Allogeneic Lymphocyte Cytotoxicity Research Articles

The early days of NK cells: an example of how a phenomenon led to detection of a novel immune receptor system - lessons from a rat model.

In this review, I summarize some of the early research on NK cell biology and function that led to the discovery of a totally new receptor system for polymorphic MHC class I molecules. That NK cells both could recognize and kill tumor cells but also normal hematopoietic cells through expression of MHC class I molecules found a unifying explanation in the “missing self” hypothesis. This initiated a whole new area of leukocyte receptor research. The common underlying mechanism was that NK cells expressed receptors that were inhibited by recognition of unmodified “self” MHC-I molecules. This could explain both the killing of tumor cells with poor expression of MHC-I molecules and hybrid resistance, i.e., that F1 hybrid mice sometimes could reject parental bone marrow cells. However, a contrasting phenomenon termed allogeneic lymphocyte cytotoxicity in rats gave strong evidence that some of these receptors were activated rather than inhibited by recognition of polymorphic MHC-I. This was soon followed by molecular identification of both inhibitory and stimulatory Ly49 receptors in mice and rats and killer cell immunoglobulin-like receptors in humans that could be either inhibited or activated when recognizing their cognate MHC-I ligand. Since most of these receptors now have been molecularly characterized, their ligands and the intracellular pathways leading to activation or inhibition identified, we still lack a more complete understanding of how the repertoire of activating and inhibitory receptors is formed and how interactions between these receptors for MHC-I molecules on a single NK cell are integrated to generate a productive immune response. Although several NK receptor systems have been characterized that recognize MHC-I or MHC-like molecules, I here concentrate on the repertoires of NK receptors encoded by the natural killer cell gene complex and designed to recognize polymorphic MHC-I molecules in rodents, i.e., Ly49 (KLRA) receptors.

Partial NK cell tolerance induced by radioresistant host cells in rats transplanted with MHC-mismatched bone marrow

We have studied the effect of radioresistant host cells in inducing tolerance and adaptation of the MHC recognition repertoire of donor-derived NK cells in stem cell allotransplanted (allo-SCT) rats. Sub-lethally irradiated PVG.1AV1 rats (RT1(av1)) were transplanted with bone marrow from fully MHC-mismatched allotype-marked PVG.7B (RT1(c)) rats; MHC-identical PVG (RT1(c)) controls were transplanted in parallel. In the PVG.7B → PVG.1AV1 allogeneic chimeras, NK cells were donor derived and showed partial tolerance toward host cells. Allogeneic chimeras failed to efficiently reject PVG.1AV1 cells by an NK-mediated mechanism in vivo (allogeneic lymphocyte cytotoxicity), and IL-2-cultured NK cells derived from these chimeras showed diminished cytolytic activity against PVG.1AV1 cells in vitro. There were corresponding changes in the phenotype and function of the highly alloreactive Ly49i2(+) NK cells, which are specifically inhibited by a donor MHC class I ligand, RT1-A1(c). The ligand-negative host MHC haplotype apparently induced expression of a second uncharacterized inhibitory MHC receptor responsible for the partial tolerance toward host-derived cells, along with a modest increase in Ly49i2 receptor levels. The host MHC haplotype did not induce a general hyporesponsiveness in Ly49i2(+) NK cells, which showed normal activation responses in a panel of MHC congenic strains. The data suggest that the MHC constitution of radiation-resistant host cells can have permanent, albeit not fully tolerogenic, effects on the development of a functional NK repertoire following allo-SCT.

