Limiting Dilution Cultures Research Articles

Regulation of cytotoxic lymphocyte precursors: I. Interactions between concanavalin A and T cell growth factors

The induction of cytotoxic T lymphocytes by concanavalin A has been analyzed under conditions of limit dilution. The dose-response curves deviate from linearity in a way that has been interpreted as revealing successive zones of suppression as the cell concentration was increased. The magnitude of suppression was influenced by both the concentration of concanavalin A and the amount of T cell growth factors added to the culture. These regulatory events involve the cytotoxic T cell clones produced by (CBA x DBA)F 1 spleen cells which are detected by DBA mastocytoma (P815) targets at a maximum detectable frequency of 1 in 2000 cells. Similar multiphase dose-response data were also obtained with syngeneic and allogeneic combinations with the same target cell. It is suggested that the successive zones of suppression and activation are a consequence of the relative frequencies of CTL-P, suppressive and helper cells, and the ease with which the cells are activated in limit dilution cultures. The experimental approach illustrates how CTL production can be manipulated to study the balance of signals required to control effector cell production.

Primary in vitro generation of cytotoxic cells specific for human minor histocompatibility antigens between HLA-identical siblings.

A limiting dilution culture system was developed for the primary in vitro detection of human minor histocompatibility antigens by cytotoxic T lymphocytes (CTL). CTL were generated in primary in vitro culture between two HLA-identical sibling pairs and propagated as stable CTL lines. Population and family studies indicate that these CTL lines recognize minor histocompatibility antigens in an HLA-restricted manner. The antigen recognized by one CTL line is detected on six (out of 37) HLA-B7-positive donors but not on 32 HLA-B7-negative donors. The cytotoxicity of this CTL line is mediated by T3+, T8+ effector cells. The antigen detected by this CTL population is different from all known human minor histocompatibility antigens. The data of this study, like those in the mouse system, suggest that a suppressor cell is diluted out in a limiting dilution culture, which allows the activation of the CTL precursors.

A method for testing the specificity of influenza A virus-reactive memory cytotoxic T lymphocyte (CTL) clones in limiting dilution cultures

This paper describes a system for determining the frequency and fine specificity of influenza A virus-immune memory cytotoxic T cell (CTL) clones from limiting dilution (LD) microcultures. We found that such experiments can only be performed (1) by analyzing the clonal response of CTL from wells of replica plates containing fractions of one original plate, (2) when it has been ascertained that splitting is possible at the clonal level, and that each fraction of a microculture well gives an identical response on target cells infected with the stimulating virus. With these requirements fulfilled we found that short term CTL clones from LD microcultures from influenza A virus (A/X-31)-immune mice (C57BL/6) occur at a frequency of f = 1:546 to f=1:6303. The effector cells carry the Lyt-2.2 marker and are specific for target cells infected with the immunizing virus (influenza virus A/X-31). They do not lyse NDV (Newcastle disease virus) or HSV (herpes simplex virus)-infected or NK (YAC) target cells.

Frequency of influenza-responsive cytolytic T-lymphocyte precursors in the thymus and spleen of unprimed mice

Limiting dilution culture conditions were established which allowed the differentiation and quantitation of influenza-specific cytotoxic T-cell precursors (CTL.Ps) from naive mouse spleen and thymus. One in 49,000 nucleated spleen cells and 1 in 40,000 thymocytes responded to stimulation with influenza-infected, anti-Thy 1.2 and complement-treated spleen cells, in the presence of ConA-stimulated cell supernatant, with the production of cytotoxic effector cells. These frequencies are 4- to 15-fold higher than those for influenza-responsive B cells in the relevant lymphoid compartments, and it is argued that major quantitative discrepancies may exist in the sizes of the B- and CTL-receptor repertoires.

