Further evaluation of the pregnancy-linked down-regulation of the paternal antigen-specific splenic cytotoxic T lymphocyte activity in allogeneically pregnant mice.

Cytotoxic T lymphocyte (CTL) activity directed against paternal alloantigen was examined in allogeneically pregnant mice using various allogeneic combinations. The spleen cells from pregnant C57BL/6 (H-2b) mice mated with BALB/c (H-2d) male mice generated less anti-H-2d CTL after in vitro sensitization than those from unpregnant or syngeneically mated C57BL/6 mice. Different allogeneic combinations including the incompatibility at only D region of H-2 or minor histocompatibility loci were effective for downregulating the anti-paternal CTL activity in pregnancy. The downregulation of anti-paternal CTL activity induced by allogeneic pregnancy occurred at day 10 to day 18 of pregnancy, most extensively at day 14. The allogeneic pregnancy also downregulated the allogeneic CTL activities that had been amplified by injecting alloantigens before mating.     

In vitro expression of secondary antitumor immunity by in vivo tumor-sensitized T cells: Inhibition by tumor-induced suppressor T cells

Summary In vitro cultivation of memory immune cells from P815- or P388-immune mice with corresponding irradiated tumor cells induced generation of cytolytic T cells (CTL). The induction of CTL generation, as well as the cytolytic activity itself, was tumor-specific. The in vitro generation of CTL from P815- or P388-immune cells was suppressed by spleen cells from mice bearing corresponding progressive tumors (tumor size 15 mm). The tumor-induced suppressor cells suppressed the in vitro generation of CTL, but did not affect their cytolytic function. The suppression was tumor-specific and was mediated by Ly1⁺2^–L3T4⁺ T cells. Treatment of suppressor cell donors with cyclophosphamide or sublethal -radiation completely abolished the ability of their spleen cells to inhibit the in vitro CTL generation.     

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.     

Regulatory T cell subpopulations in pregnancy. I. Evidence for suppressive activity of the early phase of MLR.

Previous in vivo experiments have provided evidence of suppressive activity induced by multiple allogeneic pregnancies. The reactivity of maternal spleen cells toward paternal strain alloantigens was investigated by use of MLR microculture technique. A study of the kinetics of the MLR showed an early peak of reactivity (48-hr culture) followed by a decline leading to a decreased reactivity by 96 hr when spleen cells from allogeneically pregnant mice were compared to those of virgin or even isogeneically pregnant mice, suggesting the possible action of MLR regulatory cells. A strong suppression of a H-2k (CBA) anti-H-2a (A/J) or anti-H-2d (C57BL/Ks) MLR was observed when mitomycin-treated spleen cells from CBA mice multiparous by A/J or C57BL/Ks (but not CBA) males were added to the culture. This suppression was abolished by treating the regulatory cell population with anti-theta serum plus complement or replacing the 1% normal mouse serum in the medium by a proper antiidiotypic mouse serum.     

Fas Ligand-Mediated Lysis of Self Bystander Targets by Human Papillomavirus-Specific CD8+ Cytotoxic T Lymphocytes

Mouse cytotoxic T lymphocytes (CTL) reactive with a H-2D^b-presented 9-mer peptide of the human papillomavirus type 16 protein E7^49-57 (RAHYNIVTF) were generated from the spleen cells of wild-type C57BL/6 (B6) or B6 perforin-deficient (B6.P⁰) mice. CD8⁺ B6 CTL displayed peptide-specific perforin- and Fas-mediated lysis of E7-transfected mouse RMA lymphoma cells (RMA-E7), while CD8⁺ CTL from B6.P⁰ mice lysed RMA-E7 cells via Fas ligand (FasL) exclusively. Rapid and efficient lysis of syngeneic bystander B6 blasts or RMA cells by either B6 or B6.P⁰ Ag-activated CTL was mediated by a FasL-Fas mechanism. Fas-resistant bystanders were not lysed, nor were allogeneic Fas-sensitive C3H/HeJ (H-2^k) or BALB/c (H-2^d) bystander blasts. Interestingly, however, phorbol myristate acetate-ionomycin preactivation of B6.P⁰ effectors enabled lysis of allogeneic H-2^k and H-2^d bystanders even in the absence of antigenic stimulation. Lysis of syngeneic bystander cells was always FasL-Fas dependent and required effector-bystander contact and, in particular, an interaction between CTL LFA-1 and bystander ICAM-1. Thus, in the context of major histocompatibility complex class I molecule-peptide ligation of the T-cell receptors of CD8⁺ CTL, neighboring bystander cells that are syngeneic and Fas sensitive and express the adhesion molecule ICAM-1 are potential targets of CTL attack.With the dissection of two basic cytolytic mechanisms of cytotoxic T lymphocytes (CTL) (10, 14, 20, 34), it has become possible to delineate the important criteria that determine direct (Ag-restricted) and bystander cytotoxicity. CTL use complementary cytotoxic mechanisms, one based on the granule exocytosis of a calcium-dependent pore-forming protein, perforin (8, 26), and granzymes (35) and another that depends on a calcium-independent interaction of effector T-cell tumor necrosis factor or Fas ligand (TNF or FasL) and target cell TNF receptor (TNFR) or Fas (22, 33). The function of the granule exocytosis pathway appears to be largely in non-major histocompatibility complex (MHC)-restricted NK lysis of class I molecule-defective tumor cells and in direct CTL-mediated immunity against tumor cells (37) or virus-infected cells (11, 19, 39). By contrast, the FasL-Fas and TNF-TNFR interactions are important for the maintenance of T-cell homeostasis following exposure to foreign Ag (5, 42) and Th-1 FasL-mediated B-cell apoptosis (27, 28). Blockage of both TNF and FasL is required to abrogate T-cell death: TNF mediates the death of most CD8⁺ T cells, whereas FasL mediates the death of most CD4⁺ T cells (42). While FasL-dependent lysis appears to be the primary mechanism used by CD4⁺ Th-1 effectors, CD8⁺ CTL use FasL or TNF secondarily in the absence of perforin-mediated lysis (10, 14, 20).After T-cell activation, a functional role for FasL is not apparent for several days until the T cell becomes Fas sensitive and hence susceptible to autocrine T-cell suicide (1, 5, 38). However, by using alloreactive CTL cultures or clones, it has recently become apparent that in the presence of Ag-bearing target cells (i.e., upon T-cell receptor [TCR] activation) CTL can also lyse Ag-free bystander cells via a FasL-Fas interaction (13, 34). While the specificity of CTL toward Ag-bearing target cells has been considered a hallmark of an efficient immune response, CTL do not appear to spare Ag-free bystander cells during lysis of specific Ag-bearing target cells. In this study, we have generated CD8⁺ CTL from both wild-type and perforin-deficient (P⁰) mice reactive with a high-affinity H-2D^b-binding peptide of human papillomavirus type 16 protein E7. These peptide-specific CTL have been employed to demonstrate the requirements for CD8⁺ CTL-mediated lysis of Ag-free bystander cells and in particular the different properties of CTL activated by antigen versus a nonspecific stimulus.     

