¹Toomey, J.A., ¹Gays, F., ²Foster, D., and ¹Brooks, C.G.
¹School of Cell and Molecular Biosciences, The Medical School, Newcastle, U.K.; and ²Department of Functional Cloning, ZymoGenetics Inc., Seattle, U.S.A.

Submitted, 2003.
 

NK cells arise from immature progenitors present in fetal tissues and adult bone marrow, but the factors responsible for driving the proliferation and differentiation of these progenitors are poorly understood.  Mouse NK cells had previously been thought not to express IL2Ra chains but we show here that both immature and mature mouse NK cells express IL2Ra chain mRNA, and that low levels of IL2Ra chains can be detected on the surface of immature and mature NK cells provided they are cultured in the absence of IL2.  Despite their potential expression of high affinity IL2 receptors, immature NK cells only proliferate if IL2 is present at extremely high concentrations.  Surprisingly, IL15 can also only support the growth of immature NK cells at high, presumably non-physiological, concentrations.  Although NK cells express mRNA for the high affinity IL15Ra chain, they also express a variety of alternately spliced transcripts whose protein products could potentially disrupt signaling through IL15 receptors.  The requirement for high concentrations of IL2 and IL15 suggests that if these cytokines play any role in the proliferative expansion of NK cells in vivo they either act indirectly via other cells or in cooperation with other factors.  In support of the latter possibility, we report that the recently described cytokine IL21 can markedly enhance the proliferation of immature [and mature] NK cells in the presence of doses of IL2 and IL15 that by themselves have little growth promoting activity. Key Data

¹Fraser, K.P., ¹Gays, F., ¹Robinson, J.H., ²van Beneden, K.,  ²Leclercq, G., ³Vance, R.E., ³Raulet, D.H., and ¹Brooks, C.G.
The ¹Department of Microbiology and Immunology, The Medical School, Newcastle NE2 4HH, United Kingdom, ²The Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University, University Hospital, B-9000 Ghent, Belgium, and ³Department of Molecular and Cell Biology and Cancer Research Laboratory, University of California at Berkeley, California 94720, U.S.A.

Eur. J. Immunol. 32, 868-78, 2002.
 

In the presence of appropriate cytokines and stimulants, progenitor cells present in the thymus develop into functional NK cells that express a variety of NK receptors but generally lack expression of any of the Ly49 molecules that have previously been examined.  We demonstrate here that during their development in vitro these NK cells acquire at least one previously uncharacterized member of the Ly49 family, most likely Ly49E, in a time-dependent and stochastic manner.  CD94 and NKG2 are also acquired in a stochastic manner but more rapidly than, and independently of, Ly49 molecules.  Throughout development CD94 is expressed at two different levels, the CD94^hi population accounting for all of the cells that stain strongly for NKG2 and with Qa1 tetramers.  In IL2-containing cultures CD94 is largely confined to NK1.1⁺ cells, but in cultures lacking IL2 and in vivo CD94 could be expressed in the absence of NK1.1.  IL4 displays a powerful and selective effect on the expression of NK receptors, blocking the acquisition of Ly49, CD94, and NKG2, but not NK1.1, by progenitor cells, and down regulating the expression of CD94 and NKG2 but not Ly49 or NK1.1 on more mature NK cells.  Key Data

¹Gays, F., ¹Fraser, K.P., ¹Toomey, J.A., ¹Diamond, A.G., ²Millrain, M.M., ²Dyson, P.J., and ¹Brooks, C.G.
The ¹Department of Microbiology and Immunology, The Medical School, Newcastle, and ²The Transplantation Biology Unit, MRC Clinical Sciences Centre, London, W12 0NN, United Kingdom.

J. Immunol. 166, 1601-1610, 2001.
 

CD94/NKG2 receptors on NK cells recognize the nonclassical class I molecule Qa1 and can deliver inhibitory signals that prevent NK cells from lysing Qa1-expressing cells. However, the exact circumstances under which Qa1 protects cells from NK lysis and, in particular, the role of the dominant Qa1-associated peptide, Qdm, are unclear. In this study, we examined in detail the lysis of Qa1-expressing cells by NK cells that express CD94/NKG2 receptors for Qa1 but that lack receptors for classical class I molecules. Whereas L cells and human C1R cells transfected with Qa1 were resistant to lysis by these effectors, Qa1-transfected TAP-deficient human T2 cells showed no resistance despite expressing high levels of surface Qa1. However, these cells could be efficiently protected by exposure to low concentrations of Qdm peptide or certain Qdm-related peptides. By contrast, even prolonged exposure of TAP-deficient RMA/S cells to high doses of Qdm peptide failed to induce levels of surface Qa1 detectable with a Qa1-specific mAb or to protect them from NK lysis, although such treatment induced sensitivity to lysis by Qa1-specific CTL. Collectively, these findings indicate that high surface expression of Qa1 is necessary but not sufficient for protection, and that effective protection requires the expression of sufficient levels of suitable Qa1-peptide complexes to overcome activatory signals. Results obtained with a series of substituted Qdm peptides suggest that residues at positions 3, 4, 5, and 8 of the Qdm sequence, AMAPRTLLL, are important for recognition of Qa1-Qdm complexes by inhibitory CD94/NKG2 receptors. Key Data

¹Gays, F., ¹Unnikrishnan, M., ¹Shrestha, S., ¹Fraser, K.P., ¹Brown, A.R., ¹Tristram, C.M.G., ²Chrzanowska-Lightowlers, Z.M.A., and ¹Brooks, C.G.
The ¹Department of Microbiology and Immunology, and the ²Department of Neurology, The Medical School, Newcastle, NE2 4HH, U.K.

