Ontogeny and function of B220+ L3T4+ T-cell subset of MRL/Mp-lpr/lpr mice

T-cell-enriched populations obtained from lymph nodes (LNs) of 4-month-old MRL/Mp-lpr/lpr (MRL-lpr), C3H/HeJ-lpr/lpr (C3H-lpr), and C3H/HeJ-gld/gld (C3H-gld) mice were studied for the expression of B220, L3T4, and Lyt 2 antigens. A new B220+ L3T4+ phenotype was demonstrated in T-cell populations of these mice by two-color flow cytometry with phycoerythrin-conjugated monoclonal antibodies (MoAb) to L3T4 and FITC-anti-B220 MoAb. The generation of the T subset was apparently under the influence of the lpr or gld gene, since it could not be demonstrated in T-cell-enriched populations of MRL/Mp- +/+ and normal C3H mice. The expression level of L3T4 antigen on the T subset was lower than that on B220- L3T4+ cells, while the level of B220 antigen was similar to that of B220+ L3T4- or B220+ Lyt 2- cells. The B220+ L3T4+ phenotype appeared in LNs and spleens, but not in thymuses, of MRL-lpr mice as early as 2 months of age. Its proportion to whole LN T cells at this age was equivalent to that observed in 4-month-old mice. Functional studies on FACS-sorted cell populations revealed that the T subset similar to B220+ L3T4- cells possessed deficiencies in the IL-2-IL-2 receptor system. The appearance of the T subset with an intermediate phenotype and its functional defects suggests that lpr and gld genes in these autoimmune mice exert their influences on the ontogeny and function of L3T4+ T cells and contribute to the induction of early lupus.     

Augmentation of killer activity of murine spleen cells against allogeneic and syngeneic tumor cells by inoculation of alloantigen in vivo

After C57BL/6 (B6) mice were inoculated with BALB/c spleen cells via tail vein, kinetics of cytotoxic activities in the B6 mice against sensitizing alloantigens (H-2d) and against syngeneic antigens were investigated using, as target cells, P815 mastocytoma cells (H-2d) and B16 melanoma cells (H-2b). Cytotoxic activity against P815 in the B6 spleen cells reached a peak 3 days after alloantigen inoculation, decreased drastically on day 5 and rose again thereafter. The profile of anti-B16 cytotoxic activity was similar to that of anti-P815 activity. The cytotoxic activity against P815 was inhibited partially by cold B16, but that against B16 was not inhibited by cold P815. Surface phenotype of cytotoxic cells against P815 was Lyt2+, Thy1+, Asialo GM1+ and that of cytotoxic cells against B16 was Lyt2-, Thy1+/-, and Asialo GM1+. The results indicate that inoculation of B6 mice with allogeneic BALB/c spleen cells induce two types of cytotoxic cells; one is similar to lymphokine-activated killer (LAK) cells and the other is activated natural killer cells.     

Virus-lymphocyte interaction: T cells of the helper subset are infected with lymphocytic choriomeningitis virus during persistent infection in vivo.

The lifelong persistence of lymphocytic choriomeningitis virus (LCMV) in neonatally or congenitally infected mice is accompanied by a suppression of virus-specific T-cell responses. In this study, we identified the subset of T cells infected with LCMV during persistent infection in vivo. Using specific monoclonal antibodies to separate the different lymphocyte cell populations and employing both an infectious center assay and immunofluorescence to detect the virus, we found that infection is confined primarily to T cells of the helper subset (L3T4+ Lyt2-), with minimal involvement of cytotoxic T cells (Lyt2+ L3T4-) and mature B cells. About 0.54 to 1.1% of L3T4+ T cells were producing the virus, as determined by the infectious center assay. In contrast, 9.1 to 12.2% of these L3T4+ T cells contained viral antigen, as shown by immunofluorescence studies. This finding suggested that, at any given time, a substantial number of infected T cells were not producing infectious virus. This infection of T helper cells may be involved in the suppression of LCMV-specific T-cell responses observed in persistently infected mice.     

