T cell responses to alloantigens. I. Studies of in vivo and in vitro immunologic memory and suppression by limit dilution analysis

A limit dilution technique was used to study the frequency of cytotoxic T cell precursors (f pre Tc) in the peripheral blood lymphocytes (pbl) of naive and alloimunized mice. It was found that alloimmune mice showed a 2- to 5-fold specific increase in the f pre Tc reactive to the immunizing alloantigens. This technique was further adapted for use as a sensitive in vitro assay for alloantigen-specific suppressor T cells. It was found that nonimmunosuppressed B6AF1 mice bearing surviving B10.BR cardiac allografts had circulating alloantigen-specific suppressor cells. In vitro it was shown that the culture of heat-inactivated B10.D2 spleen cells with B6AF1 spleen cells gave rise to alloantigen-specific B6AF1 suppressor cells.     

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.     

The generation of cytolytic activity in mixed leukocyte cultures: cortisone-resistant thymocytes are deficient in helper T lymphocytes

Cortisone-resistant (CR) thymocytes did not generate cytolytic activity toward H-2 K or D alloantigen unless they were also stimulated by H-2 I or non-H-2 alloantigens, even though spleen cells generated brisk cytolytic activity toward H-2 K or D alone. Backstimulation by stimulating strain T lymphocytes accounted for neither the response of spleen cells toward H-2 K or D alloantigen nor the response of CR thymocytes to a full set of alloantigens. In addition, lack of non-T accessory cells did not account for the CR thymocyte pattern of reactivity. Rather, CR thymocytes appeared to be relatively deficient in helper T lymphocytes (HTL). CR thymocytes generated specific cytolytic activity toward H-2 D alloantigen when T cell growth factors (TCGF) or cloned alloreactive helper T lymphocytes were added to culture. CR thymocytes contained fewer HTL precursors detected at limit dilution than spleen cells did. Thus spleen cells generated cytolytic activity toward class I alloantigens alone, but under the same culture conditions CR thymocytes had to be stimulated by both class I and class II alloantigens. Class II alloantigens may be required to stimulate cytolytic activity only under culture conditions in which class I-reactive HTL are not sufficient to provide a minimal threshold of help.     

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.     

Cellular and antigenic requirements for production of mixed leukocyte reaction suppressor factor

When murine spleen cells, alloantigen-sensitized previously in vivo, are incubated with spleen cells bearing the sensitizing alloantigens, a supernatant factor is produced that inhibits 3H-thymidine incorporation by responding lymphocytes in the mixed leukocyte reaction. This study evaluates the cellular and antigenic requirements during restimulation for elaboration of this suppressor factor, MLR-TsF. BALB/c spleen cells, sensitized to C57BL/6 (B6) alloantigens in vivo, produced MLR-TsF when cultured with B6 spleen cells in vitro, despite depletion of Sephadex G-10-adherent cells from factor-producing cells, stimulator cells, or from both populations. T cells were not required within the stimulating population, but a requirement for viable stimulator cells was demonstrated when heat-killed or glutaraldehyde-fixed stimulator cells failed to induce MLR-TsF production. The alloantigenic requirements for MLR-TsF production were addressed by 2 approaches. Treatment of stimulator cells with appropriate anti-I region antisera and complement did not affect MLR-TsF production, demonstrating that an absolute requirement for cells expressing I region determinants did not exist. However, spleen cells primed against entire H-2 haplotype differences produced significant quantities of MLR-TsF when they were restimulated with spleen cells homologous to the priming cells in only the I region, in the K and D regions, or in the D region alone. The additive nature of subregion-specific restimulation suggests that distinct subpopulations of K, I, and D region-specific MLR-Ts comprise the MLR-Ts population primed to entire H-2 haplotype differences.     

