Antigen-presenting Accessory Cells Research Articles

SJL and NOD macrophages are uniquely characterized by genetically programmed, elevated expression of the IL-12(p40) gene, suggesting a conserved pathway for the induction of organ-specific autoimmunity

Genetic susceptibility of the SJL mouse to experimental autoimmune encephalomyelitis (EAE) appears, in part, to be a result of genes that promote abnormal development of the pathogenic Type 1 (Th1) phenotype of neuroantigen-specific T-cells. Because antigen-presenting/accessory cells (APCs) produce cytokines that can modulate the development of Th1 and Th2 phenotypes, we addressed whether APCs from SJL mice were genetically programmed for elevated expression of the Th1-promoting cytokine, IL-12. Activated peritoneal macrophages (Mphi; i.e., APC) from naïve SJL mice produced levels of TNF-alpha, IL-1, IL-6, IL-10, and TGF-beta within the range of six normal strains. In contrast, SJL IL-12p40 (in addition to IL-12p70) production was consistently five- to 20-fold greater than that of any normal strain tested, which arose from elevated expression of the IL-12p40 but not the IL-12p35 gene, because p40 mRNA levels were eight- to 15-fold greater than those of normal strains. This aberrancy in IL-12p40 expression appears identical to that observed in the NOD mouse, another strain prone to organ-specific autoimmunity. A genetically programmed bias toward elevated expression of IL-12 in Mphi from the SJL and NOD strains of autoimmunity provides a conserved mechanism for the dominant Th1 development of naive, autoantigen-specific T-cells in these strains. This study is the first demonstration of a genetically programmed aberrant phenotype that is intrinsically expressed within a cell type in the SJL mouse and provides insight into its predisposition for EAE.

The role of accessory cell products in the regulation of T cell cytokine production.

The balance between specific cellular and humoral immune responses is determined largely by cytokines secreted by CD4+ T lymphocytes after activation upon recognition of antigen presented by antigen-presenting accessory cells (AC). Subsets of CD4+ T cells secreting high levels of the Thl cytokine IFN-γ and low levels of Th2 cytokines favor cellular immunity characterized by delayed type hypersensitivity, which is particularly efficient for the clearing of certain intracellular parasites. Secretion of low levels of IFN-γ and high levels of the Th2 cytokines IL-4 and IL-5 favors humoral immunity, characterized by immediate type hypersensitivity mediated by antigen-specific IgE (via IL-4) and late phase hypersensitivity, mediated by eosinophilia (via IL-5) and these responses are considered to be efficient for the clearing of certain helmith species. CD4+ T cells secreting balanced levels of Thl and Th2 cytokines, although less efficient in clearing intracellular parasites and the helminth types, induce mixed cellular and humoral (without IgE) responses that are often sufficient for the many other immune activities.

The anatomy of the rheumatoid lesion

Rheumatoid arthritis primarily involves the synovial membrane of the joints, together with that of the tendon sheaths and bursae. These structures are targeted in a selective and symmetrical manner. The inflammatory cell infiltrate is usually dominated by mononuclear phagocytes. These are recruited into the synovial lining as type A synoviocytes. A proportion of the mononuclear cell population has proven to be specialized antigen-presenting accessory cells. The T and B lymphocyte infiltrate is quite variable in intensity, but may become highly organized. Polymorphonuclear leukocytes pass rapidly into the joint space. The cellular characteristics of rheumatoid nodules have some similarities with those of the synovial membrane. In a minority of patients, immunoglobulin production and circulating immune complex formation reaches levels which precipitate the appearance of a complement mediated vasculitis. This may lead to tissue damage in remote organs.

