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The interpretation and application of clinical and laboratory data is an essential component of the job of a laboratory medicine practitioner or Transfusion Medicine director. Such data may be generated from assays within one’s own laboratory or may arise in the form of published studies. Ultimately, such data will often be incorporated into routine practice and therefore, a clear understanding of data interpretation is critical. This chapter will provide a review of common scenarios faced by laboratorians and physicians where data interpretation is at the forefront of the clinical challenge.

Although donor-specific transfusion (DST) plus CD154 blockade represents a robust protocol for inducing transplantation tolerance, the underlying mechanisms are incompletely understood. In a murine T-cell adoptive transfer model, we have visualized alloantigen-specific, TCR-transgenic for H2-A(b) /H2-K(d) 54-68 epitope (TCR75) CD4(+) T cells with indirect allospecificity during the course of tolerance induction. Three main observations were made. First, although the majority of TCR75 CD4(+) T cells were deleted following DST plus CD154 blockade, the surviving TCR75 CD4(+) T cells were capable of making IL-2, upregulating CD44, and undergoing cell division, suggesting that they were functionally active. Indeed, residual TCR75 CD4(+) T cells reisolated from the primary recipients given DST plus CD154 blockade were fully capable of rejecting allografts upon secondary transfer. Second, in tolerant mice, TCR75 CD4(+) T cells were not induced to express Foxp3 in the graft-draining lymph node. TCR75 CD4(+) T cells were also absent in accepted graft tissues in which endogenous Treg cells were enriched. Finally, DST plus CD154 blockade resulted in an abortive expansion of TCR75 CD4(+) T cells, a process that required the presence of endogenous Treg cells. Collectively, surviving TCR75 CD4(+) T cells are immunocompetent but kept in check by an endogenous immunosuppressive network induced by DST plus CD154 blockade.

T cell depletion strategies are an efficient therapy for the treatment of acute rejections and are an essential part of tolerance induction protocols in various animal models; however, they are usually nonselective and cause wholesale T cell depletion leaving the individual in a severely immunocompromised state. So far it has been difficult to selectively delete alloreactive T cells because the majority of protocols either delete all T cells, subsets of T cells, or subpopulations of T cells expressing certain activation markers, ignoring the Ag specificity of the TCR. We have developed a model in which we were able to selectively deplete alloreactive T cells with an indirect specificity by targeting intact MHC molecules to quiescent dendritic cells using 33D1 as the targeting Ab. This strategy enabled us to inhibit the indirect alloresponse against MHC-mismatched skin grafts and hence the generation of IgG alloantibodies, which depends on indirectly activated T cells. In combination with the temporary abrogation of the direct alloresponse, we were able to induce indefinite skin graft survival. Importantly, the targeting strategy had no detrimental effect on CD4+CD25+FoxP3+ T cells, which could potentially be used as an adjunctive cellular therapy. Transplantation tolerance depends on the right balance between depletion and regulation. For the former this approach may be a useful tool in the development of future tolerance induction protocols in non-sensitized patients.

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules - a process known as indirect recognition. As CD4(+)CD25(+) Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4(+)CD25(+) cells from C57BL/6 mice (H-2(b)) with allogeneic DCs from BALB/c mice (H-2(d)). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2K(d) presented by H-2A(b) MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential.

This review focuses on the role of antigen-specific T cells that mediate active inhibition of immune responses over the past 35 years since their initial description. The field has experienced several changes in the accepted paradigm of such suppressor/regulatory T cells, from initial indications that such cells were CD8(+), to the view that such cells did not exist, to the identification of the transcription factor Foxp3 as a key orchestrator of inhibitory function. Although most Foxp3(+) cells in a resting animal are CD4(+)CD25(+) cells, Foxp3 expression and inhibitory function can be induced by antigens in the periphery by selective cytokine conditions, particularly TGF-beta. Such induced T cells occur within both the CD4 and the CD8 T-cell lineages and appear to mediate suppression by inhibiting the costimulatory activity of antigen-presenting cells and the production of inhibitory cytokines. Recent data generated by analysis of TCR Tg T cells that do not select many Foxp3-positive cells during thymic development are reviewed, emphasizing the pattern of "linked suppression" and focus of the relative potency of different mechanisms of suppression.

Although donor-specific transfusion (DST) plus CD154 blockade represents a robust protocol for inducing transplantation tolerance, the underlying mechanisms are incompletely understood. In a murine T-cell adoptive transfer model, we have visualized alloantigen-specific, TCR-transgenic for H2-A(b) /H2-K(d) 54-68 epitope (TCR75) CD4(+) T cells with indirect allospecificity during the course of tolerance induction. Three main observations were made. First, although the majority of TCR75 CD4(+) T cells were deleted following DST plus CD154 blockade, the surviving TCR75 CD4(+) T cells were capable of making IL-2, upregulating CD44, and undergoing cell division, suggesting that they were functionally active. Indeed, residual TCR75 CD4(+) T cells reisolated from the primary recipients given DST plus CD154 blockade were fully capable of rejecting allografts upon secondary transfer. Second, in tolerant mice, TCR75 CD4(+) T cells were not induced to express Foxp3 in the graft-draining lymph node. TCR75 CD4(+) T cells were also absent in accepted graft tissues in which endogenous Treg cells were enriched. Finally, DST plus CD154 blockade resulted in an abortive expansion of TCR75 CD4(+) T cells, a process that required the presence of endogenous Treg cells. Collectively, surviving TCR75 CD4(+) T cells are immunocompetent but kept in check by an endogenous immunosuppressive network induced by DST plus CD154 blockade.

