Superior specificity of anti-citrullinated peptide antibodies in patients with chronic lymphocytic leukemia and arthritis.

OBJECTIVES: Sera from patients with lymphoid neoplasias contain rheumatoid factors (RF) so often that RF are of limited use for diagnosing arthritis in lymphoma patients. Antibodies against citrullinated peptides (ACPA) might be helpful in distinguishing between true RA and rheumatoid factor-positive conditions with arthritis. We compared the specificity of RF and of ACPA for the diagnosis of RA in patients with B-cell chronic lymphocytic leukemia (CLL). METHODS: One hundred and seven patients with CLL without any clinical signs of arthritis and five patients with RA and concomitant CLL were included in the investigation. Serum samples were tested for RF-isotypes IgM, IgG and IgA. ACPA were determined with an ELISA that detects anti-cyclic citrullinated peptide (aCCP) antibodies. RESULTS: RF well beyond the cut-off levels were detected in 50% of the CLL patients without RA. The isotype distribution was 41% IgM-RF, 20% IgG-RF and 3% IgA-RF. None of the 107 CLL patients without arthritis had Accp antibodies. Within the whole cohort of CLL patients the specificity for the diagnosis of RA was 100% for aCCP antibodies and 59% for IgM-RF. CONCLUSIONS: Only aCCP antibodies but not IgM-, IgG- or IgA-RF are useful for the diagnosis of RA in patients with CLL.

Monitoring of cardiac function by serum cardiac troponin T levels, ventricular repolarisation indices, and echocardiography after conditioning with fractionated total body irradiation and high-dose cyclophosphamide.

OBJECTIVES: Highly differing rates of cardiac complications associated with high-dose cyclophosphamide (CY) have been reported, and only one clinical study has been performed on the cardiotoxic effects of CY monotherapy following total body irradiation (TBI). PATIENTS AND METHODS: We prospectively evaluated the potential cardiotoxic effects of conditioning with fractionated total body irradiation and high-dose cyclophosphamide (TBI/CY) by serial measurement of serum cardiac troponin T (cTnT), assessment of systolic and diastolic echocardiographic parameters and analysis of ventricular repolarisation indices (QT-dispersion and corrected QT-dispersion) in 30 adult patients with haematological malignancies undergoing haematopoietic stem cell transplantation. RESULTS: There was no evidence of pretreatment cardiac dysfunction in any patient. Although cTnT was determined serially for a median of 14 d after completion of conditioning, no elevated levels were observed. Echocardiographic parameters did not show any significant change at a median follow-up of 5 months except for one patient with evidence of impaired diastolic filling. No significant differences for mean values before and after high-dose CY were noted for ventricular repolarisation indices. Two patients had a significant increase in corrected QT-dispersion after CY without any other signs of cardiotoxicity. Congestive heart failure or arrhythmias were not observed. CONCLUSIONS: These data suggest that TBI/CY is safe with respect to cardiotoxicity in patients without pre-existing cardiac dysfunction. Hitherto unknown synergistic cardiotoxic effects of CY with other cytostatic drugs may constitute the major pathogenic factor of myocardial dysfunction after high-dose chemotherapy.

The role of CD40-CD40 ligand (CD154) interactions in immunoglobulin light chain repertoire generation and somatic mutation.

To determine whether CD40 ligation influences the molecular and selective mechanisms that govern the development of the human Ig light chain repertoire, analysis of the Vkappa and Vlambda repertoires of CD19+ B cells obtained from a patient with X-linked hyper IgM syndrome (XHIM) and a nonfunctional CD154 was carried out. The nonproductive Vkappa and Vlambda repertoires were largely comparable to that of the normals with respect to V gene and J segment distribution as well as CDR3 length and VLJL joint complexity. Comparison of the nonproductive and productive repertoires indicated that a limited number of VL genes were positively and negatively selected in the XHIM patient. Although mutations were observed in the XHIM VL repertoires, the frequency of mutations was significantly lower than in normals. Typical targeting of these mutations into RGYW/WRCY motifs was significantly reduced and subsequent selection of RGYW/WRCY mutations, which is normally observed, was not found. These results indicate that CD40 ligation is not required for generation of the light chain repertoire, positive selection of some Vk rearrangements, negative selection of specific VL genes, and some degree of somatic mutation. Importantly, however, targeting of mutations to RGYW/WRCY motifs and subsequent selection of these mutated motifs does not occur in the absence of CD40 ligation.