5'-nucleotidase activity of murine T-cell subpopulations separated according to their Lyt2 and L3T4 phenotypes

Murine T lymphocytes were separated by "panning" into four subpopulations, according to their Lyt2 and L3T4 phenotypes: Lyt2+L3T4+, Lyt2-L3T4-, Lyt2+L3T4-, and Lyt2-L3T4+. The activity of ecto-5'-nucleotidase in each subpopulation was measured. 5'-Nucleotidase activity was undetectable in Lyt2+L3T4+ cortical cells but was expressed in medullary Lyt2-L3T4+ and Lyt2+L3T4- T lymphocytes. The small cortical subpopulation of thymocyte precursors with the Lyt2-L3T4- phenotype expressed levels of 5'-nucleotidase comparable to the levels of medullary, mature lymphocytes. These results suggest that the use of ecto-5'-nucleotidase as a marker of the degree of T-cell maturation is questionable.     

In vivo dynamics of pulmonary lymphoid cell subpopulations generated against pulmonary metastasis: evaluation by bronchoalveolar lavage fluid.

The dynamics of lymphoid cell subpopulations in bronchoalveolar lavage fluid (BALF) and the systemic lymphoid organs of mice after intravenous injection of B16 melanoma cells were examined with a fluorescence-activated cell sorter. The lymphoid cell subpopulations of BALF and mediastinal lymph nodes showed significant changes in numbers and proportions, while those of other lymphoid organs including inguinal lymph nodes, thymus and spleen, showed little change. In week 1, the cells with a Thy-1.2+, Lyt-1+, L3T4-, Lyt-2- phenotype and asialo-Gm1+ cells in BALF significantly increased and L3T4+ cells slightly increased in number. By week 3, the numbers of Lyt-2+ cells in BALF markedly increased in number (by about 90 times) compared with controls. The number of Thy-1.2+ cells in mediastinal lymph nodes also increased significantly by week 3. Mice that had been pretreated with an immunosuppressive dose of cyclophosphamide were also inoculated intravenously with B16 melanoma cells. In these mice, a significantly increased number of pleural tumors developed and the number of Thy-1.2+ cells in BALF was markedly reduced from week 1 to 3. The results indicate that L3T4 and Lyt-2 double negative T-cells and natural killer (NK) cells may be generated and/or mobilized to the lung in an early phase of experimental metastasis of B16 melanoma cells and that at a later stage, when multiple metastases develop, T-cells with a Lyt-2+ phenotype markedly increase, probably as an expression of a host reaction against proliferating metastatic tumor cells.     

Both cloned interleukin 2 and purified interleukin 1 are required for optimal growth of purified L3T4+ and Lyt 2+ lymphocytes initiated by concanavalin A

Concanavalin A (Con A), cloned interleukin 2 (IL-2), purified interleukin 1 (IL-1) or two different crude preparations containing IL-1 activity alone, did not induce proliferation of rigorously accessory cell (AC)-depleted splenic L3T4+ or Lyt 2+ lymphocytes. Con A together with saturating concentrations of cloned IL-2 (100 U/ml) promoted less than 40% of the proliferative responses observed in AC-supplemented L3T4+ and Lyt 2+ T-cell cultures. The three preparations of IL-1 used supported minimal proliferation of Con A-treated purified L3T4+ or Lyt 2+ lymphocytes. However, all these IL-1 preparations promoted significant growth of the T-cell populations if AC (1%) were included in the cultures. Cloned IL-2 combined with purified IL-1 promoted proliferation of Con A-treated L3T4+ and Lyt 2+ lymphocytes achieving approximately 75% of the responses observed in AC-supplemented T-cell cultures. The additive effect of IL-1 was apparent in the presence of saturating concentrations of cloned IL-2. Finally, Con A alone induced a detectable number of both L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors as determined with the anti-mouse IL-2 receptor antibody 7D4 by immunofluorescence and FACS analysis. Purified IL-1 neither induced detectable number of L3T4+ or Lyt 2+ T cells to express IL-2 receptors nor increased the number of Con A-treated T cells bearing IL-2 receptors. We have interpreted these findings to indicate the following: Con A alone is sufficient to induce highly purified L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors. Cloned IL-2 and purified IL-1 are required for optimal growth of L3T4+ and Lyt 2+ lymphocytes and these cytokines together efficiently replace AC in growth of T cells initiated by Con A. IL-1 alone does not replace AC in Con A-induced activation of mouse T cells. IL-1 exerts potentiation on IL-2-driven growth of Con A-treated L3T4+ and Lyt 2+ lymphocytes. The additive activity of IL-1 on growth of normal T cells is not due to increased production of IL-2 in the cultures or induction of normal T cells to expression of IL-2 receptors by IL-1. We propose that IL-1 optimizes the action and/or interaction of IL-2 with its receptors on the T-cell membrane (by, i.e., increasing affinity of the IL-2 receptor for its ligand and/or stabilizing the IL-2 receptor).     

