Alteration in lymphocyte recognition repertoire during aging: II. Changes in the expressed T-cell receptor repertoire in aged mice and the persistence of that change after transplantation to a new differentiative environment

Changes in the T-lymphocyte alloreceptor repertoire associated with aging by exploring the frequency of cytotoxic T-lymphocyte precursors (CTLp) available for activation by various major histocompatibility complex (MHC) haplotypes in mice of different ages have been investigated. There was no consistent pattern of change in CTLp frequencies. Thus, for instance, while the frequency of responder C57Bl/6 CTLp for ATH alloantigen decreased with age, the frequency for C3H alloantigen increased. There was no significant change in the overall frequency of splenic CTLp (assessed irrespective of antigen specificity). No evidence was found that CTL produced by activated CTLp of aged mice were less specific in their lytic capacity that CTL produced by CTLp of young mice. However, by assaying responder CTLp cultures at limiting dilution we obtained evidence that the “burst size” (mean lytic capacity per responder well assayed at limiting dilution) was diminished with age of the donor of the CTLp pool. Furthermore, we obtained evidence that the apparent affinity of CTL for their target antigen was consistently decreased when those effector cells were derived from a pool of CTLp of aged mice. All of these changes reflected in mature T cells derived from aged mice were already apparent in the bone marrow stem cell pool of aged individuals and were not due to environmental influences alone, as assessed by the phenotype of T cells derived from young or old bone marrow stem cells transplanted to young or aged recipient mice. A final study has examined evidence for more subtle changes in the T-cell alloreceptor repertoire, reflecting heterogeneity in young or aged mice in the recognition repertoire associated with a given antigenic specificity. By preparing F₁ anti (parent anti-F₁)-suppressor cells directed against CTL from young parental mice (a, b, c), or aged parental mice (x, y, z), we have explored the heterogeneity in the anti-C3H alloreceptor repertoire in individual young or aged C57Bl/6 mice. Suppression by immunized F₁ animals was assessed in tissue culture (inhibition of mixed lymphocyte culture (MLC) responses) or in vivo (inhibition of lethal GvHD induced by inoculation of parental lymphocytes into sublethally irradiated F₁ hybrid mice). Irrespective of the assay system used, the data suggests that the receptor repertoire of aged T lymphocytes uses recognition structures different from those of young individuals, and that there is less individual-to-individual variation in the receptor repertoire of aged mice than in young mice.     

Age-related changes in the capacity of bone marrow cells to differentiate in thymic organ cultures

The capacity of the bone marrow to give rise to T cells in advanced age was studied in vitro by reconstituting fetal thymic lobes from 14-day C57BL/Ka (Thy-1.1) mice with bone marrow cells from old (24-month) or young (3-month) C57BL/6 (Thy-1.2) mice. The use of these congenic strains enabled distinguishing between donor and host contribution to the developing T cells. We found that bone marrow cells from aged mice maintained their capacity to reconstitute fetal thymic explants and to differentiate into various T-cell subsets as assessed by distinct T-cell-specific surface markers (Thy-1, Lyt-1, Lyt-2, and L3T4) and functions (concanavalin A-induced proliferative and cytotoxic responses). However, when mixtures of old and young bone marrow cells reconstituted fetal thymic explants, the cells of old mice were less efficient than those of young in their capacity to give rise to T cells. These results indicate that bone marrow cells from aged mice can reconstitute the thymus and differentiate into T cells; however, their reconstituting capacity is inferior to that of bone marrow cells from young mice.     

Immune response gene control of antibody specificity

The expression of the histocompatibility-linked PLL Ir gene was investigated in guinea pig B cells. Strain 2 and F₁ (2 × 13) guinea pigs, immunized with the αDnp-Lys₉, produce both T cells and antibody which are equally discriminatory for αDnp-Lys₉. In contrast strain 13 (PLL Ir gene negative) guinea pigs immunized with αDnp-Lys₉ do not develop specific T-cell responses and the antibody produced while restricted in heterogeneity cannot differentiate the immunizing antigen from Dnp-OH. However, if in a F₁ (2 × 13) environment, PLL Ir gene-negative B cells are provided with F₁ (2 × 13) T cells they express the ability to make antibody as specific and discriminatory as the antibody produced by PLL Ir gene-positive B cells. These findings strongly suggest that in the guinea pigs the PLL Ir gene defect is localized to the T cells and that the repertoire of specificity of B cells is similar if not identical in both responder and nonresponder animals. In addition these observations support the notion that the cellular locus for the PLL Ir gene expression in the guinea pigs is limited to T cells and not to macrophages and B lymphocytes.     

