Expression pattern of CD45 RA/RO isoformic antigens in T-lineage neoplasms

The expression of CD45 RA/RO antigen was investigated in neoplasms including cases expressing CD7 antigen as the sole pan-T antigen (n = 8), T-lineage acute lymphoblastic leukemia (ALL)/lymphoblastic lymphoma (LBL) at various stages of differentiation (n = 32), peripheral stage T-lineage leukemia (n = 10) and adult T-cell leukemia (ATL) (n = 14). The p56^Ick gene expression was also investigated in selected cases. The expression pattern of CD45 RA/RO antigen was defined as of RA, mixed, or RO type. All but one CD7+ CD5? CD2? case were of the RA type. The CD7+ CD5+ CD2? prothy-mic stage included seven RA and one mixed type cases. One CD7+ CD5? CD2+ case was of the RA type, but the other was of the RO type. The CD7+ CD5+ CD2+ prothymic stage included three RA and four mixed type cases. All seven CD3? CD4+ CD8+ (double-positive) thymic cases were of the RO type. The CD3+ CD4+ CD8+ (triple-positive) stage included two RO and three mixed-type cases. One CD3+ CD4+ CD8? late thymic case was of the mixed type. The peripheral stage cases included five RA, three RO, and two mixed type cases. All ATL cases were of the RO type. The expression of p56^Ick gene in the prothymic stage was less marked than that in the thymic stage. On the basis of these results, the following sequence of pattern of the CD45 RA/RO antigen expression along with T-lineage differentiation was reconstructed: prothymic stage [RA and mixed type] → double-positive thymic stage [RO type] → triple-positive thymic stage [RO and mixed type] → peripheral stage [RA, mixed, and RO type]. While one RO-type CD7+ CD5? CD2? and one RO-type CD7+ CD5? CD2+ cases were not in accord with this sequence, the pattern of CD45 RA/RO antigen expression in most of T-lineage neoplasms could be determined by the respective stage of differentiation. The poor expression of the p56^Ick gene by the prothymic blasts compared with the thymic blasts may be related to the expression pattern of the CD45 RA/RO molecules, which exhibits phosphatase activity. The consistent RO-type expression in the ATL cases may reflect the activated status of the neoplastic T cells due to the presence of the HTLV-I gene. Alternatively, the target cells for HTLV-I-induced neoplastic transformation may possibly be of the RO type. © 1995 Willey-Liss, Inc.     

The effect of hemodialysis on the virgin CD45RA+, CD45RO- and memory CD45RO+, CD45RA- lymphocyte count in patients with chronic renal failure

The hallmark of immunological memory is a quick and effective response to a repeated antigen exposure. Virgin lymphocytes, with their surface receptors CD45RA+, CD45RO- are produced in primary lymphatic organs, then migrating to secondary lymphatic structures. Memory lymphocytes CD45RO+, CD45RA- produced in these organs migrate to non-lymphatic organs--a possible location of inflammatory process, thus enabling the immunological system to eliminate effectively the same antigen, when repeatedly present. The aim of the study was 1) to test the influence of hemodialysis on the number of virgin lymphocytes and/or memory lymphocytes; 2) whether such impact (if any) depends on the type of dialysis membrane used (cuprophan or polysulphon), 3) if the effect is different in patients with or without diabetes. Overall number of virgin T helper lymphocytes CD45RA+CD4+ was significantly lower in patients with end-stage renal disease, while the number of total CD45RO+, CD45RA- memory lymphocytes was significantly greater among patients with diabetic nephropathy, compared to normal control subjects. After 15 minutes of hemodialysis, number of virgin lymphocytes CD45RA+, CD45RO- (p < 0.001, p < 0.01) and their subclasses, as well as memory lymphocytes CD45RO+, CD45RA- were significantly decreased. After 15 minutes of hemodialysis with polysulphon membrane, the decrease in T virgin cytotoxic, B virgin CD45RA+CD4-, T memory cytotoxic as well as B memory CD45RO+CD4- lymphocytes was significantly lower, when compared with cuprophan membrane (p < 0.02). Among patients treated with cuprophan hemodialysis, the decrease of T helper memory CD45RO+CD4+ lymphocytes was significantly lower in patients with diabetic nephropathy, than in non-diabetic patients. CONCLUSIONS: In all patients with end-stage renal disease, the impact of hemodialysis on the number of memory lymphocytes CD45RO+, CD45RA-, as well as virgin lymphocytes CD45RA+, CD45RO- was shown, but the effect was less profound during hemodialysis with polysulphon membrane, compared to cuprophan. The presence of diabetic nephropathy effects the hemodialysis-induced changes in the number of T memory helper CD45RO+ CD45RO+CD4+ lymphocytes, with no impact on other subclasses of the examined cells.     