H-2K/H-2D and Mls and I region-associated antigens stimulate helper factor(s) involved in the generation of cytotoxic T lymphocytes

This study examines the antigen that stimulate production or release of a soluble helper factor(s) involved in development of cytotoxic T lymphocytes (CTL). Antigens associated with the Mls locus, I and K/D regions of the MHC were all capable of stimulating responder cells in MLC to produce helper factor. These supernatant fluids were all capable of providing "help" for the generation of cytotoxic T lymphocytes in MLC in which spleen cells are stimulated by allogeneic heat-treated thymocytes or splenocytes. Previous reports from our laboratory as well as others have shown that heat-treated cells do not stimulate a cytotoxic response. Heat-treatment of Mls, I, and H-2K/H-2D region incompatible stimulatory cells in MLC eliminated their ability to induce responder cells to produce helper factor, suggesting this is the mechanism whereby heat-treatment reduces the ability of cells to stimulate cell-mediated lympholysis (CML). The inability of supernatant fluids, from MLCs in which heat-treated cells were the stimulators, to assist in the generation of cytotoxic T cells did not appear to be the result of any suppressive factor induced by such treatment. Further, the antigens that stimulate pre-killer cells appear functionally distinct from those heat labile antigens (Mls, I, H-2K/H-2D associated) that stimulate helper factor production since heat-treated allogeneic cells served as stimulators of cytotoxicity provided helper activity was added to the MLC.     

Properties of H-2L locus products in allogeneic and H-2 restricted, trinitrophenyl-specific cytotoxic responses.

The role of the recently defined L antigen (a second D region product) in allogeneic and TNP-specific syngeneic primary CML responses has been investigated. The lysis by anti-L specific cytotoxic effector cells was not inhibited when the target cells were pretreated with an antiserum directed against K and D, whereas an antiserum against L completely abrogated this response. Therefore, H-2L products are recognized on the target cell independently of H-2K and H-2D locus products. Both A.SW cells as well as B10 cells were found to respond to Ld alloantigens, in addition to Dd alloantigens when stimulated by cells differing only in the D region. The results of cold target blocking and antiserum inhibition experiments failed to detect cytotoxic cells with specificity of L antigens in association with TNP, under conditions in which TNP-specific effectors to K and D antigens were demonstrable. These findings suggest that there is a more limited involvement of H-2L locus products than the H-2K or H-2D locus products in the induction and specificity of these responses.     

The role of HLA-DR determinants in monocyte-macrophage presentation of herpes simplex virus antigen to human T cells

Spleen cell killing of target cells can manifest through spleen cell-target cell interaction in the presence of mitogenic lectin, lectin-dependent cell-mediated cytotoxicity (LDCC). Spleen cells from C57B1/6 mice immunized with C3H mouse cells were found to be capable of cytotoxicity against autologous and other C57B1/6 spleen cells in the presence of Con A. Thus, alloimmune spleen cells are capable of an anti-self cytotoxic response in the presence of mitogenic lectin, antiautologous LDCC. Antiautologous LDCC is blocked by preincubation of cytotoxic cells with colchicine, an inhibitor of the cytotoxic effector mechanism. Analysis of alloimmune spleen cell subpopulations suggests that the antiautologous LDCC cell is an immature alloimmune cytotoxic cell (prekiller cell). Potent LDCC was found in alloimmune spleen cell preparations depleted of alloimmune cytotoxic T cells (killer-depleted) by three passes on allogeneic cell monolayers genetically identical with the immunizing cell. However, some LDCC effectors were also found to adhere to the adsorbing target, suggesting that there is some maturational diversity among LDCC effectors.     

Early antigen elimination by cytotoxic T cells results in diminished cellular immune responses to allogeneic tumor

Previous reports have suggested that repeated alloantigenic challenge increases humoral responses to alloantigens, but may cause decreasing cellular responses. We stimulated BALB/c (H-2^d) mice with intraperitoneal EL-4 tumor (H-2^b) and serially assessed cytotoxic responses in spleen and peritoneal lymphocytes using ⁵¹Cr-labeled EL-4 target cells. We observed that cytotoxicity generated in the spleen of nonimmune BALB/c mice was much greater than that in immunized mice; similar peak responses were generated in peritoneal lymphocytes in normal and immunized hosts. Complement-mediated cytotoxicity was not necessary for diminished splenic responses in hyperimmune hosts, for the same phenomenon was seen in the Hzl anti-BL/6 system which is free of humoral responses. Irradiation (2000 rad) of the EL-4 tumor challenge prevented tumor cell proliferation and markedly reduced splenic responses in nonimmune mice. We suggest that cytotoxic cells suppressed further generation of cyto-toxicity; by effecting an early elimination of tumor inoculum, tumor proliferation was abrogated and dose of cellular antigen was, consequently, markedly reduced.     

