The generation of effector T cells in influenza A-infected, cyclosporine A-treated mice

Specific effector T cells that mediate DTH to influenza virus were found to be formed in vivo in CsA-treated mice. The activity of these cells could only be measured when they were transferred into untreated naive mice. The cells mediating DTH were H-2 restricted in the I region of the MHC. When effector T cells that mediated DTH were transferred into CsA-treated recipients, no DTH activity could be detected. Influenza-specific cytotoxic T cells could not be detected in the spleens of CsA-treated mice given virus intravenously, even when drug treatment was started 3 days after virus administration. There was only a partial restoration of cytotoxic activity when spleen cells from CsA-treated infected mice were cultured in the presence of virus-infected stimulators. This seemed to indicate that Class I-restricted responses were more susceptible to CsA than the generation of Class II (or I-region-restricted) responses.     

Production by Cultured Spleen Cells of Inflammatory Substances and Other Lymphokines that Mediate Delayed-Type Hypersensitivity in Mice

In vitro cultivation of normal mouse spleen cells with human serum albumin generated effector cells that mediate the delayed-type hypersensitivity (DTH) reaction. The cultured cells, when incubated in a serum-free medium for a further 24 hr, released substances (FPRF) which caused a footpad inflammatory reaction at a maximum of 6 hr after injection into normal syngeneic or allogeneic strains of mice, as well as macrophage migration inhibition factor (MIF) and macrophage activating factor (MAF). The DTH-effector cells in the culture were fractionated in the low density layers by discontinuous bovine serum albumin density gradient centrifugation. The effector cells in the low density layers were further enriched in the Lyt 1 subpopulation of T cells when fractionated on a fluorescence activated cell sorter. Cells capable of producing the inflammatory substances (FPRF), MIF and MAF were also enriched in the same fraction containing DTH-effector cells. These results suggest that low density, Lyt 1-positive T cells mediating the DTH reaction produce FPRF as well as MIF and MAF.     

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

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

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

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

Induction of suppressor cells by a tumor-derived suppressor factor

Murine fibrosarcomas produce a factor that activates suppressor cells to inhibit expression of delayed-type hypersensitivity (DTH) responses to dinitrochlorobenzene (DNCB). This tumor-derived suppressor factor (TDSF) was partially purified by preparative isoelectric focusing of spent medium and 3 M KCl extracts of cultured methylcholanthrene-induced and spontaneous fibrosarcomas of C3H/He mice. Incubation of 1 micrograms/ml of a fraction, isoelectric pH less than 2.9, with normal syngeneic spleen cells for 1-6 hr at 37 degrees C induced suppressor cells that inhibited the primary DTH response to DNCB upon intraperitoneal transfer to normal C3H/HeJ mice. TDSF was not present in extracts of either syngeneic embryonic fibroblasts or normal spleen cells or in medium conditioned by normal peritoneal exudate cells but was present in 3 M KCl extracts of and the spent medium from four different cultured murine fibrosarcomas. TDSF activity was not restricted at the major histocompatibility complex. The suppressor cells inhibited the efferent limb of the DTH response because (1) hyporesponsive recipients of TDSF-treated spleen cells had splenic effector T cells capable of transferring DTH to DNCB into naive secondary recipients and (2) the ability of Lyt 1+,2- effector Tdth cells to transfer a secondary DTH response to DNCB was inhibited by co-incubation with macrophages or Lyt 1-,2+ T cells treated with TDSF. Preliminary biochemical analysis suggested that TDSF was an RNA- protein complex. Thus, several murine fibrosarcomas produced a soluble factor that activated splenic suppressor cells to depress the immune response to nonneoplastic antigens. These suppressor factors represent a novel group of regulatory molecules which may be ribonucleoprotein complexes.     