Neurotransmitter suppression of the in vitro generation of a cytotoxic T lymphocyte response against the syngeneic MOPC-315 plasmacytoma

We have previously shown that, as a consequence of low-dose melphalan (l-phenylalanine mustard (l-PAM) therapy, the hitherto immunosuppressed spleen cells from BALB/c mice bearing a large MOPC-315 tumor (in contrast to spleen cells from normal mice) acquire the ability to generate a greatly enhanced anti-MOPC-315 cytotoxic T lymphocyte (CTL) response upon in vitro stimulation with MOPC-315 tumor cells. Here we show that the catecholamines norepinephrine, epinephrine, and isoproterenol suppressed the in vitro generation of anti-MOPC-315 cytotoxicity by spleen cells from mice that had just completed the eradication of a large MOPC-315 tumor following low-dosel-PAM therapy (l-PAM TuB spleen cells), as well as by spleen cells from normal mice. In contrast to the marked suppression obtained with catecholamines, the cholinergic agonist carbachol had no effect on the in vitro generation of splenic anti-MOPC-315 cytotoxicity. The inhibitory effect of the catecholamines was mimicked by the membranepenetrating analog of cAMP, dibutyryl-cAMP, and by cholera toxin at concentrations that stimulate the endogenous production of cAMP. The -adrenergic receptor antagonist propranolol did not block norepinephrine-induced inhibition of the generation of anti-MOPC-315 cytotoxicity by either normal orl-PAM TuB spleen cells. Since the curative effectiveness of low-dosel-PAM therapy for MOPC-315 tumor bearers requires the participation of CD8⁺ T cells that exploit a CTL response in tumor eradication, it is conceivable that norepinephrine may reduce the therapeutic outcome of low-dose chemotherapy by inhibiting the acquisition of CTL activity.     

Immunological characterization of tumor-rejection antigens on ultraviolet-light-induced tumors originating in the CB6F1 mouse

Six ultraviolet-light(UV)-induced tumors of (BALB/c×C57BL/6)F₁ (H-2^d/b) mouse origin were analyzed for the effector T cell subsets involved in tumor rejection the MHC class I to which cytolytic T lymphocytes (CTL) are restricted, and the effect of UV radiation on tumor rejection, to characterize their tumor-rejection antigens (TRA) recognized by CTL. All tumors were rejected in syngeneic normal mice but grew progressively in nude mice. CD8⁺ T cells mediated the antitumor responses for all tumors and CD4⁺ T cells could also do so for one tumor 6.1B. Each tumor induced potent CTL that recognized the specific TRA in preferential association with MHC class I haplotypes not from H-2^b but from H-2^d; that is, K^d, D^d or L^d. Profiles of TRA expression on two tumors were obtained by the analyses of their antigen-loss variants. 1A codominantly expressed at least four distinct TRA associated with K^d, all of which induced CTL. On the other hand, UV 1 had at least two distinct TRA, one of which, associated with K^d, exclusively induced CTL. However, in the absence of the dominant TRA, another TRA associated with L^d on R95C, a variant of UV, 1, induced CTL. Unlike other tumors, R95C grew progressively in short-term-UV-irradiated syngeneic mice. Nude mice reconstituted with a combination of CD4⁺ T cells from short-term-UV-irradiated mice and CD8⁺ T cells from normal mice did not reject R95C. An increase in the former T cell population led the reconstituted mice to reject the tumor. These findings suggest some functional defects of CD4⁺ T cells rather than the generation of suppressor cells in short-term-UV-irradiated mice. The UV-induced tumors used in the present study provide a unique system for analyzing the preferential sorting of TRA as well as for elucidation of the TRA itself.     

An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia

Background

Xenotransplantation of patient-derived AML (acute myeloid leukemia) cells in NOD-scid Il2rγ ^null (NSG) mice is the method of choice for evaluating this human hematologic malignancy. However, existing models constructed using intravenous injection in adult or newborn NSG mice have inferior engraftment efficiency, poor peripheral blood engraftment, or are difficult to construct.