The influence of CD40-CD154 interactions on the expressed human V(H) repertoire: analysis of V(H) genes expressed by individual B cells of a patient with X-linked hyper-IgM syndrome.

Analysis of the V(H)DJ(H) repertoire of peripheral blood IgM(+) B cells from a patient with X-linked hyper-IgM syndrome (X-HIgM) was undertaken to determine whether the distribution of V(H) families in the productive repertoire might be regulated by in vivo CD40-CD154 interactions. The distribution of V(H) genes in the non-productive repertoire of IgM(+) B cells was comparable in X-HIgM and normals. Unlike the normal productive V(H) repertoire, however, in the X-HIgM patient the V(H)4 family was found at almost the same frequency as the V(H)3 family. This reflected a diminution in the positive selection of the V(H)3 family observed in normals and the imposition of positive selection of the V(H)4 family in the X-HIgM patient. Unique among the V(H)3 genes, V(H)3-23/DP-47 was positively selected in both normals and the X-HIgM patient. No major differences in the usage of J(H) or D segments or the complementarity-determining region (CDR) 3 were noted, although the foreshortening of the CDR3 noted in the mutated V(H) rearrangements of normals was absent in the X-HIgM patient. Finally, a minor degree of somatic hypermutation was noted in the X-HIgM patient. These results have suggested that specific influences on the composition of the V(H) repertoire in normals require CD40-CD154 interactions.

Gamma-glutamyl transpeptidase is up-regulated on memory T lymphocytes.

The ectoenzyme gamma-glutamyl transpeptidase (GGT) hydrolyzes glutathione (GSH), is required for the maintenance of normal intracellular GSH levels and modifies the activity of GSH-containing adducts. Previous data suggested that this enzyme was present on mitogen-activated T lymphocytes. However, the level of GGT protein expression on human mononuclear cell subsets has not been determined. A novel mAb to human GGT, 3A8, was developed. 3A8 was used to show that the expression of GGT is, in fact, highest on resting T cells that express markers of the memory phenotype, specifically CD45RO and decreased expression of CD45RB. The peripheral blood of patients with rheumatoid arthritis was found to have expanded numbers of T cells expressing levels of GGT up to 10-fold higher than controls. In addition, the CD4(+) T cell subset with the capacity to migrate across a human endothelial cell monolayer expresses high GGT levels. GGT expression was up-regulated on peripheral blood T cells following activation in vitro by either superantigen, phorbol ester, or IL-15, a stimulatory cytokine synthesized in rheumatoid synovium. Resting peripheral blood T cells that express GGT have higher levels of intracellular thiols than those that do not. These observations suggest that GGT may play an important role in the regulation of lymphocytes that are at a particular developmental stage.

Activated T cells acquire endothelial cell surface determinants during transendothelial migration.

Activated T cells acquire endothelial cell (EC) plasma membrane constituents during transendothelial migration. This was assessed using an in vitro model system in which human peripheral blood CD4+ T cells migrated through confluent monolayers of HUVEC. Flow cytometry of migrated CD4+ T cells demonstrated that activated, but not resting, T cells acquired a variety of endothelial surface determinants, including CD31, CD49d, CD54, CD61, and CD62E. The extracellular domains of these molecules were detected on migrated T cells with mAbs, including those directed to the ligand-binding regions. A number of approaches were employed to document that the acquisition of these molecules was uniquely accomplished by activated T cells and clearly involved transfer from both resting and TNF-alpha-activated EC. Acquisition of endothelial markers by activated T cells occurred as part of the transfer of membrane components, as migrating T cells acquired EC membranes prelabeled with the lipophilic dye, 3,3'-dihexadecyloxacarbocyanine perchlorate (DiOC-16), along with EC surface proteins. Thus, during transendothelial migration, activated T cells acquire endothelial membrane components, and as a result may deliver them to perivascular sites.

Similar characteristics of the CDR3 of V(H)1-69/DP-10 rearrangements in normal human peripheral blood and chronic lymphocytic leukaemia B cells.

