Lawrence S. Lamb

Non-MHC-restricted cytotoxic cells: Their roles in the control and treatment of leukaemias

The role of T lymphocytes in the MHC-restricted control and eradication of leukaemia after allogeneic bone marrow transplantation is established, but there is increasing evidence that the non-MHC-restricted cytotoxic lymphocytes have the capacity to lyse allogeneic or, in some cases, autologous leukaemic blasts. Recently, the field of tumour immunotherapy has been dominated by T cell-mediated approaches, probably as a result of the rapid and significant advances in our understanding of the mechanisms involved in the generation of responses from naõ Ève T cells. During the last 5 years, our knowledge of the activation and control mechanisms involved in non-MHC-restricted cell-mediated immunity has advanced enormously and we are now seeing a concomitant increase in the understanding of these cell types in tumour immunotherapy and in the immunotherapy of haematopoietic malignancies in particular. The term `non-MHC-restricted cytotoxic lymphocytes encompasses natural killer (NK) cells, NK-T cells and gd T cells, all of which derive from a common lymphoid precursor cell but differentiate along separate pathways. NK-T and gd T cells express surface CD3 and the T-cell receptor (TCR); they are thus true T cells. NK-T cells express the a and b chains of the TCR while, as their name suggests, gd T cells express the g and d chain rearrangement. In contrast, NK cells lack both CD3 and TCR expression, but mostly (. 95%) express CD56 and/or CD16. Each cell type will be described in more detail including, when known, the mechanisms of antigen recognition, but in all cases this does not require MHC compatibility between effector and target cell, hence the term `non-MHC-restricted, although NK cells do recognize MHC molecules and this interaction is involved in their control of cytolysis. Expression of appropriate MHC molecules on target cells is inhibitory to NK cell-mediated lysis. Thus, NK cells are permanently ready to lyse potential target cells and can be triggered in a variety of ways. Cells showing downregulation of MHC molecules, such as some tumours and some virally infected cells, are innate targets owing to the lack of inhibition of NK-mediated lysis. Other cells with normal MHC expression can still be NK targets if they provide appropriate stimulatory signals, for example by ligation of the Fcg receptor (CD16), that are capable of overcoming the inhibition signals. While each of the cell types discussed here are distinct entities, they share several characteristics with respect to differentiation or cell surface antigen expression (Table I), but their relationships in the ontogeny of the cellular immune system have yet to be unravelled. All these cell types are involved in primary immune responses to tumours and intracellular pathogens, but lack the ability to generate a `memory subset to mediate secondary cellular responses to recall antigens. The lack of requirement for MHC matching by these cells, indeed the potential benefit of mismatching in the case of natural killer cells, means that many clinical applications of these non-MHC-restricted cytotoxic cells are based upon allogeneic donor systems. However, the role of autologous natural killer cells in the cure of leukaemia is beginning to be unravelled and may lead to better defined trials of passive immunotherapy in the near future.

Characterization of the γδ T cell response to acute leukemia

Background: Previous work from our center has suggested a correlation between increased donor-derived Vδ1+ γδ T cells and long-term relapse-free survival following bone marrow transplantation for leukemia. Questions remain, however, as to whether this observation can be explained by a γδ T cell-based immune response against primary leukemia. Methods: We examined γδ T cell receptor (TCR) phenotype, cell proliferation, and cytolytic activity following culture with irradiated primary leukemia blasts from a haploidentical first-degree relative. Subsequently, we also studied the γδ TCR phenotype and complimentarity determining region 3 (CDR3) cDNA sequences from 17 newly diagnosed leukemia patients. Results: In 17/28 (61%) of in vitro cultures, γδ T cells proliferated in culture with primary blasts. Vδ1+ T cells were proportionally increased in all cultures and were the predominant cell population in 6/17. In the 7 cultures where cytotoxicity could be assessed, 6 (86%) showed some degree of cytotoxicity to the primary leukemia. Vδ1+ T cells were also the predominant γδ T cell subtype in pre-treatment leukemia patients principally due to loss of Vδ2+ T cells rather than expansion of Vδ1+ cells. The Vδ1 CDR3-region cDNA sequence from these patients revealed exclusive use of the Jδ1 constant region and sequence conservation in 4/11 patients. Conclusions: γδ T cells exhibit an in vitro response to primary leukemia blasts that is manifested by proliferation, an increased proportion of Vδ1+ T cells, and cytotoxicity to the primary leukemia blasts. The Vδ1+ T cell population is also predominant in newly diagnosed leukemia patients likely due to a loss of circulating Vδ2+ T cells. A small proportion of newly diagnosed patients showed Vδ1 CDR3 region similarity. These findings suggest a role for γδ T cells in the immune response to leukemia.

