The phenotype and survival of antigen-stimulated transgenic CD4 T cells in vivo: the influence of persisting antigen

Naive and primed/memory CD4 T cells are distinguished by changes in the expression of activation/adhesion molecules that correspond with an altered function. Adoptively transferred TCR transgenic (tg) CD4 T cells specific for ovalbumin peptide (OVA-pep) were analysed for changing phenotype and the speed of change in vivo following antigen challenge with alum-precipitated (ap) OVA-pep, a conjugate that stimulated a Th2-type cytokine response. The change of CD45RB in relation to number of divisions showed that the transition from CD45RB(hi) (naive) to CD45RB(low) (primed/memory) was incremental; with each cell cycle the number of CD45RB(hi) molecules on the cell surface was diluted by approximately half and replaced by the low-weight isoform. Similarly, the change to CD44(hi) expression increased gradually during four rounds of proliferation. The loss of CD62L expression occurred early and was independent of cell division. CD69 was up-regulated quickly within 1-2 cycles, but down-regulated after about seven divisions. The expression of CD49d was not altered during the early rounds of division, although it was up-regulated on 30-60% of tg T cells dividing repeatedly (>or=8 cycles). When analysed on day 3 following stimulation, CD25 was no longer up-regulated. The intra-peritoneal injection of ap-OVA-pep stimulated tg T cells in the spleen and mesenteric lymph node one day in advance of those in more distant peripheral lymph nodes. Evidence indicated that residual antigen persisted for at least 4 weeks and was able to stimulate naive tg T cells. However, residual antigen had no net effect on extending or reducing survival of the transferred population.     

CD45R CD4 T cell subset-reconstituted nude rats: subset-dependent survival of recipients and bi-directional isoform switching.

High and low molecular weight variants (CD45R) of the leukocyte common antigen (CD45) divide CD4 T helper cells into subpopulations which display distinct characteristics. In vitro and in vivo evidence suggested that the presence of the high molecular weight splice variants CD45RA or CD45RB distinguished naive CD4 T cells from memory T cells which underwent an irreversible switch to the low molecular weight isoform as a consequence of antigen encounter. In the rat monoclonal antibody MRC OX22 identifies an epitope on the CD45RB molecule. We investigated this proposed differentiation pathway by reconstituting athymic nude rats with highly purified OX22+ or OX22- CD4 T lymphocyte subsets obtained from the thoracic duct (TDL) of euthymic, congenic, allotype-marked donors. Injection of CD4+CD45RB- (45R-) cells ensured long-term survival of nude recipients; recipients of CD4+CD45R+ (45R+) cells died within 2-3 months of injection. Early after transfer (3-4 weeks) the progeny of both 45R+ and 45R- TDL were uniformly 45R-. With time (by 2 months) progeny of both parental types expressed the high molecular weight CD45RB isoform. Nude recipients of 45R- TDL always generated progeny a proportion of which bore the 45R+ phenotype; 3 months to 2 years post-injection, 30%-50% of the donor-derived CD4 T cells were 45R+, 45R- progeny isolated from primary recipients of either 45R- or 45R+ cells transferred into secondary nude recipients induced skin allograft rejection with equal effectiveness and also generated 45R+ offspring. The results indicated that CD4 T cell subsets switched between CD45R isoforms and that the change between high and low molecular weight expression was bi-directional. The splice variants apparently are not lineage or maturation markers, but rather identify CD4 T cells that exist transiently in different functional states.     

Identical expression of CD45R isoforms by CD45RC+ 'revertant' memory and CD45RC+ naive CD4 T cells.

