Natural killer cells in cutaneous malignant melanoma. - PDF Download Free (2024)

JOURNAL OF PATHOLOGY, VOL.

161: 3 5 4 0 (1990)

NATURAL KILLER CELLS IN CUTANEOUS MALIGNANT MELANOMA N. M. KERNOHAN, H. F. SEWELL AND F. WALKER

Department of Pathology. University of Aberdeen. Medical School Buildings, Foresterhill. Aberdeen AB9 2 2 0 , U.K. Received 6 September 1989 Accepted 22 November I989

SUMMARY Using immunocytochemical techniques on fresh surgical samples, a series of 16 cases of cutaneous malignant melanoma (CMM) were examined to characterize further the host inflammatory response. Antibodies to the following cluster of differentiation (CD) antigens were used: CD-3, CD-4, CD-8 (T-cell markers), CD-I lb, CD-14 (macrophage marker), CD-16 [an antigen expressed by natural killer (NK) cells and granulocytes],and CD-25. Also examined were a small number of other melanocytic lesions [two cases of lentigo maligna (Hutchinson’smelanotic freckle) and five of intradermal naevi]. The results of the study document a population of cells with the morphological and immunophenotypic characteristics of NK cells in association with 10 of the 16 cases of CMM. These cells were consistently absent from the other melanocytic lesions studied. The presence of NK cells in association with some cases of CMM bears no clear relationship to the Breslow thickness, Clark level, tumour ulceration, or the presence of activated T cells as determined by expression of the CD-25 antigen. Whilst an explanation for the significantnumbers of NK cells in some CMM lesions is unclear, their presence in intimate association with tumour cells does prompt speculation regarding a possible role in determining the biological behaviour of the turnour. Additionally, the study has confirmed and extended previous findingswith respect to the broad characterization of mononuclear cells present in the host infiltrate associated with CMM. KEY

WoRDs-Cutaneous malignant melanoma, natural killer cells. INTRODUCTION

16 is expressed by N K cells and granulocytes and is reco ized as being a reliable marker for N K cells.’>’ In view of their proposed role in host defence against tumour development and metastasisI6 and also as they are the principal cell type giving rise to lymphokine-activated killer (LAK) cell demonstration of N K cells in relation to lesions of CMM may disclose information of prognostic value.

gn

In view of the enigmatic behaviour of CMM,’ the characteristic phenomenon of tumour regression,’ and the current successful application of adoptive immunotherapeutic techniques to the management of disseminated disease,- there has been much interest concerning the potential role of the host immune response in influencing the biological behaviour ofthe tumour. There are now several reports in the literature that document the immunophenotypic characteristics of the host response to CMM, and all present broadly similar findings.%I4In our MATERIALS A N D METHODS attempts to dissect further the immunophenotypic composition of the host lymphocytic reaction to Tissue CMM, antibodies directed against the CD-I l b and CD- 16 antigens were used. The antigen CD- 1 1b is From fresh surgical excision specimens, tissue expressed on natural killer (NK) cells, putative T- bearing a representative portion of a melanocytic suppressor cells, monocytes, and granulocytes; CD- skin lesion along with adjacent normal skin was Addressee for correspondence:Dr N. M. Kernohan, Lkpart- donated for immunohistochemical study. The tismerit of Pathology, University Medical Buildings, Foresterhill, sue, which was 1-2 mm in maximum thickness, was Aberdeen AB9 2ZD. U.K. snap-frozen in Arcton (ICJ)/liquid nitrogen and 0022-34 I7/90/050035-06$05.00 0 1990 by John Wiley & Sons, Ltd.

s.M. KERNOHAN E r AL.

