T cell responses to heat-shock protein 60: differential responses by CD4+ T cell subsets according to their expression of CD45 isotypes

We demonstrate that human T lymphocytes proliferate in vitro to highly purified human heat-shock protein 60 (Hu.hsp60). The response to this self Ag was confined to the CD45RA+ RO- T cell subset, with minimal responses by adult CD45RA- RO+ T cells. Experiments using keyhole limpet hemocyanin as a prototypic novel Ag, or tetanus toxoid as a recall Ag, were consistent with the notion that CD45RA+ RO- and CD45RA- RO+ T cell subsets can be designated as naive and memory cells, respectively; thus, responses to Hu.hsp60 were confined to the putative naive subset. In contrast, both CD45RA+ RO- and CD45RA- RO+ T cell populations proliferated to bacterial hsp60 from Mycobacterium leprae, Escherichia coli, or Chlamydia trachomatis. However, only CD45RA- RO+ (memory) T cells responded to a mycobacterial hsp60-derived peptide previously defined as a major bacteria-specific epitope. Experiments with cord blood T cells, which are CD45RA+ RO- and can be considered truly naive, showed that the peptide could elicit responses from naive T cells in vitro; cord blood cells also responded to Hu.hsp60. Since bacterial hsp60 Ags contain both conserved and nonconserved epitopes, we speculate that in vivo challenge with bacterial hsp60 will activate T cells capable of seeing either type of epitope, but only those that see nonconserved epitopes maintain the CD45RA- RO+ memory phenotype. However, T cells recognizing conserved epitopes, while not apparently being recruited to the memory pool, may nevertheless play a role in immunoregulation, particularly in the context of inflammation, when expression of Hu.hsp60 is increased.     

Antigen presentation by epithelial cells induces anergic immunoregulatory CD45RO+ T cells and deletion of CD45RA+ T cells

The immunoregulatory effects of alloantigen presentation by tissue parenchymal cells to resting peripheral blood CD4+ T cells was investigated. Coculture of CD45RO+ (memory) and CD45RA+ (naive) T lymphocytes with primary cultures of MHC class II-expressing epithelial cells rendered both populations of T cells hyporesponsive to a subsequent challenge by the same MHC molecule expressed on EBV-transformed lymphoblastoid B cell lines. However, the mechanisms responsible for the allospecific hyporesponsiveness were distinct. For the CD45RO+ T cells, responsiveness was restored by subsequent culture in the presence of IL-2; the addition of IL-2 had no effect on the reactivity of the CD45RA+ T cells. In contrast, the naive T cells were protected from the induction of nonresponsiveness by the presence of a neutralizing anti-CD95 Ab during the culture with thyroid follicular cells. In addition, the hyporesponsive CD45RO+ T cells effected linked suppression, in that they inhibited proliferation against a third-party DR alloantigen when the third-party alloantigen was coexpressed with the DR Ag against which hyporesponsiveness had been induced. These results suggest that recognition of Ag by T cells on tissue parenchymal cells plays an important role in the maintenance of peripheral T cell tolerance, inducing nonresponsiveness in naive and memory T cells by distinct mechanisms.     

Age-related modifications of the human alphabeta T cell repertoire due to different clonal expansions in the CD4+ and CD8+ subsets

We have studied the effects of a life-long antigen stimulation on the clonal heterogeneity of human peripheral T cell subsets, as defined by their CD45 isoform expression. CD4+ or CD8+ T cells were obtained from healthy donors ranging in age from 20 to 100 years, and sorted into CD45RA+ and CD45RO+ populations. A modified PCR-heteroduplex analysis was then used to directly compare the TCR Vbeta clonal make up of either compartment pair. We find that the CD4+ T cell repertoire remains largely polyclonal throughout life, since CD4+ expanded clones are rare and accumulate predominantly in the CD45RO+ compartment of exceptionally old donors (100 years old). In contrast, the CD8+ T cell subset contains expanded clones which are already detectable in young adults and become very frequent in 70- to 75-year-old donors in both CD45RA+ and CD45RO+ compartments analyzed. Interestingly, some expanded clones are detectable in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments of either CD4+ or CD8+ T cells. These results indicate that the age-dependent accumulation of expanded clones starts earlier and is more pronounced in the CD8+ than in the CD4+ T cell subset, reinforcing the concept that clonal expansion in the two subsets is controlled by substantially different mechanisms. Furthermore, whereas the finding of expanded CD45RO+ T cell clones is explained by antigen-driven proliferation, the detection of expanded clones in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments would support the hypothesis of reversion from the CD45RO+ to the CD45RA+ phenotype after antigen encounter.     

