Significant frequency of cytotoxic T lymphocyte precursor cells specific for TNP-modified allogeneic cells in normal lymphocytes

It was tested whether the cytotoxic T-lymphocyte precursor (CLP) repertoire in normal mice is biased toward recognizing foreign antigen in association with self H-2 as opposed to allogeneic H-2. The frequencies of CLPs in normal mice (H-2b,k,d) specific for TNP-modified syngeneic and TNP-modified allogeneic cells have been compared by limiting dilution analysis. Normal spleen cells were cultured at a limiting dilution with TNP-modified (TNP-self) or TNP-modified allogeneic (TNP-allo) stimulator cells. Cultures were split into four aliquots and assayed against TNP-self, TNP-allo, unmodified syngeneic, and unmodified allogeneic Concanavalin A blast targets and classified for cytotoxic activity directed against TNP-self, TNP-allo, and allo H-2 determinants. In disagreement with our expectations from the literature, the frequencies of CLPs in H-2b and H-2d responder cells recognizing TNP-modified H-2k were higher than the frequencies of CLPs recognizing TNP-self. There was no clear preference for TNP-self in the case of H-2b responder and H-2d allogeneic cells, nor vice versa. Only in the case of H-2k responder cells was there a distinct preference for TNP-self. The significance of a considerable number of TNP-specific, allo H-2-restricted CLPs in normal lymphocytes is discussed.     

Continuously proliferating T killer cells specific for H-2b targets: selection and characterization.

BALB/c (H-2d) thymus-derived lymphocytes sensitized to C57BL/6 (H-2b) alloantigens have been propagated in vitro for over 9 months. These T lymphocytes are specifically cytotoxic to H-2b target cells but are stimulated to proliferate by both H-2b and H-2k spleen cells. This indicates that for these selected cells the antigen requirements for cell proliferation are different from those for cell-mediated cytotoxicity. If not continuously stimulated with allogeneic spleen cells, the cytotoxic cultures fail to divide and rapidly lose their cytotoxic activity. Allogeneic erythrocytes do not stimulate cell proliferation in "quiescent" cell cultures and allogeneic tumor cells do so only in the presence of spleen cells. However, "quiescent" cell cultures display cytotoxicity in the presence of phytohemagglutinin A as do cell cultures which have lost their cytotoxic activity although they proliferate upon allogeneic stimulation. The significance of these findings is discussed.     

Activation of nonspecific killer cells by interleukin 2-containing supernatants

In the absence of specific antigen stimulation, nonspecific killer cells were induced by culturing C57BL/6 lymph node or spleen cells with interleukin 2-containing supernatants. These supernatants were obtained from stimulation of either rat spleen cells with concanavalin A or a variant of the T cell lymphoma, EL4 (H-2b) with phorbol myristic acetate. The ability of the EL4 supernatant to induce nonspecific killer cells was abrogated by absorption with an interleukin 2-dependent T cell line or by concanavalin A-stimulated spleen cell blasts, but not by lipopolysaccharide-stimulated spleen cell blasts or by a non-interleukin 2-producing EL4 line. Partially purified interleukin 2 from EL4 supernatants could also support nonspecific killer cell induction. The induction of cytolytic cells by interleukin 2 is sensitive to gamma-irradiation and has a D omicron of 120 rad. The nonspecific killer cells induced are likely cytotoxic T lymphocytes; the majority of the precursor and effector cells bear the Thy-1 alloantigen marker. These nonspecific killer cells killed a broad spectrum of target cells, including concanavalin A- and lipopolysaccharide-induced splenic blasts of syngeneic or allogeneic mice, a syngeneic tumor, and a cloned allogeneic cytotoxic T cell line. The frequency of precursors for nonspecific killer cells in C57BL/6 lymph node and spleen cells are 1/7000 and 1/12,000, respectively. Clonal analyses revealed that these nonspecific killers exhibit heterogeneity with respect to their target cell specificities. The induction of nonspecific killers by interleukin 2-containing supernatants is partially dependent on nylon wool-adherent cells; in antigen-stimulated cultures the most specific killer cells were obtained from cultures in which nylon wool-nonadherent lymph node responder cells were stimulated with nylon wool-nonadherent allogeneic splenic stimulator cells that were treated with anti-Thy-1 antibody and complement. The relevance of these findings with respect to the frequencies and fine specificities of cytotoxic T lymphocytes generated in interleukin 2-supplemented cultures is discussed.     

