T-T cell interaction in the induction of delayed-type hypersensitivity (DTH) responses: vaccinia virus-reactive helper T cell activity involved in enhanced in vivo induction of DTH responses and its application to augmentation of tumor-specific DTH responses

The role of antigen-specific helper T cells in augmenting the in vivo development of delayed-type hypersensitivity (DTH) responses was investigated. C3H/HeN mice were inoculated i.p. with vaccinia virus to generate virus-reactive helper T cell activity. These vaccinia virus-primed or unprimed mice were subsequently immunized subcutaneously (s.c.) with either trinitrophenyl (TNP)-modified syngeneic spleen cells (TNP-self), vaccinia virus-infected spleen cells (virus-self), or cells modified with TNP subsequent to virus infection (virus-self-TNP). Seven days later, these mice were tested for anti-TNP DTH responses either by challenging them directly with TNP-self into footpads or by utilizing a local adoptive transfer system. The results demonstrated that vaccinia virus-primed mice failed to generate significant anti-TNP DTH responses when s.c. immunization was provided by either virus-self or TNP-self alone. In contrast, vaccinia virus-primed mice, but not unprimed mice, could generate augmented anti-TNP DTH responses when immunized with virus-self-TNP. Anti-vaccinia virus-reactive helper activity was successfully transferred into 600 R x-irradiated unprimed syngeneic mice by injecting i.v. spleen cells from virus-primed mice. These helper T cells were found to be antigen specific and were mediated by Thy-1+, Lyt-1+2- cells. DTH effector cells enhanced by helper T cells were also antigen specific and were of the Thy-1+, Lyt-1+2- phenotype. Furthermore, vaccinia virus-reactive helper T cell activity could be applied to augment the induction of tumor-specific DTH responses by immunization with vaccinia virus-infected syngeneic X5563 tumor cells. T-T cell interaction between Lyt-1+ helper T cells and Lyt-1+ DTH effector T cells is discussed in the light of the augmenting mechanism of in vivo anti-tumor-specific immune responses.     

T cells subsets responsible for clearance of Sendai virus from infected mouse lungs

T cell subsets responsible for clearance of Sendai virus from mouse lungs determined by adoptive transfer of immune spleen cell fractions to infected nude mice. T cells with antiviral activity developed in spleens by 7 days after intranasal infection. Spleen cell fractions depleted of Lyt-2+, Lyt-1+, or L3T4+ cells showed antiviral activity in vivo, although the degree of the activity was lower than that of control whole spleen cells. The antiviral activity of the Lyt-2+ cell-depleted fraction was consistently higher than that of L3T4+ (Lyt-1+)-depleted cells. In vitro cytotoxic activity against Sendai virus-associated, syngeneic lipopolysaccharide-blast cells was detected in stimulated cells from intraperitoneally immunized mice but was lost after depletion of Lyt-2+ cells. Multiple injection of anti-Sendai virus antibody into infected nude mice had no effect on lung virus titer. These results indicate that L3T4+ (Lyt-1+) and Lyt-2+ subsets are cooperatively responsible for efficient clearance of Sendai virus from the mouse lung.     

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.     

Cooperation between humoral factor(s) and Lyt-2+ T cells in effective clearance of Sendai virus from infected mouse lungs

