Cloned cellular reagents to define antigens encoded between HLA-DR and glyoxylase

Primed lymphocyte typing reagents have been used to define antigens encoded by genes of a locus (loci) mapping between HLA-DR and glyoxalase I. This locus, which we shall refer to as the third locus of the HLA-D region, has been variously referred to as D beta, PL beta, PL3, and SB. Generating discriminatory primed lymphocyte typing reagents which can be used to define these antigens, however, has been extremely difficult. Donors of responding and stimulating cells for the priming combinations have usually been matched not only for the DR, D, and MB/MT antigens but also for the HLA-A, -B, and -C antigens. Even under these very restricted conditions, not all bulk primed lymphocyte typing reagents that are generated are discriminatory enough to be useful for antigen definition. We have derived "clones" from bulk priming combinations in which stimulator and responder differed for known antigens of this third locus. Even though the bulk reagents that were prepared did not provide discriminatory results, approximately 7-12% of the clones derived from the bulk priming combination proved to be highly discriminatory. We have been able to obtain these results with regard to all three antigens of the third locus so far evaluated. The very ease of screening clones and deriving discriminatory reagents, as compared with screening responder-stimulator combinations, allows the ready derivation of cellular reagents that define the antigens of this third locus.     

Detection of non-HLA-DR determinants by alloreactive lymphocyte clones

Using the soft-agar colony assay, we have generated three MT3-associated clones: HJ1, HJ13, and HJ39, from an MLR combination of two unrelated individuals. Another clone, HJ37, appeared to recognize a novel HLA-D determinant. PLT inhibition studies with monoclonal anti-Ia-like antibodies (Mab) were conducted on clones HJ1, HJ39, and HJ37. Five different anti-DR Mab had no significant inhibitory effect on these clones. On the other hand, two Mab SG171 and Q5/13 which appear to react with DR and MT3 (I-A like) molecules strongly inhibited the two MT3-specific PLT clones. While SG171 and Q5/13 had little effect on HJ37, it was observed that a polymorphic Mab 17.15 had a strong inhibitory effect. These results, in concordance with biochemical data on Ia molecules precipitated by these Mab, suggest that these alloreactive clones may recognize non-DR PLT determinants. They also provide further indirect support that MT3 molecules represent the human homologue of murine I-A molecules.     

A method for the accurate determination of anti-hapten cytotoxic T lymphocyte precursors: correction for apparent 'anti-self' reactivity

A method is described for accurately determining the frequency of precursors of hapten specific cytotoxic T cells. The method is based on a standard Poisson analysis of limit dilution cultures, but makes a correction of 'anti-self' reacting clones and for spontaneously arising clones that recognise modified self. These corrections are shown to be especially important when low hapten densities are used, where there may be more than a 10-fold difference between the corrected and uncorrected frequency estimates. Determined levels of antigen specificity and of H-2 restriction are significantly enhanced by application of this method.     

Direct estimation of the frequency of human cytotoxic T lymphocytes and their precursors following in vitro allosensitization

Cell mediated lympholysis (CML) has been proposed as an in vitro model of the rejection process that results from transplantation of allogeneic tissue. To date, the absolute frequencies of cytotoxic T lymphocytes (CTL) and their precursors (CTL.P) have not been directly estimated in man because of technical difficulties. Through optimizing the conditions for radiometric detection of 51Cr release and the attendant improvement in CML sensitivity, direct CTL frequency estimates have been determined in peripheral blood (PBL), spleen (SPL), and lymph nodes (LNC) after in vitro allostimulation using unrelated human cells and limiting dilution assays. The mean frequency of CTL generated from PBL is 1 in 826 cells (0.121% +/- 0.101%) which, from preliminary experiments, is significantly greater than that generated from either LNC or SPL (p less than 0.05). With restimulation of primed cells on day 10, the frequency of CTL generated from PBL was increased 400%. The CTL.P frequency (0.0064% +/- 0.0050%) was approximately 5% of the corresponding CTL frequency. The CTL.P frequencies were found to be minimal estimates as both accessory "filler" cells and T cell growth factors increased the level of detection of CTL.P an average of threefold. The limiting cell dilution assay as detailed in this report should be a powerful tool for defining the cellular requirements and related factors necessary for optimal induction of a CTL response and should provide the means for determination of the immunogenetic requirements and the allospecificity of human cytotoxic lymphocytes.     

Brazilin inhibits mitogen induced cell proliferation despite of augmentation of T cell growth factor(TCGF) production and expression of IL-2 receptors

The present work was designed to investigate the effects of brazilin on ConA-induced TCGF release, responsiveness to standard IL-2, and mitogens-induced proliferation of splenocyte when administered intraperitoneally to 8 week-old C57BL/6 mice for 2 consecutive days. Immunological tests were performed 72 hours after the treatment of brazilin. The administration of 50 mg/kg brazilin caused a noticeable increase in TCGF release and responsiveness to standard IL-2, but inhibited mitogens-induced proliferation of splenocyte. These results indicated that brazilin is able to modulate immunological functions despite of its inhibitory effect on mitogen induced cell proliferation.     

