IgG1 variations in the colostrum of Holstein dairy cows.

High-immune quality colostrum (IgG1 concentration ⩾50 g/l) is crucial for the health and development of the young calf. Studies on colostrum quality tend to focus on external factors such as breed, parity or dry period length, but few have focused on within-cow variations. Here we ran experiments to gain a deeper insight into within-cow variation in IgG1 concentrations in dairy cow colostrum. Trials were performed in an experimental farm, located in the Western part of France. Colostrum from each quarter and a composite sample (mix of four quarters) were concomitantly collected on 77 Holstein dairy cows just after calving to assess the influence of sample type on IgG1 concentrations. Variation in IgG1 concentrations during the first milking was studied on samples from nine cows collected every minute from the start of milking. Repeatability of colostral IgG1 concentration was estimated from 2009 and 2010 data on 16 healthy cows. IgG1 concentrations were tested using a radial immunodiffusion method. Sensitivity and specificity were similar regardless of sample type tested (individual quarter or composite milk). Mean average IgG1 concentration was 54.1 g/l in composite colostrum, and was significantly higher in hind quarter teats (56.2 g/l) than front quarter teats (53.1 g/l). Average IgG1 concentration did not change significantly during colostrum milking, and the variations observed (15% or less) were likely due to the laboratory method (CV 15%). IgG1 concentrations in dam colostrum increased slightly from 2009 to 2010 due to BW and parity effects. In 56% of cases, colostrum quality could have been assessed on either individual or composite colostrum samples collected at any time during the first milking without affecting the reliability of the measurement. However, in other cases, differences were significant enough to mean that estimates of average IgG1 concentration in colostrum from any one quarter would not be reliable. It is concluded that colostrum quality, from an IgG1 concentration point of view, could be assessed with a composite sample taken at any time during the first milking.

Detection by real-time RT-PCR of a bovine respiratory syncytial virus vaccine in calves vaccinated intranasally.

Seventeen four- to five-week-old calves that were not shedding bovine respiratory syncytial virus (BRSV) were vaccinated intranasally against the disease and sampled by nasal swabbing on 16 different days for up to 20 days after vaccination. BRSV vaccine virus was detected in 15 of the 17 calves. Five of the calves were PCR positive on only one swab, eight were PCR positive on two to five swabs and two were PCR positive on more than five swabs. Twelve of the calves were positive only before day 14 and three were positive after day 14. The nasal shedding of BRSV vaccine virus was very variable.

Frequency and relative fraction of tumor antigen-specific T cells among lymphocytes from melanoma-invaded lymph nodes.

Several tumor antigens have been described as candidates for immunotherapy. Our study compared HLA-A2-restricted epitopes from 5 antigens commonly expressed by melanomas, i.e., Melan-A/MART-1 peptides (26-35 and 27-35), tyrosinase (368-376), gp-100 (280-288), MAGE-3 (271-279) and NA17-A (1-10), for their relative capacity to promote the development of cytotoxic and cytokine-producing specific CD8+ lymphocytes within melanoma-invaded lymph nodes. We used short-term cultured melanoma-invaded lymph node lymphocytes (MILLs) and tested responses developed by these cells to peptide-pulsed TAP-deficient T2 cells. We measured both the lytic response developed by MILLs and the fraction of these cells that secreted interferon-gamma (IFN-gamma), as deduced from intracellular cytokine labeling. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the expression of the 5 antigens within melanoma-invaded lymph nodes. Melan-A/MART-1, tyrosinase and gp-100 peptides were recognized by MILLs derived, respectively, from 8 of 20, 5 of 19 and 4 of 20 melanoma-invaded lymph nodes expressing these antigens. Most MILLs specific for Melan-A/MART-1 and tyrosinase exhibited both lysis and IFN-gamma responses, whereas most of those specific for gp-100 developed only lysis. Weak lysis without IFN-gamma secretion was developed against NA17-A and MAGE-3 peptides by MILLs from, respectively, 3 of 9 and 2 of 14 lymph nodes expressing these antigens. Our data show a prevalence of both cytotoxic and IFN-gamma-secreting effector T cells specific for differentiation antigens within HLA-A2 melanoma-invaded lymph nodes, which makes these antigens attractive targets for specific immunotherapy.

