T lymphocyte development and maturation in horses

Monoclonal antibodies specific for equine T lymphocyte subpopulations were produced and procedures for the continuous culture of equine lymphocytes were developed. These reagents and procedures were used to analyse the appearance, maturation and functions of T lymphocytes in normal horses and in T lymphocyte deficient horses with severe combined immunodeficiency (SCID). T lymphocytes appeared as early as the 75th day of fetal development and were normally distributed prior to birth of normal foals. Analysis of thymic T lymphocyte differentiation in SCID foals revealed the presence of both prothymocytes and mature thymocytes, but a virtual absence of cortical thymocytes. The data obtained support the hypothesis that two distinct pathways of T lymphocyte differentiation exist within the thymus. Although the gene defect in foals with SCID blocks the production of mature B and T lymphocytes, such foals do possess large granular lymphocytes which are cytotoxic following induction with interleukin 2. This suggests that lymphoid cells with natural killer cell activity are spared by the gene defect resulting in SCID in horses.     

Granules of human CD3+ large granular lymphocytes contain a macrophage regulating factor(s) that induces macrophage H2O2 production and tumoricidal activity but decreases cell surface Ia antigen expression.

CTL (cytotoxic T lymphocytes) and LGL (large granular lymphocytes) exocytose cytoplasmic granules on activation after recognition of their target, releasing granule-associated molecules. We have previously suggested that this process could release immunoregulatory molecules. In this study we investigated whether normal human LGL granules contained a factor regulating different macrophage activity. Human CD3+ LGL cells were generated by activating peripheral blood lymphocytes (PBL) for 10-12 days with recombinant human IL-2 (rhIL-2), and granules were isolated from disrupted cell homogenate by Percoll gradient fractionation. Solubilized granules were tested for macrophage-activating factor (MAF) activity in three different macrophage assays. When M-CSF-differentiated murine bone marrow-derived macrophages were incubated 9 hr with human LGL granules, they were fully activated to lyse the TNF-resistant P815 tumor cells. The granule-MAF showed a synergistic effect with rhIL-1 beta, rmTNF-alpha, and rmIFN-tau in the cytolytic assay. In addition, proteose-peptone-elicited murine peritoneal macrophages profoundly increased H2O2 production after activation with human LGL granules. However, unlike IFN-tau, no increase in peritoneal macrophage Ia antigen expression was detected after incubation with granules. Moreover, granule-MAF suppressed Ia induction by IFN-tau. These results confirm that human CD3+ LGL granules contain a molecule(s) capable of regulating macrophage function.     

Feline cytotoxic large granular lymphocytes induced by recombinant human IL-2

Large granular lymphocytes (LGL) have been characterized phenotypically and functionally as cytotoxic T lymphocytes, NK cells or lymphokine-activated killer cells. The most prominent morphologic feature of LGL is large cytoplasmic granules that are thought to contain the molecules responsible for cell lysis. In this study, we describe the morphologic and functional characteristics of IL-2-dependent cytotoxic lymphocytes derived from feline PBL. Stimulation of feline PBL with Con A followed by culturing in 50 U of gibbon monkey IL-2 human rIL-2 induced long term lymphocyte cultures. These lymphocytes are cytotoxic for the feline leukemia virus-induced T cell lymphoma (FL74), in a 4-h 51Cr release assay. All cell lines are either constitutively cytotoxic for FL74 cells, or cytotoxic in a lectin-dependent cell cytotoxic assay, the latter being a characteristic of low passage cultures. In contrast, no cell lines express self lysis or lysis for other lines. [3H]TdR uptake showed that 1 U of human rIL-2 produces a 50% maximal proliferative response by feline lymphocytes suggesting a high degree of homology between the ligand binding sites of feline and human IL-2R. Feline cytotoxic lymphocytes possess abundant cytoplasm containing large azurophilic granules characteristic of LGL. These granules are bound by a bilipid membrane and contain numerous smaller membrane-bound vesicles 50 to 60 nm in diameter. A model is proposed, whereby subsequent to binding of LGL to target cell the large granules fuse to the LGL plasma membrane and release the small vesicles into the binding pocket. The vesicles then transport the lytic molecules directly and selectively to the target cell membrane.     

A primary production deficit in the thrombocytopenia of equine infectious anemia.

