Analysis of T-cell repertoire and mixed chimaerism in a patient with aplastic anaemia after allogeneic bone marrow transplantation

We analysed 26 T-cell receptor (TCR) beta chain subfamilies (VB) of a patient with aplastic anaemia (AA) who underwent allogeneic bone marrow transplantation (allo-BMT). The patient developed pancytopenia at d 80. The patient's T cells were skewed in 10 of 26 TCR-VB on d 83. These TCR-VB, especially VB15, which were almost entirely CD8-positive cells, were skewed throughout her clinical course. Chimaerism analysis of the CD8-positive cells indicated that they were of recipient origin. Therefore, some immune responses induced by the recipient CD8-positive T cells had an important role in pancytopenia in AA patients after allo-BMT.     

Reconstitution of T-cell function after CD6-depleted allogeneic bone marrow transplantation

Patients who undergo allogeneic bone marrow transplantation (BMT) are clinically immunodeficient for a prolonged period after engraftment. In the present study, we examined immune function after BMT in a series of patients who had received HLA compatible sibling marrow grafts purged of T cells with anti-CD6 monoclonal antibody and complement. None of the patients in this analysis received immunomodulating agents and none had developed graft-versus-host disease (GVHD). Initially after BMT, natural killer (NK) cells are the predominant cell type, giving way to CD3+, CD5+ T cells after 4 to 8 weeks. Despite the return of normal numbers of T lymphocytes post-BMT phenotypic analysis reveals several long-term abnormalities, including an inverted T4:T8 ratio and a significant fraction of CD3+ T cells that do not co-express CD6. In mitogenic assays, stimulation by either nonspecific lectin (phytohemagglutinin; PHA) or antibodies to the CD2 surface structure (anti-T11(2) + anti-T11(3)) results in decreased levels of T-cell proliferation compared with controls for over 18 months post-BMT. In contrast, the ability of unstimulated peripheral blood mononuclear cells (PBMC) to respond to recombinant interleukin-2 (rIL-2) is relatively intact, most likely reflecting early functional reconstitution of the NK cell population. To further characterize the prolonged abnormalities in T-cell proliferation after PHA or CD2 stimulation, we examined more proximal events in T-cell activation such as induction of IL-2 receptor expression and stimulus-induced intracellular calcium flux. We found that the induction of IL-2 receptor (p55) after in vitro activation, although initially abnormal, recovers completely by 6 months post-BMT. We also found that, after CD2 stimulation, calcium flux in T cells was normal immediately after engraftment. In contrast, after stimulation with anti-CD3 antibodies, a large population of T cells do not develop intracellular calcium flux compared with controls. We conclude that despite the recovery of normal numbers of T lymphocytes early after engraftment of CD6-depleted marrow, these T cells exhibit several physiologic and functional abnormalities that persist for varying intervals post-BMT. At present, it is unclear which of these specific defects is most closely associated with increased susceptibility to infectious agents after BMT.     

CD3 zeta dependence of the CD2 pathway of activation in T lymphocytes and natural killer cells.

In T lymphocytes, signal transduction through the CD2 adhesion molecule requires surface expression of the CD3-Ti T-cell receptor (TCR) complex. In contrast, in natural killer (NK) cells, CD2 functions in the absence of a TCR. Because the TCR on T lymphocytes and the CD16 low-affinity IgG Fc receptor (Fc gamma receptor type III) complex on NK cells share a common CD3 zeta subunit, we reasoned that CD3 zeta may be critical for CD2 signaling in T lymphocytes and NK cells. Here we show that transfection of a cDNA encoding a transmembrane form of CD16 into TCR- variants of the Jurkat T-cell line results in CD16 expression in association with endogenous CD3 zeta homodimers and restores CD2 signaling function. To test directly the role of CD3 zeta in CD2 triggering, we then transfected human CD2 into each of two murine T-T hybridomas that express TCRs containing either a full-length CD3 zeta subunit or a truncated CD3 zeta subunit incapable of transducing signals. Despite expression of equivalent surface levels of TCR, CD2-mediated signaling is seen only in the T cells containing wild-type CD3 zeta. These findings show that (i) CD16 on NK cells is functionally equivalent to the TCR on T lymphocytes for coupling CD2 to signaling pathways and (ii) CD2 signal transduction depends upon the CD3 zeta subunit of the TCR complex and, by inference, the CD3 zeta subunit of the CD16 complex.     

