Search

The unique feature of the Ly-5 system is that it is a major cell surface glycoprotein, representing up to 10 % of the total cell surface complement, confined to the hematopoietic cells as a family of isoforms generated by alternative splicing of a single Ly-5 gene. The cytoplasmic domain of Ly-5 has protein tyrosine phosphatase activity suggesting that Ly5 is involved in signal transduction. We used Ly-5 anti-sense oligodeoxyribonucleotides (oligo) and Ly-5 monoclonal antibody (mAb) to study the functional role of Ly-5 in the concanavalin A mitogenesis response by spleen cells, as well as in the generation of lymphokine-activated killer cells and the proliferative response by spleen cells induced by recombinant human interleukin-2 (rhIL-2). Our results indicate that the Ly-5 mAb could enhance these activities whereas the anti-sense oligo was inhibitory. These data clearly suggest that Ly-5 is involved in the IL-2 and IL-2 receptor responsive circuit.

Immunogenetic cell-surface markers that distinguish sets and subsets of lymphocytes, representing different stages and different lines of maturation within the total lymphoid population, have proved to be oI great value. They have helped to elucidate the immunological functions of different lymphocyte sets, and .have led to findings of much interest in other branches of biology. A serious impediment to wider use of such serological techniques is that the antisera required are often difficult to produce. Among the main problems is that, commonly, only a few immunized mice produce antiserum of adequate titer in the cytotoxicity assay, and of these, many must be discarded because they produce too much T-cell autoantibody. Thus, the volumes of satisfactory antiserum available are grossly inadequate. For this and other reasons (discussed below), we have explored the possibility that antiserum production can be improved by adoptive transfer of lymphoid cells from satisfactorily immunized donors to irradiated syngeneic recipients. The following progress report is sufficiently promising to warrant the attention of interested colleagues. The data refer exclusively to the cytotoxicity assay performed according to Boyse and coworkers (1970) with this exception: we no longer use preabsorbed rabbit serum as the source of complement. In our experience, the best and most ample source of complement is a pool of serum from a few among many rabbits whose serum has been rigorously screened (both before and after exsanguination) for low toxicity for mouse thymocytes combined with high lytic efficiency for thymocytes in cytotoxicity assays with an Lyt antiserum which has a high demand for complement. The conditions for testing the use of adoptive immunization in the three antigenic systems named below were: (i) All mice were females. (ii) All donors had a satisfactory record of antibody production, as defined below for each system, and had reached the limit of their usefulness after many immunizations and bleedings; we could not afford to sacrifice younger donors for an untried procedure. (iii) For adoptive immunization, each syngeneic recipient (aged 8-12 weeks) was subjected to 500R total-body irradiation (Gamma Cell/40, Atomic Energy of Canada, Ltd.)

We have studied the cell-surface phenotype of natural killer (NK) cells of NZB and B6 mice which react to an MuLV+ lymphoid tumor. (a) NK cells do not express Thy1, Ly2, or Ig surface markers. (b) NK cells express an antigen recognized by C3H anti-CE antiserum ('anti-Ly1.2 antiserum'). Inasmuch as NK activity of spleen cells from B6 and B6/Ly1.1 congenic strains were both equally sensitive to C3H anti-CE antiserum, the NK antigen is distinct from Ly1.2. This point was confirmed by the observation that alphaNK activity was removed by absorption of C3H anti-CE antiserum with spleen cells from either B6 or B6/Ly1.1 congenic strains. Absorption of C3H alphaCE serum with BALB/c thymocytes and spleen cells (which are Ly1.2+NK-) removed anti-Ly1.2 activity and left anti-NK activity intact. This absorption step could be circumvented by inserting the BALB/c genotype into the recipient immunized to CE cells (i.e., (C3H X BALB/c)F1 alphaCE spleen cells). This antiserum, provisionally termed 'anti-NK', defines a new subclass of lymphocytes which may play a central role in the immunosurveillance against tumors.

