Monoclonal antibodies to feline lymphocyte membranes recognize the leukocyte-common antigen (CD45R).

By immunization of BALB/c mice with a feline T lymphoblastoid cell line, MYA-1 cells, two types of lymphocyte-specific monoclonal antibodies (mAbs) were obtained. The 220/205/190 kd protein defined by 2F11 mAb is highly expressed on the surface of MYA-1 cells and another feline T lymphoma cell line, FL74 cells. The protein is also expressed on normal feline thymocytes, splenocytes and feline peripheral blood mononuclear cells (PBMCs). Another mAb, 17B10, caused similar results as those of 2F11 except for its low reactivity with FL74 cells. The second type of mAb, 15B3, defined the 220 kd protein. The reactivities of this mAb with MYA-1 cells, FL74 cells, PBMCs and feline splenocytes were lower than the former two mAbs, and did not react to feline thymocytes. On the other hand, 17B10 and 15B3 defined partial populations of MYA-1 and FL74 cells recognized by 2F11. The cells defined by the 2F11 and 17B10 are all leukocytes in spleen and lymph node. In contrast, 15B3 defined most of the cells in B cell area and partially in T cell area. These results suggested that 2F11 and 17B10 recognized the specific antigen of 220/205/190 kd of the leukocyte-common antigen (L-CA) family, CD45R, with different epitopes, and that 15B3 defined the distinct antigen of 220 kd on CD45R.     

Effects of chicken thymic stromal cells on the growth and differentiation of thymocytes in vitro

We examined contact-mediated effects of chicken thymic stromal cells (TSC) on thymocyte differentiation by co-cultivation of these cell populations. The primary cultures of TSC isolated from thymus mainly have consisted of epithelial cells which were polygonal in shape, possessed long processes and expressed MHC class II antigen. When thymocytes were co-cultured with TSC, 60% to 70% of thymocytes attached to TSC and some of them engulfed underneath TSC. These attached thymocytes were CD4-CD8- and CD4+CD8+ subsets and expressed alpha/beta TCRhigh or gamma/delta TCRlow. Some of the thymocytes attaching to TSC showed an increase of intracellular and nuclear density, fragmentation of cytoplasm and nuclei, and DNA fragmentation. And also, thymocytes attaching to TSC contained a higher percentage of cycling (S and G2 + M phase) cells than nonattaching cells. These results indicate that specific subsets in thymocytes selectively bind to TSC and undergo apoptotic death or proliferation because of interaction with TSC. Chicken TSC may play an important role in thymic differentiation by direct contact within the thymus as in mammals.     

Characterization of CD34⁺ thymocytes in newborn dogs

Using two-color flow cytometry, we characterized CD34(+) cells in the newborn canine thymus. CD34(+) thymic cells comprised approximately 5% of cells recovered by thymus tissue teasing and both large and small thymocytes have been present in this population, the former being 7-12 times more frequent. All CD34(+) cells expressed the pan-leukocyte antigen CD45. The expression of CD44 profile on the large and small CD34(+) thymocytes differed: almost all large CD34(+) cells were CD44(+), while only 75% of small CD34(+) thymocytes co-expressed the CD44 antigen. We have previously described that CD172α is present on the surface of CD34(+) bone marrow cells in dogs. In the thymus, CD172α was expressed on 5-10% and less than 5% of large and small CD34(+) cells, respectively. Some CD34(+) thymocytes also co-expressed T-lineage-specific markers like CD3, CD4, CD8, TCR1 and TCR2. Their expression increased during the large-to-small thymocyte transition. Based on our findings we suggest that thymocyte progenitors enter their primary differentiation center as large CD34(+), CD44(+), CD45(+) and CD172α(+) cells. T-cell specific markers appear on their surface at early stages of differentiation. As the size of progenitors decreases with terminal primary differentiation, the CD34, CD44, and CD172α surface markers are down-regulated.     