36 Table I-Source,

specificity. and working dilution of the antibodies utilized in this study

Antibody

Specificity

Source

Working dilution

-~

CD-3 CD-4

Oxoid Dakopatts

CD-8 CD-IIb(Leu 15)

Dakopatts Becton-Dickinson

CD-I6(Leu Ilb)

Becton-Dickinson

CD-14 CD-25 Rabbit anti-mouse (RAM) immunoglobulins Alkaline phosphatasei anti-alkaline phosphatase (APAAP complex)

Oxoid Dakopatts Dakopatts Dakopatts

stored at - 20°C double-wrapped in 'Parafilm' until required for sectioning and staining. Immunohistochemical method Cryostat cut sections, 4 - 6 ~thick on chrome alum gelatin-subbed slides, were used throughout. The source, specificity, and working dilution of the antibodies used in this study are listed in Table I. All dilutions and washes were made using 0.005 M Tris-buffered saline (TBS), pH 7.6. Serial sections were cut from the lesions and stained using the antibodies listed in Table I in a standard three-stage APAAP technique,"-*' following fixation in acetone for 20 min, after which they were allowed to air-dry for 5 min. The alkaline phosphatase was developed as previously described'' using Fast Red TR salt (Sigma) as the chromogen, which provided excellent contrast with the light haematoxylin nuclear counterstain and endogenous melanin. Optimal demonstration of CD- 16 required the use of an enhanced APAAP technique, whereby the second and third stages (RAM and APAAP, respectively) were repeated with 15 min incubations prior to development of the enzyme reaction. Assessment of results A negative control slide where TBS was substituted for the monoclonal antibody was available for each case. This was of particular value when assessing sections stained using an enhanced APAAP technique. which, although intensifying specific positive

Pan-T T helperlinducer + some macrophageimonocytes T suppressorjcytotoxic T suppressor cells, NK cells, monocytes, granulocytes Fc IgG receptor on NK cells and neutrophils Monocytes Interleukin-' receptor -

1:200 1:lO

1:lO 1:lO

1:lO 15 1:lO 1 :20 1 :40

staining, may give rise to background staining without rigorous attention to the washing of sections between each stage of the immunohistochemical procedure. Positive control slides were available from other cases whose lymphocytic infiltrate had been characterized previously. Where cells expressing CD- 16 were identified, reference was made to other sections from the case and it was confirmed that the cell population did not express markers indicative of T-cell (CD-3, CD-4, CD-8) or macrophage (CD-14) lineage. As CD-11 b is expressed by putative T-suppressor cells, monocytes, granulocytes, and NK cells. demonstration of apparent co-expression of CD-11 b and CD-16 was taken as evidence that cells so stained were NK cells. Many more cells expressed CD-11 b than CD-16, but as expected the distribution of the excess CD1 1 b positive cells coincided with that of T-cells, macrophages, and granulocytes. As CD-I 1 b and CD- 16 may also be co-expressed by polymorphonuclear granulocytes as well as by mononuclear NK cells, the morphological appearances of the positively stained cells were carefully noted. Furthermore, to ascertain that CD-16/CD-I 1b positively stained cells were NK cells and not granulocytes, sections were exposed for 6 min to 3',3'-diaminobenzidine tetrahydrochloride solution, a substrate for the endogenous peroxidase enzyme present in granulocytes but absent in NK cells. The intense brown peroxidase-substrate reaction product in granulocytes could be easily distinguished from

NATURAL KILLER CELLS IN CUTANEOUS MELANOMA

Table 11-Distribution of CD-16/CD-11b positive cells in the inflammatory infiltrate associated with melanocytic skin lesions correlated with the presence or absence of ulceration of the lesion and CD-25 positive cells in the infiltrate

Lesion

Nos.

CD-16 positive cells

~~

CMM HMF IDN

Ulceration

+

-

Incipient

CD-25 positive cells

1 0 0

16 2 5

~

16 2 5

10 0 0

5 4 0 2 0 5

37

one showed incipient ulceration, and four were non-ulcerated. Of the remaining six cases, five were non-ulcerated and one showed incipient ulceration. Ulceration was defined histologically (Tables I1 and 111) using routinely processed haematoxylin and eosin stained tissue sections. From these sections the histogenetic type of melanoma, its Clark level, and Breslow thickness were also assessed, but none of these parameters correlated with the presence of NK cells (results not shown).