The antiserum (BALB.I-H-2j x SWR/J)F1 anti-I.29 ascites cells, in reaction with B6 lymph-node cells (LNC) in the cytotoxicity assay, defines an alloantigen system called Lna-1 (lymph-node alloantigen-l) which in normal, untreated mice is expressed on the cells of lymph nodes but not of other lymphoid organs. The T- and B-cell populations of lymph nodes evidently include Lna-1+ subpopulations representing 30-40 percent of the total population The Lna-+ phenotype could be induced on cells of thymus and spleen but not of bone marrow. Congenitally asplenic +/Dh mice have no Lna-1+ cells in their lymph nodes, but their LNC can be induced to express Lna-1; this suggests that the spleen is normally required for the differentiation of Lna-1+ cells from Lna-1- precursors. It is not yet known whether thymus is also required for the expression of Lna-1 in lymph nodes. It remains to be seen whether the existence of the Lna-1+ B-cell subpopulation of lymph nodes depends on Lna-1+ T cells, and whether the Lna-1+ phenotype of B cells may be acquired rather than intrinsic. One hypothesis which is the basis of further study is that there is a T-cell pathway in which noninducible bone-marrow cells become Lna-1-inducible in the thymus, then travel to the spleen, where they are induced to become Lna-1+, after which they reside in lymph nodes.

The technique of cell-surface iodination, followed by immuno-precipitation withPC-1.1 antiserum, was applied to normal spleen cells and to MOPC-70A BALB myeloma cells. The results indicate that the cell-surface component bearing PC-1 alloantigen has a molecular weight of from 105,000 to 110,000 daltons and does not resemble any constituent of plasma membranes or MuLV-type virus so far categorized by similar methods. The PC-1 specificity of the molecule was confirmed by comparison of the spleen cells of two mouse strains with spleen cells of their respective PC-congenic partner strains. MOPC-70A myeloma cells, but not spleen cells, yield a fainter band in the PC-1 position in controls in which antiserum is omitted, but peptide maps show that this similarly placed band has no relation to the PC-1 molecule.

Generation of anti-sheep erythrocyte plaque-forming cells (PFC) is greatly reduced in the presence of monoclonal Ly-5 alloantibody. Although Ly-5 is expressed in one of its molecular forms on T cells and macrophages (M phi ) involved in this response, the only demonstrated action of Ly-5 antibody was on B cells. Evidence from elimination of Lyt-2+ cells, and from the responses of serial proportions of Ly-5.1 and Ly-5.2 cells and of Ly-5 heterozygous cells, signifies that PFC reduction cannot be ascribed to any known mechanism of suppression or to a direct suppressive action of Ly-5 antibody on B cells. A critical distinction of Ly-5 from Lyb-2 is that Ly-5 antibody reduces PFC generation to trinitrophenylated Ficoll, a thymus-independent type 2 antigen requiring T cells and M phi for maximal PFC generation in vitro. A second distinction is that PFC reduction by Ly-5 antibody is strictly tied to the time of operation of M phi factor, whereas PFC reduction by Lyb-2 antibody relates to the time of B cell triggering by antigen. Accordingly, M phi factor competitively and quantitatively inhibits the action of Ly-5 antibody in reducing PFC generation. It is likely that the Ly-5 system is concerned in the reception or handling of M phi message by B cells.