Effects of cytokines from thymocytes and thymic stromal cells on chicken intrathymic T cell development

We have studied the ability of thymic stromal cells (TSC) and thymocytes to produce cytokines and the involvement of cytokines in intrathymic T cell development. When thymocytes were co-cultured with thymic stromal cells in absence of direct contact and mitogenic stimulation, induction of thymocyte proliferation was observed. Supernatants of cultured stromal cells (TSC-CS) promoted a high proliferative response on CD3- thymocytes but had little effect on CD3+ thymocytes. These results indicate that stromal cells have produced a cytokine which can induce immature thymocyte proliferation. Moreover, stromal cells express the MRNA for stem cell factor (SCF) and c-kit (the receptor for SCF) was detected on CD3- thymocytes but not on CD3+ thymocytes. Since SCF can enhance the proliferation of immature thymocytes in synergy with IL-7 in mammals, there is a possibility that chicken stromal cells may produce a IL-7-like factor. Thymocytes have clearly expressed interferon (IFN)-gamma. In contrast, thymic stromal cells showed no detectable expression of IFN-gamma. CD3+ thymocytes express IFN-gamma MRNA more strongly than CD3 thymocytes, suggesting that IFN-gamma from thymocytes may operate on stromal cells and then may indirectly induce clonal elimination of CD3+ cells on stromal cells. The expression of these cytokines and receptors by thymic stromal cells and thymocyte subpopulations suggests that these cytokines participate in paracrine interactions between these cell populations during thymocyte differentiation.     

A new epitope on sheep CD45R molecule detected by a monoclonal antibody

This paper describes the production and characterization of a monoclonal antibody (mAb), Co-46D5, which recognizes a new epitope on the isoform of the homologous sheep leukocyte common antigen (LCA) or CD45. This nmAb was submitted to the 3rd workshop on ruminant leukocyte antigens and was assigned to a cluster reactive with B- and T-cells subsets. Co-46D recognizes a 220 kDa molecule on peripheral blood mononuclear cells (PBMC) and spleen cells but not on thymocytes. Flow cytometry (FCM) analysis shows that Co-46D5 reacted with 30% of PBMC and 50% of spleen cells and more than 95% of cells freshly isolated from lymphoid follicles of the ileal Peyer's patches (IPP) of young lambs. By immunohistochemistry, the antigen was detected mainly on B-cell areas of lymph nodes and spleen. It was also found on a subpopulation of medullar thymocytes. Based on these results, we assume that Co-46D5 recognizes a new epitope on the largest isoform of the sheep CD45 receptor, probably on the homologous to the human CD45RA isoform.     

Canine X-linked severe combined immunodeficiency.

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma (gamma c) subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors. The most striking clinical feature is a failure to thrive or 'stunted' growth. Recurrent or chronic infections begin at the time of decline of maternal antibody, usually between six and eight weeks of age. Affected dogs rarely survive past three to four months of age. The major pathologic feature of canine XSCID is a small, dysplastic thymus. Grossly identifiable lymph nodes, tonsils, and Peyer's patches are absent in XSCID dogs. During the neonatal period, XSCID dogs have few, if any, peripheral T cells and increased number of peripheral B cells. Some XSCID dogs do develop phenotypically mature, nonfunctional T cells with age, however, the absolute number of peripheral T cells remain significantly decreased compared to age-matched normal dogs. An interesting finding is that as soon as T cells begin to appear in XSCID dogs they rapidly switch from a CD45RA+ (naive) phenotype to a CD45RA- (activated or memory phenotype). One of the characteristic findings in XSCID dogs is an absent or markedly depressed blastogenic response of T cells in response to stimulation through the T cell receptor and when the necessary second messengers for cellular proliferation are directly provided that by-pass signals delivered through ligand-receptor interaction. The proliferative defect is due to the inability of T cells to express a functional IL-2 receptor. Canine XSCID B cells do not proliferate following stimulation with T cell-dependent B cell mitogens, however, they proliferate normally in response to T cell-independent B cell mitogens. Canine XSCID B cells are capable of producing IgM but are incapable of class-switching to IgG antibody production following immunization with the T cell-dependent neoantigen, bacteriophage phiX174. The number of thymocytes in the XSCID thymus is approximately 0.3% of the thymocytes present in the thymus of age-matched normal dogs. The proportion of CD4-CD8- thymocytes in XSCID dogs is increased 3.5-fold and the CD4+CD8+ population is decreased 2.3-fold. These findings demonstrate that (1) a functional gamma c is required for normal B and T cell function, (2) early T cell development is highly dependent upon a functional gamma c, and (3) B cell development can occur through a gamma c-independent pathway.     

