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.     

Distribution of primed T cells and antigen-loaded antigen presenting cells following intranasal immunization in mice

Priming of T cells is a key event in vaccination, since it bears a decisive influence on the type and magnitude of the immune response. T-cell priming after mucosal immunization via the nasal route was studied by investigating the distribution of antigen-loaded antigen presenting cells (APCs) and primed antigen-specific T cells. Nasal immunization studies were conducted using the model protein antigen ovalbumin (OVA) plus CpG oligodeoxynucleotide adjuvant. Trafficking of antigen-specific primed T cells was analyzed in vivo after adoptive transfer of OVA-specific transgenic T cells in the presence or absence of fingolimod, a drug that causes lymphocytes sequestration within lymph nodes. Antigen-loaded APCs were observed in mediastinal lymph nodes, draining the respiratory tract, but not in distal lymph nodes. Antigen-specific proliferating T cells were first observed within draining lymph nodes, and later in distal iliac and mesenteric lymph nodes and in the spleen. The presence at distal sites was due to migration of locally primed T cells as shown by fingolimod treatment that caused a drastic reduction of proliferated T cells in non-draining lymph nodes and an accumulation of extensively divided T cells within draining lymph nodes. Homing of nasally primed T cells in distal iliac lymph nodes was CD62L-dependent, while entry into mesenteric lymph nodes depended on both CD62L and α4β7, as shown by in vivo antibody-mediated inhibition of T-cell trafficking. These data, elucidating the trafficking of antigen-specific primed T cells to non-draining peripheral and mucosa-associated lymph nodes following nasal immunization, provide relevant insights for the design of vaccination strategies based on mucosal priming.     

In vivo immunomodulation by monoclonal anti-L3T4. 1. Effects on humoral and cell-mediated immune response

The in vivo administration of monoclonal anti-L3T4 antibody has been shown to be an effective preventative and, in some cases, therapeutic treatment for several murine models of autoimmune disease. This report deals with the effect of such treatments on humoral and cell-mediated responses to T-dependent antigens. Both the primary and secondary IgG responses to tetanus toxoid were inhibited when anti-L3T4 was administered prior to immunization, but it was ineffective in modulating an ongoing IgG response. Cell-mediated immunity, as detected by in vitro antigen-specific proliferative responses, was inhibited only if anti-L3T4 was given prior to immunization. It was not effective if treatment was delayed until 48 hr prior to lymph node harvest even though greater than 90% of L3T4+ lymph node cells were depleted by this treatment. The refractory behavior of the lymph node cells to anti-L3T4 treatment was not exhibited by antigen-primed cells obtained from peripheral blood or spleen. The importance of these findings with regard to antibody therapy for chronic autoimmune disease is discussed.     

Vaginal Immunization to Elicit Primary T-Cell Activation and Dissemination

Primary T-cell activation at mucosal sites is of utmost importance for the development of vaccination strategies. T-cell priming after vaginal immunization, with ovalbumin and CpG oligodeoxynucleotide adjuvant as model vaccine formulation, was studied in vivo in hormone-synchronized mice and compared to the one induced by the nasal route. Twenty-four hours after both vaginal or nasal immunization, antigen-loaded dendritic cells were detected within the respective draining lymph nodes. Vaginal immunization elicited a strong recruitment of antigen-specific CD4⁺ T cells into draining lymph nodes that was more rapid than the one observed following nasal immunization. T-cell clonal expansion was first detected in iliac lymph nodes, draining the genital tract, and proliferated T cells disseminated towards distal lymph nodes and spleen similarly to what observed following nasal immunization. T cells were indeed activated by the antigen encounter and acquired homing molecules essential to disseminate towards distal lymphoid organs as confirmed by the modulation of CD45RB, CD69, CD44 and CD62L marker expression. A multi-type Galton Watson branching process, previously used for in vitro analysis of T-cell proliferation, was applied to model in vivo CFSE proliferation data in draining lymph nodes 57 hours following immunization, in order to calculate the probabilistic decision of a cell to enter in division, rest in quiescence or migrate/die. The modelling analysis indicated that the probability of a cell to proliferate was higher following vaginal than nasal immunization. All together these data show that vaginal immunization, despite the absence of an organized mucosal associated inductive site in the genital tract, is very efficient in priming antigen-specific CD4⁺ T cells and inducing their dissemination from draining lymph nodes towards distal lymphoid organs.     

