Cross-reactive idiotypic determinants on antibodies and antigen-specific helper T cell continuous lines

Anti-idiotypic antibodies produced in C57BL/6 mice (H-2b, Igh-1b) against (T,G)-A--L-specific antibodies of C3H.SW mice (H-2b, Igh-1j) were used to probe (T,G)-A--L-specific helper T cell lines and clones for the expression of idiotypic determinants on the cell surface of the monoclonal functional T cells. By using the fluorescence-activated cell sorter (FACS II), anti-idiotypic sera of individual mice that specifically bind C3H.SW anti-(T,G)-A--L antibodies were shown to stain significantly cells of the E-9M(+) continuous T helper line originated from C3H.SW (T,G)-A--L "educated" T cells. The same antisera did not react with a helper T cell line of C3H.SW origin specific to human gamma-globulin. They also did not stain a (T,G)-A--L-specific helper T cell line derived from CWB (H-2b, Igh-1b) mice, which differ from C3H.SW mice only in their heavy chain allotypes. Thus, the expression of the idiotypic determinants on the T cell lines appears to be antigen-specific and linked to the heavy chain allotypic marker as shown for the specific antibodies. Different clones derived from the E-9 M(+) line were tested their reactivity with the individual anti-idiotypic sera. All clones but one (1.11) were stained significantly. The clones were tested for their biologic activity and all of them except clone 1.11 were found to exert helper activity specific to (T,G)-A--L. Thus, individual anti-idiotypic sera against C3H.SW anti-(T,G)-A--L antibodies recognize cross-reactive idiotypic structures on the surface of antigen-specific monoclonal helper T cells.     

Genetic characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG₁, IgG_2a, and IgG_2b. In this communication we describe their reactivities with immunoglobulins of the inbred mouse strains. Comparison with serology charts indicates that many of these monoclonal antibodies detect allotypic specificities previously defined by conventional antisera; others define previously undescribed specificities. Strain and isotype distribution allows us to assign five new allotypic specificities to Igh-1 and three new specificities to Igh-3. In addition, on the basis of reactivity with the monoclonal antibodies, we have defined a new Igh haplotype in SWR/J mice, Igh ^p.Abbreviations used in this paper Igh immunoglobulin heavy chain - SDS sodium dodecyl sulfate     

Localization of murine Igh-1a allotypic determinants by using a panel of mouse myeloma variant immunoglobulins

A number of monoclonal antibodies are available that are reactive with distinct mouse immunoglobulin allotypic determinants. By determining which ones are present on a panel of hybrid IgG2b-IgG2a immunoglobulins, we have localized some of the allotypic determinants present on the IgG2a heavy chain of the "a" allotype (Igh-1a proteins). In particular, one group of determinants--Ig(1a)9.8 (20.6B8), 17.2 (20.19.2), and 14.4 (21.74.4)--has been placed in the CH2 domain. A second group--Ig(1a)8.3 (20.8.3), 21.2 (20.11.2), and 15.3 (21.66.3)--is located in a segment spanning the C terminal 8 residues of the CH2 domain and the complete CH3 domain.     

Characterization of the antibody response against the type II collagen induced by anti-idiotypic antibody.

We reported that rabbit anti-idiotypic antibody (Ab2) against mAb, termed 1-5 (Ab1) and reactive with human type II collagen (CII) induced antibody response to CII in DBA/1J mice susceptible to collagen-induced arthritis. In the present study, we further characterized the anti-CII antibody response elicited by Ab2 with respect to epitope specificity, putative genetic background, and IgG subclass. Most of anti-CII antibodies (polyclonal Ab3) derived from Ab2-immunized mice were of the IgG1 subclass. We purified polyclonal Ab3, using a CII-coupled immunoadsorbent column and we developed monoclonal Ab3 from Ab2-immunized mice. Both purified polyclonal Ab3 and two monoclonal Ab3s specifically reacted with a selected epitope on CII, recognized by Ab1. The anti-CII antibody response stimulated by Ab2 was observed in DBA/1J (H-2q, Igh-1c) and DBA/2 (H-2q, Igh-1c) mice, but not in the BALB/c (H-2d, Igh-1a) and C57BL/6 (H-2b, Igh-1b) strains, thereby suggesting that the anti-CII antibody response elicited by Ab2 is controlled by the Igh gene.     