J. Immunol. 164, 5094-5102,  2000.
 

As a potential means for facilitating studies of NK cell-related molecules, we examined the expression of these molecules on a range of tumor cell lines. Of the lines we initially examined, only EL4 and RMA expressed such molecules, both lines expressing several members of the Ly49 and NKRP1 families. Unexpectedly, several of the NK-related molecules, together with certain other molecules including CD2, CD3, CD4, CD32, and CD44, were often expressed in a mosaic manner, even on freshly derived clones, indicating frequent switching in expression. In each case examined, switching was controlled at the mRNA level, with expression of CD3zeta determining expression of the entire CD3-TCR complex. Each of the variable molecules was expressed independently, with the exception that CD3 was restricted to cells that also expressed CD2. Treatment with drugs that affect DNA methylation and histone acetylation could augment the expression of at least some of the variable molecules. The striking phenotypic similarity between EL4 and RMA led us to examine the state of their TCRbeta genes. Both lines had identical rearrangements on both chromosomes, indicating that RMA is in fact a subline of EL4. Overall, these findings suggest that EL4 is an NK-T cell tumor that may have retained a genetic mechanism that permits the variable expression of a restricted group of molecules involved in recognition and signaling.    Key Data

¹Toomey, J.A., ²Salcedo, M., ³Cotterill, L.A.,  ³Millrain, M.M., ⁴Chrzanowska-Lightowlers, Z, ⁵Lawry, J., ¹Fraser, K.P., ¹Gays, F., ¹Robinson, J.H., ¹Shrestha, S.,  ³Dyson, P.J., and ¹Brooks, C.G.
¹Department of Microbiology and Immunology, The Medical School, Newcastle, NE2 4HH, U.K.; ²Unite de Biologie Moleculaire du Gene, INSERM U277, Institut Pasteur, F-75015 Paris, France;  ³Transplantation Biology Unit, MRC Clinical Sciences Centre, London, W12 0NN, U.K.; ⁴Department of Neurology, The Medical School, Newcastle, NE2 4HH, U.K.; and ⁵Institute for Cancer Studies, The Medical School, Sheffield, S10 2RX, U.K..

J. Immunol. 163,  3176-3184, 1999.
 

Immature NK cells are grossly deficient in the expression of Ly49 molecules yet show a limited ability to distinguish between wild-type and MHC class I-deficient target cells. In this paper we report that during their development in vitro from immature thymic progenitors, a proportion of C57BL/6 NK cells acquires receptors for a soluble form of the nonclassical class I molecule Qa1b associated with the Qdm peptide, but not for soluble forms of the classical class I molecules Kb and Db. The acquisition of these Qa1 receptors occurs in a stochastic manner that is strictly controlled by cytokines, and in particular is strongly inhibited by IL-4. All NK clones tested, including those that lack detectable Qa1 receptors, express mRNA for CD94 and for both inhibitory and noninhibitory members of the NKG2 family.  NK cells lacking receptors for Qa1 (and also for classical class I molecules) cannot distinguish between wild-type and class I-deficient blasts but, surprisingly, distinguish efficiently between certain wild-type and class I-deficient tumor cells. A variant line that lacks several members of the NKG2 family kills both types of tumor cell equally well, suggesting the existence of NKG2-containing inhibitory receptors that recognize as yet undefined nonclassical class I molecules of restricted distribution. Key Data

Manoussaka, M.S., Smith, R.J., Conlin, V., Toomey, J.A., and Brooks, C.G.
Department of Microbiology and Immunology, The Medical School, Newcastle

J. Immunol. 160, 2197-2206, 1998.
 

NK cells obtained by exposing immature thymocytes to appropriate combinations of IL-4, IL-2, and PMA are phenotypically indistinguishable from cultured adult splenic NK cells with the exception that they generally lack measurable expression of all of the inhibitory Ly49 molecules that can currently be detected with Abs (Ly49A, -C, -G, and -I) and of the activating molecule Ly49D. Despite this deficiency, NK cells developing from immature progenitors have a similar specificity to Ly49-expressing adult splenic NK cells. Individual NK cell clones display an essentially invariant and broad specificity similar to that of polyclonal populations of immature or adult NK cells, although significant differences in the fine specificity of clones can occasionally be detected.  Most remarkably, cloned NK cell lines derived from immature progenitors in vitro display heterogeneous expression of a restricted set of surface molecules that includes 10A7, Ly6C, 3C2, CD8, certain isoforms of CD45, and also, occasionally, Ly49 molecules. This heterogeneity is not related to the cell cycle or activation status of the cells, and micromanipulation recloning demonstrates unambiguously that it is not due to a lack of a single cell origin. Diversity is generated rapidly and the capacity for diversification appears to persist indefinitely in vitro. The expression of individual variable Ags is independent and stochastic, resulting in NK "clones" derived in vitro being potentially composed of hundreds of phenotypically distinct cells. We hypothesize that the NK cells that develop in vitro from immature progenitors themselves behave as progenitor cells that are undergoing a process of rapid, extensive, and continuous diversification and that are individually capable of generating and regenerating a complex NK cell repertoire.   Key Data

¹Toomey, J.A., ¹Shrestha, S., ¹de la Rue, S.A., ¹Gays, F., ¹Robinson, J.H., ²Chrzanowska-Lightowlers, Z., and ¹Brooks, C.G.
The ¹Department of Microbiology and Immunology, and the ²Department of Neurology, The Medical School, Newcastle, NE2 4HH, U.K.

Eur. J. Immunol. 28, 47-56, 1998.
 

Using appropriate conditions natural killer (NK) cells can be cultured from the liver and thymus. These in vitro derived NK cells are phenotypically and functionally indistinguishable from adult NK cells with the exception that they lack measurable expression of all of the Ly49 molecules that can currently be detected with antibodies. Despite this, they preferentially kill tumor cells and blast cells deficient in the expression of major histocompatibility complex class I molecules, although the degree of discrimination is usually weaker than that shown by adult NK cells and varies depending on the particular combination of effector and target cells used. Polymerase chain reaction analysis revealed that although NK cells derived from immature progenitors are severely deficient in the expression of mRNA for Ly49A, B, C, D, G, H, and I they express high levels of Ly49E mRNA, raising the possibility that Ly49E may have an important and special function in the early development of the NK lineage.  Key Data

Duhindan, N., Farley, A.J., Humphreys, S., Parker, C., Rossiter, B., and Brooks, C.G.
Department of Microbiology and Immunology, The Medical School, Newcastle

Eur. J. Immunol. 27, 1704-1712, 1997.
 

Although the patterns of lymphokine (LK) secretion by CD4 and CD8 alpha beta T cells have been extensively studied, the question of whether gamma delta T cells display patterns of restricted LK production and whether these patterns are the same as seen in conventional alpha beta T cells has not been previously addressed. In this study we generated panels of gamma delta T cell clones from normal individuals using a lectin-driven system and compared their patterns of secretion of nine LK with those of CD4 and CD8 alpha beta T cell clones generated in the same system. The results showed that gamma delta T cell clones displayed nonrandom patterns of highly restricted LK production with a strong bias towards the production of type 1 LK. The dominant pattern was one of high level secretion of interferon-gamma and tumor necrosis factor (TNF), with variable production of interleukin (IL)-2, and little or none of the type 2 LK IL-4, IL-5, IL-6, and IL-10. This pattern differed significantly from that of CD4 Th1 clones in that gamma delta clones showed a striking deficiency in the production of IL-3 and granulocyte/macrophage colony-stimulating factor. A small subset of gamma delta clones displayed a novel pattern, in which the only LK produced in substantial quantity were TNF and variable amounts of IL-2. The bias of gamma delta T cells towards type 1 LK production was not an artefact associated with cloning because bulk populations of splenic gamma delta T cells behaved in the same way, even when activated in the presence of high concentrations of IL-4.   Key Data

¹Manoussaka, M., ¹Georgiou, A., ¹Rossiter, B., ¹Shrestha, S., ¹Toomey, J.A., ²Sivakumar, P.V., ²Bennett, M., ²Kumar, V., and ¹Brooks, C.G.
¹Department of Immunology, The Medical School, University of Newcastle, Newcastle upon Tyne, U.K.; and ²Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas.