DISCUSSION

In this study a significant population of cells displaying the morphological and immunophenotypic characteristics (CD-1 lb’, CD-16+, CD-3-, CD14-, peroxidase-) that correlate with functional NK cells has been demonstrated in association with 10 of 16 primary CMM lesions. In addition, the absence of such cells from both cases of lentigo RESULTS maligna, a premalignant lesion, and all cases of A population of mononuclear cells expressing benign, intradermal naevi was recorded. the CD-16 antigen was identified in association with NK cells form a distinct subset of lymphoid cells 10 of the 16 cases of CMM (Fig. 1). These cells with the morphological appearance of large granuaccounted for up to 10 per cent of the chronic lar lymphocytes, and for which the CD-16 antigen inflammatory cell infiltrate and were found princi- is recognized as being the most reliable immunopally within the substance of the tumour, either phenotypic marker, in preference to anti-HNK-1 among tumour cells or in the stroma between used in previous studies.” NK cells are capable of groups of tumour cells. A minority of the cells were non-MHC restricted killing of target tumour cells, present in the inflammatory infiltrate deep in the in the absence of specific antibody or antigenic tumour. CD-16 positive cells were also present near stimulation. The identification of such cells in the ulcerated surface of some tumours, but closer association with primary CMM lesions may thereexamination indicated that most of the stained cells fore be seen as a potentially favourable feature of in this instance were granulocytes. No case of LM or the host response to the tumour. Furthermore, the IDN displayed a similar CD- 16 positive cell popu- presence of NK cells, often in intimate association lation in the inflammatory infiltrate (Table 11). with tumour cells (Fig. 1) is of considerable interest Serial sections from each case were examined and in view of the development of adoptive immunoconfirmed that T-cell markers were not expressed by therapy in the treatment of disseminated malignant the CD-16 positive cells (Fig. 2). Co-expression of melanoma. Such therapy requires the administhe CD- 16 and CD- 1 1b antigens was, however, tration ofcombinations of the cytokine interleukin-2 noted but CD-1 l b positive cells were more abun- (IL-2) and autologous lymphokine-activated killer dant than CD-16 positive cells. Immunopheno- cells (LAK cells). NK cells are now known to be typic, histochemical, and morphological control the principal precursors of LAK cell^,'^.'^ and criteria (see above) documented the excess CD-1 l b tumours already containing a significant population cells as a mixture of T-cells, macrophages, and, in of these LAK cell precursors (10 of 16 cases in the some cases, granulocytes. present study) might be expected to respond well Lymphoid cells expressing the CD-25 (inter- to immunotherapeutic approaches as pioneered by leukin-2 receptor) antigen were present in all cases Rosenberg. Despite attempts to refine such treatof CMM, LM, and IDN studied. Of the ten cases of ment rtgimes,* adoptive immunotherapy is associCMM containing CD-16/CD-l1b positive cells in ated with si nificant morbidity and, occasionally, the inflammatory infiltrate, five were ulcerated, Therefore additional information melanin pigment present in some sections. Thus, the criteria used clearly demonstrated the presence, immunophenotypically and morphologically, of CD- 16/CD-11b positive ‘NK’ cells.

38

N. M. KERNOHAN E T A L .

Fig. I X M M associated with mononuclear cells expressing CD-16 (Leu Ilb). Many of these cells lie within a nodule of turnour cells (anti-CD-16. enhanced APAAP)

Fig. 2-Same area from the same case as in Fig. 1 to show T cells; the cell population stained by anti-CD-16 is distinct (anti-CD-3. APAAP)

39

NATURAL KILLER CELLS IN CUTANEOUS MELANOMA

Table 111-Presence or absence of ulceration in cases of CMM correlated with expression of CD-16/CD-I1b by cells in the host inflammatory response CD-I 6/CD-1 I b (+)

CD-i 6/CD-11b ( - )

