Inactivation of T4D Bacteriophage by Antiserum Against Bacteriophage Dihydrofolate Reductase

Antiserum was prepared against highly purified T4D bacteriophage-induced dihydrofolate reductase (DFR). This serum not only inactivated the enzyme but also inactivated all strains of T4D examined. T6 was inactivated to a lesser extent, and T2L, T2H, and T5 were unaffected by the antiserum. The phage-killing power of the serum could be blocked by prior incubation with partially purified T4D dfr obtained from host cells unable to make phage structural proteins. These observations confirm earlier results that the phage dfr is a structural component of the phage particle, and they offer new evidence on the manner in which this enzyme in incorporated into the tail structure.     

Contrasuppressor cells that break oral tolerance are antigen-specific T cells distinct from T helper (L3T4+), T suppressor (Lyt-2+), and B cells

Continuous gastric intubation of mice with the T cell-dependent antigen sheep erythrocytes (SRBC) leads to a state of systemic unresponsiveness to parenteral SRBC challenge, a state termed oral tolerance. The systemic unresponsiveness of mice rendered orally tolerant to SRBC, however, is converted to humoral immune responsiveness by adoptive transfer of effector T contrasuppressor (Tcs) cells. In this study, the authors have isolated and characterized the Tcs cell subset, from the spleens of orally immunized mice, which abrogates oral tolerance. This Tcs cell is a novel cell type, which can be separated from functional T suppressor (Lyt-2+) and T helper (L3T4+) cells, and the effector Tcs cell exhibits a Lyt-1+, 2-, L3T4- phenotype. Furthermore, contrasuppression is not mediated by B cells, including those of the Lyt-1+ phenotype. Adoptive transfer of splenic Lyt-1+, 2-, L3T4- T cells from C3H/HeJ mice given oral SRBC for 21 to 28 days and splenic Lyt-1+, 2-, L3T4- T cells of C3H/HeN mice orally immunized for a shorter interval abrogated oral tolerance. Furthermore, separation of Lyt-1+ T cells into L3T4+ and L3T4- subsets by flow cytometry resulted in Lyt-1+, L3T4+ T cells with helper but not contrasuppressor function, whereas the Lyt-1+, L3T4- T cell fraction abrogated oral tolerance even though it was without helper activity. This Tcs cell subset was also effective when added to cultures of tolerized spleen cells derived from SRBC-fed mice. The effector Tcs cells are antigen-specific, because Tcs cells from SRBC-immunized mice reverse tolerance to SRBC but not to horse erythrocytes (HRBC), and Tcs cells from HRBC-immunized mice reverse tolerance to HRBC but not to SRBC. When splenic T3 (CD3)-positive T cells (Lyt-1+, 2-, and L3T4-) were separated into Vicia villosa-adherent and nonadherent subpopulations, active contrasuppression was associated with the T3-positive and Vicia villosa-adherent T cell fraction. Thus, a distinct Lyt-1+, 2-, L3T4- T cell subset that contains a T3-T cell receptor complex, which can regulate oral tolerance, is present in spleens of orally immunized mice.     

Impaired autoreactive T cell-induced T cell-T cell interaction in aged mice

Self-Ia-reactive (autoreactive) L3T4+ T cell clones have been shown earlier to stimulate the proliferation of syngeneic naive L3T4+ T cells and initiate a T cell-T cell (T-T) interaction leading to the generation of immunoregulatory circuits. Since aging has been shown to be associated with a decline of the immune responsiveness, age-related alterations in the T-T interaction was investigated in the present study. Using several I-Ed-specific autoreactive T cell clones isolated from 2- to 3-month-old (young) DBA/2 mice as stimulators, it was observed that L3T4+ T cells from 22- to 24-month-old (aged) DBA/2 mice, failed to demonstrate a significant response to the autoreactive T cells. In contrast, L3T4+ T cells from young mice responded strongly to the autoreactive T cell clones. The deficient T-T cell interaction in aged mice correlated with an impaired syngeneic mixed lymphocyte reaction in these mice, thereby suggesting that aging induces a defect both in the autoreactive T cells and in T cells which react with the autoreactive T cells. When exogenous recombinant interleukin 2 (rIL-2), recombinant interleukin 4 (rIL-4), or a combination of these was added to the interaction, it was observed that rIL-4 but not rIL-2 enhanced the T-T interaction in young mice. However, rIL-4 or a combination of rIL-2 and rIL-4 failed to correct the defective T-T interaction in aged mice. Since the T cell network is believed to play an important role in the maintenance of normal immune system homeostasis, the present study suggests that age-related alterations in T and B cell functions and increased susceptibility to autoimmune diseases with age may result from a defect in the T cell network regulation.     

