Lyt-1 cells mediate acute murine experimental allergic encephalomyelitis

Adult SJL/J mice were depleted of T lymphocytes and were reconstituted with Lyt-1, Lyt-2, or Lyt-1 + Lyt-2 cells. After immunization with MSCH, severe acute EAE developed in the Lyt-1- and the Lyt-1 + Lyt-2-reconstituted mice. Only 25% of the Lyt-2-reconstituted mice developed signs of EAE, and in those EAE onset was delayed. Thus, Lyt-1 cells mediate EAE in the mouse.     

Analysis of Ly-6.2-bearing murine lymphocyte subpopulations in relation to the T-lymphocyte markers,Thy-1, Lyt-1, and Lyt-2

Using immunofluorescence with a monoclonal anti-Ly-6.2 antibody and FACS analysis we have confirmed that the Ly-6.2 antigen is present on approximately 70% of mature T cells and B cells but on few immature lymphocytes. There is a wide range of antigen density among the Ly-6.2⁺ populations, with the mean density higher on T cells than B cells. Following Con A activation of splenocytes there was a sixfold increase in Ly-6.2 antigen density though approximately 20% of the activated lymphocytes were Ly-6.2^?. The increase in Ly-6.2 density was specific since similar density increases did not occur for the closely linked antigens ThB and H $\text{9}\text{25}$. By panning a predominantly T-cell population for Lyt-2-bearing cells, it was found that Lyt-2⁺ lymphocytes were either negative or dully staining for Ly-6.2. However, activated cells bearing the Lyt-2 antigen were all Ly-6.2 positive. Double-staining experiments showed that T cells which had high Ly-6.2 antigen densities also had high Thy-1 antigen densities. Corticosteroid-resistant thymocytes were highly enriched for Ly-6.2-bearing cells compared to untreated thymocytes and had staining profiles for Ly-6.2 which were similar to peripheral T cells, supporting the idea that steroid treatment selects for a phenotypically mature thymic population.     

Age-related changes in the relative numbers of Thy-1- and Lyt-2-bearing peripheral blood lymphocytes in mice: a longitudinal approach

Longitudinal and cross-sectional analyses of Lyt-2+ and Thy-1+ cell populations in the peripheral blood of aging male CBA and C57BL mice revealed that the relative number of Thy-1+, Lyt-2+ cells in the peripheral blood of both mouse strains remained relatively constant during the entire lifespan. The proportion of Thy-1+, Lyt-2- cells decreases with age, which indicates that major changes in the T-cell compartment with age must be attributed to the Lyt-2- helper compartment. For individual CBA mice, a direct relation between the relative number of Thy-1+, Lyt-2- cells at a certain age and the time remaining to live is demonstrated. The changes in the proportion of Lyt-2+ of total Thy-1+ cells in CBA mice show a regular pattern of slow increase with age followed by a rapid increase phase preceding the death of the animal. In C57BL mice, the development of the proportion of Lyt-2+ T cells with age showed various patterns. Rapid changes both positive and negative in these mice seem to be indicative of approaching death. The predictive value of Lyt-2+/Thy-1+ ratios at a given age for the remaining lifespan of individual mice is discussed.     

Deterioration of cellular immunity during aging. The relationship between age-dependent impairment of delayed-type hypersensitivity reactivity, interleukin-2 production capacity, and frequency of Thy-1+,Lyt-2- cells in C57BL/Ka and CBA/Rij mice

The effect of aging on the delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) in vivo and the interleukin-2 (IL-2) production capacity in vitro by spleen cells from young (17 weeks) and old (125 weeks) CBA/Rij and C57BL/Ka mice were investigated. For both CBA/Rij and C57BL/Ka mice an age-related decline in the DTH response to SRBC and the IL-2 production capacity was observed. Both parameters are mediated by Thy-1+,Lyt-2- spleen cells. For both mouse strains the proportion of Thy-1+,Lyt-2- spleen cells declined less strongly with aging than the DTH reactivity and the IL-2 production capacity. From this it was concluded that not only a quantitative but also a qualitative decrease of T-cell function occurs during senescence. It was also investigated whether the proportion of Thy-1+,Lyt-2- peripheral blood lymphocytes can be used as a predictive value with regard to the decline of DTH with aging of the corresponding mouse. This was indeed found to be the case in CBA/Rij mice, but not in C57BL mice.     

