Characterization of γ/δ T cell clones isolated from human fetal liverand thymus

The origin and development of T cells bearing γ/δ T cell receptors (TcR) has been extensively studied in the mouse. By contrast, little is known about development patterns and diversity of the human γ/δ T cell lineage. To study therepertoire of human γ/δ⁺ T cells during T cell ontogeny, wehave isolated clonal populations of γ/δ⁺ T cells from 14-week fetal thymus and liver and characterized the molecular compositionof their TcR. The technique of in situ hybridization was used to identify cells expressing TcR genes in fetal liver and thymus. A panel of clones representative of developing T cell populations found in vivo was subsequently isolated from both tissues and clones expressing cell surface γ/δ receptors were identified. Although both the liver-derived γ/δ⁺ T cell clone, L2, and the thymus-derived γ/δ⁺ T cell clone, T6, had similar cell surface phenotypes, namely CD3⁺, CD7⁺, CD45⁺ and CD8^?, their reactivity with anti-CD2 and -CD4 antibodies was different. L2 was CD2^high, CD4^? whereas T6 was CD2^low, CD4^low. Both clones possessed effector functions similar to those of adult T cells as demonstrated by the synthesis and secretion of cytokines in response to stimulation through the CD3/TcR complex. Analysis of the TcR composition of the fetal clones showed both clones to possess similar or identical γ chain components, C_γ1, J_γ1/2, V_γ8, and both utilize V_δ gene segments other than V_δ1. This TcR genotype has not been previously reported in the analysis of adult γ/δ⁺ T cells. Our studies have identified a unique population of human γ/δ⁺ T cells that may be derived extrathymically and appear to be preferentially and perhaps transiently expressed during fetal life.     

T lymphocyte development in p56lck deficient mice: allelic exclusion of the TcR β locus is incomplete but thymocyte development is not restored by TcR β or TcR αβ transgenes

The protein tyrosine kinase, p56^lck, is involved in signal transduction in mature T cells and in the molecular events controlling early thymocyte differentiation. Thymuses of mice deficient for p56^lck expression (p56^lck-/-) consist of immature CD4-CD8- double-negative (DN) and CD4⁺CD8⁺ double-positive (DP) thymocytes and are severely reduced in total cell number. In this report we have studied DN thymocytes from p56^lck-/- mice and found an increase in the proportion of the CD44^?CD25⁺ subset, suggesting that transit through this stage, which is known to require T cell receptor (TcR) β expression, may be delayed in the absence of p56^lck expression. In addition, the expression of a transgenic TcR β chain or TcR αβ pair did not restore thymic development in p56^lck-/- mice. However, in contrast to mice expressing a dominant negative isoform of p56^lck in which DP thymocytes do not develop, DP thymocytes still develop in nontransgenic and TcR transgenic p56^lck-/- mice. These results demonstrate that expansion of the DP subset is impaired in p56^lck-/- mice. In contrast, allelic exclusion is not severely compromised. Although there was an increase in the number of peripheral T cells expressing more than one Vβ chain in TcR transgenic p56^lck-/- mice, we found that inhibition of endogenous TcR β gene rearrangement was almost complete in thymocytes of Vβ transgenic p56^lck-/- mice and we could not detect any peripheral T cells that expressed more than one Vβ chain in non-transgenic p56^lck-/- mice.     

Sequential appearance of γ/δ- and α/β-bearing T cells in the peritoneal cavity during an i.p. infection with Listeria monocytogenes

To search for a potential role of T cell antigen receptor (TcR) γ/β-bearing cells in host-defense against Listeria monocytogenes, we analyzed the sequential appearance of γ/δ and α/β T cell in the peritoneal exudate cells (PEC) during an i.p. infection with sublethal dose (2 × 10³) of viable Listeria organisms in mice. The PEC on day 1 after the infection consisted of 48% macrophages and 50% lymphocytes, most of which were surface IgM⁺ (B) cells. The number of PEC increased to the maximal level by day 3. The PEC at this stage contained an appreciable number of CD3⁺ T cells in addition to a large number of macrophages. Of the CD3⁺ cells, the proportion of CD4^?CD8^? cells, most of which expressed no TcR α/β, increased to the maximal level on day 3 after the infection. In correlation with an increased number of CD3⁺CD4^?CD8^?TcR α/β^? cells, high level of TcR γ/δ chain gene messages was detected in the nonadherent population of the PEC on this stage. On the other hand, the PEC on day 8 contained an increased number of CD4⁺CD8^? and CD4^?CD8⁺ cells which expressed TcR α/β chain on their surface. These results suggest that the γ/δ T cells precede the α/β T cells in appearance during listerial infection. The γ/β T cells may be involved at the first line of the host-defense against Listeria.     

