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In a previous clinical study using a continuous infusion schedule of recombinant interleukin-2 (rIL-2) we noted a nearly complete loss of activity of EuroCetus rIL-2 when dissolved in 10 ml saline and infused at a very low rate through a plastic infusion device. In the present study, we demonstrated that the loss resulted from a concentration-dependent precipitation of rIL-2 in saline and adherence of the protein to the tubing material. These phenomena were not noted for four other rIL-2 muteins tested [Glaxo, Hoffmann-LaRoche, Amgen (2 muteins)]. EuroCetus rIL-2 was found to be completely soluble in water and 5% glucose.

Dendritic cells in lymphoid tissues are of key importance as highly specialized antigen-presenting cells for the induction of T lymphocyte responses. Conflicting results have been published regarding antigen processing of intact proteins by dendritic cells. We now report that highly purified dendritic cells isolated from H-2k mouse spleens very efficiently generated immunogenic fragments of intact hen egg-white lysozyme (HEL) protein to present to an I-Ak-restricted T hybridoma cell line, specific for HEL peptide 46–61. Dendritic cells required 100 times less HEL protein than lipopolysaccharide-induced B cell blasts for effective presentation. Uptake of 125I-labeled HEL protein by dendritic cells and inhibition of presentation of HEL protein by chloroquine treatment was observed. This indicates an endocytotic process and the involvement of acidified compartments. Since the supernatant of dendritic cells, that were incubated with intact HEL protein, contained immunogenic fragments, further evidence for processing of HEL protein by dendritic cells was obtained. When HEL protein was covalently coupled to beads, dendritic cells were not able to ingest these beads, but could still process HEL protein for presentation. This suggests cell surface processing of HEL protein, although internalization of HEL protein released from the beads cannot be excluded. Taken together, these data show that H-2k dendritic cells are capable of processing and presenting intact HEL protein.

RMA-S is an antigen processing-defective cell line, obtained from a Rauscher virus-induced tumor. The cells express only a low level of cell surface major histocompatibility complex (MHC) class I molecules, which are supposed to be devoid of internally derived antigenic peptides. We investigated Rauscher virus expression and Rauscher peptide presentation to virus-specific cytotoxic T lymphocytes (CTL) by this cell line. Viral proteins are expressed properly, both intracellularly and at the cell surface of RMA-S. Rauscher peptides are presented to virus-specific CTL in the groove of both the class I H-2Kb and Db molecules, but at a low level. Culture of RMA-S cells at room temperature increases their susceptibility to CTL. The RMA-S defect thus affects, but not totally abrogates, Rauscher peptide presentation by MHC class I molecules via the endogenous pathway. This indicates that the RMA-S antigen processing deficit is not absolute.

We investigated the capacity of T lymphocytes from a leukocyte adhesion-deficient (LAD) patient to respond to alloantigen. Leukocytes of this patient completely lacked LFA-1 surface expression due to the absence of mRNA coding for the LFA-1 beta chain. Despite the absence of LFA-1, T lymphocytes obtained from this patient, cultured with allogeneic stimulator cells (lymphoblastoid B cells JY), were capable of lysing JY cells. Furthermore, two T cell clones (one CD4+ and one CD8+), generated from this lymphocyte culture, specifically lysed the allogeneic lymphoblastoid JY cells. The cytolytic capacity of LFA-1-negative T lymphocytes and T cell clones was comparable to that of control LFA-1-positive T cells with allospecificity against JY. Detailed analysis of the CD4 positive and LFA-1-negative T cell clone demonstrated that it specifically recognized HLA-DQ. Antibody inhibition studies showed that the CTL/target cell interaction was mediated through the CD2/LFA-3 adhesion pathway. LFA-1 expressed by the target cells did not participate in the CTL/target cell conjugate formation and contributed only minimally to the cytotoxic activity. Moreover, when allogeneic LFA-1-deficient B cells, bearing the appropriate HLA-DQ alloantigen, were used as target cells, significant levels of specific cytotoxicity were measured, further excluding a role for LFA-1 in this interaction. The adhesion molecules, VLA-4, CD44 and L-selectin (LECAM1) were not involved. These results demonstrate that LFA-1-negative T lymphocytes can exert allospecific cytotoxicity and that CTL/target cell contact is mediated through the CD2/LFA-3 route. This observation may explain in part why in LAD patients viral infections, cleared largely by T cells, are less frequently observed than bacterial infections, in which phagocytic cells play a major role.

Publisher Summary This chapter discusses the eradication of tumor by adoptive transfer of cytotoxic T lymphocytes. Complete and permanent eradication of tumors by histocompatability complex (MHC) class I restricted CD8 + tumor-specific cytotoxic T lymphocytes (CTLs) can be achieved in a variety of experimental murine models. In human metastatic melanoma, CD8 + tumor-specific CTLs are probably therapeutically the most active components among lymphokine-activated killer or tumor-infiltrating lymphocytes cells, with a possibly important contribution by CD4 + cells in some patients, although no clinical experience with cloned T cells exists as yet to evaluate this point. Cancer cells can avoid the induction of and destruction by tumorspecific CTLs in a number of ways, some of which can be manipulated to increase the efficacy of sensitization or adoptive therapy. T cell therapy allows targeting on minute changes in any cellular peptide that is presentable in the context of MHC molecules. CD8 + CTLs stand out as remarkably effective in tumor eradication in view of their remarkable potency, and specificity in allograft rejection and antiviral immunity.

1 página.-- Presentación de póster presentation.-- Este artículo es parte del suplemento: AIDS Vaccine 2009.