Naive and memory CD4 T cells are frequently defined by exon-specific monoclonal antibodies (mAb) which stain (or not) high- or low-molecular-weight (MW) isoforms of the leucocyte common antigen CD45. The link between isoform and the naive/memory designation is complicated by the fact that CD4 T cells with a 'memory' phenotype (CD45RA-, RB-, RC-, or CD45RO+) may revert ('revertants') and re-express the high mw isoform (CD45RA+, RB+, RC+). Isoform expression also changes during normal T-cell development. Furthermore, the picture may be incomplete since an exon-specific mAb will not detect all possible isoforms on a cell. We have used molecular techniques to determine whether revertant CD4 memory T cells were different from naive T cells with respect to CD45R isoform expression. Using the anti-CD45RC mAb OX22 to purify rat lymphocyte subsets, CD45R isoform expression was examined at the mRNA level in CD4 T cells at different stages of development and compared with that of B cells and unseparated lymphocytes. B cells contained abundant message for the highest MW 3-exon isoform ABC, the 2-exon isoforms AB and BC, and the null isoform O. Both immature CD45RC- (i.e. CD4+8- 'single positive' thymocytes, and peripheral Thy-1+ recent thymic emigrants) and mature CD45RC- 'antigen-experienced' CD4 T cells had message for single-exons B, possibly C and for the O exon. In contrast, CD45RC+ CD4 T cells contained mRNA coding for ABC (low level), AB, BC, B, C (low level) and O (low level). Importantly, there was no difference between CD45RC+ T cells that had not seen antigen ('truly native') and CD45RC+ antigen-experienced revertant memory T cells. This observation has implications for understanding long-term immunological memory.     

Reactivity of naive CD4+CD25- T cells against gut microflora in healthy mice

We have previously shown that conventional as well as germ-free CD4+ T cells depleted of CD25+ cells from the gut-associated lymphoid tissue and the periphery proliferate specifically in response to enterobacterial antigen exposure whereas unfractionated CD4+ T cells are not reactive under these conditions. Here we show that the majority of the enteroantigen-specific CD4+ CD25- T cells are naive cells expressing a CD45RB high, CD62L high and CD44 low phenotype. These cells are also present in the thymus and data from adult thymectomized mice show that they represent late (>6 weeks) thymic emigrants. Upon enteroantigen activation, the CD4+ CD25- T cells secrete IL-4, IL-5, IL-10, granulocyte macrophage colony-stimulating factor, tumor necrosis factor-alpha and IFN-gamma. Clonotype mapping of the TCRBV regions 1-18 of enteroantigen-reactive CD4+ CD25- T cells by TCR clonotype mapping revealed the polyclonal nature of this subset. In conclusion, we have for the first time demonstrated the presence of an evolutionary, functionally conserved subset of CD4+ T cells, which are reactive against enterobacterial antigens. This subset resides both in the thymus and the periphery; it is not dependent on previous antigen experience and represents late thymic emigrants, which by enteroantigen-induced activation express a mixed Th 1-Th 2 phenotype. At homeostatic conditions, CD25+ T cells maintain peripheral tolerance in this CD4+ T cell subset.     

Unusual phenotype of intestinal intraepithelial lymphocytes in the rat: predominance of T cell receptor alpha/beta+/CD2- cells and high expression of the RT6 alloantigen.

The phenotype of intraepithelial lymphocytes (IEL) from the small intestine of adult rats was studied by flow cytometry. Using appropriate monoclonal antibodies the expression patterns of the T cell receptor alpha/beta (TcR2), CD2, the alloantigen RT6 and several other T cell antigens were analyzed. The vast majority of rat IEL expressed TcR2 which is in contrast with data reported for the mouse. The comparison of IEL with lymph node cells revealed major phenotypic differences. Whereas CD2 was present on virtually all lymph node T cells it was found on only less than 5% of IEL. The T cell-specific differentiation antigen RT6 present on only a fraction of lymph node cells was found on about 99% of IEL demonstrating uniform expression with an approximately tenfold higher density. Identity of the detected molecule with RT6 was proven by using congenic controls and by the demonstration of phosphatidylinositol linkage to the IEL membrane. About 86% of IEL expressed CD8 but a substantial proportion of these cells co-expressed the CD4 molecule (34%). Two-color analysis revealed that the CD4+CD8+ double-positive subset completely lacked CD45RB suggesting that they represent memory cells. In the CD4-CD8+TcR2+ subset there was a remarkable heterogeneity of CD5 expression. A substantial number of these cells did not express CD5 despite high density of TcR2. Phenotypic peculiarities found on all or most IEL such as the lack of CD2 and the increased expression of RT6 indicate that the intestinal epithelial environment exerts strong effects on the development and maturation of these cells.     

