Studies on the in vivo induction and suppression of direct and lectin-dependent, cell-mediated cytotoxicity

Alloimmunization (C57BL/6, anti-P815) can result in the development of cytolytic effector cells capable of mediating direct antigen-specific, cell-mediated cytotoxicity (DCMC) and nonspecific lectin-dependent, cell-mediated cytotoxicity (LDCC). The induction of DCMC appeared to require challenge with large numbers (10⁸) of viable replicating P815 cells, whereas LDCC reactivity was obtained following challenge with high or low (10⁴) numbers of replicating or mitomycin C-treated P815 cells. Other alloantigen preparations, e.g., soluble antigen or membrane preparations, failed to induce DCMC or LDCC. An examination of the effects of high- and low-dose challenge using viable P815 cells demonstrated that high-dose challenge resulted in strong DCMC, LDCC, a readily detectable humoral response, and some delayed-type hypersensitivity, whereas low-dose challenge yielded LDCC, strong delayed-type hypersensitivity, and suppressor cell activity. The development of DCMC was severely suppressed in animals primed with a low-dose P815 challenge and subsequently rechallenged with 10⁸ P815. Further development of LDCC was similarly suppressed. It appears that although both DCMC and LDCC effector cells are susceptible to previously activated suppression, during a primary in vivo response a portion of the LDCC effector cells develop beyond a critical stage before suppression is expressed.     

Lectin-dependent cell-mediated cytotoxicity: induction of a unique effector cell population.

Spleen cells from C57BL/6 (H-2^b) mice were assayed for their ability to mediate lectin-dependent (Con A, PHA) cell-mediated cytotoxicity (LDCC), following immunization with erythrocytes, bovine serum albumin, Bacillus Calmette Guerin, and allogeneic (H-2^d) P815 cells. Sensitization with viable, but not formaldehyde-fixed, P815 cells resulted in lectin-dependent lysis of syngeneic EL-4 cells. All other sensitization procedures failed to produce LDCC. Spleen cells from mice challenged with high (10⁸) doses of P815 cells were capable of mediating both direct (anti-P815) cytotoxicity and LDCC, while challenge with low (10⁴) doses of P815 cells produced strong LDCC reactivity in the apparent absence of direct cytotoxicity (DCMC). Characterization of the effector cells indicated that LDCC reactivity was mediated by an activated, non-adherent T cell population. The effector cells appear to be unique in that LDCC could be induced in the absence of DCMC, LDCC activity appeared prior to DCMC, and DCMC could be removed by adsorption on P815 monolayers without depleting LDCC reactivity.     

Lectin-dependent cell-mediated cytotoxicity in hamsters bearing syngeneic tumors

Spleen cells from LSH hamsters inoculated with xenogeneic, allogeneic, or syngeneic (PARA-7) tumor cells were assayed for their ability to mediate direct cell-mediated cytotoxicity (DCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) in a 4-hr chromium release assay. Spleen cells from animals immune to xenogeneic or allogeneic cells demonstrated specific DCMC against homologous target cells in the absence of Con A and nonspecific LDCC against both homologous and heterologous target cells in the presence of Con A. Spleen cells from animals bearing syngeneic PARA-7 tumors (TBA) failed to express DCMC against homologous or heterologous target cells; however, significant lysis of all target cells occurred in the presence of Con A. LDCC was not detectable when nonsensitized spleen cells from normal animals were employed. The LDCC reaction was dependent on the concentration of Con A and the number of effector cells present in the reaction. The development of LDCC effector cells in the TBA appeared to parallel the development of both DCMC and LDCC effector cells in immune animals.     

Analysis of cytotoxic mechanisms induced by minor histocompatibility antigens in the mouse.

Following immunization of BALB/c (H-2^d) mice against the P815Y (H-2^d) mastocytoma, two populations of effector cells could be identified in the spleen, namely, the cytolytic T cell and a cytostatic effector, which was resistant to anti-T-cell serum and complement and appeared to be adherent. Quantitative comparison of the activities of both effectors has been made with the levels of activity obtained following immunization across the major H-2 barrier in C57BL10 (H-2^b) mice. While T-cell activity was significantly lower in BALB/c mice, the non-T-cytostatic activity was greater compared with C57BL mice. Therefore, H-2 antigens do not appear to be essential for the efficient induction of the cytostatic effector.     

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.     

A comparison of the role of protein synthesis in cell-mediated cytotoxic reactions.