5

4 1

5

Ulceration No ulceration Incipient ulceration

that may be gleaned by immunohistochemical analysis of pretreatment biopsies might contribute to the rational use of such therapy. NK cells have previously been recognized in as~ sociation with melanoma. Kornstein et ~ 1 . , *using another CD-16 antibody, found NK cells in 1 of 8 primary LMM lesions, 10 of 12 metastatic deposits, and only 3 of 3 1 ‘dysplastic naevi’. The conclusion drawn was that NK cells probably did not play a major role in surveillance preventing the ‘dysplastic’ lesions progressing to invasive melanoma. Using an anti-CD-16 antibody with an enhanced APAAP technique, in this study we have demonstrated NK cells in a higher proportion of primary lesions but, like Kornstein er al., failed to detect them in association with the premalignant lesion, lentigo maligna. A further small series of six cases was reported as part of a study by Cohen et ~ l . , ~where ’ NK cells were rarely found in association with metastatic melanoma lesions either before or after adoptive immunotherapy with rIL-2 and LAK cells. Yamamura et u I . , ~ ‘ however, found that in a case of metastatic melanoma treated by a similar adoptive immunotherapeutic regime NK cells were present in large numbers after treatment in the tumour deposits, a finding contrary to that reported in Cohen et al.’s series. Thus, the biological role of NK cells in relation to primary and metastatic melanoma lesions and their presence in response to immunotherapy is unclear. NK cell recruitment to the site of a tumour could be the result of the release of chemical mediators by non-specific inflammatory and immunological reactions.” Such mediators include activated T-cell derived interleukin-2 and y-interferon. Activated T lymphocytes have been found in association with melanocytic lesions, both benign and malignant, and in this study they were present in all cases. Accordingly, neither the presence of CD-25 positive T-lymphocytes per se nor prognostic factors (such as the histogenetic type of tumour, its Clark level, Breslow thickness, or ulceration) correlated with the presence of NK cells.

1

In conclusion, this study has demonstrated NK cells in association with some cases of CMM. Such cells were absent from the small number of other melanocytic lesions studied including two cases of LM, a premalignant lesion. These findings prompt speculation concerning the possible role of NK cells in determining not only the biological behaviour of melanoma, but also its response to adoptive immunotherapy regimes. ACKNOWLEDGEMENTS

This work was supported through funding by the Scottish Hospitals Endowment Research Trust and by Grampian Health Board, who generously supplied the antibodies used. The authors wish to express their thanks to Mr I. R. Kernohan, who provided fresh excision biopsy specimens for use in this study; and to Mrs I. M. Watson for typing this manuscript. The expert technical assistance of Shirley Sinclair and George King is gratefully acknowledged. REFERENCES I . Elias EG. Didolkar MS. Goel IP, e r a / . A clinico-pathologx study of prognostic factors in cutaneous malignant melanoma. SurR Gynecol Obsrer 1977; 144: 327-334. 2. McGovern VJ. Spontaneous regression of malignant melanoma. In. Melanoma Hisrolagical Diagnosis and Prognosis. New York: Raven Press. 1982: 138-147. 3. Lotzc MT. Chang AE. Seipp CA. er a / . High dose recombinant interleukin-2 in the treatment of patients with disseminated cancer. J Am MedAssoc 1986;u6:3117-3124. 4. Oliver RTD. Theclinical potential of interleukin-2. Br J Cancer 1988: 58:4oM. 5. Rosenberg SA. Adoptive immunotherapy of cancer: accomplishments and prospects. Cancer Treat Rep 1985: 68: 233-255 6 . Rosenberg SA. Lotze MT, Muul LM, er al. Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. h‘ EngfJ Med 198%313 1485-1492. 7. Rosenberg SA. Lotze MT. Muul LM. ei a/. A progress report on the treatment of I57 patients with advanced cancer using lymphokineactivated killer cellsand interleukin-? or high dose interleukin-2 alone. N €ng/ J Med 1987: 316 889-897.

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N. M. KERNOHAN ET AL.

8 West WH. Tauer KW. Yannelli JR. er a/. Constant infusion of recom-

9. 10.