Cellular basis of the immunohematologic defects observed in short-term semiallogeneic B6C3F1 -- C3H chimeras: evidence for host-versus-graft reaction initiated by radioresistant T cells

Lethally irradiated C3Hf mice reconstituted with a relatively low dose (2 × 10⁶) of B6C3F₁ bone marrow cells (B6C3F₁ → C3Hf chimeras) frequently manifest immunohematologic deficiencies during the first month following injection of bone marrow cells. They show slow recovery of antibody-forming potential to sheep red blood cells (SRBC) as compared to that observed in syngeneic (C3Hf → C3Hf or B6C3F₁ → B6C3F₁) chimeras. They also show a deficiency of B-cell activity as assessed by antibody response to SRBC following further reconstitution with B6C3F₁-derived thymus cells 1 week after injection of bone marrow cells. A significant fraction of B6C3F₁ → C3Hf chimeras was shown to manifest a sudden loss of cellularity of spleens during the second week following injection of bone marrow cells even though cellularity was restored to the normal level within 1 week. The splenic mononuclear cells recovered from such chimeras almost invariably showed strong cytotoxicity against target cells expressing donor-type specific H-2 antigens (H-2^b) when assessed by ⁵¹Cr-release assay in vitro. The effector cells responsible for the observed anti-donor specific cytotoxicity were shown to be residual host-derived T cells. These results indicate strongly that residual host T cells could develop anti-donor specific cytotoxicity even after exposure to a supralethal dose (1050 R) of radiation and that the immunohematologic disturbances observed in short-term F₁ to parent bone marrow chimeras (B6C3F₁ → C3Hf) were due to host-versus-graft reaction (HVGR) initiated by residual host T cells. The implication of these findings on the radiobiological nature of the residual T cells and the persistence of potentially anti-donor reactive T-cell clones in long-surviving allogeneic bone marrow chimeras was discussed.     

A comparison of H-2 restriction of helper function using T cells from radiation chimeras and nude mice bearing thymus grafts

T cells from nude chimeras and radiation chimeras were tested for ability to cooperate with B cells of the thymus epithelium strain and B cells of the T-stem-cell strain in nude mice of appropriate strain backgrounds and also in irradiated F₁'s together with bone marrow cells of the two relevant strains (i.e., A-nu ← B or A → A × B chimera T cells were tested in A-nu and B-nu and in irradiated A × B F₁ with A bone marrow cells or B bone marrow cells). Whether the donor chimeras were antigen primed or not, all cells, when tested in nudes, provided help for the T-stem-cell type only. In contrast, in most cases, chimera T cells, tested in irradiated F₁'S, provided help for bone marrow cells of both thymus and T-stem-cell strains. These data indicate the involvement of two helper cell populations and an hypothesis to explain their interaction is presented.     

H-2 restriction specificity of T cells from H-2 incompatible radiation bone marrow chimeras: Further evidence for the absence of crucial influence of the host/thymus environment on the generation of H-2 restricted TNP-specific T lymphocyte precursors

Experiments were conducted to answer the questions related to (a) the role played by the antigen-presenting cells (APCs) present within the thymus and (b) the effect of radiation dose to the recipients on the H-2 restriction profile of TNP-specific cytotoxic T lymphocyte precursors (CTLP) recovered from spleens and/or thymuses of H-2 incompatible radiation bone marrow chimeras (BMC). The H-2 restriction profile of intrathymically differentiating TNP-specific CTLPs was also analyzed in order to test an argument that donor-H-2 restricted CTLP detected in spleens of H-2 incompatible BMC were due to the extrathymically differentiated T cells under the influence of donor-derived lymphoreticular cells. The results indicated the following: (i) splenic T cells from B10(H-2^b)→ (B10(H-2^b) → B10.BR(H-2^k)) chimeras, which were constructed by irradiating primary BIO → B10.BR chimeras with 1100 R and reconstituting them with donor-type (B10) bone marrow cells as long as 8 months after their construction, manifested restriction specificities for both donor- and host-type H-2, (ii) splenic T cells from two types of (B10 × B10.BR)F₁→ B10 chimeras which were reconstituted after exposure of the recipients with either 900 or 1100 R with donor-type bone marrow cells generated both donor- and host-H-2 restricted TNP-specific cytotoxic T cells, and (iii) the TNP-specific CTLPs present in the regenerating thymuses of B10.BR → B10 and (B10 × B10.BR)F₁→ B10 chimeras 4 weeks after their construction were also shown to manifest both donor- and host-H-2 restriction specificities. The significance of these findings on the H-2 restriction profile of CTLP generated in BMCs is discussed.     