LFA-3 co-stimulates cytokine secretion by cytotoxic T lymphocytes by providing a TCR-independent activation signal.

T cell activation is known to depend not only on efficient antigen recognition and subsequent signaling through TCR, but also on interactions involving multiple adhesion and accessory molecules such as CD28/B7, LFA-1/ICAM-1 and LFA-3/CD2. The present study dissects the role of LFA-3/CD2 interactions in the activation of melanoma-specific CD8+ T cell clones. To this end we analyzed the influence of LFA-3 density on melanoma cells on lysis and cytokine production (TNF, IL-2, IFN-gamma) by T cells following activation by various amounts of antigenic peptides. Our results indicate that increasing LFA-3 density on melanoma cells variably affects their lysis susceptibility, but systematically and considerably enhances cytokine production by melanoma-specific cytotoxic T lymphocyte (CTL) clones. At any stimulatory antigen density, LFA-3 increased the fraction of responding cells and/or cytokine amounts produced by individual cells, without affecting TCR down-regulation. These results show that CD2 engagement increases cytokine gene activation essentially by providing to T cells a TCR-independent co-activation signal. From a practical point of view, our data demonstrate that the level of LFA-3 expressed on tumors critically affects cytokine production by specific CTL and thus the efficiency of specific immune reactions mediated by these cells.

Inhibition of antigen-induced T cell response and antibody-induced NK cell cytotoxicity by NKG2A: association of NKG2A with SHP-1 and SHP-2 protein-tyrosine phosphatases.

Subsets of T and natural killer (NK) lymphocytes express the CD94-NKG2A heterodimer, a receptor for major histocompatibility complex class I molecules. We show here that engagement of the CD94-NKG2A heterodimer inhibits both antigen-driven tumor necrosis factor (TNF) release and cytotoxicity on melanoma-specific human T cell clones. Similarly, CD16-mediated NK cell cytotoxicity is extinguished by cross-linking of the CD94-NKG2A heterodimer. Combining in vivo and in vitro analysis, we report that both I/VxYxxL immunoreceptor tyrosine-based inhibition motifs (ITIM) present in the NKG2A intracytoplasmic domain associate upon tyrosine phosphorylation with the protein tyrosine phosphatases SHP-1 and SHP-2, but not with the polyinositol phosphatase SHIP Determination of the dissociation constant, using surface plasmon resonance analysis, indicates that NKG2A phospho-ITIM interact directly with the SH2 domains of SHP-1 and SHP-2 with a high affinity. Engagement of the CD94-NKG2A heterodimer therefore appears as a protein-tyrosine phosphatase-based strategy that negatively regulates both antigen-induced T cell response and antibody-induced NK cell cytotoxicity. Our results suggest that this inhibitory pathway sets the threshold of T and NK cell activation.

Heterogeneity of biologic responses of melanoma-specific CTL.

To better understand how Ag density influences the various biologic responses of CTL, we analyzed lysis and, at the single cell level, cytokine production by a panel of melanoma-specific CTL clones. Titration experiments done with peptide-pulsed TAP-deficient T2 cells indicated that: 1) Ag density affects both the fraction of responding cells and the amount of cytokine secreted by each cell. 2) Different responses have a relative Ag requirement that may vary between clones. Lysis and secretion of IFN-gamma, and for most clones' secretion of TNF-alpha, required lower Ag densities, by one or two logs, than IL-2 and granulocyte-macrophage CSF secretion. 3) In a significant fraction of IFN-gamma-secreting cells, IL-2 production is not induced. 4) A large fraction of cloned cells is refractory to lymphokine gene activation for about 2 wk after previous stimulation. Together these data indicate that CTL biologic responses are controlled by variable Ag thresholds and by additional parameters affecting activation requirements of each cell. A similar heterogeneity of cytokine responses was observed to Ag endogenously presented by melanoma cells. As a consequence, most melanoma lines, including those with the highest Ag expression, could trigger only low CTL fractions to secrete IL-2 and, also for most clones, granulocyte-macrophage CSF. This may be an important component of the inefficiency of specific CTL in cancer patients.

HLA class II-restricted recognition of common tumor epitopes on human melanoma cells by CD4+ melanoma-infiltrating lymphocytes.

