NK-like Effector Research Articles

Invariant γδTCR natural killer-like effector T cells in the naked mole-rat

The naked mole-rat (Heterocephalus glaber) is a long-lived rodent species showing resistance to the development of cancer. Although naked mole-rats have been reported to lack natural killer (NK) cells, γδ T cell-based immunity has been suggested in this species, which could represent an important arm of the immune system for antitumor responses. Here, we investigate the biology of these unconventional T cells in peripheral tissues (blood, spleen) and thymus of the naked mole-rat at different ages by TCR repertoire profiling and single-cell gene expression analysis. Using our own TCR annotation in the naked mole-rat genome, we report that the γδ TCR repertoire is dominated by a public invariant Vγ4-2/Vδ1-4 TCR, containing the complementary-determining-region-3 (CDR3)γ CTYWDSNYAKKLF / CDR3δ CALWELRTGGITAQLVF that are likely generated by short-homology-repeat-driven DNA rearrangements. This invariant TCR is specifically found in γδ T cells expressing genes associated with NK cytotoxicity and is generated in both the thoracic and cervical thymus of the naked mole-rat until adult life. Our results indicate that invariant Vγ4-2/Vδ1-4 NK-like effector T cells in the naked mole-rat can contribute to tumor immunosurveillance by γδ TCR-mediated recognition of a common molecular signal.

Abstract 5233: Engineered NK-like NKG2C+ CD8 T cells mediate superior anti-tumor efficacy over conventional CD8 T cells and NK cells

Abstract We have recently characterized a unique CD8 T cell subset that expresses NKG2C, as well as multiple other natural killer cell markers. These NKG2C+ CD8 T cells demonstrate NK-like effector functions against various tumor targets, can be efficiently expanded and engineered using GMP-compatible techniques, and most importantly, display beneficial qualities unique to T and NK cells while minimizing the therapeutic deficits of each. NKG2C+ CD8 cells can be easily expanded using a genetically modified K562 feeder line and can be efficiently engineered via retroviral transduction. We genetically engineered NKG2C+ CD8 T cells to express an anti-CD19-28-z CAR construct to target B cell malignancies and compared effector functions of the CAR-engineered NKG2C+ CD8 T cells against CAR+NKG2C- (conventional) CD8 T cells and CAR+ NK cells in vitro and in vivo. We examined expansion, engineering efficiency, tumor control, and extent of cytokine release syndrome (CRS). We also engineered NKG2C+ CD8 T cells with a transgenic NY-ESO-1 TCR to target melanoma and compared the functional responses and efficacy of TCR-engineered NKG2C+ CD8 T cells against TCR-engineered conventional CD8 T cells. CAR+NKG2C+ CD8 T cells demonstrated superior in vitro effector function when compared to CAR+ conventional CD8 T cells and CAR+NK cells and did not upregulate characteristic T cell exhaustion markers, such as PD-1, TIM-3, and LAG-3. These CAR+NKG2C+ CD8 T cells demonstrated superior control of a CD19+ NALM6 tumor model in NRG mice, which was further enhanced with administration of IL-15. CAR+NKG2C+ CD8 T cells with IL-15 demonstrate dramatic and enhanced persistence, as well as superior cytotoxicity and activation in vivo when compared to CAR+ conventional CD8 T cells and CAR+ NK cells. Using a SCID-beige CRS mouse model, we observed reduced CRS in mice treated with CAR+ NKG2C+ CD8 T cells than in mice treated with CAR+ conventional CD8 T cells, with lower levels of proinflammatory cytokines and minimal expansion of murine myeloid subsets. Similarly, TCR-engineered NKG2C+ CD8 T cells demonstrated enhanced tumor control and in vivo persistence in an A375 melanoma-bearing NRG mouse model compared to TCR-engineered conventional CD8 T cells. We have successfully demonstrated that CAR- or TCR-engineered NKG2C+ CD8 T cells have superior anti-tumor efficacy, in vitro and in vivo, to conventional CD8 T cell and NK cell counterparts due to their innate tumor killing and greater in vivo persistence. These cells mediate less CRS and provide a safer alternative to traditional engineered T cell therapeutics. Ultimately, our NKG2C+ CD8 T cells represent a unique platform for engineered T cell therapies to target both solid tumors and hematologic malignancies, that is easily expanded ex vivo, does not require CD4 T cells for in vivo persistence, and is resistant to exhaustion. Citation Format: Kyle Lupo, M. Kazim Panjwani, Anthony Daniyan, Christopher Klebanoff, Katharine Hsu. Engineered NK-like NKG2C+ CD8 T cells mediate superior anti-tumor efficacy over conventional CD8 T cells and NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5233.