Do Human Peyer's Patches Contribute to the Intestinal Intraepithelial γ/δ T-Cell Population?

T-cell receptor γ/δ? (TcRγ/γ?) lymphocytes in human Peyer's patches (PP) adjoining ileal mucosa were studied by monoclonal antibodies with paired immunofluorescence staining in situ and by flowcytomelric phenotyping of isolated cells. The proportion of γδ? T cells in the follicle-associated epithelium outside the M-cell areas (median 4·I%, range 2·2–30·1%,) was similar to that in mucosal villous epithelium (median 4·4%, range 0·5 30·5%). Most intraepithelial °/δ cells (～90%) expressed CD45RO but only a few expressed CD8 (<10%) and none l-selectin; a dominating subset (median 46%) employed the Vδl/JδI gene product (range 22–100%). The M-cell areas lacked γ/δ cells but contained clustered CD20⁺ andCD3+ lymphocytes. Thesubepithelial PP dome area and interfollicular(T-cell) zones, as well as the mucosal lamina propria. contained very few γ/δ cells (median 1·7%, range 0·4–8·9%) which were dominated (88–100%) by the Vγ 2-encoded subset. Those in the dome area and lamina propria were often (～75%) CD45RO+ (range 44·90%) while very few (～2%) expressed i.-selectin (range 0·15%). By contrast, CD45RO expression on γ/δ cells in the PPT-cell zones was relatively low (～46%) and that of l-selectin relatively high (～43%). In conclusion, TcR γ/δ⁺ cells are quite rare in human PP and belong mainly to the Vδ²-encoded subset, thus being different from most inlraepithelial γδ cells that probably have another origin. The L-selectin ‘fraction of PPγ/δ cells presumably represent newly recruited ‘naïve’ T lymphocytes while CD45RO⁺γ/δ cells both in PP and lamina propria arc probably antigen-primed.     

CD45RA expression on γδ and αβ T cells emigrating from the fetal and postnatal thymus

We have compared the expression of CD45RA on αβ and γδ T cells emigrating from the fetal and postnatal thymus. The fetal and postnatal thymus export both CD45RA⁺ and CD45RA^- T cells. The number of γδ⁺CD45RA⁺ T cells was remarkably constant regardless of stage of ontogeny or T cell maturity. Around 5--8% of γδ thymic emigrants, thymocytes and peripheral blood lymphocytes expressed CD45RA in both fetal and postnatal animals. In contrast to γδ T cells, up to one quarter of both fetal and postnatal αβ emigrants expressed CD45RA. Post-thymic maturation of CD45RA expression on αβ emigrants, which occurred both before and after birth, appeared to be antigen independent.     

Developmental expression of the αIELβ7 integrin on T cell receptor γδ and T cell receptor αβ T cells

A novel monoclonal antibody, 2E7, was shown by immunoprecipitation to be reactive with the α_IELβ7 integrin and was employed to analyze the expression of this integrin in lymphocyte subsets and during T cell ontogeny. In adult lymph nodes, α_IEL was expressed at low levels by 40–70% of CD8⁺ T cells and < 5% of CD4⁺ T cells. However, virtually all intestinal intraepithelial lymphocytes and ?20% of lamina propria CD4⁺ T cells were 2E7⁺, indicating a preferential expression of this integrin on mucosal T cells. Examination of α_IEL integrin expression during thymus ontogeny revealed that ?3–5% of fetal or adult thymocytes were 2E7⁺. Interestingly, early in fetal thymus ontogeny, ?40% of 2E7⁺ cells expressed T cell receptor (TcR)-γδ and this subset persisted through birth. A developmental switch occurred such that 2E7⁺ TcR^? CD4^?8⁺ cells detected on fetal day 19 were followed by 2E7⁺ TcR-αβ CD4^?8⁺ cells in the neonatal thymus. The latter population persisted throughout thymus ontogeny into adulthood. Interestingly, a subset of TcR-γδ Vγ3⁺ day 16 fetal thymocyte dendritic epidermal cell (DEC) precursors were 2E7⁺, but all mature DEC expressed high levels of α_IEL integrin, suggesting that the α_IEL integrin was acquired late in DEC maturation. This possibility was strenghthened by immunohistochemical localization of the majority of 2E7⁺ γδ and αβ T cells to the medullary regions of the thymus. Overall, the results demonstrate a developmentally ordered expression pattern of the α_IELβ₇ integrin that suggests a common function for this integrin during TcR-γδ and -αβ CD4^?8⁺ T cell thymocyte development or perhaps in effector functions for these subsets.     

