Effects of in vivo administration of anti-CD3 monoclonal antibody on T cell function in mice. II. In vivo activation of T cells

Anti-CD3 mAb are known to be both immunosuppressive and mitogenic to T cells in vitro. However, only immunosuppression has been observed after in vivo administration of these mAb. The present study demonstrates that T cell activation does occur after in vivo administration of anti-CD3 mAb to mice, evidenced by increased IL-2R expression on T cells, CSF secretion, and extra-medullary hematopoiesis in the spleen. These effects required multivalent cross-linking of the mAb, since F(ab')2 fragments failed to induce them. However, the F(ab')2 fragments did induce modulation of CD3/TCR from the surface of T cells, demonstrating that TCR modulation is not sufficient to induce activation. In addition, interaction of the TCR with either intact or F(ab')2 fragments of the mAb led to increased expression of CD8 in vivo, suggesting that the F(ab')2 fragments of anti-CD3 mAb might be capable of inducing a T cell to undergo some, but not all, of the changes involved in reaching a fully activated state. Further study of the activating effects of anti-CD3 mAb might increase the understanding of the mechanisms of in vivo T cell activation and might also be exploited clinically to stimulate T cell function in immunocompromised states and to enhance hematopoiesis in myelodysplastic disorders.     

Immunosuppression and lymphoid hypoplasia associated with chronic graft versus host disease is dependent upon IFN-gamma production

We have previously shown that both IFN-gamma and IFN-beta are produced in vivo and in vitro by spleen cells obtained from mice experiencing a chronic form of graft vs host disease (GVHD). Further, we have shown that in vitro production of IFN-beta by spleen cells from GVHD mice may play a role in the suppressed in vitro mitogen responsiveness of these cells. This study was undertaken to investigate if treatment of such mice with mAb to IFN-gamma or IFN-beta could alter the immunosuppression or lymphoid hypoplasia associated with chronic GVHD. GVHD was induced across minor histocompatibilities by the i.v. injection of B10.D2 spleen cells into sublethally irradiated BALB/c mice. These mice were given daily injections for 20 days of one of the following: 1) mAb to IFN-gamma, 2) mAb to IFN-beta, or 3) control IgG. Histologic examination of these mice at 21 to 22 days post transplantation revealed that mice treated with mAb to IFN-beta or control IgG had dramatic hypoplasia of the thymus, spleen, and lymph nodes which was similar to untreated GVHD mice. Mice given mAb to IFN-gamma, however, had no lymphoid hypoplasia and had a near normal gross and histologic appearance of their thymus, spleen, and lymph node tissue when compared with syngeneic controls. In vitro mitogen-induced proliferative responses of spleen and lymph node cells obtained from GVHD mice or GVHD mice treated with mAb to IFN-beta were severely suppressed or absent. In contrast, spleen and lymph node cells from GVHD mice given mAb to IFN-gamma were capable of giving a significant in vitro proliferative response to Con A, PHA, and LPS. Further, natural suppressor cell activity and spontaneous production of IFN-beta, a characteristic of this form of GVHD, was absent in spleen cells obtained from GVHD mice treated with mAb to IFN-gamma. These results further identify the IFN as playing critical roles in the pathogenesis of GVHD.     

B lymphocytes differentially influence acute and chronic allograft rejection in mice

The relative contributions of B lymphocytes and plasma cells during allograft rejection remain unclear. Therefore, the effects of B cell depletion on acute cardiac rejection, chronic renal rejection, and skin graft rejection were compared using CD20 or CD19 mAbs. Both CD20 and CD19 mAbs effectively depleted mature B cells, and CD19 mAb treatment depleted plasmablasts and some plasma cells. B cell depletion did not affect acute cardiac allograft rejection, although CD19 mAb treatment prevented allograft-specific IgG production. Strikingly, CD19 mAb treatment significantly reduced renal allograft rejection and abrogated allograft-specific IgG development, whereas CD20 mAb treatment did not. By contrast, B cell depletion exacerbated skin allograft rejection and augmented the proliferation of adoptively transferred alloantigen-specific CD4(+) T cells, demonstrating that B cells can also negatively regulate allograft rejection. Thereby, B cells can either positively or negatively regulate allograft rejection depending on the nature of the allograft and the intensity of the rejection response. Moreover, CD19 mAb may represent a new approach for depleting both B cells and plasma cells to concomitantly impair T cell activation, inhibit the generation of new allograft-specific Abs, or reduce preexisting allograft-specific Ab levels in transplant patients.     