Leucyl-leucine methyl ester treatment of donor cells permits establishment of immunocompetent parent----F1 chimeras that are selectively tolerant of host alloantigens

Treatment of murine lymphocytes with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) selectively removes natural killer cells, cytotoxic T lymphocyte precursors, and the capacity to cause lethal graft-vs-host disease, whereas bone marrow stem cell function and alloantigen-induced L3T4+ T helper function remains intact. The present studies assess the immunocompetence of allogeneic bone marrow chimeras established by reconstituting irradiated (C57BL/6 X DBA/2)F1 (B6D2F1) mice with Leu-Leu-OMe-treated C57BL/6 (B6) bone marrow and spleen cells. Spleen cells from such chimeras were found to have normal B and T cell mitogenic responses. Furthermore, levels of natural-killer cell function were comparable to those observed in B6----B6 syngeneic radiation chimeras established without Leu-Leu-OMe treatment of donor cells. Spleen cells from B6----B6D2F1 mice were identical with B6----B6 or B6 mice in allostimulatory capacity and thus contained no discernible cells of non-H-2b phenotype. Whereas B6----B6D2F1 spleen cells demonstrated alloproliferative and allocytotoxic responses toward H-2k bearing spleen cells, no H-2d specific proliferative or cytotoxic responses could be elicited. B6----B6D2F1 spleen cells did not suppress the generation of anti-H-2d or anti-H-2k proliferative or cytotoxic responses from control B6 spleen cells. Furthermore, addition of rat concanavalin A supernatants did not reconstitute anti-H-2d responses of B6----B6D2F1 chimeric spleen cells. Thus, Leu-Leu-OMe treatment of B6 donor cells not only prevents lethal graft-vs-host disease, but also permits establishment of long-lived parent----F1 chimeras that are selectively tolerant of host H-2 disparate alloantigens, but fully immunocompetent with respect to natural killer cell function, B and T cell mitogenesis, and anti-third party alloresponsiveness.     

Abrogation of hybrid resistance to bone marrow engraftment by graft-vs-host-induced immune deficiency

Lethally irradiated F1 mice, heterozygous at the hematopoietic histocompatibility locus Hh-1, which is linked with H-2Db, reject bone marrow grafts from H-2b parents. This hybrid resistance (HR) is reduced by prior injection of H-2b parental spleen cells. Because injection of parental spleen cells produces a profound suppression of F1 immune functions, we investigated whether parental-induced abrogation of HR was due to graft-vs-host-induced immune deficiency (GVHID). HR was assessed by quantifying engraftment of H-2b bone marrow in F1 mice with the use of splenic [125I]IUdR uptake; GVHID, by the ability of F1 spleen cells to generate cytotoxic T lymphocytes (CTL) in vitro. We observed a correlation in the time course and spleen cell dose dependence between loss of HR and GVHID. Both GVHID and loss of HR were dependent on injection of parental T cells; nude or T-depleted spleen cells were ineffective. The injection of B10 recombinant congenic spleens into (B10 X B10.A)F1 mice, before grafting with B10 marrow, demonstrated that only those disparities in major histocompatibility antigens that generated GVH would result in loss of HR. Thus, spleens from (B10 X B10.A(2R]F1 mice (Class I disparity only) did not induce GVHID or affect HR, whereas (B10 X B10.A(5R))F1 spleens (Class I and II disparity) abrogated CTL generation and HR completely. GVHID produced by a class II only disparity, as in (B10 X B10.A(5R))F1 spleens injected into (B6bm12 X B10.A(5R))F1 mice, was also sufficient to markedly reduce HR to B10 bone marrow. This evidence that GVHID can modulate hematopoietic graft rejection may be relevant to the mechanisms of natural resistance to marrow grafts in man.     