Marker rescue of the adsorption properties of bacteriophage T 4 by bacteriophage T 2

Rescue of adsorption properties from UV-irradiated T4 by T2 as a helper phage, revealed progeny phage with intermediate properties. Fourteen independent progeny phages, plating onE. coli B/2, were plated on several indicator strains and their adsorption properties were also studied with specific T4 antibodies. Two of these, plating onE. coli KS/4, were not inactivated by the T4 antiserum, and were T2h without apparent T4 properties. The other 12 progeny phages did not plate on KS/4, and were inactivated, but at a slower rate than the parental T4. Their mean efficiency of plating onE. coli B/2 (0.83) was significantly lower than that of the parental T4. The efficiency of plating was positively correlated with the velocity of inactivation by T4 antiserum. The observations were explained by assuming that the progeny phages were recombinants of T4 and T2 loci for adsorption sites. Plating of these 12 progeny phages on several indicator strains showed that they were allrII mutants and all, except one, wererI mutants too. In addition, two weretu andh ⁴, respectively. The condition for the appearance of multiple mutants might be a complementation by T2 of UV-damaged functions, which otherwise fail to induce the completion of the lytic cycle in monocomplexes of extracellularly irradiated T4.     

Inactivation of hepatitis A HM-175/18f, reovirus T1 Lang and MS2 during alkaline stabilization of human biosolids

AIM: To compare the inactivation rates of male-specific bacteriophage-2 (MS2), hepatitis A HM-175/18f (HM-175) and reovirus T1 Lang (T1 L) during alkaline stabilization of wastewater residues. METHODS AND RESULTS: A bench scale alkaline stabilization model was used to evaluate the inactivation of MS2 seeded into raw sludge simultaneously with HM-175 or T1 L. Stabilization was performed in triplicate at 28 and 4 degrees C for both viral combinations. During stabilization at 28 and 4 degrees C, MS2 and T1 L concentrations were similar at each time point (t = 0.1, 2, 12 and 24 h). MS2 and HM-175 concentrations were also similar at each time point during stabilization at 28 degrees C. At 4 degrees C, MS2 and HM-175 concentrations were not similar at the first two time points (t = 0.1 and 2 h), but were similar at later time points (t = 12 and 24 h). CONCLUSIONS: The inactivation rates of T1 L at 4 degrees C and both T1 L and HM-175 at 28 degrees C were similar to the inactivation rate of MS2 at all time points. At 4 degrees C, MS2 was inactivated at a faster rate during the first two time points (t = 0.1 and 2 h) than HM-175, but was inactivated similarly at later time points (t = 12 and 24 h). SIGNIFICANCE AND IMPACT OF THE STUDY: Phages, such as MS2, would be ideal indicators for the presence of enteric viruses in wastewater residues because of their ubiquity, nonpathogenic nature, low cost and time associated with their detection. The findings of this study suggest that MS2 could serve as an indicator for monitoring the persistence of enteric viruses, such as HM-175 and T1 L, during alkaline stabilization performed at moderate temperatures (28 degrees C), but may not serve as an indicator for HM-175 at reduced temperature (4 degrees C). The utility of MS2 as an indicator of viral persistence during biosolids treatment should be further evaluated, as the increased efficiency and frequency of pathogen monitoring associated with their use may reduce the potential public health risk associated with biosolids, facilitating a greater acceptance for their land application.     

Antigen-presenting T cells. I. Class I alloantigen (bm1)-bearing T lymphoblasts efficiently stimulate a primary clonal response of Lyt-2+ cytotoxic lymphocyte precursors of B6 origin

