Karen Zier

Resection of Solid Tumors Reverses T Cell Defects and Restores Protective Immunity

We have previously reported that CTL were demonstrable early after inoculation of CMS5 fibrosarcoma cells, but that they disappeared within 3 wk. These mice were unable to reject a challenge with CMS5 tumor cells. Other studies demonstrated cell surface phenotype and signaling abnormalities of cells within the spleen. Since we assumed that such an environment would make it more difficult to elicit antitumor immune responses via immunotherapy, we asked whether resection of the tumor could reverse these abnormalities. Although early after tumor cell inoculation tumor resection leads to the development of immunity, the effect at late time points has not been studied critically. To test this, mice were inoculated s.c. with CMS5 cells and after 28 days the tumors were resected. We observed a gradual normalization of the cellular phenotype of the spleen. In particular, there was a decrease in the number of Mac1+/Gr1high cells and an increase in the number of CD3+ cells in the spleen within 24–48 h of tumor resection. By day 10, these values were normal. Levels of p56lck increased as well. The functional implications of these changes were illustrated by the reduced growth rate or the complete rejection of a challenge of tumor cells in the resected mice. Both CD4+ and CD8+ cells were involved in the restoration of tumor immunity. Our results suggested that tumor resection not only led to the reversal of immune suppression, but also unmasked a population of primed T cells able to mediate protective immunity.

Decreased Synthesis of Interleukin-2 (IL-2) in Insulin-dependent Diabetes Mellitus

Synthesis of interleukin-2 (IL-2) by lymphocytes from 26 insulin-dependent diabetic subjects (IDDM) was compared with that by lymphocytes from 24 nondiabetic control subjects. The control group produced 1.001 ± 0.071 U/ml (mean ± SEM). The IDDM group, containing patients diagnosed between 5 days and 10 yr before testing, produced only 0.59 ± 0.050 U/ml (P < 0.002). IL-2 synthesis by 6 non-insulin-dependent diabetic subjects (NIDDM) was not decreased (mean ± SEM, 1.20 ± 0.04 U/ml). Moreover, decreased levels of IL-2 production was found with lymphocytes of patients in good control, as well as those in poor control. These data suggest that decreased IL-2 synthesis is specific for IDDM, not explainable solely as a consequence of poor metabolic control, and thus, might be involved in the pathogenesis of the disease.

Supportive Programs Increase Medical Students' Research Interest and Productivity

Background Advances in biomedical research during the last decade have highlighted the necessity of attracting greater numbers of physicians to careers that include a research component. Physician participation in research is essential to increase the number of clinical and translational studies performed, as well as to educate the public about the importance of clinical trials and to assist in recruiting participants. We hypothesized that attractive research opportunities that included faculty mentoring, recognition of participation, and rewards for accomplishments would encourage medical student participation. Methods The Medical Student Research Office was created at Mount Sinai School of Medicine in 1996 to develop structured research programs and advise students looking to undertake a research project. Data from students participating in the summer research program and Medical Student Research Day, from the research section of the Medical Student Performance Evaluation, were collected from 1996 to 2004. Results For the last 4 years, the majority of medical students did research following the first year of school. Students did basic and clinical research, although most preferred clinically oriented or translational projects. Participation in Research Day and the number of publications suggest that interest is growing, including that by traditionally underrepresented groups. Conclusion Although it is too early to assess the long-term effects, the research programs offered led to greater numbers of students who did research, including those in traditionally underrepresented groups. Moreover, students were highly satisfied with their experiences, with 80% feeling that it increased their interest in applying principles they learned to the practice of medicine.

Launching a new fellowship for medical students: the first years of the Doris Duke Clinical Research Fellowship Program.

