AKR Cells Research Articles

Vimentin, inversely correlating with infiltration of CD8 + T lymphocytes, promotes nuclear translocation of PD-L1 in esophageal squamous cell carcinoma

Vimentin has been considered a canonical marker of epithelial-mesenchymal transition (EMT) and is associated with tumor escape characterized by aberrant PD-L1 expression. However, whether there is a relationship between vimentin and PD-L1 in esophageal squamous cell carcinoma (ESCC) remains poorly understood. The immunological involvement of vimentin in ESCC was first analyzed by multiplex immunofluorescence staining in ESCC tissue microarray followed by a xenografted mouse model. In vivo, C57BL/6 mice were subcutaneously transplanted with AKR cells after stable silencing of vimentin. In vivo results showed that in addition to PD-L1 and PD-L2 expression, vimentin expression was inversely correlated with CD8+ T-cell infiltration. Mechanistically, vimentin can directly interact with PD-L1 and promote nuclear translocation of PD-L1 in AKR cells. In addition, SEMA6C, STC-2 and TRAILR2 were identified as cytokines modulated by vimentin. Blockade of STC-2 and TRAILR2 in co-culture with their own primary antibodies was shown to recruit more CD8+ T cells than controls. Together, these data strongly suggest targeting Vimenin to overcome the immune cycle in ESCC.

Abstract 171: Lysosomal Response Tocholesterol Loading is Altered in DBA/2 Mouse Foam Cells Which May Explain Impairedautolysosome Formation and Lipid Droplets Clearance

In a previous study, we identified autolysosome formation as the limiting step for turnover of cholesterol esters in lipid droplets of macrophage foam cells from the athero-sensitive DBA/2 strain compared to the athero-resistant AKR mouse strain. As autophagosome formation was similar in these two strains, we hypothesized that the lysosomal response to acetylated LDL (AcLDL) loading may be defective in DBA/2 vs. AKR foam cells. For all our studies, we cultured AKR and DBA/2 macrophages with or without AcLDL for 24h. AcLDL loaded DBA/2 vs. AKR cells exhibited a 40 to 50% decrease in lysosome number as measured by staining cells with Lysotracker or an anti-Lamp1 antibody, respectively. Lysosomal degradation capacity was assayed by cell incubation with Alexa647-DQ-ovalbumin, and we observed that AcLDL loading led to a 15% decrease in lysosomal capacity in DBA/2 foam cells (p<0.001) while it had no effect in AKR cells. Also, unloaded and loaded AKR cells had higher degradation capacity than DBA/2 cells (31% and 43%, respectively, p<0.001) which couldn’t be explained by a change in lysosomal pH. As the transcription factor TFEB is a key regulator for lysosome biogenesis and function, we analyzed TFEB protein expression by western blot. Upon loading, TFEB was increased in AKR (48%) but not DBA/2 cells leading to a 45% higher TFEB level in AKR vs. DBA/2 foam cells (p<0.05). AKR and DBA/2 macrophages were labeled with a TFEB antibody and the nucleus to cytoplasm fluorescence intensity ratio was assessed. AcLDL loading led to a 37% increase in TFEB nuclear localization in AKR foam cells vs. unloaded cells (p<0.001) with no effect in DBA/2 macrophages. These results were confirmed by western blot after cellular fractionation. In conclusion, we found that DBA/2 vs. AKR foam cells have altered TFEB processing that may explain decreased lysosome number and function and impaired autolysosome formation in DBA/2 cells.