Lyt phenotype of cytotoxic T cells: shift from Lyt-1+2+3+ to a mixed population of Lyt-1+2+3+ and Lyt-1-2+3+ during in vitro culture

The Lyt phenotype of cytotoxic T cells generated in the primary H-2 response was investigated kinetically. The cytotoxicity generated in the early stage of culture was abolished by treatment with alpha Lyt-1,2,3, and complement (C), whereas that generated in the late stage was only partially eliminated by alpha Lyt-1, but was abolished by alpha Lyt-2, 3, and C. This suggested late expansion of the Lyt-1-2+3+ population. Lack of Lyt-1 antigen was confirmed with cells that were depleted of Lyt-1+ from primary culture and then stimulated in the secondary response by elimination of cytotoxicity and by direct Lyt typing. Results indicated that the response of proliferative and cytotoxic T cells of the Lyt-1+2+3+ phenotype in the early stage of culture was followed by activation of Lyt-1-2+3+ T cells. Cytotoxic T cells in the late stage were shown to be a mixture of Lyt-1+2+3+ and Lyt-1-2+3+ cells. This was confirmed with cytotoxic T cells from secondary culture and uncloned long-term T cell lines.     

Studies of experimental allergic encephalomyelitis by using encephalitogenic T cell lines and clones in euthymic and athymic mice

The role of T-T cell interactions in the clinical course of acute experimental allergic encephalomyelitis (EAE) in mice was investigated. Myelin basic protein (MBP)-reactive and encephalitogenic T cell clones were established from long-term lines derived from susceptible strain SJL/J mice and resistant strain DDD/1 mice. The lines and clones from DDD/1 mice were obtained by immunization of congenitally athymic mice of DDD/1 origin, which had been reconstituted with syngeneic Lyt-2+-depleted splenic T cells. The clones derived from both strains bore surface phenotypes of Lyt-1+, 2- and L3T4+, and proliferated well in response to rat, rabbit, bovine, and guinea pig MBP in the presence of antigen-presenting cells with I-As. Passive EAE could be induced in syngeneic normal recipients by these clones as well as by the lines from which the clones were derived. The clinical features of the clone-induced EAE were essentially the same as those of the line-induced EAE. Furthermore, DDD/1 athymic recipients developed signs of acute EAE by the adoptive transfer of I-A-compatible syngeneic and allogeneic T cell clones, in which there was no significant difference in time of onset, maximum severity, or prognosis. These results indicate that the entire clinical course of acute EAE can be elicited by a single population of MBP-reactive T cells in the absence of the thymus and other populations of primed or unprimed T cells.     

The acute inflammatory process in murine lymphocytic choriomeningitis is dependent on Lyt-2+ immune T cells

Virus-immune spleen cells induce fatal immunopathology following adoptive transfer into adult C57B1/6J mice that have been infected with lymphocytic choriomeningitis virus (LCMV) and immunosuppressed with cyclophosphamide. This is accompanied by the development of potent cytotoxic T-lymphocyte (CTL) activity of donor origin in the recipient spleen. Both the capacity to trigger the acute meningitis observed at 72 hr and to generate CTL effectors in lymphoid tissue are completely abrogated by the removal of Lyt-2+ cells from the donor population. However a lower level of inflammatory process in the central nervous system may emerge, in the absence of significant CTL function in recipient spleen, by 5 days after transfer of the Lyt-2-depleted cell population. Treatment of the transferred cells with antibody to the L3T4 marker does not reduce either the severity of inflammation or the level of CTL effector function in the recipient. Thus Lyt-2+ cells are required for the acute, fatal immunopathology characteristic of LCM, but it is not clear that in a more chronic situation, they are the sole effectors capable of triggering inflammatory process in this disease.     

