Kinetics of Ii synthesis, processing, and turnover in n-butyrate-treated Burkitt's lymphoma cell lines which express or do not express class II antigens and in hairy leukemic cells

We have sought to understand the role of the electrophoretically invariant chain (Ii) in class II antigen functions, particularly in certain transformed cells in which we have previously demonstrated hyperexpression of Ii. Molecular structures and relative kinetics of Ii synthesis, processing and turnover were compared in paired, Ia+ and Ia- Burkitt's lymphoma (BL) cell lines and in hairy cell leukemia (HCL) cells. Cells were metabolically labeled with [35S] methionine for 15 min (with or without a cold methionine chase to 3 hr) or were continuously labeled for 3 hr. One- and two-dimensional gel electrophoresis resolved immunoprecipitates formed with a) a heteroantiserum to purified class II antigen (demonstrating alpha and beta chains and Ii associated with that complex), b) a heteroantiserum to hairy cell leukemia (HCL) membranes (demonstrating principally the dominant, basic form of Ii molecules, class I antigens, and some additional proteins), and c) a monoclonal antibody to human Ii. Treatment of Ia+ Jijoye and its daughter, Ia- P3HR-1, BL cells with 4 mM butyrate for 48 hr enhanced the synthesis of the dominant, basic form of Ii but did not affect apparent turnover rates of that pool of Ii chains in either cell line. In Ia+ Jijoye cells but not in Ia- P3HR-1 cells Ii was terminally processed to more acidic, sialic acid-derivatized forms. Butyrate treatment did not alter the relative turnover rate of terminally processed Ii in Jijoye cells. The level of the dominant, basic form of Ii in HCL cells equaled that in butyrate-treated Jijoye cells, and relative turnover rates of this terminally unprocessed Ii pool were similar in HCL and Jijoye cells. However, HCL Ia-associated Ii was not terminally processed, as was Ia-associated Ii in Jijoye cells. The expression of Ia auxiliary proteins, p41 and p25, was also enhanced in Jijoye cells by butyrate treatment and was prominent in HCL cells. From these experiments, we may hypothesize the following. In lymphoblastoid cells, two pathways for Ii turnover could exist. One is through association with Ia complexes and progressive terminal processing of carbohydrate side chains and a second is not associated with Ia or, apparently, with such processing. Because Ii is not found to be terminally processed in the absence of class II antigen, Ia might be considered to direct the processing of a subset of Ii towards some function (rather than vice versa).(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of I-A and I-E,C region-coded Ia antigens on functional B cell subpopulations.

Ia antigens from specific subregions have been examined on functional B cell populations. Expression of both I-A and I-E,C region antigens was demonstrated on cells required for both lipopolysaccharide mitogenesis and polyclonal activation. Similar I-A and I-E,C subregion expression was found on cells required for response to the T-independent antigen, polyvinylpyrrolidone. TNP-specific IgM and hen egg lysozyme-specific IgG plaque-forming cells also express I-A and I-E,C region antigens. No evidence was found for an Ia- population responsive in the systems tested. Further, no evidence of preferential expression of I-A or I-E,C region antigens was observed in any system examined. Therefore, it appears that B cells express both I-A and I-E,C region-coded Ia antigens.     

Differential expression of Ia and Ia-associated invariant chain in mouse tissues after in vivo treatment with IFN-gamma

B10.BR mice were injected i.v. with varying doses of recombinant IFN-gamma on three consecutive days. In tissue sections of 13 organs, the distribution of Ia antigens and Ia-associated invariant chain (Ii) was studied by using an immunoperoxidase technique. In the control animal, Ia and Ii were shown to be co-expressed in most tissues. However, on Kupffer cells, a small number of hepatocytes, and a subset of lymphocytes in lymph nodes and in the splenic red pulp only Ii, and no Ia, was detectable. In contrast, strongly Ia+ interdigitating reticulum cells of T-dependent areas of lymph nodes and spleen were only weakly stained for Ii. IFN-gamma treatment resulted in a dramatic increase of MHC antigen expression throughout the body, with striking differences in the inducibility of certain tissues for Ia and Ii: Bronchial epithelium was clearly induced to express the invariant chain, whereas Ia antigens remained entirely absent. Moreover, in kidney tubules and colon epithelium, Ii was induced more broadly than Ia. In contrast to the induction of Ii on endothelial cells of larger vessels in kidney, heart, and lungs, no de novo expression of Ia or Ii in capillary endothelial cells was observed. The number of detectable Ia+/Ii+ interstitial dendritic cells considerably increased upon exposure to IFN-gamma. Neither neurons nor glial cells were induced to MHC antigen expression. Our data demonstrate that IFN-gamma applied i.v. is a potent inducer or enhancer of Ia antigens and invariant chain in a variety of cell types.     

