Ly Phenotype Research Articles

Demonstration of Functional Heterogeneity of T lymphocytes and Identification of Their Two Major Subsets.

Demonstration of Functional Heterogeneity of T lymphocytes and Identification of Their Two Major Subsets.

Unusual phenotype of B cells in the thymus of normal mice.

A small number of B cells are found in the thymus of normal mice. A population of B lymphocytes could be enriched to greater than 90% purity by isolating a low-density fraction on Percoll density gradients and then depleting T cells with a mixture of anti-Thy-1, CD4, and CD8 mAbs and complement. Enrichment was monitored by surface Ig staining and by functional studies (responsiveness to LPS, and to anti-mu plus IL-4). When the phenotype of these B cells was studied by flow cytometry, 60-80% had the phenotype Ly-1+ (CD5), Ia+, B220low (CD45R), and Mac-1+ (CD 11b). In contrast, splenic B cells lacked CD5 and CD11b and expressed higher levels of B220 and Ia antigens. These results indicate that most thymic B cells have the phenotype of the Ly-1 B cell subset, which was identified previously as a trace subpopulation in some peripheral tissues and is thought to play a role in autoantibody formation.

Down-regulation of murine collagen-induced arthritis by a T cell hybridoma.

T cell hybridoma cell lines were generated by somatic cell fusion of BW 5147 myeloma cells and splenic cells from mice suppressed for collagen-induced arthritis (CIA). Two cell lines were characterized for their cell surface phenotype, antigen recognition and capacity to down-regulate the erythema and edema associated with CIA. Cell line T101N was determined to portray the cell surface phenotype Ly1+2- L3T4- Thy1+ by a direct binding assay. Cell line T104B1 was determined to express only the Thy1+ alloantigen. Panning studies, measurement of IL-2 production in vitro and the suppression of antibodies to type I and type II collagen in vivo indicate that the hybridoma cells are not isotype specific in their recognition of the polymorphic interstitial collagens. Down-regulation of the erythema and edema of CIA occurred on injection of 1 X 10(5) T101N cells but not T104B1 cells. Histology of the tarsus region of the hind paw of CIA mice 33 days after the administration of T101N cells showed contrasting histopathology compared to that of CIA mice. The joints of CIA mice given T101N cells showed aligned articular surfaces resembling normal joint structure and only residual pannus. The data indicate that collagen-specific cloned T cell lines can modulate the gross pathology and joint architecture of joints exhibiting CIA.

Studies on the recovery from tolerance to tumor antigens. I. Bone marrow cells from tolerant hosts are not rendered tolerant, but provide potential to reconstitute tumor-specific effector T cell clones.

The present study investigates the potential of bone marrow cells from mice tolerant to tumor antigens to repopulate tumor-specific effector T cells. C3H/He mice were inoculated i.v. with 10(6) 10,000 R X-irradiated syngeneic X5563 plasmacytoma tumor cells three times at 4-day intervals. This regimen abrogated the ability of spleen cells from these mice to develop anti-X5563 cytotoxic and in vivo protective (tumor-neutralizing) T cell-mediated immunity as induced by i.d. inoculation of viable X5563 cells followed by surgical resection of the tumor. Since such suppression was induced in a tumor-specific way, this represented a state of antitumor tolerance. When bone marrow cells from normal or X5563-tolerant mice were transferred i.v. into 950 R X-irradiated syngeneic C3H/He mice, both groups of recipient mice generated anti-X5563 tumor immunity over a similar time course and to almost the same degree. Anti-X5563 tumor immunity induced in (C3H/He X C57BL/6) F1 mice which had been transferred with bone marrow cells from normal or X5563-tolerant C3H/He mice were mediated by T cells expressing the Ly phenotype of C3H/He, but not of C57BL/6, excluding the possibility that the antitumor effector cells were derived from recipient mice. It was also demonstrated that C3H/He mice which had been reconstituted with normal marrow were rendered tolerant when the tolerance regimen was started 7 weeks, but not 1 week after the bone marrow reconstitution. These results indicate that bone marrow cells from antitumor tolerant mice are not rendered tolerant to the tumor but can provide the potential to repopulate antitumor CTL and in vivo protective effector T cells.

Qa alloantigen expression on functional T lymphocytes from spleen and thymus.