The mechanism of cooperation between the L3T4+ and Lyt-2+ T cell subsets in effective clearance of Sendai virus from infected mouse lungs was studied by adoptive cell transfer using nude mice. Simultaneous transfer of a long-term-cultured Sendai virus-specific L3T4+ T cell line with L3T4+ cell-depleted immune spleen cell (L3T4-) fraction to infected nude mice could result in viral clearance, although single injection with either of these cells was not effective. Instead of the L3T4+ T cells, culture supernatants of the L3T4- T cell line or concanavalin A-stimulated mouse spleen cells and mouse serum immunized with the virus were also active in the cooperative viral clearance with L3T4- fraction. The role of the Sendai virus-sensitized L3T4- cell fraction in cooperative viral clearance with humoral factors could be replaced by neither T cell-deprived immune spleen cell fraction nor normal spleen cells. The 1,500 units of recombinant mouse interleukin 2 (IL-2), which was more than 12 times the IL-2 activity present in the supernatants of the T cell line or concanavalin A-stimulated spleen cells, failed to clear the virus in combination with the L3T4- fraction. Monoclonal antibodies to Sendai or mouse hepatitis viruses were also effective in the cooperative antiviral activity. IL-2 activity was not detected in these monoclonal antibodies and the mouse immune serum. Single injection of any humoral factors failed to clear the virus. These results indicate that Sendai virus-sensitized Lyt-2+ subset of T cells acts cooperatively with humoral factor(s) other than IL-2 or Sendai virus-specific antibody present in supernatants of the T cell line, of concanavalin A-stimulated spleen cells or hybridomas, and in mouse serum immunized with the virus.     

In vitro T cell-mediated killing of Pseudomonas aeruginosa. IV. Nonresponsiveness in polysaccharide-immunized BALB/c mice is attributable to vinblastine-sensitive suppressor T cells

We reported previously that BALB/c mice immunized with a polysaccharide (PS) antigen isolated from immunotype 1 Pseudomonas aeruginosa and vinblastine sulfate develop T cell-mediated protective immunity, despite their failure to produce specific antibody. In vitro, Lyt-1-,2+, I-J+ T cells from vinblastine- and PS-immunized mice kill P. aeruginosa by secretion of a bactericidal lymphokine. BALB/c mice immunized with PS alone generate neither protective antibodies nor a protective T cell response. The current studies indicate that T cells from mice immunized with PS alone significantly suppress the bactericidal activity of T cells from mice immunized with vinblastine and PS. The suppressor T cells are of the same Lyt-1-,2+, I-J+ phenotype as the bactericidal T cells. Suppression is mediated by a soluble product of these suppressor T cells which both inhibits T cell proliferation and interferes with the production or release of the bactericidal lymphokine. Cyclophosphamide, used in other systems to remove suppressor T cells, fails to enhance bacterial killing and does not inhibit suppressor cell activity. These studies indicate that immunization with PS elicits responses in two functionally distinct subgroups of Lyt-1-,2+, I-J+ T cells, and that these cells are distinguishable by their sensitivity to vinblastine sulfate.     

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.     

T helper cells in cytotoxic T lymphocyte development: role of L3T4(+)-dependent and -independent T helper cell pathways in virus-specific and alloreactive cytotoxic T lymphocyte responses

I have compared the requirements for T helper (Th) cell function during the generation of virus-specific and alloreactive cytotoxic thymus (T)-derived lymphocyte (CTL) responses. Restimulation of vesicular stomatitis virus (VSV)-immune T cells (VSV memory CTLs) with VSV-infected stimulators resulted in the generation of class I-restricted, VSV-specific CTLs. Progression of VSV memory CTLs (Lyt-1-2+) into VSV-specific CTLs required inductive signals derived from VSV-induced, Lyt-1+2- Th cells because: (i) cultures depleted by negative selection of Lyt-1+ T cells failed to generate CTLs; (ii) titration of VSV memory CTLs into a limiting dilution (LD) microculture system depleted of Th cells generated curves which were not consistent with a single limiting cell type; (iii) LD analysis of VSV memory CTLs did produce single-hit curves in the presence of Lyt-1+2- T cells sensitized against VSV; and (iv) monoclonal anti-L3T4 antibody completely abrogated CTL generation against VSV. Similar results were also obtained with Sendai virus (SV), a member of the paramyxovirus family. The notion that a class II-restricted, L3T4+ Th cell plays an obligatory role in the generation of CTLs against these viruses is also supported by the observation that purified T cell lymphoblasts (class II antigen negative) failed to function as antigen-presenting cells for CTL responses against VSV and SV. T cell lymphoblasts were efficiently lysed by class I-restricted, anti-VSV and -SV CTLs, indicating that activated T cells expressed the appropriate viral peptides for CTL recognition. Furthermore, heterogeneity in the VSV-induced Th cell population was detected by LD analysis, suggesting that at least two types of Th cells were required for the generation of an anti-VSV CTL response. VSV-induced Th cell function could not simply be replaced by exogenous IL-2 because this lymphokine induced cytotoxic cells that had the characteristics of lymphokine-activated killer (LAK) cells and not anti-viral CTLs. In contrast, CTL responses against allogeneic determinants could not be completely blocked with antibodies against L3T4 and depletion of L3T4+ cells did not prevent the generation of alloreactive CTLs in cultures stimulated with allogeneic spleen cells or activated T cell lymphoblasts. Thus, these studies demonstrate an obligatory requirement for an L3T4-dependent Th cell pathway for CTL responses against viruses such as VSV and SV; whereas, CTL responses against allogeneic determinants can utilize an L3T4-independent pathway.     