L3T4 (CD4+) cells that mediate contact sensitivity to trinitrochlorobenzene express I-A determinants

The present study has further characterized the T cell-mediated inflammatory response of contact sensitivity (CS) to the hapten trinitrochlorobenzene (TNCB) in mice. A discernible CS response was found to be induced as early as 2 days after epicutaneous application of TNCB. The response peaked on Days 4 to 5 and it then declined to a nearly undetectable level by Days 10 to 11. Examination of the draining lymph nodes demonstrated that development of CS coincided with an increase in cellular proliferation and in the total number of cells present. Despite a severalfold increase in the cellular contents of the draining lymph nodes of sensitized mice, the relative percentages of most subsets of T cells remained unchanged. Flow cytometric studies revealed that the subpopulation of T cells characterized as Thy 1.2+ L3T4+ I-A+ increased substantially in comparison to its presence in unsensitized mice. Whether the Thy 1.2+ L3T4+ I-A+ cells that increased following sensitization represented the effector population that mediates CS was then examined. Four-day immune lymph node T cells or L3T4 cells positively selected from them were capable of adoptively transferring CS to normal mice. However, these cells, after treatment with anti-Ia antibody or anti-I-A monoclonal antibody and complement, were unable to transfer CS. These findings imply that expression of I-A determinants may indicate antigen-induced T cell activation in vivo and that L3T4 cells that mediate CS are I-A positive.     

In vivo administration of Fab' fragments of anti-L3T4 (GK1.5) antibody inhibits the T helper cell function of murine lymph node cells

The effect of intravenous injection of Fab' fragments of anti-L3T4 antibody (GK1.5 monoclonal antibody) into mice was studied. This treatment depleted L3T4+ cells from the popliteal lymph nodes of keyhole-limpet hemocyanin-primed mice. The T cells that remained were unable to provide help to antigen-specific B cells in vitro. The results obtained using Fab' fragments were comparable with those using intact anti-L3T4 antibody and demonstrate that either form of GK1.5 is a potentially useful immunosuppressive agent in mice.     

Higher frequency of Leishmania major-specific L3T4+ T cells in susceptible BALB/c as compared with resistant CBA mice

In previous studies, we reported that a) the adoptive transfer of parasite-specific L3T4+ T cells enhanced rather than inhibited the development of lesions induced by Leishmania major in normal BALB/c mice, and b) the depletion in vivo of L3T4+ T cells by administration of anti-L3T4 monoclonal antibody reversed the susceptibility of BALB/c mice to L. major. To further assess the role of specific L3T4+ T cells in the development of lesions induced by L. major in BALB/c mice, the frequency of parasite-specific T cells capable of mediating specific delayed-type hypersensitivity (DTH) reactivity was determined, by limiting dilution analysis, in the lymph nodes draining the lesions of susceptible (BALB/c) and resistant (CBA) mice. The numbers of L. major-specific DTH-mediating T cells was found to be substantially increased in the lymph nodes of infected BALB/c mice as compared with CBA mice. Moreover in CBA mice, analysis of the cell surface phenotype of the L. major-specific DTH-mediating T cells showed that these cells were equally represented in the L3T4+, Lyt-2-, and L3T4- Lyt-2+ subsets, whereas the majority of these cells in BALB/c mice expressed the L3T4+ Lyt-2- surface phenotype.     

Expression of the rat CD26 antigen (dipeptidyl peptidase IV) on subpopulations of rat lymphocytes.