Identification of a distinct NK-like hepatic T-cell population activated by NKG2C in a TCR-independent manner.

The liver provides a unique niche of lymphocytes enriched with a large proportion of innate-like T cells. However, the heterogeneity and functional characteristics of the hepatic T-cell population remain to be fully elucidated. We obtained liver sinusoidal mononuclear cells from the liver perfusate of healthy donors and recipients with HBV-associated chronic liver disease (CLD) during liver transplantation. We performed a CITE-seq analysis of liver sinusoidal CD45+ cells in combination with T cell receptor (TCR)-seq and flow cytometry to examine the phenotypes and functions of liver sinusoidal CD8+ T cells. We identified a distinct CD56hiCD161-CD8+ T-cell population characterized by natural killer (NK)-related gene expression and a uniquely restricted TCR repertoire. The frequency of these cells among the liver sinusoidal CD8+ T-cell population was significantly increased in patients with HBV-associated CLD. Although CD56hiCD161-CD8+ T cells exhibit weak responsiveness to TCR stimulation, CD56hiCD161-CD8+ T cells highly expressed various NK receptors, including CD94, killer immunoglobulin-like receptors, and NKG2C, and exerted NKG2C-mediated NK-like effector functions even in the absence of TCR stimulation. In addition, CD56hiCD161-CD8+ T cells highly respond to innate cytokines, such as IL-12/18 and IL-15, in the absence of TCR stimulation. We validated the results from liver sinusoidal CD8+ T cells using intrahepatic CD8+ T cells obtained from liver tissues. In summary, the current study found a distinct CD56hiCD161-CD8+ T-cell population characterized by NK-like activation via TCR-independent NKG2C ligation. Further studies are required to elucidate the roles of liver sinusoidal CD56hiCD161-CD8+ T cells in immune responses to microbial pathogens or liver immunopathology. The role of different immune cell populations in the liver is becoming an area of increasing interest. Herein, we identified a distinct T-cell population that had features similar to those of natural killer (NK) cells - a type of innate immune cell. This distinct population was expanded in the livers of patients with chronic liver disease and could thus have pathogenic relevance.

CD4+ T cells contain early extrapulmonary tuberculosis (TB) dissemination and rapid TB progression and sustain multieffector functions of CD8+ T and CD3- lymphocytes: mechanisms of CD4+ T cell immunity.