Methods

Here, we describe an improved AML xenograft model where primary human AML cells were injected into NSG newborn pups intrahepatically.

Results

Introduction of primary cells from AML patients resulted in high levels of engraftment in peripheral blood, spleen, and bone marrow (BM) of recipient mice. The phenotype of engrafted AML cells remained unaltered during serial transplantation. The mice developed features that are consistent with human AML including spleen enlargement and infiltration of AML cells into multiple organs. Importantly, we demonstrated that although leukemic stem cell activity is enriched and mediated by CD34⁺CD117⁺ subpopulation, CD34⁺CD117^? subpopulation can acquire CD34⁺CD117⁺ phenotype through de-differentiation. Lastly, we evaluated the therapeutic potential of Sorafenib and Regorafenib in this AML model and found that periphery and spleen AML cells are sensitive to these treatments, whereas BM provides a protective environment to AML.

Conclusions

Collectively, our improved model is robust, easy-to-construct, and reliable for pre-clinical AML studies.

     

Enhanced and prolonged efficacy of superantigen-induced cytotoxic T lymphocyte activity by interleukin-2 in vivo

The bacterial superantigen, staphylococcal enterotoxin A (SEA) activates T cells with high frequency and directs them to lyse MHC-class-II-expressing cells in superantigen-dependent cell-mediated cytotoxicity (SDCC). Treatment of mice with SEA induced strong CD8⁺ T-cell(CTL)-mediated SDCC, as well as abundant cytokine production from CD4⁺ and CD8⁺ T cells. However, both cytotoxicity and cytokine release were transient. In contrast, combined treatment with SEA and recombinant interleukin-2 (rIL-2) increased peak levels and maintained CTL activity. These effects were concomitant with an increased number of SEA-reactive V11⁺ T cells. Both the CD4⁺ and CD8⁺ populations contained higher frequencies of cells expressing IL-2 receptor (IL-2R) , which suggests that continuous IL-2R signaling preserves its high expression and subsequently prevents loss of growth factor signal necessary for expansion of T cells. Although IL-2R expression was increased among both CD4⁺ and CD8⁺ cells, only the cytotoxic function of CTL, but not cytokine production from either CD4 or CD8, was augmented. These findings demonstrate that treatment with rIL-2 potentiates superantigen-induced cytotoxicity and maintains high CTL activity. rIL-2 might therefore be useful in improving superantigenbased tumor therapy.     

Human Papillomavirus Type 16 E7 Oncoprotein Expressed in Peripheral Epithelium Tolerizes E7-Directed Cytotoxic T-Lymphocyte Precursors Restricted through Human (and Mouse) Major Histocompatibility Complex Class I Alleles

Mice which coexpress human papillomavirus type 16 E7 and HLA A2.1 in peripheral squamous epithelium and thymic cortical epithelium are tolerant at the cytotoxic T-lymphocyte (CTL) level to E7 epitopes restricted through HLA A*0201 and H-2^b (T. Doan, M. Chambers, M. Street, G. J. Fernando, K. Herd, P. Lambert, and R. Tindle, Virology 244:352–364, 1998). Here we used bone marrow-reconstituted radiation chimeras to distinguish whether E7-directed CTL tolerance was mediated peripherally by E7 expression in skin or centrally by E7 expression in thymus. In chimeric mice expressing E7 in skin and reconstituted with E7-naïve bone marrow and E7-naïve thymus, CTL responses to vaccine-administered E7 epitopes were not restored, i.e., the mice remained tolerant. In contrast, chimeric mice not expressing E7 in skin and reconstituted with E7-naïve bone marrow and E7-expressing thymus had full E7-directed CTL responses. These results demonstrate that E7 protein expression in peripheral squamous epithelium is sufficient to tolerize the E7-directed CTL precursor repertoire. The data have implications for E7-mediated tumorigenesis and for the development of E7-based immunotherapeutic strategies, since peripheral immunological tolerance of tumor-associated antigens may create a barrier to effective immunotherapy.The E7 oncoprotein of human papillomavirus type 16 (HPV16) is a tumor-specific antigen when expressed in HPV16-associated cervical epithelial tumors, to which immunomanipulative strategies are being directed, both experimentally (see, for example, references 7, 9, and 29) and in E7-based therapeutic vaccine clinical trials (5). We recently reported studies with mice expressing HPV16 E7 protein, driven from the keratin 14 (K14) promoter, in basal epithelium of skin and in the thymic cortex (8). We showed that immunization-induced cytotoxic T-lymphocyte (CTL) responses to each of three CTL epitopes in the E7 protein restricted through two major histocompatibility complex (MHC) class 1 haplotypes were down-regulated in these E7-transgenic mice compared with non-E7 syngeneic control mice. However, in these studies we did not determine whether the down-regulation (i.e., tolerance) was induced centrally by E7 expressed in the thymus or peripherally by E7 expressed in epithelium. In the present study, we distinguish between these two possibilities by specific immunization of bone marrow-reconstituted thymus-transplanted chimeric E7 transgenic mice. We report that chimeric mice expressing the E7 transgene in peripheral epithelium but not in the thymus showed E7-specific down-regulated CTL responses to each of two E7 CTL epitopes restricted through a human and a mouse MHC class I allele, respectively, when compared with sham chimeric but non-E7 control mice. In contrast, chimeric mice expressing the E7 transgene in thymus, but not peripheral epithelium, showed E7-directed CTL responses indistinguishable from those of non-E7 control mice. Thus, we show that the expression of E7 in peripheral squamous epithelium is sufficient to induce and maintain a state of tolerance against E7.