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.     

Evolution of CD4 T-cell subsets following infection of naive and memory immune mice with Mycobacterium tuberculosis.

We report here that during the course of an experimental infection of mice with Mycobacterium tuberculosis, the differential expression of the cell surface antigens CD44 and CD45RB could be used to delineate CD4+ T cells into four phenotypically distinct subsets. The major subset present was designated CD44lo/CD45RBhi and is associated with naive or resting T cells. The three remaining subsets expressed increased levels of the CD44 antigen as the infection progressed and could therefore be considered to be in an activated state. These activated populations could be further divided on the basis of their variable expression of the CD45RB antigen. These populations were designated CD44hi/CD45RBhi, CD44hi/CD45RBlo, and CD44hi/CD45RBneg. Kinetic studies of the emergence of these populations indicated that these subsets arose sequentially from the naive population at times associated with the peak expression of acquired specific resistance. In further studies, in an attempt to associate either the CD44hi/CD45RBlo or the CD44hi/CD45RBneg population with acquired immunologic memory of tuberculosis infection, draining lymph nodes of challenged memory immune animals were analyzed for the accumulation of the CD4+ subsets. The accumulation of both the CD44hi/CD45RBlo and the CD44hi/CD45RBneg populations was observed, but the CD44hi/CD45RBlo population was enriched in a manner consistent with the rapid accumulation of memory T cells during the anamnestic response. While functional roles for each of these subsets remain to be determined, these data provide the first evidence for the evolution of multiple, phenotypically distinct CD4+ T-cell subsets during the in vivo response to an experimental mycobacterial infection.     

Antigen-specific T cell suppression by human CD4+CD25+ regulatory T cells

Anergic/suppressive CD4+CD25+ T cells have been proposed to play an important role in the maintenance of peripheral tolerance. Here we demonstrate that in humans these cells suppress proliferation to self antigens, but also to dietary and foreign antigens. The suppressive CD4+CD25+ T cells display a broad usage of the T cell receptor Vbeta repertoire,suggesting that they recognize a wide variety of antigens. They reside in the primed/memory CD4+CD45RO+CD45RB(low) subset and have short telomeres, indicating that these cells have the phenotype of highly differentiated CD4+ T cells that have experienced repeated episodes of antigen-specific stimulation in vivo. This suggests that anergic/suppressive CD4+CD25+ T cells may be generated in the periphery as a consequence of repeated antigenic encounter. This is supported by the observation that highly differentiated CD4+T cells can be induced to become anergic/suppressive when stimulated by antigen presented by non-professional antigen-presenting cells. We suggest that besides being generated in the thymus, CD4+CD25+ regulatory T cells may also be generated in the periphery. This would provide a mechanism for the generation of regulatory cells that induce tolerance to a wide array of antigens that may not be encountered in the thymus.     

T cell control of staphylococcal enterotoxin B (SEB) lethal sensitivity in mice: CD4+ CD45RB(bright)/CD4+ CD45RB(dim) balance defines susceptibility to SEB toxicity