CD8 is needed for positive selection but differentially required for negative selection of T cells during thymic ontogeny

During thymic development, immature thymocytes expressing major histocompatibility complex (MHC) class I-restricted T cell receptors (TcR) differentiate into CD8⁺ T cells with cytolytic functions. To evaluate the role of CD8 in positive and negative selection during thymic ontogeny, mice rendered CD8-null by gene targeting were bred with three lines of transgenic mice expressing unique MHC class I-restricted TcR. In all three instances CD8 was required for positive selection of MHC class I-restricted transgenic T cells. The efficiency of positive selection decreased in accordance with a reduced level of CD8 expression on thymocytes. Surprisingly, there was a differential requirement for CD8 expression in negative selection of MHC class I-restricted thymocytes, depending on the antigen specificity of TcR. These observations show that CD8 is essential for positive selection but is differentially required for negative selection of MHC class I-restricted T cells. Thus thymic selection, at least for negative selection, can occur in the absence of the CD8 accessory molecule.     

Control of thymus-independent intestinal intraepithelial lymphocytes by β2-microglobulin

Murine intestinal intraepithelial lymphocytes (i-IEL) comprise thymusdependent cells such as T cell receptor (TcR) α/β CD8α/β⁺ i-IEL, as well as thymus-independent ones such as TcRα/β CD8α/α⁺ and TcRγ/δ CD8α/α⁺ i-IEL. Whilst the development of the CD8α/β expressing i-IEL is strictly contingent on major histocompatibility complex (MHC) class I surface expression, that of CD8α/α i-IEL appears largely MHC class I independent. We have used β2-microglobulin (β2m)^?/? mutant mice lacking surface-expressed MHC class I and TcRα/β CD8α/β⁺ i-IEL to analyze the potential impact of MHC class I on regional activation of thymus-independent i-IEL. To analyze the role of TcRγ/δ i-IEL in regional cell interactions, these mice were treated with the anti-TcRγ/δ mAb, GL3. Whilst numbers of TcRα/β CD8α/α i-IEL were markedly reduced in βm^+/? mice, those of TcRγ/δ i-IEL were elevated. Administration of GL3 in vivo caused TcR down-modulation and functional inactivation of TcRγ/δ i-IEL in β2m^+/? mice. In contrast, TcR expression and functional activities of TcRγ/δ i-IEL from β2m^?/? mice were not impaired by GL3 treatment. The TcRα/β CD8β i-IEL from β2m^?/? mice were expanded and functionally activated as a consequence of TcRγ/δ engagement. The TcRγ/δ i-IEL and TcRα/β CD8α/α⁺ i-IEL from athymic nu/nu mice which express MHC class I, but lack TcRα/β CD8α/β⁺ i-IEL, responded to TcRγ/δ engagement as those from the β2m^+/? controls. In addition, the TcRγ/δ i-IEL from TcRβ^?/? and TcRβ^+/? mutants were equally affected by GL3. We conclude that the absence of β2m renders TcRγ/δ i-IEL resistant to TcR-mediated inactivation and promotes activation of TcRα/β CD8β^? i-IEL. The activation of TcRγ/δ i-IEL seems to be directly controlled by β2m/MHC class I expression and independent from TcRα/β CD8β⁺ i-IEL. Regulation of self-reactive thymus-independent i-IEL through β2m/MHC class I may contribute to control of autoreactive immune responses in the intestine.     