The T cell activation antigen CD26 has been recently identified as the cell surface ectopeptidase dipeptidyl peptidase IV (DPP-IV). DPP-IV is found on many cell types, including lymphocytes, epithelial cells, and certain endothelial cells. The MRC OX61 monoclonal antibody (MAb) which specifically recognises rat DPP-IV was used to examine the expression of CD26/DPP-IV on rat lymphocytes. The molecular nature of the antigen was examined by immunoprecipitation from thymocytes, splenocytes, and hepatocytes. Analysis by one- and two-dimensional gel electrophoresis indicated that the native form of CD26 includes a 220-kDa homodimer. On tissue sections MRC OX61 MAb stained nearly all thymocytes and in the spleen and lymph nodes predominantly stained the T cell areas. However, in immunofluorescence experiments OX61 stained 80 to 87% of lymph node cells and 78 to 85% of spleen cells. Furthermore, two-colour immunofluorescence analysis of the CD4+, CD8+, and Ig+ lymphocyte subsets indicated that only 2 to 5% of each of these subsets lacked OX61 staining. Spleen cells and thymocytes of both CD4+ and CD8+ subsets stained much more intensely with OX61 after these cells were stimulated with phytohemagglutinin. These findings indicate that rat CD26 antigen expression is not confined to the T cell population as has been suggested, but also occurs on B cells, and is increased on T cells following their activation.     

Generation of somatic variants of a B cell hybrid mediated by a non-cytolytic L3T4+ idiotype-specific T cell

We previously reported that idiotype (Id)-loss, stable somatic variants of a B cell hybrid, 2C3E1, are generated both in vitro and in vivo, after interaction of the Id-positive tumor cells with autologous Id-specific effector T cells. The present investigation was undertaken to elucidate further the nature and functional characteristics of the effector T cells. We report here that the idiotype-specific cells mediating the generation of Id- tumor variants are Thy1+ L3T4+ Lyt-2- cells, which respond to specific idiotypic stimulation by secreting IL-2 in vitro. No IL-2 is secreted in response to unrelated Ig or an Id/Ig-2C3E1 tumor variant. Furthermore, the Id-specific T cells exert strong suppressive effects on the expression of 2C3E1 Ig and the effects can be reversed by blocking the L3T4+ T cells with monoclonal anti-L3T4 antibody in vitro during the initial 3 days of co-culture. After 4 days, the T cell-mediated suppression of the 2C3E1-Id is irreversible. In addition to the in vitro studies we have determined that the administration of anti-L3T4 mAb to mice just before priming with idiotype-bearing tumor cells also abrogates the suppressive effects of the idiotype primed spleen cells on Ig expression of 2C3E1. To study the Id-specific effector T cells in more detail we have generated functional Id-specific L3T4+ T cell lines. These T cell lines have been shown to recapitulate the generation of Id- tumor variants that we observed with Id-primed spleen cells. It is concluded that L3T4+, Id-specific Ts cells are responsible for the generation of somatic variants of the B cell hybrid 2C3E1 and that the induction or selection of these variants progresses from a reversible phase to an irreversible phase.     

Characterization of anti-idiotypic suppressor T cells (Tsid) induced after antigen priming

The induction and fine specificity of idiotype-specific suppressor T cells (Tsid) were studied. Spleen cells from C57BL/6 mice, immunized 4 wk previously with NP-KLH, failed to express NPb3 idiotype-bearing PFC when challenged in vitro with NP-Ficoll or NP-Brucella abortus. After treatment of NP-primed responder cultures with anti-Thy-1.2 anti-serum + C, NPb idiotype-bearing B cells could be detected. This B cell subset was preferentially suppressed by the addition of T cells from NP-primed mice. With this reconstitution protocol, it was determined that suppression of the NPb idiotype-bearing portion of the B cell response was mediated by a specifically induced T cell population (Tsid) that directly suppressed NPb-bearing B cells. As with a previously described suppressor population induced with hapten-modified syngeneic spleen cells (Ts2), the Tsid population bound and was lysed by NPb idiotype-bearing serum antibodies. However, the Tsid could be distinguished from the Ts2 population because it lacked I-J determinants and functioned as an effector T cell, not an intermediary suppressor cell. Furthermore, fine specificity studies with monoclonal NP-specific antibodies expressing various levels of serologically detectable NPb idiotypic determinants indicated that unlike the Ts2, the Tsid population reacts with conventional, serologically detected members of the NPb family. The combined idiotype binding studies for the Tsid and Ts2 populations demonstrate that the fine specificity of suppressor T cell populations reflects their independent mechanisms of regulation.     

