Analysis of the cellular immune response to and adoptive immunotherapy of a BALB/c lymphoma that cross-reacts with normal DBA/2 cells

Summary We have previously shown that YC8, a Moloney virus-induced BALB/c lymphoma, is susceptible to lysis by BALB/c anti-DBA/2 effector cells. To further evaluate the relationship between non-H-2 antigens of DBA/2 background and tumor-associated determinants, we investigated the pattern of cytotoxic and proliferative responses induced in BALB/c mice by immunization with YC8 lymphoma. Spleen cells from tumor-immunized animals were restimulated in vitro with YC8 cells and tested for cytotoxicity on target cells of different strains and haplotypes. Cytotoxicity was observed only against YC8, DBA/2 blasts, and P815 (a DBA/2 mastocytoma). BALB/c anti-YC8 effectors were equally blocked by unlabeled YC8 and P815 cells when assayed on YC8 labeled targets. A clonal analysis, however, revealed the existence of at least two types of effectors, one lytic for both P815 and YC8 cells, the other lytic for YC8 cells only. BALB/c anti-YC8 cells proliferate when stimulated both with YC8 and DBA/2 cells in the presence of accessory cells. When tested in an adoptive transfer assay, anti-YC8 cells given i.v. cured 100% of i.v. and 75% of i. p. tumor-injected mice, respectively. When given i. p. anti-YC8 effectors cured 100% of i. p. tumor-injected animals. These results confirm the expression of non-H-2, DBA/2-like antigens on the BALB/c lymphoma YC8 and reveal the presence of additional tumor-associated determinants; both sets of antigens may induce a cellular immune response and elicit immune lymphocytes which can eradicate YC8 cells in an adoptive immunotherapy assay.     

Adoptive immunotherapy of a BALB/c lymphoma by syngeneic anti-DBA/2 immune lymphoid cells: Characterization of the effector population and evidence for the role of the host's non-T cells

Summary It has been previously shown that the BALB/c lymphoma YC8 is susceptible to lysis by syngeneic anti-DBA/2 lymphocytes and that YC8-bearing BALB/c mice can be cured by adoptive transfer of such immune effectors. In this study in vivo and in vitro functions of the curative immune lymphocytes have been evaluated together with the role of the host immune system in the mechanism of tumor eradication. It was found that the curative anti-DBA/2 lymphocytes were not directly cytotoxic to YC8 cells although they developed into YC8-lytic cells after in vitro restimulation with YC8. In vivo, the immune lymphocytes were able to mediate a tumor-specific delayed type hypersensitivity reaction against YC8 but had a low tumor-neutralizing activity in the Winn assay. Proliferation of infused BALB/c anti-DBA/2 lymphocytes was necessary for the in vivo therapeutic effect, since irradiation of effector cells or treatment of the donor immune lymphocytes with vinblastine abolished their curative capacity. Immunodepression of the T cell compartment of the prospective tumor-bearing animals by thymectomy plus irradiation or its abrogation in B mice (thymectomized, lethally irradiated, and reconstituted with fetal liver cells) did not interfere with the therapeutic effect of the transferred anti-DBA/2 lymphocytes. Blocking the macrophage functions of the host by carrageenan, however, abolished the therapeutic effect of immune lymphocytes. These data indicate that a radiation-resistant, non-T cell is involved in the tumor eradication induced by anti-DBA/2 lymphocytes. It was also shown that cured mice, tested 90 days after therapy, become resistant to 5×10³ LD₈₀ YC8 cells and that this resistance was due to the presence of memory cells derived from the transferred and not from the host lymphocyte population.     

Differential resistance to growth of a tumor expressing incompatible minor alloantigens reflects regulatory influences rather than differences in anti-minor-CTL-P frequencies