Tumor-specific idiotype vaccines. III. Induction of T helper cells by anti-idiotype and tumor cells

In a previous report, we have demonstrated the induction of tumor-specific immunity by monoclonal anti-idiotype antibodies generated against a monoclonal anti-tumor antibody, 11C1, that also cross-reacts with mouse mammary tumor virus envelope glycoprotein gp52. Also, we showed that whereas one anti-idiotype antibody, 2F10, could induce protective immunity, another anti-idiotype antibody, 3A4, induced nonprotective immunity. Here we demonstrated the existence of T helper cells which recognize anti-idiotypes that exert differential controls on tumor growth. The qualitative nature of idiotype recognizing T cells generated in response to 2F10, 3A4, irradiated tumor, and progressively growing tumor was compared. The reactivity pattern of idiotype recognizing T cells obtained from 2F10 and irradiated tumor immunized mice were similar in nature in the sense that Lyt-2- T cells obtained from these immunized mice responded to both 2F10 and 3A4 as antigen, although T cells from tumor immunized mice responded better to 3A4 antigen. On the other hand, the idiotype-recognizing T cells obtained from 3A4-immunized mice showed a similar reactivity pattern to T cells isolated from mice during the early phase of tumor growth (within day 4 to 5 after the inoculation of 10(4) live tumor cells). Lyt-2- T cells isolated from mice immunized with 3A4 or during the early phase of tumor growth responded only to 3A4 antigen. The inability of Lyt-2- T cells, isolated from 4- to 5-day-old tumor in mice, to cooperate with 2F10-TNP is not due to the absence of 2F10 idiotype recognizing T cells as 2F10 id recognizing T cells are present when examined at the precursor level. These data on the idiotype specificity of T helper cells show a correlation with the presence of anti-tumor immunity. This information will help in the design and application of idiotype vaccine in tumor immunotherapy.     

The role of L3T4+ and Lyt-2+ T cells in the IgE response and immunity to Nippostrongylus brasiliensis

The role of L3T4+ and Lyt-2+ T cells in protective immunity to Nippostrongylus brasiliensis (Nb) was studied in BALB/c mice that were depleted of either the L3T4+ or Lyt-2+ T cell population by injection with rat mAb specific for the appropriate determinant. Host responses to Nb infection including spontaneous elimination of adult worms, development of intestinal mucosal mast cell hyperplasia and the generation of a polyclonal IgE response were all completely blocked by 0.5 mg anti-L3T4 antibody administered simultaneously with Nb inoculation. However, administration of 0.5 mg of anti-Lyt-2 antibody at the same time and 7 days after inoculation with Nb had no effect on any of these responses. Injection of anti-L3T4 antibody as late as 9 days after Nb inoculation interfered with spontaneous cure of Nb infection and anti-L3T4 antibody injection 11 days after Nb inoculation inhibited serum IgE levels measured on day 13 by 50%. In addition, administration of anti-L3T4 antibody at the time of the peak serum IgE response, 13 days after Nb inoculation, accelerated the decline in serum IgE levels. Injection of previously Nb-infected mice with anti-L3T4 antibody at the time of a second Nb inoculation prevented the development of a secondary IgE response but did not affect immunity to Nb infection based on finding no adult worms in the intestines of these mice. These data indicate that 1) L3T4+ T cells are required for spontaneous cure of Nb infection, development of intestinal mucosal mast cell hyperplasia, and the generation and persistence of an IgE response during primary infection with Nb and 2) L3T4+ T cells are required for a considerable time after inoculation for optimal development of these responses. However, L3T4+ T cells are not required for all protective responses in immune mice. In contrast, our data indicate that considerable depletion of the Lyt-2+ T cell population has no significant effect on either worm expulsion or the generation of serum IgE responses.     