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

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

Specific and nonspecific T-cell recruitment in viral meningitis: Possible implications for autoimmunity

Specific and nonspecific T-cell invasion into cerebrospinal fluid has been investigated in the nonfatal viral meningoencephalitis induced by intracerebral inoculation of mice with vaccinia virus. At the peak of the inflammatory process on Day 7 approximately 5 to 10% of the Lyt 2⁺ T cells present are apparently specific for vaccinia virus. Concurrently, in mice primed previously with influenza virus, 0.5 to 1.0% of the appropriate T-cell set located in cerebrospinal fluid is reactive to influenza-infected target cells. This vaccinia virus-induced inflammatory exudate may thus contain as many as 500 influenza-immune memory T cells. These findings are discussed from the aspect that such nonspecific T-cell invasion into the central nervous system during aseptic viral meningitis could result in exposure of potentially brain-reactive T cells to central nervous system components.     

Different functions of subsets of effector T cells in murine influenza virus infection

Enriched preparations of secondary effector T cells to influenza virus were tested for their in vivo biological function by adoptive transfer to mice 24 hr after an intranasal inoculation of infectious influenza virus. One class of cells which were Lyt 1⁺2^?3^?, I region-restricted, and could mediate DTH reaction failed to reduce lung virus titers 5 days after transfer and caused a higher mortality rate in the recipient mice than in the controls. A second class of cells which were Lyt 1^?2⁺3⁺, K,D region-restricted, and were cytotoxic and could mediate DTH activity substantially reduced lung virus titers 5 days after transfer. The influx of mononuclear cells to the lungs after adoptive cell transfer was measured by injection of [¹²⁵I]UdR 24 hr prior to harvest of lung cells, using both infected CBA and athymic BALB/ c nude (nu/nu) mice as recipients. I region-restricted cells caused increased cellular infiltration which was very marked in athymic mice. It was concluded that this reaction significantly contributed to the observed immunopathology in infected mice. Transfer of K,D region-restricted cells reduced the cellular infiltration in infected CBA mice and caused only a slight increase in infected athymic mice. The evidence supported the concept that the second class of cells exerted their protective (antiviral) effect in vivo by direct lysis of virus-infected cells rather than by liberation of lymphokines.     

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

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

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

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

Both cloned interleukin 2 and purified interleukin 1 are required for optimal growth of purified L3T4+ and Lyt 2+ lymphocytes initiated by concanavalin A

Concanavalin A (Con A), cloned interleukin 2 (IL-2), purified interleukin 1 (IL-1) or two different crude preparations containing IL-1 activity alone, did not induce proliferation of rigorously accessory cell (AC)-depleted splenic L3T4+ or Lyt 2+ lymphocytes. Con A together with saturating concentrations of cloned IL-2 (100 U/ml) promoted less than 40% of the proliferative responses observed in AC-supplemented L3T4+ and Lyt 2+ T-cell cultures. The three preparations of IL-1 used supported minimal proliferation of Con A-treated purified L3T4+ or Lyt 2+ lymphocytes. However, all these IL-1 preparations promoted significant growth of the T-cell populations if AC (1%) were included in the cultures. Cloned IL-2 combined with purified IL-1 promoted proliferation of Con A-treated L3T4+ and Lyt 2+ lymphocytes achieving approximately 75% of the responses observed in AC-supplemented T-cell cultures. The additive effect of IL-1 was apparent in the presence of saturating concentrations of cloned IL-2. Finally, Con A alone induced a detectable number of both L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors as determined with the anti-mouse IL-2 receptor antibody 7D4 by immunofluorescence and FACS analysis. Purified IL-1 neither induced detectable number of L3T4+ or Lyt 2+ T cells to express IL-2 receptors nor increased the number of Con A-treated T cells bearing IL-2 receptors. We have interpreted these findings to indicate the following: Con A alone is sufficient to induce highly purified L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors. Cloned IL-2 and purified IL-1 are required for optimal growth of L3T4+ and Lyt 2+ lymphocytes and these cytokines together efficiently replace AC in growth of T cells initiated by Con A. IL-1 alone does not replace AC in Con A-induced activation of mouse T cells. IL-1 exerts potentiation on IL-2-driven growth of Con A-treated L3T4+ and Lyt 2+ lymphocytes. The additive activity of IL-1 on growth of normal T cells is not due to increased production of IL-2 in the cultures or induction of normal T cells to expression of IL-2 receptors by IL-1. We propose that IL-1 optimizes the action and/or interaction of IL-2 with its receptors on the T-cell membrane (by, i.e., increasing affinity of the IL-2 receptor for its ligand and/or stabilizing the IL-2 receptor).     