J. Immunol. 158, 112-119, 1997.
 

Culture of day 14 liver (FL) cells in high dose IL-2, together with appropriate combinations of IL-4 and PMA, resulted in the generation of cell lines, termed FL-A lines, that were phenotypically and functionally indistinguishable from cultured adult splenic NK cell populations with the single important exception that no Ly49-expressing cells were present. By contrast, when FL cells were cultured in low-dose IL-2 alone, a second population of slow-growing NK-like cells, termed FL-B cells, emerged. These cells expressed the NK markers asialoGM1, 10A7, 2B4, and Fc gammaRII/III but differed from FL-A and splenic NK cells in expressing IL-2R alpha and stem cell factor receptor (SCFR) but no B220. Most lines derived in this manner had minimal or no cytolytic activity and only very low levels of NK1.1. However, they could secrete substantial quantities of several lymphokines including IL-3, granulocyte-macrophage (GM)-CSF, TNF-alpha, and, most surprisingly, IL-2. A minority of FL-B lines, typified by line 903, displayed marked cytolytic activity, moderate levels of NK1.1, reduced production of IL-2, and the capacity for accelerated growth in high-dose IL-2. FL-B lines generally expressed mRNA for CD3gamma but not for other CD3 chains, whereas FL-A and thymic (FT) NK lines often expressed mRNA for all four CD3 chains. Despite many similarities to pro-T cells, FL-B cells showed no capacity to differentiate into mature T cells. Taken together, our results suggest that NK lines of different maturity can be obtained from the liver, with FL-B lines being the most immature, FL-A lines the most mature, and lines such as FL-B 903 representing an intermediate state of differentiation. Key Data

Jones, S.H., Georgiou, A. and Brooks, C.G.
Department of Microbiology and Immunology, The Medical School, Newcastle

Cell. Immunol. 154, 407-419, 1994.
 

In order to examine the functional potential of individual CD4+ T cells selected, as far as possible, in a random manner, a high-efficiency cloning system driven by Con A was utilized. Under optimal conditions, cloning efficiencies of CD4+ cells of about 50% were regularly attained. Although the relative proportion of different TH subsets varied depending on the cloning conditions, the high cloning efficiency, coupled with the analysis of over 100 clones, allowed important conclusions to be drawn regarding the general construction of the CD4+ T cell repertoire. (1) At least 50% of all splenic CD4+ T cells have the potential to produce IL4, supporting the view that TH subsets arise by an instructional or regulatory mechanism, rather than by selection. (2) TH0 clones produce amounts of IL2 and IL4 similar to those produced by TH1 and TH2 clones, respectively, but secrete much lower quantities of IFN than TH1 clones. (3) A large proportion of TH2 clones secrete measurable amounts of IFN. (4) Lymphokine secretion patterns among CD4+ T cells are clearly not determined at random, since IL2 production is always accompanied by IFN production. (5) At least 50% of all splenic CD4+ T cells have cytolytic capacity as shown by killing in a 20-hr assay, but only a proportion can also kill in 4-hr assays. Killing in 4-hr assays was strongly correlated with the ability to secrete IL2, regardless of whether IL4 was also secreted.

Brooks, C.G., Georgiou, A. and Jordan, R.K.
Department of Microbiology and Immunology, The Medical School, Newcastle

J. Immunol. 151, 6645-6656, 1993.
 

Although immature thymocytes (FTC) express IL-2R, they have generally been considered to be unresponsive to IL-2. We show here that they can in fact undergo substantial and prolonged growth in vitro provided that high doses of IL-2 are present. The ability of FTC to grow in IL-2 could be enhanced slightly by PMA and also by IL-4, but dramatically by the combination of IL-4 + PMA with IL-2. Pretreatment of FTC with IL-4 + PMA for as little as 24 h primed FTC for rapid and prolonged responsiveness to IL-2, permitting the establishment of long term lines. Kinetic and clonal analysis revealed that most individual FTC could grow under these conditions. Although proliferating cells expressed functional IL-2R of only very low affinity, and IL-2R alpha chains were undetectable by immunofluorescence, blocking experiments showed unambiguously that both IL-2R alpha and IL-2R beta were involved in signal transmission. FTC lines and clones developed in this manner lacked lineage-specific markers of mature T cells, B cells, and myeloid cells, but expressed the NK cell markers NK1.1 and asialo-GM1. They displayed potent cytolytic activity against NK-sensitive targets, and, when stimulated with PMA+ionomycin, secreted IL-3 and IFN-gamma, but not IL-2 or IL-4. After intrathymic injection they showed no evidence of growth or differentiation. These results demonstrate that most, if not all, immature thymocytes have the capacity to differentiate into cells which appear to be indistinguishable from mature NK cells. They suggest that T cells and NK cells derive from a common precursor which in the thymic environment differentiates into T cells and in the extrathymic environment into NK cells.   Key Data

Robinson, J.H., Case, M.C. and Brooks C.G.
Department of Microbiology and Immunology, The Medical School, Newcastle

Immunology 76, 593-598, 1992.
 

The effect on antigenicity of covalent attachment of lipid groups to a protein antigen was investigated. Coupling of palmitic acid to ovalbumin (OVA) enhanced major histocompatibility complex (MHC) class II-restricted presentation to most OVA-specific T-cell clones in vitro. The enhanced antigenicity of palmitoylated antigen was localized to the level of presentation of the synthetic peptide epitope, OVA 323-339. T-cell responses to palmitoylated antigen were more difficult to block with anti-MHC class II antibodies than responses to native antigen. However, T-cell proliferation to palmitoyl (p)-OVA and native (n)-OVA were blocked equally by anti-CD4 antibodies. Taken together, the results suggest that lipid conjugation of a protein antigen leads to the formation of a lipopeptide T-cell epitope with increased affinity of binding to MHC class II and/or T-cell receptor (TcR). These results have implications for the design of synthetic peptide vaccines.

