Circulating tumor DNA changes for early monitoring of anti-PD1 immunotherapy: a proof-of-concept study.

BACKGROUND: Recent clinical results support the use of new immune checkpoint blockers (ICB), such as anti-PD-1 (e.g. nivolumab and pembrolizumab) and anti-PD-L1 antibodies. Radiological evaluation of ICB efficacy during therapy is challenging due to tumor immune infiltration. Changes of circulating tumor DNA (ctDNA) levels during therapy could be a promising tool for very accurate monitoring of treatment efficacy, but data are lacking with ICB. PATIENTS AND METHODS: This prospective pilot study was conducted in patients with nonsmall cell lung cancer, uveal melanoma, or microsatellite-instable colorectal cancer treated by nivolumab or pembrolizumab monotherapy at Institut Curie. ctDNA levels were assessed at baseline and after 8 weeks (w8) by bidirectional pyrophosphorolysis-activated polymerization, droplet digital PCR or next-generation sequencing depending on the mutation type. Radiological evaluation of efficacy of treatment was carried out by using immune-related response criteria. RESULTS: ctDNA was detected at baseline in 10 out of 15 patients. At w8, a significant correlation (r = 0.86; P = 0.002) was observed between synchronous changes in ctDNA levels and tumor size. Patients in whom ctDNA levels became undetectable at w8 presented a marked and lasting response to therapy. ctDNA detection at w8 was also a significant prognostic factor in terms of progression-free survival (hazard ratio = 10.2; 95% confidence interval 2.5-41, P < 0.001) and overall survival (hazard ratio = 15; 95% confidence interval 2.5-94.9, P = 0.004). CONCLUSION: This proof-of-principle study is the first to demonstrate that quantitative ctDNA monitoring is a valuable tool to assess tumor response in patients treated with anti-PD-1 drugs.

Innate mucosal-associated invariant T (MAIT) cells are activated in inflammatory bowel diseases.

Inflammatory bowel diseases are characterized by a deregulated immune response targeting the gut bacterial flora. Mucosal-associated invariant T (MAIT) cells are major histocompatibility complex (MHC) class Ib-restricted innate-like lymphocytes with anti-bacterial functions. They display an effector/memory phenotype and are found in large numbers in the blood, mucosae and liver. They have also been implicated in inflammatory diseases such as multiple sclerosis. Therefore, we aimed to analyse the possible involvement of MAIT cells in Crohn's disease (CD) and ulcerative colitis (UC). To this end, a phenotypical and functional analysis of MAIT cells isolated from the blood of healthy subjects, CD and UC patients was undertaken. MAIT cells were also quantified in ileal biopsies of CD patients. The frequency of blood MAIT cells was specifically reduced in IBD patients compared with healthy donors, whereas it was dramatically greater in the inflamed versus healthy tissue. MAIT cells were activated as they expressed significantly more the Ki67 antigen, and this was accompanied by phenotypical changes such as increased expression of natural killer (NK)G2D and B and T lymphocyte attenuator (BTLA). Finally, in-vitro-activated MAIT cells from CD and UC patients secreted significantly more interleukin (IL)-17, together with a decreased interferon (IFN)-γ in CD but an increased IL-22 in UC. These data show that MAIT cells are activated in IBD, which results in an increased recruitment towards the inflamed tissues, an altered phenotype and a switch in the pattern of cytokine secretion. This is the first demonstration that MAIT cells are immune players in IBD, whose precise functions in this context need to be addressed.

Acute effects of postural changes and lower body positive and negative pressure on the eye.