Leishmania major: analysis of lymphocyte and macrophage cellular phenotypes during infection of susceptible and resistant mice

Genetically susceptible BALB/c and resistant C57BL/6 mice were infected with Leishmania major and the phenotypes of the responding cells in the draining lymph nodes and cutaneous lesions were analyzed. As early as 1 week, significantly increased numbers of L3T4+ cells as compared to Lyt-2+ cells were present in BALB/c mice lymph nodes (P less than 0.005). Increases in L3T4+ and Lyt-2+ cells were comparable in C57BL/6 mice, resulting in threefold lower L3T4/Lyt-2 ratio than in BALB/c mice. T cell subsets were activated in both strains to express interleukin-2 receptor (IL2R) above resting values, although greater numbers of activated L3T4+ cells were present in the draining lymph nodes from BALB/c at 1 and 3 weeks of infection than in C57BL/6 (P = 0.02). Despite the presence of activated L3T4+ cells in both strains, macrophages differed in the expression of immunologically important surface molecules during infection. Tissue macrophages from BALB/c mice were IgG1/G2b Fc receptor (FcR)+ and Ia- late in disease, whereas macrophages in C57BL/6 became FcR and Ia during healing. BALB/c mice, treated with monoclonal antibody GK1.5 to transiently deplete L3T4+ cells, became resistant to subsequent infection and developed a macrophage phenotype that was FcR- and Ia+. These differences in macrophage phenotype were closely linked to susceptibility during infection with L. major and may play a role in the pathophysiology of murine leishmaniasis.     

Inhibition of humoral immunity in vivo by monoclonal antibody to L3T4: studies with soluble antigens in intact mice

Monoclonal antibody (MAb) to the mouse "helper" T cell antigen L3T4 inhibits the T cell response to class II major histocompatibility antigens on antigen-presenting cells in vitro and in thymectomized mice. To examine the effect of MAb to L3T4 on humoral immunity in euthymic mice, we treated BALB/c mice with 1 mg of anti-L3T4 i.p. at the time of immunization with either bovine serum albumin (BSA) or chicken egg ovalbumin (OA) in complete Freund's adjuvant. Administration of MAb to L3T4 selectively depleted greater than 90% of L3T4+ cells from the blood, spleen, and lymph nodes, but it had little effect on thymocytes. Mice treated with anti-L3T4 were unable to generate an IgG response to either BSA or OA. Treatment with anti-L3T4 also prevented the antigen-specific IgM response to these antigens, although it did not prevent nonspecific stimulation of IgM anti-BSA and anti-OA antibodies induced by adjuvant in the absence of antigen. Humoral immunity was inhibited even when treatment was delayed until 48 hr after immunization. These findings indicate that T cell help for humoral immunity can be abrogated in intact mice by MAb to L3T4.     

In vivo effects of GK1.5 (anti-L3T4a) monoclonal antibody on induction and expression of delayed-type hypersensitivity

The in vivo effects of monoclonal GK1.5 antibody, directed against the L3T4a determinant expressed on Class II-restricted T cells, on the induction and expression of murine delayed-type hypersensitivity (DTH) responses were examined. Development and expression of both hapten (2,4-dinitrofluorobenzene and 2,4,6-trinitrochlorobenzene)- and protein antigen poly(Glu60Ala30Tyr10)-specific DTH are significantly inhibited by injection of monoclonal anti-L3T4a antibody. The inhibitory effects of anti-L3T4a were most pronounced when administered during the afferent (induction) phase of the DTH response, leading to the functional inhibition of the generation of both polyclonal lymph node T-proliferative cells (Tprlf) and DTH effector cells (TDH). The in vivo inhibitory effect is apparently unrelated to preferential induction of suppressor T cells as GK1.5 inhibited DTH induction in cyclophosphamide-treated as well as normal recipients. L3T4a expression on the various T-cell subsets involved in DTH induction and elicitation was also examined. The data show that three functionally distinct, antigen-specific T-cell subsets, Tprlf, TDH, and Th cells involved in DTH induction, bear the Lyt 1+2-, L3T4+ phenotype. Possible mechanisms where in vivo injection of anti-L3T4a inhibits Class II-restricted T-cell subsets involved in DTH induction and expression, including immune depletion and inhibition of T-cell-receptor/ligand interactions, are discussed.     