The possibility that CD4(+) T cells can act as "innate-like" cells to contain very early Mycobacterium tuberculosis dissemination and function as master helpers to sustain multiple effector functions of CD8(+) T cells and CD3(-) lymphocytes during development of adaptive immunity against primary tuberculosis (TB) has not been demonstrated. We showed that pulmonary M. tuberculosis infection of CD4-depleted macaques surprisingly led to very early extrapulmonary M. tuberculosis dissemination, whereas CD4 deficiency clearly resulted in rapid TB progression. CD4 depletion during M. tuberculosis infection revealed the ability of CD8(+) T cells to compensate and rapidly differentiate to Th17-like/Th1-like and cytotoxic-like effectors, but these effector functions were subsequently unsustainable due to CD4 deficiency. Whereas CD3(-) non-T lymphocytes in the presence of CD4(+) T cells developed predominant Th22-like and NK-like (perforin production) responses to M. tuberculosis infection, CD4 depletion abrogated these Th22-/NK-like effector functions and favored IL-17 production by CD3(-) lymphocytes. CD4-depleted macaques exhibited no or few pulmonary T effector cells constitutively producing IFN-γ, TNF-α, IL-17, IL-22, and perforin at the endpoint of more severe TB, but they presented pulmonary IL-4(+) T effectors. TB granulomas in CD4-depleted macaques contained fewer IL-22(+) and perforin(+) cells despite the presence of IL-17(+) and IL-4(+) cells. These results implicate a previously unknown innate-like ability of CD4(+) T cells to contain extrapulmonary M. tuberculosis dissemination at very early stage. Data also suggest that CD4(+) T cells are required to sustain multiple effector functions of CD8(+) T cells and CD3(-) lymphocytes and to prevent rapid TB progression during M. tuberculosis infection of nonhuman primates.

Development of a flow cytometric, non-radioactive cytotoxicity assay in Eisenia fetida: An in vitro system designed to analyze immunosuppression of natural killer-like coelomocytes in response to 7, 12 dimethylbenz[ a]anthracene (DMBA)

We have developed a non-radioactive cytotoxicity assay to measure cytotoxicity mediated by natural killer (NK)-like effectors in the earthworm Eisenia fetida using a two-color fluorescence assay. K562, the target cell population, was labeled with 3,3′-dioctadecyloxacarbocyanine (DiO), a green fluorescent dye that stains the plasma membrane. NK-like coelomocytes isolated from E. fetida were used as the effector cell population. DiO-labeled targets were incubated with effector cells and the membrane impermeable, red fluorescent dye, propidium iodide (PI). PI labels the double-stranded nucleic acids in membrane-damaged cells only. Killed targets were analyzed using flow cytometry and exhibited coincident green membranes (DiO) and red-nuclear staining (PI). This methodology was employed to analyze the immunosuppressive effects of 7,12 dimethylbenz[ a]anthracene (DMBA), a polycyclic aromatic hydrocarbon, on earthworm coelomocytes with NK-like activity. Flow cytometry and WinList™ Interative N-Color Compensation software revealed a significant decrease in the ability of coelomocytes to kill targets following in vitro DMBA exposure, compared to untreated controls where coelomocytes were cultured in the same medium in the absence of DMBA. These experiments demonstrate how flow cytometry can be used to measure cytotoxicity by earthworm coelomocytes cultured and exposed to DMBA in vitro to study the immunosuppressive effects of DMBA on NK-like effector cells of E. fetida.

Combined Low Dose Cyclosporine and Prednisone Down-Regulate Natural Killer Cell-Like Effector Functions of CD8brightCD56+ T Cells in Patients with Active Behçet Uveitis

Purpose: To investigate the changes of effector-related phenotypic markers and the natural killer (NK)-like effector functions of CD8brightCD56+ T cells in patients with Behçet uveitis after combined cyclosporine and prednisone (Cs/Pd) treatment. Methods: Ten patients with active Behçet panuveitis and 10 healthy controls were prospectively recruited in this study. The effector-related surface markers (CD27, CD62L, CD11b, HLA-DR, CD94, NKG2D), chemokine receptors (CXCR1, CXCR3, CCR4, CCR5), and intracellular perforin of circulating CD8brightCD56+ T cells were determined by flow cytometric analysis before and after two months' treatment. NK-like cytotoxicity of ex vivo CD8brightCD56+ T cells against K562 was measured by standard 51Cr release assay. Results: The expression levels of effector-related molecules on CD8brightCD56+ T cells normalized after treatment. The expression levels of CXCR1 and CCR5 were down-regulated on CD8brightCD56+ T cells after treatment. The amounts of preformed intracellular perforin of CD8brightCD56+ T cells were reduced to the normal levels. Furthermore, the NK-like cytolytic capacities of CD8brightCD56+ T cells were decreased after treatment. Conclusions: Our results suggest that the combined Cs/Pd treatments in active Behçet uveitis may downregulate the NK-like effector functions of CD8brightCD56+ T cells.