Variable effect of anH-21 barrier on response to skin allografts in the mouse

(AQR×B10)F₁ mice were grafted with skin from donors differing in theK, I, KI, andISD regions of theH-2 complex. A dichotomy was observed in the fate of theH-2I-disparate grafts: either they were rejected acutely within the second week or were accepted indefinitely. Acceptances were much more common among male than female hosts. Acceptor status was limited to the I group, was unpredictable in occurrence, was not well-correlated with positive serum anti-Ia titers, and did not confer protection of grafts that were alike atH-2I but different atH-2K orH-2D. Since theH-2I barrier studied here elicited such divergent responses in genetically identical hosts, it is unlikely that any histocompatibility typing test could predict graft fate.Abbreviations used in this paper are MST median survival time - MHC major histocompatibility complex - CTL cytotoxic T lymphocyte - B10 C57 BL/10 - 6R BIO.T(6R) - B10.A BIO. ASn - H-2-Ia serologically detected antigens coded in theI region ofH-2 This term is used in preference toIa, since it has recently been shown that Ia-like alloantigens may be coded outside the MHC (Dickleret al. 1975).     

Specificity or affinity of cytotoxic T cells for self H-2K determinants apparently does not change between primary and secondary responses to ectromelia virus infection.

Lysis of virus-infected target cells by virus-specific cytotoxic T cells occurs where donors of T cells and targets share either H-2K or H-2D genes. The effect of four H-2K mutations on virus-induced antigens recognized by cytotoxic T cells from in vitro secondary response to infection was studied. B10.A(5R) cytotoxic T cells (which share the K end of H-2 with the mutant strains, except for the mutated gene(s)) efficiently killed virus-infected macrophage targets from mutant strains B6-H-2bg1 and B6-H-2bg2, were less effective against B6-H-2bh and did not appear to be cytotoxic for B6.C-H-2ba target cells. Conversely, B6-H-Ibg1 and B6-H-2bg2 cytotoxic T cells were more effective in killing virus-infected B10.A(5R) macrophages than B6-H-2bh and B6.C-H-2ba cytotoxic cells respectively. In addition, B6-H-2bg1 and B6-H-2bg2 cells appeared to be only slightly different from wild-type with respect to the interaction between virus-infected cells and T cells. The data obtained suggested that virus-induced antigenic patterns on infected B6.C-H-2ba (mutant) cells are more different antigenically from those on wild-type cells than are those on infected cells from the other mutants, B6-H-2bh, B6-H-2bg1 and B6-H-2bh2. This agrees with previous data using primary cytotoxic T cells and thus suggests that no detectable change in the affinity or specificity of cytotoxic T cell receptors occurs between primary and secondary responses to infection. These findings are also discussed in relation to the exclusion of T cells with receptors for H-2K determinants that are common to the mutants and wild-type, from the response to virus-infected self cells.     

Presence of T cell-associated surface antigens on murine NK cells.

The expression of T cell-associated surface antigens on natural killer (NK) spleen cells of C57BL/6 mice was evaluated by cytotoxic depletion experiments with alloantisera prepared against the Thy 1, Ly 1, Ly 2, Ly 5, Ly 6, and NK 1 antigens. The NK activity of these nonimmunized spleen cells for YAC-1 leukemia cells was dramatically reduced by antisera to the Ly 5 and NK 1 antigens. Variable results were obtained with anti-Ly 6 sera--certain pools of this antiserum decreased the NK activity, whereas other pools showed only negligible effects. The NK activity of the same cell suspensions was not affected by antisera to the Thy 1, Ly 1, and Ly 2 antigens. In parallel tests the T cell-associated cell surface antigens of alloimmune T killer cells were similarly evaluated by cytotoxic depletion experiments. In this case, the activity of these cells was consistently diminished by antisera to the Thy 1, Ly 2, Ly 5, and Ly 6 antigens, but not by antisera to the Ly 1 and NK 1 antigens. On this basis it was concluded that the NK cells expressed a restricted subset of T cell-associated alloantigens and therefore may have been derived from the T cell lineage of lymphocytes.     