Pactamycin, an irreversible inhibitor of protein synthesis, was employed to investigate the requirement for protein synthesis during in vitro cell-mediated cytotoxic reactions. The cellular reactions examined included direct cell-mediated cytolysis (DCMC) of EL-4 tumor cells by alloimmune lymphocytes, antibody-dependent cell-mediated cytolysis of HEp-2 tumor cells (ADCC-T), and antibody-dependent cell-mediated cytolysis of chicken erythrocytes (CRBC) (ADCC-E). Pretreatment of alloimmune lymphocytes with pactamycin (PAC) did not alter the DCMC reactivity osf the effector cells even though protein synthesis was inhibited by >90%. Similarly, inhibition of protein synthesis followed by 6 hr of in vitro incubation prior to the assay did not significantly reduce reactivity. Pretreatment of normal lymphocytes failed to inhibit cytotoxic reactivity when employed in an ADCC assay against HEp-2 cells, but produced partial inhibition of ADCC reactivity against CRBC. Incubation following PAC treatment had no effect on ADCC-T, but abrogated all ADCC-E activity within 3 hr. The data presented indicates that the effector cells mediating ADCC-E and those mediating both ADCC-T and DCMC differ markedly in their requirements for continued protein synthesis.     

Restricted recognition of H-2 subregion coded alloantigens in delayed-type hypersensitivity

Subcutaneous (sc) immunization of mice with H-2K, I, or D incompatible spleen cells induces a state of host-versus-graft (HvG) delayed-type hypersensitivity (DTH). The DTH reaction is elicited by challenging the immunized mice in a hind foot with similar allogeneic spleen cells and is measured as the subsequent foot swelling. DTH effector T cells specific for H-2I-coded alloantigens, but not for H-2K/D-coded alloantigens, can be induced in a graft-versus-host (GvH) model as well. In this paper we report that under HvG as well as under GvH conditions the recognition of class II antigens by DTH effector T cells is restricted by class I molecules. Furthermore, DTH effector T cells induced by sc immunization with class I antigens appear to be restricted by class II molecules.     

Differential responsiveness to MIs locus antigens in graft-versus-host and host-versus-graft reactions

After (semi)allogeneic transplantation of lymphoid cells into lethally irradiated mice, the development of anti-host directed T effector cells can be demonstrated by means of a simple delayed-type hypersensitivity (DTH) assay. Using this assay we have shown that in H-2 compatible combinations Mls locus antigens can induce the generation of such T effector cells during a graft-versus-host (GvH) reaction. Other non-H-2 alloantigens are probably of minor importance. The capacity of Mls locus antigens to induce distinct anti-host DTH reactivity correlated with the capacity to induce a one-way mixed lymphocyte culture (MLC) response. Mls^a and Mls^c locus antigens initiated a positive MLC response as well as distinct GvH-related DTH reactivity. On the other hand, in the combination DBA/2 versus (BALB/c × DBA/2) F₁, the Mls^b locus antigen was not able to initiate in vitro proliferation, a lack of response which coincided with a marginal and short-lasting GvH-related DTH reactivity. In contrast, the host-versus-graft (HvG) DTH reaction of BALB/c and DBA/2 mice to subcutaneously injected (BALB/c × DBA/2) F₁ spleen cells was equally strong. Here antigens other than those coded for by the Mls locus were mainly responsible for the antigraft DTH response. These results suggest that T effector cells generated in GvH and HvG reactions are specific for largely different sets of minor histocompatibility antigens, with a selective stimulation by Mls locus antigens under GvH conditions.     

Mechanism of cell-mediated cytotoxicity at the single-cell level: VII. Trigger of the lethal hit event is distinct for NK/K and LDCC effector cells as measured in the two-target conjugate assay