¹Holscher, M., ²Givan, A. and ¹Brooks, C.G.
The ¹Department of Microbiology and Immunology, and the ²Department of Surgery, The Medical School, Newcastle

Immunology 73, 44-51, 1991.
 

To test the hypothesis that major histocompatibility complex (MHC) molecules protect target cells from lysis by natural killer cells (NKC), we transfected the MHC- B16 melanoma line F10 with the class I genes encoding Dd, Kb, and Kk. Only low levels of Dd expression could be obtained and there was no protection against NKC. By contrast, Kb and Kk transfectants were obtained which displayed significant resistance to NKC, and with the latter transfectants resistance was clearly related to the level of transgene expression. Various mutants of the F10 line with altered patterns of MHC expression were also obtained. These mutant lines provided evidence that (i) the Db molecule is also capable of inducing resistance to NKC and (ii) high MHC class I expression does not by itself guarantee lowered susceptibility to NKC.

¹Wood, P.M.D., ¹Jordan, R.K., ²Givan, A.L. and ¹Brooks, C.G.
The ¹Department of Microbiology and Immunology, and the ²Department of Surgery, The Medical School, Newcastle

Immunology 71, 83-89, 1990.
 

The ability of lymphokines to affect the development and differentiation of thymocytes in vitro was evaluated in a carefully controlled 3-day organ culture system. Concanavalin A (Con A)-induced supernatant (SN) from the T-cell clone D10.G4, which contains high concentrations of interleukin-3 (IL-3), IL-4 and IL-5, but lacks IL-1, IL-2 and interferon (IFN), markedly increased the proportion of CD4+CD8- cells, and decreased the proportion of CD4+CD8+ cells. These effects were unaffected by dialysing the SN, showing them to be caused by macromolecular factors. Highly purified recombinant IL-3 and IL-4 could exert similar effects, rIL-3 and rIL-4 both increasing the proportion of CD4+CD8- cells, and rIL4 in addition reducing the proportion of CD4+CD8+ cells. In conjunction with the findings of other investigators, these results indicate that at least four lymphokines (IL-1, IL-2, IL-3 and IL-4) can control T-cell development in the thymus.

Brooks, C.G. and Holscher, M.
Fred Hutchinson Cancer Research Center, Seattle, U.S.A.

J.Immunol. 138: 1331-1338, 1987.
 

Short-term treatment of cloned cytotoxic T lymphocytes (CTL) with interferon (IFN) induces lytic activity for natural killer- (NK) sensitive targets. Extended culture of CTL in high concentrations of interleukin 2 induces promiscuous lytic activity in which state both NK-sensitive and NK-resistant target cells are lysed. Cold-target competition analysis showed that the development of NK activity was associated with the acquisition of binding activity for NK-sensitive but not for NK-resistant targets, whereas the development of promiscuous lytic activity was associated with the acquisition of binding activity for both types of target. Antigen-specific cytolysis was inhibited by antibodies to Ly-2, Ly-5, LFA-1 and to the V region of the T cell antigen receptor (TCR), whereas NK and promiscuous lytic activity in the same cells was resistant to inhibition by anti-Ly-2 and anti-TCR. NK activity was expressed normally against a variant NK-sensitive cell line lacking all MHC antigens. These results show that, in contrast to antigen-specific recognition, the NK and promiscuous lytic activities of CTL are expressed without participation of effector cell Ly-2 and TCR molecules or target cell MHC molecules, and are most likely mediated through novel and distinct receptor systems.

¹Chou, H.S., ¹Behlke, M.A., ¹Godambe, S.A., ¹Russell, J.H., ²Brooks, C.G. and ¹Loh, D.Y.
¹Washington School of Medicine, St. Louis, and ²Fred Hutchinson Cancer Research Center, Seattle, U.S.A.

EMBO Journal 5: 2149-2155, 1986.
 

Both cDNA and genomic clones of the T cell receptor (TCR) alpha- and beta-chain genes of the alloreactive cytotoxic T lymphocyte (CTL) clone F3 were examined. Two distinct rearrangement events, one functional and one non-functional, were found for both the alpha and beta loci. Thus only a single functional TCR alpha beta heterodimer could be defined, consistent with allelic exclusion in the TCR genes. The V alpha gene employed by F3 is part of a six-member V alpha subfamily. Genomic clones containing each member of this subfamily were isolated and the V alpha nucleotide sequences determined. Five of these six genes are functional; these genes differ from each other by 7-14% at the amino acid level. A single dominant hypervariable region was defined within this subfamily, in contrast to the pattern of variability seen between V alpha genes in general.

¹Galli, S.J., ²Brooks, C.G., ¹Dvorak, A.M. and ¹Ishizaka, T.
¹Department of Pathology, Harvard Medical School, Boston, and  ²Fred Hutchinson Cancer Research Center, Seattle, U.S.A.

Cell Immunol. 96: 223-230, 1985.
 

We recently reported that a cloned cell line with "natural killer (NK)-like" cytolytic function and prominent cytoplasmic granules also expressed large numbers of plasma membrane receptors (Fc epsilon R) which bound immunoglobulin E (IgE) with high affinity (S.J. Galli et al., 1982, Nature (London) 298, 288). We have now performed IgE-binding studies with 31 additional cloned cell lines exhibiting "NK-like" lytic activity (defined as the ability to kill YAC-1 lymphoma cells) and three antigen-specific cytotoxic-T-cell clones. One of the NK-like clones expressed a small number of Fc epsilon R (3.0 X 10(4)/cell) on one of the two occasions it was tested. None of the other clones, which were derived by several different approaches and which had a variety of surface glycoprotein phenotypes, expressed any detectable specific binding of IgE. By contrast, mast cell clones consistently expressed large numbers of Fc epsilon R. The expression of large numbers of high-affinity Fc epsilon R would appear to represent a very uncommon characteristic of NK-like cell lines isolated under conditions similar to those described in this report.

¹Ristow, S.S, ¹Starkey, J.R., ¹Stanford, D.R., ¹Davis, W.C. and ²Brooks, C.G.
¹Washington State University, Pullman, and  ²Fred Hutchinson Cancer Research Center, Seattle, U.S.A.

Immunol. Invest. 14: 401-414, 1985.
 