T cell antigen receptors in autoimmunity

Three mAb to variable region determinants of the alpha/beta-chain TCR were used to detect discrete populations of peripheral blood T cells. T cells sharing a TCR determinant defined by such an antibody presumably use the same or similar TCR V or J genes for their alpha- or beta-chains. Thus analysis with these mAb provides a tool to investigate TCR gene usage and expression. Since autoantigen specific T cells may play an important role in initiating autoimmune diseases, TCR were analyzed in different autoimmune diseases and control groups including rheumatoid arthritis, Graves disease, idiopathic thrombocytopenic purpura, psoriasis, SLE, insulin-dependent diabetes mellitus, and in nonautoimmune control diseases and normals. Purified T cells were stained by indirect immunofluorescence with three mAb to TCR variable regions: mAb S511 stains 1.8 +/- 0.9% (mean +/- 2 SD), mAb C37 stains 3.4 +/- 1.5% and mAb OT145 stains from 0 to 6% of T cells from normal donors. Several individuals were identified with expanded subsets of positive T cells. One patient with adult ITP followed during a 12-mo period consistently had elevated percentages of T cells staining with the mAb OT145 (15.9 to 24.5%). These cells were found to be exclusively CD8+. By Southern blotting DNA prepared from these OT145+, CD8+ cells, but not DNA from the patient's OT145- T cells, revealed a clonal rearrangement using a beta-chain C region probe. Thus this patient had a monoclonal expansion of CD8+, OT145+ cells. Hyperexpression of a TCR variable region, as defined by the available mAb, could not be associated with any of the diseases studied. Examination of T cells at the site of autoimmunity, such as T cells from rheumatoid arthritis synovial fluid, revealed normal percentages of cells staining with these mAb. Immunoperoxidase staining of psoriatic lesional skin showed no striking enrichment of T cells bearing one or the other TCR type.     

Immunobiology of allograft rejection in the absence of IFN-gamma: CD8+ effector cells develop independently of CD4+ cells and CD40-CD40 ligand interactions

Both wild-type (WT) and IFN-gamma-deficient (IFN-gamma(-/-)) C57BL/6 mice can rapidly reject BALB/c cardiac allografts. When depleted of CD8(+) cells, both WT and IFN-gamma(-/-) mice rejected their allografts, indicating that these mice share a common CD4-mediated, CD8-independent mechanism of rejection. However, when depleted of CD4(+) cells, WT mice accepted their allografts, while IFN-gamma(-/-) recipients rapidly rejected them. Hence, IFN-gamma(-/-), but not WT mice developed an unusual CD8-mediated, CD4-independent, mechanism of allograft rejection. Allograft rejection in IFN-gamma(-/-) mice was associated with intragraft accumulation of IL-4-producing cells, polymorphonuclear leukocytes, and eosinophils. Furthermore, this form of rejection was resistant to treatment with anti-CD40 ligand (CD40L) mAb, which markedly prolonged graft survival in WT mice. T cell depletion studies verified that anti-CD40L treatment failed to prevent CD8-mediated allograft rejection in IFN-gamma(-/-) mice. However, anti-CD40L treatment did prevent CD4-mediated rejection in IFN-gamma(-/-) mice, although grafts were eventually rejected when CD8(+) T cells repopulated the periphery. The IL-4 production and eosinophil influx into the graft that occurred during CD8-mediated rejection were apparently epiphenomenal, since treatment with anti-IL-4 mAb blocked intragraft accumulation of eosinophils, but did not interfere with allograft rejection. These studies demonstrate that a novel, CD8-mediated mechanism of allograft rejection, which is resistant to experimental immunosuppression, can develop when IFN-gamma is limiting. An understanding of this mechanism is confounded by its association with Th2-like immune events, which contribute unique histopathologic features to the graft but are apparently unnecessary for the process of allograft rejection.     