Anti-recipient cytotoxic T lymphocyte precursors are present in the spleens of mice with acute graft versus host disease due to minor histocompatibility antigens

A model for bone marrow transplantation across minor histocompatibility barriers was developed by using mouse strains that were H-2 identical and mutually non-reactive in MLC. Acute graft-vs-host disease was induced only when donor lymphoid cells were included in the marrow inoculum, in both C57BL/6 recipients of LP cells and BALB/c recipients of B10.D2/nSN cells. GVHD was prevented by treating the lymphoid cells with anti-Thy 1.2 and C before transplantation. Spleen cells from mice with acute GVHD were not directly cytotoxic to recipient strain target cells. However, when spleen cells from mice with GVHD were boosted in vitro to recipient strain stimulator cells they generated a specific anti-recipient cytotoxic response. Spleen cells from mice without GVHD did not generate a cytotoxic response in vitro. The cytotoxic effector cells and their precursors were shown to be T lymphocytes. This model and the in vitro method described may be useful in further studies of the immunobiology of GVHD due to minor histocompatibility antigens and of transplantation tolerance.     

Lymphoid bone marrow cultures can reconstitute heterogeneous B and T cell-dependent responses in severe combined immunodeficient mice

Long-term lymphoid bone marrow cultures (LBMC) produce B lymphocytes and their precursors for several months in vitro. To assess their differentiative potential and determine their capacity to function as immune effectors, cells from the cultures were transplanted into mice with severe combined immune deficiency disease (SCID). SCID mice are deficient in T and B lymphocytes and are serum immunoglobulin (Ig) negative, but grafts of normal lymphoid precursors can expand and differentiate in them, thereby restoring immunocompetence. The results of these studies indicate that cells from LBMC are able to reconstitute splenic B lymphocytes in the SCID mice. Upon in vivo transfer, LBMC cells secreted Ig that displayed isotype distribution and a pattern of heterogeneity comparable with normal BALB/c mice, as determined by two-dimensional gel electrophoresis. The transplanted LBMC cells were functional, because reconstituted mice could respond to immunization with the T-independent antigen TNP-Ficoll. The results also indicate that cultured cells could reconstitute T cell activity in SCID mice. Splenocytes from approximately one-third of the recipients could generate a cytotoxic response to alloantigens after 5 days of sensitization in a mixed lymphocyte culture, and all reconstituted SCID mice could respond to immunization with the T cell-dependent antigen TNP-BSA. These results demonstrate that B cells, as well as T cell activity, are present in LBMC-reconstituted SCID mice, and show that LBMC cells have the capacity to mediate an immune response.     

Acquisition of an anti-idiotypic cytotoxic T lymphocyte repertoire in B cell-transferred or tetraparental bone marrow chimeric mice

In previous studies we showed that major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) specific for the cross-reactive idiotype (CRI) of MOPC-104E myeloma protein could only be induced in BALB/c or BAB-14 mice which have the ability to produce the CRI, but not in C.AL-20 or C.B-20 mice which have no ability to produce the CRI. The strong correlation between CRI-specific CTL responder strains and CRI producers supports the idea that the VH gene products are intrinsic primary antigenic stimuli for the generation of the anti-idiotypic CTL. To investigate the role of B lymphocytes in the selection of T lymphocyte repertoire, the purified B cells of CRI producer strains were repeatedly injected into anti-CRI CTL nonresponder neonatal mice. CRI-specific CTL activity was successfully induced in the CRI nonproducer mice only when they were exposed to CRI producer strain B lymphocytes from neonatal life. When the CTL nonresponder adult mice received CRI producer B lymphocytes, the nonresponder phenotype was not changed into the responder phenotype. Inducibility of CRI-specific CTL was also analyzed in tetraparental bone marrow chimeras. When CRI nonproducer bone marrow cells repopulated along with CRI producer bone marrow cells, the anti-CRI CTL of CRI nonproducer origin were generated. Adaptive differentiation of haplotype preference was also observed. When these observations are taken collectively, we see that the anti-idiotypic T lymphocyte repertoire is not a genetically determined one, but rather that the repertoire of T lymphocytes strongly depends on the postnatal selection process through the intrinsic idiotypic repertoire of B lymphocytes, i.e., internal images.     