Abstract 536: Lysosome Insufficiency in Atherosclerosis Prone DBA/2 Mouse Macrophages Associated With Impaired Autolysosome Formation and Lipid Drop Clearance

In a previous study, we identified autolysosome formation as the limiting step for turnover of cholesterol esters in lipid droplets of macrophage foam cells from the atherosclerosis sensitive DBA/2 strain compared to the atherosclerosis resistant AKR mouse strain. As autophagosome formation was similar in these two strains, we wanted to evaluate the role of lysosome biogenesis and function on autolysosome formation in AKR and DBA/2 cells. The transcription factor TFEB is a key regulator for lysosome biogenesis and function that positively regulates the expression of lysosomal enzymes and structural proteins, and controls lysosomes number. For all our studies, we cultured AKR and DBA/2 macrophages with or without acetylated LDL (AcLDL) for 24h. First, we analyzed TFEB protein expression by western blot. Upon loading, TFEB was increased in AKR (48%, p<0.01) but not DBA/2 cells leading to a 45% higher TFEB level in AKR vs. DBA/2 foam cells (p<0.05), suggesting that lysosome number and function may be impaired in DBA/2 foam cells. To assess lysosome function and number, cells were labeled with Lysotracker red DND-99 (LyT) and analyzed by flow cytometry. We found that AcLDL loading did not affect LyT intensity. However, in both unloaded and loaded conditions, DBA/2 cells exhibited a 30 to 50% lower LyT intensity suggesting that they have intrinsically decreased lysosome number/function. Lysosomal degradation capacity was assayed by incubation with DQ-ovalbumin and we observed a 27% decrease in lysosome function in DBA/2 vs. AKR foam cells (p<0.01). In addition, upon loading, the mature form of cathepsin L was increased in AKR (43%, p<0.05) but not DBA/2 cells. Together these data suggest an impairment of lysosomal degradation capacity in DBA/2 foam cells. Finally, we investigated the role of TPC2, a lysosomal membrane protein which over expression has been previously linked to a defect in autolysosome formation. We found that upon AcLDL loading TPC2 protein levels were increased by 35% in DBA/2 cells, which are defective in autolysosome formation, while they were unchanged in AKR cells. In conclusion, we found that DBA/2 vs. AKR foam cells express more TPC2 and have fewer and/or dysfunctional lysosomes that may explain the autolysosome formation defect in these cells.

Transcriptome Analysis of Genes Regulated by Cholesterol Loading in Two Strains of Mouse Macrophages Associates Lysosome Pathway and ER Stress Response with Atherosclerosis Susceptibility

Cholesterol loaded macrophages in the arterial intima are the earliest histological evidence of atherosclerosis. Studies of mouse models of atherosclerosis have shown that the strain background can have a significant effect on lesion development. We have previously shown that DBA/2 ApoE−/− mice have aortic root lesions 10-fold larger than AKR ApoE−/−mice. The current study analyzes the response to cholesterol loading of macrophages from these two strains. Macrophages from the atherosclerosis susceptible DBA/2 strain had significantly higher levels of total and esterified cholesterol compared to atherosclerosis resistant AKR macrophages, while free cholesterol levels were higher in AKR cells. Gene expression profiles were obtained and data were analyzed for strain, cholesterol loading, and strain-cholesterol loading interaction effects by a fitted linear model. Pathway and transcriptional motif enrichment were identified by gene set enrichment analysis. In addition to observed strain differences in basal gene expression, we identified many transcripts whose expression was significantly altered in response to cholesterol loading, including P2ry13 and P2ry14, Trib3, Hyal1, Vegfa, Ccr5, Ly6a, and Ifit3. Eight pathways were significantly enriched in transcripts regulated by cholesterol loading, among which the lysosome and cytokine-cytokine receptor interaction pathways had the highest number of significantly regulated transcripts. Of the differentially regulated transcripts with a strain-cholesterol loading interaction effect, we identified three genes known to participate in the endoplasmic reticulum (ER) stress response, Ddit3, Trib3 and Atf4. These three transcripts were highly up-regulated by cholesterol in AKR and either down-regulated or unchanged in loaded DBA/2 macrophages, thus associating a robust ER stress response with atherosclerosis resistance. We identified significant transcripts with strain, loading, or strain-loading interaction effect that reside within previously described quantitative trait loci as atherosclerosis modifier candidate genes. In conclusion, we characterized several strain and cholesterol induced differences that may lead to new insights into cellular cholesterol metabolism and atherosclerosis.