Acute experimental allergic encephalomyelitis in the mouse: immunopathology of the developing lesion

To investigate the sequence of immunopathologic events during lesion formation in acute experimental allergic encephalomyelitis (EAE), SJL/J mice were inoculated with isogeneic spinal cord in complete Freund's adjuvant (CFA) and with Bordetella pertussis on Days 1 and 3 postinoculation (PI). Mice were sampled at different time points PI and T cells, T-cell subsets. Ia+ cells, Ig+ cells, albumin, and Ig deposits were localized in frozen sections by the avidin-biotin complex (ABC) method and direct fluorescence. Furthermore, samples were stained for Ia antigen, myelin basic protein (MBP), and galactocerebroside (GC) localization on endothelial cells by the ABC technique. Clinical and pathologic observations were correlated with the immunopathologic results. It was found that early in the disease process myelin and Ia-antigens were demonstrable on endothelial cells within the central nervous system (CNS). Simultaneously, damage to the blood-brain barrier was apparent, as indicated by albumin deposits, and small numbers of infiltrating T cells, T-cell subsets, and Ia+ cells were found. With time PI, the density of infiltrating total T cells (Thy-1.2+), helper/inducer (Lyt-1+), and suppressor/cytotoxic (Lyt-2+) T cells increased; Lyt-1+ and Lyt-2+ cells were detectable in meningeal as well as parenchymal infiltrates, while later on, Lyt-1+ cells showed some predilection for the CNS parenchyma and Lyt-2+ cells for meninges. Ia+ cells (B cells, macrophages, activated T cells) were present in small numbers only. Ig+ cells (B cells and macrophages) appeared shortly before onset of signs and persisted in moderate numbers. These results reconfirm the importance of early T-cell involvement for the development of EAE; they might also indicate a secondary role for Ig+ cells and are consistent with the concept that presentation of myelin antigens to T cells might occur locally on Ia-bearing endothelial cells within the CNS.     

Phenotypic identification of memory cytolytic T lymphocytes in a subset of Lyt-2+ cells

Murine peripheral Lyt-2+ T cells could be subdivided according to surface expression of the Pgp-1 glycoprotein into major (71%) Pgp-1- and minor (29%) Pgp-1+ subsets. A striking correlation was observed between Pgp-1 expression and enrichment for antigen-specific memory cytolytic T lymphocyte precursors (CTLp). After immunization with the male minor transplantation antigen H-Y, virtually all the H-Y-specific CTLp were found in the minor Pgp-1+ subset of Lyt-2+ cells. In addition, after alloimmunization the frequency of allospecific CTLp resistant to inhibition by anti-Lyt-2 antibody was markedly enriched within the Pgp-1+ cells, suggesting an enrichment for CTLp bearing high avidity antigen receptors. Taken together, these data suggest that surface Pgp-1 expression is stably acquired at the time of primary antigenic stimulation by virgin T cells. As such, Pgp-1 represents an important marker for identifying a subset of Lyt-2+ T cells with the quantitative and qualitative properties of memory CTLp.     

Role of L3T4 antigen in the activation of various functions of Lyt-1+2- T cells against vaccinia virus

The present study defines assay systems for vaccinia virus-reactive Lyt-1+2- T cells mediating various functions and investigates the positivity of L3T4 antigen on these Lyt-1+2- T cells as well as the role of L3T4 antigen in the activation of these T cells with respect to their functions. C3H/He mice were immunized against vaccinia virus by inoculating viable virus intraperitoneally (i.p.). Anti-vaccinia virus reactivity in lymphoid cells from these immunized mice was assessed by proliferative response, helper T cell activities involved in cytotoxic T lymphocyte (CTL) and B cell (antibody) responses, delayed type-hypersensitivity (DTH) response, and production of lymphokines such as interleukin 2 (IL2) and macrophage-activating factor (MAF). The results demonstrate that all of the above anti-vaccinia virus responses were mediated by Lyt-1+2- T cells and that these Lyt-1+2- T cells expressed L3T4 antigens on their cell surfaces. Moreover, such anti-vaccinia Lyt-1+2- T cell responses were inhibited in the presence of anti-L3T4 antigen antibody. These results indicate that there is a reciprocal relationship between Lyt-2 and L3T4 markers, and that L3T4 antigen is closely related to the activation of various functions of anti-vaccinia virus Lyt-1+2- T cells.     