Radiation chimeras, generated by transplantation of SCID bone marrow into C3H/HeJ mice, show lethal susceptibility to staphylococcal enterotoxin B (SEB), thus constituting a valid murine model for SEB shock. This SEB sensitivity is due to the ability of the irradiated host to restore residual T cell populations, since the SCID donor bone marrow is unable to generate T cells. SCID bone marrow transplanted into irradiated nude mice does not generate SEB-sensitive chimeras, as a consequence of the inability of the recipient nude mice to develop mature T cells. Thymectomy of normal recipient mice prior to bone marrow transplantation does not affect the development of susceptibility to SEB, suggesting that postthymic, residual T cells of the host probably mediate this SEB sensitivity. In vivo depletion experiments show that CD4+ T cells are required for the SEB-triggered shock, while CD8+ cells suppress it. A further examination of the T helper subpopulations in the SEB-sensitive mice reveals a prevalence of CD4+ CD45RB(dim) cells over CD4+ CD45RB(bright) cells. This T helper balance was statistically significant when correlated with SEB-induced mortality. Our model provides a possible explanation for the SEB resistance of normal mice: they have a prevalence of CD4+ CD45RB(dim) over CD4+ CD45RB(bright) cells.     

Human CD4+CD45R0+ and CD4+CD45RA+ T cells synergize in response to alloantigens.

Alloantigens, unlike recall antigens, activate both CD45RA+ (naive) and CD45R0+ (memory) CD4+ cells to the same extent. These T cell subsets may therefore interact with each other in response to alloantigens on transplanted grafts. We have investigated if the ability of activated CD4+CD45RA+ and CD4+CD45R0+ T cells to produce and respond to interleukin 2 (IL2) and IL4 may be involved in this interaction. After activation, both subsets up-regulate their IL2 receptor (IL2R) and IL4R expression, yet IL4 substantially enhanced the proliferation of the CD4+CD45RA+ but not of the CD4+CD45R0+ T cell subset, while IL2 increased the proliferation of CD4+CD45R0+ but not of the CD4+CD45RA+ T cells. Significantly, the CD4+CD45RA+ T cells synthesized two- to threefold more mRNA for IL2 than the CD4+CD45R0+ subset, while the CD4+CD45R0+ T cells synthesized mRNA for IL4 and interferon-gamma exclusively. The addition of IL2 to alloactivated CD4+CD45R0+ T cells further up-regulated their production of all three lymphokine mRNA; in contrast, IL4 induced an increase in mRNA for IL2 in only the alloactivated CD4+CD45RA+ subset. The reciprocity in the ability of both these CD4+ T cells to synthesize and respond to IL2 and IL4 may provide a rationale for the regulation of lymphokine interactions in vivo. Furthermore, the synergy between these subsets in response to alloantigens, which was directly quantitated by co-culturing CD4+CD45RA+ and CD4+CD45R0+ cells together prior to activation, may potentiate the alloreactivity against transplanted grafts in vivo.     

Progressive changes in CD45RB phenotype and lymphokine production by murine CD4+ T cells after alloantigen exposure.

Changes in CD45R expression correlate with changes in phenotype in mouse, rat and human T cells. It has been shown in mouse that CD45RB high T cells produce mostly interleukin-2 (IL-2) while CD45RB low T cells produce more IL-4 than IL-2 after mitogen stimulation in vitro. CD45RB expression also decreases when T cells are stimulated. In this study we compared responses of CD45RB high and low CD4+ T cells to alloantigens. Although a majority of unstimulated murine T cells from unimmunized mice are CD45RB high, we were able to isolate and purify sufficient numbers of T cells to study their response to alloantigens. When separated cells were stimulated in vitro with alloantigen the CD45RB high T cells became heterogeneous for their expression of CD45RB, indicating that high cells decrease their expression of CD45RB epitopes. Surprisingly, only CD45RB high T cells from unimmunized mice were alloreactive, as measured by proliferation and lymphokine secretion. In contrast, both CD45RB high and low populations from mice primed with allogeneic spleen were responsive to alloantigens. The 'primary' response of T cells from alloantigen-primed mice to third party stimulators is 10-fold greater in the CD45RB high population than in the CD45RB low, as would be predicted by our results in the primary mixed lymphocyte/leucocyte reaction (MLR). Furthermore, the response of CD45RB low T cells from unprimed mice and the response to third party alloantigen from primed mice was reconstituted by the addition of exogenous IL-2. The response of CD45RB low cells from primed mice was specific, as the third party alloresponsive cells were again primarily contained within the CD45RB high population. In the CD45RB high population in the secondary MLR we observed an increase in the production of IL-4 relative to IL-2.     

