Cell-mediated anti-tumor response in the RL♂61 system II. Control of the T killer cells by an H-2-linked Ir gene interfering with non-H-2 factors

The generation of cytolytic T lymphocytes (CTL) during the immune response directed against the syngeneic X-ray-induced BALB/c RL♂ 1 leukemia has been described previously: T killer cells react with a tumor antigen of RL♂ 1 cells, and the H-2D^d molecules of the target cells play some role in the interaction. The study of anti-RL♂ 1 responses of [(C57BL/6 × BALB/c) × BALB/c] mice shows that the major histocompatibility complex controls the anti-RL♂ 1 CTL reaction at a second level, through an Ir gene, with dominant responsiveness, probably mapping to the right of I-B and controlling the high or low-CTL responder phenotype. Another non- H-Associated gene interferes with the H-2-linked Ir gene, a responder allele at the non-H-2 locus, being necessary for the expression of the high-responder phenotype at the Ir gene locus.     

Cell-mediated cytotoxicity. Characterization of the effector cells.

Isolated human mononuclear cells were fractionated according to their membrane characteristics or physical properties. Adherent cells were depleted by filtration through glass columns; phagocytic cells were removed by iron treatment and cell subpopulations capable of forming rosettes with sheep erythrocytes (E), erythrocyte-antibody-complement (EAC) and chicken erythrocyte-antibody complexes (CEA) were separated by centrifugation of Ficoll-Hypaque gradients. The functional activity of the cell subpopulations obtained was assayed by testing PHA-induced cytoxicity (PIC), antibody-dependent cytoxicity (ADCC) and blast transformation by PHA. The results of this study demonstrate that: (1) cells reacting in PIC and ADCC assays are different, adherent and phagocytic cells being necessary for full expression of PIC and not for ADCC; (2) PHA induces direct blast transformation of purified E-RFC in the absence of PIC cytotoxic cells; (3) cell populations specifically enriched in E or EAC rosette-forming cells are not cytotoxic neither in the PHA nor in antibody mediated cytotoxic assays; (4) cells participating in ADCC can be selectively purified by centrifugation of CEA rosettes.     

JNK2 negatively regulates CD8+ T cell effector function and anti-tumor immune response

JNK1 and JNK2 have distinct effects on activation, differentiation and function of CD8+ T cells. Our early studies demonstrated that JNK1 is required for CD8+ T cell-mediated tumor immune surveillance. However, the role of JNK2 in CD8+ T cell response and effector functions, especially in anti-tumor immune response, is unknown. To define the role of JNK2 in antigen-specific immune response, we have investigated CD8+ T cells from OT-1 CD8+ transgenic mice in response to either high- or low-affinity peptides. JNK2-/- CD8+ T cells proliferated better in response to both peptides, with more cell division and less cell death. In addition, JNK2-/- CD8+ T cells produced higher levels of IFN-gamma, which is associated with increased expression of T-bet and Eomesodermin (Eomes). Moreover, JNK2-/- CD8+ T cells expresses high levels of granzyme B and show increased CTL activity. Finally, the enhanced expansion and effector function of JNK2-/- CD8+ T cells was further evidenced by their capacity to delay tumor growth in vivo. In summary, our results demonstrate that JNK2 negatively regulates antigen-specific CD8+ T cell expansion and effector function, and thus selectively blocking JNK2 in CD8+ T cells may potentially enhance anti-tumor immune response.     

Cell-mediated immunity to herpes simplex virus: specificity of cytotoxic T cells.

This communication deals with the question of which of the viral antigens constitutes the targets for cytotoxic T lymphocytes (CTL) generated against herpes simplex virus type 1 (HSV-1). The approach used was, first, to compare cytotoxicity of CTL against target cells infected with virus in the presence of tunicamycin and 2-deoxy-D-glucose, which are known to inhibit glycoprotein synthesis, and second, to compare cytotoxicity of CTL against target cells infected with wild-type HSV-1 with that against target cells infected with a temperature-sensitive mutant of HSV-1 which, at the nonpermissive temperature, exhibits diminished glycoprotein synthesis. The results show that glycoprotein expression is required for the demonstration of cytotoxic activity of CTL. The level of cytotoxicity against the temperature-sensitive HSV-1 target at the nonpermissive temperature was reduced and correlated with the level of expression of the major envelope glycoprotein region (VP123; molecular weight = 123,000) at the target cell surface as measured serologically by antibody binding studies. The results were interpreted to indicate that HSV-1-induced glycoproteins are the target antigens for anti-HSV CTL and that the principal viral antigens recognized by the CTL may be glycoproteins of the VP123 region.     