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binant interleukin-2 in adoptive immunotherapy of advanced cancer. gYEngl J Med 1987; 316 898-905. Femandez-Bussy R, Cambazard F. Manduit G. Schmitt D. Thivolet J. T-cell subsets and Langerhans cells in skin tumours. Eur J Cancer Clin Oncol1983: 19907-913. Hersey P, Murray E. Grace J. McCartney WH. Current research on immunopathology of melanoma: analysis of lymphocyte populations in relation to antigen expression and histological features of melanoma. Purhology 1985: 17: 3E-391. Kernohan NM. Sewell HF. Interleukin-2 receptor expression in benign and malignant melanocytic skin lesions. J Purhol 1989; 157: 315-319. Kornstein MJ. Brooks JSJ. Elder DE. lmmunoperoxidase localisation of lymphocyte subsets in the host response to melanoma and nevi. Cancer Res 1983: 43 2749-2753. Poppema S, Brkher E-9. Dc-Leu L, c6 ul. In siru analysis of the mononuclear cell infiltrate in primary malimant melanoma of the skin. Clin Exp lmmunol1983; 51: 77-82. Rallkiaer E, Hou-Jensen K, Gatter KC. Dmwiecki KT. Mason DY. Immunohistological analysis of the lymphoid infiltrate in cutaneous malignant melanoma. Virrhows Arch A 1987; 410 355-361 Alvarado CS. Findley HW. Chan WC, er a/. Natural killer cells in children with malignant solid tumors. ERect of recombinant interferon-a and interleukin-? on natural killer cell function against tumor cell lines. Cunter 1989: 63:83-89. Herberman RB. Natural killercells. Annu Rev Med1986; 37:347-352. Lanier LL. Le AM. Phillips JH. Warner NL, Babcock GF. Subpopulalions of human natural killer cells defined by expression of the Leu-7(HNK-I)and Leu-I 1 (NKP-15)antigens. JImmunol1983; 131: 1789-1795. Anon. Interleukin-2: sunrise for immunotherapy? Lanrer 1989; 1: 308.

19. Herberman RB, Hixrodt J. Vujanovic N. et a/. Lymphokine-activated killer cell activity. Characteristics of effector cells and their progenitors in blood and spleen. Immunof Toduy 1987: %: 178-181 20. Cordell JL. Falini 9, Erber WN. er ul. lmmunoenzymatic labelling of monoclonal antibodies using immune complexes of alkaline phosphalase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Hisrochem Ctvochem 1984: 3 2 219-229. 21. Ponder BA. Wilkinson MM. Inhibition ofendogenous tissue alkaline phosphatase with the use of alkaline phosphatase conjugates in immuno-histochemistry. J Hisrochem Cyrochem 1981: 2 9 981-984. 22. Belldegrun A. Webb DE. Austin HA, et al. Effects of interleukin-? on renal function in patients receiving immunotherapy for advanced caner. AnnInc Med 1987; 1W817-822. 23. Ettinghausen SE, Moore JG. White DE. er ul. Hematological effects of immunotherapy with lymphokine-activated killer cells and recombinant interleukin-2 in cancer patients. Blood 1987; 69: 16%1660. 24. Ognibene FP, Rosenberg SA. Lotze M, el ul. Interleukin-? administration causes reversible hemodynamic changes and left ventricular dysfunction similar to those seen in septic shock. Chest 1988: 94: 98 1-984. 25. Webb DE, Austin HA 111, Belldegrun A. ef al. Metabolic and renal affects of interleukin-2 immunotherapy for metastatic cancer. CIin Nephrol1988; 39: 141-145. 26. Kornstein MJ. Stewart R. Elder DE. Natural killer cells in the host response tomelanoma. Canrer Res 1987;47: 141 1-1412. 27. Cohen PJ. L o w MT. Roberts JR. er a/. The immunopathology of sequential tumor biopsies in patients treated with interleukin-2. Am J P ~ t h o l1987; 1 2 9 208-216. 28. Yamamura T, Fujitani Y. Kawanchi T, er a/. Histological evidence of natural killer cell aggregation against malignant melanoma induced by adoptive immunotherapy with lymphokine-activated killer cells. J P ~ t h o 1989; l 1 5 7 201-204.

Natural killer cells in cutaneous malignant melanoma. - PDF Download Free (2024)

FAQs

What vitamin is natural killer cells? ›

Vitamin D and Exercise Are Major Determinants of Natural Killer Cell Activity, Which Is Age- and Gender-Specific.

How can I get more natural killer cells? ›

Regular cardiovascular exercise, strength training, eating more antioxidants, massage therapy, and more could all potentially increase the natural killer cell levels. These lifestyle changes may be able to stimulate natural killer cell activity and encourage the body to produce more natural killer cells.

Do natural killer cells eliminate malignant cells? ›

NK cells are first-responder immune cells. When enough of their activating receptors are triggered, they mobilize to kill infected, stressed, or cancerous cells at an early stage, before T-cell responses kick in.