The variable heavy chain (V(H)) gene segment V(H)1-69/DP-10 has been shown to be over-represented in B-cell chronic lymphocytic leukaemia (CLL). Because of certain similar characteristics of their complementarity determining region 3 (CDR3), including preferential utilization of J(H)6 elements and an extended length, it has been suggested that antigenic stimulation might be involved in leukaemogenesis. Utilizing single-cell PCR to amplify and sequence genomic DNA from individual normal human peripheral blood B cells, we have obtained 7/421 productively and 1/69 nonproductively rearranged V(H) genes that used V(H)1-69/DP-10. All productive rearrangements were unmutated, used J(H)6 and had an average CDR3 length similar to that previously found in V(H)1-69/DP-10-expressing CLL cells. These results suggest that CLL may arise from B cells commonly found in the peripheral B-cell repertoire and do not represent expansion of a unique subset of specific antigen-reactive B cells.

Computerized polymorphic marker identification: experimental validation and a predicted human polymorphism catalog.

A computational system for the prediction of polymorphic loci directly and efficiently from human genomic sequence was developed and verified. A suite of programs, collectively called POMPOUS (polymorphic marker prediction of ubiquitous simple sequences) detects tandem repeats ranging from dinucleotides up to 250 mers, scores them according to predicted level of polymorphism, and designs appropriate flanking primers for PCR amplification. This approach was validated on an approximately 750-kilobase region of human chromosome 3p21.3, involved in lung and breast carcinoma homozygous deletions. Target DNA from 36 paired B lymphoblastoid and lung cancer lines was amplified and allelotyped for 33 loci predicted by POMPOUS to be variable in repeat size. We found that among those 36 predominately Caucasian individuals 22 of the 33 (67%) predicted loci were polymorphic with an average heterozygosity of 0.42. Allele loss in this region was found in 27/36 (75%) of the tumor lines using these markers. POMPOUS provides the genetic researcher with an additional tool for the rapid and efficient identification of polymorphic markers, and through a World Wide Web site, investigators can use POMPOUS to identify polymorphic markers for their research. A catalog of 13,261 potential polymorphic markers and associated primer sets has been created from the analysis of 141,779,504 base pairs of human genomic sequence in GenBank. This data is available on our Web site (pompous.swmed.edu) and will be updated periodically as GenBank is expanded and algorithm accuracy is improved.

Pairing of variable heavy and variable kappa chains in individual naive and memory B cells.

A functional Ig consists of two heterodimers each of which is composed of a heavy and a light chain. Although there is increasing knowledge about the events that govern the rearrangement of the genes encoding each individual chain, only very limited information is available about the mechanisms governing the pairing of variable heavy (V(H)) and variable light (V(L)) chains. Using a single cell PCR, we were able to obtain V(H) and Vkappa chains from 144 individual human CD19+/IgM+ B cells. Pairing of specific V(H) or Vkappa families was not observed, nor was the length or the amino acid composition of the CDR3s of V(H) and Vkappa chains in individual B cells similar. Comparison of V(H) and Vkappa genes in B cells in which one or both contained evidence of somatic hypermutation with those with no mutations revealed a significant decrease in the mean length of the V(H) CDR3. Moreover, there was a significant correlation between the frequencies of mutations in V(H) and Vkappa gene pairs in individual B cells. These results indicate that Ag-mediated selection as opposed to V(H)DJ(H) recombination or subsequent Ig chain pairing tended to approximate the CDR3 lengths and the frequency of mutations of V(H) and Vkappa in individual B cells.

Interleukin 15 is produced by endothelial cells and increases the transendothelial migration of T cells In vitro and in the SCID mouse-human rheumatoid arthritis model In vivo.

The capacity of endothelial cells (EC) to produce IL-15 and the capacity of IL-15 to influence transendothelial migration of T cells was examined. Human umbilical vein endothelial cells expressed both IL-15 mRNA and protein. Moreover, endothelial-derived IL-15 enhanced transendothelial migration of T cells as evidenced by the inhibition of this process by blocking monoclonal antibodies to IL-15. IL-15 enhanced transendothelial migration of T cells by activating the binding capacity of the integrin adhesion molecule LFA-1 (CD11a/CD18) and also increased T cell motility. In addition, IL-15 induced expression of the early activation molecule CD69. The importance of IL-15 in regulating migration of T cells in vivo was documented by its capacity to enhance accumulation of adoptively transferred human T cells in rheumatoid arthritis synovial tissue engrafted into immune deficient SCID mice. These results demonstrate that EC produce IL-15 and imply that endothelial IL-15 plays a critical role in stimulation of T cells to extravasate into inflammatory tissue.