Evidence for selective in vivo expansion of synovial tissue-infiltrating CD4+ CD45RO+ T lymphocytes on the basis of CDR3 diversity

In this study we have analyzed the TCR V alpha and V beta regions at the DNA level in the CD4+CD45RO+ memory T cell population of synovial tissue infiltrating T lymphocytes of three rheumatoid arthritis (RA) patients and one patient with chronic arthritis. Cell lines of CD4+CD45RO+, CD4+CD45RO-, CD8+CD45RO+ and CD8+CD45RO- T lymphocyte populations were generated following FACS cell sorting of freshly isolated synovial tissue mononuclear cell infiltrates (STMC) and of freshly isolated peripheral blood mononuclear cells (PBMC) of these patients. The phenotypic and molecular analyses have revealed the following. (i) The TCR repertoires of tissue infiltrating T lymphocytes in the various subsets were extensive on the basis of TCR V gene family usage. (ii) Furthermore, each patient displayed individual specific TCR V gene expression patterns in the various STMC and PBMC derived T cell subsets. However, the majority of these arthritis patients manifested increased expression of multiple TCR V gene families in the synovial tissue derived CD4+CD45RO+ T cell population when compared with the peripheral blood derived CD4+CD45RO+ subset. Of these gene families, we found enhanced expression of the TCR V alpha 7 and V beta 11 gene segments in synovial tissue to be shared by all four patients analyzed. (iii) Nucleotide sequence analysis of the CDR3 regions of a number of TCR V regions in the CD4+CD45RO+ T cell subsets has revealed that the CDR3 regions comprised within synovial tissue derived TCR V regions differed from those found in peripheral blood derived TCR V regions. These differences in CDR3 diversity might be the consequence of a specific interaction with particular MHC-peptide complexes expressed at the site of inflammation. (iv) The CDR3 region analysis also showed individual specific amino acid motifs within the N-D-N regions of all analyzed TCR V beta genes derived from PBMC as well as STMC.     

Increase in adhesion molecules on CD4+ cells and CD4+ cell subsets in synovial fluid from patients with rheumatoid arthritis

OBJECTIVE: To elucidate the role of adhesion molecules in the pathogenesis of rheumatoid arthritis (RA). METHODS: We evaluated their expression and that of an activation marker on CD4+ cell populations and CD4+ cell subsets in specimens of peripheral blood (PB) and synovial fluid (SF) obtained from 10 patients with RA and 7 with osteoarthritis (OA). A 2 or 3-color immunofluorescent method was used for analysis. RESULTS: The SF from both groups of patients showed a greater density of adhesion molecules including LFA-1 alpha, LFA-1 beta, CD2, VLA-4 alpha and VLA-5 alpha on CD4+ cells, and a higher percentage of CD4+HLA-DR+ cells compared with their PB. IN PB-CD4+ cell subsets from the arthritic and healthy subjects, the CD4+CD45RO+ cell population showed an increased expression of adhesion molecules compared with CD4+CD45RA+ cell population. The expression of adhesion molecules on circulating CD4+ cell population and CD4+ cell subsets from the patients with RA and OA was comparable to that from healthy subjects. SF from both groups of patients showed a higher percentage of CD4+CD45RO+ cells and a lower percentage of CD4+CD45RA+ cells. In SF-CD4+ cell subsets from patients with RA, the CD4+CD45RO+ cell population had an increased expression of VLA-4 alpha compared to the CD4+CD45RA+ cell population; however, there was no significant difference in other adhesion molecule expression and the percentage of HLA-DR+ cells between the 2 cell subsets. Furthermore, the expression of VLA-4 alpha on the CD4+CD45RO+ cell population in SF from patients with RA was significantly higher than that in matched PB. In CD4+CD45RA+ cell population from both groups of patients, SF showed an enhanced expression of adhesion molecules and an increased percentage of HLA-DR+ cells compared with matched PB. CONCLUSION: Our results suggest that increased expression of adhesion molecules and increased percentage of HLA-DR+ cells on CD4+ cells in SF may be responsible for cellular interactions between these cells and synovial cells or extracellular matrix.     