Mechanisms of in vivo generation of cytotoxic activity against allograft: 1. Local differentiation of mature cytotoxic T lymphocytes in rejection of allogeneic lymphocytes

Although cytotoxic activity was not detected within the spleen and regional lymph nodes from mice immunized sc with allogeneic lymphocytes, such activity was detected consistently in glass-nonadherent and anti-θ-sensitive peritoneal exudate cells (PE cells) from Day 5 after immunization and reached a maximum by Day 7. Immunized spleen cells developed cytotoxic T lymphocytes (CTLs) earlier and more effectively than normal spleen cells when transferred ip into X-irradiated syngeneic normal mice together with immunizing antigen, while they did not become cytotoxic when transferred without antigen. These results suggest that spleen and lymph node cells which may have differentiated into some transitional state by in vivo immunization may differentiate into mature CTLs, following direct contact with antigen at the site of graft. CTLs generated there appear to be responsible for the rejection of allogeneic lymphocytes. Cytotoxicity of PE cells was also generated in X-irradiated mice and augmented cytotoxicity was generated by treatment with cyclophosphamide.     

Lymphocyte function in experimental African trypanosomiasis. VIII. Loss of suppressor T cell function in lymph nodes

The immunosuppression that occurs in mice experimentally infected with African trypanosomiasis has been examined further. In the present study we have examined lymph node cells from Trypanosoma rhodesiense-infected C57Bl/6J mice for the ability to produce mitogen induced antigen-nonspecific suppressor T cells (Ts). Inguinal, mesenteric, and brachial lymph node cells were harvested from uninfected control mice and from mice at different periods of infection. These cells were cultured with or without concanavalin A (Con A) for 48 hr to induce Ts activity. After stimulation, the control and infected lymph node cells were passed over Sephadex G-10 columns to remove suppressor macrophages that arise during the infection from Con A-induced Ts. The column passed cells were then added to normal mouse responder spleen cells in a primary in vitro antibody response culture system with sheep erythrocytes (SRBC) as antigen. The resultant plaque-forming cell responses to SRBC indicated that Ts function was not induced in infected lymph node cell populations. However, early in the infection, a stimulatory signal was provided by both the untreated and Con A-treated infected lymph node cells, which was lost in the terminal stage. Determinations of T cell subpopulations revealed that the infected Lyt 2.2-bearing subpopulation was not significantly altered from normal controls. We conclude that T. rhodesense infected mice fail to mount normal lymph node cell antigen nonspecific Ts responses and that this loss of activity may be due to an intrinsic dysfunction in the suppressor T cell population.     

Pancreatic islets engineered with SA-FasL protein establish robust localized tolerance by inducing regulatory T cells in mice

Allogeneic islet transplantation is an important therapeutic approach for the treatment of type 1 diabetes. Clinical application of this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic effector T cells regardless of chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating effector T cell responses, we tested if pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of Fas ligand protein chimeric with streptavidin (SA-FasL) and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin following efficient engineering with SA-FasL protein that persisted on the surface of islets for >1 wk in vitro. SA-FasL-engineered islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute toxicity. Most importantly, the transplantation of SA-FasL-engineered BALB/c islet grafts in conjunction with a short course of rapamycin treatment resulted in robust localized tolerance in 100% of C57BL/6 recipients. Tolerance was initiated and maintained by CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells, as their depletion early during tolerance induction or late after established tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the treatment of type 1 diabetes.     

Graft versus host-induced immunosuppression: mechanism of depressed T-cell helper function in vitro.

The cellular basis of graft versus host (GVH)-induced immunosuppression was investigated. Results showed that thymus, lymph node, and splenic T cells from normal mice and thymus and lymph node T cells from GVH mice, when cultured on one side of a cell impermeable membrane, restored the plaque-forming cell (PFC) response to sheep erythrocytes of GVH-immunosuppressed spleen cells (GVH-SC) cultured on the other side of the membrane. The restoring ability of T cells present in GVH-SC was inhibited by splenic accessory (A) cells. A direct relationship was shown between the proportion of splenic A cells and the degree of suppression of the PFC response during the first 10 days of the GVH reaction. Normal or GVH A cells reconstituted the PFC response of normal cells and GVH-SC depleted of their A-cell fraction. An optimum ratio of A: nonadherent (NA) cells (1: 10) was required for maximum reconstitution. Larger proportions of A cells inhibited the PFC response. The results suggest that GVH-induced immunosuppression is due, at least in its initial phase, to a depressed T-cell helper function caused by a marked increase of A cells in the spleen.     

