Increase of lymphocytes bearing the gamma/delta T cell receptor in the jejunum of patients with dermatitis herpetiformis.

The densities of T cells and of cells bearing the T cell receptors gamma/delta and alpha/delta and the surface antigens CD4 and CD8 in jejunal specimens from 21 patients with dermatitis herpetiformis were compared with those in specimens from 13 untreated adults with coeliac disease and 13 control subjects. In the lamina propria of the jejunum the median density of gamma/delta+ cells was significantly (p less than 0.001) greater in untreated patients with dermatitis herpetiformis than in control subjects (114 v 36 cells/mm2) and similar to that found in the patients with coeliac disease (115 cells/mm2). The difference in gamma/delta+ cell density between patients with dermatitis herpetiformis and control subjects was much greater in the surface epithelium of the jejunum: the median density for 14 untreated patients with dermatitis herpetiformis was 39 cells/mm, for seven patients with dermatitis herpetiformis on a gluten free diet 34 cells/mm, and for control subjects 2 cells/mm; the coeliac patients had the same density as the patients with dermatitis herpetiformis (45 cells/mm). The higher density of cells bearing the alpha/delta T cell receptor in the epithelium (median 77 cells/mm) of untreated patients with dermatitis herpetiformis was associated with a gluten containing diet; in specimens taken from patients with dermatitis herpetiformis on a gluten free diet the median density was similar to that in the control subjects (44 v 39 cells/mm). The increase in the number of lymphocytes bearing the T cell receptor gamma/delta, particularly in the epithelium of the jejunum, seems to be a constant marker for these closely related diseases, whereas the density of alpha/delta+ T cells is dependent on the diet.     

Analysis of junctional diversity in the preferential V delta 1-J delta 1 rearrangement of fresh T-acute lymphoblastic leukemia cells by in vitro gene amplification and direct sequencing

To define the junctional diversity of T-cell antigen receptor delta gene rearrangements in fresh T-acute lymphoblastic cells and to correlate cell phenotype with the coding potential of rearrangements, we determined the junctional nucleotide sequences of 13 T-cell antigen receptor delta gene rearrangements involving the preferentially rearranged V (V delta 1) and J (J delta 1) segments using in vitro gene amplification and direct sequencing. We showed that, as in gamma delta+ cell lines, extensive junctional diversity exists in these clones and that this diversity is due both to random nucleotide deletions/additions and to the use of at least two D delta segments. We also showed that a high percentage of these rearrangements are potentially translatable (7:13) and that such functional rearrangements occur in both surface CD3+ and CD3- cells. Comparison of alpha beta versus gamma delta surface expression demonstrates that all CD3+ T acute lymphoblastic leukemias with a functional V delta 1-J delta 1 rearrangement express a surface gamma delta receptor and are recognized by the anti-delta monoclonal antibody delta TCS1, whereas a control CD3+ gamma delta+ leukemic case that had not undergone V delta 1 rearrangement was delta TCS1-. In addition, expression of this monoclonal antibody is not restricted by V gamma or C gamma usage or by the covalent or noncovalent link between gamma and delta chains.     

Disruption of epithelial gamma delta T cell repertoires by mutation of the Syk tyrosine kinase.

Chimeric mice in which lymphocytes are deficient in the Syk tyrosine kinase have been created. Compared with Syk-positive controls, mice with Syk -/- lymphocytes display substantial depletion of intraepithelial gamma delta T cells in the skin and gut, with developmental arrest occurring after antigen receptor gene rearrangement. In this dependence on Syk, subsets of intraepithelial gamma delta T cells are similar to B cells, but distinct from splenic gamma delta T cells that develop and expand in Syk-deficient mice. The characteristic associations of certain T-cell receptor V gamma/V delta gene rearrangements with specific epithelia are also disrupted by Syk deficiency.     

The cellular infiltrate of the jejunum in adult coeliac disease and dermatitis herpetiformis following the reintroduction of dietary gluten.