Normal human peripheral blood lymphocytes (PBL) express several in vitro cytotoxic functions, among which are natural killer (NK), antibody-dependent cellular cytotoxicity (ADCC), and lectin-dependent cellular cytotoxicity (LDCC). The relationship of these various cytotoxic functions and the identity of cells involved has been a subject of controversy. Recently it was reported that NK and K for ADCC can be mediated by the same cell, suggesting that they constitute in large part a single subpopulation with multiple cytotoxic functions. The ability of this NK/K effector cell to mediate LDCC was examined here using the two target conjugate assay. The effector cells were Ficoll-Hypaque PBL or LGL-enriched fractions. The targets used were K562 or MOLT for NK, RAJI coated with antibody for ADCC, and RAJI coated with PHA or Con A or modified by NaIO₄ for LDCC. In the two-target conjugate assay, one of the targets is fluorescein labeled for identification. The results show that (a) LDCC copurifies with NK/K and is enriched in the LGL fraction, as measured in both the ⁵¹Cr-release assay and the single-cell assay for cytotoxicity; (b) single effector cells simultaneously bind to NK or ADCC and LDCC targets, revealing that single cells bear binding receptors for all targets; and (c) single lymphocytes were not able to kill both bound NK/K and LDCC targets. However, significant two-target killing was obtained when both targets were NK targets, ADCC targets, LDCC targets, or one NK and one ADCC target. These results demonstrate that the NK and LDCC effector cells are distinct subpopulations copurified in the LGL fraction. In addition, the results show that lectin is unable to trigger globally an NK effector cell to mediate cytotoxicity against a bound NK insensitive target. Thus, although both NK and LDCC effector cells are present in the LGL fraction and can bind to both types of targets, the trigger of the lethal hit event is the function of specialized effector cells.     

Herpes-simplex-virus-specific,H-2Dk-restricted T lymphocytes bear receptors for H-2Dd alloantigen

Cytotoxic T lymphocytes generated in the course of an HSV-infection of CBA (H-2 ^k ) mice not only lyse syngeneic, virus-infected target cells but also cross-react with noninfected target cells expressing the D^d alloantigen. On the effector cell level, this alloreactivity is mediated by virus-specific CTL's that are restricted to H-2D^k determinants. On the prekiller cell level, the anti-HSV-reactive T cells exhibiting cross-reactivity for D^d alloantigen could be positively selected on H-2^d spleen-cell monolayers. After differentiation into cytolytic effector cells, target cells expressing D^d alloantigens and syngeneic HSV-infected target were lysed with equal efficiency. The results imply that the phenomenon of H-2-restricted versus nonrestricted T-cell reactivity is not due to distinct T-cell subsets, but rather is dependent on the antigeneic determinants recognized.     

Two Distinct Populations of Primary Cytotoxic Cells Infiltrating into Allografted Tumor Rejection Sites: Infiltration of Macrophages Cytotoxic against Allografted Tumor Precedes That of Multiple Sets of Cytotoxic T Lymphocytes with Distinct Specificity to Alloantigens

It has been reported that the rejection of tumor allografts is mainly mediated by cytotoxic T lymphocytes (CTLs). Here, we characterized the cytotoxic effector cells of C57BL/6 (B6; H-2^b) mice infiltrating into the rejection site of the i.p. allografted Meth A fibrosarcoma (or P815 mastocytoma) cells of H-2^d origin. Two types of cytotoxic cells (i.e., CD8⁺ CTLs and macrophages (Mφs)) were identified by flow cytometric fractionation of the infiltrates or by specific in vitro elimination of cells either with antibody (Ab)-coated beads or with an Ab-plus complement. Of particular interest, these effector cells showed distinct and unique target specificities. First, the CTLs were inactive against transplanted tumor (e.g., Meth A) cells, whereas they were cytotoxic against donor-related concanavalin A (Con A) blasts as well as CTLL-2 (H-2^b) cells transfected with a class I gene of H-2^d origin. A cold target competition assay suggested that the CTLs were composed of multiple sets of T cells, each of which specifically recognized different allo-antigens. Second, the Mφs lysed the allografted tumor cells but were inert toward the Con A blasts and the CTLL-2 transfectants. Unexpectedly, the infiltration of Mφs preceded the infiltration of CTLs by several days during the course of rejection. These results indicate that two distinct populations of unique cytotoxic cells (i.e., CTLs and Mφs) are induced in the allografted tumor rejection site, and that the infiltration of cytotoxic Mφs responsible for rejection precedes that of the CTLs cytotoxic against cells expressing donor-related allo-antigens.     