Introduction: Entry into weightlessness results in a fluid shift and a loss of hydrostatic gradients. These factors are believed to affect the eye and contribute to the ocular changes that occur in space. We measured eye parameters during fluid shifts produced by lower body negative pressure (LBNP) and lower body positive pressure (LBPP) and changes in hydrostatic gradient direction (supine-prone) in normal subjects to assess the relative effects of fluid shifts and hydrostatic gradient changes on the eye. Methods: Ocular parameters (intraocular pressure (IOP), ocular geometry, and optical coherence tomography measures) were measured in the seated, supine, and prone positions. To create a fluid shift in the supine and prone positions, the lower body chamber pressure ranged from -40 mmHg to +40 mmHg. Subjects maintained each posture and LBNP/LBPP combination for 15 min prior to data collection. A linear mixed-effects model was used to determine the effects of fluid shifts (as reflected by LBNP/LBPP) and hydrostatic gradient changes (as reflected by the change from seated to supine and from seated to prone) on eye parameters. Results: Chamber pressure was positively correlated with both increased choroidal thickness (ß = 0.11 , p = 0.01) and IOP (ß = 0.06 p < 0.001). The change in posture increased IOP compared to seated IOP (supine ß = 2.1, p = 0.01, prone ß = 9.5, p < 0.001 prone) but not choroidal thickness. IOP changes correlated with axial length (R = 0.72, p < 0.001). Discussion: The effects of hydrostatic gradients and fluids shifts on the eye were investigated by inducing a fluid shift in both the supine and prone postures. Both hydrostatic gradients (posture) and fluid shifts (chamber pressure) affected IOP, but only hydrostatic gradients affected axial length and aqueous depth. Changes in choroidal thickness were only significant for the fluid shifts. Changes in hydrostatic gradients can produce significant changes in both IOP and axial length. Fluid shifts are often cited as important factors in the pathophysiology of SANS, but the local loss of hydrostatic gradients in the head may also play an important role in these ocular findings.

An invariant T cell receptor alpha chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD4-8- T cells in mice and humans.

The mouse thymus contains a mature T cell subset that is distinguishable from the mainstream thymocytes by several characteristics. It is restricted in its usage of T cell receptor (TCR) V beta genes to V beta 8, V beta 7, and V beta 2. Its surface phenotype is that of activated/memory cells. It carries the natural killer NK1.1 surface marker. Furthermore, though it consists entirely of CD4+ and CD4-8- cells, its selection in the thymus depends solely upon major histocompatibility complex (MHC) class I expression by cells of hematopoietic origin. Forced persistence of CD8, in fact, imparts negative selection. Here, we have studied the TCR repertoire of this subset and found that, whereas the beta chain V-D-J junctions are quite variable, a single invariant alpha chain V alpha 14-J281 is used by a majority of the TCRs. This surprisingly restricted usage of the V alpha 14-J281 alpha chain is dependent on MHC class I expression, but independent of the MHC haplotype. In humans, a similar unusual population including CD4-8- cells can also be found that uses a strikingly homologous, invariant alpha chain V alpha 24-JQ. Thus, this unique V alpha-J alpha combination has been conserved in both species, conferring specificity to some shared nonpolymorphic MHC class I/peptide self-ligand(s). This implies that the T cell subset that it defines has a specialized and important role, perhaps related to its unique ability to secrete a large set of lymphokines including interleukin 4, upon primary stimulation in vitro and in vivo.

Increased interleukin 4 and immunoglobulin E production in transgenic mice overexpressing NK1 T cells.

Natural Killer (NK)1.1+ (NK1) T cells are a specialized subset of alpha/beta T cells that coexpress surface receptors that are normally associated with the NK cell lineage of the innate immune system. On recognition of the conserved, major histocompatibility complex class I-like CD1 molecule, these cells are able to release explosive bursts of interleukin 4 (IL-4), a cytokine that promotes the T helper type 2 (Th2) effector class of an immune response. A unique feature of their T cell receptor (TCR) repertoire is the expression of an invariant TCR alpha chain, V alpha 14-J alpha 281, and of a restricted but polyclonal set of V beta gene families, V beta 8, V beta 7, and V beta 2. Here, we show that transgenic expression of this TCR alpha chain during thymic development is sufficient information to bias the differentiation of mainstream thymocytes towards the NK1 developmental pathway. It markedly increases the frequency of cells with the NK1 pattern of T cell differentiation and also has drastic consequences for the selection of the V beta repertoire. Transgenic CD4 cells exhibited a 10-100-fold increase in IL-4 production on mitogen stimulation in vitro and in vivo, and baseline levels of the Th2-controlled serum immunoglobulin isotypes, IgE and IgG1, were also selectively elevated in vivo.

Interleukin 2-induced proliferation of leukemic human B cells.