Characterization of a murine monoclonal antibody specific for an allotypic determinant on T cell antigen receptor

Repeated immunization of normal C57L/J (H-2b) mice with peripheral T cells from BALB.B (H-2b) mice results in the production of antibodies which react with the T cell receptor. A monoclonal antibody-producing hybridoma, F23.1, was isolated from immunized C57L/J mice showing this property. This monoclonal antibody recognizes approximately 25% of peripheral T cells in BALB mice. It stains approximately the same fraction of T cells and precipitates the same heterodimer as the rat monoclonal antibody described previously that was made against isolated receptor material. The allotypic determinant recognized by this monoclonal antibody is present in most common laboratory strains (BALB, C57BL, CBA, A, DBA, C3H) and is absent in C57L, C57BR, and SJL mice. Sorting peripheral T cells from BALB.B or (SJL X BALB/c)F1 mice for the F23.1+ and F23.1- subsets revealed that both populations contain approximately the same CTL precursor frequency for alloantigen. Thus, the T cell receptor allotype defined by F23.1 is present on CTL. Furthermore, cytotoxicity mediated by an F23.1+ CTL line could be blocked specifically by the F23.1 monoclonal antibody. Under appropriate conditions, the monoclonal antibody F23.1 bound to Sepharose 4B beads can induce resting peripheral T lymphocytes of allotype-positive strains to proliferate.     

T cell-induced Ig allotypic suppression in mice. II. Both CD4+ CD8- and CD4- CD8+ T cell subsets from sensitized Igha mice are required to induce suppression of Igh-1b allotype expression

We established the phenotype of T splenocytes (Ts) from Igha/a BALB/c mice sensitized against B splenocytes from the Ighb/b CB20 congenic mice that induce Igh-1b (IgG2a of the Ighb haplotype) suppression. This was achieved by studying the action of anti-T cell subset mAb on the capacity of Ts to induce this chronic allotypic suppression in Igha/b (BALB/c x CB20)F1 mice. The Ts were treated with cytotoxic anti-mouse CD4 or anti-mouse CD8 rat mAb in vitro before their injection into the Igha/b newborns or in vivo after their injection into the Igha/b newborns. Exposure to either anti-CD8 or anti-CD4 mAb in vitro or in vivo leads to loss of the capacity of Ts to induce Igh-1b allotypic suppression. Mixing CD4+-cell-depleted Ts and CD8+-cell-depleted Ts preparations restored the capacity of the cells to induce Igh-1b suppression. Thus, both CD4+ CD8- Ts and CD4- CD8+ Ts are required for the induction of this allotypic suppression. Bone marrow cells and B splenocytes from Igh-1b-suppressed adult Igha/b mice were shown to be able to durably express Igh-1b when transferred into irradiated Igha/a BALB/c hosts whereas whole spleen cells from such donors failed to do it. Abrogation of Igh-1b suppression by in vivo anti-CD8 mAb treatment was achieved in adult Igha/b heterozygotes but with a lower efficiency than in adult Ighb/b homozygotes, all being chronically Igh-1b suppressed. The CD4- CD8+ cell population essential for maintaining this suppression is resistant to in vivo 600 rad irradiation and seems to be slightly inhibited by in vivo administration of free Igh-1b.     

Structural characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG₁, IgG_2a, and IgG_2b. In this communication we describe their reactivity at the molecular level. A number of genetic specificities (as defined by reactivity with sera from inbred strains) were divided into subspecificities (allotopes) by these analyses. With the exception of one allotope located in the hinge region of Igh-1 ^b, all other 23 allotopes examined were preserved upon reduction and alkylation of immunoglobulin antigens. To further analyze the role of immunoglobulin conformation in presenting the allotopes, we assayed their presence on mixed Igh-1^a/Igh-4^a heavy chain molecules. The Igh-1^a determinants were maintained, but the Igh-4^a determinants were lost. Taken together, our results indicate that genetic polymorphisms at the Igh loci generate an enormous antigenic complexity, much of which relies on tertiary and quaternary protein structure for expression.     

Identification of goat allotypic determinants and generation of goat-mouse hybridomas secreting goat IgG2

Five allotypic determinants controlled by independent genes have been identified in goat. Of these determinants, four have been detected with alloimmune antisera and one with monoclonal antibodies. The specificities A1, C1 and D1 are lipoproteins; B1 is possibly an alpha 2 macroglobulin and E1 and IgG2. The specificity B1 is not expressed until the age of 3-4 months. The gene controlling the specificity E1 is present at about the same frequency (0.38-0.41) in goat, sheep, cattle and water buffalo. Stable hybridomas secreting goat IgG2 have been obtained by the fusion of goat peripheral lymphocytes with mouse myeloma cells.     