The cellular infiltrate of the jejunal mucosa has been studied in patients with both treated and untreated adult coeliac disease and dermatitis herpetiformis and serially in treated patients before and after the reintroduction of gluten to the diet. In adult coeliac disease and dermatitis herpetiformis the jejunal mucosa showed similar abnormalities of the cellular infiltrate which was characterized by an increase in intraepithelial lymphocytes and lamina propria plasma cells and eosinophils, with the greatest numbers of cells occurring in untreated patients. At 24-48 hours following a single 25-g gluten challenge there was an increase in lamina propria plasma cells, lymphocytes and eosinophils and intraepithelial lymphocytes. This rise was sustained after seven days on a gluten-containing diet for all of these cell groups except lamina propria lymphocytes. These responses were essentially similar in both adult coeliac disease and in those dermatitis herpetiformis patients who had jejunal lesions before treatment. In dermatitis herpetiformis patients with normal jejunal morphology on a normal diet there was an upward trend in lamina propria plasma cells and intraepithelial lymphocytes within one to three weeks of taking extra dietary gluten. These results are compatible with the view that more than one immunological mechanism may be responsible for the pathogenesis of the jejunal lesion of coeliac disease and dermatitis herpetiformis.     

Limited diversity of T-cell receptor gamma-chain expression of murine Thy-1+ dendritic epidermal cells revealed by V gamma 3-specific monoclonal antibody.

To study the origin of and the degree of T-cell antigen receptor (TCR) diversity of Thy-1+ dendritic epidermal cells (Thy-1+ dECs) in mice, we have developed a monoclonal antibody (mAb 536) to the gamma delta TCR. mAb 536 binds to and stimulates interleukin 2 secretion from Thy-1+ dEC but not cells that express TCR composed of alpha and beta chains. mAb 536 precipitates CD3-associated gamma and delta chains from lysates of radioiodinated Thy-1+ dECs. Analysis of a panel of hybridomas that express gamma delta TCR indicated that mAb 536 defines an epitope of the variable region (V gamma 3) gene product. Flow cytometric analysis revealed that expression of V gamma 3 in the adult mouse is restricted to cells in the epidermis, where essentially all Thy-1+ cells are V gamma 3+. The majority of CD3+ cells in the 14-day fetal thymus also express V gamma 3. These results indicate that the T-cell complement in epidermis are cells that express gamma delta TCR and that the diversity of antigens recognized by the cells might be restricted by the use of a single V gamma gene segment. Finally, the data raise the intriguing possibility that Thy-1+ dECs may arise from precursors that are among the first to emerge from the developing thymus. This suggests that V gene usage during thymocyte development is highly regulated and has important consequences on the tissue localization and function of the emerging cells. As in other developing tissues, it appears that programmed and transient gene expression determines the fate of the emerging cells.     

Increase in gamma/delta T cell receptor bearing lymphocytes in normal small bowel mucosa in latent coeliac disease.

A jejunal biopsy specimen from an asymptomatic 35 year old man was studied because of a low serum titre of reticulin antibody and the finding of coeliac disease in his son. In this specimen villous structure was quite normal as was the total number of intraepithelial lymphocytes, but the number of gamma/delta T cell receptor bearing lymphocytes was 10 times higher than the mean in control subjects. Two years later a further biopsy specimen was obtained because of clinical symptoms and an increased titre of reticulin antibody. This specimen showed villous atrophy with crypt hyperplasia and increased infiltration of intraepithelial lymphocytes compatible with coeliac disease. A control biopsy specimen taken during gluten free diet showed normalisation of the villous architecture. Latent coeliac disease may be characterised by an increase in gamma/delta positive cells similar to that seen in established coeliac disease.     