Regulation of early chicken thymocyte proliferation by transforming growth factor-beta from thymic stromal cells and thymocytes

We examined expression of TGF-betas in chicken thymic stromal cells and thymocytes and roles of TGF-betas in thymocyte development within the thymus. Thymic stromal cells expressed TGF-beta 2 and 3 genes but not TGF-beta 4 gene. Thymocytes showed expressions of TGF-beta 2, 3 and 4 genes and each TGF-beta gene was expressed more strongly in CD3- than CD3+ thymocytes. When anti-TGF-beta antibody was added with supernatants of stromal cells into thymocyte culture, only proliferative activity of CD3- thymocytes was enhanced and the cells in S and G2/M compartments of cell cycle increased. These results suggest that TGF-beta which is expressed in the thymus may regulate the ability of immature thymocytes to progress through the cell cycle and to differentiate to CD3+ thymocytes.     

The effects of florfenicol on lymphocyte subsets and humoral immune response in mice

Florfenicol is a broad-spectrum bacteriostatic antibiotic used in domestic animals. The aim of the study was to determine the effect of florfenicol on the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes and the percentage and the absolute number of T cell subsets (CD4+CD8+, CD4-CD8-, CD4+, CD8+) in the thymus and T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the peripheral lymphatic organs in non-immunized mice and humoral immune response in sheep red blood cells (SRBC)-immunized mice. Florfenicol was administered orally at a dose of 30 mg/kg six times at 24 h intervals to non-immunized mice and four or seven times at 24 h intervals to SRBC-immunized mice. SRBC was injected 2 hours prior to the first dose of the drug. Florfenicol increased the percentage of CD4CD8- thymocytes and the absolute number of CD4+ and CD8+ thymocytes on day 7. The increased percentage and absolute number of CD3+, CD4+ and CD8+ lymphocytes in mesenteric lymph nodes and decreased percentage of lymphocytes B were also observed 24 hours from the last administration of florfenicol. Florfenicol administered after SRBC immunization reduced the number of plaque forming cells (PFC) and the production of anti-SRBC antibodies on days 4 and 7 after priming.     

Lymphocyte subpopulations in peripheral blood of sheep experimentally infected with tick-borne fever.

The lymphocyte subpopulations in the peripheral blood of normal sheep and sheep experimentally infected with Cytoecetes phagocytophila, the causative agent of tick-borne fever, were analysed by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with tick-borne fever was characterised by a significant reduction in the total number of circulating lymphocytes six days after experimental infection (P less than 0.001). This lymphocytopenia was associated with a significant reduction in the number of B (LCAp220+) and T (CD5+) lymphocytes (P less than 0.001) but there was a significant increase in the number of cells which were neither T nor B (CD5-LCAp220-) cells (P less than 0.01). The reduction in the number of T lymphocytes was due to reduced numbers of circulating CD4+ (helper) T cells, CD8+ (cytotoxic/suppressor) T cells and those with the pan T cell marker (CD5+) but without CD4 or CD8 epitopes (CD4-CD8-). All lymphocytes returned to preinoculation levels 13 to 16 days after experimental infection.     

Differential expression of homing receptors and vascular addressins in tonsils and draining lymph nodes: Effect of Brucella infection in sheep

The differential expression of homing receptors (HR) and complementary vascular addressins was studied in T and B lymphocytes from ovine tonsils and draining lymph nodes (LN) in uninfected and Brucella melitensis-infected sheep. In uninfected sheep, CD4+CD25+ T cells expressed proportionally more L-selectin and beta1 integrin than beta7 integrin in pharyngeal and palatine tonsils and in parotid LN (PLN), retropharyngeal LN (RLN) and the peripheral prescapular LN (PSLN). In contrast, memory CD4+CD45RA- T cells expressed an equivalent proportion of the three HR in PLN and PSLN, whereas beta1 and beta7 integrins were proportionally more expressed than L-selectin in pharyngeal tonsil. beta7 integrin was proportionally more expressed than beta1 integrin or L-selectin in palatine tonsils, RLN and the mucosal mesenteric LN (MLN). beta1 integrin was proportionally more expressed in IgG+ and IgA+ cells than beta7 integrin and L-selectin in tonsils, PLN and RLN. The main endothelial addressin expressed on venules in both pharyngeal and palatine tonsils, the PLN and RLN, as well as in the PSLN, was the peripheral PNAd, while in the MLN it was MAdCAM-1. Conjunctival infection by Brucella resulted in an increase of CD4+CD25+ and CD4+CD45RA- T cell subsets, which was associated to modifications of HR expression. CD4+CD45RA- T cells expressed proportionally more beta1 and beta7 integrins than L-selectin in regional PLN and RLN, but also in PSLN. The infection induced an increase of IgG+ and IgA+ cell percentages expressing beta1 integrin in all LN, and also beta7 integrin in the RLN. PNAd continued to be expressed on venules of tonsils and draining LN after Brucella infection, and MAdCAM-1 was also weakly expressed on RLN venules. These results suggest that lymphocyte trafficking through tonsils and draining LN could involve L-selectin/PNAd interactions, as well as beta1 or beta7 integrin, possibly in interaction with VCAM-1 or MAdCAM-1. The homing of antigen-specific lymphocytes in these tissues could be modulated after conjunctival infection with Brucella, which induces the recruitment of lymphocytes that express both beta1 and/or beta7 integrin in regional and more distant LN.     