Genetic control of contact photosensitivity to tetrachlorosalicylanilide. II. Igh complex controls the sensitivity induced by photohapten-modified spleen cells but not epidermal cells

We studied the genetic control of murine contact photosensitivity (CPS)1 to 3,3',4',5-tetrachlorosalicylanilide (TCSA) that was induced by subcutaneous injection of TCSA-photomodified epidermal cells (photoTCSA-EC) and spleen cells (photoTCSA-SC). With regard to the H-2 locus, sensitization with both types of photohaptenated cells showed the same pattern of CPS responses: H-2k and H-2b,d haplotypes were closely associated with low and high responders, respectively. On the other hand, the Igh locus affected the CPS reaction induced by photoTCSA-SC but not -EC; the Igh-1d allotype was related to low responsiveness, while high responders possessed Igh-1a,b. Thus, the photoTCSA-SC sensitization was controlled by H-2 and Igh in a codominant manner. The photoTCSA-SC-induced responses of H-2k but not Igh-1d mice were enhanced by CY pretreatment, suggesting that the mechanisms of low responsiveness in H-2k and Igh-1d mice were different. H-2 identity between donors of photoTCSA-EC and recipients was sufficient for effective sensitization, whereas both H-2 and Igh between donors of photoTCSA-SC and recipients should be identical to obtain maximum sensitization. This further confirmed the involvement of the Igh complex in the genetic control of CPS evoked by photoTCSA-SC. B cells as well as macrophages served as an effective presentation template for the photoTCSA-SC sensitization in the high responder Igh-1a mice, whereas B cells failed in inducing the CPS reaction in the low responder Igh-1d mice. These results suggest that B cells play an essential role in the Igh control phenomenon seen in the photoTCSA-SC sensitization. The present study demonstrated that CPS induced by photohapten-modified cells are differentially regulated by the H-2 and Igh gene loci depending on the cell type used for sensitization.     

Inhibition of cytolytic T lymphocyte clones reactive with Moloney leukemia virus-associated antigens by monoclonal antibodies: a direct approach to the study of H-2 restriction

H-2 restriction in cytolytic T lymphocyte (CTL)-mediated lysis of syngeneic murine Moloney leukemia virus (MoLV)-induced tumor cells was studied at the clonal level by testing the inhibitory effect of monoclonal anti-H-2 antibodies on the lytic interaction between CTL clones and target cells. Large numbers of MoLV-specific CTL clones were generated by placing limiting numbers of C57BL/6 regressor (responder) spleen cells into micro-mixed leukocyte-tumor cell cultures. The clonal CTL populations thus obtained were split into 5 aliquots and tested for lytic activity in the presence (or absence) of 1 of 3 monoclonal antibodies or of an anti-whole H-2b haplotype antiserum. Two of the monoclonal antibodies were directed against H-2Db and one against H-2Kb determinants. Specificity of these reagents had been verified by demonstrating inhibition of lysis by CTL populations directed against H-2Db and H-2Kb alloantigens. In 44 of a total of 51 clones tested, results showed selective inhibition by the anti-H-2Db (and the anti-whole haplotype) reagents, and lack of inhibition by the anti-H-2Kb antibody., Of the remaining 7 clones, none was inhibited by the anti-H-2Db antibody, and 3 were inhibited by the anti-whole haplotype antiserum. These studies show that the recognition of MoLV-associated antigens by the majority of CTL clones was restricted to the H-2Db region, and that there exists limited heterogeneity in the H-2 restriction of such clones.     

Stimulated rat T cell-derived inhibitory factor for cellular DNA synthesis (STIF). II. Kinetics of the production and characterization of the producer

Kinetics of the production of a stimulated T cell-derived inhibitory factor for cellular DNA synthesis (STIF) (1) from Con A-stimulated SD rat spleen cells, and the cells involved in the production of the factor, were examined comparatively with those of interleukin 2 (IL 2) and interferon (IFN). STIF activity in the culture supernatants reached a plateau 24 hr after the culture, and the plateau level was maintained during an additional culture for 72 hr. Characterization of the cells involved in STIF production by means of negative selection of unfractionated or nylon-fractionated spleen cells with anti-rat T cell serum or monoclonal antibodies plus complement revealed that the cells are nylon-nonadherent T cells bearing a suppressor T cell marker. The nylon-nonadherent T cell population did not require additional macrophages for STIF production. In vivo pretreatment of rats with cyclophosphamide (25 to 100 mg/kg) reduced or abolished the production of STIF from the Con A-stimulated spleen cells of the rats. The STIF production from Con A-stimulated spleen cells was inhibited by the addition of indomethacin at 10 ng/ml, indicating a regulatory role of prostaglandin(s) in STIF production. The above characteristics distinguish a T cell subset for STIF production from those for IL 2 and IFN production.     