T-cell cytotoxicity and aging: Differing causes of reduced response in individual mice

Specific T-cell cytotoxic responses to allogeneic and hapten-modified syngeneic cells decrease with age. In order to determine the causes of these reduced T-cell cytotoxic responses, spleen cells from individual young and senescent C57BL/6J female mice were mixed in various proportions in culture with either X-irradiated BALB/c spleen cells or trinitrophenyl-modified syngeneic cells and the resultant cytotoxic responses determined in comparison to those of spleen cells from young and old mice stimulated alone. In both allogeneic and hapten-modified syngeneic cytotoxicity, it was found that a low percentage of the aged mice suffered from decreased helper-cell activity or from increase of suppressor activity, while the majority of mice showed no synergy, positive or negative, with the cells from the young donor. Studies of interleukin-2 (IL-2) activity were performed on conditioned medium from spleen cells from mice of various ages cultured for 24 h with concanavalin A. Those preparations from senescent mice that showed reduced IL-2 activity did not contain activity suppressive or competitive to IL-2 produced by spleen cells from young mice. Limiting dilution of spleen cells from mice of various ages in the presence of semi-allogeneic stimulatory cells and subsequent assay of the resultant allogeneic cytotoxicity provided a measure of the frequency of cytotoxic units. Parallel experiments in which crude IL-2 was added to the limit dilution cultures provided a measure of the frequency of cytotoxic cell precursors. Once again in these experiments, individual senescent mice demonstrated different defects. Three different types of age-related defects were observed. Certain aged mice were devoid of detectable cytotoxic units and cytotoxic T-lymphocyte precursor at the cell dilutions used. Other senescent mice demonstrated a very low frequency of cytotoxic units ( ≈1 40 000 ) as compared with young mice ( ≈1 5 000 ), and the addition of crude IL-2 to cultures from these mice did

SIgD-negative B cells from neonatal mice do not respond to the thymus-independent antigen TNP-BA in limiting dilution cultures.

The majority of B lymphocytes in adult mice express both IgM and IgD on their surface (sIgM and sIgD). A small percentage of sIgM+ splenic B cells lack (or express very low levels of) sIgD. These cells have been termed "mu-predominant" (mu p) B cells. In neonatal mice (5 to 12 days of age), mu p B cells account for more than 50% of the sIg+ cells. There is conflicting evidence concerning the immunocompetency of mup cells in vitro. To study this question further, splenocytes from neonatal BALB/c mice were depleted of sIgD+ B cells by a panning procedure. A portion of the nonadherent (mu p) cell population was analyzed for residual sIgD+ cells by using indirect immunofluorescence in conjunction with the fluorescence-activated cell sorter (FACS). Such cells were then tested for their responsiveness to the thymus-independent (TI) antigen, trinitrophenyl Brucella abortus (TNP-BA), by using a limiting dilution culture system. Results indicate that the depletion of sIgD+ B cells and the decrease in the precursor frequency of splenocytes responding to TNP-BA are very similar, suggesting that virtually all of the responding B cells bear sIgD.

A reappraisal of "T-independent" antigens. II. Studies on single, hapten-specific B cells from neonatal CBA/H or CBA/N mice fail to support classification into TI-1 and TI-2 categories.