Flow cytometric analysis of thymocyte subpopulations in mice after whole-body X-irradiation.

To determined the cellular kinetics of thymocyte subpopulations in DBA1 mice after whole-body 6.8 Gy X-irradiation, they were analyzed for the expression of several cell surface antigens using flow cytometry. The results show that i) The majority of thymocytes rapidly depleted by irradiation was CD4+8+ cells. ii) radioresistant CD4+8- and CD4-8+ survived 18-48 hr after X-irradiation were considered to be relatively mature type, since they expressed high levels of CD3 and LECAM-1. iii) CD3-positive cells were detected in CD4-8- cells at 72 hr after irradiation.     

Flow cytometric patterns in blood from dogs with non-neoplastic and neoplastic hematologic diseases using double labeling for CD18 and CD45

BACKGROUND: In dogs, flow cytometry is used in the phenotyping of immunologic cells and in the diagnosis of hemic neoplasia. However, the paucity of specific antibodies for myeloid cells and B lymphocytes and of labeled antibodies for multicolor techniques limits the ability to detect all leukocyte subpopulations. This is especially true for neoplastic and precursor cells. CD18 and CD45 are expressed on all leukocytes and are involved in cell activation, and together could be useful in helping determine cell lineage. OBJECTIVES: The purpose of this study was to double label canine blood for CD18 and CD45 and to use the differential expression of antigens to identify leukocyte populations in dogs with non-neoplastic and neoplastic hematologic diseases. METHODS: A template was developed using blood samples from 10 clinically healthy dogs and a back-gating technique. Differential leukocyte counts obtained with the template were compared with those obtained by manual and automated methods on blood samples from 17 additional healthy dogs. Blood samples obtained from 9 dogs with non-neoplastic (reactive) hematologic diseases and 27 dogs with hemic neoplasia were double stained for CD18 and CD45 using mouse anticanine CD18 monoclonal antibody (mAb) plus phycoerythrin-conjugated rat anticanine CD45 mAb and fluorescein isothiocyanate-conjugated rabbit antimouse IgG. Hemic neoplasms were diagnosed by cell morphology, and immunophenotypic and cytochemical markers. RESULTS: With the double label, neutrophils, eosinophils, monocytes, and T- and B-lymphocytes were identified. In reactive disorders, a population of activated neutrophils with high CD45 and CD18 expression was detected. In hemic neoplasia, cell lineage was easily determined, even in acute leukemia. CONCLUSIONS: Double labeling for CD18/CD45 may be useful as a screening method to evaluate hematologic diseases and help determine cell lineage, and to aid in the selection of a panel of antibodies that would be useful for further analysis.     

Immunohistochemical analysis of expression patterns of tumor necrosis factor receptors on lymphoma cells in enzootic bovine leukosis

Tumor necrosis factor-alpha (TNF-alpha) has been reported to be associated with the progression of lymphoproliferative neoplastic diseases and retroviral infections. Hence we examined immunohistochemically the expression patterns of TNF-receptors (TNF-RI and RII) on lymphoma cells derived from the 29 cases of enzootic bovine leukosis (EBL). Lymphomas obtained in 29 animals with EBL were histopathologically classified into three types: diffuse mixed type (10 cases), diffuse large type (9 cases), and diffuse large cleaved type (10 cases). Immunohistochemically using a monoclonal antibody to a bovine lymphocyte surface antigen, the lymphomas were classified into three phenotypes: B-1a (CD5+/CD11b+), B-1b (CD5-/CD11b+) and B-2 (conventional B) (CD5-/CD11b-). Interestingly, the lymphoma cells in all animals expressed TNF-RII, but not TNF-RI. Although, in EBL, lymphoma cells of which the histopathological and immunological property differs has been formed, the expression patterns of TNF-Rs had the universality in all lymphoma cells. TNF-RII, which induces cell proliferation, was expressed but TNF-RI, which induces cell apoptosis was not expressed on all lymphoma cells, suggesting that TNF-Rs play an important role in the malignant proliferation of B cells and formation of lymphomas in EBL.     