In previous studies it was found that BALB/c (H-2d) was more susceptible than (BALB/c X A)F1 (H-2d X H-2a) to a tumor bearing multiple mismatched minor histocompatibility antigens, the DBA/2 (H-2d) mastocytoma P815, and that this resistance was H-2 linked. In the present studies the immunologic basis of this effect was examined by comparing the cytotoxic-T-lymphocyte (CTL) responses of BALB/c with those of (BALB/c X A)F1. Despite the BALB/c X A)F1's 34-fold greater resistance to P815 in vivo, the numbers of effector cell precursors were found to be similar in the two hosts as shown by (a) similar anti-P815 CTL responses in vitro with T-cell growth factor, (b) similar secondary anti-DBA/2 MiHA responses after in vivo priming with irradiated P815, and (c) similar frequencies of anti-DBA/2 CTL precursors by limiting-dilution analysis. However, priming with proliferating P815 in vivo revealed a defect in the BALB/c animals: Spleen cells from such animals were unable to control the growth of contaminating P815 cells in vitro or to mount strong secondary CTL responses to DBA/2 antigens. The defective priming of BALB/c could be corrected when DBA/2 spleen cells were added to the P815 inoculum. This impaired priming by living tumor cells was not seen in (BALB/c X A)F1. It is concluded that the use of living P815 tumor cells revealed a defect in immunoregulation in BALB/c mice, which rendered them susceptible to tumor growth in spite of apparently adequate numbers of anti-minor-CTL precursors. How the additional H-2 products expressed in the (BALB/c X A)F1 might correct this defect is discussed.     

Generation of cytotoxic lymphocytes to syngeneic tumor by using co-stimulator (Interleukin 2)

Cytotoxic lymphocytes (CL) highly active against the syngeneic mastocytoma, P815, were generated from spleen cells of DBA mice cultured with co-stimulator (Interleukin 2) and P815. More CL activity was generated from spleen cells of P815 tumor-bearing mice than from spleen cells of normal mice. Thymus cells from tumor-bearing mice, however, did not produce increased CL activity. Most of the CL were Thy 1 and Ly 1 positive. The optimal culture conditions and kinetics were similar to those for the generation of allogeneic cytotoxic T lymphocytes. The cytotoxic activity against syngeneic P815 was similar in magnitude to the response of DBA spleen cells to allogeneic tumor lines and to the response of allogeneic CBA spleen cells to P815. Although CL generated from tumor-bearing mice did not lyse normal DBA cells, they did lyse, to a much lesser degree, a number of tumor cell lines other than the sensitizing P815. This nonspecific lysis was not H2 restricted nor was it restricted to tumors of lymphoid origin. Generation of nonspecific cytolytic activity was antigen independent, occurring in the presence of co-stimulator alone.     

The blocking activity of antisera raised to either tumor antigens or alloantigens in cytotoxicity assays using syngeneic or allogeneic killer cells on the P815 murine mastocytoma target

Methods have been published whereby a tumor-specific antigen associated with membranes of the P815 mastocytoma of DBA/2J mice was purified. Antiserum, raised in rabbits, to this material demonstrated specificity for P815 as opposed to other cells or materials of DBA/2J origin when tested by either complement-mediated target cell lysis or the enzyme-linked immunosorbent assay ELISA. This antiserum was tested for its ability to block killing by in vitro raised syngeneic lymphocytes cytotoxic for P815. It was found that this antiserum as well as antiserum raised in rabbits to normal DBA/2J membrane components and anti-H-2^d antiserum (raised in congenic mice) were all able to block killing when ⁵¹Cr-labeled P815 targets were pretreated with these antisera. On the other hand, only the anti-DBA/2 serum and the anti-H-2^d serum were capable of slightly blocking syngeneic killing of L1210 cells. Similarly, C57B1/6 cytotoxic lymphocytes raised against DBA/2 cells were blocked by pretreatment of ⁵¹Cr-labeled P815 targets with the rabbit anti-DBA/2 serum and the anti-H-2^d serum but not by the anti-P815 serum. The implications of these observations are discussed.     

H2-restricted recognition of cloned HLA class I gene products expressed in mouse cells

Long-term syngeneic mouse cytolytic T lymphocyte (CTL) clones were obtained from DBA/2 (H2d) mice immunized with P815 (H2d) cells transfected with cloned human class I histocompatibility genes, HLA-CW3 or HLA-A24. Three distinct patterns of specificity were defined on P815 HLA transfectant target cells. One clone lysed HLA-CW3 but not -A24 transfectants, and a second lysed HLA-A24 but not -CW3 transfectant target cells. The third clone lysed P815 targets transfected with either HLA gene. None of the CTL clones lysed L cells (H2k) transfected with the same HLA genes or human targets that expressed these HLA specificities. Several lines of evidence indicated that recognition of HLA transfectants by these CTL clones was H2 restricted. First, lysis of P815 HLA transfectants could be inhibited by anti-H2Kd monoclonal antibody. In addition, the anti-P815-HLA CTL clones could lyse a (human X mouse) hybrid target that expressed both HLA class I and H2Kd antigens, but not a clonal derivative that no longer expressed H2Kd. The most direct evidence for H2-restricted recognition of P815-HLA transfectants by the syngeneic CTL clones was obtained by double transfection of mouse L cells (H2k) with both HLA and H2 class I genes. L cells transfected with HLA and H2Kd genes were susceptible to lysis by the same CTL clones that lysed the corresponding P815-HLA transfectant targets. Thus under certain conditions, CTL recognition of xenogeneic class I histocompatibility gene products can be restricted by other class I gene products.     

