Antigen- and isotype-specific regulation of IgE responses by Lyt 1+,2,3- and Lyt 1-,2,3+ T suppressor cells

Antigen-specific, IgE isotype-selective suppression is induced following treatment of mice with a high-molecular-weight glutaraldehyde-polymerized ovalbumin preparation (OA-POL). The results show that the suppression is mediated by Lyt 1+,2,3- cells residing in the spleen. Adoptive transfer experiments indicate that Lyt 2,3+ or Lyt 1,2,3+ cells are not required for the establishment of suppression by these Lyt 1+,2,3- suppressor T cells (Ts). Treatment of OA-POL-induced Ts cells with anti-I-Jk serum and complement does not affect their ability to suppress. In marked contrast, spleen cells from animals treated with a single course of OA-POL almost 300 days previously, were shown to contain boosterable memory suppressor T cells (Tsm) which display the Lyt 1-,2,3+ phenotype. The activity of both Ts and Tsm cells appears to result from stimulation by determinants common to native OA and OA-POL rather than by idiotypic determinants expressed on anti-OA antibodies.     

Target cell heterogeneity in murine leukemia virus infection. III. Identification of susceptible Lyt 1+ and resistant Lyt 2+ T-cell subsets following in vitro infection with Friend murine leukemia virus

The susceptibility of splenic T-cell subpopulations to productive infection with Friend murine leukemia virus was determined after in vitro infection and stimulation with Con A. Con A enhanced the number of productively infected cells in unseparated spleen cells as well as in T-cell-enriched spleen cell fractions. Splenic T cells were fractionated into Lyt 1+ and Lyt 2+ subpopulations using both positive and negative selection techniques; susceptible splenic T cells were recovered in the Lyt 1+ fraction and specific cytotoxic treatment with anti-Lyt 1 antibody and complement reduced the number of infectious center-producing cells by greater than 87%. In marked contrast, Lyt 2+ splenic T cells were resistant to productive infection by Friend murine leukemia virus in vitro.     

Characterization of cytotoxic cells generated from in vitro cultures of murine bone marrow cells

Bone marrow cells cultured for 5-6 days generate cytotoxic activity against a number of natural killer (NK)-susceptible tumor cells. In this study, these bone marrow cytotoxic cells were compared to cells with NK activity obtained either from spleen cells activated in vitro with interferon (IFN-alpha/beta) or mitogen or from peritoneal exudate cells (PEC) obtained 4 days after bacillus Calmette-Guerin (BCG) infection. Splenic and PEC cytotoxic cells were shown to be Thy 1.2+, NK 1.1+, Asialo GM+1, Lyt 1.2-, Lyt 2.2-. In contrast, bone marrow cytotoxic cells were Thy 1.2+, NK 1.1-, Lyt 1.2-, Lyt 2.2- and expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Precursor cells for bone marrow cytotoxic activity were shown to be Thy 1.2-, NK 1.1-, Lyt 1.2-, Lyt 2.2- but also expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Cytotoxic activity for both bone marrow and spleen cells peaked in the low-density fractions of discontinuous Percoll density gradients. The cytotoxic activity of these bone marrow cells was augmented by pretreatment with IFN (-alpha/beta, -gamma) or soluble factors (IFN free) from activated EL-4 thymoma cells. Surprisingly, the ability of bone marrow cells to generate high levels of cytotoxic activity following in vitro culture appeared to be associated primarily with mice which were of the H-2b haplotype.     

Suppression of B-cell differentiation by natural killer (asialo GM1+) cells in mice