Core 2-containing O-glycans on CD43 are preferentially expressed in the memory subset of human CD4 T cells

Human CD4 T cells can be divided into two functionally distinct subsets: a CD45RO+ memory subset and a CD45RA+ naive subset. In an attempt to identify novel cell surface molecules on these cells, we have developed a mAb, anti-1D4. The antigen defined by anti-1D4 was preferentially expressed on the memory subset of freshly isolated peripheral CD4 T cells and 1D4+ CD4 T cells functionally corresponded to memory T cells. Retrovirus-mediated expression cloning revealed that the 1 D4 antigen is human CD43. Transfection of CHO-leu cells, which stably express human CD43, with core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT) conferred expression of the 1D4 antigen and mRNA of C2GnT was detected by RT-PCR only in 1D4+ T cells but not in 1D4- T cells, implying that the 1 D4 antigen is composed of core 2-containing O-glycans on CD43. Reactivity with anti-1 D4 was completely abolished when cells were treated with neuraminidase, while them remained weak binding of anti-T305, a previously described mAb which also reacts with CD43 modified with core 2-containing O-glycans. Moreover, anti-1D4 markedly reacted with NIH-3T3 cells expressing human CD43 and low levels of endogenous C2GnT, whereas anti-T305 reacted slightly. These results indicate that the 1D4 antigen is distinct from the epitope defined by anti-T305 and anti-1D4 is a more sensitive probe to detect core 2-containing O-glycans than anti-T305. Taken together, our results indicate that core 2-containing O-glycans, whose expression can easily be detected with anti-1D4, are preferentially expressed in the CD45RO+ memory subset of CD4 T cells.     

The CD27- subset of peripheral blood memory CD4+ lymphocytes contains functionally differentiated T lymphocytes that develop by persistent antigenic stimulation in vivo.

On the basis of expression of the T cell differentiation antigen CD27, human peripheral blood CD4+ memory cells can be divided into two subsets, a large CD45RA-CD27+ (82%) and a small CD45RA-CD27- (18%) population. Analysis of the functional properties of these memory T cell subsets showed that proliferative responses to the recall antigen tetanus toxoid (TT), shortly after booster immunization, were mainly confined to the CD27- population. Also, in atopic individuals, proliferative responses to allergens for which these individuals are sensitized, were limited to the CD45RA-CD27- population. After stimulation with CD3 monoclonal antibody and phorbol ester, CD27+ cells produced vast amounts of interleukin (IL)-2 but minimal amounts of IL-4, whereas in marked contrast, CD27- T cells secreted low levels of IL-2 and high levels of IL-4. The capacity of the vast majority of these latter cells to produce IL-4 was found to be a stable feature since high IL-4 secreting T cell clones were generated from the CD27- subset. These findings suggest that upon renewed as well as chronic antigenic stimulation in vivo, memory T cells acquire the CD45RA-CD27- phenotype and that, as a consequence, in this subset functionally differentiated CD4+ T cells are compartmentalized. Our results predict that analysis of the small CD27- subset of memory cells, that makes up approximately 10% of the peripheral blood T cell population, will provide information on the specificity and function of responding CD4+ T cells at a given point in time in healthy and diseased individuals.     

Functional characterization of the CD45R (2H4) molecule on CD8 (T8) cells in the autologous mixed lymphocyte reaction system

In the present study, we have investigated the molecular basis for the immunoregulatory function of CD8 cells after autologous mixed lymphocyte reaction (AMLR) activation. We demonstrated that the CD8+CD45R+, but not the CD8+CD45R- subset of cells effected suppression following AMLR activation. In contrast, cytotoxic activity against alloantigens resided in both the CD8+CD45R+ and CD8+CD45R- subsets of cells. Biochemical analysis showed that on CD8 cells, the 220-kDa isoform of the LCA/T200 antigen family was better represented than the 200-kDa isoform, when compared to CD4 cells. The density of the CD45R antigen increased on CD8 cells following activation in AMLR and treatment of AMLR-activated CD8 cells with either anti-CD45R antibody or anti-CD3 antibody abolished the suppressor function of these cells. In contrast, treatment of AMLR-activated CD4 cells with anti-CD45R, but not anti-CD3 antibody, abolished the suppressor/inducer function of these cells. The results suggest that the CD45R antigen as well as CD3 T cell receptor complex have an important role in the suppressor function of AMLR-activated CD8 cells.     