Strategies to limit life-long dependence on antiretroviral therapy (ART) are needed. We randomized 81 human immunodeficiency virus (HIV)-infected subjects to 4 interventional arms involving continued ART plus ALVAC vCP1452 (or placebo) with or without interleukin (IL)-2 infusions. Viral load rebound 12 weeks after ART interruption was then analyzed to assess immune control. Fifty-two subjects reached the study end point. ALVAC recipients had 0.5 log 10 lower virologic rebounds (P = .033). IL-2 plus vaccine boosted CD4 + T cell counts (P

Background. The ability to control human immunodeficiency virus (HIV) replication in vivo in the absence of antiretroviral therapy (ART) is a measure of the efficiency of antiviral immunity. In a study of patients with chronic, ART-suppressed HIV infection, AIDS Clinical Trials Group 5068 investigated the effects of immunization with an exogenous HIV vaccine and pulse exposure to the subject’s unique viral epitopes, by means of structured treatment interruptions (STIs), on the dynamics of viral rebound during a subsequent analytical treatment interruption (ATI). Methods. Ninety-seven subjects receiving stable ART with an HIV-1 RNA load !50 copies/mL and CD4 + T lymphocyte count 1400 cells/mm 3 were randomized to undergo continued ART, STIs, ALVAC-HIV vCP1452 immunization, or STIs and ALVAC-HIV vCP1452 immunization. Results. Subjects in the 2 STI arms had a significantly longer median doubling time in the period of the initial rise of viral load, a significantly lower median peak viral load, a significantly lower median end-of-ATI viral load set point, and a greater proportion of subjects with an end-of-ATI viral load set point !1000 copies/mL, compared with the subjects in the 2 arms without STIs. With an immunization schedule of 3 sets of 3 weekly injections, ALVAC-HIV vCP1452 did not affect viral load measures. Conclusions. In this randomized, controlled study of intermittent STI as a therapeutic autoimmunization strategy, evidence of enhanced immunologic control of HIV replication was demonstrated. Although antiretroviral chemotherapy raises CD4 + T

Although immunological memory is characterized by both an increase in the frequency of antigen-specific T cells and a qualitative change in the pattern of their subsequent response, it is not clear which of these components is more significant in the overall enhanced response to secondary stimulation. To address this question for the CD4+ T-cell response, T-cell receptor (TCR) Tg T cells were adoptively transferred to normal syngeneic mice that were immunized with the relevant peptide. After the initial expansion of TCR Tg T cells, the size of the subsequent memory population of T cells was approximately the same as the size of the starting population, independent of the number of TCR Tg cells initially transferred. This result was not caused by redistribution of memory cells into non-lymphoid tissues, although the relative frequency of antigen-specific T cells in these sites was increased after immunization. The fraction of the antigen specific TCR Tg cells that responded by production of either interleukin-2 or interferon-gammain vitro was substantially higher after immunization. Thus, the increased frequency of functionally responsive T cells was primarily caused by a higher fraction of responding T cells, rather than a substantial increase in the absolute number of antigen specific CD4+ TCR Tg T cells.

Understanding the mechanisms of rejection of organs transplanted between unrelated individuals is confounded by the complexity of the alloantigens and the diversity of T cells responding to these alloantigens. To circumvent these problems, we developed a transgenic (Tg) C57BL/6 model system in which the T-cell receptor (TCR) expressed by CD4 T cells is specific for a defined allogeneic H-2Kd peptide and the cardiac donor expressed H-2Kd as a transgene on the C57BL/6 background (B6.Kd). These TCR Tg T cells were previously shown to mediate rapid rejection of a B10.D2 cardiac allograft when transferred to Rag1 recipients, demonstrating that the "indirect" pathway of allorecognition is sufficient for complete rejection in the absence of other T cells or antibody. Here, we report that B6.Kd hearts were rejected in an accelerated fashion by Rag1(-/-) TCR Tg T cells adoptively transferred to normal B6 recipients. Rejection in this model was associated with large myocardial infarcts and significant coronary artery inflammation. Moreover, transferred TCR Tg CD4+ cells mediated allograft injury without the requirement for cytotoxic function from recipient-derived CD8 T cells. A non-linear relationship was observed between the initial precursor frequency of the antigen-specific TCR Tg cells and the ultimate tempo of acute rejection, which is taken as evidence for cooperativity between components of the system.

T cells from TCR transgenic mice, expressing receptors specific for an allogeneic MHC class I peptide, were used to track T cell activation and migration in normal adoptive recipients that were subsequently transplanted with heterotopic hearts that were syngeneic except for a transgenic MHC class I antigen. T cells rapidly disappeared from the blood into the lymphoid tissues where they were activated within one day after transplantation. T cells initially formed discrete clusters in the spleen and lymph nodes. After proliferating for 2-4 days in lymphoid tissues, T cells reappeared in the blood and migrated to the heart and the intestines. The T cells underwent another round of proliferation in the heart, but not the intestines, and induced cardiac rejection uniformly on 6 day.

The vast array of epitopes presented by allografts and the diversity of T cells responding to them complicates mechanistic studies of rejection. To minimize these problems, we developed a transgenic (Tg) model system limited to a single T-cell receptor (TCR)/peptide/major histocompatibility complex molecule. Two alloantigen-specific CD4 T-cell clones were used to isolate cDNA encoding the TCRalpha and TCRbeta chains that recognize the Kd54-68/I-Ab epitope. Two different TCR Tg lines were produced in C57BL/6 (B6) mice and crossed onto the B6.Rag1-/- background. B6.Rag1-/- recipients of T cells from TCR Tg Rag1-/-mice promptly rejected B10.D2, but not irrelevant B10.BR, cardiac grafts. Thus, a single allogeneic epitope presented by self-major histocompatibility complex class II is sufficient to activate TCR Tg T cells and serve as a target for rejection.

The chronically HIV-infected cellular reservoir in lymphoid tissue (LT) represents a formidable obstacle to the long-term success of antiretroviral therapy. Cytoreductive chemotherapy with cyclophosphamide (CTX) reduces cells in LT, and we hypothesized that coadministration of antiretroviral therapy with CTX may diminish the cellular reservoir over time. Ten antiretroviral treatment-naive subjects were recruited, and they received stavudine, lamivudine and nelfinavir (antiretroviral therapy, ART) until 2 consecutive plasma HIV RNA levels measured < 50 copies/ml (step 1). Five subjects then received ART alone, whereas five subjects received ART plus three escalating doses of CTX (step 2). Viral DNA was measured in LT obtained by excisional lymph node biopsy and peripheral blood mononuclear cells (PBMCs), using quantitative polymerase chain reaction at three time points in both groups (before steps 1 and 2, and after CTX). Viral DNA declined in both groups after the initiation of ART alone in step 1. During step 2 both groups experienced a modest decline compared with step 1. However, no significant differences were observed in viral DNA in LT or PBMCs between the ART alone and the ART plus CTX groups. Suppression of plasma HIV RNA levels < 50 copies/ml was not maintained in the ART plus CTX group, perhaps because of inadequate medication adherence. The group receiving ART plus CTX had lower CD4(+) lymphocyte counts and absolute total lymphocytes compared with the ART alone group. We conclude that the addition of CTX to ART did not diminish the cellular reservoir in HIV-infected persons.