Characterization of a cell surface-expressed disulfide-linked dimer involved in murine T cell activation

We have produced a hamster mAb, H1.2F3, which was derived by immunization with a murine TCR-gamma delta + epidermal T cell line. H1.2F3 immunoprecipitates a cell surface-expressed disulfide-linked dimer that has a m.w. of 85,000 under non-reducing conditions and consists of subunits of 35,000 to 39,000 m.w. This dimer is distinct from the CD3-associated TCR-gamma delta complex (CD3/TCR), inasmuch as H1.2F3 does not co-precipitate or co-modulate with the CD3/TCR complex and recognizes an Ag with a single-peptide backbone of 22,000 m.w. after N-Glycanase treatment. H1.2F3 is weakly reactive with a small percentage of cells from unfractionated thymus, spleen, or lymph node, but reactivity with both T and B lymphocytes is markedly enhanced by a brief period of stimulation with Con A or PMA in vitro. This enhancement requires de novo protein synthesis. Enhanced expression of the H1.2F3 Ag can also be induced in vivo by injection of Con A or anti-CD3. H1.2F3 is a potent stimulator of T, but not B, cell proliferation in the presence of PMA and FcR-bearing accessory cells. These functional and biochemical studies strongly suggest that the Ag recognized by H1.2F3 is the murine homologue of the human CD28 Ag recognized by mAb 9.3.     

Promotion of allograft survival by CD4+CD25+ regulatory T cells: evidence for in vivo inhibition of effector cell proliferation

Regulatory T cells preserve tolerance to peripheral self-Ags and may control the response to allogeneic tissues to promote transplantation tolerance. Although prior studies have demonstrated prolonged allograft survival in the presence of regulatory T cells (T-reg), data documenting the capacity of these cells to promote tolerance in immunocompetent transplant models are lacking, and the mechanism of suppression in vivo remains unclear. We used a TCR transgenic model of allograft rejection to characterize the in vivo activity of CD4(+)CD25(+) T-reg. We demonstrate that graft Ag-specific T-reg effectively intercede in the rejection response of naive T cells to established skin allografts. Furthermore, CFSE labeling demonstrates impaired proliferation of naive graft Ag-specific T cells in the draining lymph node in the presence of T-reg. These results confirm the efficacy of T-reg in promoting graft survival and suggest that their suppressive action is accomplished in part through inhibition of proliferation.     

Specific tumor memory induced by polyethylene-glycol-modified interleukin-2 requires both helper and cytotoxic T cells

Local polyethylene-glycol (PEG)-modified interleukin-2 (IL-2) immunotherapy of the guinea pig Line 10 (L10) tumor was previously demonstrated to evoke long-lasting systemic immunity after cure of the tumor and metastases. T cells, most likely the helper T cell subpopulation, were demonstrated to be crucial to the antitumor effects. Here we show that systemic immunity is induced within 7 days after the start of PEG-IL-2 therapy, indicating a rapid systemic priming of L10-specific T cells. No in vitro cytotoxic activity was detected in cell suspensions obtained from the primary tumor site, the regional lymph node or the spleen when isolated during (days 21 and 28) intratumoral treatment with 200 000 IU PEG-IL-2. These data confirm our carlier results obtained with 60 000 IU PEG-IL-2. Moreover, no cytolytic activity was observed in the chromium-release assay after in vitro restimulation with irradiated tumor cells. Specific L10 immunity can be transferred using spleen cell suspensions. Depletion of such a suspension of helper T cells resulted in rejection of the primary tumor in two out of four animals, but all the guinea pigs developed lymph node metastases. Removal of the cytotoxic/suppressor phenotype caused rejection of the dermal tumor in four of eight guinea pigs, but the capacity to prevent lymph node metastases was retained in all animals. Thus, depletion of either subtype reduces, but does not abrogate, the capacity to transfer L10 immunity with spleen cells. In conclusion, our data suggest that tumor cell killing through direct T cell cytotoxicity is not the main mode of action in PEG-IL-2-induced L10 tumor regression, PEG-IL-2 therapy induces early systemic immunity, resulting in rejection of a distant tumor, and the transfer of this immunity depends mainly on the presence of helper T cells, although cytotoxic T cells may also play a role.     

Association of human lymph node homing receptor (Leu 8) with the TCR/CD3 complex.