As part of its commitment to increasing the pipeline of physicians pursuing careers in clinical research, the Doris Duke Charitable Foundation launched the Doris Duke Clinical Research Fellowship (CRF) Program for medical students in 2000. The program, which is based at 10 US medical schools, provides medical students from any US medical school with the opportunity to spend 1 year obtaining both didactic and “hands-on” mentored clinical research experience. This article describes the program and summarizes the early outcomes collected during the first 3.5 years of the program. Interest in the program among medical students has been robust and widespread, with 35% of CRF program fellows matriculated at non-CRF schools. Exit surveys of the first three classes of fellows totaling 174 fellows indicated that (1) 97% of the fellows felt that participating in the fellowship was a good decision; (2) commitment to a career in clinical research was increased among the 44% of fellows reporting that they were unsure about pursuing a clinical research career when they began their fellowship; (3) there was no difference in satisfaction level between the fellows who remained at the medical schools in which they were matriculated and those who completed their fellowship at a medical school in which they were not matriculated; and (4) the majority of fellows responded that the didactic component of their fellowship was useful.

Human tumor vaccines and genetic engineering of tumors with cytokine and histocompatibility genes to enhance immunogenicity.

Genetically engineered tumor cells can be used as vaccines in order to stimulate an immune response. To date, tumor cells have been modified in vitro so that they secrete cytokines or express histocompatibility molecules that they naturally fail to express. These tumor cells differ in the types of immune responses they induce and in whether the responses have local or systemic efficacy. Many questions have been raised during the past year, including whether allogeneic or autologous tumor cells should be employed and whether there may be a risk of inducing autoimmune disease along with the antitumor response. Nevertheless, because of the paucity of available therapies for patients with advanced cancer, investigators must attempt to refine the approaches used in order to minimize patient risk while maximizing tumor cell destruction.

Regulation of HLA-DR, DP, and DQ expression in activated T cells

Class II expression following restimulation of a population of T cells which had first been activated and then allowed to revert to small resting cells via IL-2 deprivation was followed on the cell surface and mRNA levels. Our interest was to determine the kinetics of class II expression during in vitro growth, the triggers which can induce them, and also whether similar or different patterns are observed for the three class II antigens, DR, DQ, and DP. The cells responded rapidly to restimulation with conditioned medium containing IL-2 as assessed by increased incorporation of [3H]TdR into DNA. Cell surface expression of HLA-DR reached peak levels by Day 1, and greater than 90% of the cells continued to express DR until Day 4, when the number of positive cells gradually began to decline. The expression of DP also increased, though maximum levels were not reached until Day 3, when it began to decline. The percentage of cells positive for DP was, however, consistently lower than that of DR. DQ was expressed by very few cells, but appeared to increase until Day 4 and then decline. Tac expression was dramatically up-regulated following IL-2 stimulation, remaining high until Day 4, and then declining more precipitously than any of the other antigens. Class I antigen expression was relatively constant during the entire culture period, though a slight decline was noted between Day 5 and 6 when proliferation and viability were at their lowest levels. On the molecular level, DR beta mRNA accumulation peaked at Day 3, then rapidly declined. Reculture in IL-2 on Day 4 resulted in transient reaccumulation of message. Study of DP beta and DQ beta mRNA demonstrated strong expression on Days 3 and 4 and no obvious up-regulation after restimulation with IL-2 on Day 4. The kinetics of Tac mRNA accumulation and the response to restimulation with IL-2 closely resembled that of DR. Finally we compared the ability of different signals to up-regulate class II mRNA and surface expression and to restimulate proliferation. Our results indicated that PHA was more effective then medium alone, but far less effective than was IL-2. PMA was essentially no different than medium alone.