Role of L3T4+ and Lyt-2+ T cell subsets in protective immune responses of mice against infection with a low or high virulent strain of Toxoplasma gondii

In order to elucidate the role of T cell subsets in protective immunity against infection with high virulent and low virulent strains of Toxoplasma gondii, monoclonal antibodies specific for T cell subsets were injected into mice before immunization or challenge infection. Treatment of mice with monoclonal antibody to either L3T4+ or Lyt-2+ T cells before they were immunized with Toxoplasma cell homogenate prepared from high virulent RH strain tachyzoites markedly reduced survival after mice were challenged with low virulent bradyzoites of the Beverley strain. Thus, induction of protective immunity against bradyzoites of the Beverley strain requires the presence of both L3T4+ and Lyt-2+ T cells. In contrast, mice injected with living bradyzoites of the low virulent Beverley strain after immunization with Toxoplasma cell homogenate acquired protective immunity against high virulent tachyzoites of the RH strain. Lyt-2+ T cells alone appear to be final effector cells for protection against the challenge with high virulent RH strain tachyzoites, since treatment of the bradyzoite-immune mice with anti-Lyt-2 antibody, but not anti-L3T4 antibody, before challenge significantly increased mortality.     

T cell subsets and IFN-gamma production in resistance to systemic candidosis in immunized mice

In addition to previous evidence for the roles of T cell-dependent immunity and delayed-type hypersensitivity in acquired resistance to systemic candidosis in mice, in the present study we have investigated the relative contributions of L3T4+ and Lyt-2+ lymphocytes in the protective immunity induced by vaccination with low virulence Candida albicans cells. We have also addressed the issue of the mode of Candida Ag recognition by specific T cells leading to cytokine release. Spleen cells from immunized mice produced high levels of IFN-gamma in vitro in response to Candida Ag, and this activity was abolished only by the combined treatment of the responder population with anti-L3T4 and anti-Lyt-2.2 mAb plus C. Positively selected L3T4+ and Lyt-2+ cells also produced IFN-gamma in vitro, provided accessory cells (plastic-adherent and Thy-1- Ia- splenocytes, respectively) were added to the lymphocyte-yeast cell cocultures. The production of IFN-gamma by purified L3T4+ and Lyt-2+ cells was inhibited by addition of the respective anti-class II and anti-class I H-2 antibody to the cultures. In vivo, administration of anti-L3T4, anti-Lyt-2.2 mAb or a combination of both significantly impaired the resistance of immunized mice to challenge with virulent C. albicans, as manifested by increased recovery of the yeast from the mouse kidneys. A similar effect was observed upon neutralization of endogenous IFN-gamma by treatment with rat mAb. These results suggest that both L3T4+ and Lyt-2+ T cells play a role in acquired immunity to systemic C. albicans infection, and that their activity may involve IFN-gamma-mediated stimulation of candidacidal mechanisms.     