Encephalitogenic T lymphocytes develop from SJL/J hematopoietic cells transplanted into severe combined immimodeficient ( SCID) mice

Previously, we constructed chimeras by injecting hematopoietic cells from experimental autoimmune encephalomyelitis (EAE)-susceptible SJL (H-2 S) strain mice into severe combined immunodeficient (SCID) C.B-17 scid /scid (H-2 d) mice. These SCID mouse-SJL mouse hematopoietic cell chimeras developed passive EAE following adoptive transfer of PLP S139–151-specific SJL T lymphocyte line cells, but were resistant to active EAE induced by primary immunization with PLP S139–151. In order to gain an understanding of the encephalitogenic potential of transplanted hematopoietic progenitors in SCID mouse-SJL mouse chimeras, we attempted to induce EAE in hematopoietic chimeras constructed with or without an additional SJL fetal thymus implant. Chimeras with the thymus implant were susceptible to passive and active EAE while chimeras without the thymus implant were susceptible to passive but not active EAE. Encephalitogenic, CD4 +, TCR + T lymphocytes were selected in vitro from PLP S139-151-immunized, thymus-implanted chimeras. These results showed that hematopoietic SJL progenitors developed into antigen-presenting accessory cells and immunocompetent encephalitogenic T lymphocytes following transplantation into SCID mice. The development of primary immune reactivity depended on a fetal thymus implant for expression in SCID mouse-SJL mouse chimeras.

Adult Worm Homogenate of the Nematode Parasite Heligmosomoides polygyrus Induces Proliferation of Naive T Lymphocytes without MHC Restriction

The documented in vitro response of mouse T cells to parasite antigens is typically anamnestic and H-2 restricted. As yet, there have been no confirmed reports of the existence of a non-H-2-restricted, superantigen type of response to the antigens of metazoan parasites. Reported here are data which show that antigens produced by the adult stage of the nematode parasite Heligmosomoides polygyrus (= Nematospiroides dubius) can stimulate naive T cells in vitro to proliferate and produce IL-2. A series of T cell hybridomas has been used to show that adult worm homogenate of H. polygyrus can stimulate parasite antigen naive T cells. This response is independent of the H-2 haplotype of the antigen-presenting accessory cells and does not appear to be influenced by the presence or absence of an H-2 E molecule. However, successful presentation of the H. polygyrus superantigen does require the presence of metabolically active accessory cells and fixation of the accessory cells with paraformaldehyde abrogates the response of the target cells. This discovery has important implications for the study of the role of superantigens in host/parasite interactions and will also help to expand current knowledge about the relationship between chronic intestinal nematodes and the host immune system.

Histiocytic Sarcomas and Monoblastic Leukemias:A Clinical, Histologic, and Immunophenotypical Study

Eight histiocytic sarcomas, identified by examination of more than 2000 malignant lymphomas, are described. For comparison, tumor infiltrates from five monoblastic leukemias were also analyzed. The histiocytic sarcomas were all high-grade malignancies consisting of markedly pleomorphic large cells with many mitotic figures. At presentation, six of the patients had systemic symptoms (fever, fatigue, loss of weight), skin infiltrates, and lymphadenopathy. Despite aggressive chemotherapy, clinical remissions were short, and six patients died of disease .5-48 months (mean, 6.5 months) after diagnosis. The remaining two patients are alive and in partial or complete remission 7 and 12 months after diagnosis. Immunotypic examination showed that all the histiocytic sarcomas were positive for macrophage-related antigens and negative for antigens on B cells, T cells, myeloid cells, epithelial cells, and melanocytes. T-cell receptor and immunoglobulin genes were studied in three cases and were present in a germline configuration. One of the histiocytic sarcomas resembled Langerhans' cells in phenotype and morphology; it was classified as a Langerhans' cell sarcoma. The remaining histiocytic sarcomas did not express accessory cell-associated antigens, but more closely resembled "ordinary" tissue macrophages; they were positive for lysozyme and/or CD68, followed in frequency by CD11c, CD4, CD11b, CDw32, peanut agglutinin receptor, and CD13. Similar features were seen in the monoblastic leukemias. These conditions could only be distinguished from histiocytic sarcoma by clinical and morphologic, rather than immunophenotypic, criteria. Expression of oncoprotein p53 was studied in nine cases and was positive in six of six histiocytic sarcomas and one of three monoblastic leukemias. Rare malignancies show features consistent with the derivation from macrophages. Two entities may be distinguished: those that resemble antigen-presenting accessory cells and those that more closely resemble ordinary tissue macrophages. Recognition of these tumors is important clinically and requires assessment of clinical, morphologic, and immunophenotypic features, supplemented by analysis of T-cell receptor and immunoglobulin genes. Whether (or how) p53 gene mutations are implicated in their pathogenesis will be an important topic for future investigation.