The distribution of CD45R, CD29 and CD45RO (UCHL1) antigens in mature CD4 positive T-cell leukaemias

We have studied the expression of antigens characterizing functional T-cell subsets in 32 CD4+ mature T-cell leukaemias. In this analysis we used two monoclonal antibodies (McAb) of the CD45R group (2H4 and GRT22) which have been shown to identify the 'native/virgin' T-cell population that functions as 'suppressor-inducer' cells in vitro, and two McAb, CD29 (4B4) and CD45RO (UCHL1), which characterize non-identical 'memory' cells that proliferate in response to soluble recall antigens and provide help in antigen-specific IgG synthesis. Four groups of CD4+ cases were identified according to this reactivity: (a) 15 CD45R+, CD29+; (b) 13 CD45R-, CD29+; (c) three CD45R-, CD29-; and (d) one case only CD45+, CD29-. The high incidence of coexpression of CD45R and CD29 (47% of cases) is a new finding which contrasts with the mutual exclusion of these antigens on normal CD4+ T-lymphocytes. There was no correlation between subset phenotypes and pathological disease entities. None of the six cases of adult T-cell leukaemia/lymphoma (ATLL), which is known as a disorder of activated 'suppressor-inducer' cells, had the 'expected' CD45R+, CD29- phenotype. Reactivity with UCHL1 showed a good correlation with CD29 in the CD45R- CD29+ cases which included three with ATLL. These results may help in the further characterization of T-cell malignancies according to functional subgroups and may clarify further the role of T-differentiation antigens in health and disease.     

Secondary T-acute lymphoblastic leukaemia mimicking blast crisis in chronic myeloid leukaemia

A 34-year-old man with chronic myeloid leukaemia (CML) firstly developed a lymphoid blast crisis of B-cell type. After a second chronic phase which lasted for > 4 years with maintenance chemotherapy of hydroxyurea, 6-mercaptopurine and methotrexate, he developed a T-cell acute lymphoblastic leukaemia of TcR-gammadelta+ type. Cytogenetic analysis revealed disappearance of the t(9;22) translocation and appearance of new abnormalities consistent with the diagnosis secondary acute leukaemia. To our knowledge, secondary leukaemia in CML has not previously been reported.     

Ubiquitin-proteasome pathway is compromised in CD45RO+ and CD45RA+ T lymphocyte subsets during aging.

Recent reports from our laboratory have demonstrated that CD45RO+ and CD45RA+ T lymphocytes from the elderly are compromised in their response to activation-induced IL-2 receptor expression, IkappaB-alpha degradation, as well as nuclear translocation of NFkB. To understand the basis of this activation-induced dysfunction in the elderly, we have examined the role of the ubiquitin-proteasome pathway. Our results demonstrate that both CD45RO+ and CD45RA+ T lymphocytes from the elderly show significant reduction in the constitutive 26S proteasome-associated chymotryptic activity, when compared to those in the young. Additionally, anti-CD3-CD28 treatment induced enhancement of proteasome-associated enzymatic activity in cells from the young, but not in cells from the elderly. Lowered proteasome-associated activity and its effect on reduced immune responses in the elderly could be mimicked by experiments which involved pretreatment of T cells from young donors with a proteasome specific inhibitor, lactacystin. These data demonstrate that IL-2 receptor induction is clearly compromised in T cells from the young when proteasomes are inhibited by pretreatment with lactacystin. An examination of ubiquitin specific hydrolase activity, demonstrated a decrease in activated CD45RA+ and CD45RO+ T cell subsets from the elderly when compared to young. These results suggest that lowered proteasome-associated enzymatic activity in combination with compromised de-ubiquitinating activity may be responsible for lowered activation-induced NFkB and NFkB-mediated gene expression in elderly subjects.     

A rapid translocation of CD45RO but not CD45RA to lipid rafts in IL-6-induced proliferation in myeloma

CD45, a receptor-type tyrosine phosphatase, is required for interleukin-6 (IL-6)-induced proliferation in human myeloma cells, which express the shortest isoform, CD45RO, but not the longest isoform, CD45RA. Here, we showed that IL-6 induced the translocation of CD45 to lipid rafts in an isoform-dependent manner. In myeloma cells, CD45RO was translocated to lipid rafts more rapidly than CD45RB, but exogenously expressed CD45RA was not translocated. When an IL-6Ralpha-transfected B-cell line was stimulated with IL-6, CD45RA was not translocated, although CD45RB was. We further confirmed that the translocated CD45 bound to IL-6Ralpha, Lyn, and flotillin-2, and this was followed by the dephosphorylation of the negative regulatory Tyr507 of Lyn. CD45 also bound to phosphoprotein associated with glycosphingolipid-enriched microdomains (PAGs), which were subsequently dephosphorylated, resulting in the release of C-terminal src kinase (Csk) from lipid rafts. Therefore, these results indicate that a rapid translocation of CD45RO to lipid rafts may be responsible for IL-6-induced proliferation, and that the change from CD45RA to CD45RO confers the ability to respond to IL-6 in human myeloma cells.     