Mice were injected intravenously with rabbit antiserum to ganglio-n-tetraosylceramide (asialo GM1, ASGM1), a neutral glycosphingolipid present at high quantities on the surface of natural killer (NK) cells. Spleen cells prepared from the mice were then examined for NK activity against YAC-1 targets, for phagocytic cells and by flow cytometric analysis for Thy1, Lyt1, Lyt2, ASGM1 and surface Ig (SIg) phenotypes. Administration of anti-ASGM1 in mice resulted in a complete depletion of NK activity and ASGM+1 cells in the spleen, but no changes in the proportions of Thy1+ cells and their Lyt1+ and Lyt2+ subsets and phagocytic cells. Corresponding to this selective depletion of ASGM+1 cells and NK activity, the spleen cells showed an increased number of SIg+ B cells and augmented mitogenic responses to B-cell but not T-cell mitogens. These NK-depleted spleen cells also showed production of pokeweek mitogen (PWM)-driven plaque-forming cells (PFC) to much higher levels than those of control spleens. In the spleens of mice treated with varying concentrations of anti-ASGM+1, a good correlation was found between the decreased NK activity and the enhanced PFC response. To directly test the possible suppressor activity of NK cells on PWM-induced PFC response, NK (ASGM+1) cells were highly purified from the spleen by a combination of Percoll gradients and cytolysis of T cells by monoclonal antibodies followed by indirect panning. When added to NK-depleted spleen cells, they suppressed the augmented PFC response of NK-depleted spleen cells, depending on the number of cells added. These results suggest that NK (ASGM+1) cells in mice exhibit a suppressor property on B cells, which are undergoing spontaneous or mitogen-induced differentiation.     

Lymphocyte subsets involved in tumor-activated nonspecific feedback suppression

Cells from the spleen, lymph nodes, and peritoneum of DBA/2 mice bearing a subcutaneous tumor mediate nonspecific suppression of an in vitro antibody response to sheep red blood cells (SRBC) when cocultured with a normal T-cell subset(s). The spleen cells from the tumor-bearing mouse required for the suppression bear the Lyt 1 and Ala 1 surface markers characteristic of "inducer" T cells and activated cells, respectively. The activity of this cell population is also sensitive to irradiation. The normal T-cell subset which cooperates in the suppression bears the Qa-1 surface antigen which has been associated with suppressor cell precursors in several systems but lacks detectable surface Lyt 1 and 2 markers. Suppression of antibody responses in spleen cell cultures from tumor-bearing mice alone could also be elicited, but only when increased numbers of cells were cultured. These data are consistent with the theory that a tumor-activated, Lyt 1+ T-cell subset has the capacity to nonspecifically suppress immune responses by activating a Qa-1+ subset(s) of T suppressor cells, perhaps via feedback signals.     

T-cell responses induced by the parenteral injection of antigen-modified syngeneic cells. II. Mechanisms, specificity, and cellular analysis of 2,4,6-trinitrophenol (TNP)-specific cytolytic response priming by intravenous versus subcutaneous injection with TNP-modified syngeneic cells

We have examined the underlying mechanisms accounting for the enhanced in vitro TNP-specific cytotoxic T-lymphocyte (CTL) response following the parenteral injection of syngeneic hapten-modified lymphoid cells. Augmented CTL activity noted following parenteral injection (iv vs sc) of 2,4,6-trinitrophenol-modified syngeneic spleen cells (TNP-SC) is most apparent when limiting numbers of TNP-modified stimulator cells are used in the in vitro sensitization phase. Enhanced CTL responses seen following sc and iv priming is due to distinct mechanisms. Spleen and lymph node (LN) cells from sc primed mice were found to contain significant levels of radioresistant helper activity upon coculture with either viable normal spleen cells in bulk culture or with thymocytes as the source of precursor CTLs in a limiting dilution assay. The helper activity was found to be mediated by a Lyt 1+2- T cells. In addition, Lyt 2-depleted spleen and LN cells from sc primed BALB/c mice could restore the ability of tolerant spleen cells from 2,4,6-trinitrobenzenesulfonic acid (TNBS)-injected BALB/c mice to generate TNP-specific CTLs. Conversely, Lyt 2-depleted spleen and LN cells from iv primed mice provided no measurable helper activity either in bulk culture or in the limiting dilution assay and did not restore the ability of TNBS-tolerant BALB/c spleen cells to generate TNP-specific CTLs. CTL priming via the iv route was found to be completely antigen specific as iv injection of either 2,4-dinitrophenol (DNP)- or fluorescein isothiocyanatel (FITC)-modified cells caused no enhanced CTL activity. Priming via the sc route exhibited a unique specificity pattern as it was shown that sc injection of both TNP-SC and DNP-SC, but not FITC-SC, resulted in enhanced TNP-specific CTL responses. CTL T-helper (Th)-cell induction via the sc route was correlated with (1) the presence of H-2 I region determinants on the inducer cells as the sc injection of TNP-modified erythrocytes led to no enhanced CTL responses or CTL Th activity (while iv injection of TNP-erythrocytes did lead to enhanced CTL responses without detectable helper activity) and (2) the detection of both hapten-specific T-cell proliferation and Interleukin 2 (IL-2) production upon restimulation in culture. We conclude that the sc injection of TNP-SC leads preferentially to an increase of specific Lyt 1+ helper activity, while iv injection leads preferentially to an apparent expansion of Lyt 2+ prelytic effector CTLs.     