Induction and characterization of minor histocompatibility antigens. Specific primary cytotoxic T lymphocyte responses in vitro

A definite cytotoxic activity was developed in a BALB/c (H-2d) anti-DBA/2 primary mixed leukocyte culture (MLC), which received interleukin 2 (IL-2) on day 3 of culture. This cytotoxic activity was minor histocompatibility antigens (MIHA)-specific at the stimulator level, and was not developed in a syngeneic (BALB/c anti-BALB/c) MLC. The addition of IL-2 on day 3 of culture was crucial; no or very weak cytotoxic activity was developed in MLC receiving IL-2 on day 0 or on both day 0 and day 3. Only appropriate MIHA-allogeneic tumor cells were lysed as the target of the cytotoxic activity. The cytotoxic activity seemed MIHA-specific also at the target level; it lysed tumor cells of DBA/2 mouse origin but not those of BALB/c (syngeneic) origin. Phenotypes of the cytotoxic effector cell were Thy-1+ Lyt-2+. We concluded from these results that MIHA-specific cytotoxic T lymphocytes (CTL) were generated in the MIHA-allogeneic primary MLC. In this newly developed system, we studied genetic and antigenic requirements for primary anti-MIHA CTL responses in vitro. We demonstrated; among spleen cells (SC) of seven B10 H-2-congenic strains only SC of B10.D2 strain whose major histocompatibility complex (MHC) (H-2d) was compatible with the responder MHC effectively stimulated responder BALB/c (H-2d) SC for an anti-MIHA (DBA-C57BL-common) CTL response. Similarly, only SC of two out of seven C x B recombinant inbred strains (C x B.H and C x B.D), which were compatible at the MHC with responder SC, activated responder BALB/c SC for the response. The possibility that cells responding to H-2 alloantigens suppressed the anti-MIHA response was ruled out. Additional experiments showed that compatibility at the H-2K-end or the H-2D-end of the MHC was sufficient for a definite anti-MIHA response. These provided formal evidence that primary anti-MIHA CTL responses in vitro were MHC-restricted at the stimulator level. We then showed that sonication-disrupted SC or Sephadex G-10 column-passed nonadherent SC failed to stimulate responder SC for a primary anti-MIHA CTL response, whereas G-10-passed nonadherent SC responded well to adherent stimulator cells. Further study demonstrated that Ia+ adherent cells were the most active cell type as stimulator. Finally, we confirmed that the primary anti-MIHA CTL responses to adherent stimulator cells was MHC-restricted.     

Induction of cytotoxic T lymphocytes against herpes simplex virus type i: role of accessory cells and amplifying factor

Spleen cells from mice primed with herpes simplex virus type 1 (HSV-1) could be induced to differentiate into cytotoxic T lymphocytes (CTL) by in vitro culture with infectious HSV-1 but not by heat-inactivated virus. Induction of CTL failed to occur if the spleen cells were depleted of adherent cells by passage over columns of nylon wool before culture with virus. The CTL response could be restored by adding normal syngeneic peritoneal cells (PC) or L cell fibroblasts but not by allogeneic PC or BALB/c 3T3 fibroblasts. Thus, the induction of HSV-1-specific CTL was H-2 restricted. The response of HSV-1-stimulated nylon wool-depleted spleen cells could also be restored by adding amplifying factor (AF) produced in 24 hr mixed lymphocyte cultures. The addition of AF to nondepleted spleen cells also permitted the generation of CTL with heat-inactivated HSV-1 as a viral stimulant. Our results indicated that induction of a HSV-1 CTL response requires two signals, one provided by virus and a second, presumably nonspecific, by helper T cells. It was suggested that only the helper cells require H-2 restriction and need to be presented virus in the context of a macrophage.     

Alloantigen-induced T helper activity. I. Minimal genetic differences necessary to induce a positive allogeneic effect.