Lyt phenotype and H-2 compatibility requirements of effector cells in the delayed-type hypersensitivity response to dengue virus infection.

The nature of effector cells which mediate delayed-type hypersensitivity (DTH) to dengue type 2 virus and the influence of the H-2 complex on the cellular transfer of DTH was investigated. The DTH effector cells appeared to consist of two types of T cells, one Lyt-1.1+, the other Lyt-2.1+ or, alternatively, an interaction between these two T cell subsets may be required for maximal expression of DTH. Cellular transfer of DTH reactivity also required compatibility at the K and D or I region of the H-2 complex. Compatibility at the D region alone was not sufficient for transfer of DTH. The significance and implications of these findings are discussed.     

Concomitant presence of anti-tumor effector cells and suppressor cells in the spleen of tumor-bearing mice : the nature of suppressor cells

In order to investigate the immunological responsiveness of tumor-bearing hosts to tumor cells, splenic suppressor cells from Ehrlich tumor-bearing mice that inhibited anti-tumor effector cell activity were characterized. In vitro cell-mediated cytoxicity and cytostasis assays were performed to test for the existence of anti-tumor immunity. suppressive activity assayed by cell mixture experiments became apparent with decline of anti-tumor immunity and progressive tumor growth. The cells mediating the suppression were found to be nylon wool column adherent T cells and inhibited T cell dependent cytotoxicity rather than non-T cell dependent cytostasis. In vivo cell transfer experiments demonstrated that intravenous injection of suppressor cells to a host already inoculated with tumor cells mixed with antitumor effector cells resulted in significant enhancement of tumor growth. This inhibition of in vivo neutralization assay be suppressor cells was found in not only allogeneic but also syngeneic tumor system. Splenectomy at the time of tumor resection endowed the host with stronger resistance against subsequent reinoculated tumor than sham-splenectomy did, reflected by prolonged survival times. These results suggest that splenectomy combined with surgical removal of the tumor is a useful treatment of clinical malignancies.     

Influence of glycosylphosphatidylinositol-linked H-2Dd molecules on target cell protection and natural killer cell specificity in transgenic mice

The expression of certain major histocompatibility complex (MHC) class I ligands on target cells is one important determinate of their susceptibility to lysis by natural killer (NK) cells. NK cells express receptor molecules that bind to MHC class I. Upon binding to their MHC class I ligand, the NK cell is presumed to receive a signal through its receptor that inhibits lysis. It is unclear what role the MHC class I molecules of the effector and target cells play in signaling to the NK cell. We have investigated the role of the cytoplasmic and transmembrane domains of MHC class I molecules by producing a glycosylphosphatidylinositol (GPI)-linked H-2D^d molecule. The GPI-linked H-2D^d molecule is recognized by H-2D^d-specific antibodies and cytotoxic T lymphocytes. Expression of the GPI-linked H-2D^d molecule on H-2^b tumor cells resulted in protection of the tumor cells after transplantation into D8 mice (H-2^b, H-2D^d) from rejection by NK cells. In addition, NK cells from mice expressing the GPI-linked H-2D^d molecule as a transgene were able to kill nontransgenic H-2^b lymphoblast target cells. The GPI-linked MHC class I molecule was able to alter NK cell specificity at the target and effector cell levels. Thus, the expression of the cytoplasmic and transmembrane domains of MHC class I molecules are not necessary for protection and alteration of NK cell specificity.     

A monoclonal antibody recognizes a subset of the H-2Dd mouse major class I antigens

Binding studies and competition experiments have shown that a monoclonal antibody (mAb) named 28-8-6 recognizes only 5 to 10% of the cell surface Dd molecules. The molecules detected by 28-8-6 mAb appear to be genuine H-2Dd antigens on the basis of their MW and isolectric points. In addition, the detectability of the subset of cell surface Dd molecules by 28-8-6 does not depend on their degree of glycosylation nor on the presence of mouse beta-2-microglobulin. Several interpretations are discussed. mAb 28-8-6 might detect a particular conformation or a particular chemical derivatization of otherwise normal H-2Dd molecules. Also, because the epitope recognized by 28-8-6 lies close to the peptide binding site, it is possible that mAb 28-8-6 recognizes a subset of Dd molecules bearing a certain category of self peptides.     