The cytotoxic response of splenic Lyt-2+ T cells to class I H-2 alloantigen-bearing stimulator cells was analyzed under limiting dilution conditions. One of 50 to one of 200 nylon wool-nonadherent (FACS-purified), small Lyt-2+ spleen cells of B6 origin gave rise in vitro to a cytotoxic T lymphocyte clone that specifically lysed targets bearing bm1 alloantigen. This population of alloantigen-specific cytotoxic lymphocyte precursors (CLP) was activated by different types of bm1 stimulator cells with different efficiency: 2 X 10(5) nonfractionated spleen cells, 5000 normal peritoneal cells, 400 to 10(4) L3T4+ helper T blasts, or 2000 to 10(4) Lyt-2+ T blasts induced clonal growth of this CLP pool. Irradiated or mitomycin-treated small (L3T4+ or Lyt-2+) bm1-derived T cells were inefficient stimulator cells for this response. Supplementation of culture medium with (recombinant) interleukin 2 was necessary and sufficient to support clonal development of alloantigen-triggered CLP in the presence of allogeneic T blasts. Under these limiting dilution conditions, we observed comparable cloning efficiencies for (wild-type) Kb-allospecific splenic Lyt-2+ CLP from bm1 mice generated in response to either irradiated B6 spleen cells or inactivated B6-derived T cell lines (EL4 and RBL-5 lymphoma cells). The data indicate that normal T lymphoblasts as well as tumor T cell lines stimulate clonal development in vitro of class I H-2-allospecific cytotoxic T lymphocytes in the presence of interleukin 2.     

Quantitative assay for transformation of 3T3 cells by herpes simplex virus type 2.

The interaction of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) with Swiss/3T3 cells was investigated. Virus-induced cytopathic effects developed in the absence of production of infectious virus. HSV-2 inactivated with UV light (2, 4, 6, and 8 min) also induced cell death in the absence of virus replication. Cell death was not detectable after infection by HSV-2 that had been inactivated by UV irradiation for 10, 12, and 14 min. 3T3 cells infected with UV-inactivated virus (10 and 12 min) continued to replicate past the contact-inhibited monolayer normally associated with these cells. Infection of 3T3 cells with UV-irradiated USV-2 also induced the development of transformed foci. Transformed cells with an epithelioid of fibroblastoid morphology were identified and isolated. All HSV-2-transformed cell lines contained HSV-2-specific antigens detectable by immunofluorescence techniques. The maximum frequency of HSV-2-induced transformation was 3 times 105 PFU per transformed focus, and the observed transformation could be inhibited by pretreatment of the virus with specific antiserum. No type C particles were detected within five cell culture passages after transformation by HSV-2. Type C virus particles were detected after 10 cell culture passages of the HSV-2-transformed cell lines.     

Human cell membrane components bound to beta2-microglobulin in T cell-type cell lines.

Cell membrane components bound to beta2-microglobulin were isolated from Renex 30 (a nonionic detergent)-solubilized membrane materials of two human T cell-type cell lines, MOLT-4 and CCRF-CEM, by gel filtration and lectin affinity chromatography. The isolation was carried out by following the beta2-microglobulin activity by radioimmune inhibition assay. The T cell membrane components bound to beta2-microblogulin had a uniform molecular size of about 200,000 daltons and most of them showed an affinity to lentil lectin. The isolated membrane components were radioiodinated and examined for identity to HLA antigens by sequential precipitation with rabbit anti-HLA antiserum (specific to HLA large components) and with rabbit anti-beta2-microblogulin antiserum. In addition to HLA antigens, the beta2-microglobulin-bound components obtained from the MOLT-4 cells were found to contain certain membrane components that are the same in molecular size as the HLA large components but that are different antigenically from the HLA large components. On the other hand, the beta2-microglobulin-bound membrane components obtained from the CCRF-CEM cells were all HLA antigens. No other membrane components were involved in the binding.     

Actin associated with membranes from 3T3 mouse fibroblast and HeLa cells

A protein component of membranes isolated from 3T3 mouse fibroblasts and HeLa cells has been identified as actin by peptide mapping. Extensive but apparently not total coincidence was found between the peptide maps of these two nonmuscle membrane-associated actins compared to chick skeletal muscle actin. Between 2 and 4 percent of the total membrane protein appears in the actin band on sodium dodecyl sulfate polyacrylamide gels of 3T3 membranes while about 4 percent of the membrane protein appears as the actin band from HeLa membranes. These values represent approximately the same proportion of actin to total protein found in the cell homogenates. Treatment of intact cells with levels of cytochalasin B sufficient to cause pronounced morphological changes did not change the amount of actin associated with the membrane in either 3T3 or HeLa cells. However, incubation of isolated membranes under conditions favoring conversion of actin from filamentous to monomeric form resulted in dissociation of approximately 80 and 60 percent of the actin from 3T3 and HeLa membranes, respectively. Thus, approximately 20 percent of 3T3 membrane actin and 40 percent of HeLa membrane actin remained associated with the membrane even under actin depolymerizing conditions.     