Addition of histoincompatible lymphocytes can influence the course of ongoing immune responses. Such allogeneic effects may either augment or diminish immune responses. We describe here the minimal genetic differences necessary to generate positive allogeneic effects (allohelp) in a humoral immune response. The antibody response to sheep erythrocytes of T cell-depleted mouse spleen cells was reconstituted by addition of syngeneic or allogeneic nylon wool column-passaged spleen T cells. T cells were pretreated with mitomycin C before culture to prevent development of allo-suppression and cytotoxic lymphocytes. Positive allogeneic effects were operationally defined as superior helper effects (to generate greater antibody forming cell responses) with T cells allogeneic rather than syngeneic to the responding B cells. Thus, addition of allogeneic T cells resulted in many more antibody forming cells than did equal numbers of syngeneic T cells, and fewer allogeneic than syngeneic T cells were necessary to generate comparable responses. With congenic, recombinant, and mutant mouse lines, genetic differences in the H-2 complex and those associated with Mls were each sufficient to provide positive allogeneic effects. With intra-H-2 recombinants, differences at either I or D were sufficient. A disparity at H-2K alone, as provided by the H-2 mutant B6.C-H-2ba against the parental line C57BL/6By, also induced helper effects. The significance of these results is discussed.     

H2Kk can influence whether cytotoxic T lymphocytes recognize trinitrophenyl in association with H2Dk unique or H-2Kk and H-2Dk shared self determinants

The present study investigates the effect of trinitrophenyl- (TNP) modified H-2Kk (TNP-Kk) antigens on the generation of anti-TNP-Dk restricted cytotoxic T lymphocyte (CTL) responses. C3H.OH mice were primed to TNP-self by skin-painting with trinitrochlorobenzene, and spleen cells from these primed mice were subsequently stimulated in vitro with TNP-self. The effector cells generated exhibited appreciable lysis of TNP-modified C3H.OH blast target cells. Cold target inhibition studies demonstrated the generation of two effector cell populations: one that recognizes TNP in association with unique Dk self determinants, and one that recognizes TNP in association with self determinants shared between TNP-Kk and TNP-Dk. This was in contrast to primed C3H/He spleen cells, which did not generate CTL that recognized TNP in association with unique Dk self determinants. When spleen cells from (C3H/He x C3H.OH)F1 mice primed to TNP were stimulated in vitro with TNP-C3H.OH cells, unique Dk self determinants were recognized in association with TNP. However, in vitro stimulation of the same F1 responding cells with TNP-C3H/He or TNP-F1 cells failed to elicit CTL that utilized these Dk-unique self determinants. The findings of this study demonstrate that unique or shared H-2Dk determinants can be differentially utilized by CTL populations, depending on the H-2 alleles expressed by the stimulator cells.     

The allogeneic effect: M-locus differences substitute for differences in the H-2 major histocompatibility complex.

The primary immune response against sheep red blood cells in T cell-deficient spleen cell cultures from nude mice was tested in the absence and presence of allogeneic spleen cells. The allogeneic spleen cells differed either in regard to the major histocompatibility complex (H-2) or only with respect to the M-locus. Surprisingly the M-locus different spleen cells were almost as efficient in enhancing the anti-sheep red blood cell response in nude cultures as were the cells differing on the complete H-2 complex. Evidence is presented that AKR anti-theta serum sensitive T cells are responsible for the M-locus-dependent effect edscribed. This effect is shown to be mediated by a factor released from actived T cells stimulated in M-locus different mixed lymphocyte cultures. Since almost identical parameters have been observed in both the M-locus-dependent situation as in the "classical" allogeneic situation we concluded that an allogeneic effect can be induced by T cells responding to a complete set of the major histocompatibility complex (H-2) as well as to lymphocyte-activating determinants (M-locus) alone.     

Effects of sodium periodate modification of lymphocytes on the sensitization and lytic phases of T cell-mediated lympholysis.

Sensitization of mouse splenic lymphocytes in vitro with sodium borohydride, suggesting that the biologic effects of sodium periodate are-treated autologous spleen cells stimulated a one-way mixed lymphocyte reaction and led to the generation of thymus-derived cytotoxic effector cells. These effectors were capable of lysing in 4 hr periodate-treated syngeneic and, to a lesser extent, periodate-treated allogeneic target cells. These results suggest that sensitization by periodate-treated autologous cells could result either from a specific reaction to modified self components or from a nonspecific mitogenic stimulation. Effector cells generated by allogeneic sensitization were detected on periodate-modified targets, irrespective of the H-2 antigens expressed by the targets. The effects of periodate modification on both stimulator and target cells were reversible by sodium periodate are dependent on the formation of a free aldehyde group on cell surface glycoproteins. Pretreatment of stimulator cells with neuroaminidase prevented the effect of periodate treatment, suggesting that the sensitization involves oxidized sialic acid residues. During the 4-hour 51Cr-release assay periodate-treated targets could be used to detect cytotoxic effector cells of any specificity. Fresh spleen cells and lymphocytes cultured for 5 days without antigen or in the presence of lipopolysaccharide did not lyse periodate-treated targets. An increasing level of cytotoxicity was detected on periodate-treated targets when the effector cells were generated, respectively, by stimulation with concanavalin A, by sensitization with periodate-modified autologous cells. Although the lysis of periodate-treated targets is itself nonspecific, effector cell specificity could be determined by selective blocking of the lytic phase with cells syngeneic to the stimulators. These results indicate that a nonspecific interaction can occur between lymphocytes and periodate-treated target cells, but that this interaction leads to lysis only when the lymphocytes were activated to become cytotoxic effectors.     