Expression of HLA-C-specific natural killer cell receptors (CD158a and CD158b) on peripheral blood mononuclear cells after allogeneic bone marrow transplantation

We investigated the expression of natural killer cell receptors (NKRs) for HLA-C on peripheral blood mononuclear cells (PBMCs) in 23 allogeneic bone marrow transplantation (allo-BMT) patients to analyse the role of NKRs in alloresponse concerning graft-versus-host disease (GVHD). CD158a expression was low and there was little change in the expression after allo-BMT. Also, there was no difference in the proportion of CD158a+/CD3- after allo-BMT. In contrast, the proportion of CD158b+/CD3- cells, mainly NK cells, increased in the early stage (< 2 months) after allo-BMT and then gradually decreased (3.3 +/- 2.6% before BMT vs. 15.4 +/- 8. 6% in the early stage after BMT, 8.5 +/- 4.9% during the period 3-6 months after BMT and 7.0 +/- 3.0% > 6 months after BMT; P < 0.05). However, CD158b expression on CD3+ T cells increased 3 months after allo-BMT (1.1 +/- 1.1% before BMT vs. 5.1 +/- 7.7% during the period 3-6 months after BMT and 3.0 +/- 2.4% > 6 months after BMT, P < 0. 05). The highest percentages of CD158 expression in patients without chronic GVHD (cGVHD) and those with cGVHD were compared. The percentage of CD158b+/CD3+ cells and also that of CD158b+/CD8+ cells were significantly increased in patients with cGVHD compared with those in patients without cGVHD (2.6 +/- 2.0% vs. 8.0 +/- 11.2% and 2.3 +/- 1.5% vs. 8.3 +/- 11.7% respectively; P < 0.05). The exact clinical relevance of these CD158b-expressing cells is not clear. However, there is an interesting possibility that CD158b-expressing cells play some role in the regulation of GVHD after allo-BMT.     

T cells redirected against CD70 for the immunotherapy of CD70-positive malignancies

T-cell therapy with genetically modified T cells targeting CD19 or CD20 holds promise for the immunotherapy of hematologic malignancies. These targets, however, are only present on B cell-derived malignancies, and because they are broadly expressed in the hematopoietic system, their targeting may have unwanted consequences. To expand T-cell therapies to hematologic malignancies that are not B cell-derived, we determined whether T cells can be redirected to CD70, an antigen expressed by limited subsets of normal lymphocytes and dendritic cells, but aberrantly expressed by a broad range of hematologic malignancies and some solid tumors. To generate CD70-specific T cells, we constructed a chimeric antigen receptor (CAR) consisting of the CD70 receptor (CD27) fused to the CD3-ζ chain. Stimulation of T cells expressing CD70-specific CARs resulted in CD27 costimulation and recognition of CD70-positive tumor cell lines and primary tumor cells, as shown by IFN-γ and IL-2 secretion and by tumor cell killing. Adoptively transferred CD70-specific T cells induced sustained regression of established murine xenografts. Therefore, CD70-specific T cells may be a promising immunotherapeutic approach for CD70-positive malignancies.     

The expression of CD26 and CD40 ligand is mutually exclusive in human T- cell non-Hodgkin's lymphomas/leukemias