Effect of dietary copper on colonic tumor production and aortic integrity in the rat

Gastrointestinal malignancy has been associated with aortic aneurysmal disease in humans, while metabolic derangement of copper has been incriminated as a possible promotor of aneurysmal development of the aorta. An animal model utilizing the carcinogen 1,2-dimethylhydrazine (DMH) was selected to evaluate levels of dietary copper on both colonic tumor production and morphologic changes in the rat aorta. Six groups, each including 10 Sprague-Dawley rats, received 16 weekly doses (20 mg/kg) of DMH beginning at 4 weeks of age. Groups were maintained on either normal (25 ppm), low (0.6 ppm), or high (100 ppm) copper chow during the entire experimental period. After 25 weeks, all animals were sacrificed to assess colonic tumor production and to perform scanning (SEM) and transmission electron microscopic (TEM) studies of the rat aorta. Results showed a significant increase in colonic tumor production (3.14 +/- 0.39 tumors per centimeter colon) in rats treated with low-copper chow and DMH when compared with rats on normal chow and DMH (0.74 +/- 0.07 tumors per centimeter colon) and animals maintained on high-copper diets and DMH (0.76 +/- 0.08 tumors per centimeter colon). In addition, morphologic study showed disruption of the intima and media in rats maintained on low-copper diet alone, and also on low-copper diet plus DMH. The results of this study showed that DMH and low dietary copper significantly increase (P less than 0.001) the yield of colonic tumors and produce loss of aortic integrity when studied morphologically. Copper levels may be important in the association of neoplasia and aneurysm formation in the clinical setting.

Colorectal cancer in animal models—A review

Colorectal cancer remains the second leading cause of cancer death in the western hemisphere. During the past several decades information regarding epidemiology, etiology and associated factors regarding colo-rectal cancer in humans has been collected through study of experimental colonic tumors in animal models. Much of this work has been influenced by the use of 1,2-dimethylhydrazine as the inducing carcinogen in susceptible populations of animals, although other specific carcinogens have been used. Through application of this experimental model, knowledge of dietary, immunologic, and bacterial factors has been realized in the etiology of colo-rectal cancer. This review details methodology and results of developing experimental models as they pertain to human colo-rectal cancer.

CMV-Independent Lysis of Glioblastoma by Ex Vivo Expanded/Activated Vδ1+ γδ T Cells

Vδ2neg γδ T cells, of which Vδ1+ γδ T cells are by far the largest subset, are important effectors against CMV infection. Malignant gliomas often contain CMV genetic material and proteins, and evidence exists that CMV infection may be associated with initiation and/or progression of glioblastoma multiforme (GBM). We sought to determine if Vδ1+ γδ T cells were cytotoxic to GBM and the extent to which their cytotoxicity was CMV dependent. We examined the cytotoxic effect of ex vivo expanded/activated Vδ1+ γδ T cells from healthy CMV seropositive and CMV seronegative donors on unmanipulated and CMV-infected established GBM cell lines and cell lines developed from short- term culture of primary tumors. Expanded/activated Vδ1+ T cells killed CMV-negative U251, U87, and U373 GBM cell lines and two primary tumor explants regardless of the serologic status of the donor. Experimental CMV infection did not increase Vδ1+ T cell - mediated cytotoxicity and in some cases the cell lines were more resistant to lysis when infected with CMV. Flow cytometry analysis of CMV-infected cell lines revealed down-regulation of the NKG2D ligands ULBP-2, and ULBP-3 as well as MICA/B in CMV-infected cells. These studies show that ex vivo expanded/activated Vδ1+ γδ T cells readily recognize and kill established GBM cell lines and primary tumor-derived GBM cells regardless of whether CMV infection is present, however, CMV may enhance the resistance GBM cell lines to innate recognition possibly contributing to the poor immunogenicity of GBM.

In vivo expansion and activation of γδ T cells as immunotherapy for refractory neuroblastoma: A phase 1 study.