Failure of infiltrating precursor cytotoxic T cells to acquire direct cytotoxic function in immunologically privileged sites

Minor H incompatible P815 tumor cells inoculated into the anterior chamber (AC) of the eyes of BALB/c mice grow progressively, revealing this to be an immunologically privileged site. By contrast, a similar inoculation of tumor cells is rapidly rejected from nonprivileged ocular sites (subconjunctiva). Mice with progressively growing AC-tumors and those that reject their ocular subconjunctiva tumors both have expanded clones of tumor-specific cytotoxic precursor cells (pTc) in their spleens and cervical lymph nodes. In an effort to determine why the expanded pool of primed pTc is unable to effect rejection of AC intraocular tumors, we have examined the susceptibility of the tumor cells growing within the immunologically privileged AC to lysis by cytotoxic T cells and the cytotoxic function of tumor-infiltrating lymphocytes. P815 tumor cells extracted from intraocular tumors and P815 cells maintained in routine tissue culture are equally susceptible to lysis when exposed in vitro to fully differentiated, DBA/2-specific cytotoxic T cells. Thus, progressively growing tumor cells within the AC are not insensitive to immune-mediated lysis by cytotoxic T cells. We have been able to harvest significant numbers of DBA/2-specific pTc from these same intraocular tumors. When the tumor-infiltrating lymphocytes are driven in vitro with exogenous IL-2, they acquire the capacity to lyse specifically P815 tumor cells. However, no evidence of fully cytotoxic, tumor-specific T cells was found among lymphocytes harvested from intraocular tumors, i.e., when the harvested cells were tested immediately for cytolytic activity. Inasmuch as we have reported that directly cytotoxic T cells are present during tumor rejection at nonimmunologically privileged ocular sites, such as the subconjunctival space, we conclude that progressive growth of P815 tumor cells within the anterior chamber is due in part to a failure of infiltrating pTc to differentiate in situ into fully functional cytotoxic effector cells.     

Defective T helper activity in the spleen of BALB/c mice immune to a syngeneic fibrosarcoma

Summary BALB/c mice were immunized with the syngeneic 3-methylcholanthrene-induced fibrosarcoma CA-2 by the growth and excision method. When lymphoid cells from different organs of these tumor-free mice were tested in a direct ⁵¹Cr-release assay, peritoneal exudate cells but not spleen cells displayed specific cytotoxicity against the syngeneic tumor target. A cytotoxic response could be obtained by tumor-immune spleen cells when cultured in a mixed lymphocyte tumor cell culture (MLTC) at high but not low density although at the same effector/stimulator ratio. Lack of cytotoxic activity in low density MLTC was not due to an impairment of cytotoxic precursors since cytotoxicity was rescued by adding exogenous interleukin-2 in experimental conditions in which no lymphokine-activated killer cells could develop relevant anti-CA-2 lysis. When low density MLTC were supplemented with either 800 R-irradiated cells or nonirradiated, negatively selected Lyt 1⁺ cells from the same immune mice, induction of a cytotoxic response against CA-2 occurred and interleukin-2 production became detectable. Additional studies indicated that spleen cells of CA-2-immune mice were also impaired in their ability to provide help to syngeneic thymocytes for the generation of cytotoxic T lymphocytes against C57BL/6J alloantigens. Dilution effect of helper cells due to immunization procedures was excluded since spleen cells of mice immunized against another BALB/c tumor, the YC8 lymphoma, or against DBA/2 minor histocompatibility antigens provided good help to thymocytes against the same alloantigens. These results indicate that tumor-immune animals may also have selective T helper defects in an important lymphoid organ like spleen.     