CD26 and CD40 ligand (CD40L) are surface molecules on human activated T lymphocytes that play a critical role in the regulation of lymphopoiesis. Both molecules are expressed on a restricted fraction of human T-cell non-Hodgkin's lymphomas (NHL)/leukemias; however, little is known about their functional and/or clinical significance in these disorders. In this study, the pattern of expression of CD40L was compared with that of the CD26 molecule. A series of 67 human T-cell NHL/leukemias and a panel of leukemia/lymphoma T-cell lines were evaluated by immunohistochemistry, flow cytometry, and RNA studies. The overall frequency of CD26+ and CD40L+ samples was rather similar (25/67 [37%] v 18/67 [27%]). However, the majority of CD26-expressing cases clustered in the lymphoblastic lymphomas (LBL)/T-acute lymphoblastic leukemias (ALL; 12/23) and CD30+ anaplastic large-cell (ALC) lymphomas (5/8), whereas CD40L+ lymphomas included a large fraction of mycosis fungoides (11/21 [52%]). CD26 and CD40L coexpression was found only in 2 myocosis fungoides cases and 1 small lymphocytic lymphoma. Thus, the expression of the two antigens was mutually exclusive in almost all T- cell lymphomas/leukemias. Accordingly, lymphoma cell lines expressed either one of the molecules or the relative amounts of CD26 and CD40L were inversely proportional. In contrast, reactive T lymphocytes from patients with non-neoplastic T-cell expansions and in vitro activated CD3+ or CD4+ normal T cells were found to coexpress CD40L and CD26. Results of a multivariate analysis showed that the expression of CD26 in T-cell LBL/ALL patients was associated to a worse outcome in terms of survival, as compared with patients with CD26- tumors (P < or = .0001). Based on our results, it can be concluded that, (1) as opposed to activated or reactive normal T cells, the expression of CD26 and of CD40L is mutually exclusive in human T-cell lymphomas/leukemias; (2) expression of CD26 is restricted to aggressive pathologic entities, such as T-cell LBL/ALL and T-cell CD30+ ALC lymphomas, whereas CD40L is expressed on slow progressing diseases such as mycosis fungoides; and (3) within the T-cell LBL/ALL group of tumors, CD26 may identify a subset of poor prognosis patients.     

Proline-rich tyrosine kinase-2 is critical for CD8 T-cell short-lived effector fate

T-cell interactions with antigen-presenting cells are important for CD8 T-cell effector or memory fate determination. The integrin leukocyte function-associated antigen-1 (LFA-1) mediates T-cell adhesion but the contribution of LFA-1–induced signaling pathways to T-cell responses is poorly understood. Here we demonstrate that proline-rich tyrosine kinase-2 (PYK2) deficiency impairs CD8 T-cell activation by synergistic LFA-1 and T-cell receptor stimulation. Furthermore, PYK2 is essential for LFA-1-mediated CD8 T-cell adhesion and LFA-1 costimulation of CD8 T-cell migration. During lymphocytic choriomeningitis virus infection in vivo, PYK2 deficiency results in a specific loss of short-lived effector CD8 T cells but does not affect memory-precursor CD8 T-cell development. Similarly, lack of LFA-1 primarily impairs the generation of short-lived effector cells. Thus, PYK2 facilitates LFA-1–dependent CD8 T-cell responses and promotes CD8 T-cell short-lived effector fate, suggesting that PYK2 may be an interesting therapeutic target to suppress exacerbated CD8 T-cell responses.     

Comparative analysis of T-cell costimulation and CD43 activation reveals novel signaling pathways and target genes

The CD43 lymphocyte surface receptor is involved in the regulation of lymphocyte adhesion and activation. Many CD43 functions remain controversial or unclear, and it is not known to which extent CD43 signaling pathways are shared with or distinct from those used by the T-cell receptor (TCR). Here, we systematically compared signaling events and target gene expression induced by CD43 or T-cell costimulation in primary human peripheral T cells. These studies identify nuclear factor-kappaB (NF-kappaB) p65 serine 468 as a novel inducible phosphorylation site strongly induced by T-cell costimulation and only weakly triggered by CD43 ligation. We also identified CD43 as a novel Jun N-terminal kinase (JNK) activator and a comprehensive analysis of further signaling events suggests that both stimuli use overlapping but also distinct signaling pathways. Microarray analysis of inflammatory genes shows 1 group of genes coregulated by both stimuli and 2 further groups of target genes affected solely by costimulation or primarily by CD43.     