Previously, we have shown that the intravenous (i.v.) injection of allogeneic lymphocytes across an H-2 class I-mutant disparity leads to specific skin allograft tolerance caused by irradiation-sensitive donor T cells, which function as veto cells. In the present study, we show that the i.v. injection of H-2 class II-incompatible spleen cells also results in specific skin allograft tolerance. However, tolerance induction depends on the presence of irradiation-resistant non-T cells in the donor cell inoculum. Thus, different mechanisms operate in tolerance induction across an H-2 class I vs. H-2 class II mismatch. I.v. injection of allogeneic spleen cells across an H-2 class I plus class II disparity does not result in skin allograft tolerance. Finally, our data show that transfusion-induced suppression of the delayed-type hypersensitivity response against alloantigens does not correlate with skin allograft tolerance induced by i.v. injected allogeneic lymphocytes. In conclusion, the type of H-2 mismatch between transfusion donor and recipient not only determines the occurrence of allograft tolerance but also the mechanism leading to tolerization.

Cell-mediated lympholysis (CML) reactions were studied among four strains of C57BL/6 (B6) mice carrying mutant alleles (H-2ba,H-2bd,H-2bg, andH-2bh) at thez1 locus in theK end ofH-2b and the original B6 (H-2b) strain. Cross killing of target cells from lines that had not participated in the mixed lymphocyte reaction (MLR) was extensive, but usually less intense than that of target cells of stimulator cell genotype. The extent of CML crossreactivity could be limited by using cells from F1 hybrid mice as responders in MLR. In a comprehensive analysis of the cytotoxicity exerted by 20 MLR combinations with homozygous, and 10 MLR combinations with F1 hybrid responder cells, 19 different CML cytotoxicity patterns were identified, corresponding to at least 19 different CML target specificites. When the number of CML mismatches of each mutant with the originalH-2b was calculated,H-2ba was found to be most distinct fromH-2b,H-2bs andH-2bd were closest toH-2b, andH-2bh occupied an intermediate position. The validity of this sequence of relatedness is supported by published reports on skin graft survival times and on the interaction of T lymphocytes with virus-infected target cells using cells fromz1 locus mutants.

An acute graft-vs.-host reaction (GVHR) was induced by i.v. injection of 10(8) lymphoid cells from C57BL/10 (B10) donors (H-2b/b) into adult non-irradiated (B10 X DBA/2)F1 mice (H-2b/d). Previous experiments have established that spleen cells obtained from such GVHF1 mice suppress the primary antibody response of normal F1 spleen cells to sheep red blood cells. This type of suppression was termed "allosuppression", and it was shown to be mediated by Ly-2+ T cells of donor origin that react against H-2 antigens of the host. It was unclear, however, whether the actual mechanism of allosuppression was due to suppressive factors generated by donor T cells or whether the latter killed the F1 B cells. Here, we show that for their suppressive effect GVHF1 spleen cells need direct cell contact with F1 spleen cells; no suppressive effect was measured in a double-chamber culture system in which the GVHF1 spleen cells were separated from the responding normal F1 spleen cells by a cell-tight membrane filter. The suppressive effect only affected cells expressing the appropriate H-2 class I alloantigen; in mixed cultures of irradiated F1 spleen cells and GVHF1 spleen cells with third-party B cells the antibody response of the third-party B cells was not suppressed. GVHF1 spleen cells showed cytotoxic T lymphocyte (CTL) activity specific for the allogeneic F1 H-2 antigen. The suppressive effect of the GVHF1 spleen cells did not differ from that exerted by cloned CTL specific for MHC class I alloantigens; cloned CTL suppressed the primary antibody response of murine spleen cells without affecting the response of third-party B cells added to the cultures. The combined findings show that "allosuppression" in vitro is not due to suppression of F1B cells, but to a direct killing of these cells by alloreactive CTL.

Two class I major histocompatibility (MHC) mutant mouse strains, H-2bm14 and H-2bm6, differ from the strain of origin C57BL/6 (B6, H-2b) in one and two amino acids of the H-2Db and H-2Kb molecule, respectively. The bm14 Db mutation results in specific failure of female bm14 mice to generate a cytotoxic T lymphocyte (Tc) response to the male-specific antigen H-Y. The allospecific Tc response of CD8+ B6T cells against bm6 Kb mutant spleen cells, in contrast to that against other Kb mutants, is absolutely CD4+ T helper cell dependent. Purified CD8+ T cells completely fail to respond. We now report that the inability to mount these specific immune responses is restored by the use of dendritic cells (DC) as antigen-presenting cells (APC). Comparison of MHC expression on various types of APC by cytofluorimetry and quantitative immunoprecipitation showed very high expression of class I and class II MHC molecules on DC. Strikingly, examination of class I and class II molecules by isoelectric focusing revealed qualitative differences as well. We show that the surface MHC class I molecules of DC are present in greater quantity and carry on average fewer sialic acids than the same molecules isolated from other APC types such as spleen cells, lipopolysaccharide blasts or concanavalin A blasts. That sialic acids on cell surface molecules, including MHC, may play a role in antigen presentation is suggested by our finding that removal of sialic acids, by neuraminidase, can restore specific responses to nonresponder APC as well.

Cells from 32 adult patients with non-Hodgkin's lymphoma were studied with respect to surface markers and functional properties in short-term culture. Twenty-six lymphomas were of B-cell origin, including all nodular and diffuse lymphocytic lymphomas. Three tumors were of T-cell origin (one histiocytic lymphoma and two undifferentiated lymphomas). In the remaining three cases (histiocytic lymphomas) the immunological nature of the tumor cells could not be determined. All reactivity to mitogenic stimuli of cells from B-cell lymphomas was due to residual normal T cells. In follicular lymphocytic lymphomas more reactive T cells prevailed among the malignant B cells than in diffuse lymphocytic lymphomas. Heterogeneity among B-cell lymphomas was indicated by differences in intensity of fluorescence with anti-Ig reagents and in stimulatory capacity in mixed lymphocyte culture. T-cell lymphomas were characterized by high percentages of T cells together with impaired responses to stimuli. The results of immunological studies correlated well with the histological classifications of Rappaport, Lukes and Lennert. Cancer 42:1804–1817, 1978.