Cell adhesion and the immune system: a case study using earthworms.

In the earthworm's immune system, cell adhesion, which occurs by putative receptors on leukocytes, is essential after recognition of self vs. non-self. Confrontation with foreign antigens is a normal event in the environment, replete with microbial pathogens that pose a threat to survival. To better understand what happens when an effector cell first recognizes a foreign target followed by its adhesion to it, isolated leukocytes, in sufficient quantities to be subjected to various analyses, have been extremely beneficial. In vitro approaches when accompanied by biochemical, immunological, and molecular technologies, have opened up new vistas concerning the immune response of earthworms and other invertebrates. The most recent discovery includes the preliminary identification of cell differentiation (CD) markers that play vital roles in recognitive and adhesive events. Certain leukocyte effectors show characteristics of natural killer (NK) cells that may act differently depending upon their source, whether autogeneic, allogeneic, xenogeneic, or expressed under normal or varying environmental conditions including exposure to xenobiotics. At the level of earthworm evolution, there is apparently a dissociation of phagocytosis from the process of killing by NK-like effectors. There are at least three future challenges. First, it is essential to determine the precise nature of the CD markers with respect to their molecular structure. Second, once their molecular and biochemical characteristics have been defined, the role of these markers in cellular and humoral mechanisms must be clarified in order to define effector cell products and resulting immune responses. Third, there is a need to differentiate between the several lytic factors that have been found in earthworms with respect to molecular structure, and biochemical and functional characterization.

Clearance of HSV-2 from recurrent genital lesions correlates with infiltration of HSV-specific cytotoxic T lymphocytes.

The mechanisms involved in host clearance of symptomatic mucocutaneous herpes simplex virus (HSV) infection are unclear. We studied the functional properties of bulk cultures of skin-infiltrating lymphocytes from normal skin and serial biopsies of recurrent genital HSV-2 lesions, and compared HSV-specific and NK responses with viral clearance. HSV-specific CD4+ or CD8+ T cells were rarely detected in lymphocytes cultured from normal skin. The total lymphocyte count and HSV-specific and NK-like effector cell activities were markedly higher in cultures derived from lesional skin. HSV-specific CD4+ proliferative responses and NK-like cytotoxic responses were present at all stages of herpetic lesions, including biopsies early in the disease course. In contrast, cytotoxic T lymphocyte activity was generally low among cells derived from early culture-positive lesions, and increased during lesion evolution. Viral clearance from the lesion site was associated with a high level of local cytolytic activity towards HSV-infected cells. The phenotypes of cells with HSV-specific cytotoxic responses varied between patients, having CD4+ and CD8+ components. Immunotherapeutic approaches to HSV should be directed at improving in vivo cytolytic activity to HSV.

Requirement for Valpha14 NKT cells in IL-12-mediated rejection of tumors.

A lymphocyte subpopulation, the Valpha14 natural killer T (NKT) cells, expresses both NK1.1 and a single invariant T cell receptor encoded by the Valpha14 and Jalpha281 gene segments. Mice with a deletion of the Jalpha281 gene segment were found to exclusively lack this subpopulation. The Valpha14 NKT cell-deficient mice could no longer mediate the interleukin-12 (IL-12)-induced rejection of tumors. Although the antitumor effect of IL-12 was thought to be mediated through natural killer cells and T cells, Valpha14 NKT cells were found to be an essential target of IL-12, and they mediated their cytotoxicity by an NK-like effector mechanism after activation with IL-12.