Contribution of Pannexin1 to Experimental Autoimmune Encephalomyelitis

Pannexin1 (Panx1) is a plasma membrane channel permeable to relatively large molecules, such as ATP. In the central nervous system (CNS) Panx1 is found in neurons and glia and in the immune system in macrophages and T-cells. We tested the hypothesis that Panx1-mediated ATP release contributes to expression of Experimental Autoimmune Encephalomyelitis (EAE), an animal model for multiple sclerosis, using wild-type (WT) and Panx1 knockout (KO) mice. Panx1 KO mice displayed a delayed onset of clinical signs of EAE and decreased mortality compared to WT mice, but developed as severe symptoms as the surviving WT mice. Spinal cord inflammatory lesions were also reduced in Panx1 KO EAE mice during acute disease. Additionally, pharmacologic inhibition of Panx1 channels with mefloquine (MFQ) reduced severity of acute and chronic EAE when administered before or after onset of clinical signs. ATP release and YoPro uptake were significantly increased in WT mice with EAE as compared to WT non-EAE and reduced in tissues of EAE Panx1 KO mice. Interestingly, we found that the P2X7 receptor was upregulated in the chronic phase of EAE in both WT and Panx1 KO spinal cords. Such increase in receptor expression is likely to counterbalance the decrease in ATP release recorded from Panx1 KO mice and thus contribute to the development of EAE symptoms in these mice. The present study shows that a Panx1 dependent mechanism (ATP release and/or inflammasome activation) contributes to disease progression, and that inhibition of Panx1 using pharmacology or gene disruption delays and attenuates clinical signs of EAE.     

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.     

Rat T lymphocyte antigens comparable with mouse Lyt-1 and Lyt-2,3 antigenic systems: characterization by monoclonal antibodies

Rat T lymphocyte antigens were defined by using two distinct monoclonal antibodies (R1-3B3 and R1-10B5). R1-3B3 antibody, when tested for its reactivity with rat lymphoid cells by immunofluorescence, stained almost all of thymus and T cells but not the majority of B cells and bone marrow cells. The antigen defined by R1-3B3 existed more abundantly on medullary thymocytes and peripheral T cells than on cortical thymocytes. Immunochemical data showed that R1-3B3 antibody recognized a single glycoprotein with a m.w. of 67,000, showing marked electric charge heterogeneity with isoelectric points ranging from 5.4 to 7.3. R1-10B5 antibody, on the other hand, had more restricted reactivity with rat T cells and labeled approximately 85% of thymus cells and 30% of the peripheral T cells but neither B cells nor bone marrow cells. These T cells positive for R1-10B5 appeared to be negative for W3/25 antigen, which has been shown to be the marker for the rat T cell subset associated with helper function. R1-10B5 antibody detected a basic glycoprotein complex consisting of sulfhydryl-linked subunits with 30,000 and 34,000 m.w. Although the antigen defined by R1-3B3 was resistant to trypsin digestion, the one detected by R1-10B5 was much more sensitive to trypsin cleavage. All of these data obtained with either R1-3B3 or R1-10B5 are quite comparable to those reported for mouse Lyt-1 or Lyt-2,3 antigens, and thus suggest that the antigens defined by R1-3B3 and R1-10B5 antibodies represent rat homologues of Lyt-1 and Lyt-2,3 antigens in the murine system, respectively.     

Subsets of Lyt-2+ cells defined by differential expression of the 9F3 antigen: alterations in mice of the lpr/lpr genotype

Recently, we found that a novel murine cell surface antigen recognized by the 9F3 monoclonal antibody demonstrates T cell heterogeneity in normal mice and in congenic strains homozygous for the lymphoproliferation- and autoimmunity-inducing lpr gene. The objective of the present work was to further characterize this heterogeneity by studying the distribution of the 9F3 marker among Lyt-2+ T cells. By using dual parameter flow cytofluorometry analysis, at least two subsets of peripheral Lyt-2+ cells differing in 9F3 expression could be distinguished. In MRL/Mp-++, C57BL/6, and C3H/HeJ mice, 9F3- and 9F3+ cells accounted, respectively, for 65 to 80% and 20 to 35% of the whole Lyt-2+ population. Interestingly, in lpr/lpr-bearing congenic strains, a three- to fivefold selective increase in the frequency and absolute number of 9F3+ Lyt-2+ cells was found to take place during aging. Functional analysis of Lyt-2+ cells isolated by panning revealed that in +/+ mice, these cells respond better to phytohemagglutinin and concanavalin A than in lpr/lpr mice, indicating that phenotypic changes of Lyt-2+ cells correlate with functional changes. Further evidence for the functional relevance of 9F3-defined Lyt-2+ cell heterogeneity was provided by the study of sorted cells from +/+ mice, showing that 9F3- cells exhibit higher mitogenic reactivities than 9F3+ cells. This finding suggests that 9F3+ Lyt-2+ cells in +/+ and lpr/lpr mice are functionally homologous. Together, these data indicate that, in addition to the known expansion of a population of abnormal Lyt-2-T cells, mice of the lpr/lpr genotype have an alteration of their Lyt-2+ cell population, characterized by an imbalance of 9F3-defined subsets and decreased mitogenic reactivities.     