Identification of early stages of T lymphocyte development in the thymus cortex and medulla

Thymocyte subpopulations with a phenotype suggesting they are early stages of T cell development in the adult mouse thymus were characterized and isolated by using multiparameter flow cytometry and sorting, in conjunction with selective killing with antibody and complement (C). The intrathymic localization of these subpopulations was assessed by dipping the thymus in fluorescent dyes to selectively label outer-cortical cells. The main phenotypic markers used were sensitivity to C-mediated lysis by the monoclonal antibody B2A2 (which spares most prothymocytes but kills most thymocytes), the expression of the T cell lineage specific markers Ly-2 and L3T4, and the levels of the common T cell antigens Ly-1 and Thy-1. A preliminary selection for cells lacking Ly-2 and L3T4, or resistant to B2A2 and C, produced a population of large cells, only 5% of all thymocytes and distinct from the typical cortical blast cells. This population of putative early thymocytes was itself heterogeneous, consisting of eight subpopulations separable by phenotype and intrathymic localization. One group of two subpopulations (B2A2-, Ly-1++, Thy-1+ and either Ly-2+ L3T4- or Ly-2- L3T4+) appeared to be of medullary location, and their phenotype suggested they could have been early members of the medullary lineages. Another group of two subpopulations (B2A2-, Ly-1++, Thy-1-, Ly-2-, L3T4- and B2A2-, Ly-1++, Thy-1+, Ly-2- L3T4-) did not show a clear localization pattern and may have represented cells in an earlier stage of transition to medullary phenotype and location. A quite different group of three subpopulations (B2A2++, Ly-1-, Thy-1-, Ly-2- L3T4-; B2A2++, Ly-1-, Thy-1+, Ly-2-, L3T4-; and B2A2++, Ly-1+, Thy-1++, Ly-2- L3T4-) was concentrated in the outer cortex and seemed to represent a series of stages of a cortical pathway, before the typical cortical blast cells. Finally, a very minor subset (0.2% of thymocytes), lacking all these markers, was concentrated in the outer cortex; this fifth group had the phenotype expected of the earliest intrathymic precursor cells. The results suggest that the separate developmental streams of cortical and medullary thymocytes may be traced back, via these minor early blast subpopulations, to common precursor cells in the outer cortex.     

Murine interstitial nephritis. IV. Long-term cultured L3T4+ T cell lines transfer delayed expression of disease as I-A-restricted inducers of the effector T cell repertoire

In the present study we observed that long-term cultures of tubular antigen-reactive L3T4+ T cells from immune SJL mice are able to adoptively transfer interstitial nephritis by 12 wk after i.v. injection. Lesions that develop under these conditions generally occur in the absence of anti-tubular basement membrane antibody formation. These cultured T cell lines are I-A restricted, require L3T4-associative interactions, and are tubular antigen specific, but do not share the phenotype, function, or H-2-restriction characteristics of Lyt-2+ nephritogenic effector T lymphocytes. Rather, our L3T4+ T cell lines, and phenotypically similar lymphocytes harvested from renal infiltrates, are inducers of this Lyt-2+ effector T cell repertoire. Such effector T cells, typically found in immune lymph nodes 4 to 7 days after immunization, can be induced in vitro within 5 days and will acutely transfer disease within another 5 days when placed under the kidney capsule. These findings collectively indicate that the time course for optimal effector cell differentiation and potential expression is relatively short. The immunologic inertia we previously observed between immunization or i.v. adoptive transfer and the development of cellular lesions, therefore, seems to reside in other systemic or interactional events beyond the timely formation of effector T cells.     

Role of the L3T4-antigen in T cell activation. II. Inhibition of T cell activation by monoclonal anti-L3T4 antibodies in the absence of accessory cells

We have used two monoclonal antibodies (Mab) to the L3T4 antigen to reexplore the role of this molecule in the process of T cell activation. Both Mab (Gk1.5 and 2B6) were capable of inhibiting Con A-induced IL 2 production by a number of antigen-specific T cell hybridomas in an assay system that was free of major histocompatibility complex (MHC) class II antigen-bearing cells. The inhibition produced by the anti-L3T4 Mab was specific, because other Mab to cell surface antigens expressed on the hybridomas were without inhibitory effects. These studies rule out the possibility that the mechanism of inhibition by anti-L3T4 in this model is mediated by blocking interaction of L3T4 with MHC class II products. Taken together, these results and those of other groups of investigators, are most compatible with a dual function for L3T4 in T cell activation. L3T4 might first interact with MHC class II molecules or other molecules on target or accessory cells; L3T4 would subsequently transmit a signal that would regulate the activation process. Mab to L3T4 might exert inhibitory effects at one or both of these steps.     