The proliferative responses of purified leukemic human B cells from nine B cell chronic lymphocytic leukemias to recombinant interleukin 2 (IL-2), spontaneously, and after preactivation by Staphylococcus aureus Cowan I (SAC) or anti-mu antibodies were studied. Three patterns of response were observed: (a) no response (three cases); (b) a moderate spontaneous response enhanced by anti-mu (one case); (c) a high proliferative response after preactivation by anti-mu and/or SAC (five cases). IL-2 could also trigger normal B cells, purified from spleen, to proliferative after preactivation by anti-mu or SAC. These results provide evidence that IL-2 is a lymphokine that acts physiologically on both B and T cells.

Lineage relationships and differentiation of natural killer (NK) T cells: intrathymic selection and interleukin (IL)-4 production in the absence of NKR-P1 and Ly49 molecules.

In this report, we have assessed the lineage relationships and cytokine dependency of natural killer (NK) T cells compared with mainstream TCR-alphabeta T cells and NK cells. For this purpose, we studied common gamma chain (gamma c)-deficient mice, which demonstrate a selective defect in CD3- NK cell development relative to conventional TCR-alphabeta T cells. NK thymocytes differentiate in gamma c- mice as shown by the normal percentage of TCR Vbeta8+ CD4-CD8- cells and the normal quantity of thymic Va14-Jalpha281 mRNA that characterize the NK T repertoire. However, gamma c-deficient NK thymocytes fail to coexpress the NK-associated markers NKR-P1 or Ly49, yet retain characteristic expression of the cytokine receptors interleukin (IL)-7R alpha and IL-2Rbeta. Despite these phenotypic abnormalities, gamma c- NK thymocytes could produce normal amounts of IL-4. These results define a maturational progression of NK thymocyte differentiation where intrathymic selection and IL-4-producing capacity can be clearly dissociated from the acquisition of the NK phenotype. Moreover, these data suggest a closer ontogenic relationship of NK T cells to TCR-alphabeta T cells than to NK cells with respect to cytokine dependency. We also failed to detect peripheral NK T cells in these mice, demonstrating that gamma c-dependent interactions are required for export and/or survival of NK T cells from the thymus. These results suggest a stepwise pattern of differentiation for thymically derived NK T cells: primary selection via their invariant TCR to confer the IL-4-producing phenotype, followed by acquisition of NK-associated markers and maturation/export to the periphery.

An invariant T cell receptor alpha chain defines a novel TAP-independent major histocompatibility complex class Ib-restricted alpha/beta T cell subpopulation in mammals.

We describe here a new subset of T cells, found in humans, mice, and cattle. These cells bear a canonical T cell receptor (TCR) alpha chain containing hAV7S2 and AJ33 in humans and the homologous AV19-AJ33 in mice and cattle with a CDR3 of constant length. These T cells are CD4(-)CD8(-) double-negative (DN) T cells in the three species and also CD8alphaalpha in humans. In humans, their frequency was approximately 1/10 in DN, 1/50 in CD8alpha+, and 1/6,000 in CD4(+) lymphocytes, and they display an activated/memory phenotype (CD45RAloCD45RO+). They preferentially use hBV2S1 and hBV13 segments and have an oligoclonal Vbeta repertoire suggesting peripheral expansions. These cells were present in major histocompatibility complex (MHC) class II- and transporter associated with antigen processing (TAP)-deficient humans and mice and also in classical MHC class I- and CD1-deficient mice but were absent from beta2-microglobulin-deficient mice, indicating their probable selection by a nonclassical MHC class Ib molecule distinct from CD1. The conservation between mammalian species, the abundance, and the unique selection pattern suggest an important role for cells using this novel canonical TCR alpha chain.

Overexpression of natural killer T cells protects Valpha14- Jalpha281 transgenic nonobese diabetic mice against diabetes.