L3T4+ T-cell-independent reactivity of Lyt2+ T cells in vivo

The aim of this study was to analyze in vivo the L3T4+ T-cell-subset-independent reactivity of Lyt2+ T cells toward transplantation alloantigens. To this end, we depleted normal mice of L3T4+ T cells by injection of monoclonal antibodies to the L3T4 antigen. This procedure not only led phenotypically to a disappearance of L3T4+ T cells, but also effectively abolished reactivity toward class II MHC antigens in vitro and in vivo. However, L3T4+ T-cell-depleted mice still reacted to class I MHC alloantigens in vivo: after immunization with class I MHC alloantigens Il-2 receptor-bearing T cells appeared in the draining lymph nodes, and developed antigen-specific cytolytic activity. Moreover, upon in vivo priming the frequencies of class I MHC-specific precursors of Il-2-producing and cytolytic Lyt2+ T lymphocytes increased up to 20-fold. L3T4+ T-cell-depleted mice rejected class I MHC-bearing skin grafts promptly. We conclude that not only in vitro but also in vivo Lyt2+ T cells remain reactive toward class I MHC antigens in the absence of L3T4+ T helper cells.     

Perforin is present only in normal activated Lyt2+ T lymphocytes and not in L3T4+ cells, but the serine protease granzyme A is made by both subsets.

We show that among the subsets of peripheral T lymphocytes (Lyt2+ L3T4- and L3T4+ Lyt2- cells) activated in short-term cultures by stimulation with H-2 incompatible leukocytes 97% of the cytolytic activity and all detectable perforin activity resides in the Lyt2+ cells. But both populations contain approximately equal amounts of a serine protease, granzyme A, the expression of which was previously thought to be restricted to cytolytic T lymphocytes.     

Increased T gamma and T mu cells in BALB/c mice with IgG and IgM plasmacytomas and hybridomas

The results of previous studies in our laboratory have shown that mice bearing plasmacytomas and hybridomas that secrete IgA or IgE are accompanied by increased frequencies of Lyt-1-2+ T lymphocytes bearing Fc receptors (FcR) for IgA (T alpha) or IgE (T epsilon), respectively. The present study was undertaken to examine whether IgG- or IgM-secreting tumors influenced the frequency of T lymphocytes that express FcR for IgG or IgM. We studied mice bearing IgG- and IgM-secreting plasmacytomas and hybridomas. BALB/c mice injected subcutaneously with the IgG-secreting hybridoma HDP1 (gamma 1 kappa, anti-TNP) were sequentially examined for the frequencies and Lyt phenotypes of splenic lymphocytes bearing FcR for IgG (T gamma), IgM (T mu), and IgA (T alpha). A threefold increase in the frequency of T gamma lymphocytes that were Lyt-1-2+, L3T4- was seen. The frequencies of T mu and T alpha lymphocytes in these mice were not significantly altered. Similarly, mice injected subcutaneously with the IgM-secreting plasmacytoma MOPC 104E (mu lambda, anti-dextran) or the IgM-secreting hybridoma C1D1 (mu kappa, anti-ox RBC) were examined sequentially for the frequencies of T gamma, T mu, and T alpha lymphocytes. Mice with established IgM subcutaneous tumors showed a twofold increase in splenic, nylon wool-nonadherent T mu lymphocytes. This was associated with a relative increase in Lyt-2+ splenic T lymphocytes and a relative decrease in Lyt-1+ splenic T lymphocytes. No changes were observed in the frequencies of either T gamma or T alpha lymphocytes. These studies extend to IgG and IgM the observation that plasmacytomas and hybridomas secreting immunoglobulins of a specific isotype cause an expansion of T lymphocytes bearing FcR specific for the corresponding isotype. The expansion of FcR+ Lyt-1-2+ T lymphocytes likely represents an exaggerated, but otherwise normal, immunoregulatory response of the host. These cells may be an important element in the regulation of isotype expression.     

Expression of T cell receptors by thymocytes: in situ staining and biochemical analysis.