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor

We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.     

Studies on the induction and expression of T cell-mediated immunity. IV. Non-overlapping populations of alloimmune cytotoxic lymphocytes with specificity for tumor-associated antigens and transplantation antigens.

Two non-overlapping populations of alloimmune cytotoxic T cells with specificity for tumor-associated antigens (TAA) and for histocompatibility antigens (H-2) were characterized by two independent methods. The heterogeneity of cytotoxic cells was demonstrated in spleen cells derived from BALB/c (H-2d) mice sensitized to EL-4 (H-2b) tumor and from C57BL/6 (H-2b) mice sensitized to G-35 (H-2d) tumor cells. Adsorption of immune lymphocytes on monolayers prepared with cells bearing the sensitizing H-2 antigens abrogated the in vitro cell-mediated cytotoxicity (CMC) directed against 51Cr-labeled normal target cells (spleen cells or ConA-activated spleen blasts), whereas significant cytolytic activity to the corresponding 51Cr-tumor cells was still retained. Likewise, in competitive inhibition assays, CMC to 51 Cr-tumor target cells was only partially inhibited by unlabeled normal cells, whereas CMC to 51Cr-normal target cells was completely abrogated. These results suggested that alloimmune cytotoxic lymphocytes are heterogeneous and can be subdivided into two independent populations of restricted specificity. Several experiments suggested that the effector cell population directed against TAA can no longer elicit a graft-vs-host (GVH) reaction in vivo. This was demonstrated by adoptive transfer into lethally-irradiated allogeneic recipients of cytotoxic or primed spleen cells fractionated on host target cell monolayers. Furthermore, these results demonstrated that both effector cells and memory cells possess high affinity binding receptors to corresponding H-2 antigens. The potential use of fractionated immune lymphocytes sensitized to tumor allografts in adoptive immunotherapy is discussed.     

In vitro induction of immunological tolerance

IL-2 was previously shown to induce cytotoxic effectors with a broad spectrum of target specificities in thymus and spleen cell cultures. This study was designed to show whether T cells activated by H-2 allogeneic cells in MLC or by syngeneic tumor cells in MLTC are also potential targets for these cytotoxic effectors. We found that thymocytes activated in vitro for 5 days by rIL-2 were capable of killing tumor cells as well as activated T cells. Thymocytes activated by IL-2 were accordingly utilized as a means of effecting clonal deletion of T cells activated by H-2 allogeneic target cells in MLC. To establish whether the unresponsiveness is specific. IL-2-activated thymocytes were added as third party cells to MLC and MLTC. The results showed that both T cells, proliferating in response to H-2 allogeneic cells, and CTL, reactive against syngeneic tumors or H-2 allogeneic cells, are eliminated from the T cell pool. Only alloreactive T cells are specifically eliminated in MLC by IL-2-activated thymocytes, as the remaining T cells are capable of proliferating and generating CTL in response to antigenically unrelated third party allogeneic cells. The possibility that unresponsiveness might be due to soluble factors was ruled out by studies performed with a diffusable "chamber insert" culture system. The results provide evidence that IL-2-activated thymocytes induce in vitro T cell tolerance.     

Cytotoxic lymphocytes induced by soluble factors derived from the medium of leukocyte cultures.