The C gamma 1-encoded disulphide-linked and the C gamma 2-encoded nondisulphide-linked forms of the gamma/delta heterodimer use different gamma and delta variable regions

We identified two types of gamma/delta T-cell clones: (1) BB3+ clones, which express the disulphide-linked form of the gamma/delta heterodimer (type 1 receptor) and carry rearrangements of the C gamma 1 gene; and (2) delta TCS1+ clones, which express the nondisulphide-linked form of the gamma/delta heterodimer (type 2 receptor) and carry rearrangements of the C gamma 2 gene. To determine the joining (J) and variable (V) region usage of the two receptor types, T gamma and T delta gene rearrangements and expression were analyzed in each gamma/delta T-cell clone. The results showed that the J and V regions used by the two receptor types are distinct: clones that express the type 1 receptor carry potentially functional rearrangements in J gamma P-V gamma 9 for gamma and in J delta 1-V delta 2 for delta; and clones that express the type 2 receptor had potentially functional rearrangements in J gamma 2-V gamma 2/V gamma 4 for gamma and in J delta 1-V delta 1 for delta. Therefore, two clearly different gamma/delta receptors (V gamma 9-J gamma P-C gamma 1/V delta 2-J delta 1-C delta and V gamma 2/V gamma 4-J gamma 2-C gamma 2/V delta 1-J delta 1-C delta) predominate in peripheral blood.     

Crosstalk between alpha/beta T cells and gamma/delta T cells in vivo: activation of alpha/beta T-cell responses after gamma/delta T-cell modulation with the monoclonal antibody GL3.

Although gamma/delta T cells express numerous in vitro functions similar to alpha/beta T cells, little is known about their biological functioning in vivo. Furthermore, it is unclear whether alpha/beta T cells and gamma/delta T cells act independently or in a coordinated way. In the present study, gamma/delta T cells were modulated in vivo by i.p. injection of the anti-gamma/delta T-cell receptor (TCR) monoclonal antibody GL3. GL3 administration caused disappearance of the gamma/delta TCR in spleen and lymph node cells and the gamma/delta TCR was reexpressed after in vitro cultivation for a few days. When cultured in vitro for 4 days, in the absence of foreign antigens, spleen and lymph node alpha/beta T cells from GL3-modulated mice showed vigorous proliferative responses. CD4 T lymphocytes from GL3-modulated mice produced interleukin 2, and CD8 T cells developed into cytolytic T lymphocytes in vitro capable of lysing syngeneic and allogeneic targets. Treatment with heat-inactivated GL3 or with normal hamster immunoglobulin did not cause any of these effects. These findings suggest that the anti-gamma/delta TCR monoclonal antibody GL3 modulates gamma/delta T cells in vivo and that this modulation has profound effects on alpha/beta T-cell reactivity. Hence, the data suggest a role for gamma/delta T cells in the regulation of alpha/beta T-cell activation in vivo.     

TCR gamma/delta positive lymphocytes after allogeneic bone marrow transplantation.

Peripheral gamma/delta+ T cells were studied in patients following allogeneic bone marrow transplantation (BMT) by indirect immunofluorescence utilizing two monoclonal antibodies (G1 and A13) able to recognize the two major subpopulations (V delta 2+ and V delta 1+, respectively) of these cells. We found that the relative percentage of 'total' (gamma/delta+ T lymphocytes) (V delta 2 + V delta 1 positive cells), and particularly of G1+ (V delta 2+) cells, in CD3+ lymphocytes was higher in transplanted patients, and especially in those presenting with acute graft-versus-host disease (aGVHD), than in normal controls. This finding was confirmed by the analysis of the V delta 2+/V delta 1+ cell ratio which was again significantly higher in patients with aGVHD as compared to controls. Similarly, the absolute number of 'total' gamma/delta+ and V delta 2+ cells was also significantly increased in patients with aGVHD. TCR gamma/delta+ T cells increased as a function of time after BMT reaching a plateau value at about day 60 post-BMT. When patients were stratified for the presence or absence of aGVHD this correlation was maintained only for patients with aGVHD. Finally, most V delta 2+ cells expressed surface T cell activation markers such as CD25 (IL-2 receptor) and DR (MHC class II) antigens. Our results suggest a possible involvement of gamma/delta+ T cells and particularly of V delta 2+ cells in the clinical and immunological events (aGVHD) occurring after allogeneic BMT.     