Physiological Difference in Autophagic Flux in Macrophages From 2 Mouse Strains Regulates Cholesterol Ester Metabolism

DBA/2 apoE(-/-) mice have ≈10-fold larger lesions than AKR apoE(-/-) mice. The objective of this study was to determine whether macrophages from these 2 strains had altered cholesterol metabolism that might play a role in their divergent atherosclerosis susceptibility. AKR and DBA/2 macrophages incubated with acetylated low-density lipoprotein resulted in higher cholesterol ester (CE) and lower free cholesterol accumulation in the DBA/2 cells. However, these strains had equivalent acetylated low-density lipoprotein uptake and cholesterol esterification activity. Cholesterol efflux from unloaded cells to apolipoprotein A-I or high-density lipoprotein was similar in the 2 strains. However, on acetylated low-density lipoprotein loading, cholesterol efflux was impaired in the DBA/2 cells, but this impairment was corrected by loading in the presence of an inhibitor of cholesterol esterification. Thus, the cholesterol efflux capabilities are similar in these strains, but there seemed to be a defect in lipid droplet-stored CE mobilization in DBA/2 cells. Lalistat 1, a specific inhibitor of lysosomal acid lipase, completely blocked the hydrolysis of lipid droplet-stored CE, implying that lipid droplet autophagy is responsible for CE turnover in these cells. CE turnover was 2-fold slower in DBA/2 versus AKR cells. Autophagic flux, estimated by a fluorescent light chain 3-II reporter and the increase in p62 levels after chloroquine treatment, was higher in AKR versus DBA/2 macrophages, which had an apparent decrease in autophagosome fusion with lysosomes. When autophagy was activated by amino acid starvation, CE levels decreased in DBA/2 cells. Physiological regulation of autophagy in macrophages controls CE accumulation and may modify atherosclerosis susceptibility.

Abstract 284: Cholesterol Handling in Bone Marrow--Derived Macrophages from Apoe-Deficient AKR and DBA/2 Mice

It has been demonstrated in humans that about 50% of the susceptibility to atherosclerosis is heritable. Therefore, it is not surprising that mice from distinct genetic backgrounds present different susceptibility to atherosclerosis. For example, apoE -/- DBA/2 mice have aortic root atherosclerosis lesion areas >10-fold larger than apoE -/- AKR mice. In order to better understand the mechanisms underlying this difference, we studied cholesterol metabolism in bone marrow-derived macrophages from these two strains. In the unloaded state, macrophages from both strains had equivalent amounts of total, esterified, and free cholesterol (TC, CE, and FC, respectively). Cells were loaded with acetylated low density lipoproteins (AcLDL) for 48h and DBA/2 macrophages had 36% higher TC (p<0.0001) vs. AKR macrophages, mainly due to 72% higher CE accumulation (p<0.0001). In contrast the loaded DBA/2 macrophages had 6% lower FC than the AKR macrophages (p<0.05). No difference was seen in AcLDL uptake or acetyl-coenzyme A acyltransferase (ACAT) activity between the two strains. When cells were loaded with AcLDL, the DBA/2 cells released cholesterol less efficiently than the AKR cells to apoAI (18% lower, p=0.01) or HDL (25% lower, p=0.001). However when the loading was performed in presence of ACAT inhibitor, blocking the formation of CE, the efflux from the two strains was equivalent suggesting a blockage of CE hydrolysis in DBA/2 cells. In loaded cells, when a 24h chase period was done in presence of ACAT inhibitor to prevent cholesterol re-esterification, the CE was reduce by 69% in AKR cells (t 1/2 = 47h) compared to chase without inhibitor while it was only reduced by 42% in DBA/2 cells (t 1/2 = 101h). Furthermore, when lalistat, an inhibitor of lysosomal acid lipase (LAL), and ACAT inhibitor were added during the chase, CE levels were equivalent to the one observed in chase without any inhibitors, suggesting that CE hydrolysis occurs primarily in the lysosome. In conclusion, our data support the hypothesis that DBA/2 cells accumulate more CE than AKR cells due to a defect in CE hydrolysis by LAL. Autophagy has been recently described as the pathway in macrophages by which lipid droplets can be delivered to the lysosome, and we are investigating the role of this pathway in our cells.