Coexpression of GD3 ganglioside with CD45RO in resting and activated human T lymphocytes

The ganglioside GD3 is preferentially expressed on the surface of malignant T cell lymphoblasts and on resting T cells which express the memory cell phenotype, CD45RA-CD29+. However, GD3 expression in activated T cells and its potential function in proliferating normal and malignant T cells are unclear. Utilizing three-color immunostaining and flow cytometry, we examined changes in the expression of GD3 in conjunction with the RA and RO isoforms of CD45 during in vitro T cell activation. GD3 was equally expressed in resting CD4 and CD8 cells and was specifically found in the CD45RO+RA population. Activation of T cells with PHA resulted in an increased percentage of GD3+ cells. This increase was evident by the first day and was observed in the CD45RO (naive cell) population; by 2 days, GD3 was expressed heterogeneously in a large population of CD45RO+RA+ cells. Further activation of T cells with PHA or anti-CD3 monoclonal antibody (OKT3) resulted in a further increase in GD3-expressing cells, and the increase in GD3 density correlated with increased CD45RO and loss of CD45RA. In contrast, increases in GD3 and interleukin-2 receptor (CD25) expression in response to PHA or OKT3 occurred independently, indicating that the GD3/ CD45RO coexpression observed was not a general consequence of cell activation. The results provide evidence for specific comodulation of GD3 and CD45RO during T cell mitogenesis, and thus suggest that these molecules may colocalize on the T cell surface.     

Intestinal lymphangiectasia, a disease characterized by selective loss of naive CD45RA+ lymphocytes into the gastrointestinal tract

Intestinal lymphangiectasia (InL) is a disease characterized by hypoproteinemia and lymphocytopenia resulting from blocked intestinal lymphatics and loss of lymph fluid into the gastrointestinal tract. This leads to immunologic abnormalities including hypogammaglobulinemia, skin anergy and impaired allograft rejection. In the present study, we evaluated whether the above immunologic abnormalities are secondary to a quantitative or qualitative disorder of T cells. In initial studies we demonstrated that adult InL patients' peripheral blood contain strikingly (and significantly) reduced numbers of CD4+/CD45RA+ T cells, whereas the numbers of CD4+/CD45RO+ T cells were only moderately (and not significantly) reduced. In addition, the CD4+/CD45RO+ T cell population contained an increased percentage of highly differentiated and previously sensitized cells, as demonstrated by decreased CD27 and CD31 expression and increased HLA-DR and CD69 expression. In subsequent functional studies, we showed that the InL CD4+/CD45RO+ T cells, when stimulated in vitro, proliferate fivefold less than control CD4+/CD45RO+ T cells and produce fourfold more IL-4 and threefold less IFN-gamma and IL-2. Thus, this cytokine production profile also reflects the highly differentiated nature of the residual cell population. Overall, these studies provide new information on the trafficking of naive/mature and Th1/Th2 T cell populations in this disease model.     

Selective migration of highly differentiated primed T cells, defined by low expression of CD45RB, across human umbilical vein endothelial cells: effects of viral infection on transmigration