T cell subsets regulating antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in virgin and immunized nonresponder mice

T cell subsets from virgin and immunized mice, which are Ir gene controlled nonresponders to GAT, which regulate antibody responses to GAT have been characterized. Virgin nonresponder B10.Q B cells develop GAT-specific antibody responses to GAT, B10.Q GAT-M phi, and GAT-MBSA when cultured with virgin or GAT-primed Lyt-1+, I-J-, Qa1- B10.Q helper T cells. Virgin T cells are radiosensitive, whereas immune T cells are radioresistant (750 R); qualitatively identical helper activity is obtained with T cells from mice immunized with soluble GAT, B10.Q GAT-M phi, and GAT-MBSA. Responses to GAT and GAT-M phi are not observed when virgin or GAT-primed Lyt-1+, I-J+, Qal+ T cells are added to culture of virgin or GAT-primed Lyt-1+, I-J-, Qa1- helper T cells and virgin B cells; the GAT-specific response to GAT-MBSA is intact. The Lyt-1+, I-J+, Qa1+ T cells from mice primed with GAT, GAT-M phi, and GAT-MBSA were qualitatively identical in mediating this suppression. Virgin Lyt-2+ T cells have no suppressive activity alone or with virgin Lyt-1+, I-J+, Qa1+ T cells, whereas responses to GAT, GAT-M phi, and GAT-MBSA are suppressed in cultures of GAT-primed helper T cells containing GAT-primed Lyt-2+ T cells (with or without GAT-primed Lyt-1+, I-J+, Qa1+ T cells). Suppression of responses to GAT-MBSA in cultures of GAT-M phi-primed helper T cells requires both GAT-M phi-primed Lyt-1+, I-J+, Qa1+ T cells and Lyt-2+ T cells; the Lyt-1+, I-J+, Qa1+ T cells appear to function as inducer cells in this case. In cultures containing GAT-MBSA-primed helper T cells, either GAT-MBSA-primed Lyt-1+, I-J+, Qa1+ or Lyt-2+ T cells suppress responses to GAT and GAT-M phi; under no circumstances are responses to GAT-MBSA suppressed by GAT-MBSA-primed regulatory T cells. This regulation of antibody responses to GAT by suppressor T cells is discussed in the context of the involvement of suppressor T cells in responses to antigens under Ir control, and of the evidence that nonresponsiveness to GAT is not due to a defect in the T cell repertoire, but rather is due to an imbalance in the activation of suppressor vs helper T cells.     

Isotype-specific immunoregulation. Systemic antigen induces splenic T contrasuppressor cells which support IgM and IgG subclass but not IgA responses

In the present study, we have isolated and characterized the Lyt-1+, -2- T contrasuppressor (Tcs) cells from mice systemically primed with SRBC. Adoptive transfer of splenic Tcs cells from these mice abrogates oral tolerance and supports IgM and IgG anti-SRBC plaque-forming cell (PFC) responses; however, unlike the responses seen after transfer of Tcs cells derived from orally primed mice, low IgA responses were seen. Mice systemically primed with lower SRBC doses (0.01 to 1%) exhibited contrasuppression only within the L3T4- T cell subset, whereas mice primed with a high dose of SRBC (10%), harbored Lyt-1+, -2- Tcs cells in both the L3T4+ and L3T4- subsets. Both the L3T4- and L3T4+ Tcs cell subsets supported IgM and IgG responses when adoptively transferred to orally tolerized mice, and when added to tolerized spleen cell cultures. Splenic Tcs cells from systemically primed mice supported mainly IgG1 and IgG2b subclass anti-SRBC PFC responses, a pattern also seen with Tcs cells derived from orally primed mice. Both L3T4+ and L3T4- Tcs cells from systemically primed mice exhibited well established characteristics of contrasuppressor cells including binding to Vicia villosa lectin and expression of I-J. The splenic effector Tcs cells which support IgM, IgG1 and IgG2b anti-SRBC PFC responses are antigen-specific, since both L3T4- and L3T4+ Tcs cells from spleens of mice primed with 10% SRBC reverse tolerance to SRBC, but not to horse erythrocytes (HRBC). Further, both L3T4- and L3T4+ Tcs cells from HRBC-primed mice reverse tolerance to IgM and IgG anti-HRBC, but not to anti-SRBC responses. Isolation of T3-positive Lyt-1+, -2- and L3T4- Tcs cell subsets by flow cytometry followed by adoptive transfer, showed that effector Tcs cells express T3 and presumably contain an Ag-R (TCR-T3 complex). These studies show that systemic priming with heterologous RBC induces splenic Ag specific Tcs cells in a dose-dependent manner, which support IgM and IgG subclass responses, but not IgA responses.     