Abstract Spleen cells from adult CBA/H or CBA/N mice, or from neonatal CBA/H mice, were fractionated on thin layers of fluorescein (FLU)-gelatin to yield FLU-specific B lymphocytes. A single cell, or small numbers ranging from 1 to 10, were cultured in 10-microliter microcultures together with various antigens and mitogens. The results were compared with those of bulk culture or limiting dilution cultures supported by thymus filler cells. B cell growth and differentiation-promoting conditioned media (BGDA) were added to some cultures. The CBA/N results gave no support to the commonly used classification of T cell-independent (TI) antigens into TI-1 and TI-2 categories. A typical supposed TI-1 antigen, FLU-LPS, strongly stimulated normal adult single FLU-specific B cells to proliferate and form antibody, but virtually failed to trigger CBA/N B cells of comparable antigen-binding avidity. The same was true of LPS or LPS plus dextran sulfate acting as mitogens. The allegedly TI-2 antigen FLU-Ficoll, although still triggering comparatively poor responses, was actually marginally more active than FLU-LPS. FLU-Brucella abortus (FLU-BA) + BGDA gave the best results with single CBA/N B cells, but still induced only 1.27% of cells to develop into antibody-forming clones vs 12.2% with CBA/H cells. The results obtained with single neonatal B cells also lent no support to the distinction between TI-1 and TI-2. Both "TI-1" and "TI-2" stimuli caused adequate proliferation, one "TI-2" antigen stimulating 23.2% of the cells. None of the antigens caused good antibody formation, however, probably because multivalent antigens can deliver signals impeding the differentiation of immature B cells. It is therefore suggested that the classification of TI-1 antigens into two subcategories be abandoned, at least for the time being.

Specificity of in vitro anti-influenza virus antibody production by human lymphocytes: analysis of original antigenic sin by limiting dilution cultures.

The fine specificity of the antibody produced in in vitro cultures of human lymphocytes stimulated with influenza virus was investigated. The antibody was specific for the stimulating type of virus in that cultures stimulated with type B influenza virus made little or no antibody to type A viruses and vice versa. There was a degree of cross-reactivity among the type A viruses, however, both within and between subtypes. In general, more antibody was made to the stimulating strain of virus than to heterologous strains, with the exception that cultures of mononuclear cells from four of five donors stimulated with A/Bangkok/79 (H3N2) made more antibody to A/Aichi/68 (H3N2) than to the stimulating virus, likely reflecting the original antigenic exposure of these donors. The specificity of this in vitro response was further investigated by using monospecific antibodies produced in limiting dilution cultures. Approximately 30% of the antiviral antibodies produced in these cultures bound to purified hemagglutinin, but only 7% bound to purified matrix protein. When antibodies stimulated with A/Bangkok/79 were analyzed for their binding to other H3N2 viruses, a variety of reactivity patterns was observed. Some antibodies were specific for A/Bangkok/79 and some bound to common determinants found on a number of H3N2 viruses. In addition, a number of antibodies were observed that did not bind to A/Bangkok/79 but did bind to earlier H3N2 viruses, demonstrating an extreme form of "original antigenic sin." A similar variety of reactivity patterns was observed when antibodies were tested for binding to heterotypic type A viruses or hemagglutinins. The results of the present experiments demonstrate that the fine specificity of an in vitro human antibody response can be analyzed by using limiting dilution cultures, and suggest that the fine specificity of the antibody response of an individual to a strain of influenza virus is affected by previous antigenic exposure of that individual.

Loss of specificity in cytolytic T lymphocyte clones obtained by limit dilution culture of Ly-2+ T cells.

Nonspecific cytotoxicity developed reproducibly and with high frequency in limit dilution cultures consisting of low numbers of murine cells stimulated with concanavalin A in the presence of growth factors and irradiated filler cells. The individual clones in cultures showing nonspecific killing were all derived from single, Thy-1+, Ly-2+ cells. At early times of culture (day 5 or 6), clones appeared to be specific in their lytic activity, as expected of cytolytic T lymphocytes (CTL). On continued culture (day 8 or 9), most of the originally specific CTL clones became nonspecific, killing a range of murine target cells, both syngeneic and allogeneic. The lack of specificity was observed at all effector cell doses. The effector cells responsible for the nonspecific cytolysis were Thy-1+ and Ly-2+, as were most cells in the cultures. The effector cells had the normal DNA content for a dividing T cell population, and most cells in the cultures had a normal chromosome complement. In mixed cultures in which the responder cells and the irradiated filler cells were from different mouse strains, the nonspecific killers displayed the Thy-1 and H-2 allotypes of the responder, and not of the filler cells. The development of a broad cytotoxic potential appears to be a normal and rapid event when Ly-2+ T cell-derived CTL-clones are grown under these conditions; this is a caveat for the use of limit dilution cultures to determine the T cell specificity repertoire. The relationship between these nonspecific CTL, activated lymphocyte killers, and natural killer cells is discussed.