Highly active antiretroviral therapy (HAART) of HIV disease is associated with effective virologic control, immune reconstitution, and clinical improvements. This study addresses the potential for improvements in lymphocyte phenotype and virologic responses of HIV-infected persons with extensive experience with dual nucleoside reverse transcriptase (NRTI) treatment and advanced HIV disease after a change to a potent antiretroviral therapy (NRTI + protease inhibitor). The majority of participants achieved virologic success. There was a median rise in CD4+ lymphocytes of 99 cells/mm(3) by 48 weeks, because of an increase in memory CD4+ cells at 4 and 16 weeks, followed by a later increase in naive CD4+ cells between weeks 16 and 48. The proportion of activated, DR+ CD38+ CD8+ lymphocytes decreased during the 48 weeks of follow-up. The immunologic findings (increased memory and naive T cells and reduced activation levels) were significantly improved in participants with persistent suppression of viral replication over the 48 weeks of the study. Baseline HIV RNA copy number was lower (median, 14,784 copies/ml) in persons who responded virologically than in those not suppressing viral replication (median, 49,454 copies/ml). CD4+ cell counts above the median (125/mm(3)) at time 0 for the participants, was the only baseline immunologic marker significantly associated with viral suppression at week 48. Participants older than 40 years of age demonstrated less immunologic recovery. The results of the study show that patients with extensive experience with NRTIs respond both virologically and immunologically during the first 48 weeks of therapy with a potent antiretroviral regimen.

The IgM-Fc receptor (FcμR) is involved in IgM homeostasis as evidenced by increased pre-immune serum IgM and natural auto-antibodies of both IgM and IgG isotypes in Fcmr-deficient C57BL/6 (B6) mice. To determine the impact of Fcmr-ablation on autoimmunity, we introduced the Fcmr null mutation onto the Fas-deficient autoimmune-prone B6.MRL Faslpr/lpr mouse background (B6/lpr). Both IgM and IgG auto-antibodies against dsDNA or chromatin appeared earlier in FcμR(−) B6/lpr than FcμR(+) B6/lpr mice, but this difference became less pronounced with age. Splenic B2 cells, which were 2-fold elevated in FcμR(+) B6/lpr mice, were reduced to normal B6 levels in FcμR(−) B6/lpr mice, whereas splenic B1 cells were comparable in both groups of B6/lpr mice. By contrast, marginal zone (MZ) B cells were markedly reduced in FcμR(−) B6/lpr mice compared with either FcμR(+) B6/lpr or wild type (WT) B6 mice. This reduction appeared to result from rapid differentiation of MZ B cells into plasma cells in the absence of FcμR, as IgM antibody to a Smith (Sm) antigen, to which MZ B cells are known to preferentially respond, was greatly increased in both groups (B6/lpr and B6) of FcμR(−) mice compared with FcμR(+) B6/lpr or B6 mice. Mott cells, aberrant plasma cells with intra-cytoplasmic inclusions, were also increased in the absence of FcμR. Despite these abnormalities, the severity of renal pathology and function and survival were all indistinguishable between FcμR(−) and FcμR(+) B6/lpr mice. Collectively, these findings suggest that FcμR plays important roles in the regulation of auto-antibody production, Mott cell formation and the differentiation of MZ B cells into plasma cells in B6.MRL Faslpr/lpr mice.

The control mechanisms that maintain a steady state viral load during chronic HIV-1 infection are critical to understanding the pathophysiology of HIV disease. The conceptual features of the two alternative models of viral control, referred to in this article as target cell limitation and immune control, are compared to the data regarding the viral and cellular dynamics of HIV-1 infection and the pattern of changes induced by effective antiretroviral drug therapy. The available data support the model that an antigen-driven immune response is the primary mechanism that limits viral growth in vivo.

Quantitation of HIV-1 specific RNA and DNA is pivotal to understanding the pathophysiology of HIV-1 diseases. A method has been developed for quantitation of HIV-1 DNA/RNA by real-time PCR using a unique fluorogenic primer-probe adduct known as scorpion. The probe hybridises to the extension of the adjoining primer intramolecularly, a process kinetically and thermodynamically more favourable than the conventional bimolecular probe-target hybridisation. Data presented in this paper indicate that the scorpion assay is extremely robust and is quite comparable to beacon-based assays. The scorpion assay is also comparable to quantitative competitive PCR (QC--PCR) assays but requires only a fraction of time and effort. Additionally, the dynamic range of the scorpion assay is several log-fold higher than the conventional end point PCR assays. As few as ten copies of vDNA can be detected in the presence of a large excess of exogenously added genomic DNA. Limiting dilution analysis indicates that the assay is capable of detecting a single copy of the viral template. Thus, the scorpion assay presents a specific and sensitive approach for quantitation of DNA/RNA templates by real-time PCR.

Background. One of the complexities of solid organ allograft rejection is the inherent diversity of the specific T cell antigenic epitopes that participate in this response, including the role of direct alloantigen recognition and indirect recognition of donor-derived peptides in recipient antigen-presenting cells. To probe the role of distinct T cell receptor (TCR) avidity differences and the role of cytokine expression patterns of different effector T cells that may participate in allograft rejections, we have identified a dominant allopeptide derived from the H-2K d molecule, recognized by H-2 b CD4 T cells in the context of syngeneic I-A b . Methods. To identify a stimulatory peptide derived from the H-2K d molecule, a panel of synthetic overlapping peptides was screened for immunogenicity and a panel of T cell clones established. These clones were characterized for TCR Vβ usage by mAb staining and/or reverse transcribed-polymerase chain reaction analysis, peptide dose sensitivity as a marker of TCR avidity, cytokine expression phenotype in vitro, and their ability to mediate rejection of a vascularized cardiac allograft after adoptive transfer to immunodeficient mice. Results. The H-2K d 54-68 peptide was identified as a dominant stimulatory peptide by the ability of T cells from C57BL/6 (H-2 b ) mice primed by a combination of allogeneic spleen cell injection and mixed peptide immunization to mount an in vitro proliferative response and interferon-γ production by peptide stimulation. Furthermore, direct immunization with synthetic H-2K d 54-68 peptide of normal C57BL/6 mice resulted in accelerated rejection of both skin and cardiac allografts from B10.D2 (H-2 d ) mice, but not 3rd party B10.BR (H-2 k ) grafts. A panel of 15 distinct CD4 + clones specific for H-2K d 54-68 peptide were established and shown to utilize a variety of TCR Vβ and different apparent TCR avidities to H-2K d 54-68 peptide when stimulated in vitro. To characterize these clones further, two clones were chosen based on the difference of avidity to H-2K d 54-68 peptide. The cytokine expression pattern was determined and indirect alloantigen specificity confirmed by analysis of responses to purified peptide and B10.D2 spleen cells using normal H-2 b and I-Aβ chain knockout mice as APC donors. Both of these T cell clones were able to mediate rejection of B10.D2, but not B10.BR hearts, in immunodeficient mice, but the morphological pattern of T cell infiltration was distinct. Conclusions. These results demonstrate the potential importance of fine dissection of the alloantigeneic response to solid organ transplants and provide unique insights into the role of TCR avidity and cytokine expression patterns in different morphological patterns of transplant rejection.