The CD27 membrane antigen is exclusively present on a large subset of human peripheral blood T lymphocytes and on mature thymocytes. Several observations point towards a specific role of this molecule on activated T cells. Upon T cell activation induced via the T cell receptor complex, CD27 expression greatly increases, while addition of anti-CD27 monoclonal antibodies amplifies the proliferative response. Within the CD4+ subset, only CD27+ T cells provide helper activity for B cell differentiation. Interestingly, CD27 expression differs not only quantitatively, but also qualitatively between resting and activated T cells. On resting cells, CD27 is a disulfide-linked homodimer with subunits of 55 kDa molecular mass. Upon activation, also a 55-kDa monomer seems to occur, while in addition a 32-kDa component is found. The relationship between these two proteins has been investigated. The 55-kDa and 32-kDa molecules do not seem to be physically associated. The two CD27 components are structurally highly homologous as determined by two-dimensional mapping of tryptic peptides. They both express the epitopes recognized by anti-CD27 antibodies, indicating that the 32-kDa molecule is also T cell specific. Both 55-kDa and 32-kDa molecules carry N-linked carbohydrate groups. However, they differ in other, not yet specified, post-translational modifications, and do not arise from a common precursor simply by alternative N-glycosylation. The 32-kDa form may be derived by proteolytic processing from the 55-kDa protein, but most likely not from a larger (common) precursor. Alternatively, both molecules may arise from a common gene by alternative mRNA splicing, or be derived from highly homologous genes. The dramatic change in molecular composition of CD27 suggests a newly acquired function for CD27 on activated T cells.

H-2-congeneic C57BL mice with milk transmission of B-tropic murine leukemia virus (V+ mice) have a much higher lymphoma incidence than the same strains without milk-transmitted virus (V- mice). Gene(s) within the major histocompatibility complex (H-2) influence virus titers, lymphoma incidence, lymphoma type and the anti-MuLV envelope antibody response. In this paper, we report that the prevalence of cytotoxic antibodies to virus-induced lymphomas is also regulated by the H-2 complex. Milk transmission of MuLV resulted in the formation of cytotoxic antibodies against primary virus-induced C57BL lymphomas. These antibodies detect an antigen that is also present on the RADAI tumor-cell line, and on normal spleen cells of young adult B10.A (H-2a) mice of both V+ and V- sublines, but not on spleen cells of young adult B10 (H-2a) mice of either subline. These cytotoxic antibodies were detected in the sera of B10V+ and B10.A(5R)V+ animals, but not in the sera of B10.AV+ mice. This indicates that the prevalence of these antibodies is controlled by a gene in the K- and/or I-A region of the H-2 complex. The presence of these cytotoxic antibodies in serum is recessively inherited. The specificity of the cytotoxic antibodies was investigated with a standard panel of transplantable tumor-cell lines. Of these, only the RADAI cells expressed the target antigen in direct cytotoxicity tests and by absorption. The ability of B10V+ sera to lyse the B10.AV+ and RADAI tumor cells is ascribed to antibody activity against a new MuLV-related cell-surface protein: G(B10.A). Immunochemical analysis and absorption experiments with different types of purified MuLV and MuLV-infected cell lines indicate that the cytotoxic antibodies belong to low-avidity IgM antibodies that are directed to MuLV.

Classical T lymphocytes recognize foreign antigens in the context of self major histocompatibility complex (MHC) molecules by means of the T-cell receptor (TCR)alpha beta heterodimer. The genes for TCR beta-chains, like immunoglobulin genes, are subject to allelic exclusion. The introduction of a functional TCR-beta gene into the germline of mice prevents rearrangement of endogenous TCR-beta genes. Here we report that the introduction of a non-functional TCR-beta genes. Here we report that the introduction of a non-functional TCR-beta gene with a deletion of the major part of the variable region (delta V-TCR-beta), also inhibits endogenous TCR-beta gene rearrangement. This inhibition is mediated via the encoded protein because impairment of endogenous TCR-beta gene rearrangement is not found if a frameshift mutation is introduced into the DJ region of the delta V-TCR-beta transgene. The delta V-TCR-beta transgene can lead to two phenotypes, in which lymphoid development is perturbed. Phenotype A is characterized by a severe impairment of both T and B cell development as reflected by the complete absence of certain lymphoid organs. In phenotype B, lymphoid organs are macroscopically normal, but T cell differentiation is impeded. Virtually all thymocytes lack membrane expression of TCR-alpha beta, but nevertheless carry the CD4 and CD8 antigens (CD4+CD8+ phenotype); they do not, however, mature further. The defect in mice of phenotype B but not of phenotype A can be corrected by the introduction of a functional TCR-beta gene.

The neoplastic T cells from five patients with adult T cell lymphoma/leukemia (ATLL), born in the Caribbean, were studied with respect to immunoregulatory activity on pokeweed mitogen (PWM) driven immunoglobulin (Ig) synthesis as well as surface-marker phenotypes with monoclonal antibodies. The neoplastic T cells in all patients had an OKT1+4+8–11+M1-I1–3A1- phenotype, but differed in the reactivity with OKT3. None of the patients' cells exerted helper activity on PWM- induced Ig synthesis. The neoplastic cells of three patients had suppressor activity on PWM-induced Ig synthesis. All patients were positive for human T cell leukemia/lymphoma virus (HTLV) or had antibodies against HTLV antigens. It has previously been shown that the neoplastic cells in Japanese ATLL patients and in patients from the Caribbean are indistinguishable by morphology and marker phenotype. We now show them to be also similar with respect to their functional properties.