Low expression of CD45RB on CD45RO+ T lymphocytes defines a subset of highly differentiated T lymphocytes that accumulate in vivo within the affected joints of patients with rheumatoid arthritis (RA). Although it is known that CD45RO+ T lymphocytes migrate to sites of inflammation in vivo, it is not clear whether within this subset the CD45RBlo cells are selectively recruited or develop in situ within the joint. Using a transwell system we show that a small proportion of resting T lymphocytes migrated across unactivated human umbilical vein endothelial cells (HUVEC). These migrating cells were CD45RO+ and enriched for low CD45RB expression. In addition, both the CD45RO+CD45RBlo subset and migrating cells expressed increased levels of beta 1 and beta 2 integrins and CD44. The percentage of CD45RO+CD45RBlo T lymphocytes was increased in the circulation of patients with acute Epstein-Barr virus (EBV) infection. These in vivo activated cells also expressed increased levels beta 1 and beta 2 integrins and CD44, and showed an enhanced rate of transmigration compared with resting T lymphocytes. Transmigration of T lymphocytes was increased using the chemokines RANTES and lymphotactin and the cytokine interleukin-15 (IL-15). In addition, infection of the HUVEC with cytomegalovirus (CMV) led to an enhanced movement of T lymphocytes. In all of these cases the selective migration of the CD45RBlo subset was maintained. Thus although the rate of T-lymphocyte transmigration could be influenced by a number factors, the CD45RO+CD45RBlo subset has a migratory advantage suggesting that more differentiated CD45RO+CD45RBlo T lymphocytes are selectively recruited to sites of inflammation.     

The levels of memory (CD45RA-, RO+) CD4+ and CD8+ peripheral blood T-lymphocytes correlate with IgM rheumatoid factors in rheumatoid arthritis

We investigated whether, in rheumatoid arthritis (RA), the CD45 isoform expression of peripheral blood T-lymphocytes (T-PBL) is related to auto-immune processes (e.g. IgM rheumatoid factors) and to clinical manifestations. By three-colour flow cytometry, we quantified three subsets of CD4+ or CD8+ T-PBL: "naive" CD45RA+,RO-, "transient" CD45RA+,RO+, and "memory" CD45RA-,RO+ cells, in 102 patients with RA and in 41 age- and sex-matched controls. The serum levels of rheumatoid factors (RF) were determined--besides conventional agglutination tests--by ELISA (IgM-RF). Extensive clinical examination was performed at the time of blood sampling. In RA, age, sex and drug therapy did not constitute major influences on the CD45RA/RO patterns. In "healthy" men, higher age significantly' correlated with fewer naive and more memory CD4+ T-PBL (P < 0.01). In RA, distinct correlations between the T-PBL subsets, autoimmune and clinical manifestations became obvious when patients with low and high levels of RF against human IgG Fc fragments, as determined by ELISA, were analysed separately. RA patients with high IgM-RF had elevated proportions of CD45RO+ T-PBL (P < 0.05), that correlated with clinical parameters of disease activity (tender joint count, Ritchie index, P < 0.05) and outcome (Health Assessment Questionnaire, Larsen radiographic scores, P < 0.05). The proportions of memory CD4+ and CD8+ T-PBL correlated strongly (P < 0.001) with the IgM-RF levels. Within 1 year, only three of 34 patients (disease duration of 5-9 years) showed seroconversion from low to high levels of IgM-RF (and positive agglutination tests); this was paralleled by reductions in naive and increases in transient T-PBL (P < 0.02). Thus, in RA, the proportions of memory CD4+ and CD8+ T-PBL correlate with the level of IgM-RF and, together with transient T-PBL, with clinical parameters of disease activity and outcome.     

Responses to human rhinovirus in CD45 T cell subsets isolated from tonsil

Isotypes of CD45 have been used extensively as markers of memory and naive populations of T cells in peripheral blood. In this study, T cells were isolated from human tonsil and their proliferative response against human rhinovirus was measured. Unexpectedly, equivalent responses were found among the CD4+CD45RA+ and CD4+CD45RO+ populations of T cells. This response requires MHC class II-positive antigen-presenting cells. The time course of the T cell response in vitro was that of a classical recall response, and no proliferative response to the virus could be detected in human cord blood. These results suggest that tonsils contain a significant population of CD45RA+ memory cells. The presence of this population may reflect ongoing stimulation with this common infectious agent, and the anatomical location of the T cells within the major lymphoid organ draining the naso-pharyngeal epithelial surface.     