Developmental sequence of T200 antigen modifications in murine T cells

The T200 glycoproteins of T cells were analyzed at different stages of T cell development. Immunoprecipitation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that Lyt-2-L3T4-, and Lyt-2+L3T4+ thymocytes had similar T200 proteins, whereas Lyt-2+L3T4- and Lyt-2-L3T4+ thymocytes expressed a distinct set of T200 molecules. This result indicated a molecular switch in regulation of T200 protein expression upon differentiation of thymocytes to mature phenotype T cells. Further modifications were evident when the T200 proteins of peripheral T cell subsets were examined. In particular L3T4+ T cells expressed T200 proteins of m.w. 220,000, 200,000, and 175,000, whereas Lyt-2+ lymph node T cells expressed an additional T200 protein of m.w. 235,000. Antigenic differences in the T200 glyco-proteins of peripheral L3T4+ and Lyt-2+ T cells were also detected. The anti-B220 monoclonal antibody, 14.8, reacted with lymph node Lyt-2+ T cells but did not react with lymph node L3T4+T cells or with Lyt-2+L3T4- thymocytes. This finding demonstrated a lineage-specific modification of the T200 protein of Lyt-2+ T cells that occurred after exit of these cells from the thymus into peripheral lymphoid organs. This modification apparently occurred on the m.w. 235,000 and 220,000 proteins since these species were precipitated by 14.8, whereas the others were not. In vitro growth and activation also resulted in further T200 antigen alterations. The monoclonal antibody, RA3, which reacts with the B220 antigen of B cells but, unlike 14.8, does not react with any peripheral T cells, showed significant reactivity with Lyt-2+ cytotoxic T cell (CTL) clones but not with L3T4+ T helper cell clones. CTL clones were also 14.8+ but T helper cell clones were not. Immunoprecipitation by 14.8 and RA3 of T200 proteins from CTL clones yielded a single protein of m.w. 240,000 that co-migrated with the B cell form of T200. Overall, the results indicate the presence of developmentally regulated mechanisms that control T200 glycoprotein expression during T cell differentiation in the thymus and in peripheral lymphoid organs.     

Class II antigen-specific murine cytolytic T lymphocytes (CTL). II. Genuine class II specificity of Lyt-2+ CTL clones

Class II-specific allogeneic cytolytic T lymphocytes (CTL) consist of two types of cells, i.e., Lyt-2+L3T4- and Lyt-2-L3T4 T cells. The Lyt-2+L3T4- class II-specific CTL population constitutes a conspicuous exception to the general correlation observed between the class of major histocompatibility complex antigen recognized and the type of accessory molecules expressed by T cells. In order to examine the specificity of such an exceptional T cell population, CTL clones were established by limiting dilution of a bulk CTL line developed in an I region incompatible combination of mouse strains, B10.QBR anti-B10.MBR. These CTL lines showed single genetic specificity indicating their clonal nature with respect to CTL activities. Lyt-2+L3T4- (2+4-), Lyt-2-L3T4+ (2-4+) and Lyt-2-L3T4- (2-4-) clones were obtained. Among many CTL clones showing a spectrum of genetic specificities, 2+4- and 2-4+ clones with apparent I-Ak-specificity, were studied further and four lines of evidence confirmed their class II specificity: 1) genes encoding the target antigen for these CTL clones were mapped within the I-A subregion by simple genetics; 2) an I-Ak-specific monoclonal antibody readily blocked specific cytolysis by these clones; 3) the clones failed to react with cells expressing mutated I-Ak antigens; and 4) a B cell tumor transfected with alpha- and beta-chain genes of I-Ak was specifically lysed by these CTL clones. These data therefore establish the existence of Lyt-2+ CTL with genuine class II specificity. All 2-4+ CTL were sensitive to the blocking effect of an antibody to L3T4, whereas none of the 2+4- class II-specific CTL were sensitive to blocking by an anti-Lyt-2 antibody, indicating that class II-specific CTL with "wrong phenotype" is not dependent on the function of the accessory molecule. Besides true class II-specific CTL clones, 2+4- clones with a spectrum of genetic specificities were obtained, including clones recognizing a combination of an I-Ak product and the Kb molecule. Two 2-4- clones were also specific for the combination of Kb + I-Ak. These clones most likely recognize an allogeneic class II antigen in the context of a class I antigen and therefore would more appropriately be included in the class I-restricted T cell population.     