B cell maturation factor: effects on various cell populations.

B cell maturation factor (BMF) is a lymphokine that promotes the maturation of resting murine splenic B lymphocytes, and the analogous B cell tumor line WEHI-279, to the state of active immunoglobulin (Ig) secretion. All subsets of normal B cells examined, including neonatal and adult B cells, B cells from various organs, and B cells from CBA/N mice, are inducible by BMF. Induction of Ig secretion is independent of thymus-derived cells, LPS receptors, and MHC haplotype, because nude, C3H/He, and mice of many strains are equally responsive to BMF. Purified B cells prepared by using a fluorescence-activated cell sorter also respond to BMF, showing that BMF directly interacts with and triggers Ig secretion by B cells. In limiting dilution cultures, most normal resting splenic B cells or WEHI-279 B tumor cells are inducible by BMF. By using the WEHI-279 cells as a model system, specific aspects of the BMF response have been analyzed. In terms of the degree of stimulation observed, the primary mechanism for the induction of Ig secretion by BMF is an enhanced and balanced synthesis of Ig heavy (H) and light (L) chains. A less significant component of the induced Ig secretion is an increase in the ratio of secretory to membrane H chains produced. Kinetically, the shift in the ratio of secretory to membrane H chain forms occurs first, and this is followed by the increased synthesis of both L and H chains. Responding B cells also die during this induction process. Although the changes in the ratio of H chain forms, H and L synthesis, and cell viability take several days to occur, BMF will program significant later responses after only 1 or a few hr of interaction with target B cells.

The adaptation of the plaque reduction assay for measuring specific cytotoxic cells in limiting dilution cultures

The use of the original haemolytic plaque reduction technique to measure cytotoxic T lymphocytes (CTL) has been developed further as a rapid screening assay, particularly suitable for limiting dilution analyses. Using hybridoma cells as targets, the cytotoxicity has been measured by the loss of haemolytic plaque formation and by the reduction of the amount of haemolytic monoclonal antibody secreted from viable target cells into the assay supernatants. The assessment of large numbers of cytotoxic samples has been greatly facilitated by quantitating the amount of haemoglobin released in the assay with an automated microELISA multiscanner and by scoring visually using a modification of the spot test. Using these new techniques, relatively high frequency estimates of cytotoxic cell precursors in an allogeneic response (1 in 462 spleen cells) and an anti-fluorescein response (1 in 3970 spleen cells) were obtained. Since the methods are very simple, rapid, and sensitive, these assay systems provide effective alternatives to radioisotope release in limiting dilution analyses.

The glycosphingolipid globoside as a serological marker on cytolytic T lymphocyte precursors and alloantigen-responsive proliferating T lymphocytes in murine spleen.