Cell surface antigens expressed by subsets of pre-B cells and B cells

A large number of monoclonal antibodies, produced by immunizing rats with mouse pre-B cell lines, have been analyzed for their ability to define cell surface antigens expressed by B cells at early stages of differentiation. Whereas many antibodies recognized antigens on pre-B cell lines, only two clones detected cell surface antigens that were distinguished by their restricted distribution among a panel of continuous cell lines and cells from various tissues. Monoclonal antibody clone AA4.1 recognized a cell surface antigen found on all pre-B lymphomas and on one of three B lymphomas tested. This antigen was found on cells at highest frequency in the bone marrow. Adult spleen and fetal liver also have detectable numbers of AA4.1+ cells. Cells that did not express this antigen include plasmacytomas, two of three B lymphomas, T lymphomas, a stem cell line, adult liver, brain, thymus, and lymph node cells. Clone GF1.2 detected an antigen on some pre-B cell lines, one of three B lymphomas tested, and a small fraction of cells from adult bone marrow, spleen, lymph node, and fetal liver. Plasmacytomas, some pre-B lymphomas, two B lymphomas, T lymphomas, adult liver, brain, and thymus cells were negative. In adult bone marrow, AA4.1 bound to all cytoplasmic IgM+ pre-B cells, whereas GF1.2 detected one-half of these cells. Both antibodies recognized approximately 50% of surface IgM+ (sIgM+) bone marrow cells. A small population of bone marrow cells lacking any detectable Ig (surface or cytoplasmic) also reacted with these antibodies. Depletion of AA4.1 or GF1.2 antigen-bearing cells from bone marrow reduced the ability of bone marrow B cells to respond to LPS by 50 to 65%. Experiments with a cloned pre-B lymphoma demonstrate that AA4.1+ pre-B cells become sIgM+ GF1.2+ B cells after activation with LPS. These antibodies recognize cell surface determinants with restricted distribution among the B lymphocyte lineage because they detect antigens displayed by normal and transformed immature B lymphocytes.     

Murine class II (Ia) molecules associate with human invariant chain

Polymorphic class II (Ia) major histocompatibility complex (MHC) gene products associate intracytoplasmically with a third nonpolymorphic class II molecule, the invariant chain (Ii), which is encoded by gene(s) unlinked to the MHC. Although the role of the Ii chain in the expression of cell surface Ia molecules is unclear, it has been suggested that the Ii chain helps in the assembly and intracellular transport of class II antigens. In this study, we demonstrate that the murine polymorphic class II antigens of an interspecies mouse-human hybrid, which has segregated the murine invariant chain gene, associates with the human invariant chain gene intracytoplasmically. The murine Ia antigens are expressed on the cell surface and can function as restriction elements in antigen presentation to T cells. The biochemical analysis demonstrates that the regions of the Ii gene that are critical to its interaction with Ia molecules are conserved between species.     

One gene encodes two distinct Ia-associated invariant chains

Murine immune response region-encoded Ia antigens are associated not only with invariant (Ii) chain but also with several other polypeptides, most notably a 41K protein. The present report demonstrates a striking similarity between Ii and 41K. These polypeptides were found to be serologically cross-reactive, and they also display a similar peptide composition on partial enzymatic digestion. Both polypeptides are derived from distinct unglycosylated precursors, as demonstrated by tunicamycin treatment and cellfree translation. Hybrid selection of mRNA, as well as Northern blotting with an Ii cDNA, shows the presence of two mRNA coding for the Ii chain and the 41K protein. Rat fibroblasts transfected with a mouse genomic Ii clone synthesize both the murine Ii chain and the 41K protein. These observations demonstrate that a single Ii gene encodes two distinct but closely related proteins.     