Enterically induced regulation of systemic immune responses. II. Suppression of proliferating T cells by an Lyt-1+, 2- T effector cell

Peripheral lymph node cells from C3H mice that were fed and injected with bovine serum albumin (REG cells) demonstrate an impaired proliferative response to antigenic stimulation in vitro compared to cells from mice only injected with BSA. To determine whether suppressor cells contributed to this enterically induced impairment of systemic T cell responses, REG cells were pretreated with various monoclonal antibodies and complement (C), and were then co-cultured with antigen-reactive indicator T cells (IND) from parenterally immunized mice. Proliferation of IND cells [( 3H]thymidine uptake) was suppressed only if REG cells were treated with anti-Lyt-2 and C before co-culture. The ability of anti-Lyt-1 plus anti-Lyt-2 and C treatment to abrogate suppression suggested that the suppressor effect was due to an Lyt-1+, 2- REG cell. Suppression was independent of Lyt-2+ IND cells, and was observed at different antigen concentrations, cultivation times, and cell densities. The cells responsible for suppressor activity were radiosensitive, nylon wool nonadherent, and antigen specific. These data suggest that an Lyt-1+, 2- T cell could be an important component in mediating enterically induced regulation of systemic T cell responses.     

Induction and activity of class II-restricted, Lyt-2+ cytolytic T lymphocytes specific for the influenza H5 hemagglutinin

In influenza A virus infections, CTL are a significant component of the host immune response which limits viral replication and promotes recovery. To examine the CTL response to the influenza virus A/Ty/Ont/7732/66[H5N9], particularly the H5 hemagglutinin, a long term CTL line was generated from spleen cells of A/Ty/Ont-immune Balb/c [H-2d] mice secondarily stimulated in vitro with A/Ty/Cal/Hurst-2/71[H5N2]. This CTL line was highly specific for influenza viruses of the H5 subtype. From this line, clones were isolated by limiting dilution and shown to be H5 hemagglutinin-specific based on recognition of an H5 vaccinia virus recombinant (H5 Vac). The clones exhibited the classical CTL surface phenotype Lyt-1-2+L3T4-; however, unlike the typically class I-restricted Lyt-2+ CTL, they were restricted in antigen recognition by class II (I-E) MHC molecules based on target cell recognition and antibody blocking of cytotoxicity. The clones recognized both infectious and non-infectious A/Ty/Ont presented by class II+ target cells. In adoptive transfer studies to assess the biologic role of the clones in vivo, these class II-restricted clones did not appear to alter mortality. However, these cells significantly reduced both morbidity and virus titers in the lungs of infected animals at 5 days post-infection. Thus, in the immune response to this virus, class II-restricted Lyt-2+ CTL specific for the H5 hemagglutinin were readily generated and their biologic role in vivo involved viral clearance.     

Graft-vs-host reaction limited to a class II MHC difference results in a selective deficiency in L3T4+ but not in Lyt-2+ T helper cell function

Hybrid mice of the (B6 X bm12)F1 combination were inoculated i.v. with parental B6 spleen cells to induce a class II graft-vs-host disease (GVH). Such mice failed to generate in vitro cytotoxic T lymphocyte (CTL) responses that were dependent upon L3T4+ T helper cell (Th) function (e.g., anti-B6-TNP) but were capable of generating in vitro CTL responses that could be mediated by Lyt-2+ Th cells (anti-allo class I). When Th function was assayed directly by interleukin 2 (IL 2) secretion, class II GVH animals were found to be deficient in L3T4+ but not Lyt-2+ IL 2-secreting Th cells. This selective deficiency in L3T4+ Th function correlates with a selective decrease in class II GVH mice of host-derived derived L3T4+ T cells. In addition, it was found that the spleens of class II GVH mice contained cells capable of selectively suppressing L3T4+ Th function. In contrast, mice in which a class I + II GVH occurred were depleted of both L3T4+ and Lyt-2+ Th function as assessed by IL 2 production. The findings that class II GVH selectively depletes L3T4+ T cells and T cell functions are discussed with respect to the immune function of distinct T cell subsets in normal and diseased states.     