The purpose of this study was to identify the mechanisms responsible for the thrombocytopenia that develops following infection of horses by the lentivirus equine infectious anemia virus (EIAV). Immunocompetent Arabian foals and Arabian foals with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were experimentally infected with EIAV. Levels of viremia and a number of clinical and hematologic parameters were examined prior to and following infection. Thrombocytopenia was not dependent on the immune response: SCID foals were affected as severely as immunocompetent foals. Production of platelets, measured by metabolic incorporation of radioactive label, was significantly reduced. The decrease ranged from 35 to 89% in three SCID and two immunocompetent foals examined. Platelet survival, measured by 51Cr labeling, also declined following infection in both SCID and immunocompetent foals: 51 and 68%, respectively, relative to the preinfection life spans. The difference between immunocompetent and immunodeficient foals was not statistically significant. The number of megakaryocytes (MK) per square millimeter of bone marrow, determined by digitizing morphometry, was not significantly altered in either SCID or immunocompetent thrombocytopenic foals. Numbers of denuded MK nuclei per unit area increased, but the elevation was not statistically significant. No evidence for viral replication in MK was found. Three different parameters of intravascular coagulation (activated prothombin time, fibrin degradation products, and one-step prothombin time) remained normal until after platelet numbers had declined significantly, arguing against an important role for disseminated intravascular coagulation. The findings indicate that EIAV induces thrombocytopenia principally through an indirect, noncytocidal suppressive effect on platelet production, the mechanism of which is unknown. A shortening of platelet life span apparently contributes moderately to the platelet deficit as well. The shortening of platelet life span is multifactorial in origin, including both mechanisms that depend on an active immune response and those that do not.     

Immunotherapy of cryptosporidiosis in immunodeficient animal models.

Immunotherapy for persistent infection caused by Cryptosporidium parvum was attempted in two immunodeficient animal models. BALB/c Athymic (nude) mice were infected with two oral doses of 2 x 10(7) C. parvum oocysts, and subsequently treated with monoclonal antibody (MAb) 17.41 that neutralizes sporozoites and merozoites. Persistent infection was established in all exposed mice. Daily oral treatment with MAb 17.41 for 10 days significantly reduced (p less than 0.005) the number of C. parvum organisms observed by microscopic study of intestinal tracts of infected mice. Young horses with severe combined immunodeficiency (SCID) also developed persistent infection following oral exposure with 10(8) C. parvum oocysts. In contrast to nude mice, SCID foals exhibited diarrhea associated with oocyst shedding. Two foals were treated orally with MAb 18.44 and immune serum, both of which neutralized C. parvum sporozoites and merozoites. Oocyst shedding patterns did not significantly differ from those in five SCID foals treated with nonimmune reagents. The results obtained indicate that SCID foals are a useful large animal model of clinical disease associated with persistent C. parvum infection, and that nude mice are a convenient animal model for testing therapeutic potential of antibodies in persistent cryptosporidial infection.     

SCID in Jack Russell terriers: a new animal model of DNA-PKcs deficiency

We recently described the incidence of a SCID disease in a litter of Jack Russell terriers. In this study, we show that the molecular defect in these animals is faulty V(D)J recombination. Furthermore, we document a complete deficit in DNA-dependent protein kinase activity that can be explained by a marked diminution in the expression of the catalytic subunit DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We conclude that as is the case in C.B-17 SCID mice and in Arabian SCID foals, the defective factor in these SCID puppies is DNA-PKcs. In mice, it has been clearly established that DNA-PKcs deficiency produces an incomplete block in V(D)J recombination, resulting in "leaky" coding joint formation and only a modest defect in signal end ligation. In contrast, DNA-PKcs deficiency in horses profoundly blocks both coding and signal end joining. Here, we show that although DNA-PKcs deficiency in canine lymphocytes results in a block in both coding and signal end joining, the deficit in both is intermediate between that seen in SCID mice and SCID foals. These data demonstrate significant species variation in the absolute necessity for DNA-PKcs during V(D)J recombination. Furthermore, the severity of the V(D)J recombination deficits in these three examples of genetic DNA-PKcs deficiency inversely correlates with the relative DNA-PK enzymatic activity expressed in normal fibroblasts derived from these three species.     