Immune T cells can protect or induce fatal neurological disease in murine lymphocytic choriomeningitis

Adoptively transferred immune spleen cells induce fatal neurological disease in cyclophosphamide-suppressed recipients injected intracerebrally (ic) with a large, but not small, dose of neurotropic lymphocytic choriomeningitis (LCM) virus. The elimination of virus from brain in the latter group, which survives without developing symptoms, depends upon the presence of Lyt 2+ lymphocytes. However removal of Lyt 2+ subset which is cytotoxic in vitro does not diminish the severity of the inflammatory process in vivo, though the onset of clinical disease is delayed in mice given Lyt 2-depleted populations and a larger ic dose of virus. The present findings are consistent with the idea that fatal LCM results from acute, synchronous damage to key functional cells in the central nervous system by virus-immune Lyt 2+, lymphocytes. Even so, if the number of virus-infected CNS cells is still relatively small at the time of T cell invasion, neurological symptoms are not recognized and the mice survive.     

Characteristics of mouse lymphoid cells proliferating in vitro by recombinant human interleukin 2 (TGP-3): comparison between normal and nude mice

Various lymphoid cells obtained from BALB/c and BALB/c nu/nu mice were cultured in vitro with recombinant human interleukin 2 (rIL 2), and the characteristics of responder cells to rIL 2 were analyzed. Spleen cells, lymph node cells, and thymocytes except for bone marrow cells obtained from BALB/c mice remarkably proliferated in response to rIL 2. On the other hand, among lymphoid cells obtained from BALB/c nu/nu mice, only lymph node cells showed significant proliferation by rIL 2. Flow cytometric analysis revealed that mainly two types of lymphoid cells were proliferating in response to rIL 2 in BALB/c mice, i.e., Thy 1+, Lyt 1-, Lyt 2- and Thy 1+, Lyt 1-, Lyt 2+ cells. On the other hand, most of the proliferating cells were Thy 1+, Lyt 1-, Lyt 2- cells in BALB/c nu/nu mice. Treatment with various antibodies plus complement revealed that the majority of IL 2-responsive cells in BALB/c mice were Thy 1+, Lyt 1+, and Lyt 2+, although a minor part of them were Thy 1-, Lyt 1-, and Lyt 2-. On the other hand, a predominant type of the IL 2-responsive cells in BALB/c nu/nu mice were Thy 1-, Lyt 1-, and Lyt 2-, though some were Thy 1+. Nonspecific killer activity against tumor cells increased to variable extents in all of the lymphoid cells of both strains after culture with rIL 2. Our results indicate that mouse responder cells to rIL 2 have the following characteristics. First, the responder cells exist abundantly among spleen, lymph nodes, and thymus in normal mice, though their cell lineages are heterogeneous; one is of T cell lineage and the other of natural killer (NK) cell lineage. Second, nude mice are defective in the responder cells of T cell lineage but not of NK cell lineage. Moreover, the responder cells in nude mice predominantly accumulate in the lymph nodes but not other lymphoid organs.     