The existence of antibody-like activities against the weakly immunogenic minor histocompatibility antigens

BALB/c mice develop cytotoxic lymphocytes as well as produce specific antibodies against the minor histocompatibility antigens when injected with DBA/2 P815 cells. P815 cells grown in BALB/c mice have IgG antibodies on their surface as demonstrated by the binding of ¹²⁵I-labeled goat anti-mouse IgG and by complement-dependent cytotoxicity. Serum from BALB/c mice hyperimmunized with P815 cells blocked lymphocyte-mediated cytotoxicity by BALB/c immune peritoneal exudate cells. This blocking activity was removed by absorbing hyperimmune serum with DBA/2 spleen cells or P815 cells. This result suggests that specific antibodies were generated against the minor histocompatibility differences between BALB/c and DBA/2 mice. The experimental procedures described may be very useful in demonstrating minute quantities of antibody against minor histocompatibility antigens on tumor cells.     

Mapping of HLA epitopes recognized by H-2-restricted cytotoxic T lymphocytes specific for HLA using recombinant genes and synthetic peptides

Immunization of DBA/2 (H-2d) mice with syngeneic P815 tumor cell transfectants that express HLA class I genes elicits CTL that recognize HLA in the context of H-2Kd molecules. Anti-HLA-CW3 CTL cross-react to a variable extent on the related alleles A3 and A24. Using a panel of target cells expressing native or recombinant HLA genes, we could map the epitope recognized by a CTL clone specific for CW3 to the second external (alpha 2) domain of CW3. Moreover, the epitope recognized by this clone could be mimicked by incubating P815 (HLA negative) target cells with a synthetic peptide corresponding to the C-terminal 12 amino acids of the CW3 alpha 2 domain (residues 171 to 182). Other independent anti-CW3 CTL clones with different fine specificities recognized the same CW3 peptide. In contrast, CTL clones specific for HLA-A24 or HLA-A3 that did not lyse P815-CW3 transfectants did not recognize this peptide. The CW3 peptide could be recognized on other tumor cell targets that were also of H-2d origin, but not on those of H-2b or H-2k origin. The requirement for the expression of H-2Kd by the target cells was directly demonstrated using L cell Kd transfectants. Our results suggest that the CTL response of DBA/2 mice immunized with P815-CW3 transfectants is predominantly Kd restricted and focused on epitopes contained within the 12 C-terminal amino acids of the alpha 2 domain.     

Frequency and cross-reactivity od cytolytic T lymphocyte precursors reacting against male alloantigens

It is well established that cytotoxic T lymphocytes (CTL) specific for the male minor histocompatibility antigen (H-Y) are generated by restimulation in vitro of in vivo primed spleen cells from C57BL/6 (H-2b) female mice with syngeneic male spleen cells. When tested on target cells from H-2 different strains, the male-specific C57BL/6 CTL populations exhibited significant lysis of DBA/2 (H-2d), A (H-2a), but not C3H (H-2k), male and female target cells. In an attempt to document this cross-reactivity further at the clonal level, a sensitive technique of limiting dilution analysis was used to determine the specificity of C57BL/6 individual CTL precursors (CTL-P) reactive against the male antigen. The mean frequency of anti-H-Y CTL-P in spleens of primed female mice was about 1/3500. Between one-third to one-tenth of these CTL-P produced a progeny that cross-reacted with H-2d (allogeneic) female target cells. These findings were confirmed by the analysis of the reactivity pattern exhibited by male-specific CTL clones derived by limiting dilution. Of 99 clones tested, 13 were found to cross-react with female DBA/2 target cells. These results thus indicate that a relatively large proportion (greater than 10%) of H-2b CTL-P directed against the H-Y antigen cross-react with target cells expressing H-2d alloantigens in the absence of H-Y antigen.     

Ontogeny of cell-mediated cytotoxicity: induction of CTL in early postnatal thymocytes.