Selective outgrowth of CD45RO+ V gamma 9+/V delta 2+ T-cell receptor gamma/delta T cells early after bone marrow transplantation.

Before and after bone marrow transplantation (BMT) for hematologic malignancies, peripheral blood mononuclear cells from 10 patients were obtained. The relative and absolute numbers of CD3+ T-cell receptor gamma delta+ (TCR gamma delta+) cells, as defined by the reaction of monoclonal antibodies (MoAbs) directed against CD3 and the TCR gamma delta (anti-TCR gamma delta-1), were determined. Before transplantation, eight of nine patients tested had less than 10% CD3+TCR gamma delta+ cells. Consistent increased numbers of gamma delta cells up to eightfold the pretransplant level can be seen in four of nine patients tested within the first 4 months after BMT. The large majority of early posttransplant gamma delta and alpha beta T cells express the CD45RO antigen, which is usually expressed on "memory" cells only. The V-region usage of the TCR gamma delta+ T cells was analyzed using fresh mononuclear cells and MoAbs against known V gamma and V delta regions. For more detailed analysis, CD3+TCR gamma delta+ cells were sorted and cultured in bulk and cloned. Using fresh cells and bulk cultures, mainly V gamma 9+V delta 1-V delta 2+ cells were found during engraftment. Only after 6 weeks post-BMT, V gamma 9-V delta 1+V delta 2- cells appear. Analysis of the V gamma and V delta usage at the clonal level confirmed the observation that early after BMT only V gamma 9+V delta 2+ cells are present, whereas gamma delta T-cell clones expressing other gamma delta TCR phenotypes can only be detected 4 to 6 weeks post-BMT. The predominance of V gamma 9+ cells during early engraftment could be explained by several mechanisms: (A) sequential rearrangements during T-cell development, leading to an early wave of V gamma 9+ cells, or (B) selective outgrowth of preexisting V gamma 9+V delta 2+CD45RO+ TCR gamma delta cells in the bone marrow graft, possibly as a result of antigen driven expansion due to exposure to environmental antigens.     

Refractory sprue syndrome with clonal intraepithelial lymphocytes evolving into overt enteropathy-type intestinal T-cell lymphoma

INTRODUCTION: Recently, patients with refractory sprue have been shown to contain a clonal proliferation of phenotypically abnormal intraepithelial lymphocytes in their intestine. Whether this signifies early enteropathy-type intestinal T-cell lymphoma (EITCL) or a reactive condition is not clear. We report on a patient presenting with the findings of refractory sprue who subsequently developed overt EITCL. MATERIAL AND METHODS: Duodenal biopsies from 1997 (refractory sprue) and duodenal and jejunal biopsies from 1998 (intestinal T-cell lymphoma) were compared by immunohistochemistry and PCR for the detection of T-cell receptor (TCR)-gamma gene rearrangements. Clonal PCR products were sequenced. RESULTS: The duodenal biopsies from both 1997 and 1998 and the jejunal tumor biopsy showed villus atrophy and an increase of intraepithelial lymphocytes with an abnormal immunophenotype (CD3+, CD4-, CD8- and TCR-beta-). In all duodenal specimens including the one from 1997, and the jenunal tumor biopsy, an identical clonal amplificate was detected by enzymatic amplification of the TCR-gamma gene. CONCLUSION: These data suggest that refractory sprue containing a clonal proliferation of phenotypically abnormal intraepithelial lymphocytes may represent an early manifestation of EITCL. The detection of immunohistochemical negativity for several antigens normally found on intraepithelial lymphocytes such as CD8 or the TCR-beta chain in combination with clonal T-cell populations by PCR may be helpful in identifying refractory sprue with a malignant transformation.     