Histocompatibility of skin grafts and in vitro lymphocyte reactions of three new H-2 mutants (B6-H-2bg1, B6-H-2bg2, and B6-H-2 bh ) are described. Complementation tests indicated thatH-2bg1,H-2bg2, andH-2 bh were mutations at the same locus (z-1) and allelic withH-2 ba . Two of the lines, B6-H-2bg1 and B6-H-2bg2, appeared identical, although they were of spontaneous and independent origin. The third line, B6-H-2 bh , was unique. Such graft rejection was paralleled by reactivity in the MLR and by the generation of cytotoxic lymphocytes in vitro. The pattern of crossreactivity among antigens specified by these mutant genes supports the notion that thez-1 locus controls multiple specificities. The results indicate that a single point mutation can simultaneously affect histocompatibility, MLR, and CML reactions.

In 230 cases of chronic lymphocytic leukemia (CLL), marker analysis was performed with rosette techniques and a panel of xeno-antisera. A monoclonal B-cell proliferation was found in the majority of cases (94%). In most cases, the B-cells carried IgM, with or without IgD. Cytoplasmic immunoglobulin-inclusion bodies were seen in 7% of the cases of B-CLL. The number of patients with non-B/non-T-CLL was small (2%) in this series. In eight patients (4%), a proliferation of T-cells was established. These patients had a different clinical presentation and marker analysis of the lymphocytes, together with functional studies, showed that this group represented a mixture of different disease processes. Functional analysis of the B-CLL cells in 19 cases showed a poor or absent mitogen response and in nine cases the absence of the capacity to differentiate in vitro into plasma cells and/or to produce immunoglobulins.

The molecular etiology of retrovirally induced T-cell tumors has been shown in many cases to involve proviral integration near a cellular oncogene, c-myc, N-myc, Pim-1 and pvt-1 being frequent targets for insertional activation. Murine B-cell tumors induced by infection with murine leukemia virus have been studied for rearrangements in these and other loci. In contrast to the T-cell lymphomas, tumors of the B-cell lineage, either early B-cell tumors induced in nude mice or late B-cell tumors in immunocompetent mice, did not show disruption of N-myc or Pim-1 in any of the tumors studied, although those lymphomas had acquired many new proviruses. The loci c-abl, bcl-2, fis-1, c-erbB, c-myb, and neu were likewise not involved. Rearrangement of c-myc was seen in 1 out of 71 and rearrangement of the pvt-1 locus in 4 out of 73 (5%) of the B-cell tumors. Thus it appears that mechanistic differences exist in the development of T-cell tumors and B-cell tumors caused by the same etiological agent.

SUMMARY Neonatal infection of C57BL/10 mice with cloned ecotropic and/or dual-tropic mink cell focus-inducing (MCF) mouse leukaemia viruses (MuLV), induces a wide spectrum of different lymphomas of T, B, and non-T/non-B cell types. The H-2 complex has a marked influence on both the development of lymphoma incidence and lymphoma type. A study using the oncogenic MCF 1233 virus and a series of B10 congenic mice enabled the mapping of the following: (a) Resistance to the early development of T cell lymphomas is controlled by the H-21-A locus. (b) Susceptibility to early T cell lymphomagenesis is associated with an I-A-regulated low anti-MCF 1233 envelope antibody response and persistent infection of the thymus. (c) B10 (H-2b) mice, which are resistant to early T cell lymphomagenesis induced by MCF 1233 or other MuLV isolates, have high anti-MuLV envelope antibody responses which are I-A-regulated. These mice develop more B cell lymphomas late in life in contrast to the early development of T cell lymphoma in B10.A (H-2a mice. The possible response mechanisms which underlie these observations, including: (1) I-A -regulated immunoselection against MuLV antigens expressed by (pre) leukaemic T cells, (2) aberrant expression of class II MHC antigens on some B cell lymphomas and (3) I-A-regulated chronic immunostimulation of MuLV-expressing (pre) leukaemic B cells, are discussed.

Cytotoxic T lymphocyte (CTL) clones against adenovirus type 5 (Ad5) early region 1 (E1)-transformed cells were generated in C57BL/6 (B6) mice. A defined peptide encoded by Ad5 E1A is the target structure for H-2Db-restricted CTLs. Upon intravenous injection into B6 nude mice bearing Ad5 E1-induced tumors, these CTLs, if combined with recombinant IL-2, destroy subcutaneous tumor masses up to 10 cm3. The in vivo action of CTLs is highly specific, and long-term "memory" persists in treated nude mice months after tumor regression. Our data show an important role for CTLs directed against a viral nuclear oncogene product in tumor eradication.

We performed a cytogenetic study on 16 murine mature B-cell lymphomas and 10 T-cell lymphomas, using G-banding techniques. All tumors, with the exception of 3 spontaneous B-cell tumors, were induced by various slowly transforming murine leukemia viruses (MuLV). Metaphases were obtained from primary (10 B-cell tumors) and first or second transplant generation lymphomas (6 B-cell and 10 T-cell tumors), all of which were well characterized with respect to phenotypic, histologic and genotypic features. In the T-cell tumors we found relatively simple karyotypic abnormalities, including various numerical aberrations, such as trisomy 15, in line with many earlier reports. However, the majority of B-cell tumors showed a great variety of both structural and numerical chromosomal anomalies. Three B-cell lymphomas had an apparently normal karyotype. No single cytogenetic abnormality occurred commonly in the B-cell lymphomas, but some structural abnormalities were found in more than one stemline, in particular, ins (II) (A1; A2) in 3 tumors, and deletions involving the D-region of chromosome 14 in 3 other lymphomas. These cytogenetic results clearly indicate that the pathogenic mechanisms involved in MuLV-induced (long latency) B-cell lymphomagenesis and (short latency) T-cell lymphomagenesis differ considerably.