4-Hydroxy-3-nitrophenyl (NP) acetyl-hapten specific lymphocyte proliferation. I. Mice bearing Igh-1b allotype can cross-react with 4-hydroxy-5-iodo-3-nitrophenyl (NIP) acetyl hapten

Hapten specific T cell proliferation was induced in several strains of mice. When lymph node T cells from 4-hydroxy-3-nitrophenyl acetyl-keyhole lympet hemocyanin (NP-KLH)-primed mice were stimulated in vitro with NP-polymer glutamic acid-lysine-phenyl alanine (NP-GL phi) or NP-ovalbumin (NP-OVA), they displayed a good level of proliferative responses. It was observed that NP-GL phi could induce NP-hapten specific proliferation even with NP-KLH lymphocytes from GL phi nonresponder strains. NP-KLH primed lymphocytes from C57BL/6 (H-2b, Igh-1b), CKB (H-2k, Igh-1b), CWB (H-2b, Igh-1b), and B10.BR (H-2k, Igh-1b) mice showed good proliferative responses to both 4-hydroxy-5-iodo-3-nitrophenyl (NIP) acetyl-GL phi and NIP-OVA antigens. However, NP-KLH primed lymphocytes from C3H/He (H-2k, Igh-1j) and C3H. SW (H-2b, Igh-1j) mice displayed poor proliferative responses to NIP-GL phi and NIP-OVA antigen. These results suggested that the gene coding for the NIP-cross-reaction might be mapped in the Ig heavy-chain linked locus.     

Detection and characterization of cytotoxic T lymphocyte precursors in the murine intestinal intraepithelial leukocyte population

Highly purified preparations of intraepithelial leukocytes (IEL) were obtained from the small intestinal mucosa. Leukocytes from the lamina propria (LPL) were isolated and phenotypically compared with IEL to verify that IEL were minimally contaminated by LPL. Because approximately 80% of IEL expressed the Lyt-2 antigen usually associated with cytotoxic/suppressor T lymphocytes, we wished to determine if precursors for cytotoxic T cells were present in this population. In order to generate cytotoxic cells, IEL and spleen cells from CBA/J mice (H-2k) were co-cultured with irradiated allogeneic spleen cells (H-2d or H-2b) in a one-way mixed leukocyte reaction (MLR). Four to six days later, the cultured cells were assayed against 51Cr-labeled H-2d or H-2b tumor or Con A-stimulated lymphoblast target cells, and the specificity of alloantigen-stimulated IEL and spleen cells was compared. The cytotoxic cells derived from both tissues displayed antigen-specific lysis of the allogeneic targets. Treatment of effector cells, generated from intraepithelial or splenic precursors, with monoclonal antibodies against Thy-1.2, Lyt-1.1, or Lyt-2.1 antigens plus complement, decreased cytotoxicity 85 to 100%, even though only 20 to 50% of the cells were lysed. The alloantigen specificity and surface antigen phenotype of the cultured IEL cells were identical to those of spleen cells and allowed us to conclude that IEL contained a cytotoxic T lymphocyte precursor (CTLp). Further characterization showed that, like spleen, the intraepithelial CTLp was Thy-1+ and Lyt-1+ and their sedimentation velocity was the same but differed from intraepithelial natural killer cells. Although 80% of IEL were Lyt-2+, the frequency of CTLp in the IEL population was estimated to be threefold to fivefold lower than in spleen, and the Lyt-2+ cells were shown not to be an enriched source of CTLp. Thus, the function of the majority of the IEL is still not known. However, there exists within this population CTLp, which may be capable of being stimulated with luminal antigens.     