Early expression of a T-cell receptor beta-chain transgene suppresses rearrangement of the V gamma 4 gene segment.

beta transgenic mice have a T-cell receptor beta-chain gene that is prematurely expressed on the surface of CD4- CD8- thymocytes and paired with an uncharacterized non-T-cell receptor alpha-chain polypeptide. The rearrangement of the T-cell receptor variable region gamma chain gene segment V gamma 4, a component of the gamma-chain gene that is rearranged and expressed preferentially on thymocytes of normal adult mice, is severely repressed in beta transgenic mice. Consequently no gamma delta T-cell receptor heterodimers are detectable on the surface of adult thymocytes or splenic T cells. These results indicate that cells expressing alpha beta or gamma (V gamma 4)-delta TCRs originate from a common precursor in which the first productive rearrangement of either the beta or gamma locus determines the further differentiation pathway into either alpha beta or gamma delta T cells. The repression of V gamma 4 rearrangement by a preexisting beta-chain gene may be indicative of one of several mechanisms which ensure that gamma delta and alpha beta receptors do not as a rule appear on the surface of the same cell.     

Colitis in mice lacking the common cytokine receptor gamma chain is mediated by IL-6-producing CD4+ T cells

BACKGROUND & AIMS: Mice that have a truncated mutation of the common cytokine receptor gamma chain (CR gamma -/Y) are known to spontaneously develop colitis. To identify the pathologic elements responsible for triggering this localized inflammatory disease, we elucidated and characterized aberrant T cells and their enteropathogenic cytokines in CR gamma -/Y mice with colitis. METHODS: The histologic appearance, cell population, T-cell receptor V beta usage, and cytokine production of lamina propria lymphocytes were assessed. CR gamma -/Y mice were treated with anti-interleukin (IL)-6 receptor monoclonal antibody to evaluate its ability to control colitis, and splenic CD4 + T cells from the same mouse model were adoptively transferred into SCID mice to see if they spurred the appearance of colitis. RESULTS: We found marked thickening of the large intestine, an increase in crypt depth, and infiltration of the colonic lamina propria and submucosa with mononuclear cells in the euthymic CR gamma -/Y mice, but not in the athymic CR gamma -/Y mice, starting at the age of 8 weeks. Colonic CD4 + T cells with high expressions of antiapoptotic Bcl-x and Bcl-2 were found to use selected subsets (V beta 14) of T-cell receptor and to exclusively produce IL-6. Treatment of CR gamma -/Y mice with anti-IL-6 receptor monoclonal antibody prevented the formation of colitis via the induction of apoptosis in IL-6-producing CD4 + T cells. Adoptive transfer of pathologic CD4 + T cells induced colitis in the recipient SCID mice. CONCLUSIONS: Colonic IL-6-producing thymus-derived CD4 + T cells are responsible for the development of colitis in CR gamma -/Y mice.     

Monoclonal antibodies specific to native murine T-cell receptor gamma delta: analysis of gamma delta T cells during thymic ontogeny and in peripheral lymphoid organs.

Three hamster monoclonal antibodies (mAbs), all recognizing different epitopes present on the native form of the murine T-cell antigen receptor (TCR) gamma delta subunits, have been generated. mAb 3A10 is specific to a pan-murine TCR gamma delta, recognizing a C delta constant region determinant. mAb 8D6 is specific to a subset of T cells expressing V gamma 4- and V delta 5-encoded gamma delta TCR, and mAb 5C10 is clonotypic. Using these and other mAbs directed against a variety of T-cell surface markers, we quantitated and characterized gamma delta T cells present in developing thymuses as well as in the conventional lymphatic organs by flow cytometry. These studies revealed that (i) many gamma delta thymocytes and peripheral T cells bear CD4 and/or CD8 molecules, (ii) T cells bearing both alpha beta and gamma delta TCRs are scarce, and (iii) thymocyte subsets bearing TCR gamma delta encoded by different combinations of V gamma and V delta gene segments appear in waves during ontogeny.     