A simple method for the quantitation of specific cytotoxic precursors to synrgeneic tumors

Cytotoxic T lymphocytes (CTLs) to EL4, a syngeneic lymphocytic leukemia of C57BL/6 (B6) (H-2^b) mice, were obtained by culturing normal B6 spleen cells with irradiated EL4 tumor cells for 4 days in conical bottom mitrotiter trays. A much lower, though significant, amount of cytotoxicity towards EL4 was observed in B6 spleen cell cultures not stimulated with EL4. The cytotoxic effector cells were shown to be Thy-1⁺ and their cytolytic activity to EL4 tumor cells was inhibited by an anti-H-2^b serum. Cold target competition studies suggest that the B6 anti-EL4 CTLs could discriminate between EL4 tumor cells and a second H-2^b lymphoma, C1498. The converse experiment yielded similar results. Culture conditions limiting for CTL precursors (CLPs) to EL4 were attained by including interleukin 2 (IL2), a lymphokine obtained by stimulating murine or rat spleen cells with concanavalin A, in the culture medium. In the presence of IL2, the CLP frequencies in B6 spleen cells to EL4 and C1498 in antigen-stimulated cultures were 113 and 231 per 10⁶ responder cells, respectively. In non-antigen stimulated B6 spleen cell cultures, the apparent CLP frequencies to EL4 and C1498 were 6 and 33 per 10⁶ responder cells, respectively. In agreement with the cold target competition studies using CTLs generated in the absence of IL2, we found that the CTL clones produced in cultures stimulated with EL4 and C1498 in the presence of IL2 were specific for the stimulating antigen. The specificity of the CTL clones obtained from cultures stimulated with IL2 in the absence of antigen appears to be different from those derived from antigen-stimulated cultures. The potential applications and limitations of this culture system are discussed.     

Longevity of human allospecific TLCs: Mycoplasma infection as a cause of in vitro “suppression” of MLC

It has been suggested that allospecific T-cell clones lose specific reactivity after approximately 30 cell doublings and subsequently acquire suppressor and NK-like characteristics. We have tested this hypothesis by assaying paired functional and nonfunctional TLCs for suppressor activity in PLT and MLC cocultures. Two sets of clones were initially studied: the first pair consisted of clone S5.2B, a functional TLC, and S5.14A, a nonfunctional TLC; the second pair of clones tested was comprised of two different expansions of the same clone S5.5A (nnfunctional) and S5.5B (functional). These experiments yielded no evidence for suppressive activity by nonfunctional clones toward functional clones, furthermore, the addition of nonfunctional clones to primary MLC assays had no effect on the level of responsiveness. Eight clones were subcloned and 89 subclones were retested for function after approximately 50 cell doublings. Generally, the subclones failed to suppress MLC proliferation. A minority of TLCs could suppress MLC responses, but this “suppression” was reversible with the addition of 2% exogenous TCGF. However, eight subclones and two parental TLC lines did suppress MLC responses in the presence or absence of TCGF, but the suppressive effects in such cocultures were reversible in the presence of tylocine, an anti-mycoplasma antibiotic. Therefore, human T -cells, cultured for extended periods, do not inexorably and universally lose specific alloreactivity and gain suppressive characteristics due to some presumed differentiative event.     

The growth of concanavalin-A activated, Lyt selected subsets in IL-2 containing supernatants

The growth properties of Con A activated, Lyt selected splenic T lymphocytes were examined by limiting dilution analysis and clonally by single cell picking. Under the conditions used, a comparable frequency of Lyt 1+ and Lyt 2+ cells grew after Con A activation in the presence of Con A rat spleen supernatant. At a clonal level, however, the growth of these subsets differed qualitatively and quantitatively. While Lyt 2+ cells obtained clone sizes of several hundred cells, Lyt 1+ clone sizes were usually less than 100 cells, and many clones aborted their growth after a few days. Morphologically, the Lyt 1+ cell was smaller and usually showed fewer pseudopodial protusions as compared to the Lyt 2+ cell.     

HLA-Dw/LD directed cytotoxic T cell clones

T lymphocyte clones were derived by micromanipulation from an MLC between a stimulator and responder matched for class I but mismatched for class II HLA antigens; among the possible stimulating antigens were DR4 and Dw4. Two of the clones, when tested for cytotoxicity on a panel of DR4 positive and negative targets, appeared to recognize a determinant closely associated with Dw4, but did not lyse, with one exception, targets expressing other DR4 associated Dw specificities or DR4 negative targets. Blocking studies, using monoclonal antibodies directed against monomorphic epitopes on class I or class II molecules, revealed that the cytotoxic activity of these clones was strongly inhibited by an anti-class II (L-243) but not by an anti-class I (w6/32) monoclonal antibody. Both clones were T3⁺, T4⁺, T8^?. These findings show that cytotoxic T cell clones, (directed against class II antigens), may have specificity that correlates with a T lymphocyte-defined LD/Dw determinant. The blocking experiments using monoclonal antibodies give further support to the idea that these clones recognize determinant(s) on a class II molecule. The cell surface phenotyping results are in agreement with previous reports that most anti-class II cytotoxic clones have a T4⁺ T8^? phenotype.