Disparate immune responses to Mi-HAg occur in vitro and in vivo (141.1)

Abstract Historically, insights into the mechanism of alloresponses to minor histocompatibility antigen (Mi-HAg) disparities have focused on T cell activity and used in vitro assays. It was believed that prior in vivo immunization was required for T cell responses to Mi-HAg to occur in vitro. Here, we investigated the generation of adaptive immune responses to Mi-HiAg using an in vivo model. We found that AKR (H-2k) mice rejected skin grafts from MHC-matched, Mi-HAg-disparate B10.BR (H-2k) mice and generated significantly high levels of anti-donor Ab, suggesting that skin graft rejection was associated with the initiation of strong adaptive humoral responses. As B cell activation requires signaling from activated T cells, in vitro and in vivo MLR assays were performed to examine the immune response to Mi-HAg. AKR cells exhibited no reactivity to Mi-HAg disparate stimulators in in vitro MLR assays. In contrast, T cells from AKR mice exhibited strong reactivity in in vivo MLR assays to Mi-HAg in B10.BR mice. The reason for this disparity in response to Mi-HAg in vitro vs. in vivo MLR is not clear. The in vitro culture environment may lack some factors that were present in vivo and that are critical for generation of T cell immune responses to Mi-HAg. T cells were indeed activated in response to Mi-HAg, as CD44highCD62L-/low effector/memory T cells were generated. 2:02 p.m.

Erbin and the NF2 Tumor Suppressor Merlin Cooperatively Regulate Cell-Type-Specific Activation of PAK2 by TGF-β

Transforming growth factor beta (TGF-beta) family ligands are pleotropic proteins with diverse cell-type-specific effects on growth and differentiation. For example, PAK2 activation is critical for the proliferative/profibrotic action of TGF-beta on mesenchymal cells, and yet it is not responsive to TGF-beta in epithelial cells. We therefore investigated the regulatory constraints that prevent inappropriate PAK2 activation in epithelial cultures. The results show that the epithelial-enriched protein Erbin controls the function of the NF2 tumor suppressor Merlin by determining the output of Merlin's physical interactions with active PAK2. Whereas mesenchymal TGF-beta signaling induces PAK2-mediated inhibition of Merlin function in the absence of Erbin, Erbin/Merlin complexes bind and inactivate GTPase-bound PAK2 in epithelia. These results not only identify Erbin as a key determinant of epithelial resistance to TGF-beta signaling, they also show that Erbin controls Merlin tumor suppressor function by switching the functional valence of PAK2 binding.

Significant MLR but not CTL responses against recipient antigens generated in T cells from bone marrow chimeras recovered from acute GVHD.

Lethally irradiated AKR mice received BMT from H-2D and minor lymphocyte stimulatory (Mls)-1 disparate B10.A mice. No GVHD signs were detected in AKR recipients of T cell-depleted BM cells (1 x 10(7)) alone ([B10.A --> AKR] T-). When B10.A splenic T cells (1 x 10(5)) were injected in addition to T cell-depleted BM cells ([B10.A --> AKR] T+), overt GVHD was observed. [B10.A --> AKR] T+ chimeras recovered from the GVHD 8 weeks after BMT. In T cells from these [B10.A --> AKR] T+ chimeras, a substantial population of Mls-1a-reactive Vbeta6+ T cells was present, whereas the Vbeta6+ cells were deleted in [B10.A --> AKR] T- chimeras. T cells from [B10.A --> AKR] T+ chimeras showed considerable MLR but no CTL response against AKR cells (split tolerance). Upon stimulation with AKR stimulators or anti-CD3 MoAb, T cells from [B10.A --> AKR] T+ chimeras produced significantly more IL-4 but significantly less IFN-gamma compared with those from [B10.A --> AKR] T- chimeras or unmanipulated B10.A mice. The serum level of IgG1 in [B10.A --> AKR] T+ chimeras was also significantly higher than that in [B10.A --> AKR] T- or B10.A mice. The present findings suggest that the split tolerance observed in BMT chimeras recovered from GVHD is attributable to the Th2 dominant state.