The generation of plaque-forming cells (PFC) to T-dependent antigen, but not to T-independent antigen, is reduced in vitro by Lyb-2 antibody. Monoclonal Lyb-2 antibody, added to Mishell-Dutton cultures within the first 2 d, but not later, greatly reduces the generation of alpha-sheep erythrocyte (SRBC) PFC from T-depleted spleen cells whether help is provided in the form of intact T cells or as soluble factors contained in mixed lymphocyte culture (MLC) supernatants. Generation of alpha-SRBC PFC from purified B cells, assisted by soluble factors in MLC and macrophage (P388D.1 cell) supernatants, is similarly reduced by Lyb-2 antibody. The initial 2-d period, during which cultures are diminishingly sensitive to reduction of PFC generation by Lyb-2 antibody, is not affected by the time at which such soluble factors are added. Thus, Lyb-2 cell surface molecules evidently do not function as receptors for these differentiative signals. Reduction of PFC generation by Lyb-2 antibody is antigen dependent in the sense that reduction of the PFC response to one antigen (SRBC) does not affect subsequent generation of PFC to a second antigen (horse erythrocytes) from the same cell population. These findings accord with the view that the Lyb-2 molecule participates in a B cell differentiative phase, probably proliferative, which begins with binding of antigen and precedes the phase in which B cells become fully receptive to signals from T and other cells.

Killer T cells with specificity for major histocompatibility antigens have been shown in mice and rats to display idiotypic receptors allowing the lysis of such cells at the effector phase by anti-idiotypic antibodies and complement. A comparison was made between idiotypes displayed by Lyt-1-2+3+ and Lyt-1+2-3- T blasts, generated in the same mixed leucocyte culture (MLC), across an entire H-2 locus barrier. This was done by absorption of anti-idiotypic antibodies with respective T blasts, followed by estimation of the ability of the absorbed antiserum to inhibit MLC or killer T-cell function. Further, the capacity of Lyt-purified, MLC-generated T blasts to provoke specific unresponsiveness via anti-idiotypic immunity in syngeneic recipients was analyzed. Taken together, the results demonstrate that Lyt-1+2-3- T blasts responsible for the major part of MLC proliferation have distincly different idiotypes from those on the Lyt 1-2+3+ killer T cells. That the idiotypes on the killer T-cell presursors can serve as triggering sites for induction of effector T-cell function was then suggested by experiments with Lyt-1-2+3+-purified, normal T cells as precursor cells in vitro. The fact that autoanti-idiotypic antibodies may circumvent the need for helper Lyt-1+2-3- T cells in the generation of allospecific killer T cells indicates that the former cells may normally function partly via such anti-idiotypic reactions.

Applying a sensitive colorimetric method for serum lipase activity in murine serum, BALB/c were found to have high levels. By contrast. B6 mice and mice of several other strains studied showed low serum lipase activity. Selecting BALB/c and B6 mice for analysis, we studied the influence of genetic crossing and bone marrow transplantation to investigate mechanisms involved in control of serum lipase activity. Compared to BALB/c, F 1 hybrids of these two strains exhibited decreased serum lipase activity, as did B6 mice, suggesting that serum lipase levels may be genetically determined. High levels of serum lipase behaved as an apparent autosomal recessive trait; an autosomal codominant inheritance could not be ruled out, however. Bone marrow transplantation, using a method based on immunologic elimination of post-thymic cells to prevent graft-vs-host disease, showed that B 6→ BALB/c, B 6→ B 6, and BALB/c→ B 6 had low lipase levels, while BALB/c→ BALB/c had high lipase levels. These findings suggest that transplanted marrow cells can influence the level of serum lipase activity if the marrow comes from a donor bearing an apparent genetically dominant trait, even though bone marrow cells themselves, or their descendents, do not exhibit lipase activity or generate lipase.