Differential regulation of interleukin-1 gene expression in human CD3- large granular lymphocytes

Our laboratory analyzed the expression of lymphokine and cytokine mRNA in CD3- peripheral blood large granular lymphocytes (LGL). Herein we present evidence that this subset of lymphocytes can synthesize IL-1 beta mRNA constitutively and that the cytoplasmic mRNA levels of IL-1 beta can be increased rapidly by interleukin (IL)-2. IL-1 alpha mRNA is expressed constitutively very infrequently and increases in IL-1 alpha mRNA are seen only after prolonged incubation with IL-2. Furthermore, IL-1 activity could not be detected in LGL culture supernatants, indicating that other processes may be involved in releasing biologically active IL-1 from LGL. In addition, MAb to the p75 IL-2 receptor on LGL abrogated IL-2 induction of IL-1 beta mRNA, suggesting that IL-2 signaling via the p75 IL-2 receptor induced IL-1 beta gene expression in LGL. Since, in contrast to T cells, LGL are capable of mediating effector functions without prior stimulation, they are said to be already "primed" for response. Overall, these data suggest that constitutive lymphokine gene expression may be involved in the in vivo priming of LGL.     

DNA-PKcs mutations in dogs and horses: allele frequency and association with neoplasia

Previously, spontaneous genetic immunodeficiencies in mice, Arabian foals, and recently in Jack Russell terriers have been ascribed to defects in DNA-PKcs (catalytic subunit of the DNA dependent protein kinase) expression. In severe combined immunodeficiency (SCID) foals, a 5 bp deletion at codon 9480 results in a frameshift and a 967 amino acid deletion from the C terminus (including the entire PI3 kinase domain) and an unstable mutant protein. In SCID mice, a single base pair mutation results in a premature stop codon and deletion of 83 amino acids; as in SCID foals, the mutant protein is unstable. Here, we define the mutation within the canine DNA-PKcs gene that results in SCID. In this case, a point mutation results in a stop codon at nucleotide 10,828 and premature termination at a position 517 amino acids before the normal C terminus resulting in a functionally null allele. Thus, this is the third documentation of a spontaneous germline mutation in the C terminus of DNA-PKcs. Emerging data implicate DNA repair factors as potential tumor suppressors. Here, we have ascertained the carrier frequency of the defective DNA-PKcs genes in Arabian horses and in Jack Russell terriers. Our data indicate (in good agreement with a previous report) that the carrier frequency of the equine SCID allele is approximately 8%; in contrast, the carrier frequency of the canine SCID allele is less than 1.1%. We also assessed the frequency of the equine SCID allele in a series of 295 tumors from Arabian horses. We find a statistically significant correlation between the development of a virally induced tumor (sarcoid) and heterozygosity for the equine SCID allele. These data provide further support for an emerging consensus: that DNA-PK may normally act as a tumor suppressor through its caretaker role in maintaining chromosomal stability.     

Inability of large granular lymphocytes to recirculate through blood-lymph barriers

The level of large granular lymphocytes (LGL) has been determined in thoracic lymph duct and peripheral blood of cattle. It has been shown that in contrast to the blood, these cells are present in the lymph in minor quantities. Unlike blood LGL, lymph LGL have smaller granules in cytoplasm. It is concluded that LGL do not recirculate from blood to central lymph.     

Frequency of the SCID gene among Arabian horses in the USA

Severe combined immunodeficiency disease (SCID) of horses is an autosomal, recessive hereditary disease occurring among Arabian horses. The genetic defect responsible for this disease was recently identified as a 5-basepair deletion in the gene encoding DNA-protein kinase catalytic subunit (DNA-PKcs). Horses with one copy of the gene appear normal, while horses with two copies of the gene manifest the disease. The present report describes a PCR-based test for detection of the gene defect and the results from testing 250 randomly selected Arabian horses. The frequency of SCID gene carriers was 8·4% (21/250). Based on the gene frequency reported here, the authors would expect 0·18% (1 out of 567) of Arabian foals to be affected with SCID based on a random breeding population.     