Angiotensin-(1–7) regulates the levels of angiotensin II receptor subtype AT1 mRNA differentially in a strain-specific fashion

Ang-(1–7) is an effector peptide of the renin–angiotensin system with several distinct actions that are likely mediated by a specific receptor. Regulatory effects of angiotensin (Ang) peptides, Ang-(1–7) and Ang II, on Ang receptor subtype 1 (AT1) mRNA expression were investigated in vascular smooth muscle cells (VSMC) from four University of Akron (Akr) rat strains (WKY, SHR and two backcross consomic lines SHR/y and SHR/a), and in SHR and WKY cells from Charles River Laboratories (Crl). In WKY/Akr and SHR/Akr, Ang-(1–7) treatment increased the levels of AT1 mRNA. This effect was inhibited by the specific Ang-(1–7) antagonist, A-779, in WKY/Akr but not SHR/Akr. Ang II had no effect in Akr cells, but it down-regulated AT1 mRNA in WKY/Crl and SHR/Crl VSMC. Ang-(1–7) did not affect AT1 mRNA levels in Crl lines. In conclusion, Ang-(1–7) regulates the AT1 receptor either directly or indirectly in a strain-specific fashion. The Ang-(1–7) antagonist, A-779, blocks the actions of Ang-(1–7) only in VSMC from WKY/Akr rats, suggesting either that the binding sites for Ang-(1–7) have different properties in SHR/Akr and WKY/Akr cell lines, or that some of the effects of Ang-(1–7) are not receptor mediated. Further, we found differences between Akr cells and Crl cells that are consistent with their genetic heterogeneity.

Clonal deletion and clonal anergy in allogeneic bone marrow chimeras prepared with TBI or TLI.

The evolution of Vbeta6-expressing C3H (H2k, Thy 1.2, Mls a-) lymphocytes was investigated in C3H recipients mice pretreated with total body irradiation (TBI) or total lymphoid irradiation (TLI) and infusion of AKR (H2k, Thy 1.1, Mls a+) cells. After TBI (9.5 Gy) all Vbeta6+ Thy 1.2 (C3H) cells, which are capable of reacting against the Mls a antigen that like expressed by AKR mice, were deleted in the thymus and the periphery in stable bone marrow (BM) chimeras obtained by infusion of 5 x 10(6) T-cell-depleted (TCD) AKR BM cells. When, in the opposite combination, 30 x 10(6) C3H spleen cells were infused into TBI-treated AKR cells, all animals developed graft-versus-host disease (GVHD) with no clonal deletion and in contrast, showed an increase in Vbeta6+ C3H cells. After injection of 30 x 10(6) AKR BM cells into TLI-treated C3H mice no C3H cells were detected in the thymus and only a small percentage in the periphery. Within these C3H cells Vbeta6+ cells were only partially deleted and anergized as they did not respond in vitro after stimulation with Mls a+ AKR cells or anti-Vbeta6 mAb. Cells suppressing anti-Mls a-reacting C3H cells were not found. After injection of 15 x 10(6) AKR cells more C3H cells were found in the thymus, but only a minority of Vbeta6+ cells persisted in the periphery of these animals. In conclusion in TBI-prepared chimeras only clonal deletion occurred, whereas in TLI-prepared chimeras both clonal deletion and anergy occurred in maintaining tolerance.

Split tolerance between spleen and lymph node cells in severe combined immunodeficiency mice grafted with AKR fetal liver cells.