Strain-dependent expression of four structurally related rat Ly49 receptors; correlation with NK gene complex haplotype and NK alloreactivity

Natural killer (NK) cells from certain rat strains promptly kill MHC allogeneic lymphocytes in vivo, a rejection phenomenon termed allogeneic lymphocyte cytotoxicity (ALC). ALC can be reproduced in vitro, and is preferentially mediated by a subset of NK cells expressing the Ly49 stimulatory receptor 3 (Ly49s3) in PVG strain rats. Functional studies have suggested that Ly49s3 triggers NK cell alloreactivity, but its importance relative to other Ly49 receptors has not been investigated. In this study, we have characterized three rat Ly49 receptors with close sequence similarity to Ly49s3 in the extracellular region, i.e., Ly49s4, Ly49 inhibitory receptor 3 (Ly49i3), and Ly49i4. Similar to Ly49s3, Ly49s4 mediated cellular activation while Ly49i4 inhibited NK cytolytic function. Ly49s4, -i3, and -i4 all reacted with a previously described anti-Ly49s3 monoclonal antibody (mAb) (DAR13), but not a novel mAb (STOK6), which was shown to be specific for Ly49s3. Expression of these Ly49 receptors varied markedly between inbred strains, in patterns related to their NK gene complex (NKC) haplotype, and ability to mediate ALC. Three major groups of NKC haplotypes could be discerned by restriction fragment length polymorphism analysis. Ly49s3 was present in strains from one of the groups, which corresponded with the "high" ALC responders. Ly49s3 surface expression was also markedly reduced in the presence of its putative MHC class Ib ligand(s) in MHC congenic strains. These data support the notion that Ly49s3 functions as a triggering MHC receptor both in vitro and in vivo. MHC ligands for the other three Ly49 receptors remain to be determined.

Ly-49s3 is a promiscuous activating rat NK cell receptor for nonclassical MHC class I-encoded target ligands.

Previous studies of the rapid rejection of MHC-disparate lymphocytes in rats, named allogeneic lymphocyte cytotoxicity, have indicated that rat NK cells express activating receptors for nonclassical MHC class I allodeterminants from the RT1-C/E/M region. Using an expression cloning system that identifies activating receptors associated with the transmembrane adapter molecule DAP12, we have cloned a novel rat Ly-49 receptor that we have termed Ly-49 stimulatory receptor 3 (Ly-49s3). A newly generated anti-Ly-49s3 Ab, mAb DAR13, identified subpopulations of resting and IL-2-activated NK cells, but not T or B lymphocytes. Depletion of Ly-49s3-expressing NK cells drastically reduced alloreactivity in vitro, indicating that this subpopulation is responsible for a major part of the observed NK alloreactivity. DAR13-mediated blockade of Ly-49s3 inhibited killing of MHC-congenic target cells from the av1, n, lv1, and c haplotypes, but not from the u or b haplotypes. A putative ligand was mapped to the nonclassical MHC class I region (RT1-C/E/M) using intra-MHC recombinant strains. Relative numbers of Ly-49s3(+) NK cells were reduced, and surface levels of Ly-49s3 were lower, in MHC congenic strains expressing the putative Ly-49s3 ligand(s). In conclusion, we have identified a novel Ly-49 receptor that triggers rat NK cell-mediated responses.

The effect of in vivo depletion of NKR-P1+ or CD8+ lymphocytes on the acute rejection of allogeneic lymphocytes (ALC) in the rat.

We have depleted lymphocyte subsets in PVG and AO rats with MoAbs 3.2.3 (against NKR-P1 on NK and NK/T cells) and OX-8 (against CD8 on CTL and NK cells), and examined the effect on the killing of YAC-1 target cells in vitro and the effect on the acute rejection of small allogeneic lymphocytes in vivo (allogeneic lymphocyte cytotoxicity, ALC). While 3.2.3 treatment led to only a partial depletion of 3.2.3-positive cells in PVG rats, this treatment drastically reduced the number of NKR-P1+ cells in AO rats, abolished splenic NK activity against the NK-sensitive tumour target YAC-1, and markedly diminished the ALC response. Rats treated with OX-8 for 1 day showed a similar loss of NK cell function in vivo and in vitro. However, in rats treated with OX-8 for 3 days a 3.2.3+ and OX-8- population consisting of NK cells appeared, restoring ALC. The results demonstrate that NK cell responses can be greatly diminished after in vivo treatment with these MoAbs. Furthermore, they demonstrate that ALC is not necessarily linked to expression of the CD8 molecule.