Defective thymocyte maturation in horses with severe combined immunodeficiency

Six monoclonal antibodies, designated EqT2, EqT3, EqT6, EqT7, EqT12, and EqT13, which identify T lymphocyte antigens present at different stages of T cell maturation were used to examine T lymphocyte development in foals with severe combined immunodeficiency (SCID). Flow microfluorimetry demonstrated the presence of EqT12+ and EqT13+ prothymocytes and a few phenotypically mature EqT2+ and EqT3+ thymocytes within the thymic remnants of SCID foals. However, very few EqT6+ and EqT7+ resident cortical thymocytes were detected. The near absence of EqT6+ and EqT7+ cortical thymocytes was confirmed by immunofluorescence analysis of thymic tissue from SCID foals. Those cells present were larger than normal cortical thymocytes. Furthermore, their activities of adenosine deaminase, adenosine monophosphate-deaminase, and 5' nucleotidase differed from those of normal cortical thymocytes. The combined evidence of monoclonal antibody analysis, size parameters, and purine enzyme activities demonstrate the near absence of cortical thymocytes in horses with this genetically defined immunodeficiency disorder.     

A C9 related channel forming protein in the cytoplasmic granules of human large granular lymphocytes

Large granular lymphocytes (LGL) from human blood main-tained in culture for 2 to 6 weeks with IL-2 were found positive in the K562 cell killing assay. The cytoplasmic granules of the LGL were isolated, lysed and the soluble proteins were passed over a Sepharose-anti-C9 column. The retained protein was eluted with NaC1 and found to consist by SDS polyacrylamide gel electrophoresis of essentially one component with a molecular weight of approximately 70 000. The protein did not give a positive precipitation test with anti-human C9 by Ouchterlony analysis, but it reacted reproducibly with anti-human C9 by Western blot analysis. By ELISA the cross reaction with human C9 was less than 1%. The C9 related lymphocyte protein lacked C9 hemolytic activity, but it formed functional pores in liposomes in presence of Ca⁺⁺ . These results suggest that the cytoplasmic granules of human LGL that are capable of killing NK target cells contain C9 related protein which is involved in the cellular cytotoxicity reaction.     

CD3 negative "small agranular lymphocytes" are natural killer cells

We describe here that CD3-, CD16+ and/or CD56+ small lymphocytes, in a highly reproducible fashion, mediate a significant level of K562 killing that is, on a "per cell" basis, comparable to the cytolytic activity of CD3- LGL. The CD3- small lymphocytes appeared to have no granules based on light and electron microscopy and lack of right-angle scatter on the FACS; we thus refer to them as small "agranular" lymphocytes (SAL). The lytic activity against K562 is inhibited by treatment with either L-leucine methyl ester or EGTA, which are reported to effect granule-dependent killing. We suggest that the SAL have lytic molecules in their cytoplasm (which are sensitive to these treatments) but that these molecules are not organized into discrete granules as found in LGL. The CD3- SAL are phenotypically very similar to LGL and both SAL and LGL mediated equal and reproducible antibody-dependent cell-mediated cytotoxicity. These observations force redefinition of the concept of NK cells to include both CD3- LGL and CD3- SAL.     

Ultrastructure of large granule-containing lymphocytes possessing natural killer activity

By means of light and electron microscopy, ultrastructural cytochemistry and immune cytochemistry methods, contents and ultrastructure of large granule-containing lymphocytes (LGL) have been studied in human blood--this is cell population possessing natural killer and, partly, antibody-depending cytotoxicity. The LGL concentrates are isolated from blood applying successive physical-chemical methods, differential centrifugation in the density gradient of pack-phycoll and percoll included. Separate LGL populations are marked by means of rosette-forming reaction with sheep erythrocytes and monoclonal antibodies OKT4 and OKT8. Relative and absolute amount of the LGL in 1 1 of blood is 5.4 +/- 0.5% and 0.319 +/- 0.28 X 10(9), respectively. The LGL ultrastructure is characterized with a low nuclear-cytoplasmic ratio, with presence of osmiophilic (azurophilic) granules in cytoplasm and specific parallel-tubular structures, with a well developed Golgi complex, an essential number of mitochondria, vesicles with smooth wall and vacuoles, as well as multivesicular bodies and Gallo bodies. The LGL subpopulations, expressing various membrane antigens (E+, E-, OKT8+, OKT8-) differ in their ultrastructure, that is evidently stipulated by the degree of their differentiation and their function.     