Severe combined immunodeficient (SCID) mice defective in stem cells for T and B cells appear to be an ideal host for construction of chimeric mice. When bone marrow cells are used as a source of stem cells, however, host SCID mice do not always show sufficient reconstitution. In this study, fetal liver cells from AKR embryos were transplanted into SCID mice without prior irradiation. This treatment induced full reconstitution of lymphopoiesis as evaluated by flow cytometry analysis and serum Ig production 2 months after transplantation. Thus, fetal liver cells seem to be a better source for reconstitution of SCID mice than bone marrow cells. Lymph node (LN) cells of these mice (FLT mice) had no proliferative or cytotoxic activities against either host-type (C.B-17) or donor-type (AKR) spleen cells. However, spleen cells from FLT mice exhibited marked proliferative and cytotoxic activities against C.B-17 cells, with no activities against AKR cells. Split tolerance against C.B-17 cells in spleen and LN cells was not a transient phenomenon, since similar results were obtained from a cytotoxic T lymphocyte assay 4 months later. In spite of the strong host reactivity in vitro, aberration of clonal deletion or development of a graft-versus-host disease was not seen in FLT mice. As IL-2 induced the host reactivity of LN cells in a mixed lymphocyte reaction, potentially host-reactive T cells were present in LN but were rendered anergic. Tolerance in FLT mice seems to be regulated by a peripheral mechanism. We supposed that the split tolerance in FLT mice was induced by the different antigenicity between the spleen and LN.

Development of T cells in SCID mice grafted with fetal thymus from AKR mice or F344 rats.

To examine the development of T cells within an allogeneic or xenogeneic environment, we engrafted the fetal thymus from AKR mice or F344 rats under the kidney capsule of SCID mice (mTG and rTG mice). T lymphopoiesis developed in SCID mice 2 months after transplantation, although the ratio of CD4/CD8 in both experimental groups was different from that of normal control. T cells in mTG mice did not show in vitro proliferation or cytotoxicity against either host-type C.B-17 (H-2d) or donor-type AKR (H-2k) cells, while they exerted potent activities against third-party B10 (H-2b) cells. In contrast, T cells in rTG mice exhibited proliferation against both host-type C.B-17 and donor-type F344 rat cells. Consistently, graft-vs.-host disease symptoms developed in these mice and histological examination showed impressive infiltration of lymphocytes into the skin or into the mucosal layers of the stomach. Activated state of T cells in rTG mice was also evidence by the positive expression of interleukin-2 receptor. Taken together, fetal thymus appears to contain progenitor cells which are sufficient for in vivo reconstitution of T lymphopoiesis, but species-specific environment is important for the induction of tolerance. In mTG mice, V beta 6+ T cells reactive to donor Mlsa determinants and V beta 3+ T cells reactive to host Mlsc determinants were deleted, suggesting that tolerance was regulated mainly by clonal deletion. By contrast, V beta 11+ T cells reactive to Mlsf determinants were not deleted possibly due to the lack of their ligands.

Interferon action: cytoplasmic and nuclear localization of the interferon-inducible 52-kD protein that is encoded by the Ifi 200 gene from the gene 200 cluster.

Recently, we reported that an interferon (IFN)-inducible, murine 72-kD phosphoprotein (the 204 protein) that is encoded by the Ifi 204 gene from the gene 200 cluster is localized in the nucleolus and the nucleoplasm. We have now raised a polyclonal antiserum against the 202 protein that is encoded by the Ifi 202 gene from the same gene cluster and regions of which are homologous to those from the 204 protein. Using the antiserum, we established that the 202 protein is a 52-kD phosphoprotein whose level in cells from various murine lines can be increased up to 16-fold upon treatment with IFN-alpha. Experiments involving fractionation of cell lysates and indirect immunofluorescence microscopy of cultured cells revealed that the 202 protein was localized in the cytoplasm and the nucleus. Upon treatment of cells with IFN, the 202 protein first accumulated on the surface of a cytoplasmic, membranous fraction and after prolonged treatment with IFN it was localized mainly in the nucleus. In IFN-treated mitotic AKR cells, the 202 protein was colocalized with chromosomes. 202 protein extracted from IFN-treated AKR cells bound double-stranded DNA in vitro. Studies on 202 protein function should be facilitated by the availability of complete cDNA clones and the finding of cell lines and an inbred strain of mice in which the expression of this protein was impaired.