The effects of gamma interferon on the natural killer and tumor cells of children with neuroblastoma. A preliminary report

Human neuroblastoma cells lack HLA‐A,‐B,‐C molecules which can be induced in vitro by gamma interferon (γIFN). To test the hypothesis that the same induction would occur in vivo leading to tumor regression, a Phase I study was initiated. Seven patients with neuroblastoma were entered on a Phase I study of recombinant γIFN in children. Three received 0.05 mg/m2 intravenously (IV) three times a week, three received 0.1 mg/m2 for 4 weeks, and one patient withdrew from study before receiving adequate treatment for evaluation. No significant clinical response was seen. The side effects were fever and chills, and no serious toxicity occurred. Natural killer (NK) and lymphocyte activated killer (LAK) precursor activity of peripheral blood mononuclear cells was determined before and during treatment, and expression of HLA‐A,B,C molecules was looked for on the tumor cells in the bone marrow of five patients. The NK activity initially low, reached control levels in six patients, but the increase was transient. The LAK precursor activity remained normal. Expression of HLA‐A,B,C, initially absent, was induced on the neuroblastoma cells in four of six patients.

Functional and antigenic properties of cultured T cells in the cell mediated lympholysis (CML) assay

Cultured T cells (CTC) were expanded in Interleukin-2 (IL-2) and used as reagents in cell mediated lympholysis (CML). CTC were able to induce the generation of primary cytotoxic effector cells and to function as 51Cr labeled target cells and cold target inhibitors. Since large numbers of CTC can be produced from as few as 1-2 X 10(6) lymphocytes within a short period of time, these reagents will enable CML studies to be performed in a broader range of situations, including those with were heretofore impossible.

The use of cytotoxic T cell lines to detect the segregation of a human minor alloantigen within families

A cytotoxic T cell (CTL) line, which detected a minor alloantigen provisionally called W was generated in vitro with lymphocytes from a multiply transfused individual, S1. Lymphocytes from S1 were first stimulated with cells from an unrelated known from previous studies to express the minor antigen. The primary CTL were then restimulated with cells from a W +/ve HLA identical sib, S2, in the presence of IL-2. As in previous work, recognition of the W antigen by these CTL was restricted by HLA-B7. Antigen assignments of W + W -, based upon cold target inhibition studies, confirmed previous assignments which had depended upon the ability of lymphocytes either to stimulate the generation of or to be killed by anti-W CTL effectors. Testing of lymphocyte targets from members of several unrelated families in which HLA-B7 segregated showed that the CTL lines could detect the expression of W on cells of individuals in the general population. In 3 of 5 cases, members of an HLA identical sib pair differed for W. These results open up the possibility of designing studies using CTL lines to determine whether differences for minor alloantigens play a role in clinical transplantation.

Expression of class II antigens by subsets of activated T cells

Gene products coded for within the HLA complex play an important role in the control of immune responses. Class I antigens, coded for by the HLA-A, B, and C loci, are expressed by virtually all mononuclear blood cells. Class II antigens, coded for by the DR, DQ, and DP loci, have a more limited tissue distribution. They are expressed by B cells, monocytes, and by activated, but not by resting, T cells. The class II molecules of B cells and antigen-presenting cells have long been of interest to immunologists, since they are involved in the presentation of antigen, in communication between T cells and B cells and between T cells and adherent cells, and in susceptibility to certain diseases. The class II antigens expressed by activated T cells, however, remain largely uncharacterized in terms of their specificity, functional significance, and molecular nature. We have studied the expression of DR and DQ antigens by activated T cells and then examined the expression of DR versus DQ antigens by Leu 2a and Leu 3a subsets of mitogen-activated populations. Our results demonstrated that, as for class II-positive macrophages, the intensity of staining with monoclonal antibodies directed against DR antigens was much greater than that obtained with those directed against DQ antigens. Interestingly, the percentages of Leu 2a- and Leu 3a-positive cells which expressed DR antigens were quite similar, as were the percentages of Leu 2a and Leu 3a cells which expressed DQ. Thus, there does not seem to be preferential expression of DR versus DQ antigens by mitogen-activated T-cell subsets. Finally, though both DR-positive-DQ-positive and DR-positive-DQ-negative populations were detected, few or no DR-negative-DQ-positive cells were observed in these populations.