Cross-linking of the human homologue of the murine MEL-14 lymph node homing receptor (Selectin-1, LECAM-1, Leu 8) on both T and B cells results in modification of cell function. To investigate this phenomenon, we performed studies to determine if the Leu 8 molecule influences T cell activation via the TCR/CD3 complex. In initial studies, we treated T cells with immobilized anti-CD3 (OKT3 mAb) in the presence or absence of immobilized Leu 8 mAb. We found that although Leu 8 mAb alone had no effect on T cell proliferation, this antibody markedly augmented immobilized OKT3 mAb-induced proliferation. In further studies, we immunoprecipitated surface radioiodinated T cell lysates with OKT3 and Leu 8 mAb to determine if molecules in the TCR/CD3 complex associate with Leu 8 molecules. Although Leu 8 mAb immunoprecipitated only a single protein of approximately 80 kDa from T cell lysates treated with Nonidet P-40 under reducing condition, it coimmunoprecipitated additional proteins of 48, 42, 28, 24, and 22 kDa from T cell lysates treated with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. These additional proteins were identified as the alpha-, beta-, gamma-, delta-, and epsilon-chains of the TCR/CD3 complex by one-dimensional and two-dimensional diagonal SDS-PAGE. Densitometric scanning showed that, on average, 18% of the TCR/CD3 complex associates with Leu 8. In a final study, we showed by immunoblotting analysis using anti-zeta peptide antibody that Leu 8 mAb coimmunoprecipitates the zeta-chain of CD3. These results indicate that the human lymph node homing receptor homologue (Leu 8) participates in the activation of T cells, probably via its association with the TCR/CD3 complex.     

Depletion of mouse alpha beta T cell antigen receptor bearing lymphocytes by neonatal monoclonal antibody treatment.

Neonatal treatment with a monoclonal antibody specific for the alpha beta TCR results in mice with a long term, severe depletion in the number of alpha beta T cells in the periphery. Significant numbers of T cells reappear in the periphery about age 65 days, but these cells tend to lack expression of CD4 or CD8. Splenocytes of antibody-treated mice are less sensitive to mitogen stimulation or stimulation with MHC allogeneic cells. The level of serum IgG but not IgM was decreased by the treatment. Anti-alpha beta TCR antibody treatment decreased single-positive T lymphocytes that express high levels of the CD3/alpha beta TCR complex from the thymus, suggesting that the treatment could act in part by affecting negative selection of alpha beta TCR+ thymocytes. This treatment does not, however, detectably affect either the homing or the numbers of gamma delta T cells which are abundant in the intestinal epithelium, but which remain a minor population in the spleen and lymph nodes. This supports the hypothesis that gamma delta T cells are developmentally autonomous from alpha beta T cells. These mice provide an excellent model system for assessing the developmental and functional role of gamma delta T lymphocytes in vivo.     

Specific T-cell factor production and lymphocytes in the direct surroundings of a subcutaneous allogeneic tumor.

Antigen-specific T-cell factors (TCF) play a role in the initiation of cellular immune responses. In allogeneic mouse-tumor models lymphocytes from the direct tumor surroundings of both euthymic and nude mice produce TCF. These lymphocytes produce TCF when collected already 1 day after subcutaneous (sc) injection of tumor cells. In contrast to euthymic mice, draining lymph nodes and spleen of nude mice did not contain TCF-producing lymphocytes at any stage after sc tumor cell injection. In sensitized euthymic mice TCF production by lymphocytes is significantly higher in the direct tumor surroundings than in draining lymph nodes or spleen. At 2 and 5 days after tumor cell injection, the mononuclear cell infiltrate of the tissue surrounding the tumor in euthymic mice showed low expression of Thy 1, CD3, TCR alpha beta, TCR gamma delta, CD4, CD8, and asialo GM1, whereas several lymphocytes and mast cells were positive for monoclonal antibody (mAb) 14-30 (directed against TCF). In both euthymic and nude mice, sc injected tumor cells showed apoptosis. In conclusion, the direct tumor surroundings are the first (and, for nude mice, the only) site of TCF production, sc injection of tumor cells attracts mAb 14-30-positive lymphocytes and renders mast cells positive for mAb 14-30.     