Lymphocyte subsets involved in tumor-activated nonspecific feedback suppression

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

Primary and secondary delayed-type hypersensitivity to minor histocompatibility antigens in the mouse.

Immunization of mice with viable allogeneic H-2-compatible spleen cells can induce a persistent state of delayed-type hypersensitivity (DTH) to these alloantigens, as measured with the footpad swelling test. Boosting of such mice, 2–4 months after priming, induced a typical secondary-type DTH reactivity. The capacity of secondary DTH to non-H-2 alloantigens could be adoptively transferred from primed mice into irradiated syngeneic hosts by means of nylon wool-nonadherent, Thy-1.2⁺ spleen cells. Vinblastine treatment of the donor mice did not affect the adoptive DTH responsiveness. These results suggest that a population of long-lived T memory cells contributes to secondary-type DTH responsiveness to non-H-2 alloantigens. The phenomenon of persistent DTH is discussed in the light of these results. The hypothesis is put forward that persistent DTH is dependent on the continuous antigen-driven differentiation of long-lived, recirculating T memory cells into nonrecirculating, functionally short-lived DTH effector cells.     

In vitro and in vivo induction of effector T cells mediating DTH responses to a protein and a synthetic polypeptide antigen.

We have developed an in vitro system for the activation of T cells in order to get a better insight into the genetic and molecular mechanisms involved in the generation of delayed-type hypersensitivity (DTH) effector T cells. Low doses of fowl γ-globulin (FγG) as well as the synthetic polypeptide (T,G)-A-L were bound to splenic adherent cells and served as immunogens for the in vitro sensitization of lymphocytes. In parallel, (T,G)-A-L-specific T cells were activated in vivo in irradiated recipient mice. The ability of the in vitro- and in vivo-activated cells to mediate DTH responses was determined in naive recipient mice by the radioisotopic ear assay. Twenty to thirty × 10⁶ “educated” cells were sufficient to elicit significant DTH responses. Irradiation of the spleen cells prior to their transfer resulted in higher responses. The DTH reactivity was transferable by nylon wool-enriched T cells but not by a Thy 1.2-depleted population indicating the T-cell dependency of the response. The in vitro and in vivo antigen-activated T-cell population exhibited also helper-cell activity as determined by their cooperation with B cells in adoptive transfer experiments.     

Characterization and function of autoreactive T-lymphocyte clones isolated from normal, unprimed mice

Self-Ia-reactive cloned T-cell lines, designated PK, were established by long-term culture of T cells from normal DBA/2 mice with irradiated syngeneic splenic adherent cells (SAC), rich in macrophages and dendritic cells. The cell lines were Thy 1+, Lyt 1+, Lyt 2-, produced IL-2 following stimulation with syngeneic spleen cells, and did not exhibit alloreactivity when screened against six different H-2 haplotypes. Of the five cloned PK cell lines tested, four were I-Ed restricted while one was I-Ad restricted as determined by genetic mapping and blocking studies carried out with monoclonal anti-Ia sera. Extensive specificity studies suggested that the PK cells reacted to syngeneic Ia molecules alone and not to foreign antigens such as fetal calf serum (FCS) used in the culture medium, in association with self-Ia. SAC pulsed with FCS or other protein antigens such as turkey gamma-globulin (TGG) were tested for their ability to induce proliferation of autoreactive T cells and other antigen-specific T cells using culture conditions consisting of serumless medium and interleukin 2 (IL-2). The data showed that the autoreactive T cells proliferated better in response to antigen-unpulsed SAC, while FCS-specific and TGG-specific cell lines, developed independently, proliferated only in response to FCS- or TGG-pulsed SAC, respectively, but not to antigen-unpulsed SAC. These results clearly distinguished the autoreactive T-cell clones from the antigen-specific T-cell clones. Preliminary studies carried out to investigate the functions of autoreactive T cells suggested that these cells helped in the in vitro differentiation of alloantigen-specific cytotoxic T lymphocytes (CTL) from CTL precursors obtained from the thymus and augmented syngeneic, allogeneic, and antigen-specific immune responses in vitro. The autoreactive T cells were also capable of inducing both proliferation and differentiation of antigen-specific populations of B cells in the absence of antigen. The present investigation suggests that autoreactive, non-antigen-reactive T cells can be cloned from normal, unimmunized mice and that such cell lines may provide a powerful tool for analyzing the role of the syngeneic mixed lymphocyte reaction in induction and maintenance of both T-and B-cell immune responses.     