Comparable impact of mutational and selective influences in shaping the expressed repertoire of peripheral IgM+/CD5- and IgM+/CD5+ B cells.

Somatic hypermutation and subsequent selection play a significant role in shaping the peripheral B cell repertoire. This repertoire is composed of CD5+ (5%) and CD5- B cells (95%) which are known to traffic through different lymphoid compartments. Previous studies have shown that V(H) gene usage by CD5+ and CD5- B cells is similar, although mutations are more frequent in the latter. However, the effect of mutation and subsequent selection on the expressed V(H) repertoire of CD5+ and CD5- B cells has not been delineated in detail. This study, therefore, analyzed the mutational pattern of individual IgM+/CD5+ and IgM+/CD5- B cells. In both populations, mutations can occur without heavy chain isotype switching. Despite the differences in mutational frequency, the patterns of mutation and subsequent selection were comparable in CD5+ and CD5- B cells. These results imply that although mutations are more frequent in CD5- B cells, the overall mechanisms governing somatic hypermutation and subsequent positive and negative selection are similar in CD5+ and CD5- B cells.

Delineation of selective influences shaping the mutated expressed human Ig heavy chain repertoire.

After Ag exposure, somatic hypermutation and subsequent selection play significant roles in shaping the peripheral B cell repertoire. However, the disparate impact of each process has not been completely delineated. To address this, the mutational patterns of a large panel of productive V(H)DJ(H) rearrangements of individual human B cells were analyzed and compared with those of a previously reported panel of nonproductive V(H)DJ(H) rearrangements. The productive V(H) rearrangements exhibited a significantly lower mutational frequency and a significantly smaller number of replacement mutations than the nonproductively rearranged genes, suggesting that structural constraints of the Ig molecule and selective influences both impacted the repertoire, militating against replacement mutations. Positive selection favored a mean of four to six replacements in complementarity-determining region 1 (CDR1) and CDR2, and less than two replacements in the framework regions (FRs). In contrast, the negative impact of replacement mutations generated an increased number of silent mutations within both the CDRs and FRs of the productive repertoire accompanied by a net increase in the ratio of replacement to silent mutations in the CDRs compared with that in the FRs. Moreover, there was a negative influence on the distribution of amino acid changes resulting from mutations of highly mutable codons, such as AGY, TAY, GTA, and GCT, preferentially leading to conservative changes in the expressed Ig repertoire. The results are consistent with the conclusion that the expressed repertoire is limited, compared with the potential generated by the mutational machinery, by the dual requirements of avoiding autoreactivity and satisfying structural constraints of an intact Ig molecule.

Analysis of the human VH gene repertoire. Differential effects of selection and somatic hypermutation on human peripheral CD5(+)/IgM+ and CD5(-)/IgM+ B cells.

To analyze the immunoglobulin repertoire of human IgM+ B cells and the CD5(+) and CD5(-) subsets, individual CD19(+)/ IgM+/CD5(+) or CD5(-) B cells were sorted and non-productive as well as productive VH gene rearrangements were amplified from genomic DNA and sequenced. In both subsets, the VH3 family was overrepresented largely as a result of preferential usage of a small number of specific individual family members. In the CD5(+) B cell subset, all other VH families were found at a frequency expected from random usage, whereas in the CD5(-) population, VH4 appeared to be overrepresented in the nonproductive repertoire, and also negatively selected since it was found significantly less often in the productive compared to the nonproductive repertoire; the VH1 family was significantly diminished in the productive rearrangements of CD5(-) B cells. 3-23/DP-47 was the most frequently used VH gene segment and was found significantly more often than expected from random usage in productive rearrangements of both CD5(+) and CD5(-) B cells. Evidence for selection based on the D segment and the JH gene usage was noted in CD5(+) B cells. No differences were found between the B cell subsets in CDR3 length, the number of N-nucleotides or evidence of exonuclease activity. Somatically hypermutated VHDJH rearrangements were significantly more frequent and extensive in CD5(-) compared to CD5(+) IgM+ B cells, indicating that IgM+ memory B cells were more frequent in the CD5(-) B cell population. Of note, the frequency of specific VH genes in the mutated population differed from that in the nonmutated population, suggesting that antigen stimulation imposed additional biases on the repertoire of IgM+ B cells. These results indicate that the expressed repertoire of IgM+ B cell subsets is shaped by recombinational bias, as well as selection before and after antigen exposure. Moreover, the influences on the repertoires of CD5(+) and CD5(-) B cells are significantly different, suggesting that human peripheral blood CD5(+) and CD5(-) B cells represent different B cell lineages, with similarities to murine B-1a and B-2 subsets, respectively.