We have examined the in situ expression of T cell receptor (TCR) V beta 8 protein in murine thymus during ontogeny using the monoclonal antibody F23.1. Positive cells were first detected at day 15 of gestation (0.6%). By day 16 the frequency of positive cells increased dramatically (4.18%). From day 16 to day 17 positive cells doubled (8.17%). The first clusters of F23.1 positive cells were seen at day 17. In the cortex, positive cells decreased from 14% in the newborn mice to 9.8% in 8-week-old mice, whereas in the medulla the frequency remained unchanged at 20%. The antibody F23.1, as well as an antiserum raised against the constant region of the beta chain, immunoprecipitated receptor dimers from highly purified Lyt2+, L3T4+ thymocytes and from two thymic lymphomas of cortical phenotype which express full size alpha and beta mRNA. The receptor dimer could not be precipitated from Lyt2-, L3T4- thymocytes. The results are discussed with regard to intrathymic T cell repertoire selection.     

Ly1+ PRO-B lymphocyte clones. Phenotype, growth requirements and differentiation in vitro and in vivo.

Several clones obtained from the bone marrow of a BALB/c mouse were found to contain the heavy and light chain Ig genes in the germline configuration, to express Ly1 and to carry the B cell lineage markers B-220, Lyb8 and BP-1; these clones are Pgp-1+, LFA-1+, J11d+, Mac-1+ and Thy1-, Lyt2-, L3T4-, GM1.2- and Ia-. Three clones analyzed in detail (Lyd9, LyH7 and Lyb9) have receptors for interleukin (IL) 2 and IL3 as assessed with the 7D4 and CC11 monoclonal antibodies respectively. They grow in rIL3 but not in rIL2 or rIL1; both rIL4 and rIL5 also promote their proliferation, albeit to a much lesser extent than rIL3. None of the interleukins tested alone or in various combinations promoted the clones to differentiate in vitro along the B cell pathway. Treatment with 5-Azacytidine (5-Aza) induced cell surface Ia expression but not rearrangement or expression of Ig genes. However, 5-Aza-treated Lyd9, LyH7 and Lyb9 cells co-cultured with X-ray irradiated accessory cells and LPS gave rise to Ly1+, IgM+ B lymphocytes (range 14-51%) including mu + kappa + (78-93%), and mu + lambda + (9-25%) B lymphocytes. In vivo, the Lyd9, LyH7 and Lyb9 clones gave rise to IgM+ B lymphocytes (8.5-17%) including mu + kappa +, and mu + lambda +, but not to Lyt2+ or L3T4+ T lymphocytes after 4-6 weeks of transfer into Scid mice. Our results indicate that Ly1+ IgM+ cells comprise a subpopulation of B lymphocytes that is derived from IL3-responsive Ly1+ PRO-B lymphocytes.     

64Cu-labeled alpha-melanocyte-stimulating hormone analog for microPET imaging of melanocortin 1 receptor expression

The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor (melanocortin type 1 receptor, or MC1R) plays an important role in the development and growth of melanoma cells. It was found that MC1R was overexpressed on most murine and human melanoma, making it a promising molecular target for melanoma imaging and therapy. Radiolabeled alpha-MSH peptide and its analogs that can specifically bind with MC1R have been extensively explored for developing novel agents for melanoma detection and radionuclide therapy. The goal of this study was to evaluate a 64Cu-labeled alpha-MSH analog, Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys(DOTA)-NH2 (DOTA-NAPamide), as a potential molecular probe for microPET imaging of melanoma and MC1R expression in melanoma xenografted mouse models. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated NAPamide was synthesized and radiolabeled with 64Cu (t1/2=12 h) in NH4OAc (0.1 M; pH 5.5) buffered solution for 60 min at 50 degrees C. Cell culture studies reveal rapid and high uptake and internalization of 64Cu-DOTA-NAPamide in B16F10 cells. Over 90% of receptor-bound tracer is internalized at 3 h incubation. A cellular retention study demonstrates that the receptor-bound 64Cu-DOTA-NAPamide is slowly released from the B16F10 cells into the medium; 66% of the radioactivity is still associated with the cells even after 3 h incubation. The biodistribution of 64Cu-DOTA-NAPamide was then investigated in C57BL/6 mice bearing subcutaneous murine B16F10 melanoma tumors with high capacity of MC1R and Fox Chase Scid mice bearing human A375M melanoma with a relatively low number of MC1R receptors. Tumor uptake values of 64Cu-DOTA-NAPamide are found to be 4.63 +/- 0.45% and 2.49 +/- 0.31% ID/g in B16F10 and A375M xenografted melanoma at 2 h postinjection (pi), respectively. The B16F10 tumor uptake at 2 h pi is further inhibited to 2.29 +/- 0.24% ID/g, while A375M tumor uptake at 2 h pi remains 2.20 +/- 0.41% ID/g with a coinjection of excess alpha-MSH peptide. MicroPET imaging of 64Cu-DOTA-NAPamide in B16F10 tumor mice clearly shows good tumor localization. However, low A375M tumor uptake and poor tumor to normal tissue contrast were observed. This study demonstrates that 64Cu-DOTA-NAPamide is a promising molecular probe for alpha-MSH receptor positive melanoma PET imaging as well as MC1R expression imaging in living mice.     