Enhanced proliferation of murine T cell lines following interaction of Poly(Glu 60, Phe 40) (GPhe) and antigen-presenting accessory cells : III. Possible mechanisms responsible for activity of GPhe

The random copolymers (Glu 80, Phe 20) n (GPhe 20), (Glu 60,Phe 40) n (GPhe), and (Glu 50, Phe 50) n (GPhe 50) were compared for the capacity to augment proliferation of antigen-reactive murine T cell lines. GPhe 20, GPhe, and GPhe 50 showed “augmenting” activity in order of increasing potency. Phenylalanyl residues constituted a significant portion of the “active” determinant(s) in the GPhe polymers tested. High titer murine anti-GPhe (ascites fluid) inhibited augmentation by GPhe of exogenous (IL-1 + rat-conditioned media (RCM)) driven T cell proliferation, indicating that (a) the antibodies by binding to specific active determinant(s) in GPhe may have prevented critical GPhe-APC membrane interaction, and/or (b) “GPhe-anti-GPhe” complexes interfered with necessary “processing” of GPhe by APCs. Time course studies demonstrated that the appearance of increased T cell proliferation after GPhe addition occurred after proliferation to (a) nominal antigen or (b) exogenous (IL-1 + RCM) had reached peak [ 3H]thymidine incorporation ([ 3HT]). This suggested that more than GPhe-APC membrane interaction was necessary for GPhe activity. Leupeptin, a lysosomal protease inhibitor, inhibited the augmentation of T cell proliferation by GPhe, which led to the conclusion that GPhe must be “processed” by APCs to exhibit activity.

Enhanced proliferation of murine T cell lines following interaction of poly (Glu 60,Phe 40) (GPhe) and antigen-presenting accessory cells : I. GPhe-stimulated enhancement of antigen-dependent proliferative responses

Following interaction of the random polymer (Glu 60,Phe 40) n (GPhe) with antigen-presenting accessory cells (APC), unusual costimulatory activities were noted in several murine T cell systems. When GPhe, in contrast with other random copolymers (GT,GL), was added during “inhibition” and T cell “repertoire” studies as a (negative) control to GLA-reactive nonclonal T cell lines of haplotypes H-2 d (DCL-2) or H-2 bm12, augmentation of T cell proliferation ([ 3H]thymidine incorporation ([ 3HT])) to homologous antigen was observed. Augmentation by GPhe was also observed in the response of a GLPhe-reactive (H-2 s X H-2 d)F 1 T cell line and the allogeneic response of the clonal T cell line D10.G4.1. This augmentation was critically dependent on the concentration of adherent accessory cells. Although the mechanism of action of GPhe remains, as yet, undefined, the GPhe-mediated enhancement of DCL-2 (a TH2, H-2 d anti-GLA, T cell line) proliferation was not dependent upon the production of either IL-1 or IL-6 by accessory cells. In addition, enhanced DCL-2 proliferation was not accompanied by a significant increase in detectable IL-4 release.