GvHD after small bowel transplantation: the role of caspases, FAS-L, and galectin-1

IN small bowel transplantation, graft-versus-host disease (GvHD) can play a potential risk factor if the recipient organism is unable to counteract donor-derived immunocompetent cells (eg, in a semiallogeneic setting). Previously, we demonstrated that host-derived natural killer (NK) cells can prevent lethal GvHD if the F1-hybrid recipient’s NK cell population is preactivated prior to transplantation of parental small bowel, an immune mechanism also referred to as allogeneic lymphocyte cytotoxicity (ALS). The molecular mechanisms contributing to the lytic machinery of cytotoxic T lymphocytes and natural killer cells in the context of GvHD are presumed to include the interaction between Fas and Fas-ligand, one of the most potent signal transducers involved in the process of activation-induced cell death (AICD), or apoptosis. In this context, it is noteworthy that new groups of cytoplasmic proteins, such as cysteine proteases (caspases) like interleukin-converting enzyme (ICE) and the polypeptide cysteine protease-P32 (CPP-32), have been characterized as important intracellular transmitters of apoptotic signaling via the Fas pathway. CPP-32 serves as a substrate for ICE and generates enzymatically active ICE-like proteases from the inactive proenzymes. Beside the Fas/Fas-L pathway, galectin-1, a member of the family of b-galactoside binding proteins with immunomodulatory properties, has been shown to induce apoptosis of alloreactive T cells, a mechanism that could be blocked by anti-CD45 antibodies. The present study was conducted to investigate the impact of various apoptotic signal events in the context of GvHD with respect to the activation status of the F1 recipient’s NK activity prior to parental small bowel transplantation (SBTx).

INNATE IMMUNITY IN SMALL BOWEL AND HEART TRANSPLANTATION

104 NK cells comprise a unique entity of hymphocytes which act as an important effector arm of the innate immune response. Nevertheless NK cells are able to distinguish non-self MHC-class I molecules on target cells in an allospecific fashion. Target cell recognition of non-self MHC-class I molecules encoded by the RT1.C-locus of the RT1 gene complex induces activation of NK cells without the need of prior sensitization process, an immune mechanism also referred to as allogeneic lymphocyte cytotoxicity (ALC). Small bowel transplantation(SBTx) and heart transplantation (HTx) were performed between intra-MHC-class I disparate rat strain combinations which merely differed at the RT1.C-locus in order to show the impact of NK-related and T cell mediated immune responses. Methods: HTx and SBTx was performed in the following rat strain combinations (n=4): (i) LEW.1LM1 (A1B1/D1Clml) → LEW(A1B1/D1C1), (no treatment), (ii) LEW.1LM1 → LEW (recipient treatment with 100μg poly-I:C/kg [NK cell activator], i.p., at d -2,-1 and 0), (iii) LEW.1LM1→LEW (donor treatment with anti-NKR-P1 mAb 3.2.3. 100μl, i.p. at d -2, -1 and 0), (iv) LEW.1R15(A1B1/D1Cu)→LEW, (no treatment), (v) LEW.1R15→LEW (reciepient treatment with 100μg polyI:C at d -2,-1 and 0), (vi) LEW.1R15→LEW (reciepient treatment with 3.2.3 100μl at d-2,-1 and), (vii) LEW.1LV3 (A1B1/D1Clv3)→LEW,(no treatment), (viii) LEW.1LV3→LEW, (recipient treatment with poly-I:C 100μg/kg at d -2,-1 and 0). Flow cytometric analysis and immunohistology was performed from grafts and spleens with a broad panel of mAb including CD4+, CD8+ macrophages, and anti-NKR-P1 mAb 3.2.3. Results: The impact of the non-classical MHC-class I locus RT1. C significantly affected the clinical outcome following HTx and SBTx. Whereas LEW.1R15 donor organs were chronically rejected by LEW recipient rats, LEW.1LM1 donor small bowel grafts (but not hearts) induced lethal GvHD in LEW recipients. GvHD could be abrogated by preoperative depletion of donor(LEW.1LM1) NK cells with mAb 3.2.3. LEW.1LM1 donor organs are never rejected by LEW recipients. LEW.1LV3 donor hearts never induced rejection nor GvHD irrespective of NK-cell modulation. However, in contrast to LEW.1LM1 donor organs LEW.1LV3 donors induced a considerable graft infiltration of recipient NKs but without T cells. Interestingly, chronically rejected organs (LEW.1R15) always displayed a common infiltration of NK and T cells. Conclusions: There is a strong body of evidence that immunogenic eptitopes of the RT1.C locus in the absence of classical MHC class I and II disparities are able to exert strong immune responses which are mainly triggered by donor or host NK cells in the context of GvH and HvG reactions and with apparently tissue-specific differences.