Allotypic specificities of murine IgD and IgM recognized by monoclonal antibodies

Hybridomas generated from mice immunized with allotype and H-2-incompatible spleen cells were screened by flow cytometry. Monoclonal antibodies (MAb) to four of the five known specificities of IgD were identified. The location of these specificities on the IgD molecule was determined by the ability of a given MAb to bind to cells stripped of the Fab fragment by trypsin proteolysis. Igh-5.1 (present on IgDa and IgDe) and Igh-5.5 (unique to IgDe) were both located on the Fab fragment. Results from cross-blocking experiments suggest that, except for Igh-5.3, MAb which recognize the same specificity bind to the same antigenic determinant. Both the trypsin digest and blocking studies indicate that Igh-5.3 is composed of at least two antigenic determinants. AF6-78.25, a MAb specific for IgM of the b, d, and n haplotypes, was also identified; this MAb defines a new specificity, Igh-6.6. On the basis of the reactivities of these MAb, it appears that although the Igh-Ce and Igh-Cd haplotypes are virtually identical at the Igh-3(gamma 2b), Igh-1(gamma 2a), and Igh-2(alpha) loci, they are highly divergent at the Igh-5(delta) and Igh-6(mu) loci. This indicates that the Igh-Cd haplotype may have resulted from a recombination between Igh-Ce and another haplotype.     

Antigen presentation to T cell hybridomas by a macrophage cell line: an inducible function

We have investigated the ability of an Ia-, nonantigen-presenting macrophage tumor cell line, P388D, (H-2d), to present antigen to T cell hybridomas after incubation in a lymphokine-containing preparation. P388D, cells were incubated in microtiter wells with various concentrations of Con A-stimulated spleen cell supernatants. Antigen-specific stimulation of H-2d-restricted, KLH-specific T cell hybridomas was observed by P388D1 incubated with SUP.P388D1 cells incubated for 3 days in medium or control SUP did not present antigen. In addition, no stimulation of T hybridomas was seen by P388D1 in the inhibited by the appropriate monoclonal anti-Ia reagents. These results demonstrate that a macrophage tumor cell line can be induced to present antigen and provides for large numbers of readily available, homogeneous macrophages for studying the cellular biochemical requirements for antigen processing and presentation.     

Molecular localization of allogeneic and self determinants recognized by bulk and clonal populations of cytotoxic T cells

The localization of MHC-encoded determinants recognized by hapten- and allo-specific cytotoxic T cells was analyzed with the use of cell lines expressing recombinant H-2Dd and H-2Ld MHC products. Bulk cultures of CTL against TNP-self, FITC-self, and AED-self recognized self determinants associated with the N/C1 domains of both Dd and Ld products. A number of allo- and hapten-specific CTL clones were also tested for recognition of the recombinant MHC products. The allo clones specific for Ld or Dd antigens recognized the respective N/C1-associated determinants. In addition, all clones generated against H-2q and known to cross-react with H-2Dd antigens recognized determinants associated with the N/C1-associated Dd determinants. Thus, some of the results obtained with CTL parallel, whereas others contrast with, those findings obtained with monoclonal anti-H-2 antibodies. Similar to the observations made with the monoclonal antibodies, no determinant as defined by T cells has been found to be lost as a result of the interaction between the N/C1 and C2/M domains of the Ld and Dd proteins. Nor did our studies detect the presence of new antigens resulting from the interaction of these gene products. However, the present T cell findings continue to contrast previous results demonstrating that antibody interaction with class I products includes recognition of C2/M-associated epitopes.     

Specific neonatally induced tolerance to Mls locus determinants

Neonatal injection of CBA/HT6T6 (H-2k, Mlsb) mice with adult, Mls-incompatible (CBA/J [H-2k, Mlsd] X CBA/HT6T6)F1 spleen cells results in the abrogation of cell proliferation and interleukin 2 (IL 2) production in bulk mixed lymphocyte cultures, when spleen cells from the inoculated mice are tested at 6 to 8 wk of age with stimulator cells expressing the Mlsd of the tolerizing inoculum. In limiting dilution assays, this tolerant state was manifested in a 25- to 550-fold (280-fold average) decrease in the frequency of precursors of Mlsd-responsive IL 2-producing T cells. Tolerance was specific in that the frequencies of precursors of IL 2-producing cells responding to Con A, allogeneic H-2d, and self-Ia were not affected. The observed low frequency of Mls-responsive cells was due neither to extensive chimerism resulting in the dilution of Mlsd-responsive cells by the nonresponsive F1 cells of the inoculum, nor to the action of suppressor cells. These findings indicate that neonatal injection of Mls-incompatible spleen cells produces a state of specific tolerance by a clonal deletion or inactivation mechanism. This specific tolerance supports the view that 1) the Mls locus encodes or regulates the expression of defined alloantigenic determinants and 2) Mls-incompatible responder mice have specific receptors for Mls determinants on clonally distributed IL 2-producing responder T cells.     