Enhanced proliferation of murine T cell lines following interaction of poly (Glu 60,Phe 40) (GPhe) and antigen-presenting accessory cells : II. The role of accessory cells and cytokines in the activity of GPhe on antigen-independent proliferation

Our previous study (1) demonstrated the “cytokine-like” activity of poly (Glu 60,Phe 40) (GPhe) in augmenting the antigen-dependent proliferation of a variety of long-term murine T cell lines, particularly the bulk, BALB/c anti-poly (Glu 36,Lys 24,Ala 40) (GLA), interleukin-4-producing, DCL-2 T cell line. GPhe was found to also augment the antigen-independent proliferation of DCL-2 in response to exogenous cytokines ([interleukin(IL)-2 ± IL-1] in most experiments). Such exogenous cytokine-driven proliferative responses of DCL-2 were used to investigate further the role of accessory cells and of various soluble factors in the action of GPhe. GPhe did not act as a direct mitogen for T cells, rather it acted in a costimulatory fashion, requiring the presence of plastic-adherent accessory cells and a T cell growth factor (either IL-2 or IL-4). In the presence of accessory cells and exogenous IL-2, augmentation of antigen-independent DCL-2 proliferation by GPhe or by IL-1 depended upon the induction of autocrine IL-4 production. However, GPhe also augmented the response of these cells in the presence of exogenous IL-4 (±IL-2, ±IL-1), and exogenous IL-4 added in combination with exogenous IL-2 (±IL-1) failed to mimic the GPhe effect, suggesting that another signal was involved in the mechanism of action of GPhe. The ability of allogeneic accessory cells to interact with GPhe to augment proliferative responses suggested that either a soluble factor or an unusual non-MHC-restricted cell-cell interaction provided this signal. In the presence of uv-irradiated accessory cells, DCL-2 proliferation was enhanced over that observed in the presence of non-uv-treated accessory cells, mimicking the GPhe effect, and interaction of GPhe with uv-irradiated accessory cells did not result in further enhancement of DCL-2 cytokine-driven proliferation. Using monoclonal antibodies which could block the function of IA or CD4 molecules, these cell-surface “adhesion” molecules were shown not to participate in the activity of GPhe. By the addition of recombinant cytokines, neutralizing antibodies, or indomethacin, the mechanism of action of GPhe was also shown not to be dependent upon IL-1, IL-6, IL-7, TNF α, or prostaglandin production by accessory cells. However, the presence, individually, of some of these factors (IL-1, IL-7, or prostaglandins) could influence to a variable degree the magnitude of the GPhe effect.

The B-cell antigen CD22 mediates monocyte and erythrocyte adhesion

Interaction with antigen-presenting accessory cells is thought to be an important step in B-cell activation, and the B-cell receptor CD22, which is coordinately expressed with surface immunoglobulin, has been proposed to participate in the antigen response. Here we show that CD22 has a structure closely related to myelin-associated glycoprotein (MAG, a neuronal adhesion protein), and mediates monocyte and erythrocyte adhesion. Like CD2, the T-cell erythrocyte receptor, CD22 may facilitate antigen recognition by promoting antigen-nonspecific contacts with accessory cells.

Requirement for Both the Cd3/T Cell Receptor Complex and the Cd2/Lymphocyte Function-Associated Antigen-3 Adhesion System in Monocyte-Independent T Cell Activation by Oxidized Erythrocytes

Recent reports have shown that human T lymphocytes can be activated in vitro by desialyzed oxidized erythrocytes (DOE) in a monocyte-independent fashion. These findings suggest that, in addition to providing the activating stimulus, DOE may also fulfill accessory cell roles normally supplied by monocytes in T cell activation. DOE may thus be a useful substitute for antigen-presenting accessory cells in studies of T cell activation. This report describes experiments conducted to determine if T cell activation by DOE involves cell surface structures known to play a role in antigen-induced T cell activation. T cell stimulation by DOE was blocked by anti-CD3 monoclonal antibody, suggesting that perturbation of the CD3/T cell antigen receptor complex on T cells is required for activation. Activation was also blocked by anti-CD2 monoclonal antibody, indicating involvement of the CD2/LFA-3 adhesion system. Since DOE and monocytes both express LFA-3, the role of LFA-3 on DOE in T cell activation was assessed. DOE pretreated with anti-LFA-3 monoclonal antibody induced markedly lower levels of T cell proliferation. As further evidence for LFA-3 involvement, DOE prepared from 5 patients with paroxysmal nocturnal hemoglobinuria (PNH), known to exhibit deficient LFA-3 expression, were also deficient in T-cell stimulatory activity. These findings illustrate that T cell activation by DOE requires many of the surface structures involved in activation by antigen-bearing accessory cells, and may thus serve as a useful model for studies of T cell activation.