Relatively large numbers of nonimmune spleen cells do not protect against the local growth of two lymphomas. However, this heterogeneous population of splenic lymphocytes contains a subset of cells that efficiently protects against in vivo tumor growth. This cell population (cell-surface phenotype Thyl.2(-)Ig(-)Ly5.1(+)) represents less than 5 percent of the spleen cell population and is responsible for in vitro NK-mediated lysis. Although these studies clearly and directly demonstrate that Ly5(+) NK cells selected from a heterogeneous lymphoid population from nonimmune mice can protect syngeneic mice against local in vivo growth of two different types of tumor cells (in contrast to other lymphocyte sets within the spleen), they do not directly bear upon the role of NK cells in immunosurveillance. They do indicate that highly enriched Ig(-)Thyl(-)Ly5(+) cells, which account for virtually all in vitro NK activity, can retard tumor growth in vivo. It is difficult to ascribe all anti-tumor surveillance activity to NK cells, because they probably do not recirculate freely throughout the various organ systems of the body. Perhaps NK ceils may play a role in prevention of neoplastic growth within discrete anatomic compartments where there is rapid differentiation of stem cells to mature progeny (e.g., bone marrow, spleen, and portions of the gastrointestinal tract)and may normally act to regulate the growth and differentiation of non-neoplastic stem cells. Long-term observation of chimeric mice repopulated with bone marrow from congenic or mutant donors expressing very low or very high NK activity may help to answer these questions.

Purified Ly1 cells induce other T-cell sets to exert potent feedback inhibitory activity and this T-T interaction has been shown to play an important role in regulating in vivo immune responses. Approximately 2/3 of Ly1 cells also express the Qa1 surface phenotype (Ly1:Qa1+ cells). The experiments reported here indicate that Ly1:Qal+ cells are responsible for induction of feedback inhibition and that signals from both Ly1:Qal+ cells and Ly1:Qal- cells are required for optimal formation of antibody by B cells.

Antigen-stimulated Ly1:Qa1+ cells induce a nonimmune set of T-acceptor cells (surface phenotype Ly123+Qa1+) to participate in the generation of specific suppressive activity. The experiments reported here were designed to test the possibility that the interaction between T-inducer and T-acceptor cells might be governed by genes linked to the Ig locus. We find that inducer:acceptor interactions occur only if the inducer and acceptor T-cell sets are obtained from donor that are identical at the Ig locus and are independent of the Ig locus expressed on the B cells used for assay of T-helper activity. In addition, experiments using inducer and acceptor T cells from the congenic recombinant BAB. 14 strain show that T-T interactions are not governed by Ig-CH genes, per se. These data indicate that T-inducer: T-acceptor interactions are governed by Ig-linked genes that may control expression of VH-like structures on T cells, or control expression of as yet unidentified cell-surface molecules.

When B mice are supplied with Ly1 or Ly23 cells they acquire, over the next 6 mo, only the immune functions associated with each of these T-cell subclasses, respectively. The T-cell population of these "B-Ly1" and "B-Ly23" mice mice also remains restricted to the Ly1 and Ly23 subclass phenotypes. Thus the Ly1 and Ly23 populations are derived from two separate lines of differentiation and are not sequential stages of a single differentiative pathway.

Lethally irradiated AKR mice were reconstituted with C57BL/6 bone marrow cells. Though the allogeneic marrow transplantation protected AKR recipients from acute irradiation deaths, the mice given unmanipulated marrow developed severe GVHR disease, and 80% died within 50 days. The thymus and spleen from the recipient mice, following recovery of body weight between the 10th and 20th days, gradually involuted and became miniscule after Day 30. Thymocytes from recipients were found to be entirely of donor cell type by Day 15. Thereafter, however, as the graft versus host reaction (GVHR) developed, changes in sensitivity of the thymocytes to four different alloantisera directed toward donor histocompatibility antigens (H-2b, Thy 1.2, Lyt 1.2, and Lyt 2.2) were observed and these changes were associated with changes in antigen expression or quantity of Thy 1 antigens on the thymocytes. A different pattern of changes was observed in antigen expression on thymocytes in mice given B6 marrow cells that had been pretreated with anti-Thy 1 serum which prevented initiation of graft-vs-host disease and in the mice which received marrow not so treated and which regularly led to graft-vs-host disease. By contrast, the amount of H-2 antigen on the thymocytes from chimeras with or without GVHR was elevated equally. The mechanisms of these changes are discussed.