Activated T lymphocytes in the celiac lesion: Non-proliferative activation (CD25) of CD4+ α/β cells in the lamina propria but proliferation (Ki-67) of α/β and γ/δ cells in the epithelium

In order to identify any dominating subset of activated T cells in the celiac lesion, we examined CD3⁺, CD4⁺, CD8⁺ and T cell receptor (TcR) γ/δ⁺ Lymphocytes in jejunal cryosections from 25 patients with celiac disease and 10 controls by three-color immunofluorescence staining for expression of the nuclear proliferation marker detected by monoclonal antibody (mAb) Ki-67 and the p55 α chain of interleukin-2 receptor (CD25). mAb Ki-67⁺ intraepithelial lymphocytes (IEL) were exclusively observed in celiac patients. The median proportion of CD3⁺ IEL positive for Ki-67 increased from nil in controls to 4.5% in partly treated (range 0-19.0%; n = 10; p = 0.05) and 12.8% in untreated celiac disease (range 4.0-30.7%; n = 15; p 0.005). Only 1.5% of CD3⁺ subepithelial T cells expressed the Ki-67 marker in celiac disease (range 0-9.5%). Two- and three-color staining combining mAb to CD3 and Ki-67 with mAb to CD4, CD8 or TcR5 showed that both TcR α/β⁺ CD8⁺ and TcR γδ⁺ (but not CD4⁺) mucosal T cells proliferated in the epithelium. By contrast, CD25 were almost exclusively expressed on CD4⁺ T cells in the lamina propria. The percentage of CD25⁺ T cells increased significantly from 1.7% in controls (range 0-2.9%) to 7.5% in partly treated (range 0.8-17.8%, p 0.002), and to 14.65% in untreated celiac disease (range 3.9-21%, p 0.002). These results suggest that gluten ingestion in celiac disease induces proliferative activation of TcR α/β⁺ CD8⁺ and TcR γδ⁺ IEL but non-proliferative activation (lymphokine production?) of lamina propria CD4⁺ T cells.     

CD5− dendritic epidermal T cells are derived from CD5+ precursor cells

Murine Thy-1⁺, TcR Vγ3/Vδ⁺ dendritic epidermal T cells (DETC) differ from most other T cell subsets by the absence of CD4 and CD8 antigens as well as the lack of CD5 expression. To see whether negativity for those antigens is an intrinsic feature of a given T cell population or if such triple-negative T cells go through a maturational stage where they express these antigens, we determined the phenotype of TcR Vγ3⁺ fetal thymocytes which are the precursor cells of DETC. We found that TcR Vγ3⁺ fetal thymocytes phenotypically differ from mature DETC in that they are CD5⁺, mostly CD8⁺ and partly CD4⁺. The injection of fetal thymic suspensions containing TcR Vγ3⁺/CD5⁺ (but not TcR Vγ3⁺/CD5^?) thymocytes into Thy-1-disparate athymic nude mice resulted in the appearance of donor-type TcR Vγ3⁺/CD5^? dendritic cells in the recipients' epidermis, indicating that TcR Vγ3⁺ thymocytes are indeed the precursors of CD5^? DETC. Tracing CD5 expression on DETC precursors during their intrathymic maturation and their migration to the fetal skin, we found that (i) the earliest DETC precursor cells as defined by TcR Vγ3 expression express high levels of CD5 antigen (day 15 of gestation), (ii) after day 16 of gestation 70% of TcR Vγ3⁺ thymocytes express high and 30% express intermediate levels of CD5, (iii) TcR Vγ3⁺ cells in the fetal blood express low levels of CD5, (iv) the first TcR Vγ3⁺ cells entering the epidermis express very low levels of this antigen and (v) TcR Vγ3⁺ epidermal cells later than day 19 of gestation are CD5^?. A similar down-regulation of CD5 expression on DETC precursors was also noted when TcR Vγ3⁺ cells were cultured in vitro. Even the addition of PMA and ionomycin, which up-regulates CD5 expression on TcR α/β-bearing thymocytes and lymph node T cells, could not prevent down-regulation on DETC precursors. The described cell system may serve as a useful tool in further experiments aimed to clarify the function of the CD5 glycoprotein as well as the mechanism(s) regulating its expression.     

The lack of CD8α cytoplasmic domain resulted in a dramatic decrease in efficiency in thymic maturation but only a moderate reduction in cytotoxic function of CD8+ T lymphocytes