Does natural killer cells work? ›

Natural killer cells (NK cells) are white blood cells that destroy infected and diseased cells, like cancer cells. They're also a type of lymphocyte, like B-cells and T-cells. NK cells can destroy harmful cells in the early stages, preventing viruses and cancer cells from spreading.

What foods boost natural killer cells? ›

Selenium: A deficiency of this mineral reduces the number of natural killer cells, while supplemental selenium has been shown to increase their activity. You need 55 micrograms per day. Good sources include tuna, halibut, shrimp, and brown rice.

Does vitamin D increase NK cells? ›

Vitamin D Enhances Immune Effector Pathways of NK Cells Thus Providing a Mechanistic Explanation for the Increased Effectiveness of Therapeutic Monoclonal Antibodies.

What enhances natural killer cells? ›

The administration of anti-PD-1 antibody (Pembrolizumab) has been shown to enhance NK cell-mediated cytotoxicity against multiple myeloma. Pembrolizumab enhances the interaction between patient-derived NK cells and myeloma cells expressing PD-L1, which was associated with increased production of granzyme B and IFNγ.

What are the symptoms of NK cell deficiency? ›

What are the symptoms of NK cell disorders? Frequent, recurrent infections, usually lungs (pneumonia) and viral (herpes virus) are the most common symptoms of NK cell disorders. They are at increased risk for developing cancer.

What gives rise to natural killer cells? ›

NK-cell precursors (NKPs) lack typical NK-cell markers but give rise to NK cells. Immature NK cells express CD161 and natural-killer group 2, member D (NKG2D) in both mice and humans, as well as receptors required for growth and survival.

What autoimmune disease has NK cells? ›

Natural killer (NK) cells, which are key components of the innate immune system, have been implicated in the development of multiple autoimmune diseases such as systemic lupus erythematosus, type I diabetes mellitus, and autoimmune liver disease.

What is the success rate of NK cell therapy? ›

Researchers also observed durable responses with CAR NK cell treatment. One year after treatment, complete responses were seen in 83% of patients with low grade-NHL, 50% of patients with CLL and 29% of patients with DLBCL.

What is NK cell therapy? ›

Natural killer cell therapy is a type of cellular immunotherapy, just like CAR T-cell therapy. Like T-cells, NK cells form part of the immune system and attack germs and other malignant cells. Unlike T-cells, however, NK cells are not tailored to specific antigens.

How do you stimulate natural killer cells? ›

Factors that May Raise Natural Killer Cell Function
  • Exercise [16]
  • Strength training [36]
  • Antioxidants [37]
  • Massage therapy [17]
  • White button mushrooms [38]
Sep 18, 2020

What is another name for a natural killer cell? ›

Natural killer cells, also known as NK cells or large granular lymphocytes (LGL), are a type of cytotoxic lymphocyte critical to the innate immune system. They belong to the rapidly expanding family of known innate lymphoid cells (ILC) and represent 5–20% of all circulating lymphocytes in humans.

What activates NK cells? ›

Two signaling molecules that are critical for NK cell activation are Vav1 and PLC-γ2. NK cells in Vav1-deficient mice are not as impaired as T cells but show defects in tumor cell killing (77).

Does vitamin C increase NK cells? ›

Ascorbic acid (vitamin C) was found to inhibit human natural killer cell (NK cell) activity in a dose- dependent manner.

How do you treat NK cell deficiency? ›

There is no specific treatment for NK cell disorders. Children with the disease will usually require more frequent courses of antibiotics, or other medications to fight off viral or fungal infections. There appears to be evidence to suggest other therapies may enhance immune system functioning.

What is the source of natural killer cells? ›

Natural killer or NK cells are a sub-population of Large Granular Lymphocytes (LGLs) which arise from a common NK/T-cell progenitor. Accumulating evidence indicates that NK cells can develop and mature both in the bone marrow and secondary lymphoid tissues (SLTs) including tonsils, spleen, and Lymph nodes (LNs)[1].

What reduces natural killer cells? ›

Sleep disturbance, measured by either subjective report or electroencephalographic (EEG) assessment of sleep, correlates with a reduction of natural killer (NK) cell activity in major depression.

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