Trisomy of chromosome 15 is a common feature in murine T-cell leukemias of chemical, viral and spontaneous origin. It is not present in all T-cell derived lymphomas, however. In order to study whether trisomy 15 was restricted to a specific T-cell subtype, diploid and chromosome 15 trisomic M-MuLV induced leukemias and derived in vitro lines were compared with regard to histology, karyotype, Thy-1,2 and Lyt-1, 2, 3 and 5 antigen expression. Histologically trisomic lymphomas showed a higher mitotic rate, higher peri-cellular reticulum content and less small cell infiltration than diploid tumors. It was not possible to distinguish trisomic from diploid lymphomas in blind tests based on these criterias, however. Trisomic and diploid lymphoma cells were indistinguishable in morphology. Three of nine lymphomas induced by M-MuLV inoculation were trisomic; two of them were thymomas. Transplantation to syngeneic recipients of the opposite sex and study of the sex chromosome marker was used to establish the origin of the enlarged organ. Upon subcutaneous transplantation, trisomic tumors and thymomas grew locally and were of donor cell type whereas splenic lymphomas caused spleen enlargement, as a rule, and were of donor or recipient type. Transplantation of bone marrow from mice infected with M-MuLV as newborns gave rise to lymphomas with the same sex as the donor and established the presence of preleukemic T-cells in the bone marrow of the donor. In vitro lymphoma lines expressed four different patterns of Lyt antigens: (a) 1.2 + , 2.2 + , 3.2 + , 5.1 + (4 lines); (b) 1.2 + , 2.2 − , 3.2 − , 5.1 + (5 lines); (c) 1.2 + , 2.2 + , 3.2 − , 5.1 + (7 lines); (d) individually distinct patterns (3 lines). Each group contained Thy-1.2 − and Thy-1.2 + lines. The trisomic tumors were mostly in group (a), all of them were Thy-1.2 positive. Diploid tumors were randomly distributed largely between groups (b) and (c). Two of three tumors in group (d) were aneuploid (not trisomic for 15). In spite of the differences observed between trisomic and diploid lymphomas, no specific T-cell subset could be assigned to the two groups. This is in line with the finding of trisomy 15 in some B-cell lymphomas suggesting that this anomaly may contribute to the neoplastic change in a variety of lymphocyte subsets, representing different lineages and steps of differentiation.

An I-J-subregion controlled determinant is expressed on Ly-1 inducer and Ly-1,2 acceptor T cells in the feedback suppression circuit. Ly-1 T cells absorb the I-J antibody reactive with the Ly-1,2 acceptor T cell, suggesting that both inducer and acceptor T cells have the same 1-J determinant. Since less than 10 percent of Ly-1 or Ly-1,2 T cells are killed by anti-I-J plus complement treatment, the I-J determinant demarcates functionally distinct subsets of both the Ly-1 and Ly-1,2 T-cell sets. This I-J determinant is not expressed on a detectable number of Ly-1 helper T cells which induce B lymphocytes to produce anti-sheep red cell antibody in tissue culture.

We have shown that (a) purified T-helper cells induce cells of another T-cell set-, expressing the Ly123+Qa1+ surface phenotype, to exert potent suppressive activity, (b) this T-T interaction plays an important role in regulating in vivo immune responses, and (c) this interaction represents an important barrier to protocols intended to augment the immune status of individuals by adoptive (or active) immunotherapy. Our results also indicate that the Ly123+ T-cell set mediating feedback suppression in vivo is sensitive to both low doses of cyclophosphamide and removal of the thymus in adult life. The importance of this T-T interaction to normal, physiologic regulation of the immune system is emphasized by the finding that the major T-cell deficit of NZB mice (an inbred strain of mice that spontaneously develops an autoimmune disorder) is the absence or malfunction of an Ly123+ T-cell set responsible for feedback inhibition.