Age-associated increase in number of CD4+CD8+ intestinal intraepithelial lymphocytes in rats.

A significant number of CD4+CD8+ T cells were detected in intestinal intraepithelial lymphocytes (IEL) of various strains of rats including Wistar, WKA, BN, LEW and F344. The site of the CD4+CD8+ population in IEL increased with age in all strains we examined. Most IEL bearing CD8 expressed no CD5 antigen in young rats, while all CD4+CD8+ IEL and some of CD8+ IEL in aged rats were of CD5+CD45RB- phenotype. In germ-free Wistar rats, age-associated increase in the number of CD4+CD8+CD5+ IEL was not evident, indicating that stimulation by the intestinal microflora was important for expansion of the CD4+CD8+CD5+CD45RB- IEL. Aged athymic F344 nude rats contained appreciable numbers of CD4+ IEL and CD8+ IEL but few CD4+CD8+ IEL, suggesting that the CD4+CD8+ IEL may be derived from thymus-dependent populations. Unlike a majority of CD4+CD8+ thymocytes bearing a low intensity of CD3/T cell receptor (TcR) alpha/beta, the CD4+CD8+ T cells in IEL expressed a high intensity of CD3/TcR alpha/beta on their surface. The CD4+CD8+ IEL appear to contribute to the spontaneous proliferation of the IEL in aged rats as assessed by tritiated thymidine incorporation after in vitro culture with medium only. These results suggest that with aging a unique CD4+CD8+ IEL may expand at a local site of the intestine under the influence of intestinal microflora and may contribute to the first line of defense against various pathogens in the epithelium.     

Pertussis toxin as an adjuvant suppresses the number and function of CD4+CD25+ T regulatory cells

We observed a remarkable reduction in the frequency and immunosuppressive activity of splenic CD4+CD25+ T cells in C57BL/6 mice with MOG33-55-induced experimental autoimmune encephalomyelitis (EAE). Our study revealed that pertussis toxin (PTx), one component of the immunogen used to induce murine EAE, was responsible for down-regulating splenic CD4+CD25+ cells. Treatment of normal BALB/c mice with PTx in vivo reduced the frequency, suppressive activity and FoxP3 expression by splenic CD4+CD25+ T cells. However, PTx treatment did not alter the expression of characteristic phenotypic markers (CD45RB, CD103, GITR and CTLA-4) and did not increase the expression of CD44 and CD69 by the residual splenic and lymph node CD4+CD25+ T cells. This property of PTx was attributable to its ADP-ribosyltransferase activity. PTx did not inhibit suppressive activity of purified CD4+CD25+ T regulatory (Treg) cells in vitro, but did so in vivo, presumably due to an indirect effect. Although the exact molecular target of PTx that reduces Treg activity remains to be defined, our data suggests that alteration of both distribution and function of splenic immunocytes should play a role. This study concludes that an underlying cause for the immunological adjuvanticity of PTx is down-regulation of Treg cell number and function.     

Selective increase of activation antigens HLA-DR and CD38 on CD4+ CD45RO+ T lymphocytes during HIV-1 infection.