Cytotoxic T cell response to lymphocytic choriomeningitis virus: Properties of precursors of effector T cells, primary effector T cells and memory T cells in vitro and in vivo

Spleen cells from CBA/H mice pre-primed intravenously at various intervals with lymphocytic choriomeningitis (LCM) virus were tested for their capacity to respond and generate cytotoxic effector T cells on secondary stimulation both in vitro and in vivo. In vitro secondary stimulation was performed by culturing preprimed spleen cells with infected, syngeneic, peritoneal `stimulator' cells for periods of up to 7 days at 37°. Controls were incubated with uninfected `stimulators'. In vivo secondary stimulation was obtained by intravenous transfer of pre-primed spleen cells into irradiated, heavily infected CBA/H recipients at various times prior to assay of recipients' spleens. Controls were uninfected, irradiated recipients. Effectors were assayed against LCM virus-infected or uninfected, H-2 compatible L929 target cells in a ⁵¹Cr release assay.

Spleen cells gave three different patterns of cytotoxic T cell response following in vitro or in vivo stimulation, depending upon the interval between priming and secondary stimulation. Populations taken from mice approximately days 2–5 post-infection (PI) developed increasing cytotoxic activity against virus-infected targets in vitro when cultured with infected or uninfected peritoneal cells, or in vivo when transferred into irradiated, uninfected recipients. However, these early populations did not generate cytotoxic activity when transferred into irradiated, heavily infected recipients. Established primary effector populations (i.e. approx. days 7–11 PI) exhibited continuing effector activity when maintained in vitro or in vivo, more so when peritoneal `stimulator' cells or irradiated recipients were infected. Memory populations (i.e. more than approx. day 13 PI) developed highly potent secondary effectors when cultured with infected peritoneal cells, or on transfer to irradiated, infected recipients.

The three different patterns of cytotoxic T cell response of the different preprimed spleen cell populations can be interpreted as indicating three different phases of functional activity of the same population of antigen-reactive T cells from LCM virus-primed mice.

     

Generation of effector cells from T cell subsets III. Synergy between Lyt-1 and Lyt-123/23 lymphocytes in the generation of H-2-restricted and alloreactive cytotoxid T cells

Lyt-123/23 and Lyt-1 T cell subsets, positively selected by separation on the fluorescence-activated cell sorter, were tested in vitro for their role in the generation of H-2-restricted and alloreactive cytotoxic T cells. It is shown that in the proliferation assay, both T cell subsets responded equally well to H-2 and non-H-2 antigens (H-Y), respectively. In contrast, none of the selected Lyt subsets, but only the mixed population containing Lyt-1 and Lyt-123/23 lymphocytes gave rise to both H-2-restricted (anti-H-Y, anti-trinitrophenyl) and alloreactive (anti-H-2) cytotoxic lymphocytes. The data imply an essential role of Lyt-1 cells as inducers or helpers in the generation of all cytotoxic lymphocytes from their precursors in the Lyt-123/23 pool.     

Committed memory effector type 2 cytotoxic T (Tc2) cells are ineffective in protective anti-tumor immunity

Cytotoxic CD8+ T cells (Tc) are a major effector cell population in protection against tumor growth and classified into Tc1 or Tc2 based on their cytokine-secreting profiles. However, their relative tumor protective roles remain undefined. In the present study, CD8+ memory T cells were obtained from mice given with CT26-IL 12 and tumor-specific Tc1 and Tc2 cells were induced by in vitro primary stimulation (1 degrees). In vivo anti-tumor immunity and in vitro cytotoxicity of 1 degrees Tc2 memory effector cells were highly protective comparably to 1 degrees Tc1, but they secreted high level of IFNgamma as well as IL 4 and IL 5. Moreover, memory cells obtained again from tumor-protected mice by either 1 degrees Tc1 or Tc2 transfer showed indistinguishable, Tc1-like, cytokine profiles. These results strongly suggest that 1 degrees Tc2 cells are insufficiently polarized. Tc2 memory effector cells were therefore examined for their transitional anti-tumor activity during consecutive stimulation until Th2 commitment. Secondary stimulation (2 degrees) markedly reduced secretion of IFNgamma (by 94%) and in vivo tumor protection (by 83%). Tertiary (3 degrees) and further stimulation completely abrogated both of tumor protective activity and IFNgamma secretion of Tc2 cells. This progressive loss of activity following repeated stimulation was accompanied by a reduction of in vitro cytotoxicity to CT26 tumor cells. In addition, when 1 degrees Tc2 cells were trans-differentiated to Tc1 during secondary stimulation, 2 of 6 cultures recovered tumor protective activity concomitantly with IFNgamma secretion, indicating that repeated stimulation does not deteriorate tumor protective activity of 2 degrees Tc2 cells. Collectively, these data demonstrate that highly committed Tc2 cells are ineffective in tumor protection.     