High sensitivity of thymocytes of LEC strain rats to induction of apoptosis by X-irradiation

It is known that physical disruption of cell contacts induces apoptosis of thymocytes. When thymocytes from LEC and WKAH rats were incubated in vitro at 37 degrees C for 0-6 hr and then the proportion of apoptotic cells was determined using a flow cytometer, it was found that the percentages of apoptotic thymocytes from both LEC and WKAH rats increased with incubation time and that the proportion of apoptotic cells from LEC rats was significantly higher than that from WKAH rats at each incubation time. The fact that cycloheximide, an inhibitor of protein synthesis, did not show significant inhibitory effects on induction of apoptosis of thymocytes indicates that induction of apoptosis during in vitro cultivation did not require de novo protein synthesis. When thymocytes from LEC and WKAH rats were X-irradiated in vitro at 4 and 8 Gy, the percentages of radiation-induced apoptotic cells increased with post-incubation time after X-irradiation in both LEC and WKAH rat thymocytes and the proportions of apoptotic cells from LEC rats were significantly higher than those from WKAH rat cells at 2 and 4 hr post-incubation after X-irradiation. When thymocytes from LEC and WKAH rats were X-irradiated in the presence of cycloheximide, the induction of apoptosis was substantially inhibited, indicating that radiation-induced apoptosis of thymocytes from LEC and WKAH rats required de novo protein synthesis. The present results showed high sensitivities of thymocytes of LEC rats to induction of apoptosis during in vitro cultivation and by X-irradiation.     

Magnificent expression of asialo GM1 on thymus cells and spleen T cells of the musk shrew, Suncus murinus.

The expression of asialo GM1 (GA1) was observed on almost all thymocytes from young musk shrew, at the age of 4 weeks by flow cytometric analysis. In adult shrew aged 10 months, the ratio of GA1-negative thymocytes was increased. Among several anti-glycolipid antibodies used, anti-GM1 and anti-Forssman also reacted with the thymocytes. Protein fraction of the thymocytes was analyzed by SDS-PAGE followed by immunoblotting. Anti-GA1 and anti-GM1 showed two bands and one band, respectively, however, their mobilities were different from each other. Anti-Forssman did not stain any protein. The GA1-positive population in spleen T cell fraction was not detected in young shrew but most of the T cells were changed to GA1-positive cells in adult shrew. When mixed lymphocyte culture was performed, the GA1-negative spleen T cells in young shrew were changed to express GA1 marker on their cell surface by differentiation. Abbreviations used were as follows: GA1, Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc-Cer; GM1, Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4Glc-Cer; Forssman, GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer.     

Characterization of a feline T-cell-specific monoclonal antibody reactive with a CD5-like molecule.

The 43 monoclonal antibody raised against feline T cells was found to react with a single-chain glycoprotein of Mr 72,000 that is present on most thymocytes, 60% of lymph node cells, 20% of splenocytes, and 45% of blood mononuclear cells. All CD4+ and CD8+ T cells were found to express the 43-reactive determinant, as did a small subpopulation of CD4-/CD8-/IgM- lymphocytes in the periphery. The 43-reactive determinant was not detected on B cells, macrophages, or other types of blood cells. The 43 antigen was phosphorylated in resting and activated T cells. Its expression was upregulated by stimulation with phorbol myristate acetate and with phytohemagglutinin. When added to concanavalin A-stimulated T-cell cultures in low concentrations, the 43 antibody was found to augment mitogenesis. The data indicate that this antibody may identify a CD5 homologue on feline T cells.     

The bovine p150/95 molecule (CD11c/CD18) functions in primary cell-cell interaction.

The monoclonal antibody (MAb), C5B6, recognizes the CD11c/CD18 molecule on the surface of bovine peripheral blood monocytes. C5B6 was reactive with 69-83% monocytes, all granulocytes, and less than 5% of lymphocytes from cattle. Of the lymphocyte series, the antibody had specificity for large lymphocytes and two lines expressing T cell markers, but was not reactive with small lymphocytes, thymocytes, a tumor cell line of B-cell lineage, an interleukin 2 (IL2)-dependent T cell line, fibroblasts, or human, sheep, goat or pig peripheral blood mononuclear cells. No dual fluorescence was seen using C5B6 and antibodies to bovine IgM, CD2, CD4 or CD8. Immunoprecipitation of 125I labeled peripheral blood mononuclear cells with C5B6 antibody defined two bands: 150,000 and 95,000 Da. Antibody to the beta chain (CD18) of the leukocyte adhesion receptor family precipitates the 95 kDa beta subunit and the three associated alpha subunits (180, 165 and 150). The bands obtained using MAb C5B6 correlated with the p150/95 bands observed using an antibody that precipitated the alpha and beta chains of the leukocyte adhesion receptor family. Functionally, the primary but not the secondary proliferative response to alloantigens was inhibited by C5B6 MAb. No effect was seen using C5B6 MAb in cytotoxicity assays or in the secondary proliferative response to Brucella abortus or bovine herpes virus type 1.     