E7-directed bone marrow-derived precursor CTLs (pCTLs) are not tolerized in mice expressing E7 in thymus but not in skin.

(K14E7 × A2.1K^b)F₁ mice (designated KA mice) were derived by crossing male K14.HPV16E7(+/+) mice (16), which express an HPV16 E7 transgene perinatally and throughout life in skin and thymic cortical epithelium, with female HLA A2.1K^b(+/+) mice (30). (FVB × A2.1K^b)F₁ mice (designated FA) are syngeneic but do not possess the E7 transgene. KA (E7⁺) and FA (E7⁻) mice are on an H-2^b background. To inquire whether pCTLs from E7-transgenic mice were tolerized on E7-expressing thymus, we constructed thymus-transplanted radiation chimeras as described elsewhere (8) from immunologically depleted FA (E7⁻) mice reconstituted with KA (E7⁺) bone marrow cells. In half of the mice, [designated KA→FA(FA) mice], the bone marrow-derived T-cell precursors were made to mature through a thymus implant from an FA (E7⁻) donor mouse; in the other half of the mice [designated KA→FA(KA) mice], the bone marrow cells were made to mature through an E7-expressing KA thymus implant (Fig. ​(Fig.1,1, panel I). KA→FA(FA) mice, KA→FA(KA) mice, and control FA (E7⁻) and KA (E7⁺) mice were immunized for CTL response induction with a mix of peptides containing ⁸²LLMGTLGIV⁹⁰ (an HLA A*0201-restricted E7 CTL epitope [24]), ⁴⁹RAHYNIVTF⁵⁷ (an H-2D^b-restricted E7 CTL epitope [9]), and ⁵⁸GILGFVFTL⁶⁶ (an HLA A*0201-restricted influenza virus matrix CTL epitope [13]). Control mice underwent surgical procedures but without receiving cell and/or organ transplants (sham). KA (E7⁺) mice showed the previously documented (8) down-regulated CTL response to the E7 epitopes (but not to the irrelevant influenza virus matrix epitope) compared to FA (E7⁻) mice (Fig. ​(Fig.1,1, panels IIC and IID). In contrast, KA→FA(KA) mice exhibited E7 (and influenza virus matrix)-directed CTL responses of the same magnitude as those of KA→FA(FA) mice and FA (E7⁻) mice (Fig. ​(Fig.1,1, panels IIA to IIC). These data indicate that E7-directed pCTLs from E7 transgenic mice which mature through an E7-expressing thymus, and emerge into a non-E7-expressing peripheral epithelial environment, are not tolerized. Open in a separate windowFIG. 1(I) Derivation of KA→FA(FA) and KA→FA(KA) chimeric mice from immunologically ablated FA (E7⁻) mice. (II) CTL responses of splenocytes from chimeric mice and sham control FA (E7⁻) and KA (E7⁺) mice (three per group) immunized with a mix of peptides containing E7 CTL epitopes LLMGTLGIV and RAHYNIVTF and influenza virus matrix CTL epitope GILGFVFTL. Immunizations were given in Quil A adjuvant and tetanus toxoid as described elsewhere (8). Spleen cells were restimulated with individual peptides in vitro. Targets were EL4.A2 cells (8) pulsed with individual peptides as indicated. EL4.A2 cells are susceptible to specific CTL lysis through both HLA A*0201 and H-2^b restriction elements. CTL assays were conducted as described elsewhere (8). bm, bone marrow; th, thymus; sk, skin.To eliminate the possibility that bone marrow-derived precursors from KA (E7⁺) mice had somehow previously encountered E7 protein before transfer to recipient mice, thereby influencing their immunological status in the above-described experiment, we asked whether bone marrow-derived pCTLs from FA (E7⁻) mice would be tolerized during maturation in an E7-expressing thymus. We constructed chimeras from immunologically ablated FA (E7⁻) mice by reconstitution with FA (E7⁻) bone marrow cells. In half the mice [designated FA→FA(KA) mice], the bone marrow cells were made to mature through an E7-expressing KA thymus implant. In the other half of the mice [designated FA→FA(FA) mice], the bone marrow cells were made to mature through a non-E7-expressing FA thymus implant (Fig. ​(Fig.2,2, panel I). FA→FA(KA) mice, FA→FA(FA) mice, and control FA (E7⁻) and KA (E7⁺) mice were immunized for CTL induction with a mix of peptides containing the HLA A*0201-restricted and H-2^b-restricted E7 CTL epitopes and influenza virus matrix CTL epitope. FA→FA(KA) mice exhibited E7-directed CTL responses of the same magnitude as those of FA→FA(FA) mice and FA (E7⁻) mice, while KA (E7⁺) mice exhibited the expected down-regulated E7-directed (but not down-regulated influenza virus matrix-directed) CTL responses (Fig. ​(Fig.2,2, panel II). These data indicated that E7-naïve bone marrow-derived pCTLs which mature through an E7-expressing thymus and emerge into a non-E7 peripheral epithelial environment are not tolerized to E7. Open in a separate windowFIG. 2(I) Derivation of FA→FA(FA) and FA→FA(KA) chimeric mice from immunologically ablated FA (E7⁻) mice. (II) CTL responses of splenocytes from chimeric mice and sham control mice (three per group) immunized with a mix of peptides containing E7 CTL epitopes LLMGTLGIV and RAHYNIVTF and influenza virus matrix CTL epitope GILGFVFTL. Spleen cells were restimulated with individual peptides in vitro. Targets were EL4.A2 cells pulsed with individual peptides as indicated. CTL assays were conducted as described elsewhere (8). bm, bone marrow; th, thymus; sk, skin.