Cell surface thiols are required for a line of cloned natural killer lymphocytes to bind to and lyse tumor target cells. These lymphocytes neither bound to nor killed YAC-1 or G1Tc cells when the effector lymphocyte cell surface thiols were covalently coupled with the non-penetrating reagent, monobromotrimethylammoniobimane (qBBr). A limited number of thiol-bearing proteins were identified by gel electrophoresis on the cell surface using the fluorescence of the group that remains associated with the sulfur molecule. These results indicate that either one or more of these reactive proteins or different cell surface thiol-bearing molecules present at low frequencies are crucial to lymphocyte binding and killing. In contrast, we found little evidence that intracellular thiols are required for natural killer cell activity. Killing was relatively unimpaired when over 90% of lymphocyte glutathione was depleted with DL buthionine-S,R-Sulfoximine (BSO). Blocking the intracellular or the extracellular thiols of tumor targets had no effect on their ability to be lysed. Based on these data, we suggest that infrequently expressed extracellular thiols are required either for the conformation or for the disulfide crosslinking of proteins that participate in lymphocyte-mediated cytolysis.

Brooks, C.G., Holscher, M. and Urdal, D.
Fred Hutchinson Cancer Research Center, Seattle, U.S.A.

J.Immunol. 135: 1145-1152, 1985.
 

It has previously been shown that monoclonal antigen-specific CTL lines can be induced to express cytolytic activity with the same specificity as that of splenic natural killer (NK) cells following culture in high concentrations of concanavalin A-induced spleen cell supernatants. In the present experiments, we made use of this in vitro system to explore the regulation of NK activity at the clonal level. Interferon-alpha and interferon-beta and interleukin 2 (IL 2) were potent inducers of NK activity in CTL, demonstrating that these substances can activate NK functions directly without the participation of other cell types. By comparison, IFN-gamma was a poor activator of NK activity in CTL (and also in fresh spleen cells). Three major differences between induction of NK activity by IFN-alpha,beta and IL 2 were noted: IFN induced NK activity selectively without affecting specific cytolysis, whereas IL 2 also enhanced specific killing; IFN acted much more rapidly than IL 2; and IFN did not induce the cells to enter the cell cycle nor were there any obvious morphologic changes. Specific antigen was also a strong inducer of NK activity in CTL, but studies with antisera against the various classes of IFN revealed that this effect was mediated, at least in part, via the release of IFN-beta. By contrast, the same antisera had no effect on NK induction by crude TCGF or by highly purified IL 2, indicating that the regulation of NK activity by IL 2 occurs at the clonal level in an IFN-independent manner. Although, IL 2, IFN, and Ag could apparently act alone to induce NK activity, much greater (synergistic) induction was obtained by various combinations of these regulators, suggesting that the delivery of two (or more) signals to the responder cell was required for full expression of the NK state. As with fresh splenic NK cells, the induced NK state in cloned CTL was intrinsically labile as revealed by its rapid decay in the absence of inducers, but it could nonetheless be maintained indefinitely at very high levels in the continued presence of inducers. This clonal system thus displays a responsiveness to regulatory signals exactly analogous to that of splenic NK cells and provides a unique and exciting opportunity to evaluate the biochemistry of the regulation of NK activity.

Wayner, E.A. and Brooks, C.G.
Fred Hutchinson Cancer Research Center, Seattle, U.S.A.

Adv Exp Med Biol. 184:221-38, 1985.
 

Using a variety of experimental approaches we have been unable to find any evidence that monoclonal CTL line, induced to express high levels of NK activity by treatment with IFN, mediates target cell lysis by secretion of a cytoxic factor. Thus, supernatant prepared in a variety of ways by incubating cloned killer cells with mycoplasma-free YAC-1 cells, or by freeze-thawing the killer cells themselves, were essentially devoid of lytic activity even when tested in 18hr assays. These findings substantiate at the clonal level our previous observations, that splenic NK cells do not secrete detectable cytotoxic factors in the absence of mycoplasma (Wayner and Brooks, 1984). In addition, it appears that NK killing does not involve the participation of reactive oxygen intermediates. Neither catalase nor SOD were inhibitory. Inhibition observed with some OH. scavengers failed to correlate with their rate constants for reaction with OH., and was apparently due to the general toxicity of these compounds. A cloned cell line expressing potent NK activity failed to produce any luminol-reactive chemiluminescent species during incubation with target cells or with PMA.

Wayner, E.A. and Brooks, C.G.
Fred Hutchinson Cancer Research Center, Seattle, U.S.A.

J.Immunol. 132: 2135-2142, 1984.
 

Co-culture of CBA/J spleen cells and certain lines of YAC-1 stimulators resulted in the appearance of NKCF-like activity in 24- to 48-hr supernatants. Numerous other in vitro cell lines were effective stimulators of this splenic cytotoxic factor (SCF). The cells participating in SCF production were absent from normal thymocytes and were present in BALB/c nu/nu spleen, were nonadherent, asialo GM1+, and bore low levels of Thy-1.2. SCF could mediate lysis of certain NK-sensitive tumor targets in an 18-hr 51Cr-release assay. However, the induction of SCF was not correlated with the ability of a particular cell line to be lysed by NK cells, but showed an absolute correlation with the presence of mycoplasma contamination in cultured tumor cell lines. Mycoplasma negative cell lines, including an uninfected but NK-sensitive subline of YAC-1, were unable to induce SCF. Decontamination of mycoplasma-infected lines with antibiotics or by passage in vivo abrogated the ability of infected tumor cells to stimulate SCF. The ability to induce SCF could be restored by reinfection with mycoplasma. Tumor cell-free supernatants from contaminated cultures were mitogenic for CBA spleen cells and could themselves induce SCF activity in spleen cell supernatants. SCF production and the agent responsible could be removed by passing such supernatants through 0.1-micron filters. The organism apparently responsible for SCF induction from CBA spleen cells was typed and found to be Mycoplasma orale, a nonfermentative, arginine-dependent, common tissue culture contaminant. About 50 to 60% of SCF activity could be removed by 0.1-micron filters, suggesting that SCF is composed of two components: mycoplasma organisms themselves and a soluble cytotoxic factor produced in response to mycoplasma.

Brooks, C.G.
Fred Hutchinson Cancer Research Center, Seattle, U.S.A.

Nature 305: 155-158, 1983.
 

Natural killer (NK) activity is a poorly understood component of the immune system, generally identified as the ability to kill certain tumour cells. Perhaps the most controversial issue has been the lineage to which cells displaying this activity belong. Extensive studies of surface antigens on cells with NK activity have led to enigmatic results, such cells apparently bearing markers of both T-cell (Thy-1 and E receptor) and myeloid (Mac-1 and OKM1) lineages. A fresh approach to this problem would be to take cells of known lineage and test whether they express, or could be induced to express, NK cell function. Using this approach we show here that monoclonal cytotoxic T lymphocyte (CTL) lines can be induced, by culture in high concentrations of spleen cell supernatant, to express a new lytic activity apparently identical with that of splenic cells NK activity. Preliminary evidence implicates both interleukin-2 (IL-2) and interferon (IFN) as mediators of this phenomenon. These findings clearly demonstrate that cells of T cell lineage have the capacity to express NK activity.