Introduction:CD3+ &ggr;&dgr;+ T cells comprise 2% to 5% of circulating T cells with V&ggr;9V&dgr;2+ cells the dominant circulating subtype. V&ggr;9V&dgr;2+ cells recognize non-peptide phosphoantigens and stress-associated NKG2D ligands expressed on malignant cells. Strategies that incorporate the tumoricidal properties of &ggr;&dgr; T cells represent a promising immunotherapeutic strategy for treatment of solid malignancies including neuroblastoma (NB). In this prospective, non-randomized Phase I trial, we assessed whether circulating V&ggr;9V&dgr;2+ cells could be safely expanded using intravenous ZOL (Zoledronate [Zometa®]) and subcutaneous Interleukin-2 (IL-2) in patients with refractory NB. Methods:Patients 2 to 21 years of age with refractory neuroblastoma with no known curative therapeutic options received ZOL on day 1, and IL-2 on days 1 to 5 and 15 to 19 of each 28-day cycle (n = 4). Lymphocyte immunophenotyping was assessed weekly. Immunophenotyping studies from the treatment group were compared with healthy pediatric controls (n = 16; range, 5y–15y) and of untreated NB disease controls (n = 9; range, 4m–18y). Results:Treatment was well tolerated with no unexpected grade 3 and 4 toxicities. Lymphocyte subset counts did not differ significantly between volunteers and disease controls with the exception of &ggr;&dgr;+ T cell counts that were significantly higher in healthy volunteers (212u200a+u200a93 vs. 89u200a+u200a42, P = 0.05). Study patients showed increases in circulating &ggr;&dgr;+ T cell count (3–10 fold) after the first week, increasing into the range seen in healthy volunteers (125u200a+u200a37, P = 0.1940). Interestingly, all ZOLu200a+u200aIL-2 treated patients showed significant increases in CD3+CD4+CD27hiCD127dim T cells that rose weekly in 2 patients throughout the 4 weeks of observation (maximum 41% and 24% of total CD3+CD4+ T cells, respectively). Conclusions:In summary, combined ZOL and IL-2 is well tolerated and restored &ggr;&dgr;+ T cell counts to the normal range with a moderate expansion of Natural Killer cells. Progressive increases in circulating CD4+ T cells with a regulatory phenotype cells may offset beneficial effects of this therapy.

Hematopoietic cellular therapy: implications for the flow cytometry laboratory

Advances in hematopoietic stem cell transplantation, gene therapy, and immunotherapy have necessitated a host of novel monitoring procedures. Cell sorting is also coming of age as a clinical procedure designed to organize hematopoietic grafts for specificity of cellular components designed for individual patients or diseases. This article has focused on these novel developments in their historical context. The next generation of transplantation flow cytometry promises to be an exciting one.

T-Cell Lymphoblastic Leukemia/Lymphoma Syndrome With Eosinophilia and Acute Myeloid Leukemia

This case represents an example of an unusual T‐cell lymphoblastic leukemia/lymphoma syndrome associated with eosinophilia and myeloid malignancy in a young boy. This case is one of only five reported “leukemic” variants of the disease and demonstrates the importance of considering this poor prognostic diagnosis in pediatric acute lymphoblastic leukemia. This case also illustrates the importance of an interactive multidisciplinary approach to the laboratory evaluation of a leukemia patient.

Effect of HSV-IL12 Loaded Tumor Cell-Based Vaccination in a Mouse Model of High-Grade Neuroblastoma.

We designed multimodal tumor vaccine that consists of irradiated tumor cells infected with the oncolytic IL-12-expressing HSV-1 virus, M002. This vaccine was tested against the syngeneic neuroblastoma mouse model Neuro 2a injected into the right caudate nucleus of the immunocompetent A/J mice. Mice were vaccinated via intramuscular injection of multimodal vaccine or uninfected irradiated tumor cells at seven and 14 days after tumor establishment. While there was no survival difference between groups vaccinated with cell-based vaccine applied following tumor injection, a premunition prime/boost vaccination strategy produced a significant survival advantage in both groups and sustained immune response to an intracranial rechallenge of the same tumor. The syngeneic but unrelated H6 hepatocellular tumor cell line grew unrestricted in vaccinated mice, indicative of vaccine-mediated specific immunity to Neuro 2a tumors. Longitudinal analyses of tumor-infiltrating lymphocytes revealed a primary adaptive T cell response involving both CD4+ and CD8+ T cell subsets. Spleen cell mononuclear preparations from vaccinated mice were significantly more cytotoxic to Neuro 2a tumor cells than spleen cells from control mice as demonstrated in a four-hour in vitro cytotoxicity assay. These results strongly suggest that an irradiated whole cell tumor vaccine incorporating IL-12-expressing M002 HSV can produce a durable, specific immunization in a murine model of intracranial tumor.