Basic protein-specific T-cell lines that induce experimental autoimmune encephalomyelitis in SJL/J mice: comparison with Lewis rat lines

Myelin basic protein (BP)-specific T-cell lines were selected from SJL/J mice using techniques to select similar lines from Lewis rats. SJL/J BP-specific T-cell lines were composed of T cells with the helper/inducer phenotype (Lyt 1.2+, 2.2- and L3T4+) and proliferated in response to both the 1-37 and the 89-169 fragments of guinea pig BP. BP-specific T-cell lines transferred delayed-type hypersensitivity (DTH) responses to BP that persisted for over 60 days. Most recipient animals (32/41) developed acute experimental autoimmune encephalomyelitis (EAE), and most survivors (19/24) developed chronic relapsing EAE. Spinal cords of animals during both the acute and the chronic phases of illness contained plaques of demyelination and infiltrates of lymphocytes and macrophages. These findings differed from those of Lewis rat BP-specific lines which respond to a different region of BP, transfer DTH responses that last less than 12 days, and induce acute EAE in which demyelination does not occur.     

T-cells inhibit Friend murine leukemia virus infection of B-cells in vitro

The ability of splenic T-cells to regulate Friend murine leukemia virus replication in lipopolysaccharide-activated target B-cells infected in vitro was investigated. Removal of the T-cell fraction from spleen cells resulted in an 8- to 10-fold enhancement in the number of productively infected cells in the remaining B-cell-enriched fraction, as compared with unseparated spleen cells, and the addition of increasing numbers of purified T-cells to isolated B-cells prior to infection resulted in a directly proportional reduction in the number of B-cells releasing infectious progeny virus. Separation of splenic T-cells into Lyt 2- and Lyt 2+ T-cells before addition to infected B-cell cultures resulted in inhibition of infection only with the Lyt 2- T-cells; Lyt 2+ T-cells did not inhibit infection, even at high 1:1 ratios. Similarly, separation of splenic T-cells into L3T4+ and L3T4- T-cells before addition resulted in inhibition by L3T4+ but not L3T4- T-cells. Also, cytotoxic treatment of splenic T-cells with monoclonal anti-L3T4 antibody and complement before addition to B-cell cultures destroyed the regulatory effects. Finally, depletion of macrophages from both T-cells and B-cells before infection and coculture had no effect on the ability of T-cells to regulate B-cell infection. Collectively these results demonstrate that L3T4+ T-cells can inhibit Friend murine leukemia virus replication in target B-cells. Culture of isolated splenic T-cells with Friend murine leukemia virus in vitro resulted in the induction of alpha/beta but not interferon-gamma synthesis and in some experiments interferon-containing supernatants from T-cell-virus cultures were able to mediate suppression of B-cell infection with Friend helper virus; the addition of antibody specific for interferon-alpha/beta to cultures inhibited the ability of T-cells to regulate B-cell infection.     