Virus reservoirs can persist in human immunodeficiency virus type 1 (HIV-1)-infected subjects despite effective plasma virus suppression. To compare viral dynamics in the absence and presence of antiretroviral therapy, blood mononuclear cells from 19 subjects with high plasma RNA levels and 18 subjects following prolonged virus suppression were examined, by use of in situ hybridization, to detect virus RNA expression before and after in vitro T cell activation. This approach reveals circulating lymphocytes expressing HIV-1 RNA before activation and an increase in cells with detectable HIV-1 RNA transcription after in vitro activation. The frequencies of these 2 cell populations are strongly correlated with plasma virus load and appear to be stable once a new steady state is established during therapy. The frequency of viral RNA-positive cells is equivalent to the frequency of cells that produce infectious virus. Thus, in HIV-1-infected subjects there are distinct virus reservoirs comprising both latent and replication-active cells.

Previous studies proposed a dynamic, steady-state relationship between HIV-mediated cell killing and T-cell proliferation, whereby highly active antiretroviral therapy (HAART) blocks viral replication and tips the balance toward CD4(+) cell repopulation. In this report, we have analyzed blood and lymph node tissues obtained concurrently from HIV-infected patients before and after initiation of HAART. Activated T cells were significantly more frequent in lymph node tissue compared with blood at both time points. Ten weeks after HAART, the absolute number of lymphocytes per excised lymph node decreased, whereas the number of lymphocytes in the blood tended to increase. The relative proportions of lymphoid subsets were not significantly changed in tissue or blood by HAART. The expression levels of mRNA for several proinflammatory cytokines (IFN-gamma, IL-1beta, IL-6, and macrophage inflammatory protein-1alpha) were lower after HAART. After therapy, the expression of VCAM-1 and ICAM-1 -- adhesion molecules known to mediate lymphocyte sequestration in lymphoid tissue -- was also dramatically reduced. These data provide evidence suggesting that initial increases in blood CD4(+) cell counts on HAART are due to redistribution and that this redistribution is mediated by resolution of the immune activation that had sequestered T cells within lymphoid tissues.

Development of distinct CD4+ T cell cytokine phenotypes may be conditioned by the anatomic site in which activation occurs. A double-label in situ hybridization technique was used to characterize co-expression of cytokine mRNA in antigen-specific responses of Peyer's patch (PP), lamina propria (LP), and splenic (SP) CD4+ T cells isolated from alpha beta T cell receptor-transgenic mice. Interleukin (IL)-2 was the dominant cytokine expressed by antigen-stimulated PP and SP populations, though it was expressed by a minority of the activated T cells. Cells that expressed interferon (IFN)-gamma were less frequent, and IL-4, IL-5, and IL-10 were infrequent. In contrast, cells that expressed IFN-gamma or IL-10 were most frequent in the LP population, with lower frequencies of IL-2, and few IL-4- and IL-5-positive cells. Co-expression of two cytokines by the same cell was the exception, regardless of the anatomic site from which the T cells were isolated. The surface phenotype of transgene-positive T cells isolated from each anatomic site was distinct, despite the absence of in vivo exposure to antigen for which the transgenic T cell receptor is specific. These data suggest that the cytokine responses of CD4+ T cells may be conditioned by the microenvironment, independently of specific antigen, and that the LP CD4+ T population has a distinct cytokine expression pattern with counter-regulatory properties that may be important for homeostasis in mucosal immune tissues.

Several reports support the view that growth hormone (GH) promotes proliferation and cytotoxicity by T cells in a mixed leukocyte culture (MLC). The present study was undertaken to begin to determine the mechanism of action of GH on the MLC in vitro. First, we determined that peripheral blood mononuclear cells (PBMC) cultured with mitomycin-treated allogeneic PBMC in an MLC in the presence of exogenously added rhGH develop an augmented proliferative (25-100%) and cytotoxic response (50-600%). We next examined the possibility that GH may promote alloresponses by inducing gamma-interferon (IGN gamma) production. In these experiments, in situ hybridization was used to determine the frequency of cells expressing mRNA for IFN gamma. It was observed that GH increased significantly the frequency of cells expressing mRNA for IFN gamma (100-800%). To determine the site of action of rhGH, we evaluated the response of purified T cells to alloantigens in the presence of rhGH. The addition of rhGH to an MLC had no demonstrable effect when purified T cells were used as the responding population. However, when T cells were reconstituted with autologous mitomycin-treated PBMC and used as the responding population, rhGH augmented proliferation and cytotoxicity. Taken together, these data show that rhGH augments proliferation, cytotoxicity and IFN gamma production during an MLC, and at lease part of the action of rhGH appears to be on the autologous antigen-presenting cell.

To examine the role of T cell subpopulations in an acute graft-versus-host (GVH) reaction, gamma delta T cells and alpha beta T cells expressing one of the two prototypic V beta families were negatively isolated from adult blood samples and injected into allogeneic chick embryos. CD4+ alpha beta T cells expressing either V beta 1 or V beta 2 receptors were equally capable of inducing acute GVH reactions, consistent with the idea that alpha beta T cell alloreactivity is determined by CDR3 variability. By themselves, the gamma delta T cells were incapable of inducing GVH reactions. However, host gamma delta T cells were recruited into the donor alpha beta T cell-initiated lesions, where they were activated and induced to proliferate. The data suggest that gamma delta T cells may play a secondary role in GVH reactions.