Phytohemagglutinin-lnduced acquisition of T-cell surface markers by rat bone marrow precursor cells in the absence of the thymic microenvironment

Two monoclonal antibodies specific for different rat T-cell subpopulations, the anti-helper-T-cell antibody, W3/25, and the OX8 suppressor cell antibody were used to investigate lectin-stimulated T-lymphocyte differentiation of F-344 rat bone marrow cells in culture. Cytofluorometric analysis of freshly isolated lymphocytes from thymus and spleen revealed that these tissues contained both W3/25? and OX8-positive populations but differed with respect to the number of cells and receptor density distribution. By contrast, bone marrow-derived lymphocytes exhibited negligible W3/25? or OX8-associated fluorescence. However, several days after stimulation of bone marrow lymphocytes with phytohemagglutinin (PHA), cells appeared bearing these markers. Two-parameter histogram analysis of light scatter measurements with cell surface immunoflu-orescence indicated that this phenomenon represented the appearance of a new population of cells, presumably mature T cells, bearing an increased density of marker. These findings suggest an induction of differentiation of bone marrow T precursor cells by nonthymic factors (PHA) since lymphocytes lacking mature T-cell marker expression developed this characteristic after several days in culture.     

Concentration of mast-cell progenitors in bone marrow,spleen, and blood of mice determined by limiting dilution analysis

When hematopoietic cells of congenic +/+ mice were injected into the skin of genetically mast-cell-depleted (WB × C57BL/6)F₁-W/W^v mice, mast cells appeared at the injection site. The donor origin of developing mast cells was confirmed by using granules of C57BL/6-bgl/bgl mice as a marker. When the number of injected cells was decreased, the proportion of injection sites at which mast cells did not appear increased according to the expected frequency of null response in a Poisson distribution. Therefore, such proportions were used to calculate the concentration of mast-cell precursors in the bone marrow, spleen, and peripheral blood. The relative concentration of mast-cell precursors in these tissues was similar to that of spleen-colony-forming cells. The present method seems useful as a semiquantitative in vivo assay for a population of progenitor cells which are committed to differentiate into mast cells.     

Human T lymphocyte receptor for sheep erythrocytes: Characterization of a specific antiserum and its application in the detection and quantitation of the receptor in a soluble form

An anti-human T lymphocyte serum specific to the receptor for sheep erythrocytes (E) was produced by immunizing sheep with the complex autologous E-soluble E receptor (ER_s). The soluble receptor (R_s) was obtained by heating human lymphocytes at 45 °C for 1 hr. The anti-R_s serum has been shown to inhibit E-rosette formation, to be cytotoxic to T cells, to identify T lymphocytes by indirect immunofluorescence, and to stimulate blastogenesis. The reaction of anti-R_s with R_s was directly demonstrated by two newly developed methods: agglutination of complexes formed by the treatment of formolized E with R_s (EFR_s complexes) and adhesion of a protein A producer strain of Staphylococcus aureus to EFR_s treated with anti-R_s. The anti-R_s antibodies could be neutralized by R_s present in supernatant of heated peripheral lymphocytes, inhibiting the above reactions and therefore providing methods to quantitate R_s in biological preparations. The importance of these assays is that R_s plays an immunoregulatory activity, and high levels of R_s in serum are associated with depressed cell-mediated immunity.     

Immunosuppressive activity of murine newborn spleen cells. I. Selective inhibition of in vitro lymphocyte activation.

Cell-mediated immune responses to newborn lymphocyte alloantigens were investiated using mitogen activation, mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML). Spleen cells from 1- to 5-day-old (C57BL/6 × Balb/c) F₁ mice co-cultured with maternal strain (BALB/c) splenocytes did not affect DNA synthesis of maternal strain cells in the presence of concanavalin A or phytohemagglutinin. Newborn cells did inhibit the lipopolysaccharide response of maternal strain lymphocytes and these cells also depressed DNA synthesis when added to MLR cultures of BALB/c and C57BL/6 spleen cells. Newborn cells expressed poor stimulatory capacity in semiallogeneic MLR and also caused marked inhibition of DNA synthesis when added to semiallogeneic MLR containing BALB/c (responder) and CB6F₁ adult splenocytes (stimulator). The suppression of MLR by neonatal cells persisted for the first 2 weeks of life and was associated with a soluble factor released during culture. The suppressive activity was almost completely abrogated after depleting the T-cells from newborn splenocytes. However, these same cells did not interfere with the in vitro generation of cytotoxic lymphocytes in the CML assay. The selective immunosuppressive properties of newborn spleen cells may be important during pregancy by protecting the immunologically alien fetus from rejection by the mother.     

