydependent cell-mediated cytotoxicity (ADCC) 6, if they are important at all, would only be effective after a specific priming or activation period requiring days or weeks. It is not unreasonable to assume that under the selective pressure of such a lethal condition as malignancy, several independent defence mechanisms may have evolved. In principle, the hypothesis is an adaptation of the one originally proposed by Thomas v and modified by Burnett 8. However, the role of the T cell in i m m u n e surveillance has not withstood critical test (for reviews see Refs 3 and 9-11). In the scheme shown in Table I and Fig. 1, we have placed the NK cell as the foremost, but not necessarily the only, barrier to tumor development. The mechanism by which the NK cell recog© Elsevier/North-Holland Biomedical PJess 1980

nizes and destroys the developing tumor has been discussed elsewhere12,13. It is, however, instructive to consider some recent work by Collins, Patek, and Cohn 14 which shows a correlation between the tumorigenic potential of cloned, chemically transformed cell lines and their susceptibility to NK-mediated lysis in vitro. N cells, from a cloned fetal fibroblast line which does not grow in agarose, were not tumorigenic in vivo [in normal mice or lethally irradiated mice thymectomized as adults and reconstituted with fetal liver (ATxFL mice)] and were not lysed by NK cells in vitro. W h e n these cells were t r a n s f o r m e d by methylcholanthrene (MCA) they grew in agarose and after cloning could grow as tumors in A T x F L mice ~ which were N K deficient 14 but not in normal mice. This same mutation, N t>I, also made the cells susceptible to NK-mediated cytolysis in vitro. Cell lines derived from these I tumors were capable of growth in normal mice. This I DC mutation, or selection, was accompanied by a loss of N K sensitivity, so that the tumor could escape N K surveillance and grow in normal mice. Although this is a useful model it would be misleading to suggest that NK cells only lyse tumor cells since