Rejection of skin allografts by CD4+ T cells is antigen-specific and requires expression of target alloantigen on Ia- epidermal cells

The effector mechanism of skin allograft rejection has been characterized as Ag specific, rejecting cells that express the target alloantigen but sparing those that do not. However, the rejection of MHC class II disparate skin grafts, in which very few cells (Langerhans cells) actually express the target Ia Ag could conceivably proceed by either one of two distinct rejection mechanisms. One possibility is that Ia- cells are destroyed by a sequence of events in which CD4+ T cells, activated by Ia+ LC, elaborate soluble factors that are either directly cytolytic or that recruit and activate non-specific effector cells. The alternative possibility is that activated CD4+ T cells elaborate soluble factors which induce Ia expression on Ia- cell populations, and that these Ia+ cells are subsequently destroyed by effector cells specific for the induced Ia alloantigens. We found that rejection of Ia+ LC was not of itself sufficient to cause rejection of skin grafts, indicating that skin allograft rejection is contingent on the destruction not only of LC but of other graft cell populations as well. We then investigated whether CD4+ T cells rejected allogeneic skin grafts in an antigen specific fashion. To do so, we engrafted immunoincompetent H-2b nude mice with trunk skin grafts from B6----A/J allophenic mice because such skin is composed of mutually exclusive cell populations expressing either H-2a or H-2b histocompatibility Ag, but not both. The engrafted mice were subsequently reconstituted with H-2b CD4+ T cells. The CD4+ T cells destroyed keratinocytes of A/J origin but spared keratinocytes of B6 origin, even though neither cell population constitutively expresses target IAk alloantigen. The targeted rejection of A/J keratinocytes but not of B6 keratinocytes indicates that the target Ia alloantigen must have been induced on Ia- A/J keratinocytes, rendering them susceptible to destruction by anti-Iak-specific CD4+ effector cells. These data demonstrate that CD4+ T cell rejection of skin allografts is mediated by Ag-specific CD4+ cytolytic T cells and hence, requires the induction of target Ia alloantigens on epidermal cells within the graft.     

Role of macrophages as modulators but not as autonomous accessory cells in primary antibody response

The role of macrophages (M phi) in the in vitro primary antibody response of murine lymphocytes to sheep erythrocytes was investigated. Peritoneal M phi were activated to express Ia antigens either in vitro or in vivo. Nonactivated Ia- M phi were also examined. We observed that only Ia- M phi but also Ia+ M phi failed to trigger the antibody response, in contrast with splenic dendritic cells (DC) which served as potent and autonomous accessory cells, but that M phi modulated the level of response which was dependent primarily on the DC content of culture. The modulation appeared to incline to suppression rather than enhancement, when M phi were allowed to remain throughout the culture period for 4 days. A highly enhancing capacity of M phi, however, could be revealed by removing M phi 2 days after the initiation of culture, indicating that M phi exerted their suppressive effect more strongly in the late phase than in the early phase of in vitro antibody response. The modulatory activity seemed higher in Ia+ M phi than in Ia- M phi.     

Synthesis and characterization of membrane immunoglobulin, Ia, and H-2 molecules of thy 1+ AKR/J lymphomas.

Three AKR lymphomas displaying B cell and T cell characteristics have been described. Because of the proclivity of normal AKR/J mice to develop T cell lymphomas, and the rarity of lymphomas with dual characteristics, the B cell markers of these tumors were studied more intensively. Fluorescence data with class-specific anti-immunoglobulin reagents demonstrated that the tumor cells stained only with class-specific anti-IgM reagents. Because of the possibility that the surface Ig was passively acquired and of reports that certain anti-mu-chain sera react with "IgT", chemical characterization of the immunoglobulin molecules was performed. Using 3H-leucine internal labeling, we showed that all three tumor lines synthesized the immunoglobulin found on their surface, and that the immunoglobulin had the chemical and immunologic characteristics most typical of monomeric surface IgM, and was composed of mu-chains and light chains. The Ia antigens found on these cells were also examined. These antigens were also synthesized by the cells and were present in the same molecular form and in the same approximate quantity as Ia antigens on normal spleen cells.     

Soluble factors produced during an immune response regulate Ia antigen expression by murine adenocarcinoma and fibrosarcoma cells