Objective. This pilot phase II, double-blind, placebo-controlled trial of 1 month duration, with a 2–3-month open-label extension, evaluated the safety, tolerability, biologic effects, and efficacy of interleukin-2 diphtheria fusion protein (DAB486IL-2) in refractory rheumatoid arthritis (RA). Methods. Forty-five RA patients were enrolled in the trial, and were randomized, after a 3–4-week diseasemodifying antirheumatic drug washout, to receive a daily intravenous dose of either DAB486-IL-2 or placebo (saline) for 5 days. A blinded, third-party observer evaluated arthritis activity. Clinical response was defined as ≥25% improvement in swollen and tender joints and ≥25% improvement in at least 2 of 6 additional parameters. The double-blind phase was 4 weeks; placebo patients could cross over to receive open-label treatment for a maximum of 3 monthly DAB486IL-2 cycles. Results. In the double-blind phase, 4 of 22 patients (18%) in the treated group and none in the placebo group (P = 0.05) met the criteria for clinical response. During the open-label treatment phase, 11 of 36 patients (31%) and 11 of 33 patients (33%) had a clinical response after completing 2 and 3 courses of DAB486IL-2, respectively. Adverse events included transient fever/chills (45%), nause/vomiting (50%), elevated (≥13x normal) transaminases (55%), and increased joint pain (45%). Twelve patients (8 placebo, 4 DAB486IL-2) did not complete 3 treatment cycles. No apparent differences were noted in CD4+ CD25+ cells of responders versus nonresponders, or of DAB486IL-2-treated versus placebo-treated patients. Conclusion. Clinical responses were noted in patients treated with DAB486IL-2 (18%) compared with placebo (0%) in the double-blind phase. In the open-label phase, 33% of patients completing 3 monthly DAB486IL-2 cycles had improvement in arthritis activity. Further studies of IL-2 diphtheria fusion proteins are warranted to elucidate factors that may predict clinical response and define mechanism(s) of action.

BACKGROUND AND PURPOSE Improvement in axonal regeneration may lead to the development of longer nerve grafts and improved outcomes for patients with peripheral nerve injury. Although the use of acellular nerve grafts has been well documented (Groves et al, Exp Neurol. 2005;195:278-292; Krekoski et al, J Neurosci. 2001;21:6206-6213; Massey et al, Exp Neurol. 2008;209:426-445; Neubauer et al, Exp Neurol. 2007;207:163-170; Zuo et al, Exp Neurol. 2002;176:221-228), less is known about the ability of neurotrophic factors to enhance axonal regeneration. This study evaluates axonal ingrowth augmentation using acellular, chondroitinase-treated nerve grafts doped with nerve growth factor (NGF). METHODS Acellular chondroitinase-treated murine nerve grafts were placed in experimental (NGF-treated grafts) and control (carrier-only grafts) rats. Five days after implantation, axonal regeneration was assessed by immunocytochemistry along with digital image analysis. RESULTS Higher axon count was observed throughout the length of the nerve in the NGF group (P < 0.0001), peaking at 3 mm from proximal repair (P = 0.02). Although the NGF group displayed a higher axon count per slice, the mean diameter of individual NGF axons was smaller (P < 0.0001), potentially consistent with induction of sensory axons (Rich et al, J Neurocytol. 1987;16:261-268; Sofroniew et al, Annu Rev Neurosci. 2001;24:1217-1128; Yip et al, J Neurosci. 1984;4:2986-2992). CONCLUSION The simple technique of doping acellular, chondroitinase-treated nerve grafts with NGF can augment axonal ingrowth and possibly preferentially induce sensory axons.

We report the characterization of a molecule recognized on chicken T cells by the murine A19 monoclonal antibody that was generated by immunization with intestinal intraepithelial lymphocytes. Immunofluorescence analysis indicated that both alpha beta and gamma delta T cell subpopulations in the intestine express the A19 antigen, but natural killer cells and B cells do not. The A19-marked T cells were preferentially localized in the intestinal epithelium and less frequently in the underlying lamina propria. T cells appearing in the intestine during embryonic life were A19 negative but acquired the antigen within the first few days after hatching. Although rarely found on cells in non-intestinal tissues at any age, very late expression of the A19 antigen could be induced by concanavalin A stimulation of splenic and circulating T cells. Transforming growth factor beta 1 enhanced this induction of A19 expression. The A19 molecules expressed by intestinal T cells and activated splenic T cells were biochemically identical, consisting of a multi-molecular complex of proteins with approximate M(r) of 205, 145 and 75 kDa under nonreducing conditions and 120, 90 and 28 kDa under reducing conditions. The characteristics of this multimolecular complex and its differential expression suggest that the A19 antigen is a member of the integrin family which may function in the retention of intestinal lymphocytes.

This study was designed to evaluate which of several T-cell-specific, immune response assays are the most relevant in measuring the key characteristics of an effective immune response to HIV-1. Using 5 HIV-1 antigens as stimulants, we assessed lymphocyte proliferation, supernatant gamma interferon (IFN-γ) cytokine production (CP), single-cell IFN-γ production by enzyme-linked immunospot (ELISPOT) assay, with and without Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs), and intracellular cytokine production (ICC) for IFN-γ and interleukin 2 (IL-2) by flow cytometry. We used these to compare specimens from HIV-1-infected subjects who were virally suppressed with a stable antiretroviral therapy (ART) regimen (group A) with specimens from subjects not on ART but with HIV-1 viremia of

Aims: Prior studies have shown that HAART is associated with decreased HIV viral load in the lungs. The correlation between antiretroviral exposure in bronchoalveolar lavage (BAL) fluid and virologic response was evaluated in patients starting HAART and enrolled in The AIDS Clinical Trial Group Protocol 723. Materials & methods: A total of 24 subjects underwent blood and BAL sampling prior to starting HAART, and after 4 and 24 weeks of HAART. Drug concentrations and HIV RNA were measured in paired plasma and BAL samples. Results: Antiretroviral drugs, including efavirenz, were detectable in BAL fluid of HIV-infected subjects beginning HAART. Efavirenz was also associated with a higher likelihood of clearing HIV RNA from the lungs. Conclusion: These results suggest the excellent pulmonary virologic response to antiretroviral therapy may, in part, be due to penetration of antiretroviral drugs into the alveolar compartment.