Identification of transcripts of the T cell antigen receptor beta chain gene and major histocompatibility complex class II genes in antigen-presenting cloned BK-BI-2.6.C6 cells.

The cloned murine cell line BK-BI-2.6.C6 has previously been shown to exhibit T cell characteristics, to synthesize and express MHC class II molecules, and to present protein antigens to antigen-dependent T cell clones. As a more definitive proof of the T-cell nature of these cells, transcripts of the rearranged T cell antigen receptor (TcR) beta gene were assessed by Northern blot analysis. BK-BI-2.6.C6 cells constitutively transcribe mRNA for the light chain of TcR and express the disulphide-linked alpha, beta TcR heterodimer at the cell surface. In addition mRNA for the polymorphic MHC class II subunits A alpha and A beta as well as for the invariant gamma chain were detected. BK-BI-2.6.C6 T cells effectively stimulated bovine insulin-reactive T hybridoma cells to lymphokine production in the presence of this antigen. Since the antigen-presenting and the responding T cell populations are maintained in culture in the absence of feeder cells, contamination by conventional accessory cells is excluded. These data unequivocally demonstrate that cloned murine Ia-expressing T cells can act as antigen-presenting accessory cells.

The role of adherent accessory cells in the generation of effector suppressor T cells.

The role of accessory cell populations in the generation of effector suppressor (Ts3) cells was studied. By using an in vitro culture system, it was previously determined that the induction of NP-specific effector suppressor activity requires T cells, antigen, and an anti-idiotypic B cell population. We now demonstrate that the generation of Ts3 cells in this system also requires accessory cells. The accessory population appears to play a role in the processing and presentation of antigen. These antigen-presenting accessory cells are required early in the induction phase of Ts3 generation. These accessory cells can present NP coupled to immunogenic or non-immunogenic polypeptide carriers, including polymers of L-amino acids. However, NP coupled to polymers of poorly metabolized D-amino acids fail to induce suppressor T cell generation. Furthermore, the data demonstrate that an H-2 homology must exist between the Ts3 precursors and the antigen-presenting cell population if suppressor activity is to be generated. We also characterize the differential genetic restrictions that govern the induction of Ts3 cells that control suppression of either T cell or B cell responses. The data suggest that although I-J region encoded gene products control the induction and effector phases of suppressor cell activity as measured on T cell responses, the suppression of B cell responses appear to be controlled by I-A gene products. Possible cellular mechanisms that might explain these findings are discussed.

Regulation of B-cell differentiation: interactions of factors and corresponding receptors.

The antibody response to nominal antigen such as hapten-carrier con­ jugates involves the cooperation of hapten-specific B cells and carrier­ specific helper T cells (1-4). In addition to the carrier specificity, helper T cells recognize determinants encoded by genes in the class-II major histocompatibility complex (MHC) on antigen-presenting cells as well as on B cells (5-13). The interaction between helper T cells and antigen­ presenting accessory cells (APC) is genetically restricted by products of the major histocompatibility complex (14-16). However, the cellular interac­ tions between helper T cells and B cells have been reported as restricted by self-MHC products (5-7, 10-12, 17) or not restricted by the MHC elements (18, 19). Since genetic restrictions imposed by products of the MHC are considered as a requirement for the recognition by T cells of self-MHC determinants expressed by non-T cells (APC), this controversy can be explained on the basis of whether T cells must necessarily recognize the MHC determinants expressed by B cells in order to trigger them to secrete antibodies. Since the original description of a role for T cells in the antigen­ dependent stimulation of B cells, a great deal of effort has gone toward elucidating the mechanism by which helper T cells function. A series of studies demonstrated that some members of the B-cell population participate in cognate cellular interactions with helper T cells. Cognate interaction refers to the recognition of both antigen and a class-II MHC molecule on the B-cell surface as a requirement for activation. Other