LT-85 is an alveologenic adenocarcinoma of C3Hf/HeN mice. Comparisons of the in vitro and in vivo surface properties of these cells revealed that under normal conditions, they expressed I-A and I-E antigens iv vivo only. By using clonally derived cells, it was established that this phenomenon was not due to the selection of an Ia antigen-positive tumor cell subpopulation, but resulted from phenotypic conversion of Ia antigen-negative tumor cells. These tumor cells and 1053 cells (a fibrosarcoma of C3H/HeN MTV- mice) could, however, be induced to express I-A, I-E, and much higher levels of H-2 antigens in vitro by co-culturing them with spleen cells from LT-85 tumor-bearing C3H/HeN MTV- mice. In vitro induction of Ia and H-2 antigens did not result from contaminating splenocytes or from antigen transfer, because splenocytes from BALB/c (H-2d) mice immunized with A/J (H-2k/d) cells were able to induce the expression of Iak antigens by both tumor cell lines. It was found that this phenomenon was neither H-2-restricted nor antigen-specific. The results clearly indicated, however, that an immune response was required to generate phenotypic conversion of the tumor cells, both in vivo and in vitro. It was further found that soluble, rather than cellular, factors produced during an immune response induced the expression of Ia antigens by LT-85 and 1053 tumor cells. In contrast to what has been reported about the induction of Ia antigens on macrophages and normal epithelial and endothelial cells, the induction of Ia antigens on LT-85 and 1053 cells did not appear to require T cells, and did not involve gamma-interferon. These findings demonstrate that some tumor cells are capable of altering their MHC antigen phenotype in response to factors produced during an immune response in vivo or in vitro. Because of the involvement of Ia antigens in several aspects of immune phenomena, the ability of tumor cells to differentially express Ia antigens in response to environmental factors may have profound effects on host-tumor interactions. Furthermore, the differences seen in the phenotypes of tumor cells grown in vitro and in vivo suggest that in vitro methodologies of tumor cell characterization may not present a complete picture of the natural state of the tumor cell surface.     

Lymphocyte subsets differentially induce class II human leukocyte antigens on allogeneic microvascular endothelial cells

Increased expression of major histocompatibility complex class II (Ia) antigens on vascular endothelium is a common observation in allografts undergoing acute rejection. This phenomenon is generally ascribed to the host immune response directed against graft alloantigens, but its cellular and molecular basis are incompletely understood. In the present study we show that constitutively Ia-negative human microvascular endothelial cells (EC) can be induced to express surface class II human leukocyte antigens shortly after exposure to allogeneic lymphocytes in vitro. CD16+ (natural killer) and CD8+ (cytotoxic/suppressor) lymphocytes were efficient in triggering Ia antigen expression by EC, whereas CD4+ (helper/inducer) lymphocytes induced EC Ia expression only if cultured in the presence of autologous monocytes. Binding of lymphocytes to EC was shown to be essential for the subsequent induction of EC Ia, and anti-CD18 (LFA-1) antibody, which blocks lymphocyte-EC adhesion, was the only antibody of a panel of antilymphocyte antibodies that completely blocked the induction of EC Ia. Antibodies to interferon-gamma, which is a potent inducer of EC Ia, and to the CD3 T cell-surface antigen partly inhibited the induction of EC Ia by T cells, but neither antibody had any effect on Ia induction mediated by CD16+ cells, suggesting that T cells and natural killer cells utilize different mechanisms to induce Ia on EC. When combined with data from other laboratories indicating that Ia+ but not Ia- EC stimulate allogeneic T cell proliferation and cytotoxicity, our results suggest that the binding of EC by lymphocyte subpopulations followed by the induction of Ia antigen may represent the initial stage of incompatible allograft rejection.     

The role of the Ia-invariant chain complex in the posttranslational processing and transport of Ia and invariant chain glycoproteins

The invariant chain (Ii) is a nonpolymorphic glycoprotein that associates with the Ia alpha- and beta-chains of MHC class II Ag during their transport to the cell surface. Although surface expression of Ia can occur in the absence of Ii, it has not been shown whether the intracellular association of Ia and Ii affects the biosynthetic rate or specificity of posttranslational modifications to the individual molecules. Analysis of transfected cell lines carrying either Ia, Ii, or both Ia and Ii demonstrated efficient assembly of alpha-beta whether or not Ii was present. Pulse-chase studies and two-dimensional gel electrophoresis showed that Ii did not affect the addition of Ia N-linked oligosaccharide chains, or the extent or rate of their conversion to the complex form, nor did it affect the sulfation of alpha and beta glycoproteins. Ii also did not affect the rate of Ia synthesis or appearance of Ia at the cell surface. In contrast, Ia dramatically affected the posttranslational modification of Ii. Although invariant chain was modified by addition of fatty acid, N-linked oligosaccharide, and glycosaminoglycan in the absence of Ia, the processing of Ii-linked oligosaccharide into more acidic, terminally glycosylated forms was significantly less when Ia was absent, and although conversion to proteoglycan did occur, the glycosaminoglycan chains were significantly shorter than normal. The disappearance of radiolabel from the 31,000 Da form of Ii was faster when Ia was present, and the processing of Ii was more rapid when it was associated with Ia. Thus, the rate and manner in which Ii enters and passes through the Golgi is critically affected by Ia.     