Progression to destructive insulitis in nonobese diabetic (NOD) mice is linked to the failure of regulatory cells, possibly involving T helper type 2 (Th2) cells. Natural killer (NK) T cells might be involved in diabetes, given their deficiency in NOD mice and the prevention of diabetes by adoptive transfer of alpha/beta double-negative thymocytes. Here, we evaluated the role of NK T cells in diabetes by using transgenic NOD mice expressing the T cell antigen receptor (TCR) alpha chain Valpha14-Jalpha281 characteristic of NK T cells. Precise identification of NK1.1(+) T cells was based on out-cross with congenic NK1.1 NOD mice. All six transgenic lines showed, to various degrees, elevated numbers of NK1.1(+) T cells, enhanced production of interleukin (IL)-4, and increased levels of serum immunoglobulin E. Only the transgenic lines with the largest numbers of NK T cells and the most vigorous burst of IL-4 production were protected from diabetes. Transfer and cotransfer experiments with transgenic splenocytes demonstrated that Valpha14-Jalpha281 transgenic NOD mice, although protected from overt diabetes, developed a diabetogenic T cell repertoire, and that NK T cells actively inhibited the pathogenic action of T cells. These results indicate that the number of NK T cells strongly influences the development of diabetes.

CD1 recognition by mouse NK1+ T lymphocytes.

Rare major histocompatibility complex (MHC) class I-like CD1-specific T cells have been isolated from human blood, but it has not been determined whether these clones are part of a defined subset of CD1-specific T cells selected during T cell development, or whether their recognition of CD1 is a fortuitous cross-reaction. In mice, an entire subset of alpha beta thymocytes with a unique phenotype was found to be CD1-specific. This particular subset, and its human counterpart, provide evidence that CD1 has a general role in selecting and interacting with specialized alpha beta T cells.

Regulation of interleukin 2 gene expression by CD28 costimulation in mouse T-cell clones: both nuclear and cytoplasmic RNAs are regulated with complex kinetics.

T-cell receptor (TCR) signalling is required to induce expression of the interleukin 2 (IL-2) gene in mouse T cells. Additional costimulation through CD28 augments IL-2 production by 30- to 100-fold. Using IL-2 RNA accumulation and transcription reporter assays, we have addressed potential mechanisms of CD28 regulation at various time points of stimulation. The kinetic regulation of IL-2 mRNA by TCR and CD28 signals is complex: (i) at the earliest detectable time point, CD28 signalling causes a 20-fold increase compared with TCR signalling alone; (ii) both groups rapidly accumulate mRNA for the first 4 h; (iii) IL-2 mRNA then disappears from cells stimulated through the TCR alone but plateaus or increases slightly in cells costimulated through CD28; and (iv) after 8 h, the mRNA disappears in cultures with the anti-CD28 antibody. Transcription reporter assays did not show a specific effect of CD28 signalling on IL-2 enhancer driven transcription. This was true for either a 353- or a 1.9-kb enhancer, over a broad range of kinetics and TCR occupancy, and with several TCR signal mimics. The early component of CD28 costimulation is nuclear, however, since the initial enhancement of mRNA is also found in unspliced IL-2 RNA. Between 2 and 6 h, there is a marked difference in the rates of decay of IL-2 mRNA in the presence and absence of the CD28 signalling. Rapid decay of IL-2 mRNA commences after 8 h even in the presence of CD28 signals, although the decay occurs at a rate slower than that seen after 4 h of anti-TCR stimulation alone. This complexity suggests the existence of two interesting molecular mechanisms by which CD28 costimulates lymphokine gene expression.

Pre-transplant donor CD4- invariant NKT cell expansion capacity predicts the occurrence of acute graft-versus-host disease.

Clinically useful pre-transplant predictive factors of acute graft-versus-host-disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-SCT) are lacking. We prospectively analyzed HSC graft content in CD34+, NK, conventional T, regulatory T and invariant natural killer T (iNKT) cells in 117 adult patients before allo-SCT. Results were correlated with occurrence of aGVHD and relapse. In univariate analysis, iNKT cells were the only graft cell populations associated with occurrence of aGVHD. In multivariate analysis, CD4- iNKT/T cell frequency could predict grade II-IV aGVHD in bone marrow and peripheral blood stem cell (PBSC) grafts, while CD4- iNKT expansion capacity was predictive in PBSC grafts. Receiver operating characteristic analyses determined the CD4- iNKT expansion factor as the best predictive factor of aGVHD. Incidence of grade II-IV aGVHD was reduced in patients receiving a graft with an expansion factor above versus below 6.83 (9.7 vs 80%, P<0.0001), while relapse incidence at two years was similar (P=0.5).The test reached 94% sensitivity and 100% specificity in the subgroup of patients transplanted with human leukocyte antigen 10/10 PBSCs without active disease. Analysis of this CD4- iNKT expansion capacity test may represent the first diagnostic tool allowing selection of the best donor to avoid severe aGVHD with preserved graft-versus-leukemia effect after peripheral blood allo-SCT.