Normal growth in high-risk hyperlipidemic children and adolescents with dietary intervention

We have analyzed the effect of complete T cell activation (anti-CD3 plus anti-CD28) on the activation of NF-kappaB in CD45RA+ (naive) and CD45RO+ (memory/effector) T cells. Long exposure (24 h) induced stronger NF-kappaB DNA binding in CD45RA+ cells than in CD45RO+ cells. Analysis of the nuclear c-Rel protein indicated that after anti-CD3+anti-CD28 stimulation the level of c-Rel was higher in CD45RA+ cells. Analysis of the cytoplasmic inhibitor IkappaBalpha indicated that anti-CD3+anti-CD28 stimulation induced a long-lasting degradation in CD45RA+ cells but in CD45RO+ cells the degradation process was more rapid. Because the CD28 costimulus is known to induce the production of reactive oxygen intermediates (ROIs), the intracellular ROI levels in CD45RA+ and CD45RO+ cells were compared by flow cytometry. ROIs were produced in both cell types, but more strongly in CD45RA+ cells. The data presented in this study further emphasize the differences between CD45RA+ and CD45RO+ T lymphocytes in ROI-dependent signaling pathways.     

Absence of bcl-2 expression by activated CD45RO+ T lymphocytes in acute infectious mononucleosis supporting their susceptibility to programmed cell death

bcl-2 proto-oncogene encodes an inner mitochondrial membrane protein that blocks programmed cell death (apoptosis). There is now increasing evidence that regulation of bcl-2 expression is a determinant of life or death in normal lymphocytes. We have recently described that activated (CD45RO+) CD4+ and CD8+ T cells in acute infectious mononucleosis (IM) undergo apoptotic cell death on culturing, indicating an activation-driven cell death of mature T cells. In this work, we examine bcl-2 expression by activated T cells in acute IM using a flow-cytometric analysis with an anti-bcl-2 monoclonal antibody (MoAb). It was consistently observed that most T cells from acute IM patients displayed only much less bcl-2, while normal T cells expressed bcl-2 relatively strongly. Multicolor analysis showed that bcl-2-lacking T cells in acute IM were restricted to the CD45RO+ (activated) populations of CD4+, as well as CD8+ T cells. In contrast, the relatively intense levels of bcl-2 were expressed in both CD45RO+ and CD45RO- T-cell populations from normal subjects. This marked difference in bcl-2 expression of CD45RO+ T cells between acute IM and normal controls was also confirmed by Western blot analysis. Activated (CD45RO+) T cells with low bcl-2 expression, but not bcl-2-expressing CD45RO- T cells, in acute IM patients were found to die easily when cultured without added growth factors. However, in normal individuals, both CD45RO+ and CD45RO- T cells were relatively stable on culturing. These findings suggest that lack of bcl-2 expression by activated (CD45RO+) T cells in acute IM might be associated with their susceptibility to programmed cell death.     

Age-related increase in the fraction of CD27-CD4+ T cells and IL-4 production as a feature of CD4+ T cell differentiation in vivo

The influence of ageing on phenotype and function of CD4+ T cells was studied by comparing young (19-28 years of age) and aged (75-84 years of age) donors that were selected using the SENIEUR protocol to exclude underlying disease. An age-related increase was observed in the relative number of memory cells, not only on the basis of a decreased CD45RA and increased CD45RO expression, but also on the basis of a decrease in the fraction of CD27+CD4+ T cells. Our observation that the absolute number of CD45RO+CD4+ T cells was increased, while absolute numbers of CD27-CD4+ T cells remained unchanged in aged donors, indicates that the latter subset does not merely reflect the size of the CD45RO+CD4+ T cell pool. The increased fraction of memory cells in the aged was functionally reflected in an increased IL-4 production and T cell proliferation, when cells were activated with the combination of anti-CD2 and anti-CD28, whereas IL-2 production was comparable between both groups. No differences were observed with respect to proliferative T cell responses or IL-2 production using plate-bound anti-CD3 or phytohaemagglutinin (PHA). The observation that IL-4 production correlated with the fraction of memory cells in young donors but not in aged donors suggests different functional characteristics of this subset in aged donors.     