Anti-metastatic effect by in vivo administration of concanavalin A through augmentation of T-derived activated killer activity: efficacy to B16 melanoma expressed MHC antigen

We attempted to investigate if the in vivo administration of concanavalin A (Con A), a potent T cell stimulator, would render anti-metastatic activity in hosts. Assays of activity were performed 20 days after iv inoculation of two clones of the B16 melanoma, B16-H (H-2+, highly metastatic), B16-L (H-2-, low metastatic), or 3LL cells into C57BL mice by enumerating lung colonies. In some experiments, hosts treated with anti-asialo GM1 Ab were used to evaluate effector mechanisms other than NK cells. While the injection of Con A alone had no significant effect on anti-metastatic activity, in nonimmunized hosts the effect by Con A was displayed when the mice were preimmunized with B16-H cells but not in those immunized with B16-L cells. Immunization with B16-H or B16-L cells alone resulted in the generation of killer cells with promiscuous lytic activity and induced an anti-metastatic effect against B16-H, B16-L, and 3LL cells. Con A treatment significantly augmented the killer activity of spleen cells of mice preimmunized with B16-H cells but not of those immunized with B16-L cells. The effectors from mice immunized with B16-H alone or given both Con A and B16-H were mainly of Thy 1+ Lyt2+ asialo GM1- cells, on the other hand, those from mice immunized with B16-L cells expressed asialo GM1 antigen. We showed the efficacy of Con A on the anti-metastatic effect in relation to the host immune response.     

Cytotoxic T lymphocytes generated by short-term in vitro TCR stimulation in the presence of IL-4 are therapeutically effective against B16 melanoma

P14 TCR transgenic CD8+ T cells (LCMV gp33-specific) were activated by antigen in the presence of either IL-2 or IL-2+IL-4 to generate effector cytotoxic T lymphocytes (CTLs). The therapeutic effectiveness of such IL-2- or IL-2+IL-4-grown CTLs was tested in mice that had received intravenous inoculations of B16.gp33 melanoma cells 7 days previously. Administration of P14 CTLs activated by antigen +IL-2+IL-4 was significantly more effective at reducing melanoma colony formation in the lung than those grown in the presence of antigen +IL-2. Highly significant improvement in survival was observed with 80% of B16.gp33-inoculated mice showing long-term survival after therapy with 10×10⁶ antigen +IL-2+IL-4-activated P14 CTLs. Similar therapeutic effectiveness of antigen +IL-2+IL-4-activated P14 CTLs against subcutaneously inoculated B16.gp33 melanoma cells was also found. There was significant reduction in P14 CD8+ T cells in the peripheral blood of B16.gp33-inoculated mice than in mice that did not receive B16.gp33 melanoma cells, indicating possible homing of P14 CD8+ T cells to the site of tumor growth or antigen-induced apoptotic cell death. These results may have implications in tumor therapy using CTLs grown ex vivo, especially during early stages of tumor formation. They also support the concept that the therapeutic effectiveness of CTLs can be governed by the cytokine context in which they are activated.     

Analysis of effector T cells against the murine syngeneic tumor MethA in mice orally administered antitumor polysaccharide SPR-901

The growth of MethA tumor was significantly inhibited by oral administration of the -glucan SPR-901 in BALB/c (+/+) mice but not in nude mice. Mice treated orally with SPR-901 exhibited an augmentation of antigen-specific resistance against rechallenge with the tumor cells. The tumor-neutralizing activity of regional lymph node cells from MethA-bearing mice against the tumor was augmented by oral administration of SPR-901. The tumor-neutralizing activity of lymph node cells from SPR-901-treated mice mainly appeared in Lyt2+cells. Furthermore, lymphokine-activated killer activity of these cells was enhanced by administration of SPR-901. The antitumor effect of SPR-901 was abrogated in mice depleted of either L3T4+ or Lyt2+ cells, and in cyclosporin-A-treated mice. These results suggest that Lyt2+ cells are important effector cells in MethA-bearing mice orally adminstered SPR-901 and that functional exertion of both Lyt2+ and L3T4+T cells is necessary for the antitumor effect of orally administered SPR-901 in vivo.     