Antigen Presentation by B Cells and Its Significance in T-B Interactions

Publisher Summary This chapter reviews the evidence in favor of the concept that B cells can serve as antigen-presenting accessory cells to T cells of the helper/inducer subset as well as the experiments suggesting that this is a critical step in the differentiation of B cells for a T-dependent immune response. The unique characteristic of B cell antigen-presenting capacity lies in the ability of specific B cells to efficiently take up antigen via their immunoglobulin receptors and to present those antigen to antigen-specific helper T cells. Although this proposed mechanism of antigen presentation to T cells by B cells explains many features of T–B interactions, such as the major histocompatibility complex (MHC) restriction and the requirement for hapten and carrier to be on the same macromolecule to facilitate efficient T–B interaction, the relevancy of this mechanism for an in vivo immune response remains to be demonstrated.

Evaluation of accessory cell heterogeneity. II. Failure of dendritic cells to activate antigen‐specific T helper cells to soluble antigens

The activation of antigen-specific T cells requires Ia+, antigen-presenting accessory cells (AC). Dendritic cells (DC) and macrophages (M phi) isolated from spleen an peritoneal exudate were tested as AC for the activation of the activation of T helper cells and the induction of T cell proliferation. The cell separations to obtain DC and splenic M phi were performed by discontinuous bovine serum albumin gradients, adherence on petri dishes and rosetting with opsonized sheep erythrocytes. DC as well as the M phi were able to induce antigen-specific T cell proliferation, but only the M phi and not the DC activated antigen-specific T helper cells which help B cells for antibody production to soluble antigens. Keyhole limpet hemocyanin-specific T cells repeatedly stimulated with DC and antigen also did not express helper activity. The failure of DC to induce T helper cells was not due to the activation of a suppressor pathway. Thus, dendritic cells, although very efficient as AC in the induction of various T cell functions, are not able to activate T helper cells required for carrier-specific T-B cooperation and therefore cannot be the sole accessory cells. Based on these results and on previous data using Ia+ tumor cell lines as AC, we confirm the existence of functional AC heterogeneity.

Accessory cell-dependent selection of specific T-cell functions

Activation of many T-cell functions depends on their interaction with antigen-presenting accessory cells which express I region associated (Ia) products. Cells expressing accessory cell function include those of the monocyte/macrophage lineage and dendritic cells. More recently, B cells and a number of tumour cell lines of macrophage or B-cell origin were shown to act as accessory cells in certain assays. We showed previously that normal peritoneal exudate macrophages (PEC) induced both T-cell proliferation as well as T-cell help, whereas various Ia+ tumour lines of macrophage or B-cell origin, although stimulating antigen-specific T-cell proliferation, did not significantly activate T-cell help. We report here that during the initial T-cell activation in vitro accessory cells (PEC or Ia+ tumour cells) select particular T cells to express previously determined functions. Moreover, some tumour cell lines induce suppressor T cells which inhibit helper activity.

Immune response gene control of T dependent reactivity to collagen in man.

T dependent reactivity controlled by immune response (Ir) genes in rodents exhibits the following three characteristics. (1–3) First, it is dependent on interactions between T cells and antigen presenting accessory cells (conveniently referred to here as MO). Second, the reactivity is directed against determinants inherent in either synthetic polypeptides or limited regions of globular proteins. Third, the reactivity is controlled by genes present in the immune response (Ir) locus of the major histocompatibility complex.

Ia antigens on antigen-presenting cells do not carry the same Ia specificities as detected on suppressor and helper T cells.