Studies were undertaken to evaluate the cytotoxic capacity of human peripheral blood lymphocytes activated by either supernatants (CFM) derived from lymphocyte cultures or lymphocytes treated for 60 min at 45 degrees C. The effect of the addition of heat-treated cells on the cytotoxic activity of CFM-induced effector cells was also studied. CFM from either unmixed or mixed cultures of lymphocytes was capable of activating cytotoxic effector cells. These effector cells could kill any allogeneic target cells but failed to effect cytotoxicity on the target cells autologous to the responding cells. Both the heat-treated cells and CFM from cultures of these cells also activated lymphocytes to cytotoxic effector cells having specific receptors for nonself antigens. The question of whether heat-treated cells activate cytotoxic cells by themselves or through secreted soluble factor cannot yet be clearly answered. The findings of the present investigation suggest that expression of cytotoxicity induced in MLC is not necessarily restricted to the target cells syngeneic to the stimulator cells, but can be extended to any allogeneic target cells by the indirect effect of soluble factor secreted from stimulated cells that causes a polyclonal activation of cytotoxic precursors in the responding cell populations. The present findings also emphasize the need for caution in the use of heat-treated lymphocytes as innocent-bystander cells in MLC to provide additional cytotoxic specificities in the responder cells, since heat-treated cells alone can activate lymphocytes to cytotoxic effector cells that kill any allogeneic target cells.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

Xenogeneic serum-induced murine cytotoxic cells. I. The generation of effector components specific for self and allogeneic target cells.

Spleen cells from unimmunized mice cultured in vitro without the intentional addition of exogenous antigen generated cytotoxic effector cells which lysed tumor and mitogen-stimulated blast target cells in a 5-hr chromium release assay. Effectors were generated in fetal bovine serum but not in adult horse serum, although both serum sources supported the generation of allogeneic cytotoxic cells. No correlation was observed between the ability of a serum source to support and generate serum-induced effectors and its ability to support an allogeneic cytotoxic response. The effectors lysed targets which were H-2 matched and those which were not H-2 matched with the cultured spleen cells, although the H-2 matched targets were consistently lysed more efficiently. “Cold” target cell inhibition studies indicated that multiple clones of cytotoxic cells were generated—including effector cells with specificity for self-structures, and particular alloantigens. Possible roles for the xenogeneic serum in the generation of this response are considered, including (a) the provision of essential stimulating and target antigens; (b) the induction of the expression of neoself-determinants; and (c) the possession of mitogenic properties.     

Murine T-cell-mediated cytotoxicity against syngeneic and allogeneic cell lines induced by fetal calf serum

Mouse spleen cells from normal animals developed easily measurable cytotoxicity against various cell lines when cultured in vitro without deliberate sensitization. Cytotoxicity, measured by a 3-hr ⁵¹Cr-release assay, was maximum on Days 3 and 4 of culture and was dependent on the presence of fetal calf serum. Neither cell recovery nor blastogenesis, however, invariably correlated with the amount of cytotoxicity generated. Nylon wool adsorption of effector cells cultured 3 days had only a marginal effect on cytotoxicity, whereas cytolysis was markedly reduced (but not totally eliminated) by treatment with anti-T-cell serum and complement. When target cells were in relative excess to effector cells, ⁵¹Cr release was proportional to effector cell number and proceeded for at least 22 hr. The cytotoxicity was not tumor or H-2 specific, and targets without known C-type viral antigens (gp71) were killed as readily as those with easily measurable viral antigens. Nontumorigenic fibroblasts were lysed, but concanavalin A-induced blast cells were not. Cytotoxicity was not augmented in cultures of spleen cells from mice injected with fetal calf serum or with tumor fragments exposed to fetal calf serum. Mercaptoethanol was not necessary for the generation of cytotoxic activity, but T cells and Sephadex G-10 or nylon wool-adherent cells were necessary and the function of the adherent cell could not be replaced by mercaptoethanol. Removal of plastic adherent cells had no effect. Fetal calf serum retained its activity when heated for 45 min at 56 °C or when dialyzed. Dilution and reconcentration by Amicon filtration revealed that the mass of the active material was between 30,000 and 100,000 daltons.The early appearance, transient nature, and nonspecificity of this cytotoxicity distinguish it from antigen-specific reactions. The effector's stability at 37 °C and its relatively easily detectable T-cell markers distinguish it from natural killer and cytotoxic cells. This activity is like lectin-induced cytotoxicity but differs because allogeneic blast cells are not lysed. The observed cytotoxic activity may be of in vivo relevance (vis-à-vis natural killer cells) or, more likely, an in vitro expression of a stage of cell differentiation that T cells may normally pass through during their response to antigen.     