Recovery of activated cytotoxic T cells from minor H incompatible tumor graft rejection sites

In this study we determined whether minor H-specific cytotoxic T cells and their precursors (pTc) are present at the site of rejection of minor H disparate tumor allografts. Lymphocytes were retrieved from eyes of BALB/c mice that received subconjunctival injections of minor H-incompatible P815 tumor cells. The lymphocytes were then assayed for direct cytotoxic activity as well as precursor frequency by limiting dilution. Similar assays were conducted on cells obtained from the draining lymph nodes and from the spleen. As expected, tumor rejection was accompanied by significant clonal expansion of minor H-specific pTc within the draining lymph node and the spleen. A correspondingly high frequency of pTc was also detected at the graft site. More importantly, fully functional cytotoxic T cells were recovered from the tumor graft site during rejection, but no similarly active cells were found in either the draining nodes or spleen. We conclude that, after Ag stimulation, pTc are generated in draining central lymphoid compartments. From this generative site, the precursor cells then disseminate systemically, gradually reaching and infiltrating the tumor graft site. A further activation step, dependent upon Ag and T cell help, permits these cells to mature into fully active cytotoxic cells which can then effect tumor rejection. We propose that the terminal stage(s) of pTc activation is promoted by lymphokines released locally from TDH cells that are also generated during the alloimmune response and simultaneously infiltrate the site.     

Concomitant administration of hapten and IL-2-toxin (DAB486-IL-2) results in specific deletion of antigen-activated T cell clones

During the proliferative burst after Ag recognition, T cells express cell-surface, high-affinity IL-2R. The importance of IL-2R+ T cells in supporting/mediating tissue injury has been documented by the ability of mAb anti-IL2R therapies to prevent allograft rejection and autoimmunity. The delayed-type hypersensitivity (DTH) response, an experimental model of T-dependent immunity, offers the possibility of studying responses mounted against defined Ag. We previously reported that the chimeric IL-2 toxin (DAB486-IL-2) prevents DTH responses and selectively eliminates activated IL-2R bearing CD4 and CD8 T cells from lymph nodes draining the site of inflammation. We have examined the duration of immunosuppression and relative specificity of action of DAB486-IL-2 and anti-CD3 mAb for Ag-activated clones in a murine model of DTH using two different noncross-reacting haptens. Treatment with DAB486-IL-2 generates a state of selective unresponsiveness to subsequent challenge with the hapten introduced during the therapeutic period. Immediately after cessation of DAB486-IL-2 therapy, immunization with an unrelated hapten induces a normal vigorous immune response. By comparison, anti-CD3 mAb treatment causes a broad immunosuppression because unrelated haptens introduced after anti-CD3 therapy do not evoke a vigorous immune response. After cessation of DAB486-IL-2 toxin treatment response to the hapten is eventually restored probably by cells trafficking from the thymus, because thymectomized hosts remain unresponsive to the hapten. Taken together these data reinforce the role of the IL-2R as an important target for immunosuppression in T cell-mediated immune reactions. DAB-486-IL-2 treatment confers highly selective immunosuppression.     

Mice deprived of exogenous antigenic stimulation develop a normal repertoire of functional T cells.

The development of T cells and the selection of the TCR repertoire in the absence of exogenous antigenic stimulation were investigated. For this purpose germfree BALB/c mice fed an ultrafiltered solution of chemically defined low m.w. nutrients (GF-CD) were used. Previous studies on B cell development and differentiation in GF-CD mice have demonstrated a high reduction in the number of cells secreting Ig of the non-IgM isotypes but an Ig-VH gene usage and a B cell specificity repertoire that is substantially different from that observed in conventional adult mice and more closely resembles that of neonatal conventional mice. In contrast, the present comparison of the various lymphocyte populations in the thymus, lymph nodes, and spleen from GF-CD and conventional mice using flow cytometry analysis revealed no significant differences. Analysis of the TCR-V beta expression on both mature thymocytes and lymph node T cells showed a high degree of similarity between GF-CD and conventional mice. These findings indicate a marked difference in the influence of exogenous antigenic stimulation on the development of B and T cells. Additionally, development in an environment free of exogenous antigenic stimulation allows for full functional maturation of T cells to occur, because MLC showed that GF-CD splenic T cells could mount allogeneic responses in a way similar to T cells generated in a conventional environment. Most importantly, full Th cell function is generated, because activation of GF-CD spleen cells by cross-linking with mAb against CD3 resulted in the induction of cells secreting IFN-gamma and Ig of the non-IgM isotypes, which cannot be detected in GF-CD sera. These findings demonstrate that functional T and B cells develop in mice that have not been exposed to exogenous Ag, and that the TCR repertoire, in contrast to the B cell compartment, is predominantly shaped by endogenously expressed Ag.