T lymphocyte-mediated suppression of myeloma function in vitro. III. Regulation of antibody production in hybrid myeloma cells by T lymphocytes

To investigate the mechanisms by which T lymphocytes regulate myeloma function in vitro, the effects of regulatory T cells on antibody secretion by a hybrid myeloma cell line were examined. Suppressor T cells (Ts) specific for idiotypic determinants on M315 (IgA, lambda 2 anti-2,4-dinitrophenol and anti-2,4,6-trinitrophenol [TNP]) and MPC 11 (IgG2b, kappa) myeloma proteins inhibit antibody secretion by the appropriate parental myeloma cells. When cocultured with a hybrid cell line derived by fusion of MOPC 315 and MPC 11 myelomas, the idiotype-reactive Ts inhibit secretion of only the immunoglobulin (Ig) bearing the relevant idiotype. In contrast, syngeneic TNP-reactive cytolytic T lymphocytes (CTL) inhibit antibody secretion by TNP-binding MOPC 315 cells but not by MPC 11 cells in the presence of soluble TNP-keyhole limpet hemocyanin (KLH), and this inhibition probably represents a prelytic effect of the CTL. Such TNP-reactive CTL, in the presence of TNP-KLH, inhibit both IgA and IgG secretion by the MOPC 315-MPC 11 hybrid, which is consistent with a prelytic effect. Thus, myeloma hybrids are a useful tool for investigating the effector function of regulatory T cells. These results are discussed with reference to the mechanisms of action of regulatory T cells and their relevance to modulation of physiologic humoral immune responses.     

T lymphocyte-mediated suppression of myeloma function in vitro. IV. Generation of effector suppressor cells specific for myeloma idiotypes

BALB/c mice immunized intravenously with syngeneic splenocytes, to which affinity-purified IgA produced by the MOPC 315 myeloma is covalently coupled, develop suppressor T cells (Ts1) that inhibit IgA secretion by MOPC 315 cells after 3-4 d of co-culture. Immunization with M315-coupled splenocytes subcutaneously, followed by administration of a soluble extract of Ts1 cells, leads to the generation of effector Ts that are also idiotype specific and inhibit myeloma function within 1 d. Moreover, effector Ts are Lyt-1-2+, whereas Ts1 are either Lyt-1+2+ or require Lyt-1+ and Lyt-2+ cells to mature into effector Ts in vitro. Such a protocol should be useful for analyzing the interactions that result in the maturation of Ts and in defining the mechanisms of action of Ts, whose effect can be measured on a homogeneous target population and that are specific for a well-characterized myeloma idiotype.     

Separation of antigen-specific lymphocytes. II. Enrichment of hapten- specific antibody-forming cell precursors

Normal spleen cells were separated in dishes coated with thin layers of DNP-gelatin or NIP-gelatin into binding and nonbinding cells and stimulated in vitro with DNP- and/or NIP-conjugated polymerized flagellin (POL). Hapten-specific unresponsiveness was induced in the binding cell population by melting the gel at 37 degrees C or in unfractionated cells by pretreatment with soluble hapten-gelatin and could be reversed by treatment with collagenase. A specific enrichment of anti-DNP and anti-NIP antibody-forming cell precursors (AFCP) could be demonstrated in the binding cell populations after treatment with collagenase in cultures with or without "feeder" cells. However, the response of small numbers of unfractionated and purified hapten-specific spleen cells was suboptimal even in the presence of mitomycin-treated or irradiated feeder cells. Optimal numbers of anti-DNP (anti-NIP) antibody-forming cells were generated by small numbers of normal or purified spleen cells in the presence of spleen cells depleted of anti-DNP (anti-NIP) AFCP. In this system the response of only 2 times 10-4 purified hapten-specific cells was higher than the response of 10-6 unfractionated cells. Purified DNP-specific cells responded only to DNP-POL but not to NIP-POL and purified NIP-specific cells responded only to NIP-POL but not to DNP-POL. The degree of enrichment of anti-DNP AFCP decreased with increasing numbers of binding cells. NIP3-gelatin layers bound four to five times less spleen cells than DNP2-gelatin layers and the enrichment of anti-NIP AFCP (about 300-fold) was three times greater than the enrichment of anti-DNP AFCP (about 100-fold). The immunological significance of hapten-gelatin binding cells which apparently failed to respond to antigen is discussed.     