In this study we characterized the time when cytotoxic T lymphocytes (CTL) can be induced in the thymus and spleen from their immediate CTL precursors (CTL-P). In contrast to fetal or newborn thymus, the thymus of 1 to 2-day-old C57BL/6 mice contained cells that, after cultivation in vitro with allogeneic DBA/2 stimulating cells, exhibited high levels (as great or greater than that induced in adult thymocytes) of CTL activity as measured by the ability to lyse P815 (DBA/2) tumor target cells. However, CTL activity induced in spleen cells remained how during the first 5 days of life, increased sharply between 6 to 9 days, and reached adult levels at 11 to 20 days. Furthermore, early postnatal spleen cells did not suppress the adult splenic CTL response. These results suggest 1) that the full potential to generate CTL in response to an allogeneic stimulus commences in the thymus on the first day after birth and 2) a different temporal appearance of immediate CTL precursors in the thymus and spleen.     

Two epitopes and one agretope map to a single HLA-A2 peptide recognized by H-2-restricted T cells

Mice immunized with syngeneic cells transfected with cloned genes coding for HLA class I molecules could recognize the human MHC Ag in the context of their own H-2 molecules. We obtained CTL clones from DBA/2 mice (H-2d) which had been immunized with P815 cells (a mastocytoma of DBA/2 origin) expressing either HLA-A2 or HLA-A3 or two different molecules containing recombined sequences of HLA-A2 and HLA-A3. Fourteen of these clones recognized a synthetic peptide corresponding to the region 170-185 of HLA-A2 in the context of H-2Kd. Moreover, from their activity on P815 cells expressing HLA-Cw3, two subpatterns could be distinguished: subpattern Cw3+, defined by those clones which lysed P815-Cw3, and subpattern Cw3- defined by those clones which did not lyse P815-Cw3. By testing the activity of clones of each subpattern on a series of modified synthetic peptides, we were able to define two epitopes on the same 170-185 peptide of HLA-A2. One of them was dependent on amino acids at positions 173 and 177, whereas the other was dependent on amino acid 177 alone. By using competition experiments, we were also able to define an agretopic region strongly dependent on the amino acid at position 178. Furthermore, experiments with L cells expressing molecules containing recombined sequences between H-2Kd and H-2Dd demonstrated the determinant role of residues 152, 155, and 156 from H-2Kd in the presentation to murine T cells of the 170-185 peptide of HLA-A2.     

Induction and characterization of minor histocompatibility antigens. Specific primary cytotoxic T lymphocyte responses in vitro

A definite cytotoxic activity was developed in a BALB/c (H-2d) anti-DBA/2 primary mixed leukocyte culture (MLC), which received interleukin 2 (IL-2) on day 3 of culture. This cytotoxic activity was minor histocompatibility antigens (MIHA)-specific at the stimulator level, and was not developed in a syngeneic (BALB/c anti-BALB/c) MLC. The addition of IL-2 on day 3 of culture was crucial; no or very weak cytotoxic activity was developed in MLC receiving IL-2 on day 0 or on both day 0 and day 3. Only appropriate MIHA-allogeneic tumor cells were lysed as the target of the cytotoxic activity. The cytotoxic activity seemed MIHA-specific also at the target level; it lysed tumor cells of DBA/2 mouse origin but not those of BALB/c (syngeneic) origin. Phenotypes of the cytotoxic effector cell were Thy-1+ Lyt-2+. We concluded from these results that MIHA-specific cytotoxic T lymphocytes (CTL) were generated in the MIHA-allogeneic primary MLC. In this newly developed system, we studied genetic and antigenic requirements for primary anti-MIHA CTL responses in vitro. We demonstrated; among spleen cells (SC) of seven B10 H-2-congenic strains only SC of B10.D2 strain whose major histocompatibility complex (MHC) (H-2d) was compatible with the responder MHC effectively stimulated responder BALB/c (H-2d) SC for an anti-MIHA (DBA-C57BL-common) CTL response. Similarly, only SC of two out of seven C x B recombinant inbred strains (C x B.H and C x B.D), which were compatible at the MHC with responder SC, activated responder BALB/c SC for the response. The possibility that cells responding to H-2 alloantigens suppressed the anti-MIHA response was ruled out. Additional experiments showed that compatibility at the H-2K-end or the H-2D-end of the MHC was sufficient for a definite anti-MIHA response. These provided formal evidence that primary anti-MIHA CTL responses in vitro were MHC-restricted at the stimulator level. We then showed that sonication-disrupted SC or Sephadex G-10 column-passed nonadherent SC failed to stimulate responder SC for a primary anti-MIHA CTL response, whereas G-10-passed nonadherent SC responded well to adherent stimulator cells. Further study demonstrated that Ia+ adherent cells were the most active cell type as stimulator. Finally, we confirmed that the primary anti-MIHA CTL responses to adherent stimulator cells was MHC-restricted.     