Molecular mechanisms and selective influences that shape the kappa gene repertoire of IgM+ B cells.

To analyze the human kappa chain repertoire and the influences that shape it, a single cell PCR technique was used that amplified Vkappa Jkappa rearrangements from genomic DNA of individual human B cells. More than 350 productive and 250 nonproductive Vkappa Jkappa rearrangements were sequenced. Nearly every functional Vkappa gene segment was used in rearrangements, although six Vkappa gene segments, A27, L2, L6, L12a, A17, and O12/O2 were used preferentially. Of these, A27, L2, L6, and L12a showed evidence of positive selection based on the variable region and not CDR3, whereas A17 was overrepresented because of a rearrangement bias based on molecular mechanisms. Utilization of Jkappa segments was also nonrandom, with Jkappa1 and Jkappa2 being overrepresented and Jkappa3 and Jkappa5 underrepresented in the nonproductive repertoire, implying a molecular basis for the bias. In B cells with two Vkappa Jkappa rearrangements, marked differences were noted in the Vkappa segments used for the initial and subsequent rearrangements, whereas Jkappa segments were used comparably. Junctional diversity was generated by n-nucleotide addition in 60% and by exonuclease trimming in 75% of the Vkappa Jkappa rearrangements analyzed. Despite this large degree of diversity, a strict CDR3 length was maintained in both productive and nonproductive rearrangements. More than 23% of the productive rearrangements, but only 7% of the nonproductive rearrangements contained somatic hypermutations. Mutations were significantly more frequent in Vkappa sequences derived from CD5- as compared with CD5+ B cells. These results document that the gene segment utilization within the Vkappa repertoire is biased by both intrinsic molecular processes as well as selection after light chain expression. Moreover, IgM+ memory cells with highly mutated kappa genes reside within the CD5- but not the CD5+ B cell compartment.

Analysis of the frequency and pattern of somatic mutations within nonproductively rearranged human variable heavy chain genes.

Somatic hypermutation plays an essential role in avidity maturation of Ab. To characterize the effects of hypermutation without the imposed bias of Ag-mediated selection, the mutational pattern of 37 nonproductively rearranged VH genes amplified from individual human B cells was analyzed. A high frequency of mutations as well as frequent replacement mutations were observed in the complementarity-determining regions (CDR) and in the framework regions of nonproductive VHDJH rearrangements. Comparison with 57 productive VH rearrangements indicated that replacement mutations, especially those occurring in the framework regions, were less frequent in productively rearranged VH genes, suggesting that they were deleted from the expressed repertoire. A number of factors contributed to the nonrandom localization of mutations, including: the targeting of specific motifs, such as AGY, GCY, GTA, TAY, and RGYW; an increased frequency of some commonly mutated motifs in the CDRs; and an apparent increased likelihood of mutations of CDR nucleotides. Each of these appeared to bias the mutational machinery, resulting in an increased frequency of replacement mutations in the CDRs of nonproductive VH rearrangements.

Expression of the beta 7 integrin by human endothelial cells.

Integrin adhesion receptors mediate fundamental intercellular interactions of many cell types as well as cellular interactions with specific extracellular matrix molecules. To date, the beta 7 integrin has been shown to be expressed by leukocyte subsets and to mediate interactions of these cells with extracellular matrix molecules as well as with endothelial and epithelial cells. The data presented here indicate that human endothelial cells also express the beta 7 integrin both in vitro and in situ. Analysis of cDNA indicated that endothelial beta 7 was identical to that expressed by leukocytes. Cell surface expression of beta 7 was increased by exposure of the endothelium to the pro-inflammatory cytokines, tumor necrosis factor-alpha and interleukin-1 beta. In leukocytes, beta 7 complexes with alpha 4 or alpha E integrin chains. Endothelial cells also expressed a number of alpha-integrin chains, including alpha 4, but not alpha E. The expression and utilization of beta 7, presumably complexed with alpha 4, by endothelial cells may be instrumental in the maintenance of the function or phenotype of endothelial cells.

Enrichment of differentiated CD45RBdim,CD27- memory T cells in the peripheral blood, synovial fluid, and synovial tissue of patients with rheumatoid arthritis.