H-2-restricted cytolytic T lymphocytes specific for HLA display T cell receptors of limited diversity.

We previously showed that H-2Kd-restricted cytotoxic T lymphocyte (CTL) clones specific for a single nonapeptide derived from the Plasmodium berghei circumsporozoite (PbCS) protein displayed T cell receptors (TCRs) of highly diverse primary structure. We have now analyzed the TCR repertoire of CTLs that recognize a peptide derived from the human class I major histocompatibility complex (MHC) molecule HLA-Cw3 in association with the same murine class I MHC molecule H-2Kd. We first sequenced the TCR alpha and beta genes of the CTL clone Cw3/1.1 and, based on this genomic analysis, the TCR alpha and beta cDNA junctional regions of 23 independent H-2Kd-restricted CTL clones specific for HLA-Cw3. The results show that the TCR chains display very limited heterogeneity, both in terms of V alpha, J alpha, V beta, and J beta segments, and in terms of length and sequence of the CDR3 alpha and beta loops. The TCR repertoire used in vivo was then analyzed by harvesting CTL populations from the peritoneal cavity of immune mice. The peritoneal exudate lymphocytes (PELs) displayed HLA-Cw3-specific cytolytic activity in the absence of any stimulation in vitro. Remarkably, most of these freshly isolated PELs expressed TCRs that shared the same structural features as those from HLA-Cw3-reactive CTL clones. Thus, our results show that a peptide from HLA-Cw3 presented by H-2Kd selects CTLs that bear TCRs of very limited diversity in vivo. When taken together with the high diversity of the TCRs specific for the PbCS peptide, these findings suggest that natural tolerance to self peptides presented by class I MHC molecules may substantially reduce the size of the TCR repertoire of CTLs specific for antigenic peptides homologous to self.     

T lymphocytes specific for immunoglobulin allotype. I. Igh- 1(b)-specific T cells demonstrated by suppression in vivo and cytotoxicity in vitro

We show that determinants of IgG(2a) of C57BL/6 mice (Igh-1(b)) stimulate allotypespecific T cells in BALB/c mice. Such cells are detected in two different functional assays; chronic allotype suppression and T cell-mediated cytotoxicity. A population of suppressor T cells capable of inducing chronic Igh-1(b) suppression was demonstrated by rosetting procedures to possess Igh-1(b)-specific receptors, a result interpreted as indicating that suppressor T cells may act directly upon allotype-bearing B cells. From similar populations we were also able to demonstrate Igh-1(b)-specific cytotoxic T cells. Such cells were lytic for target myeloma cells expressing the Igh-1(b) allotype of IgG28, and were ineffective against a variant cell line failing to express Igh-1(b), and other target cell lines expressing different allotypes or isotypes. The similar specificity of suppressor T cells and cytotoxic T lymphocytes for Igh-1(b) allotype raises the possibility that the target in allotype suppression is a B cell, and that allotype-specific cytotoxic T cells may play some role in regulation of allotype expression in the suppressed state.     

Cytotoxic T lymphocytes reactive against a syngeneic murine tumor and their specific suppressor T cells are both elicited by in vitro allosensitization