Bone marrow-derived pCTLs are specifically tolerized in mice expressing E7 in skin but not in thymus.

To inquire whether E7-directed pCTLs were tolerized in mice expressing E7 in skin but not in thymus, we constructed chimeric mice in which bone marrow-derived precursors were made to mature through a non-E7-expressing thymus and to emerge into an E7-expressing peripheral epithelial environment. In a first experiment, immunologically ablated KA (E7⁺) mice were reconstituted with KA (E7⁺) bone marrow cells which were made to mature through a thymus implanted from an FA (E7⁻) mouse. The recipient mice, designated KA→KA(FA) (Fig. ​(Fig.3,3, panel I), and control FA (E7⁻) and KA (E7⁺) mice were immunized for CTL response induction with a mix of peptides containing the HLA A*0201-restricted and H-2^b-restricted E7 CTL epitopes and the influenza virus matrix CTL epitope. In KA→KA(FA) mice, E7-directed CTL responses to both E7 epitopes were down-regulated to the level seen in control KA (E7⁺) mice (Fig. ​(Fig.3,3, panel IIB), while control FA (E7⁻) mice showed the expected high responses to both E7 CTL epitopes. Open in a separate windowFIG. 3(I) Derivation of KA→KA(FA) chimeric mice from immunologically ablated KA (E7⁺) mice. (II) CTL responses of splenocytes from chimeric mice and sham control mice (three per group) immunized with a mix of peptides containing E7 CTL epitopes LLMGTLGIV and RAHYNIVTF and influenza virus matrix CTL epitope GILGFVFTL. Spleen cells were restimulated with individual peptides in vitro. Targets were EL4.A2 cells pulsed with individual peptides as indicated. CTL assays were conducted as described elsewhere (8). bm, bone marrow; th, thymus; sk, skin.In a second experiment, immunologically ablated KA (E7⁺) mice were reconstituted with bone marrow from FA (E7⁻) mice, which was made to mature through a non-E7-expressing FA thymus. These mice, designated FA→KA(FA) mice (Fig. ​(Fig.4,4, panel I), were immunized for CTL response induction with a mix of peptides containing the HLA A*0201-restricted and the H-2^b-restricted E7 CTL epitopes and the influenza virus matrix CTL epitope. As with KA→KA(FA) mice in the previous experiment, E7-directed CTL responses to both E7 epitopes were down-regulated as in KA (E7⁺) controls and in contrast to FA (E7⁻) controls, while the influenza virus matrix response confirmed adequate reconstitution. Open in a separate windowFIG. 4(I) Derivation of FA→KA(FA) chimeric mice from immunologically ablated KA (E7⁺) mice. (II) CTL responses of splenocytes from chimeric mice and sham control mice (three per group) immunized once with a mix of peptides containing E7 CTL epitopes LLMGTLGIV and RAHYNIVTF and influenza virus matrix CTL epitope GILGFVFTL. Spleen cells were restimulated with individual peptides in vitro. Targets were EL4.A2 cells pulsed with individual peptides as indicated. CTL assays were conducted as described elsewhere (8). bm, bone marrow; th, thymus; sk, skin.The results from these two experiments indicate that bone marrow-derived E7-directed pCTLs which mature through a non-E7-expressing thymus and emerge into an E7-expressing epithelial environment are specifically tolerized to E7.We have previously reported pCTL tolerance to epitopes of the HPV16 E7 oncoprotein in KA mice expressing a K14 promoter-driven E7 transgene perinatally and throughout life in the thymus and in basal and/or suprabasal cells of peripheral epithelium (8). In the present experiments, we demonstrate that the E7-directed pCTL repertoire is tolerized in mice expressing E7 in peripheral epithelium in the absence of thymic expression. Conversely, the repertoire is not tolerized in mice expressing E7 in the thymus, in the absence of E7 expression in peripheral epithelium. These data indicate that expression of E7 in peripheral epithelium, and not the thymus, is sufficient to induce and maintain a state of pCTL tolerance to E7. In the thymus, the K14 promoter directs transgene expression to the cortical epithelial compartment (19), which, in other mouse models, has been shown to contribute to the shaping of the T-cell repertoire by positive rather than negative selection (for example, see reference 20). Melero et al. (21) observed no functional down-regulation of the CTL responses induced by immunization with E7 peptide epitope RAHYNIVTF in H-2^b mice expressing HPV16 E7 from a K14 promoter and concluded that the mice remain immunologically ignorant of this epitope. This result contrasts with ours. Together, they provide further examples of T-cell tolerance to peripheral antigens in some systems (see, for example, references 1, 2, and 23) and T-cell ignorance in others (see, for example, references 14, 17, and 25). While determinants of immunological outcome of peripheral antigen expression are clearly complex (22), the level of expression (as well as timing and site of expression) can determine whether an antigen induces tolerance or is ignored by naïve T cells. This consideration may explain the difference between the results of Melero et al. and ours. The effect of the level of E7 expression on peripheral tolerance induction is under investigation in our laboratory.Specific CTL tolerance has implications for E7-mediated tumorigenesis. Nascent E7-expressing tumor cells will escape surveillance where little or no positive priming of cognate pCTLs by endogenous E7 occurs. Additionally, specific CTL tolerance which inhibits the generation of an immunization-induced CTL response will detract from effective immunotherapy (26). We have previously reported that (K14.E7 × C57)F₁ mice fail to control a challenge with an E7-expressing tumor following immunization with E7 CTL epitope RAHYNIVTF, whereas in immunized non-E7-transgenic control mice the tumors did not become established (12). Failure to control the tumor was correlated with a lack of an inducible RAHYNIVTF-directed CTL response in E7-transgenic mice, in contrast to non-E7-transgenic control mice, where a powerful CTL response was observed. In further experiments, multiple immunization of KA mice with E7 CTL epitopes or whole E7 protein failed to arrest the development of E7-associated endogenous tumors (8), again being correlated with a lack of E7-directed CTL responses.The current therapeutic vaccine strategy for HPV16-associated cervical carcinoma targets the E7 tumor-specific antigen by CTL induction (5, 28). The possibility arises that chronic expression of E7 in transformed cervical epithelial cells during the life of the tumor functionally tolerizes E7-directed pCTLs.Ongoing experiments in our laboratory will distinguish between presentation of E7 to pCTLs directly by keratinocytes and cross presentation of exogenously acquired E7 by bone marrow-derived professional antigen-presenting cells. Presentation of antigen by either of these routes can be tolerogenic (3, 6, 15, 27). Additionally, we will determine whether loss of functional E7-directed CTLs results from pCTL deletion (4, 11) or anergy (10, 25). Peripheral tolerance of tissue-specific antigen depends, at least in some cases, on the generation of regulatory CD4⁺ cells (see, for example, reference 18). That E7-directed CTL tolerance in KA (E7⁺) mice reflects an impairment of cognate CD4⁺ help is unlikely in view of our finding that (K14.E7 × C57)F₁ mice immunized with full-length E7 and displaying E7-specific pCTL tolerance showed concomitant enhanced E7-directed CD4⁺ T-helper responses (12).The data reported in the present study demonstrate the induction of peripheral tolerance in E7-directed pCTLs by HPV16 E7 expressed in squamous epithelial cells, in the context of human (and mouse) MHC class 1 haplotypes. There are direct implications for the development and progression of cervical cancers which express E7 in transformed squamous epithelium and for the design of E7-based immunotherapeutic strategies for cervical cancer. In the broader context, there are implications for CTL response induction to any foreign or aberrant protein expressed constitutively in squamous epithelial cells as a result of infection, tumorigenesis, or appearance of autoantigen.     