In vitro effects of certain polycyclic aromatic hydrocarbons on mitogen activation of mouse T-lymphocytes: action of histamine

T lymphocytes were isolated from the spleens, thymuses, and bone marrow of three inbred mice strains, and the effects of two carcinogenic polycyclic aromatic hydrocarbons (PAH) on the mitogen activation of these cells were assessed. Benzanthracene (BA) and 3-methylcholanthrene (MCA) enhanced mitogen activation of splenic T cells in a strain-related fashion: C3H greater than C57BL greater than DBA/2 (P less than 0.025). This pattern of strain relatedness was not observed in T cells from the other lymphoid organs. Mitogen activation was suppressed by histamine to a greater degree in T cells from PAH-responsive mice (C3H and C57BL) than in the nonresponsive strain (DBA/2). Histamine inhibited rosette formation between T cells and histamine-conjugated sheep red blood cells. A histamine suppressor factor (HSF), isolated from splenic lymphocytes grown in the presence of histamine or histamine plus MCA, was significantly higher in activity in culture supernatants from T cells derived from responsive mice than from nonresponsive mice. With the use of Lyt 1 and Lyt 2 monoclonal antibodies, it is shown that the baseline percentage of T helper and T suppressor cells was not significantly different in all three strains. Further, histamine and MCA had no effect on the expression of the Lyt 1 and Lyt 2 surface antigens on splenic lymphocytes. These results suggest that PAH-responsive mice may have more T-cell H2 receptors than T cells from nonresponsive mice. Histamine and PAH compounds may act on the same T-cell subsets, as evidenced by the fact that BA and MCA enhance blastogenesis, histamine suppresses mitogen activation, and these PAH compounds enhance histamine and HSF activity.     

Cell-mediated cytotoxicity against HLA-D-Region products expressed in monocytes and B lymphocytes

The cytotoxic effector cells that recognize HLA-D-region determinants and their precursors were characterized using monoclonal antibodies against human T lymphocytes and T-cell subsets. These studies were performed using MLC combinations giving rise to cytotoxic cells specific for both class I (HLA-A, B, C) and class 11 (HLA-D-region) antigens, and then tested against target cells displaying relevant antigens of only one class. Both class I and class II specific CTL (cytotoxic T lymphocytes) were inhibited by treatment with the OKT3 monoclonal antibody and complement, indicating that the effector cells were T lymphocytes. A major portion.of class II specific CTL, and their precursors, were inhibited by OKT4 and complement, while class I specific CTL from the same cultures were not. The T4+T8 — cell subset has previously been associated with helper or inducer functions, but not with cytotoxicity. The present findings indicate that class I and class 11 specific CTL, and their precursors, are different on the basis of the class of target antigen recognized and on the basis of surface phenotype detected by monoclonal antibodies.     

The presence of Ia-like determinants on a subpopulation of human T lymphocytes

By planned immunization within HLA-A-, and -B-compatible and HLA-D-disparate combinations, we have raised two antisera which are cytotoxic in complement-dependent cytotoxicity (CDC) tests with B lymphocytes, but not with T lymphocytes, from the immunizing donor and other donors sharing the immunizing HLA-D phenotype. The sera were found previously to inhibit the stimulating capacity of cells in MLC and the Fc receptor of cells producing EA rosettes, suggesting that they may detect alloantigens analogous to Ia antigens in mice. Although apparently non reactive with T cells in CDC tests and immunofluorescence, these sera were investigated further for their potential interference with some T-cell functions. After pretreatment with the appropriate antiserum and complement, the cells behaved normally as responding cells in mixed lymphocyte culture, as precursors to the cytotoxic cells in cell-mediated lympholysis, and as cells responding to the purified protein derivative (PPD). However, the response to phytohemagglutinin (PHA) was reduced at low concentrations of this mitogen, and the response to concanavalin A was strongly reduced at all concentrations, indicating that some subpopulations of human T cells also carry Ia-like specificities.     