¹Brooks, C.G., ²Burton, R.C., ³Pollack, S.B. and ¹Henney, C.S.
¹Fred Hutchinson Cancer Research Center, Seattle, U.S.A., ²Department of Surgery, University of New South Wales, Australia, and ³Department of Cell Biology, University of Washington, U.S.A.

J.Immunol. 131: 1391-1395, 1983.
 

A panel of sera raised against NK-1.1 and NK-2.1 alloantigens was tested for reactivity against a panel of cloned antigen-dependent CTL lines. By using indirect immunofluorescence and flow cytofluorimetry, weak, but clear and consistent, reactivity was found on all CTL. Concordant with the genetics of NK alloantigens, C57BL/6-derived clones were reactive with anti-NK-1.1 and anti-NK-2.1 sera, whereas CBA-derived clones were reactive with anti-NK-2.1 sera but not with anti-NK-1.1 sera. Cloned CTL lines were also able to partially and specifically absorb the antibodies from NK alloantiserum that reacted with splenic NK cells. These results indicate that cloned CTL lines express at least some of the NK alloantigen determinants present on splenic NK cells and have important implications regarding the relationship of CTL and NK cells.

Olabuenaga, S., Brooks, C.G., Gillis, S. and Henney, C.S.
Fred Hutchinson Cancer Research Center, Seattle, U.S.A.

J.Immunol. 131: 2386-2391, 1983.
 

Interleukin 2 (IL 2) has been strongly implicated as the agent responsible for the continuous growth of T cell lines in vitro. In the present study we confirmed that IL 2 alone could support the growth of a widely used cytotoxic T cell line. In contrast, we found that IL 2 was not sufficient to support the long-term growth of cloned NK-like cytotoxic lymphocyte cell lines. Whereas such lines would grow indefinitely in concanavalin A-induced spleen cell supernatant, they would only grow for short periods (2 to 3 days) in the IL 2-containing supernatant of phytohemagglutinin-stimulated LBRM-33 tumor cells, or in IL 2 partially purified from spleen cell or LBRM-33 supernatants. The addition of concanavalin-A or interferon (type beta or gamma) to these supernatants did not improve growth. By contrast, the NK-like cells proliferated equally well in a short-term (24-hr) assay, irrespective of the source of IL 2 (spleen or LBRM-33 supernatant, or partially purified IL 2). Furthermore, the NK-like cells readily depleted IL 2 from the medium, either during growth at 37 degrees C or by absorption at 4 degrees C. It is concluded that at least some cytotoxic cell lines require both IL 2 and other, as yet unidentified, spleen cell-derived factors for long-term growth.

Kawase, I., Brooks, C.G., Kuribayashi, K., Newman, W., Gillis, S. and Henney, C.S.
Fred Hutchinson Cancer Research Center, Seattle, U.S.A.

J.Immunol. 131, 288-292, 1983.
 

Interleukin 2 (IL 2) has been shown to be a potent stimulator of natural killer (NK) cells. In the present studies, partially purified and human IL 2 preparations were also found to induce interferon (IFN) from spleen cells. By the criteria of sensitivity to treatment at pH 2 and failure to be neutralized by a potent anti-alpha, beta IFN serum, the species of IFN produced was of type gamma. Cooperation between two types of cell, a macrophage and an NK-like cell, was required for IFN production by spleen cells treated with IL 2. The requirement for macrophages could be replaced with supernatant obtained by incubating macrophages for 24 hr with lymphokine preparations containing IL 2. Interestingly, mature T cells apparently played no role in the process. Furthermore, the beige (bg/bg) mutation, which severely impairs NK cell lytic activity, had no effect on the ability of NK-like cells to participate in IFN production. Cell fractionation experiments revealed no dissociation between the requirements for augmentation of NK cytotoxic activity and for IFN production, and it is concluded that at least a portion of the NK boosting induced by IL 2-containing preparations is mediated through gamma-IFN.

Brooks, C.G., Kuribaryashi, K., Sale, G.E. and Henney, C.S.
Fred Hutchinson Cancer Research Center, Seattle, U.S.A.

J.Immunol. 128: 2326-2335, 1982.

Abstract

Cantrell, D.A., Robins, R.A., Brooks, C.G. and Baldwin, R.W.
Cancer Research Laboratories, University of Nottingham, U.K.

Immunology, 45: 97-104, 1982.
 

The fluorescence-activated cell sorter and a rosette-depletion technique were used to separate spleen lymphocytes and BCG-activated lymphocytes into subpopulations with and without the antigens defined by W3/13, W3/23 and OX8 monoclonal antibodies. The resultant populations were then tested for natural killer (NK) activity in a quantifiable 6 hr 51Cr release assay. The data establish that natural killer cells are heterogeneous with respect to their surface antigen expression and that subpopulations of NK cells express the OX8 and W3/13 defined antigens. However, NK cells do not express the antigen defined by W3/24 monoclonal antibody.

Flannery, G.R. and Brooks, C.G.
Cancer Research Laboratories, University of Nottingham, U.K.

Int.J.Cancer 28: 747-756, 1981.
 

Natural killer (NK) cell activity was measured in a quantitative 6 h chromium release assay using sarcoma MC7 cells as targets. The total NK lytic activity present in the spleen and blood of tumour-bearers was compared with the corresponding values for age/sex/parity-matched animals. Individuals with primary spontaneous tumours in the breast or subcutaneous sites showed normal levels of NK activity, while individuals with primary spontaneous kidney tumours had elevated NK activity, the degree of augmentation being greater with increasing tumour size. A similar elevation of NK activity was generally found in individuals with large, transplanted, spontaneous or chemically-induced tumours. This augmentation could only detected when total lytic activity was considered: when NK activity was measured merely on a cell-for-cell basis, it often appeared to be depressed in such animals, in agreement with previous reports. However, with one rapidly metastasizing spontaneous tumour, a real depression of both spleen and blood NK activity was found. Small inocula of cells from non-immunogenic spontaneous mammary tumours or from other non-immunogenic spontaneous tumours caused no early increase in systemic NK activity when injected into the mammary pad, a site where spontaneous tumours frequently arise. However, cells from one immunogenic spontaneous tumour and 2/3 immunogenic chemically-induced tumours did occasionally stimulate significant early increases in NK activity when placed at this site. Early changes in peritoneal exudate NK activity were also investigated using small inocula of these tumours injected intraperitoneally. Augmentation of NK activity occurred with a 3-fold greater frequency following inoculation with immunogenic tumour cells than with non-immunogenic cells in this system. It can be concluded from these studies that: (1) spontaneous tumours do not selectively arise in members of an inbred strain with subnormal NK activity; (2) most large tumours stimulate rather than depress NK activity; (3) early boosting of NK activity by small inocula of tumour cells placed in the mammary pad does not occur with non-immunogenic spontaneous tumours; (4) early boosting of NK activity in the peritoneal site does occur with non-immunogenic tumours, but with a very low frequency. The latter findings suggest that developing spontaneous tumours are unlikely to stimulate the NK system, and emphasize the importance of using syngeneic, spontaneous tumours for studying tumour-host relationships in animals.