Sensitization of C57BL/6 (B6, H-2b) splenocytes against normal BALB/c (H-2d) leukocytes (B6 a/BALB) in bulk MLC induced CTL reactive against the syngeneic (H-2b) nonimmunogenic lymphoma PIR-2, in addition to the CTL directed against the corresponding (H-2d) allotargets. However, MLC-derived lymphocytes did not directly exhibit anti-PIR-2 cytotoxicity in spite of the high anti-PIR-2 CTL frequency (up to 1/20) among them, as demonstrated by the limiting dilution culture (LDC) technique. The present study was undertaken to resolve this contradiction. We found that anti-PIR-2 cytotoxicity could be detected only when B6 a/BALB MLC-derived responding cells were plated in LDC at low numbers (less than 200) of cells/well. In contrast, increasing the number of the plated cells to 500-5,000 resulted in a gradual decrease in the percentage of wells cytotoxically reactive against PIR-2, whereas the percentage of wells exhibiting cytotoxicity against the allotargets remained unchanged (100%). This decrease of anti-PIR-2 cytotoxicity in LDC and the lack of anti-PIR-2 reactivity among MLC-derived lymphocytes were shown by mixing experiments to result from the activity of radioresistant Thy-1+, Lyt-2+, L3T4- suppressor cells, blocking the anti-PIR-2 cytotoxicity at the effector phase. The suppression was specific as indicated by the following observations: (a) freshly obtained B6 splenocytes, cultured unsensitized B6 splenocytes, mitogen-induced B6 lymphoblasts, B6 LAK cells, or B6 a/B6 MLC-derived lymphocytes were not suppressive; (b) anti-PIR-2 cytotoxicity elicited in B6 a/BALB LDC was suppressed only by lymphocytes derived from B6 a/BALB MLC and not from B6 a/C3H (H-2k) MLC; and (c) B6 a/BALB MLC-induced suppressor cells could be adsorbed on monolayers of BALB/c but not of C3H lymphoblasts. Since both syngeneic tumor and allogeneic target cells were lysed by the same clonal cell population but only the antisyngeneic activity was suppressed, we suggest that a single CTL can exhibit two cytotoxic activities that are differentially affected by the described suppressor cells. This mode of suppression may play a role in controlling autoimmune reactivity.     

Isolated hapten-binding receptors of sensitized lymphocytes. III. Evidence for idiotypic restriction of T-cell receptors

The primary antibody response of C57BL/6 mice to the 4-hydroxy-3- nitrophenylacetyl (NP) hapten is restricted to antibody molecules expressing the NPb idiotype. This idiotype is a genetic marker for V genes in the heavy chain linkage group. In the secondary response, the frequency of NPb idiotype-positive molecules within the antibody population drops to very low values. Accordingly, isolated NP binding receptors from NP-sensitized B lymphocytes are largely devoid of this idiotype. In contrast, the NPb idiotype is expressed on the majority of the receptor fractions which we consider T-cell derived. This finding suggests that the antigen receptors of T lymphocytes may be restricted to the expression of major (germ-line encoded?) heavy chain idiotypes.     

Evidence for genetic restriction in the suppression of erythropoiesis by a unique subset of T lymphocytes in man.

The suppression of erythropoiesis by lymphocytes from patients with a T cell lymphoproliferative syndrome and pure erythrocyte aplasia has been previously demonstrated. To study the nature of the suppressor cell and possible genetic restriction of this suppression, we investigated a patient with severe anemia, splenomegaly, lymphocytosis, and erythroid aplasia. A 3-mo course of low-dose daily oral cyclophosphamide achieved a complete remission for over 12 mo. The surface phenotype of his lymphocytes was analyzed by means of antibodies to lineage, differentiation, and activation-specific surface antigens. The cells expressed mature T cell antigens T3, T8, and T11, while lacking T1. Immature T cell, B cell, and the monocyte-specific antigen Mo2 were absent, while Mo1, a monocyte-associated antigen not normally expressed on T cells, was present. T10 and Ia expressed as activation antigens were also present. The cells, cryopreserved at diagnosis, were thawed and co-cultured in plasma clot with patient remission marrow samples at T cell/bone marrow ratios of 1:1 and 2:1. There was nearly 90% suppression of erythroid colony-forming unit expression and 60% suppression of erythroid burst-forming unit expression at 2:1 T cell to bone marrow ratios and somewhat less suppression at 1:1. Granulocyte/macrophage progenitor expression was unaffected. Erythroid progenitor differentiation in the marrows of two HLA identical siblings was similarly suppressed. The cells were co-cultured with the marrows of nine nonrelated donors to investigate the potential genetic restriction of this suppression. Colony suppression equal to that observed in the marrow of the patient and his siblings was found in studies of two partially HLA identical individuals. No suppression was detected in marrow co-cultures of two entirely HLA dissimilar individuals. These results show that suppression of erythropoiesis by a unique subset of T8, Mo1, Ia-positive lymphocytes isolated from a patient with lymphocytosis and erythrocyte aplasia is genetically restricted.     