Identification of an Inhibitory MHC Receptor on Alloreactive Rat Natural Killer Cells

Studies of allogeneic lymphocyte cytotoxicity have shown that the rat NK allorecognition repertoire is controlled by genetic elements in both the MHC (RT1) and the NK gene complex (NKC). DA rats, possessing NK cells that are unable to lyse allogeneic lymphoblasts, were immunized with alloreactive NK cells from MHC-matched PVG.1AV1 rats, and two mAb, STOK1 and STOK2, were generated. STOK1 and STOK2 stained identical subsets of NKR-P1+ T and NK cells from certain strains of rats. Relative numbers varied markedly in a panel of MHC congenic strains, however, implicating a role for self MHC genes in their development. Both STOK1 and STOK2 immunoprecipitated a 110-kDa disulfide-linked homodimeric molecule, with extensive N-linked glycosylations, encoded by a gene that mapped to the NKC. NK cells expressing this glycoprotein displayed an increased ability to lyse allogeneic lymphoblasts, while syngeneic targets were spared. However, blockade of the STOK2 Ag with F(ab')2 of STOK2 permitted the NK lysis of syngeneic targets, but did not affect NK allorecognition. These results indicate that mAb STOK1 and STOK2 identify an NKC-encoded MHC receptor in the rat that acts as a negative regulator of cytotoxicity.

Impact of RT1.C-encoded MHC antigens on host-versus-graft and graft versus-host reactions in a model of small bowel transplantation in the rat

Non-classical MHC-class I antigens encoded by rat RT1.C region genes have been demonstrated to have a strong in vitro and in vivo potential to trigger natural killer (NK) cell activity. 1,2 NK cells comprise a unique entity of lymphocytes that act as an important effector arm of the innate immune response. Nevertheless, NK cells distinguish non-self MHC-class I molecules on target cells in an allospecific fashion. 3 Target cell recognition of non-self MHC-class I molecules encoded by the RTI.C-locus of the RT1 gene complex induces activation of NK cells without the need of a prior sensitization process, an immune mechanism also referred to as allogeneic lymphocyte cytotoxicity (ALC). 4 By virtue of its vast mass of lymphatic tissue, heterotopic small bowel transplantation (SBTx) was performed between intra-MHC class I recombinant inbred rat strains, differing only at the RT1.C-locus, in order to show the impact of NK-related immune responses for this model of solid organ transplantation.

Positive and negative MHC class I recognition by rat NK cells.

The prompt rejection of transplanted allogeneic lymphocytes by rat NK cells in non-sensitized recipients (allogeneic lymphocyte cytotoxicity or ALC) is determined by MHC genes as well as by genes located in the NK complex. The same genetic control is found when NK alloreactivity is measured by an in vitro assay, and we have employed this assay to delineate the specificity of NK cells for the MHC. The MHC of the rat, RT1, contains class I genes situated on either side of the class II/class III region. The majority of these class I genes are located in the RT1.C region and expressed class I products usually behave as non-classical (class Ib) molecules. They do not serve as restriction elements for the vast majority of conventional alpha/beta T-cells, in contrast to those class I molecules encoded by one or more loci in the classical (class Ia) region, RT1.A. However, NK cells appear to recognize the products of either class I region. Immunogenetic studies suggest that NK cells are inhibited by RT1. A molecules, whereas RT1.C region molecules may have a dual role in regulating NK cytolytic activity, i.e. they either inhibit or activate natural killing. Based on these premises, a model is proposed in which identification of a target as self or non-self depends on different receptors for class I in single NK cells, interpreting coincident positive and negative signals from the various target class I molecules. The putative role of peptides presented by class I, the biological implications, and the evolution of the NK receptors and their ligands are discussed.