Stimulation of splenic T cells on syngeneic monolayers of epidermal basal cells (EBC) in mice. Regulation by Lyt 1+ and Lyt 2+ cell subsets

Syngeneic proliferative response of splenic T cells against monolayers of epidermal basal cells (EBC) was obtained with C57BL/6 and DBA/2 mice. Optimal response, as assessed by [³H]thymidine uptake, occurred on the 6th day of coculture. The level of [^3H]thymidine uptake by unseparated spleen cells was lower than by fractionated T cells from C57BL/6 mice, and null for DBA/2 mice. It was not significantly different when lymphocytes were cocultured with syngeneic or allogeneic epidermal cells. Ia antigens did not appear to be involved in the syngeneic response, since it was not prevented by pretreating stimulator monolayers with monoclonal anti-Ia^k antibody or by adding this antibody directly to the cultures. When the proliferative responses of separated Lyt 1⁺ and Lyt 2⁺ cell subsets were compared, the prominent role of Lyt 1⁺ cells was demonstrated. Enhancement of the T-cell reactivity by eliminating Lyt 2⁺ cells and suppression of the response of a constant number of Lyt 1⁺ cells by adding Lyt 2⁺ cells suggested that Lyt 2⁺ cells could suppress and modulate the Lyt 1⁺ cell proliferation.     

Involvement of L3T4+, LYT2.2+ T cell subsets and non-T cells in the resistance of mice against Trypanosoma cruzi infection

Cellular populations involved in resistance against T. cruzi infection were characterized from mice chronically infected with the parasite. Mice transfused with spleen cells (SC), nylon-wool-non-adherent spleen cells (NWNA) or sera from mice chronically infected with T. cruzi, showed an enhanced resistance against challenge with the parasite. The protective activity of NWNA but not of SC was completely abrogated by treatment with anti-Thy1.2 monoclonal antibodies (mAb) and complement (C). Pretreatment of NWNA cells from chronically infected mice with either anti-L3T4 or anti-Lyt 2.2 mAb partially reduced the transfer of resistance. When both L3T4+ and Lyt2.2+ cells were depleted from NWNA populations, transfer of resistance was abolished. These results appear to indicate that L3T4+, Lyt2.2+ T cell subsets and non-T cells are involved in the immunity to T. cruzi.     

Lyt phenotype analysis of the effector cells responsible for evoking lymphocyte-induced angiogenesis (LIA)

Lymphocyte-induced angiogenesis (LIA) is a vascular response observed when allogeneic or semiallogeneic immunocompetent lymphocytes are inoculated intradermally into immunosuppressed or irradiated host mice. The reported experiments were carried out to characterize the effector cell population(s) responsible for causing LIA. Lyt 1.2, Lyt 2.2, and monoclonal Thy 1.2 antisera were used for negative selection with complement (C′) to investigate the ability of selected subsets of lymphocytes to evoke angiogenesis. Treatment of C57BL/6 spleen cells with either anti-Lyt 1.2 or anti-Thy 1.2 and C′ resulted in an almost complete abrogation of the LIA reaction. In contrast, depletion of Lyt 2+ cells, under conditions which fully abrogated their ability to generate cell-mediated cytotoxicity in allogeneic mixed leukocyte cultures, resulted only in a partial (45%) reduction in the induced vascular response. Synergistic interaction between cell preparations treated separately with either anti-Lyt 1.2 or anti-Lyt 2.2 serum was not observed. We conclude that (i) Lyt 1 + 2?T lymphocytes can induce a significant LIA reaction; (ii) lymphocytes resistant to negative selection with anti-Lyt-1.2 serum are incapable of inducing such a reaction; and (iii) Lyt 1 + 2+ cells directly or indirectly play an additional role in generating a maximal LIA response.     

Augmentation of killer activity of murine spleen cells against allogeneic and syngeneic tumor cells by inoculation of alloantigen in vivo

After C57BL/6 (B6) mice were inoculated with BALB/c spleen cells via tail vein, kinetics of cytotoxic activities in the B6 mice against sensitizing alloantigens (H-2d) and against syngeneic antigens were investigated using, as target cells, P815 mastocytoma cells (H-2d) and B16 melanoma cells (H-2b). Cytotoxic activity against P815 in the B6 spleen cells reached a peak 3 days after alloantigen inoculation, decreased drastically on day 5 and rose again thereafter. The profile of anti-B16 cytotoxic activity was similar to that of anti-P815 activity. The cytotoxic activity against P815 was inhibited partially by cold B16, but that against B16 was not inhibited by cold P815. Surface phenotype of cytotoxic cells against P815 was Lyt2+, Thy1+, Asialo GM1+ and that of cytotoxic cells against B16 was Lyt2-, Thy1+/-, and Asialo GM1+. The results indicate that inoculation of B6 mice with allogeneic BALB/c spleen cells induce two types of cytotoxic cells; one is similar to lymphokine-activated killer (LAK) cells and the other is activated natural killer cells.     