Targeting canarypox (CP)-HIV vaccine to dendritic cells (DCs) elicits anti-HIV-1 immune responses in vitro. We conducted a phase I/II clinical trial to evaluate whether adding DC to a CP-HIV vaccine improved virologic control during analytic treatment interruption (ATI) in HIV-1-infected subjects. Twenty-nine subjects on suppressive antiretroviral therapy were randomized to vaccination with autologous DCs infected with CP-HIV + keyhole limpet hemocyanin (KLH) (arm A, n = 14) or CP-HIV + KLH alone (arm B, n = 15). The mean viral load (VL) setpoint during ATI did not differ between subjects in arms A and B. A higher percentage of subjects in the DC group had a VL setpoint

The murine BP-3 antigen was initially described as a variably glycosylated cell surface protein of Mr 38,000 to 48,000 on lymphoid and myeloid cells. In the present experiments we found that this antigen is released from the surface of pre-B cells and macrophages by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), suggesting a glycosyl-phosphatidylinositol (GPI) linkage with the plasma membrane. When the tissue distribution of the BP-3-reactive cells was examined by immunohistology, high levels of the antigen were observed on brush borders of the intestinal epithelial cells, within collecting tubules of the kidney and on a subpopulation of reticular cells located on lymph nodes. Peyer's patches and the white pulp areas of the spleen. In contrast, reticular cells located in the thymus, bone marrow and splenic red pulp did not express the BP-3 antigen. Ontogenic studies revealed that BP-3 was expressed by the reticular cells in peripheral lymphoid tissues in the neonatal period near the time of lymphocyte immigration into these organs. BP-3+ reticular cells were observed in the collapsed periarterial lymphatic sheaths of adult mice depleted of T and B cells by cyclophosphamide treatment and in mice with severe combined immunodeficiency (scid), indicating that development of this reticular network is lymphocyte independent. The BP-3 antigen on the splenic reticular cells was also GPI anchored but its glycosylation pattern differed from that of the BP-3 molecules on pre-B cells. A specific subpopulation of reticular cells is thus marked by the BP-3 antigen, and the distribution and biochemical properties of the molecule make it an attractive candidate for a role in lymphocyte-stromal interactions in the peripheral lymphoid tissues.

The role of CD4+ T cells in the gastrointestinal (GI) immune system in vivo was studied in mice selectively depleted of this subset by treatment with monoclonal anti-L3T4 (GK1.5) mAb. Treatment of young BALB/c mice with weekly injections of anti-L3T4 mAb resulted in a selective depletion of CD4+ T cells in both IgA effector (lamina propria regions of the intestine; LP) and inductive (Peyer's patch; PP) sites. However, levels of CD3+CD4-CD8+ and CD4-CD8- (double negative) T cells remained constant or increased. When sections of small intestine were assessed for the isotype of Ig-containing cells, normal mice contained predominantly IgA plasma cells with small numbers of IgM and IgG plasma cells while anti-L3T4 treatment dramatically reduced the numbers of IgA plasma cells. When numbers of IgA-producing cells were assessed by the isotype-specific ELISPOT assay, the LPL of anti-L3T4 mAb-treated mice showed an 80% reduction in the number of IgA spot-forming cells. The effect of anti-L3T4 mAb treatment on IgA inductive sites was also studied and this treatment reduced the overall size of PP as well as the germinal centers in this tissue. Although anti-L3T4 treatment depleted CD3+CD4+ T cells in PP, the relative frequency of surface IgA-positive (slgA+) B cells in this tissue did not change. These results show that repeated injection of anti-L3T4 mAb results in a CD4+ T cell deficiency in both IgA inductive (PP) and effector (LP) sites. The depletion of CD4+ T cells resulted in reductions in the numbers of mature IgA plasma cells present in the LP of gut-associated tissues, and reduced the overall size of PP including germinal centers, but did not affect the frequency of sIgA+ B cells in this IgA inductive site.

Background. Human immunodeficiency virus (HIV) is readily detectable in the lungs of infected subjects and leads to an accumulation of CD8 + lymphocytes in the alveolar space. Although highly active antiretroviral therapy (HAART) is effective in reducing viremia, less is known about its effect on tissue compartments. The AIDS Clinical Trials Group Protocol 723 Team evaluated the effect of HAART on lung viral load and cellular constituents. Methods. Bronchoalveolar lavage (BAL) fluid and blood were collected before initiation of HAART and again at 4 and 24 weeks after initiation of therapy. The BAL cell differential was determined, lymphocyte phenotyping was performed, and acellular BAL fluid, plasma HIV RNA load, and BAL cell and peripheral blood mononuclear cell HIV RNA and DNA loads were measured. Results. HAART induced a rapid decrease in HIV that was detectable in acellular BAL fluid and a slower decrease in the HIV RNA and DNA loads in BAL cells. HAART was associated with a significant decrease in the absolute number and percentage of CD8 + alveolar lymphocytes. There was a significant correlation between residual BAL cell DNA at 24 weeks and the absolute number of CD4 + lymphocytes in the alveolar space. Conclusion. HAART is associated with a significant decrease in the pulmonary HIV burden and a return of alveolar cellular constituents to normal.

BACKGROUND: Interleukin 2 (IL-2) administration increases CD4 counts in persons with higher counts. This study investigated persons with moderately advanced human immunodeficiency virus infection receiving highly active antiretroviral therapy (HAART). METHODS: Two hundred four patients with CD4 T-cell counts from 50/microL to 350/microL who were treatment naive or had been treated only with reverse transcriptase inhibitors began a specified protease inhibitor HAART regimen. Virologic responders (< or =5000 copies/mL) at 12 weeks were randomized to open-label continuous-infusion IL-2 (IV IL-2), subcutaneous IL-2 (SC IL-2), or HAART alone. Thirty were not randomized and 15 enrolled in a substudy, leaving 159 for analysis. Subjects continued HAART alone for 72 weeks (n = 52) or with IV IL-2 (n = 53) or SC IL-2 (n = 54) for 5 days every 8 weeks. The IV IL-2 subjects could switch to SC IL-2 if their CD4 T-cell count increased by 100/microL or by 25%. RESULTS: Patients receiving IV or SC IL-2 had greater increases in CD4 cell counts. At week 84, median increases were 459/microL, 312/microL, and 102/microL. Increases of greater than 50% at week 60 (primary end point) were achieved in 39 patients (81%) and 32 (67%) in the IV and SC IL-2 arms, respectively, compared with 13 (29%) in the HAART arm (P