"Physiological interaction" does not explain the H-2 requirement for recognition of virus-infected cells by cytotoxic T cells.

B10.A (H-2Kk, H-2Dd) ectromelia-immune T cells from secondary responses in vitro were protent killers of both infected L929 (H-2Kk H-2Dk) and infected P-815 (H-2Kd, H-2Db) target cells. Specific competition with unlabelled targets showed that two separate T cell subsets were responsible for lysis of infected L929 and infected P-815 cells. One hypothesis to account for this (a form of "physiological interaction") is that T cells which kill one target e.g. infected L929) display only one out of two possible self-complementary recognition structures, in this example the H-2Kk alloantigen, not H-2Dd, whereas T cells that lyse infected P-815 targets display only H-2Dd, not H-2Kk. This hypothesis was tested and seems untenable because of the following results: A.TH (H-2Ks, H-2Dd) ectromelia-immune, secondary cytotoxic T cells which killed infected SJL/J (H-2Ks, H-2Ds) targets were themselves inactivated by pre-incubation with SJL/J cytotoxic T cells generated in one-way mixed lymphocyte reaction (MLR) against BALB/c (H-2Kd, H-2Dd). A.TL (H-2Ks, H-2Dd) ectromelia-immune secondary cytotoxic T cells which killed infected BALB/c targets were themselves inactivated by BALB/c cytotoxic T cells generated in MLR against SJL/J. Thus, virus-immune T cells which lyse infected targets by virtue of shared H-2K are also displaying H-2D alloantigen, and vice versa.     

Studies on temperature-dependent ultraviolet light-sensitive mutants of bacteriophage T4: the structural gene for T4 endonuclease V.

Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation specifically at high temperature were isolated after mutagenesis with hydroxylamine. At 42 °C the mutants are twice as sensitive to ultraviolet light as T4D, whereas at 30 °C they exhibit survival curves almost identical to that of the wild-type strain. Complementation tests revealed that the mutants possess temperature-sensitive mutations in the v gene.Evidence is presented to show that T4 endonuclease V produced by the mutants is more thermolabile than the enzyme of the wild-type. (1) Extracts of cells infected with the mutants were capable of excising pyrimidine dimers from ultraviolet irradiated T4 DNA at 30 °C, but no selective release of dimers was induced at 42 °C. (2) Endonuclease V produced by the mutant was inactivated more rapidly than was the enzyme from T4D-infected cells when the purified enzymes were incubated in a buffer at 42 °C. From these results it is evident that the v gene is the structural gene for T4 endonuclease V, which plays an essential role in the excision-repair of ultraviolet light-damaged DNA.The time of action of the repair endonuclease was determined by using the mutant. Survival of a temperature-sensitive v mutant, exposed to ultraviolet light, increased when infected cells were incubated at 30 °C for at least ten minutes and then transferred to 42 °C. It appears that repair of DNA proceeds during an early stage of phage development.     

L3T4+ T-cell-independent reactivity of Lyt2+ T cells in vivo

The aim of this study was to analyze in vivo the L3T4+ T-cell-subset-independent reactivity of Lyt2+ T cells toward transplantation alloantigens. To this end, we depleted normal mice of L3T4+ T cells by injection of monoclonal antibodies to the L3T4 antigen. This procedure not only led phenotypically to a disappearance of L3T4+ T cells, but also effectively abolished reactivity toward class II MHC antigens in vitro and in vivo. However, L3T4+ T-cell-depleted mice still reacted to class I MHC alloantigens in vivo: after immunization with class I MHC alloantigens Il-2 receptor-bearing T cells appeared in the draining lymph nodes, and developed antigen-specific cytolytic activity. Moreover, upon in vivo priming the frequencies of class I MHC-specific precursors of Il-2-producing and cytolytic Lyt2+ T lymphocytes increased up to 20-fold. L3T4+ T-cell-depleted mice rejected class I MHC-bearing skin grafts promptly. We conclude that not only in vitro but also in vivo Lyt2+ T cells remain reactive toward class I MHC antigens in the absence of L3T4+ T helper cells.     