Biochemical analyses of murine lymphocytes have shown that the glycosphingolipid globoside (Glo) is present exclusively on alloantigen-stimulated murine T lymphocytes (Gruner, K. R., Van Eijk, R. V. W. and Mühlradt, P. F., Biochemistry 1981. 20: 4518). An anti-Glo antibody has now been raised in rabbits immunized with purified antigen. Most activity was recovered in the IgM fraction. The specificity of the antibody was ascertained in an enzyme-linked immunosorbent assay with purified glycosphingolipids bound to the solid phase. In antibody-dependent complement lysis experiments the anti-Glo eliminated about 20% of nylon wool-nonadherent splenic T cells of CBA/J mice. To determine the functional identity of these Glo+ cells, the effects of Glo+ cell elimination on mitogen stimulation with concanavalin A and lipopolysaccharide, as well as the effects on the mixed lymphocyte culture (MLC) reaction and cell-mediated lympholysis with mitomycin-treated DBA/2 splenocytes as stimulator cells were studied. Whereas lipopolysaccharide stimulation was not affected by elimination of Glo+ cells, there was a slight inhibitory effect on the concanavalin A stimulation, and a severe inhibition of the MLC reaction and the generation of H-2d-specific cytolytic T lymphocytes. Addition of interleukin 2 increased the MLC reaction, but interleukin 2-saturated cultures were also severely inhibited by anti-Glo and complement treatment. Combined treatment with anti-Glo and anti-Lyt-1 or anti-Lyt-2 antibodies, and determination of cytolytic T lymphocyte precursor frequencies in limiting dilution cultures after Glo+ cell elimination showed that a large proportion of T cells proliferating in a primary MLC are Lyt-1+,2+,3+Glo+, whereas in secondary MLC they are Lyt-1+,2-,3-,Glo+. Fifty % of the cytolytic T lymphocyte precursors in primary as well as secondary MLC are Glo+. The Glo marker is lost upon differentiation to cytolytic T lymphocyte effector cells. It is discussed herein that Glo is a marker for alloantigen-stimulated precursor T lymphocytes of both helper and cytolytic T cells.

T-T cell interactions during in vitro cytotoxic T cell responses. VI. The role of T cell-derived colony-stimulating factor in helper T cell activation.

A limiting dilution culture system has been used to analyze the role of T cell-derived colony stimulating factor (CSF) during the activation of IL 2-producing helper T lymphocytes (HTL). EL4 thymoma-derived supernatant (EL4-SN) increased about 4-fold the frequency of HTL precursors responding to metabolically active allogeneic stimulator cells. Upon biochemical separation this biological activity within the EL4-SN segregated from interleukin 2 (IL 2) and gamma interferon but was associated with or identical to CSF. Further, a comparable rise in HTL precursor frequencies was observed when semipurified interleukin 1 (IL 1) derived from the P388 D1 cell line was added to limiting dilution cultures. In contrast to IL 1, semipurified CSF failed to facilitate the activation of HTL precursors when heat-treated allogeneic spleen cells were used as stimulator cells. Because EL4-derived CSF was found to induce IL 1 production by macrophages we conclude that T cell-derived CSF amplifies, via stimulation of antigen-presenting cells the number of inducible HTL precursors. Therefore, the CSF/IL 1-dependent increase in HTL precursor frequencies reported here may reflect the differential activation threshold of HTL-precursors, most of which will not be activated by antigen per se but only in presence of additional cytokines.

Quantitative representation of all T cells committed to develop into cytotoxic effector cells and/or interleukin 2 activity-producing helper cells within murine T lymphocyte subsets.

A limiting dilution culture system based on stimulation with concanavalin A (Con A) has been used to study the quantitative distribution of helper and of cytotoxic precursor cells in Lyt-2-defined subpopulations of murine T cells. Virtually all of the selected Lyt-2+ and Lyt-2-T cells grow and expand to large clonal colonies within an 8-9-day culture period. Our data show that upon stimulation with Con A, 90% of the Lyt-2-T cells were capable to produce interleukin 2 (IL 2) activity. In addition, IL 2 activity is produced by 8-10% of Lyt-2+ T cells. However, at the clonal level, the average of the IL2 activity produced by Lyt-2+ T cells is about 8-fold less as compared to Lyt-2-T cells. Precursors of cytotoxic T cells were almost exclusively found in the Lyt-2+ population, of which about 70% displayed lytic activity in a lectin-dependent cytolysis test. For the vast majority of clones analyzed the capacity to produce IL 2 activity and the capacity to express lytic activity, was found to be mutually exclusive. A minority of clones (less than 3%) was found to simultaneously produce IL 2 activity and to express cytotoxicity. These latter cells are therefore considered as bifunctional T cells.

Cytotoxic T-cell precursors revealed in neonatally tolerant mice.