CDw60: a marker for human CD8+ T helper cells

The existence of helper cells among the CD8+ T cell subset has been recognized for a long time. However, the phenotype of these cells has remained elusive. In this study, we provide evidence that the expression of the CDw60 antigen on human CD8+ T cell allows one to distinguish between CD8+ T helper cells and CD8+ T cells with cytotoxic and suppressor capacity. CDw60 monoclonal antibodies (mAb) recognize the 9-O-acetylated disialosyl group on ganglioside GD3 expressed on 20-40% of CD8+ cells. By use of the direct and indirect mAb-rosetting technique, we were able to isolate the CDw60+CD8+ and CDw60-CD8+ cells at high purity. The alloantigen-specific cytotoxic activity of CD8+ cells resided entirely in the CDw60- population. Helper and suppressor capacity of both CD8 subsets was assayed by the pokeweed mitogen-induced differentiation of B cells into immunoglobulin-secreting cells. These studies clearly indicate that the CDw60+CD8+ subset provided substantial help to B lymphocytes, whereas the CD8+ cells with the CDw60- phenotype were suppressing B cell differentiation. Both subsets produced similar amounts of interleukin 2 (IL-2) after stimulation with phytohemagglutinin. Activation with phorbol myristate acetate in combination with Ca-ionophore induced IL-4 secretion in both populations, but preferentially in the CDw60+ subset, whereas the vast majority of interferon gamma was produced by the CDw60-CD8+ cells. When used in combination with other markers, CDw60 may prove to be useful in defining CD8+ subsets with reciprocal functional activities.     

CD45RA(bright)/CD11a(bright) CD8+ T cells: effector T cells

An important aspect of peripheral T cell development is the differentiation from naive into memory cells. To distinguish naive from memory cells, CD45RA and CD11a are commonly used: CD45RA+ or CD11a(dim) T cells are regarded as naive, while CD45RA- or CD11a(bright) T cells are thought to be of memory type. There is, however, a CD8+ T cell subset which is CD45RA+ and at the same time CD11a(bright). It increases with age and in patients with systemic viral infections, though its functional role in the immune response is unknown. In the present study, we give evidence that this subset is related to memory-like T cells as it produces IFN-gamma and tumor necrosis factor-alpha, contains high levels of perforin, and expresses CD95 in the same way as memory-type CD45RA-/CD11a(bright) CD8+ T cells. Since it contains a high percentage of CD28- and CD57+ cells, is increased in size and granularity, and is transiently expressed following in vitro stimulation of naive CD8+ T cells, we speculate that this subset mainly represents recently activated effector T cells that are able to interact with CD80 and CD86 (B7-1 and B7-2 respectively) negative tissue cells.     

The CD7- T cell subset represents the majority of IL-5-secreting cells within CD4+CD45RA- T cells

Absence of CD7 is a stable phenotype in a subset of normal human T cells. Most circulating CD7- T cells express the CD4CD45RO+CD45RA- memory phenotype. We analysed CD4+CD45RA- peripheral blood lymphocytes that were separated into CD7+ and CD7- for their in vitro cytokine secretion in response to different stimuli. The CD4+CD7- subpopulation was found to secrete significantly higher levels of IL-5 compared with the CD4+CD7- subset upon stimulation with ionomycin/phorbol myristate acetate (PMA) plus anti-CD28 MoAbs. In contrast to IL-5 secretion, IL-4 and interferon-gamma (IFN-gamma) secretion was not significantly different in CD7+ and CD7- T cells upon stimulation in vitro. The data indicate that the CD4+CD7- T cell represents the majority of IL-5-secreting cells within the population of CD4+CD45RA- memory T cells. Since CD4+CD7- T cells were found to be enriched in various skin lesions associated with eosinophilic infiltration, the results of our study support the hypothesis that skin-infiltrating CD7- T cells are one of the major sources of IL-5 responsible for the development of eosinophilic inflammation in certain skin diseases.     

Definition of the specificity of monoclonal antibodies against porcine CD45 and CD45R: report from the CD45/CD45R and CD44 subgroup of the Second International Swine CD Workshop