Limiting dilution analysis of alloantigen-reactive T lymphocytes. IV. High frequency of cytolytic T lymphocyte precursor cells in MLC blasts separated by velocity sedimentation

A sensitive limiting dilution microculture system was used to obtain minimal estimates of the frequency of cytolytic T lymphocyte precursor cells (CTL-P) directed against DBA/2 alloantigens, after priming of spleen cells in unidirectional mixed leukocyte cultures (MLC, C57BL/6 anti-DBA/2). The mean CTL-P frequency in day 4 to 5 MLC populations was found to be approximately 50- to 100-fold greater than the frequency in normal spleen, and up to 25% of the cells present in such MLC could be identified operationally as CTL-P. Even higher frequencies (up to 50%) of CTL-P were obtained in a population of large-sized cells separated from day 4 MLC by velocity sedimentation. Furthermore, since a strikingly quantitative correlation was observed between CTL activity and CTL-P frequency in such separated MLC populations, it is likely that mature CTL in MLC are not end cells, but can further proliferate and thus behave operationally as CTL-P.     

Hierarchy of cytotoxic T cell clones. II. Intraclonal triggering of lysis by hapten-specific CTL

Cells from clones of anti-hapten murine cytotoxic T lymphocytes (CTL) can act as both target and effector cells, but will not lyse members of the same clone. The effect of haptenation on the cytolytic activity of anti-fluorescein (FL) and anti-trinitrophenol (TNP) CTL clones was examined. Treatment of anti-FL clones with fluorescein isothiocyanate or anti-TNP clones with trinitrobenzene sulphonic acid induces these clones to kill in an antigen-independent fashion. Targets killed by the haptenated CTL included syngeneic and allogeneic B lymphocyte blast cells, P815, YAC-1 and in one case human GM 4072 tumor cells. The importance of CD8 and T cell receptor (TCR) occupancy is demonstrated by the ability to block autotriggering by antibody directed against Ly 2 and the TCR. The results demonstrate that effects other than antigen recognition of the target play a role in the final outcome of effector-target cell interactions and provide a mechanism which could lead to autodestruction and immunosuppression particularly in some types of viral infection.     

Limiting dilution analysis of alloantigen-reactive T lymphocytes. III. Effect of priming on precursor frequencies.

The effect of specific priming with alloantigens on the frequency of cytolytic T lymphocyte precursors (CTL-P) has been investigated. Alloimmune lymphoid cells were obtained from the spleen of C57BL/6 (H-2b) mice primed with DBA/2 (H-2d) tumor cells or from 14-day unidirectional mixed leukocyte cultures (C57BL/6 anti-DBA/2). CTL-P frequencies directed against H-2d alloantigens were estimated by limiting dilution analysis in a sensitive micro MLC system. Under these conditions, an apparent increase of 3 to 4-fold in CTL-P frequency was observed in alloimmune (as compared with normal) C57BL/6 spleen cells. Evidence was obtained suggesting that this increase was specific for the priming alloantigens. A much greater increase in CTL-P frequency (25 to 100-fold) was observed after alloimmunization of C57BL/6 spleen cells in unidirectional MLC. Under the latter conditions, 5 to 20% of the surviving splenic MLC cells could be identified operationally as CTL-P. A similar enrichment in CTL-P frequency was obtained when lymph node, peripheral blood, or thymus cells were cultured for 14 days in MLC. These studies provide direct evidence that the pool of specific CTL-P can be expanded after alloimmunization. Furthermore, the very high frequencies observed after in vitro priming indicate that this system should be particularly useful for future studies of the progeny of individual CTL-P.     

Cytolytic T lymphocytes from the BALB/c-H-2dm2 mouse recognize the vesicular stomatitis virus glycoprotein and are restricted by class II MHC antigens.