Brooks, C.G, Flannery, G.R., Willmott, N., Austin, E.B., Kenwrick, S. and Baldwin, R.W.
Cancer Research Laboratories, University of Nottingham, U.K.

Int.J.Cancer. 28: 191-198, 1981.
 

If natural killer (NK) cells play a role in immunosurveillance it might by expected that, during the metastatic process, selection would occur for tumour cells with reduced NK sensitivity. This hypothesis was tested by measuring the NK sensitivity of cells freshly isolated from metastases of syngeneic transplanted spontaneous mammary carcinomas. Lysis was measured in a 6-h chromium release assay using normal syngeneic spleen cells as effectors. Our studies led to the following conclusions. (1) Metastases developing at certain tissue sites (draining lymph node and lung, but not pericardium) were frequently composed of tumour cells with markedly reduced sensitivity to NK cells. (2) This resistance could generally be detected only if freshly isolated tumour cell population were studied; after a few days in culture, resistant metastasis-derived tumour cells usually regained normal NK sensitivity. (3) Resistance to NK cells was not always due to the loss of NK target structures; it could also result from an innate resistance to the NK lytic mechanism. (4) The tissue distribution of NK-resistant metastases suggested that if NK cells exerted an immunoselective pressure they did so at the tissue site rather than in the primary tumour or in the bloodstream.

Gray, J.D., Brooks, C.G. and Baldwin, R.W.
Cancer Research Laboratories, University of Nottingham, U.K.

Immunology 42: 561-568, 1981.
 

The nature of the cytotoxic cells present in the peritoneal cavity of individuals treated with Bacille Calmette-Guerin (BCG) or Corynebacterium parvum was investigated using a 6 hr chromium release assay and a quantitative method of analysis based on consideration of target-cell killing as an enzyme-substrate reaction. When the results of cell-fractionation experiments were evaluated in terms of recovery of total lytic units and when appropriate target cells (such as sarcoma Mc7) were used, the simultaneous presence of both cytotoxic macrophages and NK cells in peritoneal exudates could be readily demonstrated. With certain other target cells different results were obtained. Thus, with normal thymocytes, normal hepatocytes, or myeloma P3NSI as targets, NK cells were preferentially detected, whereas with leukaemias L5178Y, P815, and EL4 as targets, cytotoxic macrophages were preferentially detected. These findings resolve the previously conflicting reports concerning the nature of cytotoxic cells in activated peritoneal exudates.

Brooks, C.G., Flannery, G.R., Webb, P.J. and Baldwin, R.W.
Cancer Research Laboratories, University of Nottingham, U.K.

Immunology 41: 673-680, 1980.
 

Cell fractionation techniques were used to identify the cells in spleen responsible for natural killing of a syngeneic sarcoma cell in short-term (6 h and 18 h) and long-term (72 h) cytotoxicity assays. Cytotoxicity was quantified precisely using a method previously derived from consideration of natural cytotoxicity as an enzyme-substrate reaction, and by analysing results in terms of lytic units. Killing in all three assays displayed 'single-hit' kinetics implying that a single effector cell was sufficient to lyse a single target cell. The fractionation studies, using glass adherence, carbonyl iron, nylon wool, EA and EAC monolayers and congenitally athymic individuals, revealed two populations of cytotoxic cells. In the 6 h assay most of the activity was due to cells with similar characteristics to the NK cells previously defined using leukaemic targets, but in the 18 h and 72 h assays macrophages played an important role. The activity exerted by the macrophages was cell lysis and not cytostasis. No evidence that the macrophages acted by releasing factors which stimulated NK cells could be found.

Brooks, C.G. and Flannery, G.R.
Cancer Research Laboratories, University of Nottingham, U.K.

Immunology 39: 187-194, 1980.
 

Consideration of cell-mediated cytotoxicity as an enzyme-substrate reaction provided a theoretical and practical basis for the quantification of natural immunity to solid tumours. The natural cytotoxic activity of spleen cells from normal individuals towards cultured target cells from solid tumours, measured in a 6 h chromium release assay, developed between 2 and 5 weeks after birth and, in contrast to previous reports of natural cytotoxicity against lymphoid tumour cells, showed no sign of decline up till at least 22 months of age. Both virgin and breeder (multiparous) females showed equally good maintenance of natural immunity, and the apparent specificity of this natural immunity on a panel of four target cells did not change between 8 weeks and 18 months of age. The low level of natural immunity in infant spleen, and also in adult lymph nodes and thymus, was not caused by suppressor cells but rather by an absence of appropriate effector cells.

¹Brooks, C.G., ¹Rees, R.C. and ²Leach, R.H.
¹Cancer Research Laboratories, University of Nottingham, and ²Mycoplasma Reference Laboratory, Public Health Service, Norwich, U.K.

Eur.J.Immunol. 9:  159-165, 1979.
 

Several tumor cell cultures were deliberately infected with three species of mycoplasma commonly found as contaminants of cell lines grown in vitro, and the effect of mycoplasma infection on the results of cytotoxicity assays was examined. Lymph node cells and spleen cells from normal individuals showed an apparently high spontaneous cytotoxic activity against tumor cells infected with either M. arginini or M. hyorhinis, but the reactivity against cells infected with M. orale was not significantly higher than that against uninfected cells. The high reactivity towards tumor cells infected with M. arginini and M. hyorhinis bore a close resemblence to natural cell-mediated immunity in that spleen cells were much more reactive than lymph node cells, T cell deficient spleen cells were as effective as normal spleen cells, and the reaction crossed both strain and species barriers. However, closer examination revealed that the cytotoxic effects were directly caused by depletion of arginine or other essential nutrients from the medium. These findings imply that a cautious approach should be taken when interpreting certain aspects of spontaneous cell-mediated cytotoxicity, and that the greatest care be taken to ensure that the cells used as targets in any cytotoxicity test are mycoplasma-free.