Cloned blood lymphocytes that do not express the alpha- and beta-chains of the T-cell receptor show MHC-unrestricted cytotoxicity. These cells carry the gamma-protein, disulphide-linked either to another molecule or to itself, and associated with the CD3 complex. These observations may help to solve the mystery posed by the discovery of the gamma gene.

The antibody response against the envelope proteins of a variety of cloned highly and poorly oncogenic dualtropic mink cell focus-inducing (MCF) murine leukemia viruses (MuLV) was studied and compared with the antibody response against ecotropic isolates. MCF viruses evoke stronger antibody responses than ecotropic MuLV isolates. Although these MCF viruses are highly polymorphic with respect to their gp70 and p15(E) envelope proteins, generally a similar H-2-linked immune response gene control of anti-viral antibody responses is observed. Neonatally infected BALB/c (H-2d) and C57BL/10 (B10,H-2b) mice are high responders and B10.A (H-2a) mice, congenic at the major histocompatibility complex (H-2) with B10, low responders. No correlation was found between the expression of any single gp70 or p15(E) epitope of the infecting MCF virus and the magnitude of the antibody response. This indicates that the level of the H-2 regulated anti-MuLV envelope antibody response is most likely determined by a MuLV antigen shared by all MuLV isolates examined. The magnitude of the antibody response against highly oncogenic and against poorly oncogenic MCF virus does not differ significantly. The combined data indicate that the intrinsic oncogenic potency encoded by the viral genome is a more important feature of oncogenesis than the level of antiviral envelope antibody response evoked by the MCF virus. However, the oncogenic properties of a single murine leukemia virus may vary among H-2 congenic mice, correlated with their H-2-dependent capacity to produce antiviral antibodies.

The mouse H-2Kd gene gives rise to several transcripts by alternative splicing. In addition to encoding the known Kd antigen, it could thus encode at least one minor hypothetical Kd-like molecule, with a distinct NH2 terminus. The existence of this "24" product can be inferred from a cDNA clone which was previously isolated. We have engineered both this cDNA and its canonical counterpart into a eukaryotic expression vector. After transfer of these constructs into mouse fibroblasts, we obtained cells expressing either one of the transcripts, but not both. In cytotoxicity tests, we found no expression of the "24" product on the cell surface, nor did we obtain any clue concerning its function. In contrast, cells which express Kd antigen, but none of the possible Kd-like molecules produced by alternative splicing, were functional in all aspects examined. We conclude that alternative splicing does not contribute to the known function of the Kd antigen.

In antigen-specific cytotoxic T-lymphocyte (CTL) responses H-2 class I mutations usually result in a decreased recognition of the antigen in association with the mutant molecule by CTL from the strain of origin. However, the influence of class I mutations on the magnitude and specificity of CTL responses in the mutants has been studied in only a few instances, in which usually a partial or complete loss of responsiveness was found. We now report that class I mutants extensively use gained (novel) CTL restriction sites, generated by the mutations in the CTL response against the hapten trinitrophenyl (TNP), demonstrated both at the population level and in limiting dilution. TNP-specific CTL clones, restricted by mutant-specific determinants, were detected in all mutants. The percentages mutant-specific CTL clones in limiting dilution experiments were 43, 40, 35, and 13 in the K b mutants bm1, bm8, bm3 and bm5, respectively, and 35 in the D b mutant bm14. It is concluded that H-2 class I mutations led to changes in the TNP-specific CTL repertoire resulting in gain of CTLs uniquely restricted to the mutant molecule.

A review is presented of functional studies performed with the expanded T cells in Sézary syndrome, T-cell chronic lymphocytic leukemia (T-CLL), T-prolymphocytic leukemia (T-PLL), adult T-cell leukemia lymphoma (ATLL), T-cell acute lymphoblastic leukemia (T-ALL) and T gamma lymphocytosis. The immunological and clinical relevance of immunological studies of functional properties of T-cell expansions is discussed.

The prevalence of different host-range classes of murine leukemia virus (MuLV) was studied in C57BL mice with (V+) and without (V-) milk transmission of a naturally occurring B-tropic ecotropic MuLV. Virus isolates were studied with respect to growth properties, XC-plaque formation, antigen profiles of their envelope proteins (gp70 and P15(E)), gp70 tryptic-peptide maps, and their potential to induce lymphomas after inoculation into newborn mice. B-tropic ecotropic MuLV with the capacity to cause plaques in XC cells was isolated from almost all lymphomas of both V+ and V- sublines. The reaction patterns of these ecotropic isolates with monoclonal antibodies reactive with MuLV-env proteins and the tryptic-peptide maps of the gp70 molecule indicate that they are similar to each other and differ only slightly from the ecotropic MuLV in the spleens of young V+ animals, which is identical to the milk-transmitted virus. XC − , B-tropic dualtropic mink cell focus-inducing (MCF) viruses were isolated from the majority of different types of lymphoma (B cell, T cell, or neither B nor T cell derived), but not from the spleens or milk of young V+ or V- animals. The env proteins of the MCF isolates are highly heterogeneous, but most isolates originating from B10.AV+ T-cell lymphomas share MCF-related epitopes in their gp85 envelope precursor with AKR MCF-247 virus. Most MCF viruses isolated from non-T lymphomas do not possess these epitopes. The results indicate that also in this model the generation of dualtropic MCF viruses might be important in lymphoma induction, although only some of the cloned MCF viruses show enhanced oncogenic properties in comparison with ecotropic isolates. A cloned oncogenic MCF virus induced different lymphoma types in C57BL/10 (=B10, H-2 b ) and B10.A (H-2 a ) mice, similar to what was found earlier with the milk-transmitted virus. Hence, the lymphoma-type differences are not due to differences in the B-tropic ecotropic viruses transmitted through the milk in these strains, but reflect an influence of the H-2 complex on the phenotype of the virus-induced lymphomas.