A study of the inability of subcellular fractions to elicit primary anti-H-2 cytotoxic T lymphocytes

An analysis was undertaken to understand the inability of H-2 containing plasma membranes and partially purified H-2 antigens incorporated into lipid vesicles to elicit primary anti-H-2 CTLs. It was found that in the presence of supernatants from concanavalin A stimulated spleen cells H-2 containing-subcellular fractions could elicit anti-H-2 CTLs. The result suggests that cytotoxic T lymphocyte precursor cells are available for interaction with alloantigens within the subcellular fractions and that the defect is the inability of these H-2 antigen-containing subcellular fractions to stimulate T helper activity.     

Cell-mediated immune responses in vitro. II. Simultaneous generation of cytotoxic lymphocyte responses to two sets of alloantigens of limited cross-reactivity.

The conditions for generation of simultaneous and independent cytotoxic lymphocyte (CL) responses to each of two sets of alloantigens of limited cross-reactivity by mouse spleen cells in vitro have been investigated. Responder spleen cells were incubated with mitomycin C-treated C57BL/6 (H-2b) or DBA/2 (H-2d) stimulator spleen cells and day 5 CL responses were assayed with 51Cr-labeled EL-4 leukemia (H-2b) and P815 mastocytoma (H-2d) as target cells. Spleen cells from mice of the various H-2 haplotypes tested differed greatly in their ability to develop specific CL responses against alloantigens on the stimulator spleen cells and in the degree of cross-reactive cytotoxic activity against target cells bearing alloantigens not present on the stimulator spleen cells. In contrast to the other strains examined, DBA/1 (H-2q) spleen cells developed specific CL responses to either H-2b or H-2d alloantigens without exhibiting significant cross-reactive activity on the inappropriate target cell. The CL responses to H-2b and H-2d alloantigens by DBA/1 spleen cells were comparable in magnitude and had similar stimulator cell-dose requirements. Further, DBA/1 spleen cells developed CL responses of normal magnitude simultaneously against both target cells when incubated with both mitomycin C-treated C57BL/6 and DBA/2 stimulator cells.     

Transplantation of cultured thymic fragments. VI. H-2 recombinant donors

Athymic (nude) mice were transplanted with cultured thymic fragments from syngeneic, allogeneic, and partially allogeneic (recombinant) mice. Lymphocyte proliferation and cytotoxicity in vitro were measured to assess immunologic reconstitution. Transplanted nude mice were immunocompetent whether donor and recipient were disparate for class I, class II, or both H-2 gene types. Furthermore, allotolerance for thymic H-2 class I antigens was achieved independently of class II antigen allotolerance. Class I antigen tolerance was not broken during lymphocyte responses to unrelated alloantigens, ruling out insufficient help as the tolerance mechanism. Splenocytes, isolated from nude mice transplanted with fully allogeneic or syngeneic thymic fragments and stimulated in vitro with trinitrophenyl-modified cells, displayed H-2-restricted, hapten-specific cytotoxicity. Cytotoxic cells from allotolerant mice were restricted to either host or thymic H-2 antigens, depending on the stimulating cell haplotype. Response levels for thymic and host trinitrophenyl-modified cells were comparable. We have shown that allogeneic thymic epithelium transplanted into adult nude mice can induce allotolerance to class I and II H-2 antigens equally, and permits T lymphocyte interaction with cells bearing thymic donor or host H-2 antigens. Our results are consistent with a model wherein T lymphocyte self-receptors retain their genomic repertoire but can be selectively mutated or expanded by appropriate H-2 antigen presentation by the thymus.     