Although CD4+CD25+FoxP3+ regulatory T cells play a role in allograft tolerance, the role of CD8+ cells with immunosuppressive function is less clear. To address this issue, spleen cells from Rag-1-deficient TCR transgenic (Tg) mice expressing a receptor for ovalbumin (OVA) in the context of MHC class I (OT1) were activated with OVA expressing antigen-presenting cell (APC) in the presence or absence of exogenous transforming growth factor beta (TGFbeta). TGFbeta inhibited the expression of IFN-gamma, granzyme B and the lytic activity of the OT1 T cells while inducing FoxP3 expression in 5-15% of the cells. By contrast, FoxP3 expression was not detected in naive OT-1 T cells or OT-1 T cells activated without exogenous TGFbeta. TGFbeta-activated OT1 cells inhibited the activation of Kd-specific CD8+ CTL responses by normal B6 T cells and the proliferation by Kd-specific CD4+ TCR Tg T cells, but only if the OVA epitope was co-expressed by Kd+ APC. This antigen-specific inhibitory activity, referred to as linked suppression, was neither mediated by residual lytic activity within the activated OT1 T cells nor did it depend upon IL-10 or TGFbeta. Suppression correlated with inhibition of CD86 expression on CD11c+ APC. TGFbeta-activated OT1 T cells also delayed the rejection of heterotopic, vascularized cardiac allografts mediated by anti-Kd-specific CD4+ TCR Tg T cells, but only if the cardiac allograft expressed both OVA and Kd as transgenes. Prolonged survival of allografts was associated with rapid migration of the FoxP3+ OT1 T cells into the donor heart raising the possibility that suppression may be mediated within the allograft. These data show that TGFbeta-activated CD8+ T cells mediate antigen-specific, APC-focused patterns of suppression in vitro and in vivo.

The aim of this study was to evaluate the tolerability and activity of intratumoral administered human interleukin 12 encoded by a vector derived from the canarypox virus (ALVAC-IL-12). Nine patients with surgically incurable metastatic melanoma who had subcutaneous nodules available for injection were enrolled. ALVAC-IL-12 was administered by intratumoral injection on days 1, 4, 8, and 11. Tumor nodules greater than 2 cm in diameter were injected with 2 x 10(6) median tissue culture infectious doses (TCID(50)), and smaller tumors were injected with 1 x 10(6) TCID(50). The total dose per patient per time point ranged from 1 x 10(6) to 4 x 10(6) TCID(50). Toxicity was mild to moderate and consisted of inflammatory reactions at the injection site and fever associated with chills, myalgia, and fatigue. No dose-limiting toxicities occurred. Increases in IL-12 mRNA, and also increases in interferon gamma mRNA, were observed in ALVAC-IL-12-injected tumors compared with saline-injected control tumors in four of the nine patients. ALVAC-IL-12-injected tumors were also characterized by T cell infiltration. Three patients demonstrated increases in serum IL-12 and in interferon gamma levels. All patients developed neutralizing IgG antibody to the canarypox vector. One patient manifested a complete response of injected subcutaneous metastases and uninjected in-transit metastases. The intratumoral injection of ALVAC-IL-12 at these dose levels and according to this schedule was well tolerated and resulted in measurable biologic response in patients with metastatic melanoma.

Clinical and virologic consequences of temporary interruption of HIV therapy are incompletely understood.To describe a febrile illness that was consistent with the acute HIV syndrome and occurred after interruption of antiretroviral therapy.Case report.University clinic.HIV-infected man.Plasma viral load, lymphocyte subsets, diagnostic evaluation (including cultures and serologic tests), and analysis of lymph node tissue.The patient began antiretroviral therapy 3 months after initial HIV exposure and had sustained viral suppression, except during a brief scheduled treatment interruption. One hundred sixty-nine days after resuming therapy, the patient discontinued it again immediately following an influenza vaccination. Eleven days later, he presented with a febrile mononucleosis-like syndrome associated with dramatic shifts in plasma HIV RNA level (50 to1 000 000 copies/mL) and CD4 cell count (0.743 x 10(9) cells/L to 0.086 x 10(9) cells/L). Evaluation for alternative causes of fever was unrevealing. Symptoms resolved rapidly with resumption of HIV therapy.Therapeutic interruption may be associated with profound viral rebound and recurrence of the acute HIV syndrome.

The purpose of this study was to determine whether thymic transplantation in addition to highly active antiretroviral therapy (HAART) will restore T cell function in HIV infection. Eight treatment-naive HIV-infected patients with CD4+ T cell counts of 200-500/mm3 were randomized into thymic transplantation and control arms. All patients received HAART (zidovudine, lamivudine, and ritonavir) for 6 weeks prior to transplantation. Thymic transplantation was done without immunosuppression, using postnatal HLA-unmatched cultured allogeneic thymus tissue. Patients were immunized every 6 months with the neoantigen keyhole limpet hemocyanin (KLH) and the recall antigen tetanus toxoid (TT). T cell phenotype and function and T cell receptor rearrangement excision circles (TRECs) were assessed. Thymic allografts were biopsied at 2 months. Six HIV-infected patients completed the study. Four patients received cultured allogeneic postnatal thymic grafts, two others were controls. CD4+ T cell counts increased and T cell-proliferative responses to Candida antigen and TT normalized in all patients. Proliferative responses to KLH developed in three of four transplant recipients and one of two controls. Patients responding to KLH after secondary immunization had greater TREC increases compared with the patients who did not respond. All thymic allografts were rejected within 2 months. In summary, four of six patients developed T cell-proliferative responses to the neoantigen KLH over the first 2 years of HAART. The transplanted thymus tissue, however, was rejected. There was no clear difference in restoration of T cell function in the transplant recipients compared with the controls. Increases in TRECs after initiation of HAART may correlate with improved immune function.

Stromal cell-derived factor 1 (SDF-1) is the natural ligand that recognizes CXCR4, which also serves as a coreceptor for some strains of HIV-1. In this study, we explored SDF-1 blood levels among HIV-1-infected individuals exhibiting a wide range of CD4+ cell counts. Plasma or serum concentrations of SDF-1 protein were measured by ELISA in samples from 31 HIV-1-seronegative individuals and 79 HIV-1-infected subjects. Although SDF-1 protein levels were stable for months among seronegative individuals (mean intrasubject variation, 17%), the absolute values varied widely (0.28 to 106.5 ng/ml; mean, 25.6 ng/ml). In HIV-1-infected subjects, there was a direct correlation between SDF-1 level and CD4+ cell count. Subjects with fewer than 50 CD4+ cells per cubic microliter of blood had significantly lower mean SDF-1 levels (+/-SD) than did either HIV-1-infected subjects with higher CD4+ cell counts or uninfected controls: CD4+ cell count50, mean SDF-1 level of 10.7+/-33.7, 50CD4+ cell count200, mean SDF-1 level of 12.9+/-19.0, 200CD4+ cell count500, mean SDF-1 level of 19.3+/-36.8; CD4+ cell count500, mean SDF-1 level of 18.5+/-25.2; uninfected control mean SDF-1 level, 25.6+/-34.7. No significant change in SDF-1 level was detected after administration of antiretroviral therapy in nine subjects with advanced disease (mean intrasubject variation, 43%). Analysis of SDF-1 mRNA expression in lymph nodes from HIV-1-infected subjects at different disease stages revealed that the medullary cords contained stromal cells that express SDF-1 mRNA. This preliminary analysis suggests a possible link between lower SDF-1 levels and disease progression.