Upregulation of CD9 expression during TPA treatment of K562 cells.

The CD9 antigen, a major platelet glycoprotein, is a member of the tetraspan superfamily. We show that treatment of K562 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) which induces megakaryocytic differentiation, leads to a seven-fold increase in CD9 expression, which becomes associated with the integrin beta1, suggesting that it is functionally relevant. The upregulation of CD9 expression precedes the appearance of the megakaryocytic-specific marker GPIIb (CD41) as well as integrins beta3 (GPIIIa/CD61), alpha v (CD51) and VLA-2 (CD49b). Both GPIIb/IIIa expression and CD9 upregulation are dependent on protein kinase C (PKC) activation since they are blocked by the specific inhibitor GF109203X. Steady-state levels of CD9 and GPIIb mRNA were also measured by quantitative RT-PCR. Both messengers were detected on resting cells and were shown to accumulate during TPA treatment. However, the increase of the CD9 mRNA was detected much earlier than the increase of GPIIb mRNA (1-2 h vs 24-48 h). Using different constructs of the 5'-flanking domain of the CD9 gene cloned ahead of the CAT reporter gene, we could demonstrate that a responsive element was located in a 52 bp fragment of the promoter of the CD9 gene. Altogether, these data suggest that CD9 upregulation in the megakaryocytic lineage could occur at early stages of differentiation.

Persistent alterations in T-cell repertoire, cytokine and chemokine receptor gene expression after 1 year of highly active antiretroviral therapy.

OBJECTIVES: To examine T-cell repertoire modifications, the evolution of T-helper (TH)1/TH2 cytokine imbalance and modifications in chemokine receptor expression when the viral load is decreased by 2-3 log10 copies/ml under highly active antiretroviral therapy (HAART). DESIGN: Sixteen patients previously treated with zidovudine and lamivudine, with CD4 cells below 300 x 10(6)/l and viraemia above 30000 copies/ml were treated by saquinavir and ritonavir together with both reverse transcriptase (RT) inhibitors (ANRS 069 trial). T-cell repertoire, chemokine receptor and lymphokine expression were studied from peripheral blood mononuclear cells sampled at weeks 0, 24 and 48. METHODS: T-cell repertoire study was carried out using the Immunoscope method. Interleukin (IL)-12 receptor beta2, CC-chemokine receptor (CCR)-3, CXC-chemokine receptor-4 and CCR-5 expression in CD4+ cells was measured by kinetic quantitative PCR and IL-2, IL-4, IL-10, IL-13, interferon (IFN)-gamma were measured using a quantitative RT-PCR assay with homologous internal standards. RESULTS: Repertoire alterations were more frequent in CD4- than in CD4+ cells and persisted despite undetectable viraemia. Increased CCR-3 expression and spontaneous IFN-gamma as well as mitogenic induced IL-13 were observed at baseline and decreased slightly under HAART. CONCLUSION: The CD8+ cell repertoire alterations were profound, whereas the CD4+ cell alterations were moderate and both persisted unchanged under HAART. The TH1/TH2 imbalance was more related to TH2 over-expression than to TH1 deficiency and persisted for at least 1 year under HAART.

EBV cell-lines (LCL) and E- cells as stimulator cells for limiting dilution analysis (LDA) of alloreactive IL-2-secreting cells (IL-2-SC) and cytotoxic precursors (CTLp). Comparison of their stimulator capacity and ability to generate specific alloreactivity.