The Lyb-2 cell-surface alloantigens of the mouse are selectively and perhaps exclusively expressed in the B lymphocyte lineage, but not on antibody- forming cells. Thus if the Lyb-2 molecule is concerned in specific B cell function, it must participate in the generative phase of the antibody response. Accordingly, monoclonal Lyb-2 antibody was found to depress the plaque- forming cell (PFC) response to sheep erythrocytes in 5-d Mishell-Dutton assays when added within the first 3 d of culture, but not later. The rate of PFC generation was not affected, signifying an absolute reduction in the number of PFC generated. Because reduction of PFC counts by Lyb-2 antibody was not affected by exclusion of Lyt-2(+) T cells, it is unlikely that the reduction depends on augmented suppression by T cells. Augmented B cell- mediated suppression is also unlikely, because the PFC response of serial combinations of congenic Lyb-2.1 and Lyb-2.2 cells, in the presence of monoclonal Lyb-2.1 antibody, was reduced only in direct proportion to the number of Lyb-2.1 cells present. The PFC response of Lyb-2.1/Lyb-2.2 heterozygous cells was not reduced by Lyb-2.1 antibody, presumably because generation of PFC is impeded only if most Lyb-2 sites are blocked. Further evidence that the molecule identified by Lyb-2 plays a critical role in the generation of antibody-forming cells (AFC) in response to T-dependent antigen comes from the finding that Lyb-2 antibody does not reduce the PFC response to the T-independent antigens trinitrophenylated (TNP) Brucella abortus and TNP-FicolI, although elimination of Lyb-2(+) cells from the starting population by Lyb-2 antibody and complement reduces the PFC response to T- dependent and T-independent antigens alike.

Publisher Summary This chapter explores that when living cells in free suspension are exposed to antibody, together with complement, cells bearing the respective antigen on their surfaces are lysed, while antigen-negative cells remain viable. The cytotoxicity assay is the starting-point of selective immunocytolysis with cell-discriminating antibody and complement. Understanding the cytotoxicity assay is an aid to best use of immunocytolysis because the principles and ingredients are the same. The chapter discusses that certain genes and gene products are expressed only in thymus-processed (T) lymphocytes, normal or maliginant, and the expression of these genes and products on the cell surface is further restricted to particular stages and sublincages of T cell differentiation. The chapter also reviews that complement-dependent antibody-mediated cytolysis became the first procedure whereby T cell populations could be divided into their otherwise relatively inseparable and generally indistinguishable functional compartments. Much the same applies to the other (B) category of lymphocytes and the advent of monoclonal antibodies has led to the description of homologous human lymphocyte compartments characterized immunogenetically by surface phenotype.

Spleen cells obtained from C57BL/Ks (Ks, H-2d) mice carrying passively enhanced Sarcoma I (Sa I, H-2a) tumors were tested for alloantibody formation, lymphocyte blastogenesis, antibody-dependent cellular cytotoxicity, and cell to cell cytotoxicity. Assays were usually performed approximately 6 weeks after tumor inoculation. The results of these assays indicate that spleen cells from tumor-bearing mice are actively synthesizing alloantibody, but have a depressed blastogenic response to phytohemagglutinin and allogeneneic cells, and manifest no detectable cytotoxic activity in 51Cr release assays for antibody-dependent or cell to cell cytotoxicity. The absence of cell to cell cytotoxicity was specific and could not be attributed to the activity of suppressor cells acting in vitro, or to immunoglobulin secreted during the in vitro assay. These results indicate that Ks mice carrying immunologically enhanced Sa I tumors have a strong humoral response but a defective cellular response to the alloantigens of their tumors. These results are compatible with a mechanism of immunological enhancement which involves suppression of the development of the cellular immune response throughout the course of tumor growth.