Decidual immunocyte placental interactions

The effect of cyclosporin A (CsA) on the growth of mixed leukocyte culture-derived human cloned T-cells with different functions has been studied. Interleukin 2-dependent clones with allospecific cytotoxic, natural killer-like cytotoxic, lymphoproliferation suppressive, or primed lymphocyte typing function, were used as test cells in short term (36 h) tritiated thymidine incorporation assays. CsA inhibited growth of these cells in a dose-dependent manner, and the lowest concentration of CsA tested (0.1 μg/ml) was already inhibitory for the majority of clones. Differences between clones in their susceptibility to CsA as measured in this assay did not correlate with their defined functions. Long term (2–3 week) growth of cloned cells cultured in the presence of CsA was also depressed in a dose-dependent manner; moreover, in the case of a suppressor cell clone, as well as a natural killer-like cytotoxic, and a primed lymphocyte typing clone, function of cells surviving after preculture with CsA was suppressed too. It may be important that NK-like effector function and growth of an NK-like T-cell clone showed more resistance to the inhibitory action of CsA than a PLT clone. These data show that previously alloactivated continuously proliferating Interleukin 2-dependent human T-cell clones are fully susceptible to CsA-mediated inhibition of their function as well as their proliferation. Thus, CsA can exert a strong immunosuppressive effect not only by preventing initial activation of T-cells.

Precursor frequency analysis of human cytolytic T lymphocytes directed against autologous melanoma cells

Limiting numbers of peripheral-blood mononuclear cells (PBMC) from melanoma patients were stimulated with irradiated autologous tumor cells in the presence of interleukins-2 and -4 and in the absence of feeder cells. The responder cells were restimulated every week. After 2 to 4 weeks, the microcultures were tested for their lytic activity against the autologous tumor cells. Significant lysis of the tumor cells was observed with a fraction of these microcultures, whereas no lysis was observed with control microcultures seeded without stimulator melanoma cells. Because our aim was to measure the precursor frequency of CTL showing specificity for the tumor, and not that of NK-like effectors that were also capable of lysing the melanoma cells, we used cold-target inhibition with an excess of NK target K562 to inhibit the NK-like activity. Microcultures whose lysis on the tumor cells was not abolished by K562 competition were observed. The specificity of these CTL clones was confirmed by the absence of lytic activity on autologous T-cell blasts. The numbers of microcultures with anti-tumor CTL activity fitted the zero-order of the Poisson distribution equation, indicating that they resulted from the activity of single T-cell clones. The frequency of anti-tumor CTL precursor cells (CTL-P) of 7 melanoma patients ranged from 1/900 to 1/33,000. Frequencies of anti-tumoral CTL-P were higher and NK-like effectors were less frequent when sorted CD8+ T lymphocytes were used as responder cells.

Identification of an evolutionarily conserved, function-associated molecule on human natural killer cells.

Nonspecific cytotoxic cells (NCC) in teleost fish are analogous to human natural killer (NK) cells and spontaneously lyse a variety of transformed human cell lines sensitive to human NK cell lysis. Monoclonal antibodies (mAbs) made against these NK-like effector cells inhibited their lytic activity. These anti-NCC mAbs were examined with human NK cells for their effects. It was observed that these mAbs bound specifically to a small percentage of peripheral blood lymphocytes (5-15%) and to the majority (greater than 85%) of CD3- NK cells. The mAbs inhibited human NK cell lysis against a variety of transformed cell lines. Single-cell assays showed that the mAbs exerted their effects through inhibition of conjugate formation (recognition). However, the mAbs did not inhibit NK cell-mediated antibody-dependent cell-mediated cytotoxicity. Biochemical analysis of the NK cell molecule revealed that a dimeric structure was involved (distinct from antigen receptors on T cells). Thus, this molecule appears to be a candidate NK cell antigen receptor.

Induction of murine lymphokine-activated killer cells by recombinant IL-7.