BALB/c-H-2dm2 mice (H-2KdI-AdI-EdDd), a congenic strain of BALB/c mice, have a deletion of the class I MHC Ag, H-2Ld. This gene encodes the exclusive class I MHC-restricting gene product for vesicular stomatitis virus-specific cytolytic T lymphocytes. When dm2 mice were immunized with infectious vesicular stomatitis virus, a specific CTL response was generated. These CTL lysed VSV-infected targets that expressed Iad gene products, but not VSV-infected Iad- targets. The CTL were used initially as long term cytolytic lines; 13 CTL clones were derived by limit dilution. All of the clones expressed the phenotype CD3+, CD4+, CD8-; some clones expressed TCR that are members of the V beta 8 family, others did not. The clones were restricted by class II MHC Ag, both I-Ad and I-Ed serving as restricting elements for individual clones of the panel. All of the clones derived from dm2 mice were specific for the immunizing serotype, Indiana, of VSV and did not lyse syngeneic cells infected with VSV of the New Jersey serotype. Studies using defective interfering virus particles, UV light-inactivated virus, and purified micelles of the viral glycoprotein indicated that infectious virus was not required for sensitization of target cells for immune recognition by the class II MHC-restricted CTL clones. Additional studies using recombinant vaccinia virus vectors to sensitize targets confirmed the specificity of the clones for the viral glycoprotein. These studies also demonstrated a cryptic population of class II-restricted CTL in BALB/c lines specific for VSV G. Naturally occurring variant viruses and mutant viruses, selected for escape from neutralization by mAb, were used in an effort to map the determinant(s) recognized; on the basis of patterns of target cell lysis, three groups of epitopes recognized by the clones were defined. Therefore, in the absence of the class I MHC Ag required for a CTL response to VSV, dm2 mice generated CTL with the CD4+ phenotype that recognized different epitopes on the viral glycoprotein, and lysed cells in a class II-MHC restricted, Ag-specific manner.     

Avidity dictates the lytic capacity of human cytolytic T lymphocyte clones with similar fine specificity against murine cells expressing HLA-B27 antigen

The role of the avidity of human CTL in the recognition and lysis of murine P815 cells expressing HLA-B27.1 Ag has been examined. Seven B27-specific alloreactive CTL clones were tested for their ability to lyse a B27.1+-P815 transfectant clone 1-7E, obtained after cotransfection of P815-HTR cells with HLA-B27.1 and human beta 2-microglobulin genes. The expression level of HLA-B27.1 on 1-7E cells was comparable to that on a human lymphoblastoid cell line, as determined by flow cytometry. Of the seven CTL clones used, CTL 1, 26, and 29 displayed the same fine specificity as established with a panel of target cells expressing six structurally different HLA-B27 variants. However, CTL 1 and 29 were of higher avidity than CTL 26, in that the lysis of human target cells by only this latter clone was inhibited by an anti-CD8 mAb. Based on the same criteria, CTL 2, 15, and 48 possessed the same or very similar fine specificity, but CTL 48 was of higher avidity than CTL 2 or 15. The seventh clone, CTL 40, was of a different fine specificity and its lysis of human target cells was also inhibited by the same anti-CD8 mAb. Only those clones whose lysis of human targets could not be inhibited by anti-CD8 antibody were able to lyse the 1-7E murine transfectants. These results indicate that, for human CTL clones with identical or very similar fine specificity, only those of higher avidity are able to lyse P815 murine cells expressing the HLA-B27 antigen. The lysis of HLA-B27.1+-murine transfectants by relevant clones was inhibited by anti-CD8 antibody. This result strongly suggests that the relative contribution of CD8 in stabilizing the interaction between human CTL and HLA-B27+-murine target cells is more significant than with human target cells.     

Mechanism of loss of natural killer activity in P815 ascites tumor bearing DBA/2 mice

P815 tumor cells (10(7] were administered intraperitoneally to DBA/2 mice. As the ascites tumor grew in the syngeneic host, a decline leading to a total loss of host spleen natural killer (NK) activity could be demonstrated. Removal of T and B cells or macrophages from the tumor-bearing (TB) mouse spleen cells did not raise the level of NK activity. Spleen cells from TB mice did not inhibit the NK activity of normal spleen cells. Comparable target (YAC cells) binding capacity could be demonstrated in spleen cells derived from normal or TB mice, but interferon failed to significantly stimulate the NK activity of TB mouse spleen cells. In adoptive transfer experiments, transfer of spleen or bone marrow cells from TB mice resulted in the development of significant levels of spleen NK activity in lethally X-irradiated recipient DBA/2 mice. These results indicate that the impairment of NK cell differentiation pathway rather than active suppression at the level of effector cells may be the mechanism of loss of NK activity in P815 TB DBA/2 mice.