Functional CD4 T cells after intercellular molecular transfer of 0X40 ligand.

OX40/OX40 ligand (OX40L) proteins play critical roles in the T cell-B cell and T cell-dendritic cell interactions. Here we describe the intercellular transfer of OX40L molecules by a non-Ag specific manner. After 2-h coculture of activated CD4(+) T cell (OX40L(-), OX40(+)) with FLAG peptide-tagged OX40L (OX40L-flag) protein-expressing COS-1 cells, the OX40L-flag protein was detected on the cell surface of the CD4(+) T cells by both anti-OX40L and anti-FLAG mAbs. The intercellular OX40L transfer was specifically abrogated by pretreatment of the COS-1 cells with anti-OX40L mAb, 5A8. The OX40L transfer to OX40-negative cells was also observed, indicating an OX40-independent pathway of OX40L transfer. HUVECs, allostimulated monocytes, and human T cell leukemia virus type I-infected T cells, which all express OX40L, can potentially act as the donor cells of OX40L. The entire molecule of OX40L was transferred and stabilized on the recipient cell membrane with discrete punctate formation. The transferred OX40L on normal CD4(+) T cells was functionally active as they stimulated latent HIV-1-infected cells to produce viral proteins via OX40 signaling. Therefore, these findings suggest that the intercellular molecular transfer of functional OX40L may be involved in modifying the immune responses.     

Up-regulation of interleukin 4 receptor expression on immature (Lyt-2-/L3T4-) thymocytes

In this report, the effect of interleukin 4 (IL-4) on the growth and differentiation of Lyt-2-/L3T4-(2-4-) thymocytes was investigated. It was found that these thymocytes proliferated extensively when cultured in the presence of IL-4 + phorbol myristate acetate without apparent differentiation to Lyt-2+ or L3T4+ cells. We also demonstrated that 2-4- thymocytes constitutively express a high affinity (dissociation constant of 20 to 40 pM) receptor for IL-4. Freshly isolated 2-4- thymocytes expressed on average about 100 IL-4 receptors per cell, but the number of receptors increased approximately 8-fold within 3 days after activation by IL-4 + phorbol myristate acetate. These findings suggest that IL-4 may play an important role in T cell ontogeny by promoting self-renewal of stem cells within the thymus.     

Role of interleukin 1 in early T cell development: Lyt-2-L3T4- thymocytes bind and respond in vitro to recombinant IL 1

Thymocytes that bear neither Lyt-2 nor L3T4 differentiation antigens (2-4- thymocytes) contain the precursors for mature L3T4+Lyt-2- and Lyt-2-L3T4+ T cells. In the present study we determined the capacity of 2-4- cells to respond to recombinant interleukin 1 (rIL 1) in vitro. The presence of rIL 1 enhanced IL 2-dependent proliferation to the lectins Con A and PHA by threefold to eightfold. In a second assay, rIL 1 enhanced proliferation and IL 2 production by 2-4- cells in response to phorbol myristate acetate (PMA) and the calcium ionophore, ionomycin. Using a direct IL 1 binding assay, we were able to detect both high-affinity (Kd approximately 5 pM) and low-affinity (Kd approximately 200 pM) classes of IL 1 receptors on freshly isolated 2-4- cells. Bound IL 1 was rapidly internalized, suggesting that such receptors were functional. These results are compatible with a role for interleukin 1 during thymocyte maturation.     

Generation of helper cell-independent cytotoxic T lymphocytes is dependent upon L3T4+ helper T cells

The role of L3T4+ (CD4+) Th cells in generation of CTL specific for discrete minor histocompatibility Ag was investigated. Suppression of the function of Th cells in vivo by chronic treatment with anti-L3T4 mAb prevented congenic strains of mice from being primed and from generating CTL specific for Ag encoded by the minor histocompatibility loci--H-3, H-1, and B2m. Analysis of proliferative responses and lymphokine secretion of cells from animals primed with one of these minor H Ag, beta 2-microglobulin, but not treated with anti-L3T4 antibodies, indicated that L3T4- class I MHC-restricted T cells were themselves responsible for the very great majority of the observed minor H Ag-specific proliferation and secretion of lymphokines associated with both T cell proliferation and activation of CTL. All together, the data indicate that in responses against discrete minor H Ag, L3T4+Th-independent CTL are generated through an L3T4+Th-dependent pathway.