In vitiligo, cytotoxic T cells infiltrating the perilesional margin are suspected to be involved in the pathogenesis of the disease. However, it remains to be elucidated whether these T cells are a cause or a consequence of the depigmentation process. T cells we obtained from perilesional skin biopsies, were significantly enriched for melanocyte antigen recognition, compared with healthy skin-infiltrating T cells, and were reactive to melanocyte antigen-specific stimulation. Using a skin explant model, we were able to dissect the in situ activities of perilesional T cells in the effector phase of depigmentation. We show that these T cells could infiltrate autologous normally pigmented skin explants and efficiently kill melanocytes within this microenvironment. Interestingly, melanocyte apoptosis was accompanied by suprabasal keratinocyte apoptosis. Perilesional T cells did, however, not induce apoptosis in lesional skin, which is devoid of melanocytes, indicating the melanocyte-specific cytotoxic activity of these cells. Melanocyte killing correlated to local infiltration of perilesional T cells. Our data show that perilesional cytotoxic T cells eradicate pigment cells, the characteristic hallmark of vitiligo, thereby providing evidence of T cells being able to mediate targeted autoimmune tissue destruction.

Moloney leukemia virus-specific cytotoxic T lymphocytes (CTL), generated by secondary in vitro stimulation of spleen cells with syngeneic virus-infected cells, frequently lysed not only syngeneic virus-infected cells, but also noninfected allogeneic target cells. This phenomenon was studied with B6(H-2b) responder cells and a series of H-2Kb-mutant responder cells. Thus, B6 Moloney-specific CTL lysed noninfected Kb-mutant cells, but not B6 cells, whereas Kb-mutant Moloney-specific CTL lysed noninfected B6 cells and not noninfected cells of the same mutant. Cold-target-inhibition studies showed that the CTL reactions against different allogeneic cells were mediated by different subpopulations of virus-specific CTL: lysis of allogeneic target cells was fully inhibited only by the same allogeneic and by syngeneic virus-infected cells, but not by another allogeneic cell, also lysed by the same effector-cell population. Lysis of syngeneic virus-infected cells could not be inhibited by allogeneic target cells. These data imply that a minority of virus-specific CTL shows cross-reactivity with a given allogeneic target cell. It is concluded that limited amino acid substitutions in the Kb molecule alter the repertoire of Moloney virus-specific CTL, as reflected in alloreactive CTL populations, even though the virus-specific CTL response. of B6 and all Kb mutants is mainly Db-restricted. Thus, the development of tolerance to self class-I major histocompatibility complex (MHC) molecules affects the repertoire of self-restricted cytotoxic T cells.

The in vivo importance of class I MHC regulation of the cytotoxic T cell (Tc) response to a natural pathogenic agent of high virulence was studied on the basis of our demonstration of a major difference in the capacity to generate a Sendai virus-specific Tc response between C57BL/6 (B6, H-2b) mice and H-2Kb mutant B6.C-H-2bm1 (bm1) mice. These two mouse strains differ from each other only in three amino acids in the crucial H-2Kb restriction element for this response. Bm1 mice, in contrast to B6 mice, are Tc nonresponders against this virus, but show Sendai-specific T cell proliferation, antibody production, and delayed type hypersensitivity (DTH) reactions, as well as natural killer (NK) cell activity, equal to those of B6 mice. Another H-2b mouse strain, the 129/J, also shows equal Sendai virus-specific Tc, T helper cell (Th), B cell and DTH responses compared to B6, but is virtually deficient in generating an NK cell response. B6, Sendai Tc-deficient bm1, T cell-deficient B6 nu/nu and NK low-responder 129/J mice differ from each other in susceptibility to lethal pneumonia induced by intranasal (i.n.) inoculation of virulent Sendai virus. The lethal dose (LD50) in B6 mice averages 152 TCID50, in bm1 mice 14 TCID50, in B6 nu/nu mice 0.5 TCID50 and in 129/J mice 0.2 TCID50. The importance of Tc is shown by the difference in susceptibility between B6 and bm1 mice and also by the complete protection of B6 nu/nu mice against infection with a lethal virus dose by i.v. injection of a Sendai virus-specific, IL-2 dependent and H-2Kb restricted B6 Tc clone. In vivo protection by this Tc clone is H-2Kb restricted. Apart from Tc, an important role for virus-specific Th cells is evident from the difference in susceptibility between bm1 and B6 nu/nu mice. This conclusion is supported by the demonstration that the mean survival time of B6 nu/nu and bm1 nu/nu mice can be significantly prolonged, in an I-Ab restricted manner, by the injection of in vitro-propagated, Sendai-specific B6 or bm1 Th clones after a lethal dose of Sendai virus, and by the demonstration that inoculation of these Th clones provides help to virus-specific Tc by means of IL-2 production. Strikingly, Th and Tc cooperate in anti-Sendai virus immunity, since permanent survival of lethally infected nu/nu mice is only achieved by inoculation of a mixture of Tc and Th clones or a mixture of a Tc clone and rIL-2. Furthermore, the difference in susceptibility to Sendai virus infection between B6 and bm1 mice provides a unique model for the study of MHC-disease associations. The importance of NK cells is revealed by the high susceptibility of 129/J mice to Sendai virus infection, although all other immune parameters measured appear to be normal.

The immunoregulatory activity of T cell subsets on human B cell differentiation has been amply studied in the pokeweed mitogen (PWM)-driven culture system (1,2). PWM-driven immunoglobulin (Ig) synthesis is strictly T cell-dependent and helper activity is only provided by T4+8−cells, whereas T4−8+ cells have suppressor activity (1,2). We demonstrated that interleukin-2 (IL-2) plays an important role in T cell-dependent Ig synthesis (3). It was found that PWM-induced IL-2 synthesis is an early and necessary event in PWM-driven Ig synthesis. This was confirmed by the finding that anti-Tac, an anti-IL-2 receptor antibody (4), fully abrogated PWM-driven Ig synthesis, and moreover, by the innate Iginducing capacity of IL-2 itself, dependent on T4+8− cells (3).