The data demonstrate that IL-7, a cytokine that was originally identified, purified, and cloned based upon its ability to support the growth of pre-B cells in vitro, also induces proliferation and promotes the generation of lymphokine-activated killer (LAK) cell activity in populations of resting peripheral lymphoid cells. Although the kinetics of LAK induction by IL-7 (which peaked at days 6 to 8 of culture) was slower than that detected in cultures containing IL-2 (which peaked at day 4), IL-7 was significantly more effective at maintaining cytotoxic activity over longer periods of time, and greater viable cell recoveries, than was IL-2. A wide range of murine tumor target cells were found to be lysed in an MHC-unrestricted fashion by IL-7 induced LAK, but syngeneic Con A-induced lymphoblasts were not; nor were target cells from the human tumors K562 or Daudi lysed by IL-7 LAK. IL-7 LAK were induced in populations of lymphoid cells obtained from secondary lymphoid tissues (peripheral lymph nodes and spleen), but not from primary lymphoid tissues (thymus and bone marrow). LAK induced by IL-7 from unfractionated populations of lymphoid cells were completely eliminated by treatment with anti-CD8 or anti-Thy-1+C, and unaffected by treatment with anti-CD4, anti-asialo GM1 or anti-NK1.1+C. Interestingly, although no detectable CD4+ effector cells could be detected in populations of LAK generated from unfractionated populations of lymphoid cells stimulated by IL-7, they were found to be generated from populations of lymphoid cells from which CD8+ cells had been eliminated before being cultured in medium containing IL-7. These data suggest that CD4+ T cells do not normally give rise to IL-7-induced LAK unless they are first separated from CD8+ T cells. LAK induced by IL-7 appear to be distinct from LAK activity induced by IL-2 in that there is no detectable involvement of NK-like effector cells at either the precursor or effector cell stages.

Ultrastructural and Immunohistologic Characterization of Guanethidine-Induced Destruction of Peripheral Sympathetic Neurons

Repeated daily injections of the adrenergic blocking agent guanethidine in high doses (50 mg/kg) to either newborn or adult rats produces a permanent sympathectomy which is thought to involve an immune-mediated mechanism. Ultrastructural examination of the acute phase of guanethidine-induced neuronal degeneration in the superior cervical ganglion demonstrated a prominent lymphocytic infiltrate in the ganglionic parenchyma and perivascular spaces. Within the satelliting cell sheath there were lymphocytes with delicate processes in intimate contact with abnormal or degenerating principal sympathetic neurons. The lymphocytic infiltrate and early evidence of neuronal degeneration developed within three days of onset of treatment and reached a maximum on the 7th day. Immunocytochemical studies of the ganglion demonstrated that the mononuclear infiltrate was composed primarily of CD-8 positive, OX-19 negative lymphocytes, which may represent a natural killer (NK) or NK-like effector cell, as well as scattered monocytes and macrophages. Guanethidine showed little effect on the prevertebral (superior mesenteric and celiac) sympathetic ganglia compared to the paravertebral (superior cervical) chain ganglia of the same animals. Considerable rat strain selectivity was noted with Lewis and Sprague-Dawley strains showing a greater degree of neuronal destruction than Fischer 344 rats. Neonatal thymectomy failed to prevent neuronal degeneration and the lymphocytic infiltrate. These features of immune neuronal killing are not typical of a characterized antibody or T-lymphocyte mediated effector mechanism, but may represent a selective NK cell or NK-like cell mediated pathogenetic pathway. Guanethidine-induced neuronal destruction appears to be a unique type of drug-induced, cell-mediated, autoimmune attack on sympathetic neurons and may provide insight into autoimmune disorders and possibly into diseases characterized by selective neuronal degeneration.

Specific inhibition of hybrid resistance in F1 hybrid mice pretreated with parent-strain spleen cells. III. Study of the mechanism.