Ultrastructural study of spontaneous bone marrow rosette-forming cells

Mouse bone marrow contains spontaneous rosette-forming cells (RFC) which include more than 70% T-cell precursors, as assessed by their transformation into theta-positive cells after incubation with thymic hormone. Such spontaneous RFC, examined in C57B1/6 mouse bone marrow by electron and scanning electron microscopy, have consistently been shown to be small, inactive mouse lymphocytes when macrophages have been eliminated by cell preincubation. These data suggest that thymic hormone target cells include small quiescent lymphocytes.     

Attempt to restore lymphocytes with defective T-cell function by mixing with normal cells in vitro

Blood leukocytes from a patient with T-cell immunodeficiency failed to respond to stimulation with phytohemagglutinin (PHA) in vitro although ample lymphocytes were present. Leukocytes from this patient were mixed in various proportions with normal leukocytes, and the mixtures were stimulated with PHA and cultured for three days. There was no restoration of PHA responsiveness of the patient''s cells in the presence of normal cells. This would indicate that the defect in the patient''s cell population was not due to the absence of any facilitating factor or cooperative function that could be provided by normal T cells or other blood leukocytes. Since in thymic aplasia the patient''s own cells are rapidly restored to normal PHA responsiveness by an unknown facilitating factor after thymic transplantation, we suggest that this mixed-cell culture method might differentiate between patients who need a bone marrow transplant and those who will respond to a thymus transplant.     

Lethal graft-vs-host disease across major histocompatibility barriers: requirement for leucyl-leucine methyl ester sensitive cytotoxic T cells

L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) is selectively toxic for human natural killer (NK) cells and cytotoxic T lymphocytes (CTL) at both the precursor and effector stage of differentiation. The present studies explored the effects of Leu-Leu-OMe on murine spleen cell function. Leu-Leu-OMe exposure removed NK function from murine spleen cells but spared their capacity to proliferate in response to lipopolysaccharide and Con A. The capacity to generate CTL from both L3T4 (+) and Lyt-2 (+) precursors was lost after Leu-Leu-OMe treatment, whereas alloantigen-induced proliferation and interleukin 2 (IL 2) production by L3T4 (+) T helper cells remained intact. Lethal graft vs host disease (GVHD), which developed in irradiated (C57BL/6 X DBA/2)F1 recipients of C57BL/6 bone marrow and spleen cells was completely prevented by Leu-Leu-OMe treatment of donor cells. In contrast depletion of Lyt-2 positive cells from the donor inoculum did not prevent acute GVHD in this fully major histo-compatibility complex (MHC) incompatible strain combination. However, Leu-Leu-OMe treatment of the Lyt-2 depleted inoculum completely prevented lethal GVHD, although the treated cells retained the capacity to proliferate and secrete IL 2 normally after in vitro stimulation with (C57BL/6 X DFA/2)F1 spleen cells. These findings indicate that L3T4 (+) T helper cells alone are unable to initiate lethal GVHD in this H-2 incompatible strain combination. Rather, lethal GVHD requires the transfer of a Leu-Leu-OMe sensitive T cell subset, likely to be thymus educated pre-CTL. Leu-Leu-OMe treatment should provide a useful way to delineate subpopulations of cells involved in the production of lethal GVHD and an approach to preventing this complication of bone marrow transplantation.     

High expression of the Thy-1 antigen on natural killer cells recently derived from bone marrow

The subset of murine natural killer (NK) cells that kills lymphoma targets contains about 50% cells expressing the Thy-1 antigen and this has been one of the reasons for assigning NK cells to the T-cell differentiation lineage. It has now been shown that the proportion of the Thy-1+ NK cells is not constant: ca. 90% of the NK cells appearing in the spleens of irradiated mice injected 10-14 days previously with bone marrow cells (anti-Thy-1 plus complement treated) express this antigen. The donor origin of these Thy-1+ NK cells was demonstrated by using semisyngeneic bone marrow cells in transfers but this same phenomenon could also be observed after entirely syngeneic transfers, excluding the possibilities that this Thy-1+ NK activity is due to activated T cells or to the effect of T-cell activation products on NK cells. Additionally, these early NK cells expressed the asialo-GM1 antigen, which is found on murine NK cells but not on cytotoxic T cells. These data suggest that the precursors for NK cells in the bone marrow are Thy-1-, and that the first splenic NK cells derived from these progenitors express this antigen.