Characterization of endogenous and recombinant proviral elements of a highly tumorigenic AKR cell line

As an approach to evaluating the contribution of classes of endogenous viral sequences to leukemogenesis, a genomic library was prepared from the highly tumorigenic AKR SL12.3 cell line and screened for env-containing proviruses. An extensive battery of virus-derived probes and specific oligonucleotide probes were used to segregate 83 positive clones into related groups. The nonecotropic endogenous retroviruses were identified as members of the polytropic, modified polytropic, or xenotropic groups. At least three unique xenotropic proviruses were detected that differed from the published xenotropic sequence within a variable region of the 5' portion of env. Changes among the xenotropic proviruses included relative insertions and/or deletions that maintain an open reading frame and hence the potential to encode viable envelope gene products. Several recombinant viruses were also detected. Recombination was not random and primarily involved the formation of mink cell focus-inducing class I retroviruses via recombination between polytropic elements and ecotropic virus. One other recombinant was detected which contained ecotropic virus sequences in the 5' region encoding p15 of an otherwise xenotropic provirus. An interesting observation was the finding that certain clones contained more than one provirus within the average 20-kb cloned insert. This would not be expected if integration were totally random. The de novo recombinant proviruses identified here provide a series of potential candidates to be evaluated for their contribution to the tumorigencity of the SL12.3 cell line.

Specific manipulation in vivo of immunity to skin grafts bearing multiple minor histocompatibility differences

Naive CBA mice injected with AKR spleen cells via the portal vein (p.v.) subsequently showed decreased stimulation in vitro in a primary MLR or cell-mediated lympholysis assay with irradiated AKR stimulator cells. No inhibition of stimulation by B10.BR cells is seen. These mice also show specific prolongation of survival of AKR skin grafts in vivo and diminished capacity for in vivo priming for (secondary) anti-AKR responses in vitro. These effects are not seen if initial challenge is with AKR cells injected subcutaneously (s.c.) or via the lateral tail vein (i.v.). Moreover, if immune CBA anti-AKR mice are similarly challenged with AKR cells via the portal vein, no suppression of anti-AKR immunity is elicited, as determined by subsequent in vitro assays or in vivo graft rejection. However, spleen cells from CBA anti-AKR immune mice can be used to induce, in further naive CBA mice, a specific suppression of subsequent anti-AKR graft reactivity (assayed in vitro or in vivo). Active T cell-mediated suppression can be documented using both these protocols though the additional involvement of specific serum-mediated suppression cannot be eliminated.

The requirement of intrathymic mixed chimerism and clonal deletion for a long‐lasting skin allograft tolerance in cyclophosphamide‐induced tolerance

Mechanisms of cyclophosphamide (CY)-induced tolerance were studied. When C3H/He Slc (C3H; H-2k, Mls-1b) mice were primed i.v. with 1 x 10(8) viable spleen cells from H-2-identical AKR/J Sea (AKR; H-2k, Mls-1a) mice and treated with 200 mg/kg of CY 2 days later, a long-lasting skin allograft tolerance to AKR was established. When [C57BL/6 Sea (B6; H-2b, Mls-1b) x AKR]F1 (B6AKF1) cells were used as the tolerogen, however, only a moderate, but not long-lasting, skin tolerance to AKR was observed. In the C3H mice treated with AKR cells and CY, the intrathymic clonal deletion of V beta 6+ T cells, which are strongly correlated with reactivity to Mls-1a antigens, was observed in the chimeric thymus on day 35, although neither the clonal deletion of V beta 6-bearing T cells nor the mixed chimerism was observed in the thymus on day 14. In the C3H mice treated with B6AFKF1 cells followed by CY, however, neither the clonal deletion of V beta 6+ T cells nor the mixed chimerism was observed in the thymus throughout the test period. In the lymph nodes of the C3H mice treated with AKR cells and CY, only CD4+ V beta 6+ T cells, bur not CD8+V beta 6+ T cells, had selectively decreased by day 14, and they were hardly detectable on day 35. The selective decrease of CD4+V beta 6+ T cells in the lymph nodes was also observed by day 14 when B6AKF1 cells were used as the tolerogen, although CD4+V beta 6+ T cells gradually increased on day 35, at which time almost all skin grafts from AKR had already been rejected. These results strongly support the necessity of the intrathymic mixed chimerism and clonal deletion of donor-reactive T cells for a long-lasting skin allograft tolerance in CY-induced tolerance.