T lymphocytes specific for immunoglobulin allotype. II. Cloned Igh-1(b)- specific cytotoxic T cells

This study describes long-term-cultured lines and clones of cytotoxic T cells (Tc) with specificity for determinants of the Igh-1(b) immunoglobulin allotype. These Tc clones were initiated by repeated stimulation of immune spleen cells from BALB/c mice with an Igh-1(b)-producing myeloma, and then they were maintained in medium supplemented with mitogen-induced growth factors in the absence offurther antigenic stimulation . The lytic potency of these clones was 30-100-fold greater than the primary cultures from which they were derived, and specificity studies showed them to be lytic for Igh-1(b) targets and not for targets expressing Igh-1(a) or Igh-4(b), nor the lipopolysaccharide blasts . Finally, soluble preparations of Ig were tested for their ability to block lysis of labeled Igh-1(b)-expressing targets. The results showed that Igh-1(b) and not other immunoglobulin allotypes or isotypes could block lysis, and that the mechanism of lytic inhibition is due to Igh-1(b)-induced autolysis of the killer cells.     

Mechanisms of mouse spleen dendritic cell function in the generation of influenza-specific, cytolytic T lymphocytes.

We have evaluated the capacity of dendritic cells to function as antigen-presenting cells (APCs) for influenza and have examined their mechanism of action. Virus-pulsed dendritic cells were 100 times more efficient than bulk spleen cells in stimulating cytotoxic T lymphocyte (CTL) formation. The induction of CTLs required neither exogenous lymphokines nor APCs in the responding T cell population. Infectious virus entered dendritic cells through intracellular acidic vacuoles and directed the synthesis of several viral proteins. If ultraviolet (UV)-inactivated or bromelain-treated viruses were used, viral protein synthesis could not be detected, and there was poor induction of CTLs. This indicated that dendritic cells were not capable of processing noninfectious virus onto major histocompatibility complex (MHC) class I molecules. However, UV-inactivated and bromelain-treated viruses were presented efficiently to class II-restricted CD4+ T cells. The CD4+ T cells crossreacted with different strains of influenza and markedly amplified CTL formation. Cell lines that lacked MHC class II, and consequently the capacity to stimulate CD4+ T cells, failed to induce CTLs unless helper lymphokines were added. Similarly, dendritic cells pulsed with the MHC class I-restricted nucleoprotein 147-155 peptide were poor stimulators in the absence of exogenous helper factors. We conclude that the function of dendritic cells as APCs for the generation of virus-specific CTLs in vitro depends measurably upon: (a) charging class I molecules with peptides derived from endogenously synthesized viral antigens, and (b) stimulating a strong CD4+ helper T cell response.     

Induction of specific immune unresponsiveness with purified mixed leukocyte culture-activated T lymphoblasts as autoimmunogen. III. Proof for the existence of autoanti-idiotypic killer T cells and transfer of suppression to normal syngeneic recipients by T or B lymphocytes

Specific immune unresponsiveness against a given set of histocompatibility antigens can be induced by immunization with autologous, antigen-specific T lymphoblasts. Such unresponsiveness can be transferred by lymphoid cells from autoblast-immunized donors to normal syngeneic recipients. The cells being most efficient in transferring the selective suppression are T lymphocytes from the spleen, especially if of Ly 1-2+3+ phenotype. By using such T lymphocytes we deem it likely that the actual underlying mechanism is one of actual transfer of autoanti-idiotypic killer T cells. In support for this view is the fact that such T cells endowed with exquisite specific, cytolytic reactivity towards autologous idiotype-positive T target cells exist in autoblast immune animals. Significant suppression may also be transferred with T cells of Ly 1+2-3- phenotype or with B cells. Here, we consider the suppressive mechanism to be one of production of autoanti-idiotypic antibodies. By using affinity fraction procedures, it was finally possible to prove that all T-cell suppressive activity resides in a population with true antigen-binding-specific receptors for the relevant idiotypes.     