Soluble factors in tolerance and contact sensitivity to DNFB in mice. VIII. Regulation of T suppressor cell function by autoreactive T helper cells

When cultured with DNP-labeled I-A+ cells, Lyt 2+ T suppressor cells (Ts) from 2,4,-dinitrobenzene sulfonate (DNBS)-tolerized mice are activated to synthesize and release a suppressor factor (SSF) which suppresses the transfer of contact sensitivity to DNFB. The signals required to activate the DNBS-primed Ts to produce SSF were studied in greater detail. As previously observed with fixed DNP-labeled spleen cell stimulators, the supernatants from cultures of DNBS-primed spleen cells and glutaraldehyde-fixed DNP-labeled P388D1 cell monolayers did not contain SSF. When the tolerant cells were harvested from these monolayers and were treated with IL-1, the Ts released the synthesized SSF. Synthesis and release of SSF required Ts recognition of DNP/class I MHC on the hapten-presenting cells followed by interaction with the costimulator IL-1. When the tolerant cells were cultured with fixed DNP-labeled I-A+ or I-A- stimulators to induce SSF synthesis, release was induced by adding either unlabeled or TNP-labeled unprimed spleen cells to the cultures. The release of SSF was blocked when the second stimulators were pretreated with anti-I-A antibody but not with anti-DNP or anti-class I MHC antibodies. These results indicate that the release of SSF by DNBS-primed Lyt 2+ Ts is regulated by the activity of a self-I-A-reactive (i.e., autoreactive) T cell in the tolerant spleen cell population.     

T-cell responses induced by the parenteral injection of antigen-modified syngeneic cells. III. Dissociation of primed cytolytic T-cell and efferent suppressor-T-cell activity following intravenous injection of trinitrophenol-modified syngeneic spleen cells

The parenteral injection of ligand-coupled syngeneic spleen cells has profound effects on immune responsiveness. In this regard, it was examined whether the primed in vitro trinitrophenol (TNP)-specific cytotoxic T-lymphocyte (CTL) responses observed in splenic T-cell populations from mice injected intravenously (iv) with syngeneic TNP-modified spleen cells (TNP-SC) are related to the efferent-acting suppressor-T-cell (Ts) activity observed in splenocytes from iv primed mice. Treatment of mice with cyclophosphamide, adult thymectomy, or monoclonal anti-I-J antiserum prior to the iv injection of TNP-SC was found to eliminate the ability of splenic Ts from these mice to suppress the passive transfer of delayed-type hypersensitivity (DTH) mediated by trinitrochlorobenzene-immune T cells. In contrast, spleen cells from these pretreated mice showed no impairment in the development of augmented TNP-specific CTL responses upon in vitro restimulation with TNP-SC. Separation of the two activities was also achieved in a kinetic analysis. It is concluded that specific enhancement of CTL responsiveness induced by the iv injection of TNP-SC is related to the expansion of a population prelytic Lyt 2+ CTL effector cells which does not appear to contain efferent-acting Lyt 2+ Ts active in suppressing DTH expression.     

Characteristics of 2 types of antigen-binding T suppressors formed during an immune response using T-T hybridomas and their extracts

Antigen-binding T cells of mice immunized with low doses of syngenic spleen cells modified by 2,4,6-trinitrophenyl sulphonic acid, were fused with BW 5147.3.13 thymoma subclone. Suppressor hybridomas were not identical in their differentiating antigens and functional activity. Extract from Lyt 1-2+ hybridoma suppressed efferent and afferent limbs of delayed type hypersensitivity (DTH) in recipients sensitized with subcutaneous injection of 3 X 10(7) TNP-SC. Extract from Lyt 1+2+ I-hybridoma suppressed only afferent DTH limb. It is suggested that during DTH induction with low doses of hapten-modified cells the generation of different types of antigen-binding DTH T suppressors takes place.     