Bovine T-lymphocyte lines reactive with Brucella abortus

Bovine T-cell lines reactive with Brucella abortus were established by repeated stimulation with B abortus and mitomycin C-treated autologous antigen-presenting cells. Representative results were obtained, using 33 cell lines from 14 cows. Cultures responded to the virulent laboratory strain 2308, the vaccine strain 19, and the rough mutant strain RB51 in thymidine-incorporation assays. The cells in these cultures required antigen-presenting cells for their response to B abortus. Autologous antigen-presenting cells were optimal for most lines tested, although some T-cell lines could respond to B abortus in the presence of some, but not all, allogeneic antigen-presenting cells. The cell lines expressed cell surface markers characteristics of activated bovine T cels. Of the cell lines tested for expression of cluster-determinant (CD) 4 and CD8 cell surface antigens, no cells in any cultures expressed the bovine CD8 equivalent, but all cultures included CD4+ cells in variable amounts. Some cell lines consisted of up to 50% CD2+CD4-CD8- cells. None of the cell lines tested expressed surface immunoglobulin or other bovine B-cell markers. Thus, these long-term cell lines appear to include 2 T-lymphocyte subsets: the helper/inducer subset and a second subset expressing a phenotype similar to major histocompatibility complex-unrestricted cytolytic cells in other species.     

Adhesion molecule and homing receptor expression on blood and milk polymorphonuclear leukocytes during the periparturient period of dairy cattle

Adhesion molecule and homing receptor expression on blood and milk polymorphonuclear leukocytes (PMN) from periparturient dairy cattle was studied. Both percentages and the mean fluorescence intensity (MFI) of PMN expressing CD11a, CD44, CD62L, and LPAM-1 (alpha4 beta7) were evaluated at seven time points during the twenty-one day period post calving. CD11a and CD62L were expressed on 94-100% of PMN in both blood and milk and there were no significant differences in these percentages at any time point. LPAM-1 was expressed on 3-10% of the PMN in the blood and 13-45% in the milk and the percentage of cells expressing LPAM-1 in milk was significantly (P<0.05) greater than in blood at 0, 4, 10, 14, 18 and 21 days after calving. CD44 was expressed on 11-39% of the PMN in blood and 33-69% in the milk and the percentage of cells expressing CD44 in milk was significantly (P<0.05) greater than in blood at all time points. The MFI of CD11a on milk PMN was consistently higher than that of blood PMN throughout the study period and significantly (P<0.05) higher at days 4, 10 and 18 after calving.     

Ex vivo γ-retroviral gene therapy of dogs with X-linked severe combined immunodeficiency and the development of a thymic T cell lymphoma

We have previously shown that in vivo γ-retroviral gene therapy of dogs with X-linked severe combined immunodeficiency (XSCID) results in sustained T cell reconstitution and sustained marking in myeloid and B cells for up to 4 years with no evidence of any serious adverse effects. The purpose of this study was to determine whether ex vivo γ-retroviral gene therapy of XSCID dogs results in a similar outcome. Eight of 12 XSCID dogs treated with an average of dose of 5.8 × 10(6) transduced CD34(+) cells/kg successfully engrafted producing normal numbers of gene-corrected CD45RA(+) (na?ve) T cells. However, this was followed by a steady decrease in CD45RA(+) T cells, T cell diversity, and thymic output as measured by T cell receptor excision circles (TRECs) resulting in a T cell lymphopenia. None of the dogs survived past 11 months post treatment. At necropsy, few gene-corrected thymocytes were observed correlating with the TREC levels and one of the dogs was diagnosed with a thymic T cell lymphoma that was attributed to the gene therapy. This study highlights the outcome differences between the ex vivo and in vivo approach to γ-retroviral gene therapy and is the first to document a serious adverse event following gene therapy in a canine model of a human genetic disease.