CTL induction by DNA vaccine with Toxoplasma gondii-HSP70 gene

Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) is a tachyzoite-specific virulent molecule. The DNA vaccine with T.g.HSP70 gene targeting peripheral epidermal or dermal dendritic cells (DC) induces in vivo DC maturation and successive early Th1 polarization at the draining lymph nodes (dLN) of C57BL/6 mice. In the present study, induction of cytotoxic T lymphocytes (CTL) has been explored. The CTL specific for a syngeneic DC line, DC2.4, either transfected with T. g.HSP70 gene or pulsed with recombinant T. g.HSP70 are induced in the spleen of the vaccinated mice. This CTL lyses T. gondii-infected, but not uninfected, DC2.4. Both CD8⁺ and CD4⁺ CTL are induced by the vaccine, and Fas/Fas ligand-mediated cytolysis dominantly participates in their CTL activities. Adoptive transfer experiments reveal that the vaccine-induced CD8⁺ or CD4⁺ T cells possess a protective role for toxoplasmosis at both acute and chronic phases of infection.     

Using the natural evolution of a rotavirus-specific human monoclonal antibody to predict the complex topography of a viral antigenic site

Background

Immunity against the bovine protozoan parasite Theileria parva has previously been shown to be mediated through lysis of parasite-infected cells by MHC class I restricted CD8⁺ cytotoxic T lymphocytes. It is hypothesized that identification of CTL target schizont antigens will aid the development of a sub-unit vaccine. We exploited the availability of the complete genome sequence data and bioinformatics tools to identify genes encoding secreted or membrane anchored proteins that may be processed and presented by the MHC class I molecules of infected cells to CTL.

Results

Of the 986 predicted open reading frames (ORFs) encoded by chromosome 1 of the T. parva genome, 55 were selected based on the presence of a signal peptide and/or a transmembrane helix domain. Thirty six selected ORFs were successfully cloned into a eukaryotic expression vector, transiently transfected into immortalized bovine skin fibroblasts and screened in vitro using T. parva-specific CTL. Recognition of gene products by CTL was assessed using an IFN-γ ELISpot assay. A 525 base pair ORF encoding a 174 amino acid protein, designated Tp2, was identified by T. parva-specific CTL from 4 animals. These CTL recognized and lysed Tp2 transfected skin fibroblasts and recognized 4 distinct epitopes. Significantly, Tp2 specific CD8⁺ T cell responses were observed during the protective immune response against sporozoite challenge.

Conclusion

The identification of an antigen containing multiple CTL epitopes and its apparent immunodominance during a protective anti-parasite response makes Tp2 an attractive candidate for evaluation of its vaccine potential.     