Class II molecules on rat alveolar type II epithelial cells

Class II (Ia) molecules of the major histocompatibility complex are important in the presentation of antigen to T cells and in the regulation of the immune response. Recent studies have suggested that many epithelial cell types can express class II molecules. We examined rat alveolar type II epithelial cells, a cell which can synthesize and secrete pulmonary surface-active material, for the expression of class II antigens. Using an indirect immunofluorescent technique with a mouse anti-rat class II monoclonal antibody (OX-4), the majority of type II cells isolated from pathogen-free Sprague-Dawley rats expressed Ia antigens as determined by fluorescent microscopy and cell sorter analysis. In culture, the Ia expression was lost from type II cells. The addition of recombinant interferon-gamma to cultures of type II cells induced the expression of class II antigens. These findings suggest that class II antigen expression on type II cells may have relevance to immune responses occurring in the lung.     

Astrocytes as antigen-presenting cells. I. Induction of Ia antigen expression on astrocytes by T cells via immune interferon and its effect on antigen presentation

Ia antigens seem to control immune responses on at least two levels. First, they influence the antigen recognition repertoire of the T cells. Second, their variable expression on certain antigen-presenting cells is a powerful regulatory mechanism for the local immune reaction. This is particularly important in the central nervous system (CNS) in which no Ia antigens are normally expressed. Recent experiments in this context have shown that astrocytes are able to express Ia antigens during interaction with T cells, and that they function as antigen-presenting cells. The Ia-inducing activity is produced by activated T cells, and can be replaced by immune interferon (IFN-gamma). In this study we report on the functional and kinetic relationship between Ia antigen expression on astrocytes and the immune-specific activation of T cells by astrocytes. Normal resting astrocytes were found to be negative for Ia antigens by immunofluorescence and by biochemical criteria. Moreover, they are only able to stimulate T cells after they have been induced to express Ia antigens by a signal from the T cells, which is probably mediated by IFN-gamma. In conclusion, the immune-specific interaction between astrocytes and T lymphocytes is a sensitively controlled system that might be pivotal to the development of immune responses in the brain. Malfunction of the system could be an important factor in the pathogenesis of aberrant immune reactions in the CNS, e.g., in multiple sclerosis.     

The ontogeny of murine B lymphocytes. I. Induction of phenotypic conversion of Ia-to Ia+ lymphocytes.

When bone marrow and spleen cells of 4 week-old mice are fractionated on a discontinuous BSA gradient, a small fraction of Ia- cells is obtained which can be induced in vitro to express the Ia alloantigen within 2 hr. This is in precise parallel to the prothymocyte induction system of Komuro and Boyse. Ia specificity is ascertained by the use of two reciprocal antisera, A.TH anti-A.TL (anti-Iak) and A.TL anti-A.TH (anti-Ias), which yield the expected reaction pattern on induced bone marrow cells of (B6 X A)F1 (Iak) and SJL/J (Ias) mice. Induction can be effected by a number of agents, such as catecholamines, prostaglandin PGE1, cAMP, bacterial endotoxin, lipid A, ubiquitin, and thymopoietin. The last requires a 100-fold higher concentration for Ia+ induction as compared to prothymocyte induction, thus implying a lower affinity for the B cell receptor than for the thymocyte receptor. Ia- to Ia+ conversion involves cells different from prothymocytes, as indicated by: 1) the specific cytolytic effect of our anti-Ia sera which were shown to be free of activity against thymocytes; 2) an additive cytolytic effect of anti-Ia and anti-Thy-1 sera; and 3) the fact that the Ia inducible cells are sensitive to pretreatment with anti-immunoglobulin and C. This finding that Ia- precursor cells are already Ig+ is of interest for the B cell ontogeny, as it implies that Ig expression precedes Ia expression.     