Intrathymic differentiation and tolerance induction of lymphokine-secreting Lyt-2+ T helper cells

The present study has assessed thymic influence on the differentiation and recognition specificity of developing Lyt-2+ lymphokine-secreting T cells, and compared it with those of developing Lyt-2+ CTL. It was demonstrated that development of Lyt-2+ lymphokine-secreting Th cells requires an intrathymic differentiation step, and that peripheral Lyt-2+ lymphokine-secreting Th cells, unlike peripheral Lyt-2+ CTL, are profoundly tolerant to intrathymically expressed alloantigens. These data are interpreted as demonstrating that functionally distinct Lyt-2+ T cell populations are heterogeneous in their requirements for differentiation and/or activation.     

Heterologous antisera to Lyt-1+, 2- -derived antigen-binding factor detect a subfactor of an antigen-specific suppressor factor and cell surface proteins on Lyt-1+, 2- and Lyt-1-, 2+ T cells

Rabbits were immunized with TNP-specific Lyt-1+, 2- T cell-derived, antigen-binding proteins (PCI-F) released by T cells sensitized by skin painting with picrylchloride. The resulting antiserum (anti-PCI-F) bound to PCI-F and TNP-specific factors that suppressed delayed hypersensitivity (TSF) known to be comprised of PCI-F and Lyt-2+ -derived polypeptides released by cells sensitized by injection of trinitrobenzenesulfonic acid (TNBSF). Anti-PCI-F bound to T lymphocytes and 68,000 to 72,000 m.w. T cell surface proteins but not B cells on their surface proteins. Anti-PCI-F bound to both Lyt-1+ and Lyt-2+ T cells and surface proteins. A comparison of anti-PCI-F with anti-TSF indicates that anti-TSF contains specificity for Ly-2+ T cell-derived components of TSF and T cells not present in anti-PCI-F. The possibility of multiple isotypes of T cell receptors and antigen-binding molecules is discussed.     

Autoimmune thyroiditis induced in mice depleted of particular T cell subsets. I. Requirement of Lyt-1 dull L3T4 bright normal T cells for the induction of thyroiditis

T cell-depleted C3H/He or (C57BL/6xC3H/He)F1 (B6C3F1) mice were prepared by adult thymectomy and injection of antithymocyte serum, followed 3 wk later by lethal x-irradiation and bone marrow reconstitution. When these T cell-depleted mice were not injected or injected i.v. with normal spleen and lymph node cells treated with either anti-Thy-1, -L3T4 or -Lyt-2 antibody plus C or C alone, none of the groups of mice developed thyroiditis. In contrast, the adoptive transfer of normal cells treated with anti-Lyt-1 plus C resulted in high incidence of the production of antithyroglobulin antibody and the induction of typical thyroiditis lesion. The thyroid was the sole organ involved, because neither typical inflammatory lesion in other organs nor autoantibody such as anti-DNA antibody was detected in mice that exhibited thyroiditis. Analyses of surface phenotypes of cells required for inducing thyroiditis by the adoptive transfer revealed that an appreciable percentage of Lyt-1 dull T cells remained after the treatment of normal lymphoid cells with anti-Lyt-1 plus C. Almost all of these Lyt-1 dull T cells expressed magnitudes of L3T4 or Lyt-2 Ag comparable to those detected on Lyt-1 bright T cells. More important, the induction of thyroiditis was almost completely prevented by either in vitro or in vivo elimination of Lyt-1 dull L3T4+(bright) but not of Lyt-1 dull Lyt-2+(bright) T cells. These results indicate that Lyt-1 dull L3T4+ T cells existing in normal healthy individuals have potential to induce typical thyroiditis which is associated with the production of antithyroglobulin autoantibody, and that the activation and/or function of this T cell subset is regulated by the Lyt-1 bright T cell population coexisting in normal lymphoid cell population.     