Development and characterization of an Fv-1-sensitive retrovirus-packaging system: single-hit titration kinetics observed in restrictive cells

We have constructed an RNA-packaging-deficient mutant of N-tropic murine leukemia virus WN1802N by removal of 330 nucleotides located between the upstream long terminal repeat and the start of the gag gene region. Transfection into mink CCL64 cells produced a cell line capable of packaging retrovirus vectors into ecotropic, Fv-1 N-tropic virions. Using retrovirus vectors that confer resistance to the antibiotic G418, we demonstrated that the magnitude of restriction in BALB/3T3 and SIM.R cells (both Fv-1b/b) and in RFM/3T3 cells (Fv-1nr/nr) is approximately 100-fold compared with that in AKR or NIH 3T3 cells (both Fv-1n/n). Furthermore, titration kinetics were single hit in restrictive cells. Colonies of antibiotic-resistant cells recovered after infection of genotypically restrictive cultures were phenotypically restrictive when reinfected, ruling out selection of stably nonrestrictive subpopulations. These results suggest that the ability to infect some fraction of cells in a genotypically restrictive culture does not require specific abrogation and that multihit kinetics may not be an essential feature of Fv-1 restriction.

Antibody-mediated routing of diphtheria toxin in murine cells results in a highly efficacious immunotoxin.

The chemical coupling of diphtheria toxin to an antimurine Thy1 antibody resulted in the most efficacious immunotoxin to date. At 1 micrograms/ml the immunotoxin inhibited protein synthesis of a Thy+ AKR murine cell at a rate of 1.4 logs/h, within the order of magnitude of the efficacy of native toxins. This is unusual since murine cells are highly resistant to diphtheria toxin. The conjugate is highly specific; Thy- AKR cells display no intoxication at 1 microgram/ml even after 18 h. The effects of ammonia, acid pulsing of external media, and low temperature reveal some similarities and some differences between intoxication of sensitive cells by toxin and of murine cells by the antibody-toxin conjugate. The differences that result in the high efficacy of the antibody-toxin conjugate appear to result from the antibody-mediated routing. These results imply that murine cells possess an acidic compartment which can mediate toxin cytosolic entry. Unlike the Thy antigen, the toxin receptor on murine cells is unable to route the toxin to this cellular site.

Molecular and cytogenetic studies on nucleolar cistrons (rDNA) in mouse leukemia cells

The gene dosage change of nucleolar cistrons (rDNA) in tumor cells has not been extensively studied. The present studies showed that increased dosage, as well as abnormal distribution of rDNA, was frequently associated with leukemia cells of SL/Ni and AKR mice. In normal SL cells, 37%, 39%, and 25% of rDNA was located in nucleolar organizer regions (NOR) of chromosomes #12, #18, and #19, respectively. Increase of rDNA/DNA was shown by hybridization on filter membranes in SL1, SL2, SL3, and M1 leukemia cells. Direct measurement of rDNA/DNA in G 1 cells revealed an 11% increase in synchronized M1 cells. The increased rDNA dosage was explained by trisomy 12 in SL1 and SL2, the ectopic NOR of #9 in SL3, and the double t(X;19) marker chromosomes in M1. On the other hand, in normal AKR cells, 27%, 29%, and 45% of rDNA was assigned to NORs of chromosomes #15, #16, and #18, respectively. The relative rDNA distribution among NORs estimated by autoradiographic grain counting was suggested to be abnormal in AKR leukemia cells despite their normal karyotype; 36% rDNA was shown to be in chromosomes #15 and #16, respectively, by relative reduction in chromosome #18 in AKR1; the trisomy 15 explained the increased rDNA in AKR2; a relative increase was found in chromosome #15 in AKR3. These results were discussed with reference to the reported NOR involvement in chromosome translocation and amplification in tumor cells.