Activation of specific suppressor cells with heat-treated allogeneic tumor cells

The ability of heat-treated allogeneic cells to induce suppressor cells was examined. The tumor cell lines EL-4 (H-2^b) and P815-X2 (H-2^d), were heated to 56 °C for 10 min and injected intravenously into mice of the DBA/2J (H-2^d) and C57BL/6J (H-2^b) strains, respectively. After 4 days, the splenocytes of the treated mice were mixed with normal spleen cells and cultured for 5 days with allogeneic tumor cells. The cytotoxic T-cell response was reduced in cultures of these cell mixtures. An allogeneic difference was required to induce suppression because the syngeneic combination did not induce suppressor cell activity. Furthermore, the induction of cytotoxic T cells to the C118 cell line (H-2^k) was not suppressed by this procedure, which suggests that the suppression was haplotype specific. These suppressor cells were sensitive to anti-Thy 1.2 and complement, cortisone, and cyclophosphamide, but insensitive to irradiation. These are characteristics similar to suppressor cells activated by intact cells. Heat treatment abrogated the tumor cell's ability to induce a proliferative and a primary, but not a secondary, cytotoxic T-cell response. The heat-treated cells also lost their ability to function as cold target inhibitor cells, but retained the same quantity of serologically detected antigens as the intact cells. These results suggest that the serologically detected antigens are responsible for the activation of the suppressor cells of the cytotoxic T-cell response.     

Effect of anH-2 mutation on cytotoxic T cell recognition. Specific antigen can determine the pattern ofH-2 restriction

Hz1 (H-2 ^bm1 ) mice, an H-2 mutant strain derived from C57BL/6(H-2 ^b ), were either injected with vaccinia virus or had their spleen cells sensitized in vitro with syngeneic TNP-modified cells. The cytotoxic cells generated were tested for their activity against target cells that were either infected with vaccinia virus, TNP-modified, or both vaccinia infected and TNP-modified.Hz1 anti-TNP cytotoxic cells specifically lysed syngeneic target cells that were trinitrophenylated but not infected with vaccinia virus, while anti-vaccinia cells specifically lysed vaccinia infected target cells but not TNP-cells. Hz1 (H-2K ^bm1 D ^b ) anti-TNP effector cells killed B10.A(5R)-TNP (H-2K ^b D ^d ) targets, indicating that there is cross-reactivity between TNP-H-2K^b and TNP-H-2K^bm1. On the other hand, there is no cross-reactivity between vaccinia-H-2K^b and H-2K^bm1, since Hz1 anti-vaccinia effector cells did not kill vaccinia infected B10.A(5R) targets.Since Hz1 anti-TNP effector cells lysed B10.A(5R) target cells that were first infected with vaccinia virus and then derivatized with TNP, virus does not mask cross-reactive determinants shared by TNP-H-2K^b and H-2K^bm1. Additional experiments showed that Hz1 anti-TNP effector cells lysed TNP-modified and vaccinia infected B10.A(5R) target cells irrespective of the virus concentration used for infection or the time of addition of virus. Further, there are no detectable quantitative differences between C57BL/6 and Hz1 anti-TNP effector cells in their ability to kill TNP-5R targets.The cytotoxic effect of Hz1 anti-TNP effector cells on B10.A(5R)-TNP targets could not be blocked with TNP derivatized inhibitor cells that carry theH-2D ^d region allele. Thus, the ability of anti-TNP H-2K^b effector cells to cross-react with H-2K^bm1 cannot be explained by a cross-reaction between H-2K^bm1 and H-2D^d.Abbreviations used in this paper TNP trinitrophenol - PFU plaque forming unit - Con A Concanavalin A - BSS balanced-salt-solution - FCS fetal calf serum - TNBS trinitrobenzene sulfonic acid - PBS phosphate-buffered-saline     

Mechanisms of genetic control of immune responses

We examined multiple genetically regulated Immoral and cell-mediated immune (CMI) responses to poly(glu⁶⁰ala³⁰tyr¹⁰) (GAT) using a panel of mouse strains. We show that assignment of responder/nonresponder status depends upon the assay method. In addition, two distinct categories of nonresponder mice were found: (1) those which are unresponsive by all parameters tested (H-2 ^q and H-2 ^s haplotypes) and (2) those which are partially nonresponsive [H-2 ^bm12 mutant strain—a low/nonresponder by splenic plaque-forming cell (PFC) and delayed-type hypersensitivity (DTH) responses, but exhibits B6 parental levels of high GAT-specific T-cell proliferation (Tprlf) and interleukin-2 production]. The distinction between these two nonresponder types was confirmed by complementation tests in which significant GAT-specific PFC and DTH responses were seen in (H-2 ^q × H-2 ^bm12)F₁ hybrids, but not in (H-2 ^q × H-2 ^s )F₁ hybrids. Suppressor T cells (Ts) also play a selective role in nonresponsiveness to GAT. Cyclophosphamide treatment of nonresponders (to eliminate Ts activity) as well as immunization with GAT coupled to the immunogenic carrier MBSA result in the development of GAT-specific humoral, but not CMI responses. Our results indicate that the T cell is the cellular site of Ir gene expression and that Tprlf responses do not correlate with functional helper T-cell activity and suggest distinct, multi-step Th/Ts regulatory pathways in the development of humoral and CMI effector functions.     