The cell-surface expression of the class I alloantigen Qa-2 was analyzed on resting and activated spleen and thymus cells using cytotoxic elimination and immunofluorescence and flow cytometry. Spleen cells activated by mitogens or alloantigen were homogeneously positive for cell surface Qa-2, but activated splenic T cells expressed only about one-third as much Qa-2 per cell as did nonstimulated T cells. These data correlated with the ability to perform cytotoxic elimination with Qa-2-specific monoclonal antibodies (mAbs) in that cytotoxic T lymphocyte (CTL) activity was completely abrogated by pretreatment of spleen cells prior to in vitro culture but was only partially eliminated by treatment of CTL effectors. Qa-2-positive cells constituted only a small subpopulation of fresh normal thymocytes, but were enriched (greater than 40% positive) among cortisone-resistant thymocytes (CRT). These Qa-2-positive CRT contained mature thymocytes as defined by Ly phenotype Ly-2-, Ly-1hi. When normal thymocytes were treated with Qa-2-specific mAb and complement prior to in vitro sensitization for generation of allogeneic CTL, CTL activity was completely abrogated despite the fact that the fraction of cells eliminated were undetectable as assessed by cell recovery. CTL effectors from alloantigen-stimulated thymocytes were also susceptible to cytotoxic elimination with Qa-2-specific mAb. These data suggest that the Qa-2 molecule may serve not only as a marker on resting and activated peripheral T cells, but also as a unique marker for functionally mature T cells in the thymus.

The augmentation of tumor-specific immunity by virus help. III. Enhanced generation of tumor-specific Lyt-1+2- T cells is responsible for augmented tumor immunity in vivo.

The role of vaccinia virus-reactive helper T cells (Th) in augmenting in vivo generation of antitumor protective immunity and the Ly phenotype mediating the enhanced in vivo tumor immunity were investigated. C3H/HeN mice were inoculated i.p. with viable vaccinia virus to generate vaccinia virus-reactive Th activity. The mice were subsequently immunized i.p. with virus-infected syngeneic X5563 and MH134 tumor cells, and spleen cells from these mice were tested for in vivo tumor neutralizing activity. Immunization of virus-primed mice with virus-uninfected tumor cells and of virus-unprimed mice with virus-infected tumor cells failed to result in in vivo protective immunity. In contrast, spleen cells from mice immunized with virus-infected tumor cells subsequent to virus-priming exhibited potent tumor-specific neutralizing activities. Such an augmented generation of in vivo protective immunity was accompanied by enhanced induction of tumor-specific cytotoxic T lymphocyte (CTL) and antibody activities in X5563 and MH134 tumor systems, respectively. However, analysis of the effector cell type responsible for in vivo tumor neutralization revealed that enhanced in vivo immunity was mediated by Lyt-1+2- T cells in both tumor systems. Moreover, the Lyt-1+2- T cells exerted their function in vivo under conditions in which anti-X5563 tumor-specific CTL or anti-MH134 tumor-specific antibody activity was not detected in recipient mice. These results indicate that augmenting the generation of a tumor-specific Lyt-1+2- T cell population is essential for enhanced tumor-specific immunity in vivo.

The antimetastatic function of concomitant antitumor immunity. I. Host Ly-1+2+ effector T cells prevent the enumeration of metastatic tumor cells in a biological assay.

A mouse survival assay was evaluated for its suitability to enumerate metastatic P815 tumor cells in the draining lymph node and spleen of a B6D2 F1 (H-2b X H-2d) host bearing a primary intradermal P815 tumor. The mouse survival assay is based on the linear relationship between the log10 number of P815 tumor cells (H-2d) injected i.p. into mice and their mean survival time. It was found that the assay is capable of quantifying as few as 10 tumor cells in lymph node and spleen, but only if cell suspensions of these organs are treated with anti-H-2b serum and complement, in order to selectively destroy H-2bd host cells. This was necessary because host cells from the lymph node and spleen of a tumor-bearing host possessed antitumor functions, in that they were capable of destroying the H-2d P815 tumor cells when admixed with the tumor cells and injected i.p. into 800-rad irradiated test recipients. The kinetics of acquisition and loss of host cells with antitumor function and the Ly phenotype of these host cells suggest that they are the same cells that give the tumor-bearing host the capacity to express concomitant immunity against a tumor implant.

The role of tumor-specific Lyt-1+2- T cells in eradicating tumor cells in vivo. I. Lyt-1+2- T cells do not necessarily require recruitment of host's cytotoxic T cell precursors for implementation of in vivo immunity.