Increased expression of the CD45RO (memory) antigen on T cells in HIV-infected children.

Expression of the CD45RO putative memory cell antigen on CD4 (helper) and CD8 (cytotoxic/suppressor) lymphocytes of children born to HIV-infected women was investigated using the UCHL1 antibody. Significantly raised numbers of CD45RO+ CD8 lymphocytes were found in all nine of the infected children compared with uninfected and control children. Expression of CD45RO on CD4 lymphocytes was variable; absolute numbers were not increased, although the percentage was increased in four out of nine infected children. All the infected children except two (who had comparatively low numbers of CD45RO+ CD8 cells) were clinically well, which suggests that an increase in CD45RO+ CD8 cells may be indicative of a functionally active immune response against HIV.     

Effect of didanosine, stavudine, and hydroxyurea therapy on apoptosis in CD45RA+ and CD45RO+ T lymphocyte subpopulations

The effect of aggressive antiretroviral therapy on spontaneous apoptosis (AP) in CD4+ and CD8+ lymphocytes expressing CD45RO (memory cells) and CD45RA (naive cells) and their relationship to cellular activation and viral load were examined. Ten patients receiving simultaneous treatment with d4T, ddI, and HU were evaluated. Flow cytometric analysis showed significant levels of AP (measured by TUNEL assay) among memory and naive T cells and an enhanced expression of CD38 and HLA-DR activation markers. The percentage of apoptotic CD4+CD45RO+ and CD4+CD45RA+ cells decreased, respectively, from 34 +/- 3.3 and 29 +/- 3.6 prior to treatment to 20.5 +/- 4 and 22 +/- 3.8 at week 8 into therapy. The percentage of apoptotic CD8+CD45RO+ and CD8+CD45RA+ cells similarly decreased, respectively, from 20 +/- 2.5 and 24 +/- 3 prior to treatment to 14.5 +/- 2.7 and 16 +/- 3 at week 8 into treatment. The percentage of CD4+ cells expressing the activation markers CD38 and HLA-DR decreased from 27 +/- 6 to 13 +/- 2 and from 26 +/- 4 to 13.5 +/- 3, respectively. The percentage of CD8+ cells expressing either CD38 or HLA-DR fell from 22 +/- 3 to 10 +/- 2 for the former and from 39 +/- 5 to 22 +/- 4 for the latter. This was associated with a significant decrease in viral load (mean, 1.4 log10), and a decline in circulating plasma TNF-alpha and sIL-2R levels from 50.5 +/- 10 to 21 +/- 6 and 92.5 +/- 11 to 68 +/- 9, respectively. These data indicate that short-term therapy with ddI, d4T, and HU in combination diminished AP, immune activation, and partially restored naive and memory T cell subpopulations.     

Expression of CD26 and DPP IV in T-acute lymphoblastic leukemia: comparison of immunocytochemistry with enzyme cytochemistry

Tile possible identity ofdipeptidyl peptidase IV (DPP IV) enzymatic activity and CD26 antigen expression in phenotypically defined T-acute lymphoblastic leukemia cells (T-ALL) was examined. For comparative studies, the combination of immunocytochemistry and enzyme cytochemistry methods was used. T he strong correlation between the CD26 antigen expression and DPP IV positivity in the majority of T-lymphoblasts in T-ALL patients was evident. No CD26 antigen was expressed on DPP IV negative T-cells. The variable CD4 and/or CD8 antigen expression, frequent CD5 and CD7 positivity and absence of surface membrane CD3 antigen were the characteristic immunophenotypic features of CD26/DPP IV positive T-lymphoblasts. Moreover, the clear CD71 and CD26/DPP IV coexpression suggested the association of CD26/DPP IV positive cells with proliferation. The immunophenotype of CD26/DPP IV positive T-lymphoblasts seems to be characteristic for the relative immature cell population. In addition, noteworthy was the slight disassociation between the very high CD26 antigen expression and moderate DPP IV activity in cells of some T-ALL patients. The possible existence of enzymatically inactive structures of CD26 antigen or inactive precursors of DPP IV detectable only by immunocytochemistry was discussed. Our study indicates that CD26 antigen expression is tended to identify cells with DPP IV enzymatic activity in T-ALL patients. The results provide some more information of CD26 antigen involvement in the pathology of leukemic cells via its DPP IV enzyme activity.     