Adoptive transfer of delayed-type hypersensitivity to Sendai virus. I. Induction of two different subsets of T lymphocytes which differ in H-2 restriction as well as in the Lyt phenotype

This paper investigates kinetics and antigenic and genetic requirements for transfer of delayed-type hypersensitivity (DTH) reactions against Sendai virus. Kinetics of sensitization of effector cells are similar to those described in other virus systems. Maximum DTH response is elicited in the footpad 7 days after sensitization; maximal increase in footpad thickness is found approximately 24 hr after injection of virus and transfer of immune lymphocytes. DTH effector cells are Ig^? and are susceptible to treatment with anti-theta antibody and complement. After transfer of immune lymphocytes a DTH reaction can be elicited by infectious virus, uv light-inactivated virus or cell-cultured Sendai virus which lacks fusion capacity. Requirements of H-2 compatibility are dependent on the biological nature of the virus preparation used for challenge: High doses of infectious or uv light-inactivated Sendai virus require K, D, or IA region compatibility; low concentrations of infectious virus require K and/or D region compatibility, while cell-cultured fusion-negative Sendai virus requires IA region homology. At the level of induction K, D region-restricted DTH-effector T lymphocytes (Td) and cytolytic T lymphocytes (Tc) are stimulated to a greater extent by infectious virus than by uv light-inactivated virus. Furthermore, stimulation of these T lymphocytes is dependent on the route chosen for immunization: intravenous (iv) injection is superior to intraperitoneal (ip) injection; subcutaneous (sc) injection causes the lowest degree of sensitization. IA region-restricted Td lymphocytes are stimulated to comparable amounts by infectious or uv light-inactivated Sendai virus independent of the route of immunization. Td lymphocytes, which require IA region compatibility and Td lymphocytes which require K or D region compatibility can be distinguished by their Lyt phenotype, e.g., IA region-restricted Td lymphocytes are characterized by Lyt-1⁺2^? antigens, while K or D region-restricted Td lymphocytes are phenotypically Lyt-1(⁺)2⁺.     

Immunodominance in the immune response to “multiple” histocompatibility antigens

Cytotoxic effector T cells putatively specific for multiple non-H-2 histocompatibility (H) antigens were generated by immunizing and boosting C57BL/6 and B6.C-H-2 ^dmice with BALB.B and BALB/c stimulator cells, respectively. The generated effectors were tested for cell-mediated lympholysis on a panel of targets whose BALB/c-derived non-H-2 H antigens were donated by CXB recombinant inbred mice. The spectrum of reactivity of cytotoxic effector T cells with CXB targets demonstrated that the effectors did not recognize multiple H antigens but rather preferentially recognized a single immunodominant non-H-2 H antigen. The identity of the immunodominant H antigen was determined by the H-2 genotype of the stimulator cells when (B6 × B6.C-H-2 ^d)F ₁ cytotoxic effectors were tested. These observations indicate that despite the fact that responders were challenged with more than 40 individual non-H-2 H antigens, they preferentially responded to a single immunodominant antigen.     