The present study determines the Ly phenotype of T cells mediating tumor cell rejection in vivo and investigates some of cellular mechanisms involved in the in vivo protective immunity. C3H/HeN mice were immunized to syngeneic X5563 plasmacytoma by intradermal (i.d.) inoculation of viable X5563 tumor cells, followed by the surgical resection of the tumor. Spleen cells from these immune mice were fractionated by treatment with anti-Lyt antibodies plus complement, and each Lyt subpopulation was tested for the reconstituting potential of in vivo protective immunity in syngeneic T cell-depleted mice (B cell mice). When C3H/HeN B cell mice were adoptively transferred with Lyt-1-2+ T cells from the above tumor-immunized mice, these B cell mice exhibited an appreciable cytotoxic T lymphocyte (CTL) response to the X5563 tumor, whereas they failed to resist the i.d. challenge of X5563 tumor cells. In contrast, the adoptive transfer of Lyt-1+2- anti-X5563 immune T cells into B cell mice produced complete protection against the subsequent tumor cell challenge. Although no CTL or antibody response against X5563 tumors was detected in the above tumor-resistant B cell mice, these mice were able to retain Lyt-1+2- T cell-mediated delayed-type hypersensitivity (DTH) responses to the X5563 tumor. These results indicate that Lyt-1+2- T cells depleted of the Lyt-2+ T cell subpopulation containing CTL or CTL precursors are effective in in vivo protective immunity, and that these Lyt-1+2- T cells implement their in vivo anti-tumor activity without inducing CTL or antibody responses. The mechanism(s) by which Lyt-1+2- T cells function in vivo for the implementation of tumor-specific immunity is discussed in the context of DTH responses to the tumor-associated antigens and its related Lyt-1+2- T cell-mediated lymphokine production.

The role of T cell subsets in the generation of secondary cytolytic responses in vitro against class I and class II major histocompatibility complex antigens.

Strain combinations generating cytotoxic T lymphocytes (CTL) specific for a single class I (K or D) or class II (A or E) MHC molecule were set up. The responder cells were separated into Ly subsets (Ly-1+2-, Ly-1-2+, and Ly-1+2+) on day 5 of culture by using lytic or non-lytic selection techniques and monoclonal Ly-specific antibodies. The separated subsets were restimulated on day 8 and tested for secondary CTL activity on day 12. Class II-specific secondary CTL could be generated from all three subsets, whereas class I-specific CTL developed only in the Ly-1+2+ and Ly-1-2+ subsets. The Ly-1+2+ cells underwent a phenotypic shift to Ly-1-2+ by day 12, whereas CTL generated from the Ly-1+2- and Ly-1-2+ subsets retained their phenotype up to the secondary effector stage. The cells separated according to their Ly phenotypes on day 5 were the progeny of unprimed progenitors expressing the same Ly phenotypes. Unprimed Ly-1+2+ cells gave rise to CTL in the absence of the other subsets, while unprimed Ly-1+2- and Ly-1-2+ cells required the help of Ly-1+2+ cells (or soluble factors) during priming to become non-lytic CTL precursors by day 5, and cytolytic cells after restimulation. The Ly-1+2- subset could generate class II-specific secondary CTL only in the absence of the other two subsets. Apparently, alloantigen-primed Ly-1+2+ and Ly-1-2+ cells suppressed the development of cytolytic activity in the Ly-1+2- subset. The combined data provide a comprehensive pathway of CTL differentiation from T cell subsets.

Different forms of Ly-5 within the T-cell lineage.