Expression of CD45RO on circulating CD19+ B-cells in Crohn's disease.

Crohn's disease is an immunoregulatory disorder of the intestine that can be associated with systemic manifestations. This study analysed B-cell differentiation antigens to identify B-cell subpopulations unique to patients with Crohn's disease. CD45 isoform expression was used as an indicator of B-cell differentiation stage. This work shows that B-cells in blood and gut of patients with Crohn's disease are at an advanced stage of differentiation based on their unusual presentation of transitional (RA+ RO+) and late stage (RO+)CD45 isoforms on lamina propria lymphocytes, whereas normal intestinal lamina propria lymphocytes B-cells express primarily CD45RA. Crohn's disease patients had heightened expression of the CD45RO isoform on CD19+ lamina propria lymphocytes, and was found in a statistically significant proportion of Crohn's peripheral blood mononuclear cells (PBMC) where CD19+ PBMC had an expression pattern affecting an unexpectedly high proportion of these differentiated or late stage CD45RO+ B-cells. The expression of CD45RO varied greatly among CD19+ PBMC from patients with Crohn's disease, so multiple regression analysis was performed between these CD45 isoforms and several clinical parameters. After grouping high and low CD45RO expression on CD19+ B-cells, a significant statistical difference was found between high Crohn's disease activity index (CDAI) and low CDAI Crohn's disease patients respectively.     

CD45RA+ and CD45RO+ lymphocyte subpopulations in patients with monoclonal gammopathies: correlations with diagnosis, clinical stage and treatment status

Alterations in blood lymphocyte subsets may be involved in the development of overt myeloma. Naive (CD4+CD45RA+) and memory (CD4+CD45RO+) helper T-cell subsets are important effectors of immune T-cell regulation. We analyzed the distribution of these blood lymphocyte subpopulations in patients with monoclonal gammopathies, considering the type of disorder, clinical stage, and treatment status.     

Heterogeneity of the human CD4+ T-cell population: two distinct CD4+ T- cell subsets characterized by coexpression of CD45RA and CD45RO isoforms

Activation of unprimed CD4+CD45RA+/RO- T cells results in a gradual loss of CD45RA expression concomitant with the acquisition of CD45RO. It has been suggested that this conversion occurs in vivo through a CD45RAbright/RObright stage. Next to this small CD45RAbright/RObright subset (Dbright), a larger subpopulation that expresses both RA and RO isoforms at low levels (Ddull) can be found in the circulating CD4+ T- cell population of all donors. The properties of the latter population are largely undefined. Here, we show that Ddull cells have an intermediate phenotype for antigens such as CD31, CD621, CD58, and CD95 that are differentially expressed on unprimed versus primed T cells. In addition, they are able to provide help for B-cell differentiation and contain substantial numbers of tetanus toxoid (TT)-specific precursor cells. Remarkably, both intracellular cytokine staining and analysis of T-cell clones showed that Ddull cells and CD45RO+ T cells produce comparable high amounts of both interferon (IFN)-gamma and interleukin (IL)-4, which clearly distinguishes them from CD45RA+ and Dbright T cells. Finally, prolonged culture of sorted Ddull cells in a mixture of IL-2, IL-6, and tumor necrosis factor (TNF)-alpha showed that about half of the population retained the Ddull phenotype. Part of the cells upregulated the CD45RA isoform, whereas only a minority switched to single CD45RO expression. Our findings indicate that the Ddull population contains primed T cells, some of which may reacquire an "unprimed" phenotype in the absence of antigenic stimulation.     

Clinical grade expansion of CD45RA, CD45RO, and CD62L-positive T-cell lines from HLA-compatible donors: high cytotoxic potential against AML and ALL cells