Resistance to cell-mediated cytotoxicity is correlated with reduction of H-2K gene products in AKR leukemia

AKR leukemia cell lines differing in the amount of H-2K and H-2D antigens expressed on the cell surface were used to assess cell-mediated immune responses in syngeneic mice against Gross/AKR murine leukemia virus (MuLV)-induced tumors. Leukemic cells with reduced expression of H-2K^k antigens were inactive as inducers of Gross-MuLV/H-2^k-specific cytotoxic T lymphocytes (CTL) and resistant to lysis by CTL raised against H-2K^k positive AKR leukemia cells. H-2K^k positive leukemias induced cytotoxic effectors, which upon restimulation in vitro, lysed the stimulating and other H-2K^k positive leukemia cells. In antibody inhibition experiments, T-cell-mediated cytotoxicity to these leukemias could only be inhibited by antisera and monoclonal antibodies specific for the H-2K^k antigens. Due to this specific role of H-2K^k antigens in T-cell cytotoxicity to Gross/AKR MuLV-induced tumors, reduced expression of H-2K^k antigens on spontaneous AKR leukemic cells could have important implications for surveillance of these neoplastic cells.Abbreviations used in this paper CTL cytotoxic T lymphocytes - MuLV murine leukemia virus     

Recognition of Rous sarcoma virus-induced tumor antigens by cytotoxic T lymphocytes (CTL): studies on specificity of killing by CTL employing H-2 congenic and recombinant mouse tumor cells

The specificities of cytotoxic T lymphocytes (CTL) were studied for the analysis of CTL against tumor-specific cell surface antigen(s) (TSSA) of non-virus-producing tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. Eight CTL clones were established from immune spleen cells of B10.A(5R) mice. These clones demonstrated six patterns of cytotoxic reactivity in vitro: Two clones showed H-2 restriction in tumor cell lysis. Two other clones had the capacity to lyse syngeneic, H-2K-compatible B10 and H-2-incompatible B10.A(4R) tumor cells, but not YAC-1 cells. One clone had cytotoxic activity against syngeneic, H-2D-compatible B10.D2 tumor cells and YAC-1 cells, but not against H-2-incompatible tumor cells. One clone had cytotoxic activity against syngeneic and YAC-1 tumor cells, but not against either H-2-compatible or H-2-incompatible tumor cells. One clone had lytic activity to syngeneic, H-2-compatible, H-2-incompatible, and YAC-1 tumor cells. Another clone killed H-2-incompatible B10.A(4R) tumor and YAC-1 cells, but not syngeneic or H-2-compatible tumor cells. All these clones strongly expressed surface Thy-1.2 antigens, whereas the expression of Lyt-1.2 and Lyt-2.2 antigens was different from clone to clone. These results demonstrate heterogeneity of both lytic specificity and phenotype of CTL against RSV-induced mouse tumor cells, suggesting the existence of multiple antigenic sites on the RSV TSSA recognized by CTL populations.     

Transplantation antigen expression on murine trophoblast detection by induction of specific alloimmunity

Cell-mediated immune responses to murine embryonic trophoblast cells were investigated using lymphocyte trophoblast cultures (LTC) and cell-mediated lympholysis (CML). Spleen cells from CBA (H-2^k) or C57BL/6 (H-2^b) mice hyperimmunized with 3.5-day-old Balb/c (H-2^d) blastocysts did not undergo DNA synthesis after in vitro exposure to Balb/c blastocyst outgrowths nor were cytotoxic lymphocytes (CTL) generated against H-2^d alloantigens. Splenocytes from Balb/c mice presensitized with semiallogeneic (Balb/c female × C57BL/6 male) trophoblast cells derived from 17- to 20-day placental tissue expressed a weak proliferative response in the presence of semiallogeneic placental trophoblast and produced a moderate number of CTL against H-2^b (paternal strain) alloantigens when compared to mixed lymphocyte cultures (MLC) between Balb/c responder and semiallogeneic (stimulator) spleen cells. CTL were also generated in vitro after splenocytes from Balb/c mice hyperimmunized with semiallogeneic spleen cells were restimulated in vitro with placental trophoblast cells. These studies showing that early-stage trophoblast cells fail to evoke transplantation immunity and placental trophoblast is capable of generating alloimmunity only after combined in vivo hyperimmunization with in vitro restimulation suggest that these trophoblast cells are poorly immunogenic due in part to the relatively weak functional expression of major transplantation antigens.