The Ly-5 system, defined both by alloantibodies (Komuro et al. 1975) and by xenoantibodies (Omary et al. 1980, Siadak and Nowinski 1980), is noted for molecular differences that distinguish various hematopoietic cell lineages (Michaelson et al. 1979). A 200K (Mr 200000) is expressed by T cells, a 205K form occurs on macrophages, and a 220K form [which can be separately identified by xenoantibody (Coffman and Weissman 1981, Dalchau and Fabre 1981, Kincade et al. 1981) and may therefore identify an epitope lacking in the smaller forms] is characteristic of B cells. This relation of molecular form to cell lineage has been confirmed by ascertaining the Ly-5 molecular phenotypes of cloned cell lines representing T, B, and macrophage lineages (Tung et al. 1981). Extending our survey to a further range of cloned T-cell lines, we now find that the T lineage itself is diverse in expression of distinguishable Ly-5 molecular forms. Each of the 10 cell lines, shown in Table 1, seven of which have the phenotype Ly-23 and three the phenotype Ly-1, was examined in the usual way by cell surface radioiodination, immunoprecipitation (with monoclonal Ly-5.1 alloantibody; Shen 1981), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Figure 1 shows that all three Ly-1 lines express the familiar 200K form previously found to typify T cells, whereas all seven Ly-23 lines expressed two forms, 210K and 215K, neither of which has previously been seen in any lineage. These two newly identified forms bring the number of distinguishable Ly-5 forms to five, and they are shown in Figure 2 to be separable from the three other previously known forms. All five forms were recognizable in combined lysates of spleen cells and CTLL-R8 Ly-23 cells (Fig. 2). The reason why the 210K and 215K forms were not observed before is probably that Ly-23 is a small cell set that requires cloning or other enrichment to provide enough material.

Alloreactive T-cell clones. Ly phenotypes predict both function and specificity for major histocompatibility complex products.

We have studied the association of Ly phenotype with function and specificity for major histocompatibility complex (MHC) products by examining the properties of 21 T-cell clones derived from B10 anti-B10.D2 and B10.A anti-B10.D2 mixed lymphocyte cultures (MLC). T cells were selected after MLC solely on the basis of Ly phenotype, cloned by limiting dilution, and tested for stability of Ly phenotype, function and specificity for class I or class II MHC products. Sixteen Ly-1+2- and five Ly-1-2+ T-cell clones were tested. The clones selected for the Ly-1+2- phenotype maintained this phenotype, expressed helper but not lytic function, and recognized class II MHC products (I-Ad or I-Ed). All Ly-1-2+ clones maintained this phenotype, possessed cytolytic but not helper activity, and recognized class I MHC products (Dd and Ld). Our data therefore confirm at the clonal level the original observations of a remarkably consistent correlation between Ly markers, MHC specificity, and function. They suggest that the expression of Ly antigens on T-cell clones forms part of a genetic program for each of these specialized cells that also determines their function and MHC specificity.

Adult thymectomy does not alter the proportion of T cells of the Ly 123 subclass

The murine T cell antigens Ly 1, 2 and 3 have been useful in defining functionally distinct T cell subpopulations1. However, the relationship between Ly 123+ cells and Ly 1+23− cells is not clear2–4 It has been claimed5,6 that all cells leaving the thymus are Ly 123+ the implication being that these must be the precursors of Ly 1+23− cells and that maturation involves the loss of Ly 2 post-thymically. Previous work has shown a 50% drop in the proportion of T cells of the Ly 123+ subclass following adult thymectomy5. The interpretation was that the Ly 123+ cells differentiated into Ly 1+23− cells and that no Ly 123+ migrants from the thymus were replacing them. However, when we directly measured the Ly phenotype of migrant cells within a few hours of their exit from the thymus7, we found they were mature in phenotype and already contained Ly 1+23− cells in the same proportion (∼60%)8 as peripheral T cells. This result suggests that adult thymectomy should not affect the subclass distribution of T cells. We have therefore repeated the adult thymectomy experiments using more precise techniques (flow cytometry and monoclonal antibodies) and the results reported here, in contrast to those of the earlier study5, did not show any change in the proportion of Ly 123+ cells after adult thymectomy, even though total T cell numbers were substantially reduced.

T cell-mediated immunity in malaria. I. The Ly phenotype of T cells mediating resistance to Plasmodium yoelii.

CBA mice that recover from Plasmodium yoelii 17X infection are resistant to reinfection. T cells from these mice transfer immunity to nonimmune recipients. To analyze the nature and mode of action of these T cells, we transferred selected subsets into T cell-deprived recipients. Treatment of the T cells with anti-Ly-1 but not anti-Ly-2 serum and C abrogated their ability to transfer immunity. A mixture of anti-Ly-1 and anti-Ly-2 serum-treated cells (i.e., a population devoid of Ly-123 cells) transferred a level of immunity comparable to that of unselected T cells. Hence, the T cells mediating resistance to P. yoelii 17X were primarily of the Ly-1+23- phenotype. T cell-deprived mice reconstituted with these immune Ly-1 cells developed a) high levels of IgM and IgG antibodies, b) DTH responses to parasitized RBC, and c) enhanced blood monocyte responses. The addition of immune B cells to the Ly-1 population dramatically increased its ability to transfer immunity and induce antibody production. B cells from immune CBA/N mice had no such effect. Thus, the transfer of optimal protective immunity against malaria stems from an interaction between Ly-1 cells and a select B cell subset that CBA/N mice lack. This "selective synergy" is a protective mechanism against pathogens that has not been previously appreciated.