Neonatally tolerant rats actively eliminate donor‐specific lymphocytes despite persistent chimerism

Rats from the allotype-marked PVG-RT7b and PVG-RT1u-RT7b strains were injected at birth with semi-allogenic F1 bone marrow (BM) cells from athymic nude rats (PVG-rnu/rnu x PVG-RT1u-rnu/rnu) to induce neonatal tolerance. As adults, 97% of the animals accepted donor-specific allogeneic skin grafts and a majority (65%) of rats were chimeric, expressing the major histocompatibility complex class I and allotype marker of the donor strain. Similar results were obtained when PVG-RT1u-RT7b rats were injected at birth with fully allogeneic PVG-rnu/rnu nude BM cells: as adults, 94% accepted donor-specific skin allografts and 76% of recipients were chimeric. Donor derived CD4 T cells, CD8 T cells and B cells were found in low numbers (less than 2%) in peripheral blood of rats made tolerant by F1 BM cells. A large proportion of T cells bore the phenotype of recent thymic emigrants, suggesting that they were newly produced. All the evidence was consistent with clonal deletion tolerance, induced centrally within the thymus. The thymus was chimeric and thymocytes failed to respond in vitro to alloantigens of the donor-specific haplotype; donor-specific skin allografts survived indefinitely on athymic nude recipients reconstituted with CD4+CD8- thymocytes or peripheral CD4 T cells from tolerant animals. The chimeric state was interesting, since the PVG and PVG-RT1u rat strains contain a natural killer (NK) cell system that rapidly eliminates (within 24 h) intravenously injected allogeneic or semi-allogeneic lymphocytes--a phenomenon known as allogeneic lymphocyte cytotoxicity or ALC. When neonatal tolerant rats were tested, the ALC index (a measure of cell killing) was unchanged in nonchimeric tolerant rats and significantly altered (reduced killing), but not abolished in chimeric animals. Hence, the injection of allogeneic BM cells which induced specific tolerance in the T cell population failed to tolerize the NK cell system, allowing the constant killing of newly produced donor-derived lymphocytes and putting at risk the very survival of the allogenic BM cells. This has interesting implications for clinical transplantation.

Genes in two MHC class I regions control recognition of a single rat NK cell allodeterminant.

We have previously presented evidence suggesting that the non-classical class I region of the rat MHC, RT1.C, encodes polymorphic molecules which induce the cytolytic activity of alloreactive NK cells. Those studies used target cells from a panel of MHC congenic rat strains possessing recombined portions of the RT1a, RT1l and RT1u MHC haplotypes. We have now examined in addition a set of rat strains bearing MHC haplotypes recombinant between RT1av1 and RT1c, and a more complex picture of the MHC control of rat NK alloreactivity has emerged. The expression of a major NK allodeterminant [the allogeneic lymphocyte cytotoxicity (ALC) determinant 2 or ALC-2, defined operationally using cold-target inhibition assays], appears to be under the control of both the RT1.C and the classical class I RT1.A region. Similarly, the alloreactive repertoires of NK cells from these recombinant strains are influenced by elements encoded within these two MHC class I regions. We present a model in which the classical class I autoantigen RT1.Ac exhibits dominant inhibition of NK cytotoxicity specific for the stimulatory determinant ALC-2 shared by the nonclassical class I molecules RT1.Cav1, RT1.Ca and RT1.Cc, and also prevents the deletion of NK cells of this specificity during their development.