The choice of stimulator cells is crucial in determining the frequency of alloreactive cells by limiting dilution analysis (LDA). In humans, E- cells or EBV-lymphoblastoid cell lines (LCL) are available for this purpose. The E- cells have been mostly used until now, but they appear to stimulate much less than LCL in other models. Consequently, we undertook a systematic comparison of the efficiency of both types of cell for the LDA of alloreactive IL-2-secreting cells (IL-2-SC) and cytotoxic precursors (CTLp). We show that LCL are stronger stimulator cells both for IL-2 secretion and for cytotoxicity induction. However, high levels of non-allogeneic reactivity were also induced. Therefore, to measure the frequency of specific alloreactive IL-2-SC and CTLp, T cells were depleted of such irrelevant reactive cells by a suicide technique: culture with autologous LCL in the presence of BUDR and use of the remaining live cells as responding effectors in LDA. Using this methodology, no non-allogeneic reactive T cells remain in the responding cells: after restimulation by autologous LCL, no IL-2-SC could be seen and no cytotoxic activity could be observed against autologous, irrelevant or LAK sensitive targets. In contrast, the number of IL-2-SC and CTLp against allogeneic targets was preserved. Therefore with this methodology, the strong stimulator capacity of LCL could be used whereas non-specific activation could be avoided in order to assess the frequency of allo-specific IL-2-SC and CTLp.

Quantification of porcine cytokine gene expression using RT-PCR, a homologous internal control and chemiluminescence for microplate detection.

The polymerase chain reaction (PCR) has proved to be a sensitive and versatile method for the analysis of human and murine cytokine mRNA expression. This paper describes for the first time a reverse transcription-polymerase chain reaction (RT-PCR) at end-point for the quantification of five porcine cytokines: interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-10 and IL-18. The main features of the methodology are: (1) a unique RT for all quantifications, (2) the addition of homologous DNA internal controls (IC) of equal length to the corresponding cytokine and consequently co-amplification of the target cytokine and the IC with equivalent efficacy, (3) PCR and detection of amplicons for all cytokines simultaneously, (4) cytokine quantification in relation to a housekeeping gene control (glyceraldehyde-3-phosphate dehydrogenase, GAPDH), (5) detection of the amplicons by enzyme linked immunosorbent assay (ELISA) using a chemiluminescent substrate with high sensitivity and wide dynamic range, (6) automation of the detection system for analysis of a large number of samples. This highly sensitive quantitative RT-PCR assay (able to detect 100-200 cytokines mRNA copies/75x10(3) cells) was validated on peripheral blood mononuclear cells (PBMC) from pigs infected or not with pseudorabies virus (PRV), re-stimulated in vitro by a mitogen or antigens.

A possible role for specific "anergy" in immunologic hyporeactivity to donor stimulation in human kidney allograft recipients.

The low immunological reactivity toward donor cells usually observed in transplant recipients has been linked to clonal deletion or suppression of alloreactive cells. However, the anergy of donor-specific reactive cells is another possibility not extensively tested until now in humans. In this case, donor-specific reactive cells would be present and eventually be activated without becoming effector cells (i.e., without secreting IL-2 or becoming cytotoxic) after donor-specific stimulation. We studied 8 patients under low-dose immunosuppressive drugs who displayed hyporeactivity toward donor stimulation. IL-2 production, proliferative response, and cytotoxic activity toward donor cell stimulation was decreased (respectively 22, 53, and 19% of response toward third-party stimulation). In order to evidence anergy, we studied two activation markers (cell size increase and expression of IL-2 receptor [CD25]) in allografted recipient T cells after autologous (background), donor (experimental), and third-party cell stimulation (positive control). We showed that the percentage of CD25+ cells and the cell size increase were similar after donor or third-party cell stimulation and clearly above the background as early as days 1-2 after the beginning of the mixed lymphocyte culture. Moreover, CD4+ and CD8+ cells similarly expressed CD25 after donor or third-party stimulation. Thus, donor-specific reactive cells not only were present but could be activated without becoming effector cells. These data suggest that anergy may be an important phenomenon in allograft tolerance.

Evidence that antihuman tumor necrosis factor monoclonal antibody prevents OKT3-induced acute syndrome.