The glycoprotein CD8 is believed to play an important role in the maturation and function of MHC class I-restricted T lymphocytes. CD8 has been proposed to function as a co-receptor of the TcR to participate in signal transduction, possibly through its cytoplasmic domain that binds to protein tyrosine kinase p56^lck. A T cell-specific transgene encoding CD8α truncated at the cytoplasmic domain (“tailless CD8α”), was introduced into CD8α-deficient mice. This animal model was used to study the role of the CD8 cytoplasmic domain in T cell ontogeny and function. “Tailless CD8α” was expressed on the cell surface of thymocytes and peripheral T cells. A small population of peripheral CD4^? T cells (6% of T lymphocytes) was found to have cell surface expression of “tailless CD8α” and endogenous CD8β indicating that these cells may belong to the CD8⁺ T cell lineage. A consistent result was obtained from CD8α-deficient mice bearing the “tailless CD8α” and the MHC class I-restricted 2C TcR transgenes. A small population of CD4 T cells expressing CD8β the “tailless CD8α” and the 2C TcR transgenes was present in the periphery of these mice in a selecting background, but was absent in a deleting background. When “tailless CD8α” mice were infected with lymphocytic choriomeningitis virus (LCMV), the peripheral CD8⁺ CD4^? T cell subset expanded dramatically and a significant LCMV-specific cytolytic activity was detected. The results suggest that the cytoplasmic portion of CD8α is not absolutely required but dramatically enhances the eficiency of thymic maturation of CD8⁺ T cells. The lack of CD8α cytoplasmic domain in peripheral CD8⁺ T cells does not abolish the generation of cytotoxicity in response to an in vivo LCMV infection, although the cytolytic activity is slightly reduced compared to that in control mice.     

Murine T Cell Determination of Pregnancy Outcome: I. Effects of Strain, αβ T Cell Receptor, γδ T Cell Receptor,and γδ T Cell Subsets

PROBLEM: T cells bearing αβ T cell receptor (TcR) and γδ TcR are present at the fetomaternal interface, and the latter, which express surface activation markers, can react with fetal trophoblast cell antigens. What is the role of these cells? METHOD: Using stress-abortion-prone DBA/2-mated CBA/J and abortion-resistant C57/B16 mice, αβ, γδ, and CD8^+/- T cell subsets were measured in spleen and uterine decidua. The effect of immunization against abortion and administration of anti-TcR antibody in vivo was examined. Cytokine synthesis was measured by intracellular staining of Brefeldin A-treated cells. RESULTS: Abortion-prone matings showed an unexpected accumulation of γδ T cells beginning in the peri-implantation period and this was suppressed by immunization against abortion. The immunization deleted γδ T cells producing the abortogenic cytokines, TNF-α and γ-interferon, and increased production of the anti-abortive cytokines, IL-10 and transforming growth factor-β2 (TGF-β2). Immunization also boosted the number of αβ T cells which were present in the decidua as early as 2 days after implantation. In vivo injection of GL4 (anti-δ) depleted γδ T cells producing Th1 cytokines in the peri-implantation period, and prevented abortions, whereas H57 (anti-β) decreased the number of αβ T cells and led to 100% abortions. CD8⁺ T cells present in peri-implant decidua before onset of abortions were mostly αβ TcR⁺, although some were γδ⁺. Changes in γδ and αβ T cells in pregnancy were most dramatic in uterine tissue. CONCLUSION: Although decidual γδ T cells after formation of a distinct placenta and fetus produce anti-abortive TGF-β2-like molecules and IL-10, prior events can lead to abortion. High local production of TNF-α and γ-interferon develop during the peri-implantation phase because of an excessive increase in the Th1 cytokine⁺ subset of γδ cells; these cytokines may be contributed by other tissues in decidua, and the contribution of bioactive factors by γδ T cells may augment the cytokine pool. In contrast, αβ T cells (which may be inactivated by stress that causes abortions) may mediate the anti-abortive effect of alloimmunization. Alloimmunization involves a shift from a Th1 to a Th2 pattern in the γδ T cells in decidua.     

Early degenerate selection of thymocytes by class I major histocompatibility complex

Ontogeny of T cells is accomplished in the thymus by a process of positive selection, in which interaction of the T cell receptor (TcR) expressed on CD4⁺8⁺ thymocytes with self major histocompatibility complex (MHC), expressed on cortical epithelial cells, determines the progress along the maturation pathway and confers self restriction to T cells. Conversely, cells behaving as self reactive by interaction with bone marrow-derived antigen-presenting cells are negatively selected by apoptosis. We show here that the presence of a class I-restricted soluble TcR (sTcR) in the fetal thymic microenvironment, early in T cell ontogeny, determines an enhanced negative selection of a sizeable number of CD4⁺8⁺ thymocytes, which have been previously subjected to a positive-selection event. We hypothesize that the generation of the mature thymic T cell repertoire stems from an interaction of TcR, under a critical affinity threshold, with a self peptide-MHC complex which is common to a great number of TcR specificities using the same restriction element. A shift in this affinity threshold, caused by sTcR, results in the generation of cells acting in a self-reactive manner, which are then deleted. In extended fetal thymus organ culture in the presence of sTcR, we have also observed the appearance of mature CD8⁺ T cells, which once adoptively transferred to syngeneic nude mice are expanded in the periphery, consistent with an enhanced avidity of these cells for self MHC.     