The origin of cytotoxic T lymphocyte precursors (CTL-P): MHC-restricted and alloreactive CTL-P in the spleen during regeneration after a sublethal dose of cyclophosphamide (Cy)

The regenerating spleen 8 days after an injection of a sublethal dose (300 mg/kg) of cyclophosphamide (Cy) had a defective capacity to give rise to cytotoxic T lymphocytes (CTL) in response against allogeneic cells, whereas the cytotoxicity against 2,4,6-trinitrophenyl-(TNP) modified syngeneic cells was at the normal level. Alloresponse was first obtained 2 wk after the Cy treatment. The limiting dilution analysis showed this at the clonal level: the frequency of anti-TNP-specific CTL precursors (CTL-P) in the spleen treated with Cy 8 days previously was the same as the frequency in the normal spleen. The defective alloresponse was due to a decreased number of allospecific CTL-P that was later increasing. The regenerative capacity was not abolished by adult thymectomy or treatment of the mice with a bone marrow-seeking isotope, 89Sr, suggesting that these CTL-P are derived from Cy-resistant splenic precursors rather than from the thymus or bone marrow. These precursors have probably been under thymic education: the dominance of H-2k-restricted CTL over H-2d-restricted CTL in the response of (H-2k X H-2d)F1 mice to TNP-self is known to be influenced by the H-2 genotype of the thymus, and this dominance was also demonstrated with anti-TNP CTL derived from these F1 mice pretreated with Cy. The CTL-P in the regenerating spleen (day 8) were not hydrocortisone sensitive, and nylon wool-purified T cells from this stage had a lactate dehydrogenase (LDH) isoenzyme pattern of the mature T cell type (rather than of the thymocyte type). Thus, in these aspects the T cells of the regenerating spleen resembled normal splenic cells. These data suggest that the Cy-resistant spleen population contains cells that can give rise to CTL-P that have a defective specificity repertoire at the beginning of the regeneration, but later mature to a normally alloreactive population.     

Recognition of protozoan parasites by murine T lymphocytes. II. Role of the H-2 gene complex in interactions between antigen-presenting macrophages and Leishmania-immune T lymphocytes

The proliferative response of nylon wool purified, primed lymph node cells to L. tropica parasites in vitro was found to be restored by the addition of either syngeneic or allogeneic adherent spleen cells as a putative source of macrophages. These results suggested a lack of H-2 restriction in Leishmania-specific T cell responses. However, when T cell blasts generated in vitro in response to the parasite were separated on Percoll density gradients and subsequently maintained for 4 days in the presence of TCGF, their response to L. tropica was found to be strictly dependent on the presence of syngeneic spleen cells. Further studies using congenic recombinant mice demonstrated that proliferation of a parasite-specific blasts required the presence of spleen cells compatible with the responding cells in the I-A region of the MHC. This requirement for I-A compatible adherent cells in the spleen cell populations was further confirmed by a lack of proliferative responses in the presence of spleen cells treated with monoclonal anti-la antibodies and complement. Leishmania-immune F1 blasts responding to the parasite in the context of either parental la-bearing accessory cell could be obtained by positive selection from a F1 hybrid responding cell population. Using flow microfluorometry, the T cell phenotype of the L. tropica-specific blasts was determined to be Thy-1+, lyt-1+, and Lyt-2-.     

Trypanosoma cruzi: Responses by cells from infected mice to alloantigens

In unidirectional mixed lymphocyte cultures containing (as responders, stimulators, or regulators) spleen cells from mice infected with Trypanosoma cruzi, alloantigen responses were less than in cultures containing normal spleen cells only. Depletion of plastic adherent cells from infected spleen cells (stimulators or regulators) reversed their inhibitory effect on normal spleen cells (responders); removal of adherent responder cells and/or B lymphocytes did not alter the low alloantigen responses of normal spleen cells (stimulated by infected spleen cells) or infected spleen cells (stimulated by normal spleen cells). Infected spleen cells were effective in regulating mixed lymphocyte cultures only when added at the initiation of the culture. Serum from infected mice suppressed mixed lymphocyte cultures containing responder spleen cells syngeneic to the serum donor if added up to 24 hr after initiation of cultures, whereas the “suppressor serum” had to be present at the initiation of cultures when responder cells were allogeneic to the serum donor. Cultures of infected spleen cells (whole or macrophage enriched) produced a factor which was suppressive when added to mixed lymphocyte cultures containing syngeneic responder cells at initiation. It is proposed that the serum suppressor substance regulates cell-mediated immune responses directly by suppressing the response-potential of cells and indirectly by triggering the release of a factor from adherent splenic cells which induces a hyporesponsive state in T lymphocytes.     

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor

Summary We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.This paper was presented in part at the annual meeting of the American Association of Immunologists, Chicago, Illinois, 10–15 April 1983