Gamma/delta lineage relationship within a consecutive series of human precursor T-cell neoplasms

We analyzed the gene rearrangements associated with the newly described delta T-cell receptor (TCR) gene from a series of 19 consecutive precursor T-cell (lymphoblastic) neoplasms that represent discrete stages surrounding the TCR gene rearrangement process. Significantly, the delta TCR gene showed rearrangement in most (13 of 19) of these T cells, and in addition it was rearranged in two cells displaying no rearrangement for any other TCR gene. Our survey showed three types of delta gene rearrangements associated with cell-surface TCR expression that presumably represent usage of three V delta genes. This analysis demonstrates (1) a major subclass of human precursor T-cell neoplasms belonging to the gamma/delta T-cell receptor-rearranging subtype; (2) a narrow repertoire of human V delta gene usage; and (3) the utility of delta gene rearrangements as a diagnostic clonal marker in precursor T lymphoblastic neoplasms.     

Cytotoxic and interferon gamma-producing activities of gamma delta T cells in the mouse intestinal epithelium are strain dependent.

We have analyzed the cytolytic activity of freshly isolated intraepithelial T cells (i-IEL) from the intestines of several different mouse strains in an anti-T-cell receptor monoclonal antibody-mediated redirected lysis assay. The cytolytic activity of gamma delta i-IEL but not that of alpha beta i-IEL was strain dependent. Mouse strains could be divided into high (H), marginal (M), and null (N) strains. The anti-gamma delta T-cell receptor monoclonal antibody-induced interferon gamma production showed the same strain-dependent variability, but the proliferative responses to gamma delta T-cell receptor crosslinking did not show this variability. The N phenotype of gamma delta i-IEL was found to be dominant in (H x N)F1 mice. In radiation bone-marrow chimeras the H/N phenotype was determined by the genotype of the reconstituting bone-marrow-derived cells but was not determined by the genotype of the radioresistant host cells. Analysis of (H x N)F1 backcross animals indicated that at least two genes are involved in determination of the H/N phenotype. One of these genes is major-histocompatibility-complex linked. No difference in the use of the variable region segment of the gamma-chain or delta-chain was seen between the gamma delta i-IEL from H and N strains. Various models that might explain the strain-dependent gamma delta i-IEL phenotypes are discussed.     

Small intestinal function and dietary status in dermatitis herpetiformis.

Small intestinal morphology and function were assessed in 82 patients with dermatitis herpetiformis, 51 of whom were taking a normal diet and 31 a gluten free diet. Methods used were histopathological evaluation of jejunal mucosal biopsy specimens, quantitation of intraepithelial lymphocytes, cellobiose/mannitol permeability test, tissue disaccharidase values, serum antigliadin antibodies, and formal assessment of dietary gluten content by a dietician. There was no correlation between dietary gluten intake and the degree of enteropathy in the 51 patients taking a normal diet, whereas biopsy specimens were normal in 24 of the 31 patients on a gluten free diet, all previously having been abnormal. Eighteen patients on gluten containing diets had normal jejunal histology and in seven of these all tests of small intestinal morphology and function were entirely normal. Intestinal permeability was abnormal and serum antigliadin antibodies were present in most patients with enteropathy. Studies of acid secretion in seven patients showed that hypochlorhydria or achlorhydria did not lead to abnormal permeability in the absence of enteropathy. This study shows that a combination of objective tests of small intestinal architecture and function will detect abnormalities in most dermatitis herpetiformis patients, including some with histologically normal jejunal biopsy specimens. Nevertheless there is a small group in whom all conventional intestinal investigations are entirely normal.