Publisher Summary This chapter focuses on selective expression of gene products on the cell surface of mouse lymphocytes. It discusses the expression of the Gix molecule by cells and mice that do not produce virus. Expression of Gix on the cell surface, serologically determined by Gix phenotyping of thymocytes, depends on alleles at two independently segregating Mendelian loci, Gv-1 and Gv-2. The Gix antigen is not confined to T cells. Gix+ gp70 is secreted abundantly by the testis, and it circulates freely in blood. B-cell surface components include gene products identified as alloantigens and others identified by properties other than genetic variation, notably as receptors for immunoglobulin (Fc) and for complement (CR). The products of the genes that are together programmed to compose the surface features of the cell are conveniently called the cell's surface phenotype. The chapter discusses whether and how the surface phenotype may be related to one another by spatial configuration in the plasma membrane.

Cells of the Lyl subclass generate helper activity in both primary and secondary responses to sheep erythrocytes (SRBC). In contrast, after priming with SRBC, cells of the Ly-2+ subclasses, in particular Ly23 cells, have suppressive activity. The degree of Ly23-mediated suppression is directly proportional to the amount of antigen (SRBC) used for priming. Suppression by Ly23 cells is specific, because Ly23 cells from SRBC-primed animals do not suppress the response to horse erythrocytes, and vice versa. Thus, both cytotoxic and specific suppressor functions are mediated by T cells of a subclass, provisionally designated TCS, which can be distinguished from helper T cells (TH), by their Ly phenotypes. It remains to be determined whether killing and suppression are functionally interrelated properties of a single Ly23 subclass, or whether the Ly23 population comprises two subclasses whose surface phenotypes are not yet distinguishable by immunogenetic criteria.

Specific cell-mediated cytotoxicity (CMC) of lymph node cells from immunized C3Hf mice, against syngeneic C3H/Umc mammary tumor cells, assayed in vitro, is effected by T lymphocytes. This CMC response is biphasic, with an early peak attained within 6 hr and a second major peak beginning at about 18 hr. Effector cells of both the early minor and late major phases of the response belong to the Ly23 set. Other T cell sets evidently play no part in the early effector response. But specifically activated Ly1 cells help or amplify the major late-phase response. Nevertheless, the mixture of specifically activated Ly1 and Ly23 sets still does not completely reconstitute the late response, which implies that the Ly123 set is also needed for maximal expression of CMC in this system. These Ly123 cells must come from specifically immunized donors. It appears, therefore, that maximal CMC is achieved by the participation of specific Ly123 cells which in the late phase directly or indirectly give rise to Ly23 killer cells. Thus, although killing of syngeneic mammary tumor cells in the CMC assay is invariably effected by cells of the Ly23 set, specifically activated cells of the Ly1 set, and probably of the Ly123 set also, are participants in the interactions needed to produce a maximal CMC response.

Experiments were carried out to determine the antigen specificity of two distinct helper T cells (Th) that act synergistically in adoptive secondary in vivo anti-hapten antibody responses. Both Th were present in anti-Ly-2 and complement-treated spleen T cell populations, implying that both Th are Ly-1+,23−. Adding normal T cells or T cells primed to other carriers to specific carrier-primed T cells, using a variety of different protocols did not affect the helper activity of the specifically primed Th. Thus, both Th apparently are antigen-specific. Furthermore, Th primed with one carrier and boosted with that carrier plus hapten linked to a noncross- reactive carrier cannot help B cells. However, if a mixture of Ly-1 T cells from mice primed with two different carriers is transferred along with B cells, and the mice are boosted with hapten coupled to one of the two priming carriers, then giving the other carrier induces a significant increase in antibody production. Thus, only one of the two Th (Th1) requires a hapten-carrier bridge, while the other does not (Th2). However, both Th 1 and Th2 are clearly antigen-specific and require stimulation with antigen to exert helper activity. Furthermore, these experiments strongly suggest that Th 2 cannot express helper function in vivo in the absence of Th 1. These findings, and the absence of Th2-like cells in agammaglobulinemic mice, were correlated with other studies in which two helper activities have been described. It was concluded that in vivo responses require an effective Th 1-B cell interaction, whereas Th2, if stimulated with antigen, will augment certain portions of the antibody response, such as idiotype or allotype, and thus influence the quality of the antibody response directly.