Regulatory functions of hapten-reactive helper and suppressor T lymphocytes. III. Amplification of a generation of tumor-specific killer T-lymphocyte activities by suppressor T-cell-depleted hapten- reactive T lymphocytes

2,4.6-trinitrophenyl (TNP)-reactive T-cell activities were raised in mice by immunization with TNP-isologous mouse gamma globulin. After establishing that TNP-reactive T lymphocytes can serve as amplifier cells for induction of killer T lymphocytes in allogeneic system, we explored the possibility of this hapten-reactive T-cell system to amplify tumor-specific killer T-lymphocyte activity in the syngeneic system. We utlized relatively weak immunogenic syngeneic plasmacytoma X5563 in C3H/He mice. Analysis of the TNP-reactive T-cell activities revealed that such T lymphocytes express the biological functions of both major subtypes of regulatory T cells, namely suppressors and helpers, and that TNP-reactive suppressor and helper T lymphocytes, respectively, differ in their relative susceptibility to specific inactivation by TNP conjugates of the nonimmunogenic D-amino acid copolymer, D-glutamic acid, and D-lysine (D-GL). By taking advantage of the relative susceptibility-difference to TNP-D-GL, selective inactivation of TNP-reactive suppressor T cells was induced by appropriate treatment with TNP-D-GL, and the generation of TNP-reactive helper T-cell activity was amplified. The supplement of augmented TNP-reactive helper T-cell activity to the system at the immunization with syngeneic X5563 with TNP-haptenation, resulted in a striking augmentation of induction of tumor-specific killer T-lymphocyte activity, and a considerable number of hosts survived after the challenge with lethal dose of viable tumor cells. Thus, appropriate manipulations designed to induce potent hapten-reactive helper T-lymphocytes provided the potential for a very effective mode of immunoprophylaxis against tumor.     

Elimination of syngeneic sarcomas in rats by a subset of T lymphocytes

Established subcutaneous Moloney sarcomas (MST-1) of large size and long duration were eliminated from syngeneic rats by intravenous infusion of varying numbers of specific syngeneic effector T lymphocytes. Spleen cells from BN rats in which tumor had regressed were cultured in an in vitro mixed lymphocyte tumor cell culture (MLTC) to augment cytotoxicity of effector cells. In the MLTC a T cell subset was expanded in response to MST-1 antigens and transformed into blast elements. With these changes, there was an increase in the W3/25 antigen on the T cell surface, a decrease of W3/13 antigen, and an increase in the number of T cells with Ia antigens. The subset associated with elimination of established tumors was a blast T cell W3/25+, W3/13+, as detected by monoclonal antibodies to rat T antigens. The W3/25+ subset was poorly cytotoxic in vitro for MST-1 and apparently functioned in vivo as an amplifier or helper cell in the tumor-bearing host. The W3/25- population was a melange of cells that included (W3/13+, W3/25-) T cells, null cells, Ig+ cells, and macrophages, and was associated with enhancement of tumor in vivo, suggesting the presence of suppressor cells.     

Mechanisms of idiotype suppression. I. In vitro generation of idiotype- specific suppressor T cells by anti-idiotype antibodies and specific antigen