Early development of CD8-negative and CD8-positive MHC-unrestricted cytotoxic cells induced by alloantigen inoculation in mice

Peritoneal cells (PC) in C57B1/6 (B6, H-2b) mice receiving an intraperitoneal (i.p.) injection of allogeneic BALB/c (H-2d) spleen cells demonstrated potent cytotoxic activity against syngeneic, xenogeneic, third-party allogeneic tumors as well as H-2d derived tumors. Maximum cytotoxic activity against various tumors other than H-2d derived tumor, B16 (H-2b) was elicited on day 3 post allosensitization and decreased drastically thereafter, whereas cytotoxic activity against P815 (H-2d) peaked 3 days after the inoculation and maintained the peak activity thereafter. Surface phenotype of PC responsible for the cytotoxic activity against B16 was Thy-1+/-, Lyt-2-, L3T4-, asialo GM1 (AGM1)+, and that of PC against P815 was Thy-1+, Lyt-2+ (or Lyt-2+/-), L3T4-, AGM1+. These phenotypes showed similar phenotypes to the counterparts against B16 and against P815 in spleen cells induced by intravenous inoculation of alloantigen. When mice were pretreated i.p. with anti-AGM1 antibody before the allosensitization, anti-P815 cytotoxic-activity in PC was completely diminished. Similar activity in spleen, however, was enhanced by i.v. treatment with the antibody before the i.v. inoculation of alloantigen. The data clearly demonstrate that in vivo inoculation of B6 mice with normal allogeneic cells induces "NK-like" CD8- cytotoxic cells and "anomalous" CD8+ cytotoxic cells in PC.     

Intrathymic T cell repertoire after prenatal trinitrobenzene-sulfonic acid-treatment

It is still a matter of debate, whether tolerance toward self-non-MHC antigens is due to intrathymic deletion or to regulatory processes in the periphery. To further pursue this question, responsiveness toward TNP and an anti-TNP monoclonal antibody (Sp6) carrying a recurrent idiotype was evaluated in prenatally trinitrobenzenesulfonic acid (TNBS)-treated mice. In prenatally untreated as well as in TNBS-treated mice, thymocytes proliferating in the absence of nominal antigen were double negative (L3T4-/Lyt2-), but antigen-specific thymocytes were single positive (L3T4+/Lyt2- or L3T4-/Lyt2+). TNBS-treated mice differed from controls inasmuch as in their first week of life T cells proliferating in response to TNP were found in the thymus and detected at increased frequencies in the spleen. The frequency of TNP-specific thymocytes and spleen cells declined rapidly, finally reaching in the spleen a level of 20-30% of controls. Furthermore, after antigenic stimulation, the frequency of thymocytes and spleen cells proliferating in response to TNP was found to be increased in control mice, but TNP-specific T cell were no more recovered in the thymus or the spleen of tolerized mice. The same accounted for thymic and splenic T cells proliferating in response to Sp6. They were expanded in control mice after antigenic stimulation, but were undetectable in TNBS-treated mice. Thus, T cells with specificity for an internal (Sp6) and an external (TNP) antigen, provided the latter was present during ontogeny, were detected in the thymus of control and, transiently, in the thymus of tolerized mice. But, the fate of antigen-specific thymocytes was different in prenatally untreated and TNBS-treated mice. The data are interpreted in the sense that tolerance toward non-MHC antigens may be acquired subsequently to tolerance toward self-MHC antigens and possibly after imprinting of antigen specificity.     