Infection with HIV results in a progressive depletion of CD4+ T cells and leads to significant in vivo lymphocyte phenotype changes. In this regard, the expression of HLA-DR and CD38 on CD8+ T cells has been shown to increase dramatically with disease progression. We investigated the expression of both activation markers on CD4+ T cells in HIV-1-infected subjects at different clinical stages of infection and compared the in vivo activation of CD4+ T cells with parameters of viral activity and CD8+ T cell activation. Fresh peripheral venous blood was obtained from 54 HIV-infected subjects and from 28 uninfected healthy controls. Three-colour immunophenotyping of the CD4+ T cell subset showed that the proportion of CD4+ T cells expressing HLA-DR (10% in HIV-negative controls) or CD38 (62% in HIV-negative controls) was higher in asymptomatic (P < 0.05 for CD38) and symptomatic (P < 0.001 for HLA-DR and CD38) HIV-infected subjects than in controls, whereas the proportion of CD4+ T cells expressing CD45RO (54% in controls) remained relatively unchanged. Simultaneous expression of HLA-DR and CD38 on CD4+ T cells increased from 2.3% in controls to 11% (P < 0.001) in asymptomatic and 22% (P < 0.001) in symptomatic HIV-infected subjects. This relative increase of CD38 and HLA-DR expression occurred mainly on CD4+ T cells co-expressing CD45RO. Changes in expression of HLA-DR and CD38 on CD4+ T cells correlated with similar changes on CD8+ T lymphocytes, with the presence of HIV antigen in the circulation, and with the disease stage of HIV infection.     

CD45RA antibodies split the CD3bright T cell subset.

Thymocyte subsets have been well characterized on the basis of CD4 and CD8 antigen expression. Recently, the use of anti-CD3 antibodies has allowed more precise phenotyping of these subsets. The most immature T cell precursors are largely CD3-CD4-CD8-, while the most mature are CD3brightCD4+CD8- or CD3brightCD4-CD8+. Moreover, the expression of CD45RA on thymocytes appears to define a progenitor population and may define a continuous lineage of cells. Using a panel of CD45RA antibodies, we have further characterized the CD45RA+ thymocyte population in the murine system. The size of this subset is greatly enhanced in cortisone-treated mice and in sublethally irradiated mice. Moreover, the CD45RA+ population is present early in foetal life and is maintained thereafter. Using three-colour immunofluorescence, we show that (i) while most CD45RA+ cells are present amongst the CD4-CD8- thymocyte subset in the normal thymus, after cortisone treatment or irradiation, all four thymocyte subsets co-express significant amounts of CD45RA. This suggests that not only progenitor cells but also the mature population which can survive such manipulation are CD45RA+; and (ii) a large proportion of CD45RA+ cells are CD3bright and this subset is represented in the thymus at all stages of maturation tested. These data suggest that a proportion of TCR-gamma delta + CD3+ cells in the fetus as well as of TCR-alpha beta+ CD3+ cells in the adult co-express CD45RA.     

CD26 induces T-cell proliferation by tyrosine protein phosphorylation.

CD26 antigen distribution among lymphoid cells and its participation in the process of lymphocyte activation and proliferation has been widely documented. However, the molecular and biochemical mechanisms coupled to the CD26 molecule are not yet known. With different monoclonal antibodies (mAb) we have detected that approximately 56% of CD4+ and 35% of CD8+ cells from peripheral blood lymphocytes express CD26 and the expression of this antigen is required for antigen- but not for mitogen-induced proliferation unless exogenous interleukin-2 (IL-2) is added to the culture. The stimulation of nylon wool-separated T cells and T-cell clones by the anti-CD26 mAb, 134-2C2, induced tyrosine phosphorylation on a subset of proteins of 50,000, 46,000, 26,000, 24,000 and 21,000 MW. This pattern of phosphorylation was not affected by the presence of 12-myristate 13-acetate (PMA), although this cofactor is required for CD26-mediated IL-2 mRNA expression and T-cell proliferation. When a specific tyrosine kinase inhibitor, Tyrphostin, was used in CD4+ cells cultures stimulated with 134-2C2 and PMA, the proliferation and the expression of IL-2 mRNA were inhibited. Thus, protein tyrosine phosphorylation seems to play a major role in CD26-mediated T-cell proliferation.