Type II Collagen Induces Peripheral Tolerance in BALB/c Mice via the Generation of CD8+ T Regulatory Cells

Antigens introduced into the anterior chamber (AC) of the eye induce a potent form of antigen-specific peripheral immune tolerance termed AC-associated immune deviation (ACAID), which prevents inflammatory immune responses and is characterized by impaired delayed-type hypersensitivity (DTH) responses. Type-II collagen (CII) is a fibrillar protein expressed exclusively in cartilage tissues. Although of its clinical relevance to Rheumatoid arthritis, aging, and osteoarthritis, there have been no studies to date to test if CII has the ability to induce ACAID. We hypothesized that ACAID could be generated via AC injection of CII in BALB/c mice. Using a DTH assay, the hypothesis was supported and led to another hypothesis that CII is capable of inducing specific immune tolerance via CD8⁺ T regulatory cells (Tregs). Thus, we performed functional local adoptive transfer (LAT) assays to examine the regulatory roles of spleen cells, T cells, and CD8⁺ T cells in the specific immune regulation induced by CII injection into the AC. Results indicated that CII induced ACAID when injected into the AC. Spleen cells of mice injected with CII in the AC significantly suppressed DTH responses. The T cell compartment of the spleen was capable of expressing this suppression. CD8⁺ Tregs could solely express this CII-driven suppression and even exerted more noticeable suppression than spleen cells or splenic T cells. This study suggests a crucial role for CD8⁺ Tregs in mediating CII-driven ACAID-mediated immune tolerance. This could have therapeutic implications in Rheumatoid arthritis, aging, osteoarthritis, and other diseases in which CII is involved.     

In vivo effects of GK1.5 (anti-L3T4a) monoclonal antibody on induction and expression of delayed-type hypersensitivity

The in vivo effects of monoclonal GK1.5 antibody, directed against the L3T4a determinant expressed on Class II-restricted T cells, on the induction and expression of murine delayed-type hypersensitivity (DTH) responses were examined. Development and expression of both hapten (2,4-dinitrofluorobenzene and 2,4,6-trinitrochlorobenzene)- and protein antigen poly(Glu60Ala30Tyr10)-specific DTH are significantly inhibited by injection of monoclonal anti-L3T4a antibody. The inhibitory effects of anti-L3T4a were most pronounced when administered during the afferent (induction) phase of the DTH response, leading to the functional inhibition of the generation of both polyclonal lymph node T-proliferative cells (Tprlf) and DTH effector cells (TDH). The in vivo inhibitory effect is apparently unrelated to preferential induction of suppressor T cells as GK1.5 inhibited DTH induction in cyclophosphamide-treated as well as normal recipients. L3T4a expression on the various T-cell subsets involved in DTH induction and elicitation was also examined. The data show that three functionally distinct, antigen-specific T-cell subsets, Tprlf, TDH, and Th cells involved in DTH induction, bear the Lyt 1+2-, L3T4+ phenotype. Possible mechanisms where in vivo injection of anti-L3T4a inhibits Class II-restricted T-cell subsets involved in DTH induction and expression, including immune depletion and inhibition of T-cell-receptor/ligand interactions, are discussed.     

Antigen-specific augmentation factor involved in murine delayed-type footpad reaction. IV. Effect of delayed-type hypersensitivity augmentation factor on in vitro induction of DTH

We found an antigen-specific factor capable of augmenting delayed-type hypersensitivity (DTH) in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DFR), or in the culture supernatant of the mixture of sensitized T cells and specific antigens. This factor (DTH augmentation factor; DAF) was confirmed to augment DTH in transferred recipients. In this paper, such an activity of DAF was further investigated using the system with in vitro induction and local transfer of DTH. DAF also augmented the primary in vitro induction of DTH, when spleen cells from mice transferred with the DAF-containing serum 12 hr previously or spleen cells incubated with the DAF-containing serum on ice for 2 hr were cultured with heterologous erythrocytes. DAF acted on the induction phase of DTH and augmented a typical DTH which was dependent on Thy-1-positive T cells. DAF showed antigen specificity, but was not assigned to conventional immunoglobulin. The activity of DAF was detected when nylon-wool nonadherent cells were incubated with DAF prior to the culture of those cells and antigens, but not detected when only nylon-wool adherent cells were incubated with DAF. Thus, DAF exerted its effect through binding to acceptor cells which were included in nylon-wool nonadherent spleen cells from normal mice.