Induction of neonatal tolerance leads to a profound reduction in cytotoxic T-lymphocyte precursor frequencies against the tolerated alloantigen ("tolerogen") as evaluated by limiting-dilution analysis. The curves obtained were linear within the range tested and, thus, did not yield evidence for any dissociation of cytotoxic precursors from regulatory cell populations. However, it could be shown that cytotoxic T-lymphocyte precursor frequencies against the tolerogen increased drastically if the tolerant spleen cells were adsorbed, prior to limiting-dilution culture, on monolayers of syngeneic blasts expressing receptors for the tolerogen but not if they were adsorbed on syngeneic blasts against third-party antigens. This finding implies that cytotoxic precursor cells against the tolerogen are not clonally deleted in tolerant animals but rather are suppressed by a regulatory cell population that is present at high frequency and presumably acts in an anti-idiotypic fashion.

Degradation of specificity in cytolytic T lymphocyte clones: mouse strain dependence and interstrain transfer of nonspecific cytolysis.

Limiting-dilution culture of murine Ly-2+ T cells with concanavalin A (Con A) and irradiated spleen filler cells produces, with high efficiency, cytolytic T lymphocyte (CTL) clones. With most mouse strains (including CBA and C57BL/6) the specificity of these CTL clones drops after day 6 of culture, so that by day 9 the majority of clones can lyse most murine target cells, whether syngeneic or allogeneic. The rate of specificity degradation and relative target cell preference varies with the mouse strain. Some strains (e.g. BALB/c) do not show this effect and CTL clones remain specific to day 9. Many low natural killer (NK) cell strains (e.g. C57BL/6J.bg) maintain CTL specificity in such cultures, but the correlation between CTL specificity and NK status is not absolute. Growth of BALB/c precursor cells on CBA filler cells leads to specificity degradation in the BALB/c CTL clones; however, the specificity of CBA-derived CTL clones is not maintained by growth on BALB/c fillers. The results suggest that specificity degradation is induced in the developing CTL-clone by factors in the culture environment, perhaps a soluble lymphokine (a differentiation factor) or by an infectious agent (an endogenous mouse virus). Although such CTL specificity loss may render many limiting dilution studies of the CTL specificity repertoire invalid, the problem may be bypassed by an appropriate choice of mouse strain.

Functional clonal deletion and suppression as complementary mechanisms operative in adult hapten-induced cytotoxic T cell tolerance.

Immunologic tolerance to the hapten TNP was induced in adult mice through the i.v. injection of reactive TNBS. To probe the cellular basis of the tolerant state, splenic cytotoxic T lymphocyte precursors (CTL-P) were stimulated in vitro with haptenated, x-irradiated syngeneic spleen cells in the presence or absence of exogenously added growth factors derived from Concanavalin A-stimulated spleen cell conditioned medium (CAS). The cultures were either conventional bulk cultures or limit dilution cloning cultures. For the latter, cytotoxicity was assessed through a semi-automated, radioautographic 111In-release assay. Suppressive potential was assessed by mixing spleen cells from tolerant mice with normal spleen cells before culture. In the absence of CAS, bulk cultures showed profound tolerance, and suppressive capacity was clearly evident. Suppression was dependent on the presence of TNP-self during culture and affected the generation of CTL from CTL-P and not the effector function of CTL. Cyclophosphamide treatment did not prevent tolerance induction. In the presence of CAS, bulk cultures still showed marked tolerance, but mixing experiments now yielded no evidence of suppression. As documented previously, limit dilution cultures of tolerant spleen cells in the presence of CAS showed a functional clonal deletion of hapten-specific CTL-P. In the absence of CAS, limit dilution cultures became dependent on helper T cells as the limiting element. Tolerant populations showed a diminution of activatable helper T lymphocyte precursors (HTL-P), which may have been due to a functional clonal deletion of HTL-P and/or a concomitant activation of suppressor T cells. Adoptive transfer studies showed that cells from tolerant mice did not detectably influence the number of hapten-specific CTL-P in the spleens of host animals. Taken together, the results suggest that both functional clonal deletion of CTL-P and suppression of HTL-P contribute to the tolerant state induced.