敲低肿瘤细胞来源的颗粒体蛋白前体抑制C57BL/6J小鼠黑色素移植瘤的生长

颗粒体蛋白前体 (progranulin, PGRN)在多种肿瘤中过表达。但PGRN在黑色素瘤发生发展中的作用尚无报道。为探究PGRN在黑色素肿瘤中的作用,本研究采用CRISPR-Cas9基因编辑技术建立了稳定敲低PGRN的小鼠黑色素瘤B16细胞株B16-PGRNlow。MTS法和BrdU掺入结合流式细胞（计量）术分析证明,敲低PGRN不影响B16细胞的细胞周期和增殖。将B16-ctrl（对照）和B16-PGRNlow细胞分别皮下接种野生型（WT）和PGRN敲除（KO）的C57BL/6J小鼠,比较观察黑色素移植瘤体积大小。移植瘤形成20 d后,与B16-ctrl细胞接种的移植瘤比较,无论在WT还是在KO荷瘤小鼠,B16-PGRNlow形成的移植瘤体积明显减小（WT鼠：P<0.05;KO鼠：P<0.01）。然而,比较B16-PGRNlow或B16-ctrl在WT鼠与KO鼠形成的移植瘤体积大小,并无显著差异,提示B16肿瘤细胞PGRN而非宿主PGRN影响移植瘤的生长。流式细胞术分析显示,在荷B16-PGRNlow移植瘤的WT型小鼠脾和淋巴结中,CD4+、CD8+T细胞数（百分比）比荷B16-ctrl移植瘤的WT鼠脾和淋巴结的CD4+、CD8+T细胞数明显增多（P<0.05,P<0.01）,而在KO鼠却未见明显差异。上述结果证明,敲低肿瘤细胞PGRN可抑制黑色素移植瘤的生长。上述结果还提示,抑制PGRN在黑色瘤的表达可引起脾和淋巴结CD4+和CD8+T细胞增加,提高宿主的细胞免疫能力。其机制尚待进一步研究。本文的发现为PGRN作为黑色素瘤治疗的潜在靶点提供了新证据。     

Involvement of L3T4+, LYT2.2+ T cell subsets and non-T cells in the resistance of mice against Trypanosoma cruzi infection

Cellular populations involved in resistance against T. cruzi infection were characterized from mice chronically infected with the parasite. Mice transfused with spleen cells (SC), nylon-wool-non-adherent spleen cells (NWNA) or sera from mice chronically infected with T. cruzi, showed an enhanced resistance against challenge with the parasite. The protective activity of NWNA but not of SC was completely abrogated by treatment with anti-Thy1.2 monoclonal antibodies (mAb) and complement (C). Pretreatment of NWNA cells from chronically infected mice with either anti-L3T4 or anti-Lyt 2.2 mAb partially reduced the transfer of resistance. When both L3T4+ and Lyt2.2+ cells were depleted from NWNA populations, transfer of resistance was abolished. These results appear to indicate that L3T4+, Lyt2.2+ T cell subsets and non-T cells are involved in the immunity to T. cruzi.     

Skin tumor responsiveness to interleukin-2 treatment and CD8 Foxp3+ T cell expansion in an immunocompetent mouse model

Recombinant human interleukin-2 (rhIL-2) therapy is approved for treating patients with advanced melanoma yet significant responses are observed in only 10–15% of patients. Interleukin-2 induces Foxp3 expression in activated human CD8 T cells in vitro and expands circulating CD8 Foxp3+ T cells in melanoma patients. Employing IL-2 responsive (B16-F1, B16-BL6, JB/MS, MCA-205) and nonresponsive (JB/RH, B16-F10) subcutaneous tumor mouse models, we evaluated CD8 Foxp3+ T cell distribution and changes in response to rhIL-2 (50,000 U, i.p. or s.q., twice daily for 5 days). In tumor-free mice and subcutaneous tumor-bearing mouse models, CD8 Foxp3+ T cells were a rare but naturally occurring cell subset. Primarily located in skin-draining lymph nodes, CD8 Foxp3+ T cells expressed both activated T cell (CD28⁺, CD44⁺) and Treg (CTLA4⁺, PD1^lo/var, NKG2A^+/var) markers. Following treatment with rhIL-2, a dramatic increase in CD8 Foxp3+ T cell prevalence was observed in the circulation and tumor-draining lymph nodes (TD.LNs) of animals bearing IL-2 nonresponsive tumors, while no significant changes were observed in the circulation and TD.LNs of animals bearing IL-2 responsive tumors. These findings suggest expansion of CD8 Foxp3+ T cell population in response to rhIL-2 treatment may serve as an early marker for tumor responsiveness to immunotherapy in an immune competent model. Additionally, these data may provide insight to predict response in patients with melanoma undergoing rhIL-2 treatment.