OBJECTIVE: To delineate in greater detail the phenotype of T cells that reside in the synovial tissue (ST) and synovial fluid (SF) of patients with rheumatoid arthritis (RA), in order to determine their precise differentiation status, and to determine whether the accumulation of these specific T cell subsets in these synovial compartments could be related to their capacity for transendothelial migration. METHODS: Lymphocytes from normal subjects or from the peripheral blood (PB), ST, and/or SF of RA patients were phenotypically analyzed by flow cytometry. Normal PB CD4+ T cells were also characterized using an in vitro assay of transendothelial migration. RESULTS: ST and SF were found to be enriched with memory (CD45RA-,CD45RO+,CD11abright,CD44bright and activated (CD69+) T cells. Moreover, ST and SF cells from RA patients were enriched in differentiated CD4+,CD45RBdim,CD27- T cells, a subset of mature memory T cells that develops after prolonged antigenic stimulation. In addition, PB of some RA patients contained an increased number of CD4+,CD45RBdim,CD27- T cells. The CD4+,CD11abright,CD44bright memory T cells, which included the CD45RBdim,CD27- more mature memory cells, exhibited an enhanced capacity for transendothelial migration that is likely to contribute to their enrichment in the rheumatoid synovium. CONCLUSION: RA patients manifest an increased number of mature memory T cells in the SF and ST, and some also have an increased number of these cells in PB that is likely to reflect chronic antigenic stimulation. The enrichment of these cells in the SF and ST reflects, in part, an enhanced capacity to migrate from the vascular space into inflamed tissue.

Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction.

The VH gene repertoire of human peripheral B cells was analyzed using PCR analysis of individual blood B cells. Because genomic DNA of single B cells was analyzed, data from both productive and nonproductive VDJ rearrangements were obtained. Nine out of 75 B cells contained both functional and nonfunctional rearrangement products, whereas 62/75 had a single productive VDJ rearrangement. The distribution of VH families was ordered in accordance with the germline complexity, although a bias toward VH3 and some of its members was found. This bias was noted in both the productively and nonproductively rearranged repertoires, indicating that it resulted from molecular and not selective processes. Evidence for negative selection of certain VH3 and VH4 family members was noted in that they were found less often as productive than nonproductive VDJ rearrangements. In addition, evidence for positive selection based on CDR3 was obtained, in that JH6 and DXP'1 were found at a higher frequency in the productively compared with the nonproductively rearranged repertoire. The CDR3 segments were markedly heterogenous, with a much greater degree of variability noted in the nonproductive VDJ rearrangements. Finally, a much greater frequency of mutations was noted in the nonproductively rearranged VH genes within individual B cells. These results have begun to delineate the human peripheral B cell repertoire and identify processes that shape it both positively and negatively.

Phenotypic characterization of CD4+ T cells that exhibit a transendothelial migratory capacity.

The phenotype of CD4+ T cells capable of transendothelial migration was determined using an in vitro model system, in which cells migrate through a monolayer of endothelial cells (EC) on collagen gels. A specific subset of resting CD4+ memory T cells was found to migrate. T cells within this subset can be defined by the bright expression of CD11a, CD26, CD44, and CD49d. Additionally, the migratory CD4+ T cell population is largely CD58bright, CD31-, CD62L-, and is also enriched in cells that brightly express CD49c, CD49e, and CD49f. Only a minority of the cells are activated, as indicated by expression of CD69. The EC were found to play a central role in facilitating migration of this subset because selective enrichment of CD11abright, CD26bright, CD44bright, CD4+ T cells was not observed when cells migrated in the absence of EC. Activation of the T cells induced a modest degree of migration of an additional subset of CD45RA+, CD31+ naive T cells. In contrast, TNF-alpha activation of the EC increased the transendothelial migration of an additional subset of activated memory T cells that expressed CD69 and CD62L. Neither activation of the T cells, stimulation of the EC, nor the presence of macrophage inflammatory protein-1 alpha (MIP-1 alpha) or RANTES, however, altered the phenotype of the majority of the migratory CD4+ T cell population, which is characteristic of a particular stage of memory cell differentiation. These results suggest that CD4+ T cells acquire the capacity for transendothelial migration at a specific phase of maturation that is only minimally altered by the activation of either the T cell or the EC, or by the presence of specific chemokines in the subendothelial matrix.