Specificity repertoire of splenic Lyt-2+/F23+ cytotoxic lymphocyte precursors from B6 mice

As revealed by flow cytometric analysis, about 30% of nylon wool nonadherent Lyt-2+ B6 spleen cells were F23+, i.e., were stained with the monoclonal antibody F23.1 directed against an allotypic T-cell receptor determinant. The specificity repertoire of splenic Lyt-2+/F23+ cytotoxic lymphocyte precursors (CLP) from B6 mice was investigated in a limiting dilution (LD) system designed to support clonal expansion in vitro of a representative fraction of this T-cell subset: in highly purified Lyt-2+ responder cells cocultured with mitomycin-treated F23 hybridoma cells in the presence of (recombinant) interleukin 2 under LD conditions, one out of three Lyt-2+/F23+ CLP gave rise to a functional cytotoxic T lymphocyte (CTL) clone. The split-well analysis of individual CTL populations demonstrated a clear-cut segregation of the lytic reactivities toward different allogeneic Con A blast targets. A large fraction of B6-derived CTL clones (3-10%) specifically lysed fully H-2 allogeneic (H-2k, H-2d), or H-2K mutant (bm1) targets. Self-reactive and allorestricted lytic patterns were not found.     

The cellular basis of adjuvant arthritis: II. Characterization of the cells mediating passive transfer

It has previously been reported that lymph node or spleen cells from rats with adjuvantinduced arthritis can transfer the disease to normal recipients after being cultured with concanavalin A (Con A). In this report, it is shown that a subpopulation of cells that (1) lack surface Ig and the antigen reactive with the monoclonal antibody OX8, (2) are largely nonadherent and esterase negative, and (3) are predominantly marked by the monoclonal antibody W3/25 can transfer arthritis after stimulation with Con A. Adjuvant-sensitized lymph node or spleen cells stimulated with Con A but not PHA transfer arthritis, and this difference correlates with relatively higher levels of interleukin 2 secretion by Con A-stimulated cells. A synthetic adjuvant, CP-20961, a substituted propanediamine, induces arthritis that is passively transferable under the same conditions as arthritis induced by classical mycobacterium-containing adjuvant. The data support the hypothesis that adjuvant inoculation in the rat results in the induction of a unique subpopulation of T cells that initiate the inflammatory joint disease.     

Origin of autoreactive T helper cells. I. Characterization of Thy-1+, Lyt-, L3T4- precursors in the spleen of normal mice

A new population of dull Thy-1+, Ly-1-, Lyt-2-, L3T4- PNA- cells, resistant to a double cytotoxic treatment by monoclonal antibodies to these T cell markers plus complement, has been isolated from the spleen of normal adult BALB/c and DBA/2 mice (Tkr cells). These cells exhibit no spontaneous autoreactivity or alloreactivity but can be activated with concanavalin A (Con A). Once activated, they differentiate into bright Thy-1+, Ly-1+, Lyt-2-, L3T4+ PNA- T lymphocytes. Con A-activated Tkr cells also strongly proliferate in the presence of allogeneic or syngeneic dendritic cells in secondary cultures. Moreover, contrary to other Con A-stimulated T cell populations, they induce B lymphocytes to proliferate and to differentiate into Ig-secreting cells at a very high level. Con A-activated Tkr cells are therefore very potent polyclonal B cell activators. Restimulated of Tkr cells by syngeneic dendritic cells can be inhibited by anti-L3T4 or anti-class II monoclonal antibodies. The results suggest that Tkr cells are the precursors of class II-specific autoreactive T helper cells. Tkr cells are absent in the spleen of B6 animals. This indicates that their expression might be genetically controlled. It also suggests that Tkr cells may not be the unique splenic precursors of autoreactive T cells. Con A activation of Tkr cells in Click's medium is 2-mercaptoethanol dependent and highly sensitive to pCO2, like the response of thymocytes. Tkr cells are also absent in the spleen of nude mice. We conclude that Tkr cells represent splenic precursors of autoreactive T helper cells equivalent to Thy-1+, Ly-2-, L3T4- PNA- cortical thymocytes.