Induction of nuclear contour irregularity during T-cell activation via the T-cell receptor/CD3 complex and CD2 antigens in the presence of phorbol esters

To determine whether specific T-cell activation pathways could produce nuclear contour irregularity in normal human lymphocytes, purified T cells were stimulated in vitro and subsequently analyzed by electron microscopy. The degree of nuclear contour irregularity was determined with the use of a computerized planimeter. Stimulation via the T-cell receptor (TCR)/CD3 complex using anti-CD3 monoclonal antibodies induced Sezary-like morphology (nuclear contour indices > 6.5) in a significant portion (9% to 28%) of T cells derived from different normal donors. T- cell activation via CD2 antigens showed induction of Sezary-like nuclear morphology in a lower percentage of cells in comparison with stimulation via the TCR/CD3 complex. In contrast, mitogenic stimulation in vitro did not induce alterations of nuclear morphology in T cells. Immunoelectron microscopy showed that nuclear contour irregularity induced in vitro did not correlate with surface-antigen expression of T- cell subpopulations. The results indicate that Sezary-like morphology is associated with cell activation in normal human T cells.     

Strong CD28 costimulation suppresses induction of regulatory T cells from naive precursors through Lck signaling

CD28 costimulation is required for the generation of naturally derived regulatory T cells (nTregs) in the thymus through lymphocyte-specific protein tyrosine kinase (Lck) signaling. However, it is not clear how CD28 costimulation regulates the generation of induced Tregs (iTregs) from naive CD4 T-cell precursors in the periphery. To address this question, we induced iTregs (CD25(+)Foxp3(+)) from naive CD4 T cells (CD25(-)Foxp3(-)) by T-cell receptor stimulation with additional transforming growth factorβ (TGFβ) in vitro, and found that the generation of iTregs was inversely related to the level of CD28 costimulation independently of IL-2. Using a series of transgenic mice on a CD28-deficient background that bears wild-type or mutated CD28 in its cytosolic tail that is incapable of binding to Lck, phosphoinositide 3-kinase (PI3K), or IL-2-inducible T-cell kinase (Itk), we found that CD28-mediated Lck signaling plays an essential role in the suppression of iTreg generation under strong CD28 costimulation. Furthermore, we demonstrate that T cells with the CD28 receptor incapable of activating Lck were prone to iTreg induction in vivo, which contributed to their reduced ability to cause graft-versus-host disease. These findings reveal a novel mechanistic insight into how CD28 costimulation negatively regulates the generation of iTregs, and provide a rationale for promoting T-cell immunity or tolerance by regulating Tregs through targeting CD28 signaling.     

Highly activated T-cell receptor AV2S3(+) CD4(+) lung T-cell expansions in pulmonary sarcoidosis

Sarcoidosis is a systemic disorder of unknown origin, primarily affecting the lungs. The granulomatous inflammation is driven by the interplay between various molecules and cells, including T cells. Previously, our group reported a close correlation between lung-restricted T-cell receptor (TCR) AV2S3 CD4-positive T-cell expansions and HLA-DR17 in active sarcoidosis. The aim of this study was to characterize phenotypically such AV2S3 lung T cells, to obtain more information about the state of activation of this intriguing T-cell subset. Bronchoalveolar lavage (BAL) was performed on sarcoidosis patients with active disease and on healthy control subjects (HC). The expression of activation and subset markers was evaluated and compared between BAL AV2S3-positive and AV2S3-negative T cells of patients with lung-restricted AV2S3 T-cell expansions, and between BAL and peripheral blood lymphocytes (PBL) of patients and HC. The frequency of cells expressing activation markers CD26, CD28, CD69, and HLA-DR was enhanced in AV2S3-positive versus AV2S3-negative BAL CD4(+) T-cell subsets. In contrast, CD25 (Il-2R) and CD27 were expressed at lower levels by the AV2S3-positive CD4(+) lung T cells. Our data confirm a substantial activation of BAL CD4(+) T cells of patients with sarcoidosis. Furthermore, the AV2S3 CD4-positive lung cells display a pattern of activation markers, suggesting that they are significantly more activated compared with lung CD4(+) T cells expressing other TCR V gene segments as well as compared with BAL CD4(+) T cells of HC. These results support our hypothesis of an ongoing and selective stimulation of AV2S3 T cells by a specific antigen and the participation of this subset in the inflammatory process in the lungs of patients with sarcoidosis.     