Analysis of Ir gene function using monoclonal antibodies: independent regulation of GAT and GLPhe T cell responses by I-A and I-E subregion products on a single accessory cell population

T cell proliferative responses to the synthetic polypeptides GAT and GLPhe are under Ir gene control. GAT responses are regulated by gene(s) in the I-A subregion, and GLPhe responses are controlled by a pair of complementing genes mapping to the I-A and I-E subregions. We demonstrate that monoclonal antibody to the I-A gene product inhibits GAT proliferation but not the GLPhe response, whereas a monoclonal antibody to the I-E associated Ia-7 determinant inhibits GLPhe but not GAT proliferation, which indicates independent involvement of each Ia determinant in antigen presentation for the T cell response to these antigens. Use of the same subregion-specific monoclonal antibodies in complement-dependent lysis demonstrates that the antigen-presenting cells for GAT and GLPhe express both I-A and I-E products. The possibility that an Ia subregion-specific "self-receptor" functions on the reactive T cells as a regulatory element is discussed.     

Long-term-cultured mouse B-lymphocyte line. II. Modulation of Ia-antigen expression on B-lymphocyte line

Mouse B-cell line, established by culturing anti-Thy-1 and complement-treated splenic B cells with concanavalin A-stimulated conditioned medium, expressed immunoglobulins and Ia antigens on its surface. The long-term-cultured B-cell line was split in two and maintained with or without 3300 R X-irradiated T-cell-depleted syngeneic splenic adherent cells (SAC). Interestingly, the B-cell line cultured without SAC lost its Ia antigen but not its Ig expression, whereas the cell line with SAC maintained both Ia and Ig expression. The ability to express Ia antigens was restored by culturing them only in the presence of Ia-positive feeder cells. Neither recombinant interferon-gamma or lectin-stimulated conditioned medium nor cell-free culture supernatant SAC had the ability to restore Ia antigen expression on the B-cell line. Incubation of Ia-negative B-cell line with phorbol esters restored the Ia expression. It is suggested that the expression of Ia antigen on B lymphocytes was controlled differently from that on macrophage lineage. The B-cell line expressing Ia antigens acts as stimulator cells for alloantigen-activated T lymphocytes and as antigen-presenting cells on the KLH-specific Ia-restricted proliferative T-cell clone in the presence of a specific antigen.     

Natural killer-like activity mediated by activated T lymphocytes

Diverse types of lymphocytes mediate in vitro cytotoxic activity. In addition to CTLs (cytotoxic T lymphocytes) and NK (natural killer) cells which differ in their activation requirements, target specificities, and lytic mechanisms, a natural killer-like activity of activated cells (A-NK) has recently been described. The data presented here suggest that an activated T lymphocyte can mediate A-NK activity. A-NK activity can be separated from resting NK activity by its requirement for activation and an effector phenotype (T12+,Ia+,Mol-) which includes the presence of the T12 and Ia antigens and the absence of the Mol antigen. In contrast, resting NK activity is mediated by T12-,Ia-,Mol+ cells. Cells that mediate A-NK activity can be differentiated from CTLs by their differing kinetics of activation and susceptibility to inhibition by monoclonal antibodies. An additional distinguishing feature is the fact that A-NK cells are predominantly Ia+ and are derived from either the T4+ or T8+ T-cell subsets whereas CTLs generated under similar conditions are predominantly T8+,T4-,Ia-. The in vivo relevance of this newly defined T-cell cytolytic activity remains to be defined.     

Subpopulations of the T4+ inducer T cell subset in man: evidence for an amplifier population preferentially expressing Ia antigen upon activation

Prior studies demonstrated that Ia molecules were expressed on a fraction of human peripheral T4+ inducer cells upon stimulation by soluble antigen. In the present study, we utilized a fluorescence-activated cell sorter to separate antigen-activated T4+,Ia+ and T4+,Ia- populations and characterized their function. It was found that the T4+,Ia+ population contained the majority of proliferating T cells as assessed by tritiated thymidine incorporation. This proliferation largely appeared to be nonspecific. Despite macrophage repletion, elimination of the Ia+ subset of T cells with monoclonal anti-Ia antibody and complement treatment equally diminished subsequent proliferation to both the triggering antigen and an unrelated antigen. Moreover, the antigen-induced Ia+ subset of T cells alone produced a nonspecific helper factor, LMF. In contrast, the the T4+,Ia- population showed minimal proliferation to soluble antigen and did not generate LMF. Nevertheless, both T4+,Ia+ and T4+,Ia- inducer T cells were required to generate maximal immunoglobulin production by B cells in an antigen-driven system. We conclude that the human T4+ inducer T cell subset is comprised of at least 2 functionally distinct subpopulations, which are capable of acting in a synergistic fashion to provide help to B cells.