Differentiation from precursors in athymic nude mouse bone marrow of unusual spontaneously cytolytic cells showing anti-self-H-2 specificity and bearing T cell markers.

We describe an in vitro limiting dilution culture system that supports growth and differentiation of nylon wool nonadherent bone marrow cells from athymic nude mice. Cells were seeded at low cell numbers (5-120 cells per 20-microliter microculture well) in the absence of added filler or feeder cells but in the presence of conditioned medium. Microwells positive for growth appeared to contain a single clone of cells that adhered together to form a tight cluster referred to here as a colony. A fraction of colonies contained cells that expressed an unusual spontaneous cytolytic activity. They lysed syngeneic or semisyngeneic Con A blast or tumor cell targets but seldom lysed H-2-incompatible Con A blast or tumor target cells, even in a lectin-facilitated assay. A large fraction of colonies contained lymphoid cells that expressed the T cell markers Thy-1 and Lyt-1. Colonies expressing spontaneous cytolytic activity and also containing cells with Thy-1+ and/or Lyt-1+ markers could be grown from nylon wool nonadherent nude marrow cells depleted rigorously by cell sorting of cells expressing either of these markers. Expression of Thy-1 and spontaneous cytolytic activity in a particular colony was significantly correlated. Short-term lines established from cytolytic colonies with T cell markers maintained both characteristics. The cytolytic effector cells observed in these cultures may represent an early stage in the development of the T cell repertoire.

Frequency analysis of functional immunoglobulin C and V gene expression in murine B cells at various ages.

The frequencies of lipopolysaccharide- (LPS) reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of mice at different ages. A limiting dilution culture system was employed that allows the growth and development of every LPS-reactive B cell into a clone an IgM-secreting cells that are capable of switching to other Ig heavy chain isotypes (C gene expression). The secretion of IgM and IgG1 was assessed in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V gene expression) were detected with the use of plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The frequencies of LPS-reactive B cells in the spleen and BM of C3H/Tif, C57BL/Ka, BALB/c, and CBA/Rij mice appeared to be similar in 6- to 12- and 100-wk-old animals, as was the switch frequency to IgG1 secretion in three strains tested. Moreover, no age-related changes were observed in the frequencies of antigen-specific B cells within the pool of LPS-reactive B cells in the spleen and BM of C57BL/Ka mice. The frequencies ranged from 1 in 10 to 1 in 20 for NIP4- and NNP2-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 4000 for SRBC, HRBC, and GRBC. The specificity repertoire of the "spontaneously" occurring ("background") IgM-secreting cells in the spleen and BM, on the other hand, did differ between young and old C57BL/Ka mice. During aging the frequencies of the tested specificities decreased in the spleen but increased in the BM. Our data indicate that in unintentionally immunized mice the clonal selection of B lineage cells by antigen takes place at the level of the mature, antigen-reactive B cell.

Genetic and molecular analyses of lymphocyte-defined HLA-D region specificities

Determinants encoded in the HLA-D regin have been studied with both cellular (PLT) and molecular (SDS-IEF) methods. When the PLT response against a lymphoblastoid cell line was analyzed by limiting dilution culture and determination of the reactivity of individual cultures against a panel of loss mutants of the initial stimulating LCL, a large fraction of the cultures showed the same pattern, apparently recognizing a determinant associated with DR. In two-dimensional gel analysis of several DR4-positive HLA-D region homozygous cells, the IEF pattern of the DR beta chain correlated with the Dw specificity expressed by the cell. These two pieces of evidence suggest that, although many determinants may contribute to reactivity in mixed leucocyte culture or PLT, an immunodominant determinant associated with the DR beta chain may be the most important single factor in the assignment of Dw specificity.