Mechanisms of genetic control of immune responses

The mechanisms underlying Ir gene control of CMI were addressed by examining the DTH and Tprlf responses specific for the synthetic polymers GT, GAT, and GA. We show that BALB/c mice (GAT/GA responders, GT nonresponders) primed with GT fail to develop DTH and Tprlf responses specific for GT, GAT, or GA. GAT immunization resulted in DTH responses that could be elicited not only with GAT and GA but also with GT, demonstrating that GT-specific T_DH are present in nonresponder mice. GT-specific DTH was transferred with Thy-1⁺ Lyt-1⁺2^–, H-2 Irestricted, nylon wool nonadherent cells. GA-primed BALB/c mice developed GAT- and GA-, but not GT-apecific DTH responses, indicating that GA and GT do not cross-react at the T-cell level. The ability of GAT [but not a mixture of GA plus GT, or GT electrostatically complexed to the immunogenic carrier MBSA (GT-MBSA)] to induce GT-specific DTH suggested a requirement for covalent linkage of stimulatory GA and nonstimulatory GT determinants present on the GAT molecule. Similarly, GT-specific in vitro Tprlf responses could be demonstrated in GAT-primed mice exhibiting significant levels of GT-specific DTH but not in GT- or GT-MBSA-primed mice. Tolerization experiments also suggested that GT-specific Th were involved in the development of GT-specific DTH in GAT-primed mice. The GT nonresponsiveness of BALB/c mice for DTH and Tprlf responses could not be reversed by treatments designed to abrogate Ts activity (priming with GT-MBSA and CY injection), nor could GT-primed cells be shown to inhibit the development or elicitation of GT-specific CMI in GAT-primed mice during the afferent and/or efferent stages of DTH. Our results suggest that GT nonresponsiveness does not result from the absence of GT-specific T cells or preferential induction of Ts. The results are discussed in the context of hole-in-the-repertoire and antigen presentation (determinant selection) models of Ir gene control.Abbreviations used in this paper APC antigen-presenting cells - BSA bovine serum albumin - BSS Mishell-Dutton balanced salt solution - CFA complete Freund's adjuvant - CMI cell-mediated immunity - CY cyclophosphamide - DTH delayed-type hypersensitivity - GA poly(Glu⁶⁰Ala⁴⁰) - GAT poly (Glu⁶⁰Ala³⁰Tyr¹⁰) - GT poly(Glu⁵⁰Tyr⁵⁰) - GT-MBSA GT complexed to methylated bovine serum albumin - It immune response - LN lymph node - PPD purified protein derivative of tuberculin - TDH DTH T cells - Th helper T cells - Tprlf T-cell proliferation - Ts suppressor T cells - TsF T-cell suppressor factor(s)     

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 of the suppressor cell(s) responsible for anterior chamber-associated immune deviation (ACAID) induced in BALB/c mice by P815 cells

Anterior chamber-associated immune deviation (ACAID) is a complex set of immune responses induced by the inoculation of antigens into the anterior chamber of the eye. Histocompatibility antigens, tumor-specific antigens, reactive haptens, and viral antigens have been shown to induce this phenomenon, which comprises the following specific host responses: high titer humoral antibodies, primed cytotoxic T cells, but specifically, impaired skin graft rejection and delayed-type hypersensitivity (DTH). Using the model system of ACAID induced by inoculation of P815 mastocytoma cells into the anterior chambers of H-2-compatible, but minor H-incompatible, BALB/c mice, we demonstrate that the impaired capacity of these animals to develop and express DTH is due to the activation of suppressor T cells. Generation of these cells requires an intact spleen, is not inhibited by cyclophosphamide pretreatment, and is abrogated by systemic treatment of the host with anti-I-J monoclonal antibodies. This splenic suppressor cell(s) can transfer suppression of DTH adoptively to naive syngeneic mice. One suppressor cell is Thy-1.2, Lyt-2.2, and I-Jd positive. A minority of these cells (or a second population of suppressor cells) also expresses the L3T4 surface marker. Suppression is exerted on the efferent limb of DTH expression, although afferent suppression is not excluded. P815-induced ACAID suppressor cells resemble similar cells induced by haptenated spleen cells inoculated into the anterior chamber of the eye. We propose that induction of these suppressor cells, whose target of action is selective for T DTH cells, but not for other types of T cells, is responsible for the phenomenon of immune privilege in the anterior chamber of the eye.     

Ongoing hapten-specific delayed-type hypersensitivity prevents generation of cytolytic T lymphocytes to the same hapten

Cytolytic T lymphocytes (CTL) specific for trinitrophenyl (TNP)-altered self antigens can be generated in vivo through the simultaneous injection of TNP-modified syngeneic spleen cells and H-2-compatible, minor histocompatibility locus (Mls)-disparate auxiliary spleen cells into the footpads of mice. The latter stimulates host helper cells to produce differentiative and proliferative signals required for the generation of CTL. Advent of this protocol allowed investigation of the initiation of two different cell-mediated immune responses, delayed-type hypersensitivity (DTH) and the generation of CTL, in the same experimental animal. Mice presensitized for CTL were able to develop DTH as well as normal controls. However, when mice were first sensitized for DTH, they were thereafter incapable of generating CTL. This effect was hapten specific, relatively long lasting, and preventable by treating mice with cyclophosphamide before sensitizing for DTH. Adoptive transfer of lymphoid cells from DTH-immune mice conferred DTH reactivity upon naive recipients but not a suppressed CTL response. Therefore, cells mediating DTH were not responsible for suppression of CTL. The mechanism for suppression has been discussed from the viewpoint of the suppressor-T-cell circuits that are known to be generated when animals are sensitized for DTH and which are susceptible to treatment with cyclophosphamide.