Proliferation of theileria annulata and Theileria parva macroschizont-infected bovine cells in scid mice

Theileria annulata and Theileria parva macroschizont-infected bovine cells formed tumours at the inoculation site when injected subcutaneously into C.B.-17 scid mice. T. annulata tumours showed more vigorous growth than T. parva tumours. The tumours did not regress and infected cells spread to other tissues. Intraperitoneal injection of high doses of T. annulata-infected cells resulted in the development of ascites; the infected cells colonized abdominal organs, in particular mesenteric tissue. Low doses of cells did not establish when administered by this route. Evidence for a role for macrophages in controlling proliferation of Theileria-infected cells was provided by finding (i) that uninfected bovine cells did not survive for as long in the peritoneal cavities of scid mice as in Balb/c mice; (ii) peritoneal macrophages both proliferated in vivo in the presence of infected cells and were activated as assessed by production of interleukin-1. Evidence against a role for NK cells was provided by (i) the failure of an in vivo assay for allogeneic lymphocyte cytotoxicity to reveal any activity against bovine cells in the lungs or liver, i.e. the sites usually associated with NK cell cytotoxicity, and (ii) the lack of correlation between tumour regression and NK cell activity in the spleens of mice with chronic T. annulata tumours.

Non-adaptive cellular immune responses as studied in euthymic and athymic nude rats

The athymic nude (rnu) rat lacks a functional thymus and normal alloreactive T cells. These animals, therefore, have been widely used as tools for studying thymus-independent immune responses. The absence of functional T cells would, to most investigators, indicate that these rats have a defective cellular immune defence. However, although rnu rats accept organ allografts infinitely, they are nevertheless capable of rejecting allografts consisting of lymphocytes or bone marrow cells with increased vigour, and this via antibody-independent mechanisms. These rejection phenomena have operationally been termed allogeneic lymphocyte cytotoxicity (ALC) and allogeneic bone marrow cell cytotoxicity (ABC). Unlike organ allograft immunity this kind of rejection requires no presensitization of the recipient and is surprisingly rapid: it commences within a few hours of i.v. injection of the allogeneic cells and is usually complete by 24 h. Moreover, products coded for by genes within, or closely linked to the major histocompatibility complex (MHC), are clearly involved in the interaction between effector and target cells, as grafted cells from MHC-congenic rat strains are vigorously rejected. In contrast to the defective T cell immune responses in athymic nude rats, the natural killer (NK) cell function is not impaired, and it has been suggested that the spontaneous rejection of MHC-incompatible lymphohematopoietic cells is in fact mediated by NK cells. If the MHC antigens themselves serve as targets in this kind of allorejection, this hypothesis is in apparent contrast with the prevailing view that recognition by NK cells is not guided by, or directed against, MHC-antigens on the target cell surface. Aging athymic nude mice and rats generate cells that rearrange and express T cell receptor (TCR) genes, and this has raised the possibility that T cells or T-like cells in athymic nude animals are responsible for ALC and ABC. This possibility urged the device of an in vitro test system for identification and further characterization of the effector cells in these rejection phenomena. Under appropriate conditions, cells from the rnu rat spleen or liver with natural killer function, i.e. the ability to lyse certain kinds of tumor cells in vitro, are also spontaneously cytotoxic for allogeneic small lymphocytes and bone marrow cells in vitro. Furthermore, these cells can be grown in vitro in the presence of interleukin 2 (IL-2) to generate populations of lymphokine-activated killer (LAK) cells, and these cells have the same spectrum of alloreactivity in ALC and ABC as the native NK cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Major histocompatibility complex control of NK-related allogeneic lymphocyte cytotoxicity in rats. The contributions of strong and medial transplantation antigens.

Allogeneic lymphocyte cytotoxicity (ALC) describes the elimination of allogeneic lymphocytes in vivo by an NK-related activity. There is evidence that ALC is demonstrable between donor and recipient when these are incompatible at MHC gene loci alone. Since ALC is a property of T cell-deficient nude rats, the role of the MHC in this rejection process needs further study. We have determined the contribution of the MHC to ALC using congenic and recombinant rats. In our analysis we have assumed that ALC involves the recognition of classic alloantigens by clonally distributed effector cells as for other examples of transplant rejection, although this is not yet proved. Strong ALC was measured between congenic rats that differed for MHC genes only. Non-MHC incompatibility alone did not elicit ALC. In the presence of MHC incompatibility the strength of ALC generated in a recipient was dependent on non-MHC genes. The PVG background generated high ALC responses whereas ALC was not measured in the DA rat. However ALC was measured in the congenic PVG-RT1avl (DA) rat. The contributions of classic class I (RT1.A), class II (RT1.B/D), and medial transplantation (RT1.C) regions of the rat MHC were determined by comparing different recombinant donors into the same recipient strain. Single region differences alone in any of these three MHC regions did not elicit full ALC. In two sets of transfers a combination of RT1.B/D and RT1.C region incompatibility was sufficient to generate a full allogeneic response. It can be concluded that the controlling element for allogeneic lymphocyte cytotoxicity is in the RT1.B/D-RT1.C region of the rat MHC.