OBJECTIVE: Identification of a clinical grade method for the ex vivo generation of donor-derived T cells cytotoxic against both myeloid and lymphoblastic cells still remains elusive. We investigated rapid generation and expansion of donor derived-allogeneic T-cell lines cytotoxic against patient leukemic cells. MATERIALS AND METHODS: Acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts were cultured 5 days in Stem Span, granulocyte macrophage colony-stimulating factor, interleukin-4, and calcium ionophore. All B-precursor ALL (N22) and AML (N13), but not T-cell ALL (N3), differentiated into mature leukemia-derived antigen-presenting cells (LD-APC). All but one LD-APC generated cytotoxic T lymphocyte (CTL) from adult human leukocyte antigen (HLA)-identical (N8) or unrelated donors (N2). RESULTS: Upon in vitro culture, donor-derived CTL acquired a memory T phenotype, showing concomitant high CD45RA, CD45RO, CD62L expression. CD8(+) cells, but not CD4(+) cells, were granzyme, perforine, and interferon-gamma-positive. Pooled CD4(+) and CD8(+) cells were cytotoxic against leukemic blasts (32%, 30:1 E:T ratio), but not against autologous or patient-derived phytohemagglutinin blasts. LD-APC from five ALL patients were used to generate CTL from cord blood. A mixed population of CD4(+) and CD8(+) cells was documented in 54% of wells. T cells acquired classical effector memory phenotype and showed a higher cytotoxicity against leukemia blasts (47%, 1:1 E:T ratio). Adult and cord blood CTL showed a skewing from a complete T-cell receptor repertoire to an oligo-clonal/clonal pattern. CONCLUSIONS: Availability of these cells should allow clinical trials for salvage treatment of leukemia patients relapsing after allogeneic stem cell transplantation.     

Peripheral decrease and pulmonary homing of CD4+CD45RO+ helper memory T cells in cystic fibrosis

Interstitial lung disease, although of prognostic impact for patients with cystic fibrosis (CF), remains difficult to assess without histopathologic investigations. As changes of peripheral blood lymphocyte subsets (LS) may accompany severe systemic lymphocyte immune responses, we compared peripheral LS of 44 patients with CF, 23 non-CF patients with recurrent pulmonary infections and 83 healthy controls (flow cytometry; CD3, CD19, CD16, CD56, CD4, CD8, CD11b, CD45RA, CD45RO, HLA-DR and CD25 antigens). Additional immunohistochemistry was performed on lung tissue of four CF patients aged 0.5, 12, 17 and 20 years, respectively. Patients with CF showed low absolute counts of CD4+CD45RO+ memory helperT cells, CD16+CD56+ NK cells, CD8+ and interleukin-2 receptor-positive T cells in peripheral blood (P < 0.001). Similar changes were registered in the non-CF patients with pulmonary infections, indicating that those were not specific for CF. Immunohistochemistry showed activation of bronchus-associated lymphoid tissue with interstitial accumulation of CD4+CD45 RO+ T cells in the three older patients. Patients with CF show marked changes of peripheral blood LS which are presumably not CF-specific and may mirror homing to lung tissue in the course of interstitial lung disease. Further research should evaluate its usefulness in monitoring progression of lung disease in CF.     

Expression of the leucocyte common antigen (LCA,CD45) isoforms RA and RO in acute haematological malignancies: possible relevance in the definition of new overlap points between normal and leukaemic haemopoiesis

Summary. The membrane expression of CD45RA and CD45RO on fresh leukaemic cells taken from 529 cases of acute haemopoietic malignancies, including 117 B-origin acute lymphoblastic leukaemia (B-origin ALL), 3 7 T-origin acute lymphoblastic leukaemia (T-origin ALL), 297 de novo acute myeloid leukaemia (AML), 42 refractory anaemia with excess of blasts in transformation (RAEB-T) and 36 myeloid blastic phase of chronic myelogenous leukaemia (CML-BP-my), was analysed. B-origin ALLs were characterized by the lack of the RO isoform along with the consistent presence of RA. Conversely, a differential expression of the two isoforms was detected in different subsets of T-origin ALL, in that T-stem cell leukaemias (T-SCL: CD7⁺, CD4^?, CD8^?, CD1^?) preferentially expressed CD45RA whereas conventional T-acute lymphoblastic leukaemias (T-ALL: CD7⁺, CD4⁺ and/or CD8⁺ and/or CD1+) were consistently marked by CD45RO. Within myeloid malignancies, most of AMLs displayed CD45RA, while a substantial group of CML-BP-my preferentially exhibited CD45RO. As a general rule, a reciprocal exclusion of the two isoforms was observed in AML as well as in ALL. Nevertheless, a frequent coexpression of CD45RA and CD45RO was observed in CD14⁺ AML. In vitro treatment with all-trans retinoic acid (ATRA) was able to promote a switch from CD45RA to CD45RO expression in 2 7 de novo AML, independently from morphological subtyping. To our knowledge, this is the first report on CD45 isoform expression in a large series of patients with acute leukaemia. The knowledge of the differential expression of CD45RA and CD45RO can ameliorate our classificative approach to haematological malignancies, as well as disclose new multiple overlap points between normal and leukaemic cell differentiation.