The passage of spleen cells through a tightly packed nylon wool column partially separated two populations of cells required for the antigen-induced secondary proliferative response of T cells. The proliferating T cells were in the nylon wool column-passed cell population, but the addition of a small number of nylon-wool-adherent cells was required for their maximal proliferative response. Such adherent cells were non-T, non-B, Ia antigen-positive cells serving as antigen-presenting cells. Ia determinants on this cell type are encoded in two separate I subregions, I-A and I-E/C, and they are expressed simultaneously on the same cells. Antisera specific for I-J subregion gene products having as a known activity the killing of antigen-specific suppressor and helper T cells were unable to kill antigen-presenting adherent cells. The results indicated that at least I-J subregion gene products expressed on suppressor and helper T cells are not present on antigen-presenting accessory cells. The Ia specificities detected on accessory cells follow the cross-reactive pattern found in Ia molecules of B cells, and thus antigen-presenting cells carry Ia antigen identical to those of B cells, and not those of T cells.

Role of accessory cells in B cell activation. III. Cellular analysis of primary immune response deficits in CBA/N mice: presence of an accessory cell-B cell interaction defect.

The effect of the X-linked CBA/N genetic defect on the ability of mice to generate primary responses to thymic-dependent and thymic-independent antigens was assessed by comparing the ability of abnormal (CBA/N x DBA/2)F1 male mice and normal (DBA/2 x CBA/N)F1 male mice to generate 2,4,6-trinitrophenyl (TNP)-specific plaque-forming cell responses to TNP-keyhole limpet hemocyanin (KLH), TNP-conjugated Ficoll (TNP-Ficoll), TNP-Brucella abortus (BA), and TNP-lipopolysaccharide (LPS). The reciprocal F1 combinations used in this study differ genetically only in the origin of their X chromosome, but differ immunologically in that (CBA/N x DBA/2)F1 male mice express all the CBA/N immune abnormalities, whereas (DBA/2 x CBA/N)F1 male mice are immunologically normal. Analysis of thymic-dependent responses to TNP-KLH revealed that abnormal F1 mice were capable of generating primary responses in vivo to high doses of TNP-KLH, but failed to generate responses to suboptimal doses of TNP-KLH that were still immunogenic for normal F1 mice. Furthermore, under limiting in vitro micro-culture conditions, the abnormal F1 mice failed to generate primary thymic-dependent responses to any dose of TNP-KLH, even though under the identical conditions normal F1 mice consistently responded to a wide antigen dose range. The cellular basis of the failure of abnormal F1 mice to respond in vitro to TNP-KLH was investigated by assaying the ability of purified populations of accessory cells, T cells, and B cells from these mice to function in responses to TNP-KLH. The results of these experiments demonstrated that helper T cells and antigen-presenting accessory cells from abnormal F1 mice were competent and functioned as well as the equivalent cell populations from normal F1 mice. Instead, the failure of CBA/N mice to generate primary in vitro responses to TNP-KLH was solely the result of a defect in their B cell population such that B cells from these mice failed to be triggered by competent helper T cells and/or competent accessory cells. Similarly, the failure of abnormal F1 mice to respond either in vivo or in vitro to TNP-Ficoll was not the result of defective accessory cell presentation of TNP-Ficoll, but was the result of the failure of B cells from these mice to be activated by competent TNP-Ficoll-presenting accessory cells. In contrast to the failure of B cells from abnormal F1 mice to be activated in vitro in response to either TNP-KLH or TNP-Ficoll, B cells from abnormal F1 mice were triggered to respond to TNP-BA and TNP-LPS, antigens that did not require accessory cell presentation. The specific failure of B cells fron abnormal F1 mice to be activated in responses that required antigen-presentation by accessory cells suggested the possibility that the X-linked CBA/N genetic defect resulted in B cell populations that might be deficient in their ability to interact with antigen-presenting accessory cells...