Protective effects of broadly neutralizing immunoglobulin against homologous and heterologous equine infectious anemia virus infection in horses with severe combined immunodeficiency

Using the equine infectious anemia virus (EIAV) lentivirus model system, we previously demonstrated protective effects of broadly neutralizing immune plasma in young horses (foals) with severe combined immunodeficiency (SCID). However, in vivo selection of a neutralization-resistant envelope variant occurred. Here, we determined the protective effects of purified immunoglobulin with more potent broadly neutralizing activity. Overall, protection correlated with the breadth and potency of neutralizing activity in vitro. Four of five SCID foals were completely protected against homologous challenge, while partial protection occurred following heterologous challenge. These results support the inclusion of broadly neutralizing antibodies in lentivirus control strategies.     

Seasonal changes in the white blood cell system, lyzozyme activity and cortisol level in Arabian brood mares and their foals

In 34 pure-breed Arabian horses divided into four groups (Gr. I, ten pregnant mares; Gr. II, seven barren mares; Gr. III, ten foals born in 1981; Gr. IV, seven foals born in 1982) seasonal changes in the white blood cell system, cortisol level and lyzozyme activity were studied. Seasonal periodicity was found in all groups for the number of lymphocytes, segmented neutrophils and eosinophils and cortisol level. Leukocyte periodicity was found in three groups, but not in the barren mares. In lyzozyme activity there was periodicity in three groups but not in the youngest foals. In the stab neutrophils, basophils and monocytes no cycle was observed. The behaviour of the indices studied showed the influence of age of the horses (mature vs young) and the physiological state of the mares (pregnancy or barrenness).     

Modulation of in vitro myelopoiesis by LGL: different effects on early and late progenitor cells

We describe here the modulatory activity of human peripheral blood natural killer (NK) cells on the growth and differentiation of myeloid progenitor cells at different stages of maturation. NK-enriched cell fractions containing 54 to 75% large granular lymphocytes (LGL) and displaying high levels of NK activity significantly inhibited the growth of late (7 day) granulocyte-macrophage colony-forming cells (CFU-GM) from about 50% of normal human bone marrow samples. However, the same fractions strongly enhanced the growth of early (14 day) stem cells from peripheral blood. Enhancing activity on early CFU-GM from blood was greater in highly purified NK cell preparations containing 96% LGL than in NK-depleted T cell preparations from the same donors. Analogous to the results when using the NK-enriched fractions, the NK-purified preparations inhibited late CFU-GM and stimulated the early ones. We conclude from these observations that human LGL have a modulatory effect on myelopoiesis depending on the maturation stage of the progenitor cell.     

Adaptive immunity is the primary force driving selection of equine infectious anemia virus envelope SU variants during acute infection

Equine infectious anemia virus (EIAV) is a lentivirus that causes persistent infection in horses. The appearance of antigenically distinct viral variants during recurrent viremic episodes is thought to be due to adaptive immune selection pressure. To test this hypothesis, we evaluated envelope SU cloned sequences from five severe combined immunodeficient (SCID) foals infected with EIAV. Within the SU hypervariable V3 region, 8.5% of the clones had amino acid changes, and 6.4% had amino acid changes within the known cytotoxic T lymphocyte (CTL) epitope Env-RW12. Of all the SU clones, only 3.1% had amino acid changes affecting potential N-linked glycosylation sites. In contrast, a much higher degree of variation was evident in SU sequences obtained from four EIAV-infected immunocompetent foals. Within V3, 68.8% of the clones contained amino acid changes, and 50% of the clones had amino acid changes within the Env-RW12 CTL epitope. Notably, 31.9% of the clones had amino acid changes affecting one or more glycosylation sites. Marked amino acid variation occurred in cloned SU sequences from an immune-reconstituted EIAV-infected SCID foal. Of these clones, 100% had amino acid changes within V3, 100% had amino acid changes within Env-RW12, and 97.5% had amino acid changes affecting glycosylation sites. Analysis of synonymous and nonsynonymous nucleotide substitutions revealed statistically significant differences between SCID and immunocompetent foals and between SCID foals and the reconstituted SCID foal. Interestingly, amino acid selection at one site occurred independently of adaptive immune status. Not only do these data indicate that adaptive immunity primarily drives the selection of EIAV SU variants, but also they demonstrate that other selective forces exist during acute infection.     