Amniotic Fluid Enhances Allogeneic Cytotoxic T Cell Responses,Whereas It Suppresses Mitogen-Stimulated Lymphocyte Proliferation

Mouse amniotic fluid has been shown to suppress T lymphocyte proliferation and suggested to be important in regulating immunity during pregnancy. In allogeneic pregnancy, cytotoxic T cells in pregnant lymphocytes against paternal transplantation antigen are impaired. We examined the effect of amniotic fluid to the alloreactive CTL responses. Although the amniotic fluid suppressed Con A or LPS stimulated lymphocyte proliferation as previously reported, the amniotic fluid taken from syngeneic C57BL/6 pregnant mice or allogeneic C57BL/6 × BALB/c pregnant mice enhanced the anti-H-2^d or anti-H-2^k CTL responses dose-dependently. We speculate that amniotic fluid contains not only immunosuppressive factors but also immunoenhancing factors which upregulate the allogeneic CTL responses.     

Antitumor effects and immunoregulation mechanisms of IL-23 gene in mouse mammary cancer mediated by retrovirus

Background: Interleukin (IL)-23, composed of p19 and p40 subunits, has diverse functions in regulating immune systems, enhancing cell-mediated immunity. In the present study, we investigated whether forced expression of the p19-linked p40 gene in murine mammary cancer cells (MA891) produced antitumor effects in vivo. Tumor growth of MA-891 cells expressing IL-23 (IL-23/MA891) in mice was retarded compared with parental and vector DNA-transduced tumors and survival of the mice inoculated with IL-23/MA-891 cells was prolonged. Expressions of the CD4⁺ T cells and CD8⁺ T cells were up-regulated not only in IL-23/MA-891 tumor specimens but also in spleen cells of mice inoculated with IL-23/MA-891 as compared with those of mice inoculated with parental or vector DNA-transduced tumors. Cytotoxic CD8⁺ T lymphocyte (CTL) activity of spleen cells from mice inoculated with IL-23/MA-891 was also significantly higher than the other two groups. Th1-type cytokines such as interferon-γ, TNF-α and IL-12p70 secreted from spleen cells of mice bearing IL-23/MA-891 tumors were increased while Th2-type cytokine IL-4 was negative regulated. Moreover, we have identified that the quantity of DC in spleen cells of mice bearing IL-23/MA-891 tumors was increased as compared with those mice bearing parental or vector DNA-transfected tumors.     

Mycobacterium tuberculosis Multidrug Resistant Strain M Induces an Altered Activation of Cytotoxic CD8+ T Cells

In human tuberculosis (TB), CD8⁺ T cells contribute to host defense by the release of Th1 cytokines and the direct killing of Mycobacterium tuberculosis (Mtb)-infected macrophages via granule exocytosis pathway or the engagement of receptors on target cells. Previously we demonstrated that strain M, the most prevalent multidrug-resistant (MDR) Mtb strain in Argentine, is a weak inducer of IFN-γ and elicits a remarkably low CD8-dependent cytotoxic T cell activity (CTL). In contrast, the closely related strain 410, which caused a unique case of MDR-TB, elicits a CTL response similar to H37Rv. In this work we extend our previous study investigating some parameters that can account for this discrepancy. We evaluated the expressions of the lytic molecules perforin, granzyme B and granulysin and the chemokine CCL5 in CD8⁺ T cells as well as activation markers CD69 and CD25 and IL-2 expression in CD4⁺ and CD8⁺ T cells stimulated with strains H37Rv, M and 410. Our results demonstrate that M-stimulated CD8⁺ T cells from purified protein derivative positive healthy donors show low intracellular expression of perforin, granzyme B, granulysin and CCL5 together with an impaired ability to form conjugates with autologous M-pulsed macrophages. Besides, M induces low CD69 and IL-2 expression in CD4⁺ and CD8⁺ T cells, being CD69 and IL-2 expression closely associated. Furthermore, IL-2 addition enhanced perforin and granulysin expression as well as the degranulation marker CD107 in M-stimulated CD8⁺ T cells, making no differences with cells stimulated with strains H37Rv or 410. Thus, our results highlight the role of IL-2 in M-induced CTL activity that drives the proper activation of CD8⁺ T cells as well as CD4⁺ T cells collaboration.     

Mesenchymal stromal cell exosome–enhanced regulatory T-cell production through an antigen-presenting cell–mediated pathway

Background aims

The immunomodulatory property of mesenchymal stromal cell (MSC) exosomes is well documented. On the basis of our previous report that MSC exosomes increased regulatory T-cell (Treg) production in mice with allogenic skin graft but not in ungrafted mice, we hypothesize that an activated immune system is key to exosome-mediated Treg production.

Phorbol ester-induced enhancement in lytic activity of CD8+ splenic T cells from low-dose melphalan-treated MOPC-315-tumor bearers