Normal BALB/c spleen cells are unresponsive in vitro to the phosphorylcholine (PC) determinant in the presence of anti-idiotype antibodies specific for the TEPC-15 myeloma protein (T15) which carries an idiotypic determinant indistinguishable from that of most anti-PC antibodies in BALB/c mice. The possibility that idiotype-specific suppressor cells may be generated during the culture period was examined by coculturing the cells with untreated syngeneic spleen cells. Cells that had been preincubated with anti-T15 idiotype (anti-T15id) antibodies and a PC-containing antigen, R36a for 3 d, were capable of specifically suppressing the anti-PC response of fresh normal spleen cells, indicating that idiotype-specific suppressor cells were generated during the culture period. The presence of specific antigen also appeared to be necessary because anti-T15id antibodies and a control antigen, DNP-Lys-Ficoll, were not capable of generating such suppressor cells. Suppressor cells were induced only in the population of spleen cells nonadherent to nylon wool and the suppressive activity was abrogated by treatment with anti-Thy 1.2 serum and complement. These results indicate that anti-idiotype antibodies and specific antigen can generate idiotype-specific suppressor T cells in vitro. These in vitro results may reflect in vivo mechanisms of idiotype suppression.     

Mechanisms of anti-tumor action of Corynebacterium parvum. II. Potentiated cytolytic T cell response and its tumor-induced suppression

It was shown that subcutaneous implantation of P815 tumor cells admixed with Corynebacterium parvum resulted in the emergence of a tumor that grew for 9-10 d and then regressed. The onset of tumor aggression was preceded by the substantial generation in the draining lymph node and spleen of T cells capable of specifically lysing P815 target cells in vitro. The finding that the magnitude of this cytolytic response was much greater than the cytolytic response to a control tumor that grew progressively is consistent with the hypothesis that the anti-tumor action of C. parvum is based on its capacity to augment the production of T cells sensitized to tumor-specific transplantation antigens. This adjuvant action of C. parvum was revealed by additional experiments in which irradiated, nonreplicating tumor cells were substituted for living tumor cells in the admixture. The results support the conclusion that the potentiated cytolytic response to subcutaneous injection of an admixture of irradiated tumor cells and C. parvum is responsible for the ability of this admixture to cause the regression of a test tumor growing at a distant site. Finally, it was shown that the failure of the therapeutic admixture to cause the regression of distant test tumors above a certain size was associated with a failure of the admixture to cause a potentiated, anti-tumor cytolytic response. We discussed the possibility that this failure was caused by the presence of a tumor-induced state of immunosuppression.     

Cross-reactive lysis of trinitrophenyl (TNP)-derivatized H-2 incompatible target cells by cytolytic T lymphocytes generated against syngeneic TNP spleen cells

Normal spleen cells, when cultured with irradiated trinitrophenyl (TNP)-derivatized syngeneic spleen cells, develop cytotoxic effectors that lyse most effectiviely a TNP-derivatized target that is H-2 compatible with the effector. However, these effectors also lyse to a lesser extent TNP tumor and TNP spleen targets that are H-2 incompatible. This cross-reactive lysis correlates with the degree of cytolysis seen on the TNP-derivatized syngeneic target; it appears to be medicated by Thy 1.2-bearing cells and is inhibited by antisera to the K and/or D loci of the target cell and not by antisera to non-K or non-D surface antigens. Nonradiolabeled TNP-derivatized lymphoid cells syngeneic to either the stimulator or the target are able to competitively inhibit cross-reactive lysis, while TNP chicken red blood cells are unable to specifically inhibit lysis. These data on cross-reactive lysis of TNP-conjugated targets are most consistent with the altered-self hypothesis.     

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. II. T lymphocyte-mediated cytolysis

Tumor cell variants that were rejected by syngeneic mice (tum-) were obtained from mastocytoma P815 by mutagenesis (as described in the accompanying report (13). A considerable T lymphocyte-mediated lysis was observed upon incubation of these tum- variants with peritoneal exudate cells collected a few days after an intraperitoneal challenge of immune animals. Spleen cells from these animals were cytolytic after stimulation in vitro with the immunizing variant. New antigens, absent from the original P815 tum+ cells, were detected on 15 of the 21 tum- variants that were tested. All these antigens appeared to be different. No new antigen was detected on any of 10 mutagenized P815 clones that had retained their ability to form tumors. We compared the evidence obtained in vivo and in vitro for the presence of specific antigens on five tum- variants. Three variants were shown both in vivo and in vitro to carry an individual antigen. One showed no specificity either in vivo or in vitro. However, for one variant, no specificity was observed in vivo, although cytolysis tests demonstrated the existence of a singular antigenic specificity.     