The rejection of H-2b parental bone marrow graft by lethally irradiated F1 recipients, that is known as hybrid resistance (HR), is a multistep process. In a first step a 5-fluorouracil (5-FU)-sensitive T cell recognizes the parental bone marrow cells and stimulates a macrophage-like cell to secrete IFN-alpha/beta (recognition phase). IFN-alpha/beta in turn activates a cyclophosphamide-sensitive NK-like cell that is the effector cell for HR (effector phase). In a previous paper we described that HR is specifically abrogated by the pretreatment of the F1 recipient with H-2b parental spleen cells. This abrogation is due to a Thy-1+CD5+CD4+CD8- nylon adherent suppressor cell of F1 origin. The aim of the present work was to study during which of the different phases of HR the activity of the suppressor cell is exerted. Our results showed that abrogation of HR in (C57BL/6 x C3H)F1 (B6C3F1) hybrids pretreated with B6 spleen cells results from: 1) the suppression of the 5-FU-sensitive T cell; 2) the suppression of the cyclophosphamide-sensitive NK-like cell; and 3) the disappearance of a humoral factor that is present in the serum of normal B6C3F1 hybrids and which seems to be involved in the effector phase of HR. The 5-FU-sensitive T cell is the only target of Thy-1+CD5+CD4+CD8- suppressor cell. The mechanisms responsible for the suppression of the NK-like effector cell and the disappearance of the humoral factor are discussed.

B cells from chronic lymphocytic leukemia (CLL) patients are strong inducers of proliferation and major histocompatibility complex (MHC)-unrestricted [natural killer (NK)-like] cytotoxicity in normal T-lymphocytes

We studied the ability of chronic lymphocytic leukemia (CLL) B cells to stimulate proliferation and major histocompatibility complex (MHC)-unrestricted [natural killer (NK)-like] cytotoxicity in a mixed lymphocyte tumor-cell culture (MLTC). CLL-derived B cells induced a significantly higher proliferative response than did B cells from healthy normal donors. Comparable levels of interferon and interleukin-2 production were detected in the mixed lymphocyte cultures (MLC) set up with normal B cells from healthy donors and in MLTC. Higher levels of NK-like cytotoxic activity were induced after stimulation with CLL than with normal B cells in nonlytic precursors. Inhibition experiments with specific monoclonal antibodies indicate that the NK-like response in MLTC is attributable to HLA class II antigens, which are expressed at comparable levels on CLL and normal B cells. Percoll gradient centrifugation of the MLC and MLTC recovered cells demonstrated that the NK-like effectors in both types of cultures were blast-transformed, highly mitotic cells.

Gut mucosal immunization with reovirus serotype 1/L stimulates virus-specific cytotoxic T cell precursors as well as IgA memory cells in Peyer's patches.

In this report we have shown that reovirus 1/L is an effective mucosal immunogen capable of generating a cytotoxic T cell (CTL) and associated helper T cell response to the nominal antigens associated with reovirus 1/L. The effectors that mediate reovirus-specific cytotoxicity are Thy-1+, Lyt-2+, and major histocompatibility complex (MHC)-restricted in their recognition of reovirus antigens, and can therefore be classified as CTLs. Frequency analysis of precursor CTLs occurring in Peyer's patches (PP) and peripheral lymph nodes (PLN) 6 d and 6 mo after intraduodenal stimulation have demonstrated that a persistent gradient of precursors is established, with higher frequencies present in PP. The generation of a CTL response in PP may be important in preferentially repopulating mucosal tissues with effector CTLs that could result in the local containment of infections in the gut. We also found that reovirus 1/L generates a virus-specific B cell response that is dominated by IgA memory cells after intraduodenal immunization. We hypothesize that the efficacy of reovirus 1/L at stimulating T and B cells in the gut mucosa is related to its ability to selectively enter PP via microfold (M) cells after enteric application. In this study we have also demonstrated that PP cells, upon in vitro culture and unrelated to prior reovirus priming, can generate natural killer-like (NK) cytotoxic activity. This may be an in vitro correlate of the in vivo generation of effectors that may populate mucosal tissues (i.e., the intestinal epithelium) with NK-like effector cells.