In June of 1908, Paul Ehrlich, the founder of immunology, lectured at the University of Amsterdam on the subject of cancer. His remarks included the following: “the understanding of natural immunity really represents the key to carcinoma.... I have acquired the firm conviction that natural immunity does not depend on the presence of anti-microbial materials, but rather, it depends on purely cellular activities. I am certain that, in the enormously complicated course of fetal and post-fetal development, aberrant cells become unusually common. Fortunately, in the majority of people, they remain completely latent thanks to the organism’s positive mechanisms. If such mechanisms did not exist, one might expect carcinomas to have an enormous frequency. These cells may live in a latent state for 20, 30 or 40 years before changing to a significant tumor. This means, in light of my theory, that there has been a diminution of some vital cell activity [which] allows the rapid, parasitic growth of certain cells” (Ehrlich, 1957).*

Characteristics of murine leukemia viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 A. General aspects and scope of review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 B. Host-range classes of MuLV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 C. Endogenous MuLV-related proviral sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

The role of interleukin 2 (IL 2) in the induction of human B cell differentiation in vitro was studied. IL2 was unable to induce immunoglobulin (Ig) production in non-T cells either in the presence or absence of pokeweed mitogen (PWM). However, IL2 alone could induce Ig production in non-T cells when irradiated T cells were present. Similar to the PWM-driven system helper activity was delivered by T4+ but not T8+ cells. Apparently, IL2 acts on T4+ cells and induces these cells to deliver the actual helper signal(s) for Ig production by B cells. Whereas in the PWM-driven system only T8+ cells suppress Ig synthesis, ILZdriven Ig synthesis was suppressed by both T4+ and T8+ cells added to a mixture of non-T cells and irradiated T4+ cells. This suppressor activity could be abrogated by irradiation. PWM was shown to induce IL2 production in both T4+ and T8+ cells. Moreover, PWM-induced Ig synthesis, like IL2-induced Ig synthesis, could be totally abrogated by a monoclonal antibody against the human IL2 receptor (anti-Tac). These findings, coupled to the innate Ig-inducing capacity of IL 2, indicate a role for IL 2 in the PWM-driven system. The mechanism of suppression in both the PWM- and the IL 2-driven systems was not shortage of IL 2 in the culture due to consumption or inhibition of production of IL 2. Moreover, the T8+ cells produced IL2, despite their failure to help Ig synthesis. Helper T cell activity can thus be divided into two distinct activities: (a) IL2 production and (b) the ability to deliver the actual helper signal such as helper factors for B cell differentiation. This insight allows a better evaluation of the immunoregulatory activities of T cell subsets in Gealth and disease.

Neonatal infection of C57BL and BALB/c mice by cloned ecotropic and dualtropic mink cell focus-inducing (MCF) murine leukemia viruses (MuLV) induces a wide spectrum of different lymphomas of T, B, and non-T/non-B cell types. Oncogenic dualtropic MCF viruses and poorly oncogenic ecotropic MuLV act synergistically in lymphomagenesis. Within one mouse strain virus-induced T-cell lymphomas arise earlier than B-cell lymphomas after neonatal inoculation of a single-cloned MuLV. The host genetic constitution, notably the H-2 complex has a marked influence on lymphoma type. This H-2 influence can be explained by an H-2-linked difference in penetration of the thymus early in life by oncogenic thymotropic MuLV, which in turn is correlated with, but not necessarily due to the magnitude of the anti-MuLV antibody response.

It was shown previously that B6.C-H-2bm14 (bm14) mice, carrying a mutation in the H-2Db locus, are unable to generate cytotoxic T cells (CTL) against Moloney murine sarcoma virus (M-MSV). We now report an analysis of tumor induction and regression kinetics and of immunity to the virus, following the injection of graded doses of M-MSV into C57BL/6 (B6) and bm14 mice. Contrary to expectation, bm14 mice showed slightly less tumors than wildtype B6 mice. Moreover, all bm14 mice that developed a tumor were able to reject this tumor, even after injection of the highest virus dose tested. From the spleen cells of bm14 mice that had rejected tumors, no secondary in vitro CTL responses could be generated, in contrast to strong CTL responses generated from B6 spleen cells. Although bm14 mice were unable to generate virus-specific CTL, they showed normal antibody and T-cell proliferative responses against Moloney virus, suggesting an intact T-helper-cell function. It is concluded that in bm14 mice, under the conditions tested, virus-specific CTL are not generated despite excellent tumor immunity. Therefore, this CTL response is not necessary for protection against M-MSV-induced tumors. Protection is likely to be mediated by a normal T-cell proliferative response.

Functional activities and surface markers of the neoplastic T cells of three patients with Sezary syndrome were studied and compared with the functional activities of OKT4+ and OKT8+ T-cell subsets from healthy donors. The neoplastic T cells of the patients had the following marker phenotype: OKT1+3+4+6-8-11+17+I1-M1-3A1-. In contrast to healthy donor OKT4+ T cells, none of the neoplastic T cells had significant helper activity on pokeweed-mitogen-driven Ig synthesis. In one patient the neoplastic T cells suppressed pokeweed-mitogen-driven Ig synthesis. The suppression by the patient cells did not require the presence of radiosensitive OKT4+ suppressor-inducer cells in the culture. It is concluded that OKT4+8-3A1- neoplastic T cells from Sezary-syndrome patients lacking helper activity are more common than previously thought and that in some cases the OKT4+8-3A1- Sezary cells can mediate suppressor activity.