Robins, R.A., Rees, R.C., Brooks, C.G. and Baldwin, R.W.
Cancer Research Laboratories, University of Nottingham, U.K.

Br.J.Cancer 39, 659-666, 1979.
 

Incubation in vitro of lymph node cells (LNC) from individuals bearing a transplanted syngeneic methylcholanthrene-induced sarcoma (Mc7) resulted in the generation of a potent cytotoxic activity. Four to seven days' culture was required for development of cytotoxic activity, which was shown to be mediated by a heat-stable soluble factor. The cytotoxicity was not detectable in a 3 h or 15 h ⁵¹Cr-release assay, but was demonstrated in a 48 h microcytotoxicity assay, where post-labeling with isotopically labelled cell precursors was used to quantitate cell survival. The cytotoxicity of the cultured tumour-bearer LNC and their supernatant factor was shown to be cross-reactive for tumour cell lines other than sarcoma Mc7, and was also expressed against adult or embryonic fibroblasts.

Brooks, C.G.
Cancer Research Laboratories, University of Nottingham, U.K.

J.Immunol.Meth. 22: 23-36, 1978.
 

Four intracellular radioisotope labels, [³H]proline, Na ₂ ⁵¹CrO₄, [⁷⁵Se]selenomethionine and [¹²⁵I]iododeoxyuridine, were evaluated for use in a pre-labelling long-term microcytotoxicity assay for cell-mediated immunity. Adherent tumour cells established in tissue culture were used as targets and the basic variables studied were labelling efficiency, toxicity and spontaneous release rates. [¹²⁵I]Iododeoxyuridine was found unsuitable on account of its high toxicity and correspondingly high spontaneous release rate, and Na ₂ ⁵¹CrO₄ for its toxicity and low labelling efficiency. Of the two other radiolabels, [⁷⁵Se]selenomethionine had the advantage over [³H]proline of higher labelling efficiency (especially in Ham's F10 medium), lower toxicity, and being a gamma-emitter. Furthermore, released ⁷⁵Se was shown to be non-reutilisable and its retention by target cells provided an accurate measure of cell survival in an alloimmune system. Methods of calculating the results of pre-labelling cytotoxicity tests based on the total radioactivity in target cells at the beginning of the assay were found to be invalid.

Brooks, C.G., Rees, R.C. and Robins, R.A.
Cancer Research Laboratories, University of Nottingham, U.K.

J.Immunol.Meth. 21: 111-124, 1978.

We have studied the suitability of various commonly used radioactive materials for the direct post-labeling of adherent target cells in long-term cytotoxicity tests. The use of nucleosides at high concentration avoids the necessity of adding fluorodeoxyuridine to enhance nucleoside uptake by target cells, and reduces the degree of non-specific inhibition of nucleoside uptake caused by products released from effector lymphoid cells. However, when [¹²⁵ I]iododeoxyuridine was used for labelling, such inhibition was not completely avoided even at very high nucleoside concentration, necessitating the washing of target cells prior to labelling. Similarly, without prewashing, the uptake of ⁵¹CrO₄ ions frequently failed to correlate well with the numbers of surviving target cells as assessed by cell counting. On the other hand, radiolabelled amino acids, when present at semi-saturating concentrations, were taken up quantitatively by target cells under all conditions tested. Furthermore, in comparison to [¹²⁵I]iododeoxyuridine, radioactive amino acids showed little if any toxicity to target cells. The use of the gamma-emitting amino acid analogue, [⁷⁵Se]selenomethionine, is particularly recommended.

Brooks, C.G., Rees, R.C. and Baldwin, R.W.
Cancer Research Laboratories, University of Nottingham, U.K.

Int.J.Cancer 18: 778-786, 1976.
 

Lymph node cells from normal individuals were found to affect the growth/survival of syngeneic chemically-induced solid tumour cells in the microcytotoxicity test. Whether inhibition or enhancement of tumour cell numbers occurred depended on the particular tumour cell type and, in some cases, on the particular in vitro subline used. Fractionation of LNC on nylon wool columns revealed that the two effects were mediated by distinct subpopulations of lymphoid cells: column-retained cells showed predominantly an inhibitory effect and column-eluted cells predominantly an enhancing effect. When column-retained and column-eluted cells were cultured under the conditions of the microcytotoxicity test but in the absence of tumour cell growth. The inhibitory activity was maximal within one hour of lymphocyte culture, while the enhancing effect developed slowly during incubation. Furthermore, the tumour cells themselves were found to produce growth-enhancing activity. It is proposed that interaction between these various supernatant activities accounts at least in part for the non-specific effects of normal lymphoid cells in the microcytotoxicity test.

Brooks, C.G.
Department of Immunology, St. Mary's Hospital Medical School, London, U.K.

Eur.J.Immunol. 5: 741-747, 1975.

Mixed lymphocyte culture (MLC) tests on the cells of individuals made fully tolerant to allogeneic tissues in neonatal life showed an absence of specific antigen-reactive cells. Similary, no cells cytotoxic in a chromium release test could be detected. The absence of reactivity in the tolerant populations could not be accounted for by either suppressor cell activity or serum blocking factors. By contrast, individuals in which partial tolerance had been deliberately or inadvertently induced often possessed detectable numbers of MLC-reactive cells and, after grafting, their sera inhibited the MLC reactivity of normal cells. These results are discussed in terms of possible mechanisms of self tolerance.

Brooks, C.G.
Department of Immunology, St. Mary's Hospital Medical School, London, U.K.

J.Immunol.Meth. 9: 171-184, 1975.
 

Various parameters affecting the quantitative assessment of mixed lymphocyte response (MLR) in microplates were investigated. In agreement with the findings of others cell density was found to be particularly critical, maximal proliferative efficiency occurring at 3 x 10⁶ viable cells/cm². Slight reduction in the degree of viable cell-cell interaction caused by either reduction in cell density or insertion of irradiated cells into the cultures greatly reduced the efficiency of the proliferation. An incubation temperature of 33-34 degrees C was found to have distinct advantages over 37 degrees C, there being improved cell survival and reproducibility at this lower temperature. Transformation of lymphocytes was strongly inhibited by low concentrations of normal syngeneic serum, the degree of inhibition depending on the concentration of foetal calf serum (FCS) in culture and on the method of preparation of serum. Inhibition of cell responses was also caused by syngeneic red blood cells (RBC), in contrast to the pronounced enhancement of guinea pig lymphocyte responses to phytohaemagglutinin (PHA) in the presence of syngeneic RBC.