Prostaglandin I2 analogs inhibit Th1 and Th2 effector cytokine production by CD4 T cells

An anti-inflammatory effect of PGI(2) has been suggested by increased inflammation in mice that are deficient in the PGI(2) receptor (IP) or in respiratory syncytial viral- or OVA-induced CD4 T cell-associated responses. To determine the mechanism of the anti-inflammatory effect, we hypothesized that PGI(2) analogs inhibit CD4 T cell effector cytokine production. To test this hypothesis, we activated purified CD4 T cells with anti-CD3 and anti-CD28 antibodies under Th1 and Th2 polarizing conditions for 4 days and restimulated the T cells with anti-CD3 in the presence of PGI(2) analogs for 2 days. We found that PGI(2) analogs (cicaprost and iloprost) inhibited the production of Th1 cytokines (IFN-gamma) and Th2 cytokines (IL-4, IL-10, and IL-13) in a dose-dependent pattern. The inhibitory effect was partially dependent on the IP receptor signaling and was correlated with elevated intracellular cAMP and down-regulated NF-kappaB activity. Pretreatment of the CD4 T cells with 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, to inhibit a key signaling molecule in the cAMP pathway, protein kinase A (PKA), attenuated the suppressive effect of PGI(2) analogs significantly, suggesting that PKA, in part, mediates the inhibition of the cytokine production. These data indicate that PGI(2) analogs have an immune-suppressive effect on previously activated and differentiated CD4 T cells in vitro and suggest that PGI(2) may have a similar function in vivo.     

Cell-mediated cytotoxicity against ectromelia virus-infected target cells. III. Role of the H-2 gene complex

The role of the H-2 gene complex in expression of cytotoxicity exerted by specific ectromelia-immune thymus-derived (T) cells against ectromelia-infected target cells was examined. A repertoire of inbred mouse strains (some congenic) including the H-2 haplotypes k, d, b, s, q, the recombinant H-2^a (k|d) and F₁ hybrids (k/b and d/b) were immunized with virus and their spleen cells tested 6 days later, at the peak of the primary response, against H-2^k, H-2^d and H-2^b target cells. Significant specific cytotoxicity occurred only when the immune cell donors and the target cells shared all or part of the same H-2 gene complex. For example, H-2^a (k|d) immune cells killed both H-2^k and H-2^d target cells. There wasno detectable effect of the non-H-2 genetic background, H-2 public specificities, or the M-locus Target cells infected with ectromelia virus exhibited quantitative or qualitative changes (or both) in expression of normal H-2 antigens as indicated by reduced susceptibility to killing by T cells activated against H-2 antigens in mixed lymphocyte culture. These data are consistent with the hypothesis that T cells in this system are responding to virus-induced, specific changes in antigens on infected cells which are controlled by genes in the H-2 complex; these genes seem likely to be those coding for H-2 private specificities, or genes closely linked to them.     

Tissue distribution, antigen specificity and effector functions of gamma delta T cells in human diseases

Conclusions  In conclusion, the large number of studies on human γδ T cells have shown that these lymphocytes share several characteristics in common with αβ T cells, and also embody many unique properties. Some investigations have perhaps suffered a constant analogy with the αβ T cell population, which has precluded new and original experimental approaches. Nevertheless, this gigantic amount of work has provided solid clues for defining the role of human γδ T cells in diseases. In addition, we have also learnt more about the extreme plasticity of the immune system and its polymorphic capacity to adapt and recognize foreign molecules.     

