A novel approach to identify antigens recognized by CD4 T cells using complement-opsonized bacteria expressing a cDNA library.

In patients with hematological malignancies receiving HLA-matched stem cell transplantation, T cells specific for minor histocompatibility antigens play a major role in graft rejection, induction of graft-versus-host disease and beneficial graft-versus-leukemia reactivity. Several human minor histocompatibility antigens recognized by T cells have been identified, but only two are presented by HLA class II molecules. In search of an efficient approach to identify antigenic peptides processed through the HLA class II pathway, we constructed a cDNA library in bacteria that were induced to express proteins. Bacteria were opsonized with complement to enforce receptor-mediated uptake by Epstein-Barr virus immortalized B cells that were subsequently used as antigen-presenting cells. This approach was validated with an HLA class II-restricted antigen encoded by gene DBY. We were able to identify bacteria expressing DBY diluted into a 300-fold excess of bacteria expressing a nonrelevant gene. Screening of a bacterial library using a DBY-specific CD4 T cell clone resulted in the isolation of several DBY cDNAs. We propose this strategy for a rapid identification of HLA class II-restricted antigenic peptides recognized by CD4 T cells.

TCR gamma delta+ cells expressing Vgamma 9V delta2, which normally predominate the blood, are found in the spleens of patients with hairy cell leukemia.

T cell receptor gamma delta+ (TCR gamma delta+) cells are present in all lymphoid tissues, but at lower frequencies than TCR alpha beta+ cells. In normal spleens, they account for about 15-20% of CD3+ cells. In hairy cell leukemia (HCL), a B cell malignancy with a characteristic involvement of the spleen, splenic T cells of approximately 40% of the examined patients showed a remarkable increase in CD3+ TCR gamma delta+ cells. Therefore, we studied the TCR gamma delta phenotypes in blood and spleen of 12 HCL patients in comparison with normal samples. In normal blood (n= 6) the majority of TCR gamma delta+ cells expressed the Vgamma 9V delta2 phenotype, while in normal spleens (n = 9) a different population was found expressing Vdelta1. In contrast, six of eight paired blood and spleen samples of individual HCL patients demonstrated the same phenotype with predominantly Vgamma 9V delta2 expression. This change in splenic TCR gamma delta usage in patients with HCL might be the result of cell-trapping, or an infiltration of TCR gamma delta+ cells from the blood.

T-cell dysfunction in hairy cell leukemia: an updated review.

Hairy cell leukemia (HCL) is clinically associated with severe T-cell dysfunction. Several new observations have given more insight into the abnormal T-cell responses seen in this disease. T-lymphocytes in the spleen of patients with HCL seem to be abnormally activated. On the other hand, they are non-responsive, possibly as a result of monocytopenia which may lead to inadequate antigen presentation. This, together with the lack of CD28 on T-cells, may cause T-cell dysfunction. Furthermore, there is a very restricted repertoire of the T-cell receptor-beta family, which may also result in non-responsiveness. Otherwise, T-cell clonal excess may be indicative for activated, possibly autoreactive T-cells.

Hairy cell leukemia-specific recognition by multiple autologous HLA-DQ or DP-restricted T-cell clones.

We studied in patients with hairy cell leukemia (HCL) whether autoreactive T cells could be isolated with specific reactivity to the HCL cells. HCL cells were activated via triggering of CD40 on the cell membrane and used as stimulator cells to generate autologous T-cell clones. Two types of CD4(+)BV2(+) T-cell clones with different CDR3 rearrangements and one type of CD4(+)BV8S3(+) T-cell clone were generated from the spleen or blood. These clones specifically recognized the autologous HCL cells, without reactivity to autologous peripheral blood mononuclear cells (PBMC), phytohemagglutinin blasts, or Epstein-Barr virus-transformed B cells in a primed lymphocyte test. Blocking and panel studies using HCL cells from 11 other patients showed that recognition of the HCL cells by the BV2(+) T cells was restricted by HLA-DQA1*03/DQB1*0301, and the BV8S3(+) T cells were restricted by DPB1*04. The T-cell clones did not recognize DPB1*04(+) or DQ3(+) PBMC from healthy donors or DP/DQ matched malignant cells from patients with other hematologic malignancies, except for one patient with acute lymphoblastic leukemia. These HCL-specific T-cell clones may be used for the detection of an HCL-specific tumor antigen.

Impaired expression of CD28 on T cells in hairy cell leukemia.

T cells from patients with active hairy cell leukemia (HCL), a chronic B cell malignancy, show poor proliferation in response to allogeneic peripheral blood mononuclear cells (PBMC). In order to study the T cell dysfunction, the expression of several adhesion and costimulatory molecules was analyzed by flow cytometry. Circulating T cells from HCL patients showed increased percentages of CD28(-) in all T cell subsets. In some patients the percentage of CD28(-) T cells within the CD4(+) subset was increased up to 80%. These CD4(+)CD28(-) T cells did not proliferate in a mixed lymphocyte culture (MLC) against allogeneic PBMC. After enrichment for CD4(+)CD28(+) T cells, the proliferative response in the MLC was recovered, but this response was still lower than the proliferative response from control T cells. In conclusion, lack of CD28 on T cells and a restricted T cell repertoire may contribute to immune deficiency in patients with HCL.

Persistent clonal excess and skewed T-cell repertoire in T cells from patients with hairy cell leukemia.

Hairy cell leukemia (HCL) is characterized by a severe T-cell-mediated immune deficiency. At the same time, spontaneous T-cell activation is noted when splenic T cells are studied in vivo and in vitro. Therefore, we searched for oligoclonal T-cell populations in the blood and spleens of 25 patients with HCL using a T-cell receptor gamma-polymerase chain reaction (TCR gamma-PCR). Subsequently, in 6 patients, the CDR3 length and conformation from 22 different TCRBV subfamilies were analyzed after PCR amplification of cDNA using TCRBV subfamily-specific primers. T cells from 15 of 25 HCL patients showed clonal excess by the TCR gamma-PCR. In fluorescence-activated cell sorted T-cell subsets, more clonal bands were observed than in the unseparated T cells, with most of these in CD8+ subsets, but also in CD4+, CD3+ gamma/delta+, and a double-negative CD3+ alpha/beta+ subset. In other B-cell malignancies, 6 of 16 samples showed oligoclonal T cells, whereas only 2 of 18 normal spleen and blood samples showed abnormal bands. Analysis of the TCRBV subfamilies disclosed in all 6 HCL patients a markedly abnormal pattern, with many clonal bands in 5 to 15 subfamilies, and absent or abnormal weak patterns in another 1 to 8 subfamilies. In comparison, 6 normal samples (2 spleens and 4 blood samples) showed in only 1 blood donor 1 clonal band. Two patients with active HCL but without infections or treatment were tested several times during the course of the disease and showed a complete identical skewed T-cell repertoire with the same oligoclonal T-cell populations. In conclusion, T cells in the blood and spleen of HCL patients show impressive abnormalities with many oligoclonal T-cell populations and a very restricted and skewed TCRBV repertoire.