Immunopathology of the lesion in chronic relapsing experimental autoimmune encephalomyelitis in the mouse

To analyze immunopathologic events within the central nervous system (CNS) during various stages of actively induced chronic relapsing EAE in SJL/J mice, animals were sampled at various timepoints post inoculation (PI) and T cells, T-cell subsets, Ia+ cells and Ig+ cells, albumin, and Ig deposits were localized in frozen sections by immunocytochemical techniques. Furthermore, sections were stained for the demonstration of Ia antigen, myelin basic protein (MBP), and galactocerebroside (GC) on endothelial cells and astrocytes. During the acute phase of the disease, large numbers of all types of inflammatory cells studied (Lyt-1.2+, L3T4+, Lyt-2+, Ia+, Ig+) were randomly distributed throughout lesions, a finding similar to that described previously for acute EAE. A more distinct distribution pattern of infiltrating T cells was found during active chronic disease in that L3T4+ cells predominated within the CNS parenchyma, while Lyt-2+ cells were more numerous in meningeal and perivascular areas. During all chronic stages, a low-grade diffuse infiltration of the neuraxis by hematogenous cells was present. Ia and myelin antigens were detectable on some endothelial cells and astrocytes. Damage to the blood-brain barrier, as indicated by albumin and Ig deposits, was more extensive during the acute than during chronic stages of the disease. Taken in concert, the results further support the possibility of local antigen presentation on endothelial and astroglial cells and an essential involvement of helper (L3T4+) T cells in CNS lesion formation. These findings correlate well with events reported previously in acute and chronic multiple sclerosis lesions.     

Lyt-2- T cell-independent functions of Lyt-2+ cells stimulated with antigen or concanavalin A

Approximately 30% of cytolytic Lyt-2+ clones from primed mice are able to proliferate autonomously, i.e., independent of IL 2 derived from Lyt-2- cells after antigenic stimulation. H-2K- or -D-restricted induction of Lyt-2+ cells to autonomous proliferation requires Ia+ stimulator cells. A strict correlation was observed between the ability of Lyt-2+ clones to proliferative autonomously and to induce DH. Eventually, the growth of all Lyt-2+ cytolytic clones becomes dependent on exogenous IL 2, and their ability to induce DH is lost. Small Lyt-2+ cells can also be induced in primary cultures by antigen or concanavalin A to proliferate in the absence of exogenous IL 2. The frequency of autonomously proliferating small Lyt-2+ cells is the same as that of small Lyt-2+ cells proliferating in the presence of exogenous IL 2. IL 2 derived from Lyt-2- cells can augment proliferation of Lyt-2+ cells, but is not obligatory.     

The allogeneic effect: the mechanism of allosuppression by Lyt-1, Ia- T cells

The kinetics of allohelp mediated by diffusable factors revealed that help by nonirradiated T cells (TOR) peaked at 48 to 72 hr, followed by a sharp decline if the T cells remained in the cultures. The temporal decrease in help after 72 hr was not mediated by suppressor lymphokines because mixtures of early (24 to 48 hr) and late (120-hr) allogeneic supernatants enhanced help synergistically. Lyt-1, Ia- T cells mediated the temporal decline in help and suppressed allogeneic B cell activation in co-cultures, and this "down-regulatory" activity (allosuppression) was radiosensitive. Help by irradiated T cells (T1000R) increased gradually until it plateaued between 96 and 120 hr. The helper activities of the allogeneic supernatants were directly proportional to their T cell growth factor (TCGF) activities. In addition, their kinetics were identical, and the removal of TCGF from 48-hr allogeneic supernatants by adsorption with TCGF-dependent HT-2 cells depleted both helper and TCGF activities. Help was restored to depleted 48-hr and 120-hr allogeneic supernatants by preparations of TCGF obtained from concanavalin A (Con A)-stimulated FS6-14.13 hybridoma cells that were adsorbed with lipopolysaccharide (LPS)-activated B cells or normal spleen cells (NS), but not with HT-2 cells. These results indicate that allohelp is dependent on TCGF. Moreover, help was dependent on at least one factor in addition to TCGF, because a high level of synergy occurred between TCGF and the "help-deficient" 120-hr allogeneic supernatant. In conclusion, the mechanism whereby Lyt-1, Ia- T cells regulated B cell activation with positive and negative allogeneic effects was through the production and subsequent exhaustion of TCGF, respectively. The production of TCGF and help was radioresistant, but exhaustion of TCGF and suppression was radiosensitive.