Generation of non MHC restricted killing in non-cytotoxic T-lymphocytes from leukaemia patients.

Experiments were undertaken to determine whether the depression of natural killer (NK) activity previously observed in the peripheral blood lymphocytes (PBL) of leukaemia patients in remission extended to NK-like precursors in the blood. T-lymphocytes (E+) from leukaemia patients and normal subjects were depleted of IgG Fc-receptor-bearing (gamma FcR) fresh NK cells by passage over immune complex-coated monolayers. gamma FcR - E + PBL were cultured alone or with DAUDI cells. On day 5 of culture, cytotoxicity toward the NK-sensitive cell lines K562 and MOLT-4 was evaluated in the responder lymphocytes of leukaemia patients and controls. Negligible NK-like cytotoxicity was found in both FcR - E + PBL responder populations cultured alone. By contrast, stimulation with DAUDI induced high levels of K562 and MOLT-4 cytotoxicity in leukaemia as well as in normal responder cells. Complement-mediated cytotoxicity experiments using various McAb demonstrated that in both normal and leukaemia cultures NK-like effectors react with the pan-T OKT3 McAb and with the OKT11 McAB directed to the SRBC receptor, but not with Leu 1 1b and OKM1 McAbs, directed against antigens expressed on peripheral blood NK cells. Fractionation of the responder cells on discontinuous Percoll gradients showed that most of this activity was present in the highly dividing blast cell fraction.

The functional and histological basis for graft-versus-host-induced immunosuppression.

The GvH reaction resulting from the injection of parental strain cells into adult F1 hybrids suppresses both cell-mediated and humoral immune responses and is dependent on the donor-host combination and the number of parental cells used to induce the GvH reaction. The early suppression is due, at least in part, to the increased number of macrophages and the activation of suppressor macrophages which act directly on the T-helper cell and perhaps the B-cell as well. The macrophage suppression is associated with an increase in PGE production. The long-term T-cell immunodeficiency is mediated by GvH-induced thymic dysplasia resulting in a block or an arrest in T-cell differentiation and deficient IL-2 production. The B-cell immunodeficiency is associated with both a decrease in B-cell production from lymphoid progenitors and a decrease in CFU-s production. The GvH reaction induces 2 types of thymic lesion, a stress-related effect causing atrophy of the thymic cortex and a cytolytic process causing severe-to-moderate lesions in the thymic medulla as a consequence of injury to medullary epithelial cells and a loss of Hassall's corpuscles (thymic dysplasia). By employing the NK-cell-deficient beige mutation, it was shown that the severe-to-moderate thymic medullary lesions occurred in F1 mice only in those transplant situations in which the donor inoculum was of the +/bg genotype, regardless of the genotype of the recipient. It is proposed that activation of parental T cells may contribute to the early immunosuppressive events; however, the relatively permanent immunosuppression appears to be associated with NK-like effector cells which are capable of causing injury to lymphoid and epithelial tissue, especially epithelium of the thymic medulla. These studies raise the possibility that the GvH reaction may contribute to some T- and B-cell immunodeficiencies observed in the SCID and AIDS syndromes, as well as in patients following bone marrow transplantation.

NK-like effector cell heterogeneity of peripheral blood mononuclear cells stimulated in vitro with autologous lymphoblastoid cells : David Scrivner, Neville Lee, and Glenn Rodey; American Red Cross, St. Louis, MO

NK-like effector cell heterogeneity of peripheral blood mononuclear cells stimulated in vitro with autologous lymphoblastoid cells : David Scrivner, Neville Lee, and Glenn Rodey; American Red Cross, St. Louis, MO