We have used an antihuman tumor necrosis factor monoclonal antibody, CB006 (murine IgG1), to prevent the OKT3-induced acute clinical syndrome. This syndrome is due to the massive, although transient release in the circulation of various cytokines (TNF, interferon gamma, interleukin 2, interleukin 6) and represents one important side effect linked to in vivo use of OKT3. Fourteen kidney allograft recipients undergoing prophylactic OKT3 therapy were treated with CB006 in a single i.v. injection of either 0.4 mg/kg (group I, 7 patients) or 2 mg/kg (group II, 7 patients), 1 hr before the first OKT3 administration. Nineteen consecutive patients formed a historical control group. None of the CB006-pretreated patients showed any of the common, severe OKT3-associated symptoms (hypotension, respiratory distress, or neurotoxicity), which were observed in 10% of the historical controls. In addition, CB006-treated patients showed a lower frequency of pyrexia (> or = 39 degrees C) and gastrointestinal symptoms. None of the CB006-treated patients presented severe vomiting or diarrhea, defined as repeated episodes inducing significant fluid and electrolyte loss. Two out of the 7 patients in group I and group II had mild transitory diarrhea. Mild single vomiting episodes occurred in 2 group I patients and 3 group II patients. At variance in all controls, gastrointestinal symptoms were long lasting and associated with major prostration due to electrolyte and fluid loss. Importantly, CB006-treated patients who presented mild symptoms had detectable bioactive circulating TNF, showing incomplete inactivation of OKT3-induced TNF by CB006. CB006 was perfectly well tolerated, did not induce xenosensitization, and did not affect the biological or clinical effectiveness of OKT3.

Decreased lymphokine-activated killer cells in kidney transplant recipients. Correlation with a diminished number of CD3-/NKH1+ cells.

The activity of lymphokine-activated killer cells, measured either by a clonal or polyclonal technique, was assessed in 30 kidney transplant recipients (TX), in 13 hemodialyzed patients (HD-CRI), and in 18 normal (N) controls. A highly significant decrease of the LAK activity in TX in comparison with HD-CRI or N (P = 0.0001) was observed. Moreover, the percentage of CD3-/NKH1+ cells was decreased in TX in comparison with N (P = 0.01). LAK activity was strongly correlated (r = 0.72; P = 0.0001) with the percentage of CD3-/NKH1+ cells and not with that of double-positive CD3+/NKH1+ cells. Multivariate analysis showed that the sole independent variable that determined the LAK activity was the percentage of CD3-/NKH1+ cells: the pathological status (TX, HD-CRI, and N) variable was statistically not significant. On the other hand, two T cell-specific functions (IL-2 secretion and specific cytotoxic activity) were, on the whole, preserved in TX. Altogether, these results suggest that TX are LAK deficient predominantly because they have a decreased number of CD3-/NKH1+ cells. The normality of T cell functions suggests that the high rate of malignancies seen in TX is related to this LAK deficiency. Moreover, our study indicates that, in vivo, CD3+ cells do not significantly contribute to the LAK precursors.

A versatile ELISA-PCR assay for mRNA quantitation from a few cells.

Gene expression studies require a sensitive and quantitative assay of mRNA amounts present in small samples. We describe a general method of quantifying specific mRNA quickly and easily from purified RNA or directly from a few cells by PCR and enzyme-linked immunosorbent assay (ELISA) revelation of the resulting products (sensitivity of the last step: < 0.1 fmol). Cells are digested and the total cellular RNA is reverse-transcribed and then amplified with 5' and 3' primers; the former being 5' biotinylated. The amplification product is captured on avidin-coated microplates and quantified by hybridization with a digoxigenin-labeled internal oligonucleotide probe. After revelation with an anti-digoxigenin alkaline phosphatase coupled antibody (anti-DIG-AP1), the amount of hybridized probe is determined by optical reading. The results can be easily converted to absolute values by comparison with an external DNA standard curve. An internal DNA or RNA standard can also be used. The method we describe can be adapted to any cellular or viral gene of known sequence in a matter of days. Since it uses nonradioactive probes, commercially available reagents and standard microplate readers, it is inexpensive and could be automated easily. In this study, interleukin-2 mRNA expression could be studied in as few as 40 Jurkat cells. It was also possible to quantify human immunodeficiency virus (HIV) DNA from 1500 to 1.5 copies out of 1.5 x 10(5) human genomic DNA copies.