Stimulation of murine T cells via the Ly-6C antigen: lack of proliferative response in aberrant T cells from lpr/lpr and gld/gld mice despite high Ly-6C antigen expression

Cross-linking of cell surface Ly-6C molecules with the 6C3 rat monoclonal antibody (MAb) followed by anti-rat immunoglobulin antibody acts in concert with phorbol myristate acetate (PMA) as a potent mitogenic stimulus for normal T cells. Specificity of this stimulation was demonstrated by its absence in T cells from NZB, NOD, or STb/J mice which lack the 6C3 determinant. In 6C3+ normal strains, the extent of 6C3-mediated stimulation varied, depending on the level of 6C3 antigen expression. Analysis of this stimulation in purified T cell subsets revealed that in Ly-6.1 strains (e.g., BALB/c, CBA/J), Lyt-2+ cells responded, but not L3T4+ cells, whereas in Ly-6.2 strains (e.g., C57BL/6, MRL-+/+), both subsets produced IL 2 and proliferated, although with different kinetics. Moreover, in adult MRL-+/+ mice, the minor Lyt-2-/L3T4- subset from the lymph nodes gave low responses to 6C3 cross-linking, whereas that from the thymus reacted strongly. Stimulation via Ly-6C therefore provides a pathway for differential activation of normal T cells. In contrast, the expanding population of Lyt-2-/L3T4- T cells from lpr/lpr or gld/gld mice did not proliferate in response to 6C3 antigen cross-linking plus PMA despite high levels of 6C3 antigen expression. Responsiveness of lpr/lpr T cells could not be restored with IL 1, IL 2, or both. These T cells also failed to be triggered by conjunction of PMA with either Thy-1 antigen cross-linking or concanavalin A. Moreover, they were not stimulated, in the presence of PMA, by doses of ionomycin that were optimal for normal T cells, but did respond to higher ionomycin concentrations (2 micrograms/ml), and this response was not altered by Ly-6C cross-linking. It is concluded that the Ly-6C pathway of T cell activation is not functional in the aberrant lpr/lpr (and gld/gld) T cells, and that this defect may reflect abnormalities of intracellular signaling.     

Higher frequency of Leishmania major-specific L3T4+ T cells in susceptible BALB/c as compared with resistant CBA mice

In previous studies, we reported that a) the adoptive transfer of parasite-specific L3T4+ T cells enhanced rather than inhibited the development of lesions induced by Leishmania major in normal BALB/c mice, and b) the depletion in vivo of L3T4+ T cells by administration of anti-L3T4 monoclonal antibody reversed the susceptibility of BALB/c mice to L. major. To further assess the role of specific L3T4+ T cells in the development of lesions induced by L. major in BALB/c mice, the frequency of parasite-specific T cells capable of mediating specific delayed-type hypersensitivity (DTH) reactivity was determined, by limiting dilution analysis, in the lymph nodes draining the lesions of susceptible (BALB/c) and resistant (CBA) mice. The numbers of L. major-specific DTH-mediating T cells was found to be substantially increased in the lymph nodes of infected BALB/c mice as compared with CBA mice. Moreover in CBA mice, analysis of the cell surface phenotype of the L. major-specific DTH-mediating T cells showed that these cells were equally represented in the L3T4+, Lyt-2-, and L3T4- Lyt-2+ subsets, whereas the majority of these cells in BALB/c mice expressed the L3T4+ Lyt-2- surface phenotype.