Cytokine- and Ig-producing T cells in mucosal effector tissues: analysis of IL-5- and IFN-gamma-producing T cells, T cell receptor expression, and IgA plasma cells from mouse salivary gland-associated tissues.

The present study has focused on the analysis of cytokine- and Ig-producing mononuclear cells (MC) that reside in the salivary glands and their associated tissues (SGAT) in the oral region. The SGAT are located under the mandibular area and consist of submandibular glands, periglandular lymph nodes, and cervical lymph nodes. MC were isolated from individual SGAT and examined for T cell subsets and TCR expression, in comparison with T cells obtained from other mucosa-associated and systemic tissues. Forty to fifty percent of MC in submandibular glands were CD3+ T cells, equally divided into CD4+ CD8- and CD4- CD8+ T cell subsets. On the other hand, the intestinal lamina propria and Peyer's patches possessed a approximately 2 to 3:1 ratio of CD4+ CD8- to CD4- T cells. A high frequency of CD4- CD8- (double negative) (DN) T cells (approximately 6 to 10%) was also isolated from submandibular glands. In contrast, approximately 70 to 90% of MC in periglandular lymph nodes and cervical lymph nodes were CD3+ T cells and like the peripheral lymph nodes consisted of fivefold higher numbers of CD4+ CD8- than CD4- CD8+ T cells, with low numbers of DN cells (less than 5%). When expression of gamma/delta and alpha/beta TCR was examined in individual T cell subsets of submandibular glands, the CD4- CD8+ and DN T cell fractions contained 25% and 100% gamma/delta TCR+ cells, respectively. On the other hand, essentially all CD4+ CD8- T cells in SGAT as well as CD4- CD8+ cells in periglandular lymph nodes and cervical lymph nodes were alpha/beta TCR+ T cells. When cytokine production was examined by using IFN-gamma- and IL-5-specific enzyme-linked immunospot assays, the CD3+ CD4+ CD8- T cells in submandibular glands contained T cells spontaneously producing IFN-gamma and IL-5. Further, IL-5 spot-forming cells (SFC) were two- to threefold greater in number, compared with IFN-gamma SFC. The periglandular lymph node T cells contained cytokine producing cells with a ratio of 2:1 for IL-5 and IFN-gamma SFC cells, whereas cervical lymph node T cells did not produce cytokines unless stimulated with T cell mitogens. When the isotype distribution of Ig-producing cells was examined among SGAT, submandibular glands contained large numbers of IgA-producing cells, with few IgM- and IgG-producing cells, a pattern similar to that of the lamina propria. Further, elevated numbers of IgA-secreting cells were also seen in periglandular lymph nodes but not in cervical lymph nodes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of L3T4+ and Lyt-2+ T cell subsets in protective immune responses of mice against infection with a low or high virulent strain of Toxoplasma gondii

In order to elucidate the role of T cell subsets in protective immunity against infection with high virulent and low virulent strains of Toxoplasma gondii, monoclonal antibodies specific for T cell subsets were injected into mice before immunization or challenge infection. Treatment of mice with monoclonal antibody to either L3T4+ or Lyt-2+ T cells before they were immunized with Toxoplasma cell homogenate prepared from high virulent RH strain tachyzoites markedly reduced survival after mice were challenged with low virulent bradyzoites of the Beverley strain. Thus, induction of protective immunity against bradyzoites of the Beverley strain requires the presence of both L3T4+ and Lyt-2+ T cells. In contrast, mice injected with living bradyzoites of the low virulent Beverley strain after immunization with Toxoplasma cell homogenate acquired protective immunity against high virulent tachyzoites of the RH strain. Lyt-2+ T cells alone appear to be final effector cells for protection against the challenge with high virulent RH strain tachyzoites, since treatment of the bradyzoite-immune mice with anti-Lyt-2 antibody, but not anti-L3T4 antibody, before challenge significantly increased mortality.     