Characterization of lymphocytes from rejected and nonrejected islet xenografts.

A procedure is described for obtaining lymphocytes from xenografts of rat islets transplanted beneath the renal capsule of diabetic mice. In acute rejection of transplants of fresh rat islets, the lymphoid reaction was composed of 90% T lymphocytes with a predominance of Ly-2 cells. The Ly-2 cells were presumably cytotoxic T lymphocytes. On the other hand, if the islets are pretreated to avoid rejection, by culture in 95% O2 and administration of antilymphocyte serum to the recipients, the lymphocytes that are attracted by the graft are quite different. First, the percentage of T lymphocytes decreased, although they continue to be the most common cell. Second, however, the Ly phenotype was altered. Early after transplantation the Ly-2 population was decreased relative to Ly-1 cells. By day 70, the proportion of Ly-2 cells had returned to that of infiltrates actively rejecting the grafts, even though no rejection was evident. It is possible that the Ly-2+ cells present in nonrejected, established islet xenografts may be suppressor lymphocytes.

Interactions between molecules (subfactors) released by different T cell sets that yield a complete factor with biological (suppressive) activity.

T cells that have been immunized to express optimal levels of contact hypersensitivity upon adoptive transfer to normal animals can be inhibited from doing so by incubating them with an antigen-specific T suppressor factor. This factor is composed of at least two subunits which come from cells expressing different Ly phenotypes; an antigen-specific antigen-binding "subfactor" is made by an Ly-1 cell and a non-antigen-binding one is made by an Ly-2 cell. Neither of these cells nor their products express detectable amounts of major histocompatibility gene products. The mode of immunization plays an important role in determining which of these subfactors will be produced. Painting the skin with a reactive hapten immunizes Ly-1 cells that secrete antigen-binding material, whereas intravenous injection of trinitrobenzenesulfonic acid activates Ly-2 cells to produce a second subunit that does not see antigen. There is reason to believe that the molecule that does not bind to antigen does have some antigen specificity. An analysis of the data at hand suggests that the antigen specificity stems from an interaction of the two subunits described with yet another subunit and that biological activity is dependent upon three macromolecules. Thus, the complex level of cellular interactions that regulate immunity may also be reflected in a similar type of complexity in the interaction between their biologically active cell-free products.

T cell development. Precursors and inducer requirements of helper effector and feedback suppression inducer cell sets.

The surface phenotypes and differentiative history of specific helper-effector (HE) and specific feedback suppression-inducer (FBSI) cell sets were further defined in reference to the Qa-1 and I-J marker systems by culture of selected sets of cortisone-resistant nylon-purified thymocytes with antigen on primed macrophages. The generation of Ly-1:HE and Ly-1:FBSI cell sets required, in each case, two initiating sets: a precursor set and a differentiation-inducing set. Precursor sets were distinguished from inducer sets by genetic markers. Accordingly, HE cells, phenotype Ly-1:Qa-1-:I-J-, differentiated from Ly-123:Qa-1- cells in the presence of Ly-1:Qa-1+:I-J+ inducer cells; and FBSI cells, phenotype Ly-1:Qa-1+:I-J+, differentiated from Ly-123:Qa-1- in the presence of Ly-1:Qa-1+:I-J+ inducer cells. The Ly-123:Qa-1-precursors of HE and FBSI cells have been distinguished from one another previously but there is as yet no evidence whether differentiation of these precursor sets requires the same or different Ly-1:Qa-1+:I-J+ inducer sets.

Contrasuppression. A novel immunoregulatory activity.