活化B淋巴细胞诱导健康人外周血HBcAg 短肽特异性细胞毒性T淋巴细胞

目的 探讨负载乙型肝炎病毒核心抗原18-27(HBcAg 18-27)序列肽的活化B淋巴细胞诱导自体外周血单个核细胞(PBMC)产生HBcAg 18-27特异性细胞毒性T淋巴细胞(CTL)的能力.方法 免疫磁珠法分选B淋巴细胞,与CpG寡核苷酸(CpG-ODN)(2 μg/mL)和白细胞介素-4(IL-4)(2 ng/mL)共培养48 h,再加入人工合成的HBcAg 18-27肽(50 μg/mL),继续温育12 h.将负载抗原肽的活化B淋巴细胞与自体PBMC进行混合淋巴细胞培养5 d,采用Pro5TM MHC Pentamers技术检测HBcAg 18-27特异性CTL.结果 免疫磁珠法分选B淋巴细胞纯度为89.60%.荧光显微镜观察到HBcAg 18-27抗原肽进入活化B细胞,流式细胞仪定量检测抗原负载率为41.3%.以此作为HBcAg 18-27特异性抗原递呈细胞(APC)能够从自体PBMC中诱导出HBcAg 18-27特异性CTL.对照组(不加APC)HBcAg 18-27特异性CTL为(0.20±0.10)%,实验组(加APC)为(0.50±0.19)%,2组差异有统计学意义(P<0.01).结论 负载了HBcAg 18-27肽的活化B淋巴细胞能够诱导自体PBMC产生HBcAg 18-27特异性CTL.     

活化B淋巴细胞诱导健康人外周血HBcAg短肽特异性细胞毒性T淋巴细胞

目的探讨负载乙型肝炎病毒核心抗原18-27(HBcAg 18-27)序列肽的活化B淋巴细胞诱导自体外周血单个核细胞(PBMC)产生HBcAg 18-27特异性细胞毒性T淋巴细胞(CTL)的能力。方法免疫磁珠法分选B淋巴细胞,与CpG寡核苷酸(CpG-ODN)(2μg/mL)和白细胞介素-4(IL-4)(2 ng/mL)共培养48 h,再加入人工合成的HBcAg 18-27肽(50μg/mL),继续温育12 h。将负载抗原肽的活化B淋巴细胞与自体PBMC进行混合淋巴细胞培养5 d,采用Pro5TMMHC Pentam ers技术检测HBcAg 18-27特异性CTL。结果免疫磁珠法分选B淋巴细胞纯度为89.60%。荧光显微镜观察到HBcAg 18-27抗原肽进入活化B细胞,流式细胞仪定量检测抗原负载率为41.3%。以此作为HBcAg 18-27特异性抗原递呈细胞(APC)能够从自体PBMC中诱导出HBcAg 18-27特异性CTL。对照组(不加APC)HBcAg 18-27特异性CTL为(0.20±0.10)%,实验组(加APC)为(0.50±0.19)%,2组差异有统计学意义(P<0.01)。结论负载了HBc...     

Antigenic drift as a mechanism for tumor evasion of destruction by cytolytic T lymphocytes

It is established that mutations in viral antigenic epitopes, or antigenic drifts, allow viruses to escape recognition by both Ab's and T lymphocytes. It is unclear, however, whether tumor cells can escape immune recognition via antigenic drift. Here we show that adoptive therapy with both monoclonal and polyclonal transgenic CTLs, specific for a natural tumor antigen, P1A, selects for multiple mutations in the P1A antigenic epitope. These mutations severely diminish T cell recognition of the tumor antigen by a variety of mechanisms, including modulation of MHC:peptide interaction and TCR binding to MHC:peptide complex. These results provide the first evidence for tumor evasion of T cell recognition by antigenic drift, and thus have important implications for the strategy of tumor immunotherapy.