Cellular interactions and the role of interleukin 2 in the expression and induction of immunity against a syngeneic murine sarcoma

We have previously demonstrated that following the adoptive transfer of immune cells, the regression of established pulmonary metastases from a weakly immunogenic sarcoma, MCA 105, required the collaboration of two T cell subsets. In this study, we found that the critical role played by L3T4+ immune cells was to provide a helper function since tumor regression proceeded in the absence of L3T4+ immune cells if exogenous interleukin 2 (IL-2) was administered. To extend these observations, we analyzed the events leading to the induction and generation of L3T4+ and Lyt-2+ immune T cells after immunization of mice with viable tumor cells admixed with Corynebacterium parvum. The basic protocol involved immunization, surgical excision of the immunization site on day 7, and challenge with viable tumor cells on day 21. The ability of mice to reject tumor challenge provided a means to evaluate the occurrence of a systemic antitumor immunity. With the use of this experimental protocol, we have found that depletion of T cell subsets in vivo with either L3T4 or Lyt-2 monoclonal antibodies after active immunization abrogated the development of antitumor immunity. Mice immunized and depleted of L3T4+ but not Lyt-2+ T cells were able to reject tumor challenge if exogenous IL-2 was given for 7 days. However, the rejection of tumor challenge required 3 days of additional exogenous IL-2 administration. These results indicate that the induction of Lyt-2+ immune T cells depended on the helper function of L3T4+ T cells via the secretion of IL-2. In the absence of L3T4+ immune lymphocytes, the expression of antitumor immunity by Lyt-2+ immune cells could be facilitated by in vivo administration of exogenous IL-2. The induction of L3T4+ immune T cells, on the other hand, occurred independently of the Lyt-2+ T cell response because the transfer of spleen cells from Lyt-2+ cell-depleted, immunized animals was able to restore antitumor reactivity in L3T4+ cell-depleted, immunized mice. These results demonstrate the intricate cellular interactions leading to the induction as well as the expression of antitumor immunity.     

Depletion of L3T4+ and Lyt-2+ cells by rat monoclonal antibodies alters the development of adoptively transferred experimental autoimmune thyroiditis

To delineate the contribution of L3T4+ and Lyt-2+ cells in the pathogenesis of experimental autoimmune thyroiditis (EAT), synergistic pairs of monoclonal antibodies (mAb) to the T cell subsets were used in conjunction with the adoptive transfer of mouse thyroglobulin (MTg)-activated cells from immunized mice. Initial experiments verified the important role of L3T4+ cells in the transfer of EAT. Subsequent experiments pointed to the relative contribution of both L3T4+ and Lyt-2+ cells, depending on the stage and extent of disease development. Treatment during disease with L3T4, but not Lyt-2, mAb alone significantly reduced thyroiditis. However, in situ analysis of the cellular infiltrate in thyroid sections revealed that, after treatment with mAb, the appropriate subset was eliminated without altering the amount of the other subset in the remaining lesion. In addition, treatment during severe thyroiditis following the transfer of MTg-activated lymph node cells showed that Lyt-2 mAb alone also reduced thyroid infiltration. When the recipients were pretreated with either pair of mAb before transfer, disease development was only moderately affected. We conclude that (i) donor L3T4+ cells are the primary cells responsible for the initial transfer and development of thyroiditis; and (ii) previous in vitro cytotoxicity data, plus current monoclonal antibody treatment of disease and in situ analysis, further implicate a role for Lyt-2+ cells in EAT pathogenesis.     