Summary We have previously shown that while spleen cells from untreated mice bearing a large MOPC-315 tumor are not cytotoxic in vitro for MOPC-315 tumor cells, spleen cells obtained from such mice on day 7 after low-dose melphalan (l-phenylalanine mustard);l-PAM therapy exert a substantial anti-MOPC-315 cytotoxicity [Mokyr et al. (1989) Cancer Res 49: 4597]. Here we show that this anti-MOPC-315 lytic activity is evident by day 5, and peaks on day 7 after the low-dose chemotherapy, at a time when the mice are actively engaged in tumor eradication. Short-term exposure of spleen cells from mice bearing a MOPC-315 tumor and treated with low-dosel-PAM (l-PAM TuB mice) to phorbol 12-myristate 13-acetate (PMA) was found to enhance greatly the ability of these spleen cells to lyse MOPC-315 tumor cells. The highest level of anti-MOPC-315 cytotoxicity was obtained when spleen cells from tumor-bearing mice that had received chemotherapy 7 days earlier were exposed to PMA at a concentration of 1–10 ng/ml. The exertion of the enhanced anti-MOPC-315 lytic activity byl-PAM TuB spleen cells exposed to PMA was found to require CD8⁺, but not CD4⁺, T cells. The apparent specificity of the lytic activity exerted by the PMA-stimulatedl-PAM TuB spleen cells was illustrated not only by the inability of the spleen cells to lyse an allogeneic, antigenically unrelated thymoma (EL4), but also by their relatively weak lytic activity for two antigenically related syngeneic plasmacytomas. In addition, when EL4 target cells were admixed with MOPC-315 tumor cells, the lytic activity triggered in thel-PAM TuB spleen cells by the MOPC-315 tumor cells plus PMA was not effective in lysing the antigenically unrelated target cells. Moreover, even in the presence of the calcium-specific ionophore, ionomycin,l-PAM TuB spleen cells exposed to PMA were unable to lyse the EL4 target cells. Thus, fresh CD8⁺ splenic T cells froml-PAM TuB mice that are in the process of eradicating a large MOPC-315 tumor as a consequence of low-dosel-PAM therapy can be triggered with PMA to exert enhanced lytic activity against MOPC-315 tumor cells. Since the curative effectiveness of low-dose chemotherapy for MOPC-315 tumor-bearing mice requires the participation of CD8⁺ T cells that exploit a cytotoxic T lymphocyte type lytic activity for tumor eradication, it is feasible that in some situations PMA-like stimulants could be used to augment the antitumor cytotoxic activity of the CD8⁺ T cells, which in turn could improve the therapeutic outcome of low-dose chemotherapy.Supported by research grant IM-435A from the American Cancer Society and research grant B-8806 from the Bane EstateIn partial fulfillment of the requirements for the Doctor of Philosophy DegreeRecipient of Career Development Award CA-01 350 from the National Cancer Institute     

The Spleen Plays No Role in Nephrotoxic Serum Nephritis,but Constitutes a Place of Compensatory Haematopoiesis

Background

The spleen has been implicated in the pathogenesis of immune-complex glomerulonephritis by initiating and resolving adaptive immune responses. Thus, we aimed to evaluate the role of the spleen in experimental nephrotoxic serum nephritis (NTS).

Methods

In order to accelerate the disease, animals were subjected to NTS by preimmunizing male C57BL/6J mice with rabbit IgG three days before injecting the rabbit anti-glomerular basement antiserum, or were immunized only. A group underwent splenectomy before NTS induction.

Results

We observed enlargement of the spleen with a maximum at 14 days after NTS induction or immunization only. Splenectomized mice were found to develop albuminuria and renal histological changes comparable to sham-operated controls. Nevertheless, anaemia was aggravated in mice after splenectomy. During the course of NTS, we detected CD41⁺ megakaryocytes and Ter119⁺ erythroid precursor cells in the spleen of mice with NTS and of immunized mice. Ter119⁺Cxcr4⁺ cells and the binding partner Cxcl12 increased in the spleen, and decreased in the bone marrow. This was accompanied by a significant systemic increase of interferon-gamma in the serum.

Conclusions

In summary, splenectomy does not influence the course of NTS per se, but is involved in concomitant anaemia. Extramedullary haematopoiesis in the spleen is probably facilitated through the migration of Cxcr4⁺ erythroid precursor cells from the bone marrow to the spleen via a Cxcl12 gradient and likely arises from the suppressive capacity of chronic inflammation on the bone marrow.     

Induction of tumor-specific T lymphocyte responses in vivo by prothymosin α

We have recently reported that administration of Pro T to DBA/2 mice before the inoculation of syngeneic L1210 leukemic cells prolonged the survival of these animals by (a) inducing tumoricidal peritoneal macrophages, (b) enhancing natural killer (NK) and inducing lymphokine-activated killer (LAK) activities in splenocytes and (c) inducing the production of interleukin-2 and tumor necrosis factor [Papanastasiou et al. (1992) Cancer Immunol Immunother 35:145; Baxevanis et al. (1994) Cancer Immunol Immunother 38:281]. In this report we demonstrate that Pro T , when administered simultaneously with L1210 tumor cells, is capable of generating in DBA/2 animals tumorspecific CD8⁺ cytotoxic T lymphocytes (CTL). The Pro T -induced CD8⁺ CTL lysed their syngeneic L1210 targets in a major histocompatibility complex (MHC)-restricted fashion since monoclonal antibodies (mAb) against the H-2K^d allelic product could inhibit the cytotoxic response. Mice receiving only Pro T developed non-MHC-restricted cytotoxic activity (NK, and LAK activities) whereas those receiving Pro T and L1210 tumor cells developed both MHC-restricted (CTL) and non-MHC-restricted cytotoxic activities and survived longer. The Pro T -induced CD8⁺ CTL activity was regulated by Pro T -induced L1210-specific syngeneic CD4⁺ cells. This was shown in two different ways: first, CD8⁺-cell-mediated cytotoxic responses against L1210 targets were associated with L1210-specific and MHC-restricted proliferative responses of syngeneic CD4⁺ cells and, second, CD4⁺ cells from mice that had received both Pro T and L1210 tumor cells could enhance in vitro the otherwise weak, MHC-restricted and L1210-specific cytotoxicity of syngeneic CD8⁺ cells from mice that had received only L1210 cells. Our data suggest that Pro T is capable of inducing nonspecific, as well as tumor-specific CTL responses in vivo. This is of importance since Pro T may prove to be useful in clinical protocols aimed at cancer immunotherapy.This work was supported by a CEC grant to Dr. M. Papamichail