CD4+ T cell clones derived from lymphocytes infiltrating four human melanomas specifically recognized melanoma-derived tumor epitopes as shown by secretion of tumor necrosis factor (TNF) in vitro upon interaction with autologous melanoma cells, whereas they did not recognize HLA class II-expressing autologous lymphoblasts or HLA class II mismatched allogeneic melanoma cells. Specificity was further established by demonstrating that TNF responses to tumor cells were inhibited by HLA-DR or HLA-DQ monoclonal antibodies. Most of these clones cross-reacted with allogeneic melanoma cells expressing a potentially restricting HLA allele or a structurally similar one. These data show that shared epitopes of human melanoma cells presented on HLA class II molecules are frequently recognized by autologous CD4+ T lymphocytes.

T cell activation by antigens on human melanoma cells--co-stimulation by B7-1 is neither sufficient nor necessary to stimulate IL-2 secretion by melanoma-specific T cell clones in vitro.

B7-1 expression, induced by transfection in poorly immunogenic murine tumours, was shown to elicit a T cell-mediated rejection of these tumours and further active immunity against the non-transfected tumour. We therefore asked to what level similarly induced expression of B7 on human melanoma cells would affect the antigen-dependent responses of tumour-specific T cell clones in vitro. Data presented show that B7-1 expression by melanoma lines: (i) significantly induced, or improved, an IL-2-dependent proliferative response of such clones to the antigen; (ii) increased the amount of IL-2 produced by two clones in response to the parental non-transfected tumour cells; and (iii) increased the TNF responses of all the CD4+ clones. However, despite these clear co-stimulatory effects on antigen-induced responses of all T cell clones, which demonstrated an effective interaction of the B7-1 transfected molecule with one or the other of its counter-receptors expressed on T cell clones, B7 co-stimulation did not correct the defect of IL-2 secretion exhibited by many of these clones in response to in vitro antigen presentation by melanoma cells. We further show that defective IL-2 secretion in response to melanoma antigens was not due to a T cell clone refractoriness induced by the culture, since one of these clones could be induced to secrete IL-2 by an antigen-expressing melanoma line, upon increased lymphocyte function associated antigen-3 expression induced by gene transfection. Together these data suggest that defective IL-2 secretion by many tumour-infiltrating lymphocytes clones in response to antigen presentation by melanoma cells in vitro is not exclusively due to the inability of these cells to provide an appropriate co-stimulation through the B7-1 molecule.

Defective lymphokine production by most CD8+ and CD4+ tumor-specific T cell clones derived from human melanoma-infiltrating lymphocytes in response to autologous tumor cells in vitro.

Human melanomas are infiltrated by tumor-reactive T lymphocytes. However, the ability of these cells to elicit a specific anti-tumor response in vivo remains to be established. Because lymphokine production is critical for T cell functions, we have analyzed the capacity of melanoma-specific tumor-infiltrating lymphocyte (TIL) clones to produce major lymphokines: interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), as well as tumor necrosis factor (TNF), in response to direct antigen presentation by autologous and allogeneic tumor cells. We report here that, upon stimulation by autologous melanoma cells, all TIL clones secreted TNF but only a few of them produced significant amounts of IL-2, IL-4 or IFN-gamma. Nonetheless, all these clones consistently produced two or three of these last lymphokines upon stimulation with phorbol myristate acetate and calcium ionophore, as well as IL-2 upon CD3 stimulation, showing the existence of three lymphokine profiles among them: Th1, Th0 and a profile characterized by IL-2 and IL-4, but not IFN-gamma secretion. Stimulation of TIL clones by allogeneic melanoma lines sharing the appropriate HLA-peptide complexes revealed that defective IL-2 production seemed to be a constant feature for some clones, while it was, for other clones, dependent on the antigen-presenting tumor cells. For this last type of clone, we further showed that defective IL-2 induction resulted from an LFA-3 defect of some melanoma cells or from distinct yet undefined defects of other melanoma lines. Our data suggest that defective lymphokine secretion may be an essential component of the in vivo failure of melanoma-reactive TIL to control tumor development. Interestingly both CD4+ and CD8+ TIL clones from one patient were fully activated by the autologous melanoma cells in vitro, supporting a potential role of such TIL in spontaneous or induced tumor rejection.