CD43 interferes with T-lymphocyte adhesion.

CD43 is a cell-surface sialoglycoprotein of uncertain physiologic function expressed to various degrees by most leukocytes. We tested whether or not CD43 participates in intercellular adhesion by comparing the binding of human T lymphocytes to transfected HeLa cells stably expressing CD43 and sham-transfected HeLa cells (CD43-negative). Significantly fewer T lymphocytes adhered to the CD43-positive HeLa cells than to the CD43-negative HeLa cells. Diminished T-cell adherence to the CD43-positive HeLa cells was seen for all T lymphocytes tested, irrespective of their source or derivation. Antibody-blocking experiments revealed that CD43 interference with T-cell adhesion largely represented interference with T-cell leukocyte function-associated antigen 1 binding to HeLa cell intercellular adhesion molecule 1. The CD43 anti-adhesion effect was not overcome by treating cells with phorbol 12-myristate 13-acetate, a chemical that increases the binding avidity of leukocyte function-associated antigen 1 for intercellular adhesion molecule 1. However, neuraminidase treatment of the HeLa cell transfectants diminished the CD43 antiadhesion effect. These data indicate that CD43 expression by opposing cells can interfere with cell-cell adhesion. The data also suggest that CD43 might regulate T-cell adhesion by interfering with leukocyte function-associated 1 binding to intercellular adhesion molecule 1, a major activation-induced adhesion pathway among lymphocytes.     

Internalization of CD26 by mannose 6-phosphate/insulin-like growth factor II receptor contributes to T cell activation

CD26 is a T cell activation antigen known to bind adenosine deaminase and have dipeptidyl peptidase IV activity. Cross-linking of CD26 and CD3 with immobilized mAbs can deliver a costimulatory signal that contributes to T cell activation. Our earlier studies revealed that cross-linking of CD26 induces its internalization, the phosphorylation of a number of proteins involved in the signaling pathway, and subsequent T cell proliferation. Although these findings suggest the importance of internalization in the function of CD26, CD26 has only 6 aa residues in its cytoplasmic region with no known motif for endocytosis. In the present study, we have identified the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR) as a binding protein for CD26 and that mannose 6-phosphate (M6P) residues in the carbohydrate moiety of CD26 are critical for this binding. Activation of peripheral blood T cells results in the mannose 6 phosphorylation of CD26. In addition, the cross-linking of CD26 with an anti-CD26 antibody induces not only capping and internalization of CD26 but also colocalization of CD26 with M6P/IGFIIR. Finally, both internalization of CD26 and the T cell proliferative response induced by CD26-mediated costimulation were inhibited by the addition of M6P, but not by glucose 6-phosphate or mannose 1-phosphate. These results indicate that internalization of CD26 after cross-linking is mediated in part by M6P/IGFIIR and that the interaction between mannose 6-phosphorylated CD26 and M6P/IGFIIR may play an important role in CD26-mediated T cell costimulatory signaling.     

CD56-positive peripheral T-cell lymphoma primarily presenting with tonsillar swelling

A 51-year-old woman was admitted to our hospital with tonsillar swelling. After tonsillectomy was performed, she was diagnosed as having CD56-positive T-cell lymphoma, mainly composed of small and medium-sized atypical cells. An immunohistochemical study showed that the malignant lymphocytes were positive for CD3, CD8, CD56, TIA-1 and granzyme B, while negative for CD20, CD5 and CD10. Flowcytometry demonstrated the lymphocytes were positive for CD56. Southern blot analysis revealed a rearrangement of the T-cell receptor gamma chain. The disease stage by Ann Arbor staging classification was II B. We provided MCEC therapy followed by autologous peripheral blood stem cell transplantation, and complete remission (CR) was achieved. Two months after CR, however, the patient relapsed with peritonitis due to perforation of an ileal tumor, and died of sepsis. It is rare for CD56-positive T-cell lymphoma to occur primarily in the tonsils. Because small bowel ulcers were revealed during the course of induction chemotherapy, we report a valuable case in which suspected CD56-positive enteropathy-type T-cell lymphoma (ETL) occurred primarily in the tonsils.     