Specific inhibition of natural killer (NK) activity against different alloantigens

Allogeneic lymphocyte cytotoxicity (ALC), i.e., rapid rejection of i.v. injected allogeneic lymphocytes in unprimed hosts, is an example of NK activity. Apparently anomalous rejection patterns, such as acceptance of F1 hybrid cells by parental hosts and rejection of parental cells by F1 hybrid hosts in many strain combinations, would fit the hypothesis that the effector cells in ALC recognize the absence of certain self-molecules (passwords) rather than the presence of nonself determinants. However, cold target inhibition studies showed that ALC displays allospecificity: when a mixture of radiolabeled AO and DA cells were injected i.v. into euthymic or athymic PVG rats, adding a surplus of cold DA cells reduced killing only of labeled DA cells and vice versa. Furthermore, semiallogeneic cold target cells were ineffective in inhibiting elimination of fully allogeneic cells, which supports the argument against a modification of the hypothesis that self-determinants inhibit a postbinding stage of lysis. Finally, (DA x AO)F1 cells injected into (DA x PVG)F1 hosts were rapidly rejected, despite the fact that donor and host shared expressed DA determinants. In sum, our results show that a hypothesis based on inhibition of killing by self-determinants can only be sustained with extensive modifications, and favor the alternative mechanism that the effector cells positively recognize the presence of allospecific determinants on the target cell surface.

The Rapid Elimination of Allogeneic Lymphocytes: Relationship to Established Mechanisms of Immunity and to Lymphocyte Traffic

Allogeneic lymphocyte cytotoxicity (ALC) refers to the destruction of lymphocyte beginning within a few hours of intravenous injection into non-sensitized, allogeneic recipients. Usually this has been detected in rats and mice by comparing the localization of 51Cr-labelled lymphocytes in the tissues of allogeneic and syngeneic recipients. In a particular strain combination the existence of ALC is supported by deficient localization of allogeneic lymphocytes in the LN, lungs and blood mononuclear population and an excess of the label that had been associated with allogeneic cells in the lymph plasma, blood plasma and kidneys. As the destruction of the allogeneic cells occurs in the lymphatic tissues, especially the spleen, it is paradoxical that there is sometimes an excess of the label associated with allogeneic cells in the spleen but evidence is presented that most of the isotope is no longer associated with living cells in that organ at 24 h after transfer. The data cannot be explained by an altered distribution of allogeneic lymphocytes between different organs. Experiments on the early migration of lymphocytes from the blood of syngeneic and allogeneic recipients point unequivocally to the conclusion that the adhesion of lymphocytes to specialized vascular endothelium in LN and their consequent entry into LN does not require that the lymphocytes and the endothelial cells share MHC products. The characteristics of ALC stressed in this review include the following: 1) it is independent of T-cell activation, either of host T-cells or of donor T-cells: 2) B- and T-cells are about equally vulnerable to ALC; 3) it varies greatly between different strain combinations regardless of other indices of cellular and humoral immunity; 4) F1 hybrid donor cells are vulnerable but the effect is always less marked than with allogeneic cells; 5) ALC is less radio-sensitive than primary cellular or humoral immune responses; 6) adoptive transfer of ALC can be achieved with TDL from nude rats. The possible mechanism underlying ALC has been debated in terms of natural killer cells recognizing certain allo-antigens or alternatively pre-existing "natural" antibody with low affinity for allogeneic cells leading to their elimination by ADCC. The argument hinges on the necessity for antibody and cannot be resolved by current data.