Ontogeny of fetal CD8lo4lo thymocytes: Expression of CD44, CD25 and early expression of TcR α mRNA

CD8^lo 4^lo cells are the immediate precursors of immature CD8^hi4^lcTcR^lo, CD8^hi 4^hiTcR^lo and CD8^hi4^hiTcR^lo double-positive (DP) thymocytes in the adult murine thymus. These cells are the first subset in the adult thymus to express accessory CD8 and CD4 molecules, to rearrange the T cell receptor (TcR) a chain genes and to express the TcR αβ heterodimer at low levels at the surface. Here, we investigate the fetal ontogeny of CD8^lo 4^lo cells. We detect these cells on day 15 of fetal development. They dominate the thymus on day 15.5, to become progressively less prominent thereafter. An important characteristic of fetal CD8^lo 4^lo cells is the early expression of TcR α mRNA (on fetal day 15, 36–48 h earlier than reported previously). Our results also suggest, but do not prove, that the receptor may be expressed on the surface as early as day 15.5. Fetal CD8^lo 4^lo cells, like their adult counterparts, become DP in vitro. However, early fetal CD8^lo 4^lo thymocytes express both CD44 and CD25 – unlike the adult subset - and that links them to their putative precursors, fetal CD44⁺CD25⁺ double-negative cells. This finding underscores the difference between adult and fetal thymocytes in turnover of membrane molecules and/or the kinetics of progression through phenotypic stages.     

T lymphocyte development in the absence of Fcϵ receptor Iγ subunit: analysis of thymic-dependent and independent αβ and γδ pathways

During fetal development, early thymocyte progenitors transiently express low affinity Fc receptors for IgG (FcγR) of both FcγRII and III isoforms. Only the FcγRIII isoform requires association of an FcγRIII (CD16) α subunit with an FcϵRIγ homodimer for surface expression. To address the role of FcγR in ontogeny, we studied thymic development in FcϵRIγ^−/− mice. We find that day 14.5 CD4⁻CD8⁻ double-negative (DN) fetal thymocytes of FcϵRIγ^−/− mice express mRNA of both FcγRIIb₁ and FcγRIII. Surface expression of FcγRII/III is readily detected on these cells. It appears that FcγRIIb₁, whose surface expression is FcϵRIγ independent, replaces FcγRIII during thymic development in these animals. Moreover, subsequent development into CD4⁺CD8⁺ double-positive and CD4⁺CD8⁻ and CD4⁻CD8⁺ single-positive subsets appears normal even in the absence of FcϵRIγ. However, alterations were noted in adult animals among the DN αβ TCR⁺ thymocytes and peripheral splenic DN T cells as well as CD8αα⁺ intestinal intraepithelial lymphocytes (iIEL). In contrast to conventional T lymphocytes, which do not express either FcγRIII or FcϵRIγ, DN αβ TCR⁺ thymocytes and extrathymically derived αβ TCR⁺ and γδ TCR⁺ CD8αα⁺β⁻ iIEL express TCR which incorporate FcϵRIγ as one of their subunits. Consistent with this, the TCR levels of these cells are lower than the TCR levels on cells from wild-type C57BL/6 mice. Despite the reduction in the level of surface TCR, the development of these cells was unaltered by the absence of FcϵRIγ. Thus, we observed alterations in adult DN αβ TCR⁺ thymocytes, splenic DN αβ TCR⁺ and DN γδ TCR⁺ large granular lymphocytes (LGL), and αβ TCR⁺ and γδ TCR⁺ CD8αα⁺β⁻ iIEL, but no detectable changes in their major fetal thymic developmental pathways. Cultivation of peripheral DN αβ TCR⁺ and DN γδ TCR⁺ cells from FcϵRIγ^−/− mice with interleukin-2 generates LGL which mediate natural killer activity. Unlike LGL from wild-type C57BL/6 mice, LGL from FcϵRIγ^−/− mice lack FcγRIII expression and could not mediate antibody-dependent cellular cytotoxicity through FcγRIII.