Significant enlargement of a specific subset of CD3+CD8+ peripheral blood leukocytes mediating cytotoxic T-lymphocyte activity during human immunodeficiency virus infection.

We have obtained a monoclonal antibody termed BY55 that defines an 80-kDa cell-surface structure on a subset of circulating peripheral blood mononucleocytes. This structure, which was not detected on most cell lines or activated lymphocytes, was expressed exclusively on 15-25% of CD2+ circulating lymphocytes, including a major subset within the CD3- and the T-cell receptor gamma delta + lymphocytes and a small percentage of the CD3+CD8+ cells. Moreover, we have shown that the BY55 molecule delineated the competent killer circulating lymphocytes. In the present report, additional two- and three-color immunofluorescence studies of peripheral blood lymphocytes were done to precisely determine BY55 expression within the T-cell population. In normal individuals, peripheral blood CD3+CD8+BY55+ cells represented only 5-6% of the lymphocytes, and these cells possessed cytolytic activity. Interestingly, we found that the percentage of total BY55+ lymphocytes as well as the percentage of CD3+CD8+BY55+ was significantly increased in peripheral blood lymphocytes of human immunodeficiency virus-seropositive individuals.     

Activated human B lymphocytes express three CTLA-4 counterreceptors that costimulate T-cell activation.

Signaling via the T-cell receptor complex is necessary but not sufficient to induce antigen-specific T lymphocytes to expand clonally. To proliferate, T cells must receive one or more costimulatory signals provided by antigen presenting cells (APCs). One such critical costimulatory signal is delivered by the CD28/CTLA-4 counterreceptor, B7, expressed on APCs. B7 costimulation induces CD28 signaling, resulting in interleukin 2 (IL-2) secretion, and T-cell proliferation. Conversely, T-cell receptor signaling in the absence of B7 costimulation results in induction of antigen-specific tolerance. Here, we show that activated human B lymphocytes express two additional CTLA-4 counterreceptors also capable of providing T-cell costimulation. At 24 hr postactivation, B cells express a CTLA-4 counterreceptor not recognized by anti-B7 or -BB-1 monoclonal antibodies (mAbs), which induces detectable IL-2 secretion and T-cell proliferation. At 48 and 72 hr postactivation, B cells express both B7 and a third CTLA-4 counterreceptor identified by the anti-BB-1 mAb. BB-1 appears to be a molecule distinct from B7 by its expression on B7- cells and its capacity to induce T cells to proliferate without significant accumulation of IL-2. As observed for B7, costimulatory signals mediated by these alternative CTLA-4/CD28 counterreceptors are likely to be essential for generation of an immune response and their absence may result in antigen-specific tolerance. We propose the following terminology for these CTLA-4 counterreceptors: (i) B7, B7-1; (ii) early CTLA-4 binding counterreceptor, B7-2; and (iii) BB-1, B7-3.     

Incomplete T-cell immune reconstitution in two major histocompatibility complex class II-deficiency/bare lymphocyte syndrome patients after HLA-identical sibling bone marrow transplantation.

To study the effects of major histocompatibility complex (MHC) class II expression on T-cell development, we have investigated T-cell immune reconstitution in two MHC class II-deficiency patients after allogeneic bone marrow transplantation (allo-BMT). Our study showed that the induction of MHC class II antigen expression on BM graft-derived T cells in these allo-BMT recipients was hampered upon T-cell activation. This reduction was most striking in the CD8(+) T-cell subset. Furthermore, the peripheral T-cell receptor (TCR) repertoire in these graft-derived MHC class II-expressing CD4(+) and in the CD8(+) T-cell fractions was found to be restricted on the basis of TCR complementarity determining region 3 (CDR3) size profiles. Interestingly, the T-cell immune response to tetanus toxoid (TT) was found to be comparable to that of the donor. However, when comparing recipient-derived TT-specific T cells with donor-derived T cells, differences were observed in TCR gene segment usage and in the hydropathicity index of the CDR3 regions. Together, these results reveal the impact of an environment lacking endogenous MHC class II on the development of the T-cell immune repertoire after allo-BMT.