Quantitative analysis of the relationship between virus expression and disease outcome has been critical for understanding HIV-1 pathogenesis. Yet the amount of viral RNA contained within an HIV-expressing cell and the relationship between the number of virus-producing cells and plasma virus load has not been established or reflected in models of viral dynamics. We report here a novel strategy for the coordinated analysis of virus expression in lymph node specimens. The results obtained for patients with a broad range of plasma viral loads before and after antiretroviral therapy reveal a constant mean viral (v)RNA copy number (3.6 log10 copies) per infected cell, regardless of plasma virus load or treatment status. In addition, there was a significant but nonlinear direct correlation between the frequency of vRNA+ lymph node cells and plasma vRNA. As predicted from this relationship, residual cells expressing this same mean copy number are detectable (frequency

Recombinant human growth hormone (rhGH) improves growth in children after renal transplantation, but may be associated with augmented immune responses. We previously demonstrated that rhGH augments proliferative and cytotoxic responses and interferon-gamma (IFN-gamma) mRNA expression during a mixed leukocyte culture (MLC). In this study, we evaluated 12 pediatric patients after receiving a renal allograft from one of their parents. Peripheral blood mononuclear cells (PBMC) were isolated from patients and cultured with either donor or unrelated third-party PBMC in an MLC. Patients developed significant donor-specific hyporesponsiveness (DSH), however, no correlation was seen between the amount of DSH and graft function. Of the 12 patients, 2 developed augmented responses in the presence of rhGH. rhGH augments proliferation, cytotoxicity, and IFN-gamma expression during an MLC. Some patients develop increased responses to donor-specific alloantigens after renal transplantation. Further study is needed to better determine the significance of this finding.

A sensitive in situ hybridization procedure using both digoxigenin and 35S-labeled riboprobes is described that allows detection of single T cells expressing cytokine mRNA species in both single and double label formats. Modifications to existing procedures have been developed that allow in situ hybridization to be performed in either fresh frozen tissue sections or cytocentrifuge preparations of cultured cells. For single label studies, the digoxigenin labeling technique is equivalent to 35S labeling for sensitivity of detection and is superior with respect to precise localization and ease of use. A procedure to detect two cytokine mRNA species in individual cells can be performed using one digoxigenin-labeled riboprobe and one 35S-riboprobe, with equivalent sensitivity between the two labels and no non-specific mixing of the two signals. Since production of many T cell cytokines are controlled by transcriptional mechanisms, the use of in situ hybridization will be useful to investigate the biology of T cell activation, patterns of cytokine phenotype development, and histological localization of cytokine expressing cells in inflammatory lesions. Initial studies using this method to examine cytokine expression by a panel of T cell clones reveals that individual cytokine genes are not necessarily expressed in coordination in individual cells and relatively few individual cells in a Th0 clone express Th1-like and Th2-like cytokines simultaneously.

Natural killer (NK) cell activity appears to be conserved throughout vertebrate development but NK cells have only been well characterized in mammals. Candidate NK cells have been identified in the chicken as cytoplasmic CD3+ and surface T cell receptor (TCR)/CD3- (TCRO) lymphocytes that often express CD8. The fact that the TCRO cells are abundant in the embryonic spleen before T cells enter this organ allowed us to cultivate the embryonic TCRO cells using growth factors derived from activated adult lymphocytes. These TCRO cells were cytotoxic for an NK target cell line. They expressed cell surface CD8, a putative interleukin-2 receptor, CD45 and a receptor for IgG, but did not express CD4, major histocompatibility complex class II or immunoglobulin. Biochemical analysis of the cytoplasmic CD3 antigen revealed two of the three CD3 gamma, delta and epsilon homologues, and RNA transcripts for the third. The CD3 monoclonal antibody also precipitated a 32-kDa dimer that may represent a heterodimer of different CD3 constituents. TCR alpha and beta gene transcripts were not detected in the TCRO cells. These results indicate that the avian TCRO cell is the mammalian NK cell homologue. The shared evolutionary features of T cells and NK cells in birds and mammals support the idea that they derive from a common progenitor.

We have analyzed the embryonic development of a population of lymphoid cells that express a CD3 antigenic determinant in the cytoplasm but not on the cell surface. Since these cells lack T cell receptor (TcR) molecules, we have provisionally named them TCRO cells. Their development, expansion and distribution was investigated following transplantation of splenic and bursal fragments from chicken embryos into quail embryos. Since quail cells are not recognized by our panel of monoclonal antibodies against chicken TcR1, TcR2, TcR3, CD3, CD4 and CD8 molecules, these antibodies provided reliable markers for donor chick lymphocytes in the tissues of the quail recipients. Transplanted spleen and bursa both generated CD3+ cells, the number of which increased as a function of age. Notably, approximately half of these CD3+ cells expressed surface CD8, but none acquired TcR1 (gamma/delta), TcR2 (alpha/beta) or TcR3 expression. Since TCRO cells normally appear first in the spleen of 8-day chick embryos (E8), their generation in E6 splenic transplants indicated an extrathymic origin. The TCRO cells of chick splenic origin migrated to the spleen, bursa and thymus of the quail recipients. In six of seven chimeras acquiring CT3+ cells in the recipient thymus, these cells were restricted to the medulla and displayed the typical TCRO phenotype: CD3+CD8+TcR1-TcR2-TcR3-. These intrathymic TCRO cells also lacked the CT1 thymocyte antigen. We conclude that the TCRO cells represent a thymus-independent lineage of lymphoid cells that can migrate into a receptive thymus by rarely, if ever, differentiate into conventional T cells.