IgG or IgM monoclonal antibodies reactive with different determinants on the molecular complex bearing Lyt 2 antigen block T cell-mediated cytolysis in the absence of complement

Five rat monoclonal antibodies have been derived that express specificities for determinants present on the molecular complex bearing the Lyt 2 antigen. SDS-polyacrylamide gel electrophoresis of 125I-labeled polypeptides precipitated by each of these antibodies reveal 3 components (150,000, 75,000, and 33,000 daltons), and 2 components (44,000 and 33,000 daltons) when analyzed under nonreducing and reducing conditions, respectively. Two of these antibodies are IgG and are specific for the Lyt 2.2 determinant; the other 3 are IgM and react with determinants other than Lyt 2.2, which are nonpolymorphic. Each of the 5 antibodies can block the cytolytic activities of 5-day MLC cells or of cloned cytolytic T cells in the absence of C. Treatment of responding spleen cells with any of these antibodies and C inhibits the generation of cytolytic activity in MLC.     

Intrathymic T cell repertoire after prenatal trinitrobenzene-sulfonic acid-treatment

It is still a matter of debate, whether tolerance toward self-non-MHC antigens is due to intrathymic deletion or to regulatory processes in the periphery. To further pursue this question, responsiveness toward TNP and an anti-TNP monoclonal antibody (Sp6) carrying a recurrent idiotype was evaluated in prenatally trinitrobenzenesulfonic acid (TNBS)-treated mice. In prenatally untreated as well as in TNBS-treated mice, thymocytes proliferating in the absence of nominal antigen were double negative (L3T4-/Lyt2-), but antigen-specific thymocytes were single positive (L3T4+/Lyt2- or L3T4-/Lyt2+). TNBS-treated mice differed from controls inasmuch as in their first week of life T cells proliferating in response to TNP were found in the thymus and detected at increased frequencies in the spleen. The frequency of TNP-specific thymocytes and spleen cells declined rapidly, finally reaching in the spleen a level of 20-30% of controls. Furthermore, after antigenic stimulation, the frequency of thymocytes and spleen cells proliferating in response to TNP was found to be increased in control mice, but TNP-specific T cell were no more recovered in the thymus or the spleen of tolerized mice. The same accounted for thymic and splenic T cells proliferating in response to Sp6. They were expanded in control mice after antigenic stimulation, but were undetectable in TNBS-treated mice. Thus, T cells with specificity for an internal (Sp6) and an external (TNP) antigen, provided the latter was present during ontogeny, were detected in the thymus of control and, transiently, in the thymus of tolerized mice. But, the fate of antigen-specific thymocytes was different in prenatally untreated and TNBS-treated mice. The data are interpreted in the sense that tolerance toward non-MHC antigens may be acquired subsequently to tolerance toward self-MHC antigens and possibly after imprinting of antigen specificity.     

Analysis of a fetal calf serum-induced T cell line. I. H-2 restricted growth and expression of membrane-bound and released molecules bearing 5936-idiotypic determinants

It was previously established that a fetal calf serum-induced C57BL/6 T cell line that induces T and B cell differentiation could be kept proliferating in vitro only if cultured in the presence of irradiated syngeneic spleen cells and FCS. The present experiments were performed in order to investigate a) whether this cell line was a pure T cell line, b) whether the cells in this cell line (called line 12) were homogeneous with regard to Lyt phenotype, c) whether its growth was H-2 restricted, and d) whether line 12 cells reacted with our anti-idiotype (5936) and anti-T cell receptor allotype/isotype (6036) antisera. The results showed that line 12 consisted of T cells of Lyt 1+, 2.3- and phenotype. Its growth and proliferation was restricted to Kb and/or IAb alloantigen, and this phenomenon was observed with isolated Lyt 1+, 2.3- T cells. Line 12 cells reacted with both 5936 and 6036 antisera, and the positive cells were of Lyt 1+, 2.3- phenotype. Thus, our data indicate that Lyt 1+, 2.3- line 12 T cells interact with FCS and Kb/IAb alloantigen via receptors, which may bear 5936 and 6036 antisera-defined determinants. However, because these antisera only react with a subpopulation of Lyt 1+, 2.3- cells, proof that the same T cell has both MHC specificity and B cell idiotypic determinants will require further experimentation. 5936 and 6036 antisera-reactive molecules could be isolated from the supernatants of line 12 cells. Such molecules had characteristics similar to the 50,000 m.w. form of receptor molecules isolated from B6 anti-CBA T cell supernatants: a single chain polypeptide carrying both 5936 and 6036 antisera-defined determinants.     