Swine cell binding analyses of a set of 48 monoclonal antibodies (mAbs), including eleven standards, assigned to the CD44 and CD45 subset group of the Second International Swine CD Workshop yielded 13 clusters. Although none of these corresponded to CD44, seven mAbs formed a cluster which was identified as being specific for restricted epitopes of CD45 (CD45R). In addition, a T-cell subset specific cluster comprised of four mAbs was also identified. Two mAbs (STH106 and SwNL 554.1) reacted exclusively with CD8 bright lymphocytes, the other two (2B11 and F01G9) with a subset of CD4 lymphocytes. The other 10 clusters were either specific for MHC-class I like molecules or overlapped with clusters identified by the adhesion molecule subgroup and are therefore just briefly discussed in this report. The specificity of all the mAbs in the CD45R cluster was verified by their ability to immunoprecipitate distinct proteins and to react with CHO cells expressing individual isoforms of CD45. Three CD45R mAbs (3a56, MIL5, -a2) did react with a 210 kDa isoform(s), while another three (STH267, FG2F9, 6E3/7) only recognized a 226 kDa isoform(s). The remaining one (MAC326) precipitated both a 210 and 226 kDa protein. The specificity of all the mAbs in the CD45R cluster, and of the CD45 common mAbs, was confirmed by their reactivity with CHO cells transfected with cDNAs encoding the extracellular and transmembrane portions of distinct CD45R isoforms. Those mAbs recognizing a 210 kDa protein reacted with CHO cells expressing the CD45RC isoform, while those capable of precipitating a 226 kDa, but not the 210 kDa, polypeptide recognized CHO cells expressing either the CD45RAC and the relatively rare CD45RA isoform. MAC326 was unique in its inability to react with CHO cells engineered to produce the CD45RC and CD45RAC isoforms. Thus, three mAbs (6E3/7, STH267, and FG2F9) appear to be specific for an epitope(s) encoded by the A exon, while one (MAC326) recognizes a determinant encoded by the C exon. The remaining three mAbs (3a56, -a2, MIL5) are apparently specific for an epitope(s) which results from the fusion of the C exon to the invariant leader sequence and is destroyed by inclusion of the A exon. All three CD45 common mAbs, K252.1E4, MAC323 and 74.9.3, did react with the CHO cells lines expressing either the CD45RA, CD45RC, CD45RAC or CD45RO isoforms, but not with untransfected CHO cells. When the natural expression of CD45 isoforms was examined by reacting lymphocytes with CD45R mAbs, a high level expression of isoforms containing the A exon-generated domain was detected in all B cells while the majority of CD4+ T cells had undetectable or lower expression density of this protein than B cells. In contrast, the density of expression of the CD45 isoform(s) containing the C exon-generated domain ranged from undetectable to high in CD4+ T cells whereas the amounts were approximately ten-fold lower in B cells. Overall this panel of CD45 mAbs will be very useful in analyzing the maturation and differentiation of swine lymphoid cells subsets.     

Inhibition of the progression of multiple sclerosis by linomide is associated with upregulation of CD4+/CD45RA+ cells and downregulation of CD4+/CD45RO+ cells

In a recent double-blind, phase II study, conducted in our department, we showed that Linomide-treated MS patients had significantly less active lesions (in serial monthly MRI tests) and a tendency for clinical stabilization. Here we present the immunological evaluation of the patients who participated in this study and propose a novel mechanism by which Linomide downregulates autoreactivity. Peripheral blood leukocytes (PBLs), serum, and CSF samples were obtained at two to four time points over the 6 months of the trial. Flow cytometric analysis (FACS) of the CD5/CD19, CD4/CD8, CD14/CD3, CD16/CD3, CD45RA/CD4, and CD45RO/CD4 surface markers on PBLs was performed and the levels of the IL-1beta, IL-2, IL-4, IL-6, IL-10, TNF-alpha, IFN-gamma, and IL-2R were also examined. White blood counts of Linomide-treated patients were consistently elevated throughout the treatment period (P = 0.002-0.04). Cytokines levels in serum and CSF were highly fluctuating and we could not detect any clear trend as a result of Linomide treatment. FACS analysis showed that Linomide treatment significantly increased the percentage of the CD4+/CD45RA+ cells (from 35.5% at baseline to 42.3% at week 24; P = 0.02), and decreased CD4+/CD45RO+ lymphocytes (62.6% at baseline vs 53.7% at week 24, P = 0.02). Linomide also induced a transient increase in the NK-cells, the NK 1.1 cells, and the CD5 B-cells (P = 0.02). Upregulation of naive CD45RA T-lymphocytes and parallel downregulation of memory CD45RO cells seems to be one of the main mechanisms by which Linomide inhibits MS activity and may represent an alternative immunomodulating approach for the treatment of MS and autoimmunity in general.