We have described an interaction between two T cells subsets that results in interference with the expression of Ly-1-, 2+ (Ly-2) T cell-mediated suppression. We refer to this novel immunoregulatory activity as contrasuppression. The T cell responsible for the induction of contrasuppression (inducer cell) expresses the phenotype Ly-1-, 2+;I-J+;Qa-1+. This phenotype distinguishes it from the suppressor effector cells which we find to be I-J-2.3. An I-J+ soluble mediator from the contrasuppressor inducer cell acts on another cell (acceptor cell) that expresses the phenotype Ly-1+, 2+; I-J+; Qa-1+. This phenotype distinguishes it from T helper cells. Both the inducer cell (or its biologically active mediator) and its acceptor cell are required for the expression of contrasuppression. Because contrasuppressor cells can block the suppressive activity of cell-free mediators released by Ly-2 suppressor T cells, the mechanism of contrasuppression is either separated from or in addition to the inactivation of suppressor cells themselves. The potential importance of contrasuppressor activity in the regulation of suppressor T cell activity in allowing immunologic memory to be expressed and in permitting microenvironmental immune regulation is discussed.

Cells mediating graft rejection in the mouse. I. Lyt-1 cells mediate skin graft rejection.

The Ly phenotype of cells mediating skin graft rejection was determined using monoclonal anti-Lyt-1.1 and Lyt-2.1 antibodies in CBA mice that received CBA lymphoid cells from mice sensitized to C57BL/6; i.e., alloantigenic differences arising from the H-2 and non-H-2 loci. It was clear that graft rejection was due wholly to the presence of Lyt-1 cells in the inoculum and that Lyt-123 or Lyt-23 cells had no effect. Furthermore, no synergism was noted between Lyt-1 and Lyt-2 cells. In this model, both the cytotoxic T cell and cytotoxic lymphocyte precursors were shown to be Lyt-123 and these could be depleted from sensitized Lyt-1 populations that mediated graft rejection. Thus cytotoxic T cells are not responsible for skin graft rejection, but rather, this is mediated by an Lyt-1 cell. Whether this T cell is distinct from other Lyt-1 cells (T helper, T cells mediating delayed hypersensitivity) is not clear at present, but other evidence, and traditional concepts, link graft rejection and delayed type hypersensitivity as being different manifestations of the same mechanism.

Analysis of intrathymic differentiation patterns during the course of AKR leukemogenesis

Thymocytes from AKR mice in different stages of leukemia development were analyzed with the fluorescence-activated cell sorter (FACS) using monoclonal antisera to Lyt-1, Lyt-2, Thy-1.1, H-2K k, and Ia k. In addition, the number of cells bearing receptors for peanut agglutinin (PNA) was assessed. The results were correlated with the expression of murine leukemia virus (MuLV) antigen. Thymocytes from late preleukemic and leukemic stages were found to have a phenotype characteristic of a more mature cell population in that there was an increase in the expression of determinants encoded within the K end of H-2 k and Ia k. This was associated with a decrease in the number of thymocytes bearing receptors for PNA during the leukemic stage. Simultaneously, a shift from a Lyt-1 + 2 + thymocyte population to cells with varying expressions of Ly antigens was observed. Analysis of Lyt determinants on thymomas indicated that they could arise from cells bearing any of the different possible combinations of Ly phenotypes. The cell surface antigen changes occurred in temporal correlation with an increased expression of MuLV antigens.

Analysis of antigen-specific, Ig-restricted cell-free material made by I-J+ Ly-1 cells (Ly-1 TsiF) that induces Ly-2+ cells to express suppressive activity.

A set of T cells defined by a unique profile of cell surface alloantigens (phenotype Ly-1+2-; Qa-1+; I-J+) produces biologically active cell-free material(s) (Ly-1 TsiF) which induces another T cell set (cell surface phenotype Ly-1,2+; I-J/; Qa-1+) to participate in the suppression of primary immune responses to heterologous erythrocytes. The suppression is specific for the inducing antigen, and the Ly-1 TsiF binds antigen in a specific way. The activity of Ly-1 TsiF can be removed by anti-I-J immunosorbents and will not be expressed if the functional producer and acceptor cells do not share gene products that are encoded in or are tightly linked to the VH portion of the Ig complex. There is no requirement for the Ly-1 TsiF and its acceptor cell(s) to share major histocompatibility complex gene products. Thus, for optimal induction of antigen-specific suppression by cell-gree materials from Ly-1 T cells, three necessary conditions must be met: (a) antigen recognition by Ly-1 TsiF; (b) the expression of I-J gene products and (c) identify of VH-linked Ig locus gene products (or other products influenced by those genes) on both the inducer molecule and its acceptor cell. The role of the Ig-linked restriction is particularly intriguing, and its possible meaning is considered in detail.