In vivo and in vitro synergistic antitumor effect of interleukin-2-cultured tumor-bearer spleen cells and immune fresh spleen cells

Summary The synergistic antitumor effect of interleukin-2(IL-2)-cultured tumor-bearer spleen cells (cultured lymphocytes) and immune fresh spleen cells was examined. Tumor-bearer cultured lymphocytes were obtained by culturing BALB/c spleen cells from syngeneic MOPC104E-tumor-bearing mice for 11 days with crude IL-2 and a soluble tumor extract. These cultured lymphocytes had weak antitumor activity when transferred i.p. into tumor-bearing mice that had been inoculated i.p. with 10⁵ tumor cells 5 days previously. Immune fresh spleen cells, obtained from mice in complete remission after the treatment with cyclophosphamide, also had weak antitumor activity when transferred at the same schedule. The cultured cells and the fresh cells, mixed together before transfer, significantly augmented the therapeutic effect. At least 1×10⁷ tumor-bearer cultured lymphocytes and 4×10⁷ immune cells were needed for the synergistic effect. A tumor-specific combination was needed for both cultured and fresh cells. The effective subpopulation of tumor-bearer cultured lymphocytes was a cytotoxic one from an Lyt2⁺ precursor, and that of the immune fresh spleen cells was noncytotoxic, Lytl⁺ and Lyt2⁺ T-cells.A similar synergistic effect was also observed during in vitro coculture of tumor-bearer and immune cells. Cytotoxicity, as assessed by the ⁵¹Cr-release test, of tumor-bearer IL-2-cultured lymphocytes was maintained most effectively after 3 or 4 days of culture without IL-2 when the lymphocytes were cocultured with immune fresh spleen cells and tumor cells.     

Thymus cell differentiation and in vivo T-cell migration. I. Migration of lectin-selected thymocytes

The in vivo quantitative distribution and tissue positioning of mouse thymocytes selected in vitro by Lyt phenotype and lectin binding properties were examined. Lyt 1+2- thymocytes were selected for by cytotoxic elimination; peanut agglutinin (PNA) and soybean agglutinin (SBA) binding and nonbinding thymocyte fractions were separated by an agglutinin technique. Selected cell suspensions were labelled in vitro with 51chromium (51Cr) or [3H]adenosine. Labeled washed cells were injected intravenously into syngeneic recipients which were killed at 1, 24 or 48 hr. In recipients of 51Cr-labeled cells, tissues were collected for gamma counting, and the overall percentage recovery of injected radiolabel from the various tissues was assessed. Tissues collected from recipients of [3H]adenosine-labeled cells were fixed, sectioned, and processed for autoradiography; the positioning of labeled cells within the tissues was determined. Selected Lyt 1+2-, PNA-, and SBA- sets all showed significantly enhanced entry into lymph nodes and intestinal lymphoid tissues. Entry of SBA+ cells into these tissues was comparable to that of peripheral T cells. PNA- and SBA- selected sets, but not Lyt 1+2- selected cells, also showed increased localization to the spleen and lungs, and decreased localization to the liver. By autoradiography, PNA- cells entered lymphoid tissues much more than PNA+ cells, and at 1 hr fewer PNA+ cells in spleen were associated with lymphoid follicles. At 24 and 48 hr almost all labeled cells in lymphoid tissues were positioned in T-dependent areas. These results suggest that enrichment for thymocyte subpopulations described as "mature" also enriches for cells with the ability to enter lymphoid tissue. They also suggest that interactions at other tissue sites are important in the determination of in vivo migration, and that surface carbohydrate composition is an important factor in this determination.