Comparative evaluation of CD8+CTL responses following gene gun immunization targeting the skin with intracutaneous injection of antigen-transduced dendritic cells

The skin is an attractive target for antigen-specific vaccination. Particle bombardment of the epidermis with plasmid DNA using the gene gun results in antigen expression in keratinocytes of the epidermis leading to antigen presentation in the draining lymph nodes by migratory dendritic cells (DC). In order to better understand the role of the skin in stimulating antigen-specific CD8+cytotoxic T cells (CTL), we compared gene gun immunization with intracutaneous injections of antigen-transduced DC. A single intracutaneous injection of antigen-transduced DC was able to induce in vivo expansion of CD8+CTL specific for the model antigen chicken ovalbumin while four simultaneous shots with the gene gun were not effective. Antigen-transduced DC were much more efficient than particle bombardment of the epidermis in stimulating adoptively transferred TCR-transgenic CD8+CTL in the draining lymph nodes. Employing the novel technique of in vivo bioluminescence imaging, we demonstrated efficient gene transfer to the skin following gene gun bombardment and confirmed that a similar amount of antigen reached the lymph node when compared with injection of antigen-transduced DC. Our results suggest that direct transfection of the skin does not optimally reach and activate appropriate antigen-presenting DC. We believe that this reflects the immunological function of the epidermis which must balance immunity and tolerance to foreign antigens. Further investigations will have to address the role of Langerhans cells for the activation of cellular immunity in the skin.     

Homing of antigen-specific T cells in the Lewis rat. I. Accumulation of antigen-reactive cells in the perithymic lymph nodes

A permanent ovalbumin-specific T cell line of "helper/inducer" cell phenotype (W 3/13+, W 3/25+, OX 8-) was used to study the homing pattern in normal untreated Lewis rats. After i.v. injection, the migration of these cells was followed directly by using 51Cr- or [14C]thymidine-labeled cells. In addition, I tried to retrieve the cells from different lymphatic tissues by antigen restimulation. I found that most of the radiolabeled cells migrate to the lung, liver, kidney, and spleen. Other lymphoid tissues such as the thymus and the cervical and mesenteric lymph nodes were almost devoid of such cells with one exception: the perithymic lymph nodes (pt-LN). Twenty-four hours after the cell transfer, viable antigen-specific cells could be recovered from these organs. Within 9 days the pt-LN enlarged, the percentage of W 3/13+ and W 3/25+ T lymphocytes was enhanced, and both relatively high spontaneous and antigen-driven responses were measurable in cell cultures of these lymph nodes. All the effects were observed if viable but not irradiated antigen-specific T blasts were transferred. Moreover, after active immunization, antigen-reactive cells appeared to accumulate not only in the draining but also in the pt-LN. In both experimental situations, the adoptive transfer and the in vivo activation of antigen-specific lymphocytes, the pt-LN appear to play an important role in the homing of such cells.     

Tumor-specific idiotype vaccines. II. Analysis of the tumor-related network response induced by the tumor and by internal image antigens (Ab2 beta)

In this study the tumor-specific immuneresponse induced by irradiated tumor cells (L1210/GZL) and by anti-idiotype antibodies was analyzed. The anti-idiotype antibodies (Ab2) were made against the paratope of a monoclonal antitumor antibody (11C1) that recognizes a tumor-associated antigen which cross-reacts with the mouse mammary tumor virus-encoded envelope glycoprotein 52. Two Ab2, 2F10 and 3A4, induced idiotypes expressed by the monoclonal antitumor antibodies 11C1 and 2B2. Cytotoxic T cells, generated by immunization with irradiated tumor cells, lyse 2F10 and 3A4 hybridoma cells. Furthermore, immunization with Ab2 induces tumor-specific cytotoxic T lymphocytes. The frequency of tumor-reactive cytotoxic T lymphocyte was found to be similar in mice immunized with Ab2 or irradiated tumor cells when examined at the precursor level. However, only 2F10 induces protective immunity against the growth of L1210/GZL tumor cells. The depletion of a L3T4+ T cell population from 2F10 immune mice was found to increase the effectiveness of transferred T cells to induce inhibition of tumor growth. The inability of 3A4 to induce antitumor immunity could be correlated with the presence of a population of Lyt2+ regulatory T cells. Collectively, these results demonstrate the existence of a regulatory network controlling the expression of effective tumor immunity. Our results demonstrate that selection of binding site-related Ab2 may not be a sufficient criteria for the development of an idiotype vaccine. A better understanding of the regulatory interactions induced by anti-idiotypes is needed for the design of effective antitumor immunotherapy.