A region of the influenza A/NT/60/68 PB2 protein containing an antigenic determinant recognized by murine H-2Dd restricted cytotoxic T lymphocytes

We have used a recombinant vaccinia virus to investigate the recognition of the PB2 protein of influenza A/NT/60/68 (H3N2) by murine polyclonal CTL populations. PB2 is recognized as a major cross-reactive target antigen. Recognition of PB2 is under strict genetic control, since BALB/c (H-2d) but not CBA (H-2k) mice are responders. We also demonstrate, by use of cell lines transfected with individual genes encoding class I molecules of the H-2d haplotype, that recognition of PB2 occurs in conjunction with the H-2Dd but not the H-2Kd or H-2Ld molecules. In contrast, recognition of the nucleoprotein of A/PR/8/34 by BALB/c-derived polyclonal CTL is restricted via the H-2Kd molecule. By using three recombinant vaccinia viruses expressing deleted forms of the PB2 protein we show that at least one epitope of the PB2 protein resides within the amino-terminal 256 amino acids. This approach offers an effective method to map the regions of large proteins containing epitopes recognized by CTL.     

Immunological activity of a T hybrid line. I. Production of an H-2-related suppressor factor with specificity for sheep red blood cells.

T lymphocyte hybrid lines have been produced by fusion of the thymoma BW 5147 with spleen cells of C57BL/10 mice primed to sheep red blood cells (SRBC). The supernatant (culture fluid) of a T hybrid designated A 1 was able to suppress the primary (IgM) and secondary (IgM and IgG) antibody responses to SRBC in vitro. The suppressive activity of supernatants could be titrated to 50% end points at final dilutions of up to 1 : 270. The suppression affected only SRBC and haptens coupled to SRBC, except when used at high concentration when some nonspecific suppression was observed. Absorption of the A 1 supernatant with SRBC removed all activity, while several other species of red cells failed to do so. The suppressor factor present in A 1 supernatant was not removed by anti-Ig adsorbents, but was removed by anti-H-2 antibodies reacting specifically with the haplotype of the spleen cells used in the fusion (H-2b). The cellular target of action of the factor was apparently a B cell, based on absorption with different cell populations. No genetic restrictions in the activity of the factor were found. A 1 cells carried H-2 and Thy-1 alleles of both parental cells and formed rosettes with SRBC.     

Absence of four H-2d antigenic specificites in an H-2d sarcoma.

MCG4 is a BALB/c sarcoma induced as a solid tumour with 0.2 mgs of methylcholantrene. The primary tumour was serially transplanted subcutaneously in syngeneic mice. The ascites form obtained was sued to study the expression of H-2 antigeneic specificities in a postlabelling radioassay. MCG4 did not express H-2D.4 (private specificity of H-2d haplotypes) as well as H-2.3, H-2.8 and H-2.13 (public specificities). In addition it expressed H-2.5 (a public specificity not present in H-2d cells). These results were confirmed by quantitative absorption analysis using MCG4 and positive-negative normal lymphoid cells for a particular specificity. Results are discussed with regard to the control of expression of H-2 antigens by regulatory genes.     

Fine specificity of the H-2 linked immune response gene for the gallinaceous lysozymes.

An immune response (Ir) gene is described which controls the ability of mice to respond to seven very closely related gallinaceous egg white lysozymes (GEL). This Ir-GEL gene locus is linked to the major histocompatibility locus of the mouse and operates at the level of the T cell. Responsiveness to the nonimmunogenic prototype hen egg white lysozyme (HEL) is dominant and is unrelated to age or sex of the animals, or to dose of protein administered. Ninety percent of C57BL/6 mice are absolute nonresponders to the nonimmunogenic lysozymes in complete Freund's adjuvant. The remaining mice exhibit severely restricted responses, with different anti-HEL clonotypes appearing in individual mice. The fine specificity of the Ir-GEL locus is evident in the discrimination of as few as two amino acid differences in a single region of the lysozyme molecule. This very precise distinction determines whether there will, or will not, be any response to the multideterminant molecule.     

Properties of T cells synthesizing macrophage migration inhibition factor in the H-2 system

It was shown by means of the indirect macrophage migration inhibition test in the H-2 system, using fractionated lymphocytes, that macrophage inhibition factor (MIF) is produced by T but not by B cells. Cells producing MIF are more sensitive to the action of anti--antibodies than T-killer cells. MIF formation by lymph node cells was detected at earlier periods after immunization than the cytotoxic activity of these cells. The results obtained are evidence of differences between the subpopulation of T cells synthesizing MIF and cytotoxic T lymphocytes.Laboratory of Immunochemistry and Diagnosis of Tumors, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 6, pp. 701–704, June, 1978.