Lysis of fresh human tumour cells by autologous tumour-associated lymphocytes: Two distinct types of autologous tumour killer cells induced by co-culture with autologous tumour

Summary The specific and natural killer (NK)-restricted nature of auto-tumour cytotoxicity of tumour-associated lymphocytes was studied in cancer patients with malignant pleural effusions. Large granular lymphocytes (LGL) and small T lymphocytes were isolated from carcinomatous pleural effusions by centrifugation on discontinuous Percoll gradients. Tumour cells freshly isolated from pleural effusions were classified according to their susceptibility to lysis by Percoll-purified LGL from the blood of normal donors in a 4-h ⁵¹Cr release assay. Of 12 NK-sensitive tumour samples, 11 were killed by autologous fresh effusion LGL, whereas only 2 were lysed by autologous T cells. Neither LGL nor T cells were cytotoxic to NK-resistant autologous tumour cells. T cells and LGL were each cultured in vitro with autologous tumour cells for 6 days. Effusion LGL maintained their auto-tumour killing activity in 10 of 12 autologous mixed lymphocyte-tumour cultures (MLTC) with NK-sensitive tumour, while LGL lost the activity when cultured alone. Removal of high-affinity sheep erythrocyte-rosetting cells from Percoll-purified LGL enriched effector cells. Autologous MLTC-derived LGL could also kill NK-sensitive allogeneic effusion tumour cells and K562 cells, as did fresh LGL. In autologous MLTC LGL failed to acquire lytic function to NK-resistant autologous tumour cells. In contrast, in vitro activation of effusion T cells with autologous tumour cells induced auto-tumour killer cells in 9 of 12 NK-sensitive tumour samples and 3 of 6 NK-resistant tumour cases. However, cultured T cells were incapable of killing allogeneic tumour cells and K562 cells. In the autologous MLTC effusion T cells proliferated vigorously in response to autologous tumour cells, whereas LGL showed no proliferation. The enrichment of blasts from cultured T cells on discontinuous Percoll gradients resulted in an enhancement of auto-tumour cytotoxicity, with no reactions recorded in blast-depleted, small, resting T cells. These results indicate that two distinct types of auto-tumour-recognising lymphocytes, LGL and T cells, are present in carcinomatous pleural effusions of cancer patients and that each effector type recognises different membrane moieties of autologous effusion tumour cells.     

NK cell function in severe combined immunodeficiency (SCID): evidence of a common T and NK cell defect in some but not all SCID patients

The immunologic work-up of eight infants with the clinical diagnosis of severe combined immunodeficiency (SCID) was performed with special emphasis on natural killer (NK) cell function and ontogeny. Contrary to previous reports, our study shows that not all SCID patients lack NK activity; some may even express very high NK- and antibody-dependent cellular cytotoxicity (ADCC). The present group of eight SCID infants was homogeneous with respect to normal levels of the purine metabolism enzymes adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP). They all had low serum Ig levels and were defective for specific antibody formation against BSA and diphtheria toxin (DiT). None of the infants' peripheral blood mononuclear cells (PBMC) proliferated significantly upon in vitro stimulation with PHA, concanavalin A (Con A), pokeweed mitogen (PWM), and irradiated allogeneic lymphocytes. Seven of eight patients, however, responded significantly to mitogenic factors present in a lectin-free interleukin 2 (IL 2) preparation, and two exhibited a positive costimulation as well with simultaneous exposure to IL 2 + Con A. The lymphocyte marker analysis revealed high percentages of OKT10+ cells in seven of eight infants, whereas peripheral T cells (OKT3+) with suppressor/killer (OKT8+) or helper/inducer (OKT4+) phenotypes were abnormally low in all infants with one exception. The PBMC of two patients formed low to normal percentages of E rosettes but expressed no B cell markers (B-/SCID). The six other infants had high percentages of B cells (B+/SCID) but lacked E rosette-forming cells. High NK and ADCC activity was found in the two B-/SCID patients. The B+/SCID infants either totally lacked NK and ADCC function (four of six) or expressed low to normal NK activity together with some T cell markers as revealed by monoclonal antibody staining but not by E rosette formation (two of six). From the data presented, an ontogenic model is proposed that assumes the status of an independent cell lineage in between T cells and monocytes for human NK cells, or that places these cells in close proximity to early differentiation steps of the T cell lineage. In any case, NK cell function clearly constitutes an additional parameter of heterogeneity in the immunologic analysis of SCID.