Intravenous injection of spleen cells across mutant class I H-2 incompatibility results in a drastic donor-specific prolongation of skin allograft survival and a marked decrease in the donor-specific cytotoxic T lymphocyte precursor (CTLp) frequency. This immunosuppressive effect depends on the presence of radiosensitive T cells in the donor cell inoculum. It was excluded that a graft-vs.-host reaction was responsible for the observed effects. In mixing experiments, spleen cells from animals transfused with allogeneic lymphocytes could not suppress a normal CTL response against the alloantigen, despite an excess of putative recipient-derived spleen suppressor cells. The data are compatible with the idea that donor T cells function as veto cells which inactivate recipient CTLp directed against the alloantigen expressed by the veto cell.

The recently discovered B6.C-H-2bm12 (bm12) histocompatibility mutant bears a mutation of the I-A subregion of the H-2b haplotype. We have studied cytotoxic T lymphocytes (CTL) generated in secondary mixed lymphocyte culture (MLC) between the bm12 mutant and the strain of origin C57BL/6 (B6). In both directions, strong CTL responses were generated against lipopolysaccharide-stimulated, but not against concanavalin A-stimulated target cells. Studies of the specificity of the bm12 anti-B6 CTL response, using target cells from a panel of H-2 recombinants, showed that the CTL were directed against specificities of the I-Ab subregion. This conclusion was confirmed by the observation that the bm12 anti-B6 CTL could be specifically blocked by anti-Iab, but not by anti-Kb antisera. EL4 tumor cells, which express Kb and Db but not Iab antigens, were not lysed by these CTL. The combined data confirm that bm12 is an I-A subregion mutant and indicate that the I-A subregion antigens, recognized by antibody and those recognized by CTL, are probably present on the same molecule. The CTL effector cells generated in secondary MLC across the I-Ab mutant difference had the surface phenotype Lyt 1−2+.

Summary. In this report a simple procedure for the production of murine monoclonal antibodies (MoAb) against the idiotype of malignant B cells is described. Mice were immunized with lymphoid cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL). After fusion of the spleen cells, hybridoma supernatants were screened for anti-idiotypic MoAb in ELISA with immunoglobulins obtained from tumour-cell lysates, xenohybridomas and patients' sera. The anti-idiotypic MoAb were used to study tumour cells and serum immunoglobulins (Ig) from four different patients with B-CLL. It was found that the serum IgM and IgD in one patient shared the same idiotype. Evidence is presented that IgG-secreting cell populations are not restricted to λ-Ig-light chain-expressing B-CLL cells. With the help of anti-idiotype MoAb accurate measurements of total and idiotype-positive serum immunoglobulin levels during chemotherapy were possible.

A method for infection of lymphocytes with Moloney(Abelson) murine leukemia virus [M(A)-MuLV] is described. Only lymphoblasts obtained after stimulation of normal spleen cells by the B cell mitogen lipopolysaccharide (LPS) were satisfactory targets for virus-specific, secondary cytotoxic T lymphocytes (CTL), whereas spleen cells stimulated by the T cell mitogen concanavalin A were not. The secondary CTL response against M(A)-MuLV could be efficiently measured using M(A)-MulV-infected LPS blasts as stimulating cells for secondary in vitro restimulation and as target cells for virus-specific destruction. Cold target inhibition demonstrated virus specificity of CTL. The T cell character of the cytotoxic cells was demonstrated by their sensitivity to anti-Thy-1.2 treatment. Using syngeneic virus-infected LPS blasts as target and stimulator, CTL responses were measured with effector cells from C57BL mice of the H-2b haplotype and of recombinant haplotypes sharing either K or D alleles with H-2b. In analogy with previous studies on Moloney virus-specific CTL, it was observed that C57BL/6 (H-2b) effector cells predominantly lysed Db-compatible, virus-infected target cells; B10.A(5R), (KbDd) effector cells showed a poor CTL response against syngeneic, virus-infected target cells. The combined findings indicate the existence of an Ir gene in the H-2D region regulating the CTL response against Moloney leukemia virus.

A monoclonal antibody, designated CLB-LFA-1/1, directed to the human lymphocyte-function-associated antigen 1 (LFA-1) was raised by immunization of mice with the peripheral blood lymphocytes of a T gamma lymphocytosis patient. The monoclonal antibody was selected by inhibition of the natural killer cell and the antibody-dependent killer cell activity of the patient's T gamma lymphocytes. In addition, the monoclonal antibody was shown to inhibit the cytotoxic activity of T cell clones specific for either class I or class II HLA molecules. The antigen recognized by CLB-LFA-1/1 consisted of three polypeptide chains with molecular weights of 180 000 (alpha), 155 000 and 94 000 (beta). The antibody reacted with T cells, B cells, monocytes and granulocytes, and stained normal T gamma cells and T gamma cells of patients with T gamma lymphocytosis two- to threefold stronger than normal T cells. It was shown that LFA-1 and the Fc receptor on T gamma cells did not comodulate and it is therefore concluded that Fc receptors and LFA-1 are independent membrane structures, both required for the killer cell activity of T gamma cells.

We produced and characterized a panel of anti-HLA class II monoclonal antibodies (mAb) to study the inhibition of T cell-dependent B cell differentiation. The newly produced mAb belonged to different isotypes and subclasses, and recognized at least two different nonpolymorphic HLA class II determinants. We found that anti-HLA class II mAb inhibited the pokeweed mitogen (PWM)- and interleukin 2-driven T cell-dependent IgM synthesis, but did not affect the phorbol myristate acetate-, phytohemagglutinin-, anti-T3- or PWM-induced T cell proliferation. Moreover, when the mAb were tested in a monocyte-independent B cell differentiation assay in which T cells are activated by insoluble anti-T3 mAb in the absence of accessory cells, no effect was seen on IgM synthesis. These data indicate that inhibition of T cell-dependent B cell differentiation by anti-HLA class II mAb is caused by interference with an accessory cell function essential for B cell differentiation.